Characterization of Drug-Protein Interactions using Non-Linear

Chromatography and Surface Plasmon Resonance Technologyndash a Comparative Study

JOHANNA AringSTRAND

Masterrsquos Degree ProjectStockholm Sweden 2004

TRITA-NA-E04071

Numerisk analys och datalogi Department of Numerical AnalysisKTH and Computer Science100 44 Stockholm Royal Institute of Technology

SE-100 44 Stockholm Sweden

JOHANNA AringSTRAND

TRITA-NA-E04071

Masterrsquos Thesis in Biomedical Engineering (20 credits)at the School of Mechanical EngineeringRoyal Institute of Technology year 2004

Supervisor at Nada was Erik FranseacutenExaminer was Anders Lansner

Characterization of Drug-Protein Interactionsusing Non-Linear Chromatography

and Surface Plasmon Resonance Technologyndash a Comparative Study

Abstract In this work High-Performance Liquid Chromatography HPLC and Biacore 2000 have been used to measure the interaction between proteins (large molecules) and selected drugs with small molecules (ibuprofen alprenolol and propranolol) The aim of this work was to evaluate what can be determined using the different methods how good the agreements are and how the methods complement each other We also compared the parameters from Biacore HPLC and micro calorimetric techniques With micro calorimetric technique it is possible to measure heat changes during different experiments The reason to this comparative study was to see if the different parameters could be used in all three systems Chromatography is an important separation process in chemistry HPLC is a more developed and a faster type of chromatography A solute containing different components (mobile phase) flows down a column (stationary phase) that separates the components In Biacore the technique is based on Surface Plasmon Resonance SPR This detects a mass concentration of a certain component on a microchip The protein we used is human serum albumin HAS purified from human blood The albumin was bounded to both a chromatography stationary phases and to a Biacore CM5 microchip With this we studied small molecules with chiral properties With Biacore the enantiomers were studied one at a time while racemic mixtures have been studied with chromatography (using a perturbation method and numerical tools) Very high concentrations were used so that the immobilised proteins were close to saturation The two methods and their data acquired were compared Unfortunately this study was interrupted because of instabilities in the chromatographic study and we did not have enough time to repeat the experiments The instabilities depend on several factors The protein was perhaps denaturised and therefore became inactive This happens naturally after a while Physical stress too high concentration of modifier or a bad environment for active proteins could also be factors Furthermore we could see that we more easily got a separation of the enantiomers in ibuprofen than with the two beta-blockers alprenolol and propranolol The reason for that is that albumin more easily binds to acids

En jaumlmfoumlrande studie av interaktion mellan laumlkemedel och proteiner med hjaumllp av icke linjaumlr kromatografi och ytplasmaresonansteknik Sammanfattning I det haumlr arbetet har High-Performance Liquid Chromatography HPLC och Biacore 2000 anvaumlnts foumlr att maumlta interaktionen mellan proteiner (stora molekyler) och utvalda laumlkemedel bestaringende av smaring molekyler (ibuprofen alprenolol och propranolol) Vi har utfoumlrt experiment foumlr att ta reda paring hur transportproteinet albumin binder olika laumlkemedel Maringlet med arbetet aumlr att jaumlmfoumlra de tvaring metoderna hur bra resultaten oumlverensstaumlmmer samt hur det tvaring systemen kompletterar varandra Vi har aumlven jaumlmfoumlrt parametervaumlrden mellan Biacore HPLC samt mikrokalorimetri Kalorimetern har till uppgift att maumlta vaumlrmefoumlraumlndringar i olika experiment Detta foumlr att se om det aumlr moumljligt att dra paralleller och daumlrmed vara moumljligt att anvaumlnda parametrarna mellan de olika systemen Kromatografi aumlr en viktig separationsprocess inom kemin och garingr ut paring att en loumlsning (mobil fas) passerar en kolonn (stationaumlr fas) och daumlr med kan loumlsningens olika komponenter separe- ras HPLC aumlr en mer utvecklad och snabbare variant av kromatografi Biacore 2000 bygger i sin tur paring en helt annan teknik Surface Plasmon Resonance SPR vilket innebaumlr en detektion av masskoncentrationen av ett visst aumlmne immobiliserat paring ett mikrochip Maumlnskligt albumin (HSA) fraringn renat blod har anvaumlnts Albuminet har bundits till en kromato- grafisk stationaumlr fas samt till ett mikrochip CM5 i Biacoresystemet Laumlkemedel saring som det verksamma aumlmnet ibuprofen i vaumlrktabletter samt betablockerarna alprenolol och propranolol har studerats Samtliga tre har kirala egenskaper Med Biacore har enatiomererna studerats var foumlr sig medan racematen har studerats samtidigt med kromatografi Data fraringn de tvaring separa- tionsmetoderna har jaumlmfoumlrts Maringlet med arbetet var att vaumlrdera de olika resultaten bedoumlma eventuella skillnader och hur de tvaring metoderna kompletterar varandra Tyvaumlrr fick studien avbrytas pga att det kromatogra- fiska systemet var instabilt och vi hade ingen tid kvar foumlr att upprepa foumlrsoumlken Instabiliteten kan bero paring en maumlngd orsaker men vi utesluter inte att proteinerna kan ha denaturerat dvs skrumpnat ihop och blivit inaktiva Orsaken till detta kan vara att detta helt enkelt haumlnder naturligt efter en tid Andra orsaker kan vara fysik stress av naringgon sort foumlr houmlg koncentration av modifierare eller annan ej gynnsam miljouml foumlr aktiva proteiner Vi kunde aumlven konstatera att separation uppnarings laumlttare med ibuprofen aumln de tvaring betablockerarna alprenolol och propranolol Orsaken till detta aumlr att albumin laumlttare binder till syror

Table of contents 1 INTRODUCTION 1

11 Human Serum Albumin HSA 1 12 Chiral drugs 1 13 Adsorption chromatography 2

2 HPLC ndash HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 3

21 The history of HPLC 3 22 Mobile Phase 3 23 Stationary Phase 4 24 Injectors for HPLC 4 25 Pumps 4 26 Detectors 5 27 Theory of HPLC 5

3 BIACORE 2000 10

31 SPR 10 32 Biosensor chip 11 33 Optical system 11 34 IFC - integrated micro-fluidic cartridge 12 35 Sensorgram 12 36 Theory 12

4 CALORIMETRY 15

41 Thermodynamics and kinetics 15 42 The thermopile heat-conduction principle 15 43 The heat flow signal and the heat quantity 16

5 METHODS 17

51 HPLC 17 52 Biacore 2000 18

6 RESULTS 19

61 HPLC 19 62 Biacore 2000 30

7 DISCUSSION 32 8 REFERENCES 33

1

1 Introduction Proteins are molecules consisting of long chains of amino acids For a certain protein the chain is folded in a certain way and therefore has a unique structure The pharmaceutical industry is interested in these different structures because the information in the structures can give them clues how the protein works In this way a drug can be constructed which interact with the protein in a good way Interaction of a drug with and competition for the binding site on a plasma protein might strongly affect its distribution elimination as well as pharmacodynamics and toxic properties Competition between two drugs for their binding to a plasma protein can strongly affect the drug disposition of both drugs with possible serious physiological consequences One problem is that many potential drug molecules tightly bind into the protein that they loose their efficiency in the human body By knowing how different substances binds to proteins and how strong the bindings are the pharmaceutical industry is able to construct drugs with higher effect and optimal compability with the body and other drugs [10] Human serum albumin (HSA) is an interesting protein from that approach

11 Human Serum Albumin HSA One of the most important drug transport protein in human plasma is human serum albumin (HSA) This transport protein is a monomeric protein made up only of amino acids Albumin plays a fundamental role in the transport of drugs metabolites and endogenous ligands HSA contributes to osmotic pressure in the blood stream and provides most of the acidbase buffering action of the plasma proteins In general acidic drugs bind mainly to albumin while basic drugs such as most beta-blocking agents have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma) [4]

12 Chiral drugs Chiral compounds contain at least one asymmetric carbon atom or are molecularly asymmetric They exist in two enantiomorphic forms related as object and mirror images like left and right hand This type of molecules have the same physical and chemical properties and differ only in their interaction with plane-polarised light such as dextrorotatory (+) and the other laevorotatory (-) There are a number of conventions for indicating the spatial configuration as opposed to optical properties of enantiomers The classical D and L system for monosaccharide and amino acids can not be applied easily to other structures and the Cahn-Ingold-Prelog systems which assign R (rectus) or S (sinister) configurations to an enantiomer are of more general use Until recently it has not been possible to resolve mixtures of enantiomers and this has created problems for the pharmaceutical industry in its development and clinical use of drugs many of them chiral Although enantiomers have identical chemical and physical properties they are distinguishable biologically Thus they differ in their ability to interact with the receptors involved in a range of physiological responses and they are often metabolised and excreted at different rates Chromatographic techniques have now been developed that allow mixtures of enantiomers to be dissolved [14]

2

13 Adsorption chromatography In 1903 M Twett first applied adsorption chromatography to the separation of plant pigments using a hydrocarbon solvent and inulin powder (a carbohydrate) as stationary phase [15] The separation of coloured bands led to the name chromatography from the Greek word chromatos meaning ldquocolourrdquo Adsorption chromatography operates on the basis of polarity Compounds that possess functional groups capable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compounds Thus less polar compounds will elute from the column faster than compounds that are highly polar The classic chromatography builds on the principal that one phase is stationary while the other one moves past it Figure 11 shows a solution containing solutes A and B placed on top of a column packed with solid particles and filled with solvent When the outlet opens solutes A and B flow down into the column Fresh solvent applies to the top of the column and the mixture washes down the column by continuous solvent flow If solute A more strongly adsorbs than solute B on the solid particles the solute A spends a smaller fraction of time free in the solution Solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components The mobile phase (the solvent moving down the column) in chromatography is either a liquid or a gas The stationary phase is most commonly a viscous liquid coated on the inside of a capillary tube or on the surface of solid particles packed into the column Alternatively as in fig11 the solid particles themselves may be the stationary phase Columns are either packed or open tubular A packed column is filled with particles containing stationary phase as in fig11 An open tubular column is a narrow hollow capillary with stationary phase coated on the inside walls

Fig 11 A solution containing solutes A (light grey band) and B (dark grey band) flows trough a column Here solute A adsorbs more strongly to the solid particles than solute B Therefore solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components

3

2 HPLC ndash High Performance Liquid Chromatography At present high performance liquid chromatography HPLC is the most important separation method and analytical technique The wide applicability speed and high sensitivity of HPLC has made this the most popular form of chromatography and virtually all types of biological molecules have been assayed or purified using this technique Modern HPLC has many applications including separation identification purification and quantification of various compounds HPLC is widely considered to be a technique mainly for biotechnological biomedical and biochemical research as well as for the pharmaceutical industry Currently HPLC is used by a variety of fields including cosmetics energy food and environmental industries

21 The history of HPLC HPLC has undergone a period of rapid development Until the 1970 only gas chromatography could provide the same high performance as HPLC does today High-pressure liquid chromatography was developed in the mid-1970 and quickly improved with the development of column packing materials and the additional convenience of on-line detectors In the 1980s HPLC began to be commonly used for the separation of chemical compounds New techniques improved separation identification purification and quantification far above the previous techniques Computers and automation added to the convenience of HPLC Improvements in type of columns and thus reproducibility were made as such terms as micro-column affinity columns and Fast HPLC began to immerge [2]

22 Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied into the column or stationary phase The mobile phase acts as a carrier for the sample solution A sample solution is injected into the mobile phase of an assay through the injector port As the sample solution flows through a column with the mobile phase the components of that solution migrate according to the non-covalent interactions of the compound with the column The chemical interactions of the mobile phase and sample determine the degree of migration and separation of components contained in the sample A sample that bounds stronger to the mobile phase than with the stationary phase elutes faster from the column This gives a shorter retention time [12] The range of solvents that can be used in HPLC is wide Water and aqueous buffer solutions are common and some non-aqueous solvents of low viscosity are also used High viscosity solvents are avoided because they require longer time to pass through the column which results in peak broadening and poorer resolution This also requires a higher pressure to force the solvent through the column Solvents for use in HPLC must be free of particles and dissolved air To ensure that sample is free from particles it is usually passed through a Millipore filter Degassing the sample is made in a water bath with UV-sound [16]

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

Numerisk analys och datalogi Department of Numerical AnalysisKTH and Computer Science100 44 Stockholm Royal Institute of Technology

SE-100 44 Stockholm Sweden

JOHANNA AringSTRAND

TRITA-NA-E04071

Masterrsquos Thesis in Biomedical Engineering (20 credits)at the School of Mechanical EngineeringRoyal Institute of Technology year 2004

Supervisor at Nada was Erik FranseacutenExaminer was Anders Lansner

Characterization of Drug-Protein Interactionsusing Non-Linear Chromatography

and Surface Plasmon Resonance Technologyndash a Comparative Study

Abstract In this work High-Performance Liquid Chromatography HPLC and Biacore 2000 have been used to measure the interaction between proteins (large molecules) and selected drugs with small molecules (ibuprofen alprenolol and propranolol) The aim of this work was to evaluate what can be determined using the different methods how good the agreements are and how the methods complement each other We also compared the parameters from Biacore HPLC and micro calorimetric techniques With micro calorimetric technique it is possible to measure heat changes during different experiments The reason to this comparative study was to see if the different parameters could be used in all three systems Chromatography is an important separation process in chemistry HPLC is a more developed and a faster type of chromatography A solute containing different components (mobile phase) flows down a column (stationary phase) that separates the components In Biacore the technique is based on Surface Plasmon Resonance SPR This detects a mass concentration of a certain component on a microchip The protein we used is human serum albumin HAS purified from human blood The albumin was bounded to both a chromatography stationary phases and to a Biacore CM5 microchip With this we studied small molecules with chiral properties With Biacore the enantiomers were studied one at a time while racemic mixtures have been studied with chromatography (using a perturbation method and numerical tools) Very high concentrations were used so that the immobilised proteins were close to saturation The two methods and their data acquired were compared Unfortunately this study was interrupted because of instabilities in the chromatographic study and we did not have enough time to repeat the experiments The instabilities depend on several factors The protein was perhaps denaturised and therefore became inactive This happens naturally after a while Physical stress too high concentration of modifier or a bad environment for active proteins could also be factors Furthermore we could see that we more easily got a separation of the enantiomers in ibuprofen than with the two beta-blockers alprenolol and propranolol The reason for that is that albumin more easily binds to acids

En jaumlmfoumlrande studie av interaktion mellan laumlkemedel och proteiner med hjaumllp av icke linjaumlr kromatografi och ytplasmaresonansteknik Sammanfattning I det haumlr arbetet har High-Performance Liquid Chromatography HPLC och Biacore 2000 anvaumlnts foumlr att maumlta interaktionen mellan proteiner (stora molekyler) och utvalda laumlkemedel bestaringende av smaring molekyler (ibuprofen alprenolol och propranolol) Vi har utfoumlrt experiment foumlr att ta reda paring hur transportproteinet albumin binder olika laumlkemedel Maringlet med arbetet aumlr att jaumlmfoumlra de tvaring metoderna hur bra resultaten oumlverensstaumlmmer samt hur det tvaring systemen kompletterar varandra Vi har aumlven jaumlmfoumlrt parametervaumlrden mellan Biacore HPLC samt mikrokalorimetri Kalorimetern har till uppgift att maumlta vaumlrmefoumlraumlndringar i olika experiment Detta foumlr att se om det aumlr moumljligt att dra paralleller och daumlrmed vara moumljligt att anvaumlnda parametrarna mellan de olika systemen Kromatografi aumlr en viktig separationsprocess inom kemin och garingr ut paring att en loumlsning (mobil fas) passerar en kolonn (stationaumlr fas) och daumlr med kan loumlsningens olika komponenter separe- ras HPLC aumlr en mer utvecklad och snabbare variant av kromatografi Biacore 2000 bygger i sin tur paring en helt annan teknik Surface Plasmon Resonance SPR vilket innebaumlr en detektion av masskoncentrationen av ett visst aumlmne immobiliserat paring ett mikrochip Maumlnskligt albumin (HSA) fraringn renat blod har anvaumlnts Albuminet har bundits till en kromato- grafisk stationaumlr fas samt till ett mikrochip CM5 i Biacoresystemet Laumlkemedel saring som det verksamma aumlmnet ibuprofen i vaumlrktabletter samt betablockerarna alprenolol och propranolol har studerats Samtliga tre har kirala egenskaper Med Biacore har enatiomererna studerats var foumlr sig medan racematen har studerats samtidigt med kromatografi Data fraringn de tvaring separa- tionsmetoderna har jaumlmfoumlrts Maringlet med arbetet var att vaumlrdera de olika resultaten bedoumlma eventuella skillnader och hur de tvaring metoderna kompletterar varandra Tyvaumlrr fick studien avbrytas pga att det kromatogra- fiska systemet var instabilt och vi hade ingen tid kvar foumlr att upprepa foumlrsoumlken Instabiliteten kan bero paring en maumlngd orsaker men vi utesluter inte att proteinerna kan ha denaturerat dvs skrumpnat ihop och blivit inaktiva Orsaken till detta kan vara att detta helt enkelt haumlnder naturligt efter en tid Andra orsaker kan vara fysik stress av naringgon sort foumlr houmlg koncentration av modifierare eller annan ej gynnsam miljouml foumlr aktiva proteiner Vi kunde aumlven konstatera att separation uppnarings laumlttare med ibuprofen aumln de tvaring betablockerarna alprenolol och propranolol Orsaken till detta aumlr att albumin laumlttare binder till syror

Table of contents 1 INTRODUCTION 1

11 Human Serum Albumin HSA 1 12 Chiral drugs 1 13 Adsorption chromatography 2

2 HPLC ndash HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 3

21 The history of HPLC 3 22 Mobile Phase 3 23 Stationary Phase 4 24 Injectors for HPLC 4 25 Pumps 4 26 Detectors 5 27 Theory of HPLC 5

3 BIACORE 2000 10

31 SPR 10 32 Biosensor chip 11 33 Optical system 11 34 IFC - integrated micro-fluidic cartridge 12 35 Sensorgram 12 36 Theory 12

4 CALORIMETRY 15

41 Thermodynamics and kinetics 15 42 The thermopile heat-conduction principle 15 43 The heat flow signal and the heat quantity 16

5 METHODS 17

51 HPLC 17 52 Biacore 2000 18

6 RESULTS 19

61 HPLC 19 62 Biacore 2000 30

7 DISCUSSION 32 8 REFERENCES 33

1

1 Introduction Proteins are molecules consisting of long chains of amino acids For a certain protein the chain is folded in a certain way and therefore has a unique structure The pharmaceutical industry is interested in these different structures because the information in the structures can give them clues how the protein works In this way a drug can be constructed which interact with the protein in a good way Interaction of a drug with and competition for the binding site on a plasma protein might strongly affect its distribution elimination as well as pharmacodynamics and toxic properties Competition between two drugs for their binding to a plasma protein can strongly affect the drug disposition of both drugs with possible serious physiological consequences One problem is that many potential drug molecules tightly bind into the protein that they loose their efficiency in the human body By knowing how different substances binds to proteins and how strong the bindings are the pharmaceutical industry is able to construct drugs with higher effect and optimal compability with the body and other drugs [10] Human serum albumin (HSA) is an interesting protein from that approach

11 Human Serum Albumin HSA One of the most important drug transport protein in human plasma is human serum albumin (HSA) This transport protein is a monomeric protein made up only of amino acids Albumin plays a fundamental role in the transport of drugs metabolites and endogenous ligands HSA contributes to osmotic pressure in the blood stream and provides most of the acidbase buffering action of the plasma proteins In general acidic drugs bind mainly to albumin while basic drugs such as most beta-blocking agents have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma) [4]

12 Chiral drugs Chiral compounds contain at least one asymmetric carbon atom or are molecularly asymmetric They exist in two enantiomorphic forms related as object and mirror images like left and right hand This type of molecules have the same physical and chemical properties and differ only in their interaction with plane-polarised light such as dextrorotatory (+) and the other laevorotatory (-) There are a number of conventions for indicating the spatial configuration as opposed to optical properties of enantiomers The classical D and L system for monosaccharide and amino acids can not be applied easily to other structures and the Cahn-Ingold-Prelog systems which assign R (rectus) or S (sinister) configurations to an enantiomer are of more general use Until recently it has not been possible to resolve mixtures of enantiomers and this has created problems for the pharmaceutical industry in its development and clinical use of drugs many of them chiral Although enantiomers have identical chemical and physical properties they are distinguishable biologically Thus they differ in their ability to interact with the receptors involved in a range of physiological responses and they are often metabolised and excreted at different rates Chromatographic techniques have now been developed that allow mixtures of enantiomers to be dissolved [14]

2

13 Adsorption chromatography In 1903 M Twett first applied adsorption chromatography to the separation of plant pigments using a hydrocarbon solvent and inulin powder (a carbohydrate) as stationary phase [15] The separation of coloured bands led to the name chromatography from the Greek word chromatos meaning ldquocolourrdquo Adsorption chromatography operates on the basis of polarity Compounds that possess functional groups capable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compounds Thus less polar compounds will elute from the column faster than compounds that are highly polar The classic chromatography builds on the principal that one phase is stationary while the other one moves past it Figure 11 shows a solution containing solutes A and B placed on top of a column packed with solid particles and filled with solvent When the outlet opens solutes A and B flow down into the column Fresh solvent applies to the top of the column and the mixture washes down the column by continuous solvent flow If solute A more strongly adsorbs than solute B on the solid particles the solute A spends a smaller fraction of time free in the solution Solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components The mobile phase (the solvent moving down the column) in chromatography is either a liquid or a gas The stationary phase is most commonly a viscous liquid coated on the inside of a capillary tube or on the surface of solid particles packed into the column Alternatively as in fig11 the solid particles themselves may be the stationary phase Columns are either packed or open tubular A packed column is filled with particles containing stationary phase as in fig11 An open tubular column is a narrow hollow capillary with stationary phase coated on the inside walls

Fig 11 A solution containing solutes A (light grey band) and B (dark grey band) flows trough a column Here solute A adsorbs more strongly to the solid particles than solute B Therefore solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components

3

2 HPLC ndash High Performance Liquid Chromatography At present high performance liquid chromatography HPLC is the most important separation method and analytical technique The wide applicability speed and high sensitivity of HPLC has made this the most popular form of chromatography and virtually all types of biological molecules have been assayed or purified using this technique Modern HPLC has many applications including separation identification purification and quantification of various compounds HPLC is widely considered to be a technique mainly for biotechnological biomedical and biochemical research as well as for the pharmaceutical industry Currently HPLC is used by a variety of fields including cosmetics energy food and environmental industries

21 The history of HPLC HPLC has undergone a period of rapid development Until the 1970 only gas chromatography could provide the same high performance as HPLC does today High-pressure liquid chromatography was developed in the mid-1970 and quickly improved with the development of column packing materials and the additional convenience of on-line detectors In the 1980s HPLC began to be commonly used for the separation of chemical compounds New techniques improved separation identification purification and quantification far above the previous techniques Computers and automation added to the convenience of HPLC Improvements in type of columns and thus reproducibility were made as such terms as micro-column affinity columns and Fast HPLC began to immerge [2]

22 Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied into the column or stationary phase The mobile phase acts as a carrier for the sample solution A sample solution is injected into the mobile phase of an assay through the injector port As the sample solution flows through a column with the mobile phase the components of that solution migrate according to the non-covalent interactions of the compound with the column The chemical interactions of the mobile phase and sample determine the degree of migration and separation of components contained in the sample A sample that bounds stronger to the mobile phase than with the stationary phase elutes faster from the column This gives a shorter retention time [12] The range of solvents that can be used in HPLC is wide Water and aqueous buffer solutions are common and some non-aqueous solvents of low viscosity are also used High viscosity solvents are avoided because they require longer time to pass through the column which results in peak broadening and poorer resolution This also requires a higher pressure to force the solvent through the column Solvents for use in HPLC must be free of particles and dissolved air To ensure that sample is free from particles it is usually passed through a Millipore filter Degassing the sample is made in a water bath with UV-sound [16]

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

Abstract In this work High-Performance Liquid Chromatography HPLC and Biacore 2000 have been used to measure the interaction between proteins (large molecules) and selected drugs with small molecules (ibuprofen alprenolol and propranolol) The aim of this work was to evaluate what can be determined using the different methods how good the agreements are and how the methods complement each other We also compared the parameters from Biacore HPLC and micro calorimetric techniques With micro calorimetric technique it is possible to measure heat changes during different experiments The reason to this comparative study was to see if the different parameters could be used in all three systems Chromatography is an important separation process in chemistry HPLC is a more developed and a faster type of chromatography A solute containing different components (mobile phase) flows down a column (stationary phase) that separates the components In Biacore the technique is based on Surface Plasmon Resonance SPR This detects a mass concentration of a certain component on a microchip The protein we used is human serum albumin HAS purified from human blood The albumin was bounded to both a chromatography stationary phases and to a Biacore CM5 microchip With this we studied small molecules with chiral properties With Biacore the enantiomers were studied one at a time while racemic mixtures have been studied with chromatography (using a perturbation method and numerical tools) Very high concentrations were used so that the immobilised proteins were close to saturation The two methods and their data acquired were compared Unfortunately this study was interrupted because of instabilities in the chromatographic study and we did not have enough time to repeat the experiments The instabilities depend on several factors The protein was perhaps denaturised and therefore became inactive This happens naturally after a while Physical stress too high concentration of modifier or a bad environment for active proteins could also be factors Furthermore we could see that we more easily got a separation of the enantiomers in ibuprofen than with the two beta-blockers alprenolol and propranolol The reason for that is that albumin more easily binds to acids

En jaumlmfoumlrande studie av interaktion mellan laumlkemedel och proteiner med hjaumllp av icke linjaumlr kromatografi och ytplasmaresonansteknik Sammanfattning I det haumlr arbetet har High-Performance Liquid Chromatography HPLC och Biacore 2000 anvaumlnts foumlr att maumlta interaktionen mellan proteiner (stora molekyler) och utvalda laumlkemedel bestaringende av smaring molekyler (ibuprofen alprenolol och propranolol) Vi har utfoumlrt experiment foumlr att ta reda paring hur transportproteinet albumin binder olika laumlkemedel Maringlet med arbetet aumlr att jaumlmfoumlra de tvaring metoderna hur bra resultaten oumlverensstaumlmmer samt hur det tvaring systemen kompletterar varandra Vi har aumlven jaumlmfoumlrt parametervaumlrden mellan Biacore HPLC samt mikrokalorimetri Kalorimetern har till uppgift att maumlta vaumlrmefoumlraumlndringar i olika experiment Detta foumlr att se om det aumlr moumljligt att dra paralleller och daumlrmed vara moumljligt att anvaumlnda parametrarna mellan de olika systemen Kromatografi aumlr en viktig separationsprocess inom kemin och garingr ut paring att en loumlsning (mobil fas) passerar en kolonn (stationaumlr fas) och daumlr med kan loumlsningens olika komponenter separe- ras HPLC aumlr en mer utvecklad och snabbare variant av kromatografi Biacore 2000 bygger i sin tur paring en helt annan teknik Surface Plasmon Resonance SPR vilket innebaumlr en detektion av masskoncentrationen av ett visst aumlmne immobiliserat paring ett mikrochip Maumlnskligt albumin (HSA) fraringn renat blod har anvaumlnts Albuminet har bundits till en kromato- grafisk stationaumlr fas samt till ett mikrochip CM5 i Biacoresystemet Laumlkemedel saring som det verksamma aumlmnet ibuprofen i vaumlrktabletter samt betablockerarna alprenolol och propranolol har studerats Samtliga tre har kirala egenskaper Med Biacore har enatiomererna studerats var foumlr sig medan racematen har studerats samtidigt med kromatografi Data fraringn de tvaring separa- tionsmetoderna har jaumlmfoumlrts Maringlet med arbetet var att vaumlrdera de olika resultaten bedoumlma eventuella skillnader och hur de tvaring metoderna kompletterar varandra Tyvaumlrr fick studien avbrytas pga att det kromatogra- fiska systemet var instabilt och vi hade ingen tid kvar foumlr att upprepa foumlrsoumlken Instabiliteten kan bero paring en maumlngd orsaker men vi utesluter inte att proteinerna kan ha denaturerat dvs skrumpnat ihop och blivit inaktiva Orsaken till detta kan vara att detta helt enkelt haumlnder naturligt efter en tid Andra orsaker kan vara fysik stress av naringgon sort foumlr houmlg koncentration av modifierare eller annan ej gynnsam miljouml foumlr aktiva proteiner Vi kunde aumlven konstatera att separation uppnarings laumlttare med ibuprofen aumln de tvaring betablockerarna alprenolol och propranolol Orsaken till detta aumlr att albumin laumlttare binder till syror

Table of contents 1 INTRODUCTION 1

11 Human Serum Albumin HSA 1 12 Chiral drugs 1 13 Adsorption chromatography 2

2 HPLC ndash HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 3

21 The history of HPLC 3 22 Mobile Phase 3 23 Stationary Phase 4 24 Injectors for HPLC 4 25 Pumps 4 26 Detectors 5 27 Theory of HPLC 5

3 BIACORE 2000 10

31 SPR 10 32 Biosensor chip 11 33 Optical system 11 34 IFC - integrated micro-fluidic cartridge 12 35 Sensorgram 12 36 Theory 12

4 CALORIMETRY 15

41 Thermodynamics and kinetics 15 42 The thermopile heat-conduction principle 15 43 The heat flow signal and the heat quantity 16

5 METHODS 17

51 HPLC 17 52 Biacore 2000 18

6 RESULTS 19

61 HPLC 19 62 Biacore 2000 30

7 DISCUSSION 32 8 REFERENCES 33

1

1 Introduction Proteins are molecules consisting of long chains of amino acids For a certain protein the chain is folded in a certain way and therefore has a unique structure The pharmaceutical industry is interested in these different structures because the information in the structures can give them clues how the protein works In this way a drug can be constructed which interact with the protein in a good way Interaction of a drug with and competition for the binding site on a plasma protein might strongly affect its distribution elimination as well as pharmacodynamics and toxic properties Competition between two drugs for their binding to a plasma protein can strongly affect the drug disposition of both drugs with possible serious physiological consequences One problem is that many potential drug molecules tightly bind into the protein that they loose their efficiency in the human body By knowing how different substances binds to proteins and how strong the bindings are the pharmaceutical industry is able to construct drugs with higher effect and optimal compability with the body and other drugs [10] Human serum albumin (HSA) is an interesting protein from that approach

11 Human Serum Albumin HSA One of the most important drug transport protein in human plasma is human serum albumin (HSA) This transport protein is a monomeric protein made up only of amino acids Albumin plays a fundamental role in the transport of drugs metabolites and endogenous ligands HSA contributes to osmotic pressure in the blood stream and provides most of the acidbase buffering action of the plasma proteins In general acidic drugs bind mainly to albumin while basic drugs such as most beta-blocking agents have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma) [4]

12 Chiral drugs Chiral compounds contain at least one asymmetric carbon atom or are molecularly asymmetric They exist in two enantiomorphic forms related as object and mirror images like left and right hand This type of molecules have the same physical and chemical properties and differ only in their interaction with plane-polarised light such as dextrorotatory (+) and the other laevorotatory (-) There are a number of conventions for indicating the spatial configuration as opposed to optical properties of enantiomers The classical D and L system for monosaccharide and amino acids can not be applied easily to other structures and the Cahn-Ingold-Prelog systems which assign R (rectus) or S (sinister) configurations to an enantiomer are of more general use Until recently it has not been possible to resolve mixtures of enantiomers and this has created problems for the pharmaceutical industry in its development and clinical use of drugs many of them chiral Although enantiomers have identical chemical and physical properties they are distinguishable biologically Thus they differ in their ability to interact with the receptors involved in a range of physiological responses and they are often metabolised and excreted at different rates Chromatographic techniques have now been developed that allow mixtures of enantiomers to be dissolved [14]

2

13 Adsorption chromatography In 1903 M Twett first applied adsorption chromatography to the separation of plant pigments using a hydrocarbon solvent and inulin powder (a carbohydrate) as stationary phase [15] The separation of coloured bands led to the name chromatography from the Greek word chromatos meaning ldquocolourrdquo Adsorption chromatography operates on the basis of polarity Compounds that possess functional groups capable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compounds Thus less polar compounds will elute from the column faster than compounds that are highly polar The classic chromatography builds on the principal that one phase is stationary while the other one moves past it Figure 11 shows a solution containing solutes A and B placed on top of a column packed with solid particles and filled with solvent When the outlet opens solutes A and B flow down into the column Fresh solvent applies to the top of the column and the mixture washes down the column by continuous solvent flow If solute A more strongly adsorbs than solute B on the solid particles the solute A spends a smaller fraction of time free in the solution Solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components The mobile phase (the solvent moving down the column) in chromatography is either a liquid or a gas The stationary phase is most commonly a viscous liquid coated on the inside of a capillary tube or on the surface of solid particles packed into the column Alternatively as in fig11 the solid particles themselves may be the stationary phase Columns are either packed or open tubular A packed column is filled with particles containing stationary phase as in fig11 An open tubular column is a narrow hollow capillary with stationary phase coated on the inside walls

Fig 11 A solution containing solutes A (light grey band) and B (dark grey band) flows trough a column Here solute A adsorbs more strongly to the solid particles than solute B Therefore solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components

3

2 HPLC ndash High Performance Liquid Chromatography At present high performance liquid chromatography HPLC is the most important separation method and analytical technique The wide applicability speed and high sensitivity of HPLC has made this the most popular form of chromatography and virtually all types of biological molecules have been assayed or purified using this technique Modern HPLC has many applications including separation identification purification and quantification of various compounds HPLC is widely considered to be a technique mainly for biotechnological biomedical and biochemical research as well as for the pharmaceutical industry Currently HPLC is used by a variety of fields including cosmetics energy food and environmental industries

21 The history of HPLC HPLC has undergone a period of rapid development Until the 1970 only gas chromatography could provide the same high performance as HPLC does today High-pressure liquid chromatography was developed in the mid-1970 and quickly improved with the development of column packing materials and the additional convenience of on-line detectors In the 1980s HPLC began to be commonly used for the separation of chemical compounds New techniques improved separation identification purification and quantification far above the previous techniques Computers and automation added to the convenience of HPLC Improvements in type of columns and thus reproducibility were made as such terms as micro-column affinity columns and Fast HPLC began to immerge [2]

22 Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied into the column or stationary phase The mobile phase acts as a carrier for the sample solution A sample solution is injected into the mobile phase of an assay through the injector port As the sample solution flows through a column with the mobile phase the components of that solution migrate according to the non-covalent interactions of the compound with the column The chemical interactions of the mobile phase and sample determine the degree of migration and separation of components contained in the sample A sample that bounds stronger to the mobile phase than with the stationary phase elutes faster from the column This gives a shorter retention time [12] The range of solvents that can be used in HPLC is wide Water and aqueous buffer solutions are common and some non-aqueous solvents of low viscosity are also used High viscosity solvents are avoided because they require longer time to pass through the column which results in peak broadening and poorer resolution This also requires a higher pressure to force the solvent through the column Solvents for use in HPLC must be free of particles and dissolved air To ensure that sample is free from particles it is usually passed through a Millipore filter Degassing the sample is made in a water bath with UV-sound [16]

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

En jaumlmfoumlrande studie av interaktion mellan laumlkemedel och proteiner med hjaumllp av icke linjaumlr kromatografi och ytplasmaresonansteknik Sammanfattning I det haumlr arbetet har High-Performance Liquid Chromatography HPLC och Biacore 2000 anvaumlnts foumlr att maumlta interaktionen mellan proteiner (stora molekyler) och utvalda laumlkemedel bestaringende av smaring molekyler (ibuprofen alprenolol och propranolol) Vi har utfoumlrt experiment foumlr att ta reda paring hur transportproteinet albumin binder olika laumlkemedel Maringlet med arbetet aumlr att jaumlmfoumlra de tvaring metoderna hur bra resultaten oumlverensstaumlmmer samt hur det tvaring systemen kompletterar varandra Vi har aumlven jaumlmfoumlrt parametervaumlrden mellan Biacore HPLC samt mikrokalorimetri Kalorimetern har till uppgift att maumlta vaumlrmefoumlraumlndringar i olika experiment Detta foumlr att se om det aumlr moumljligt att dra paralleller och daumlrmed vara moumljligt att anvaumlnda parametrarna mellan de olika systemen Kromatografi aumlr en viktig separationsprocess inom kemin och garingr ut paring att en loumlsning (mobil fas) passerar en kolonn (stationaumlr fas) och daumlr med kan loumlsningens olika komponenter separe- ras HPLC aumlr en mer utvecklad och snabbare variant av kromatografi Biacore 2000 bygger i sin tur paring en helt annan teknik Surface Plasmon Resonance SPR vilket innebaumlr en detektion av masskoncentrationen av ett visst aumlmne immobiliserat paring ett mikrochip Maumlnskligt albumin (HSA) fraringn renat blod har anvaumlnts Albuminet har bundits till en kromato- grafisk stationaumlr fas samt till ett mikrochip CM5 i Biacoresystemet Laumlkemedel saring som det verksamma aumlmnet ibuprofen i vaumlrktabletter samt betablockerarna alprenolol och propranolol har studerats Samtliga tre har kirala egenskaper Med Biacore har enatiomererna studerats var foumlr sig medan racematen har studerats samtidigt med kromatografi Data fraringn de tvaring separa- tionsmetoderna har jaumlmfoumlrts Maringlet med arbetet var att vaumlrdera de olika resultaten bedoumlma eventuella skillnader och hur de tvaring metoderna kompletterar varandra Tyvaumlrr fick studien avbrytas pga att det kromatogra- fiska systemet var instabilt och vi hade ingen tid kvar foumlr att upprepa foumlrsoumlken Instabiliteten kan bero paring en maumlngd orsaker men vi utesluter inte att proteinerna kan ha denaturerat dvs skrumpnat ihop och blivit inaktiva Orsaken till detta kan vara att detta helt enkelt haumlnder naturligt efter en tid Andra orsaker kan vara fysik stress av naringgon sort foumlr houmlg koncentration av modifierare eller annan ej gynnsam miljouml foumlr aktiva proteiner Vi kunde aumlven konstatera att separation uppnarings laumlttare med ibuprofen aumln de tvaring betablockerarna alprenolol och propranolol Orsaken till detta aumlr att albumin laumlttare binder till syror

Table of contents 1 INTRODUCTION 1

11 Human Serum Albumin HSA 1 12 Chiral drugs 1 13 Adsorption chromatography 2

2 HPLC ndash HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 3

21 The history of HPLC 3 22 Mobile Phase 3 23 Stationary Phase 4 24 Injectors for HPLC 4 25 Pumps 4 26 Detectors 5 27 Theory of HPLC 5

3 BIACORE 2000 10

31 SPR 10 32 Biosensor chip 11 33 Optical system 11 34 IFC - integrated micro-fluidic cartridge 12 35 Sensorgram 12 36 Theory 12

4 CALORIMETRY 15

41 Thermodynamics and kinetics 15 42 The thermopile heat-conduction principle 15 43 The heat flow signal and the heat quantity 16

5 METHODS 17

51 HPLC 17 52 Biacore 2000 18

6 RESULTS 19

61 HPLC 19 62 Biacore 2000 30

7 DISCUSSION 32 8 REFERENCES 33

1

1 Introduction Proteins are molecules consisting of long chains of amino acids For a certain protein the chain is folded in a certain way and therefore has a unique structure The pharmaceutical industry is interested in these different structures because the information in the structures can give them clues how the protein works In this way a drug can be constructed which interact with the protein in a good way Interaction of a drug with and competition for the binding site on a plasma protein might strongly affect its distribution elimination as well as pharmacodynamics and toxic properties Competition between two drugs for their binding to a plasma protein can strongly affect the drug disposition of both drugs with possible serious physiological consequences One problem is that many potential drug molecules tightly bind into the protein that they loose their efficiency in the human body By knowing how different substances binds to proteins and how strong the bindings are the pharmaceutical industry is able to construct drugs with higher effect and optimal compability with the body and other drugs [10] Human serum albumin (HSA) is an interesting protein from that approach

11 Human Serum Albumin HSA One of the most important drug transport protein in human plasma is human serum albumin (HSA) This transport protein is a monomeric protein made up only of amino acids Albumin plays a fundamental role in the transport of drugs metabolites and endogenous ligands HSA contributes to osmotic pressure in the blood stream and provides most of the acidbase buffering action of the plasma proteins In general acidic drugs bind mainly to albumin while basic drugs such as most beta-blocking agents have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma) [4]

12 Chiral drugs Chiral compounds contain at least one asymmetric carbon atom or are molecularly asymmetric They exist in two enantiomorphic forms related as object and mirror images like left and right hand This type of molecules have the same physical and chemical properties and differ only in their interaction with plane-polarised light such as dextrorotatory (+) and the other laevorotatory (-) There are a number of conventions for indicating the spatial configuration as opposed to optical properties of enantiomers The classical D and L system for monosaccharide and amino acids can not be applied easily to other structures and the Cahn-Ingold-Prelog systems which assign R (rectus) or S (sinister) configurations to an enantiomer are of more general use Until recently it has not been possible to resolve mixtures of enantiomers and this has created problems for the pharmaceutical industry in its development and clinical use of drugs many of them chiral Although enantiomers have identical chemical and physical properties they are distinguishable biologically Thus they differ in their ability to interact with the receptors involved in a range of physiological responses and they are often metabolised and excreted at different rates Chromatographic techniques have now been developed that allow mixtures of enantiomers to be dissolved [14]

2

13 Adsorption chromatography In 1903 M Twett first applied adsorption chromatography to the separation of plant pigments using a hydrocarbon solvent and inulin powder (a carbohydrate) as stationary phase [15] The separation of coloured bands led to the name chromatography from the Greek word chromatos meaning ldquocolourrdquo Adsorption chromatography operates on the basis of polarity Compounds that possess functional groups capable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compounds Thus less polar compounds will elute from the column faster than compounds that are highly polar The classic chromatography builds on the principal that one phase is stationary while the other one moves past it Figure 11 shows a solution containing solutes A and B placed on top of a column packed with solid particles and filled with solvent When the outlet opens solutes A and B flow down into the column Fresh solvent applies to the top of the column and the mixture washes down the column by continuous solvent flow If solute A more strongly adsorbs than solute B on the solid particles the solute A spends a smaller fraction of time free in the solution Solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components The mobile phase (the solvent moving down the column) in chromatography is either a liquid or a gas The stationary phase is most commonly a viscous liquid coated on the inside of a capillary tube or on the surface of solid particles packed into the column Alternatively as in fig11 the solid particles themselves may be the stationary phase Columns are either packed or open tubular A packed column is filled with particles containing stationary phase as in fig11 An open tubular column is a narrow hollow capillary with stationary phase coated on the inside walls

Fig 11 A solution containing solutes A (light grey band) and B (dark grey band) flows trough a column Here solute A adsorbs more strongly to the solid particles than solute B Therefore solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components

3

2 HPLC ndash High Performance Liquid Chromatography At present high performance liquid chromatography HPLC is the most important separation method and analytical technique The wide applicability speed and high sensitivity of HPLC has made this the most popular form of chromatography and virtually all types of biological molecules have been assayed or purified using this technique Modern HPLC has many applications including separation identification purification and quantification of various compounds HPLC is widely considered to be a technique mainly for biotechnological biomedical and biochemical research as well as for the pharmaceutical industry Currently HPLC is used by a variety of fields including cosmetics energy food and environmental industries

21 The history of HPLC HPLC has undergone a period of rapid development Until the 1970 only gas chromatography could provide the same high performance as HPLC does today High-pressure liquid chromatography was developed in the mid-1970 and quickly improved with the development of column packing materials and the additional convenience of on-line detectors In the 1980s HPLC began to be commonly used for the separation of chemical compounds New techniques improved separation identification purification and quantification far above the previous techniques Computers and automation added to the convenience of HPLC Improvements in type of columns and thus reproducibility were made as such terms as micro-column affinity columns and Fast HPLC began to immerge [2]

22 Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied into the column or stationary phase The mobile phase acts as a carrier for the sample solution A sample solution is injected into the mobile phase of an assay through the injector port As the sample solution flows through a column with the mobile phase the components of that solution migrate according to the non-covalent interactions of the compound with the column The chemical interactions of the mobile phase and sample determine the degree of migration and separation of components contained in the sample A sample that bounds stronger to the mobile phase than with the stationary phase elutes faster from the column This gives a shorter retention time [12] The range of solvents that can be used in HPLC is wide Water and aqueous buffer solutions are common and some non-aqueous solvents of low viscosity are also used High viscosity solvents are avoided because they require longer time to pass through the column which results in peak broadening and poorer resolution This also requires a higher pressure to force the solvent through the column Solvents for use in HPLC must be free of particles and dissolved air To ensure that sample is free from particles it is usually passed through a Millipore filter Degassing the sample is made in a water bath with UV-sound [16]

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

Table of contents 1 INTRODUCTION 1

11 Human Serum Albumin HSA 1 12 Chiral drugs 1 13 Adsorption chromatography 2

2 HPLC ndash HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 3

21 The history of HPLC 3 22 Mobile Phase 3 23 Stationary Phase 4 24 Injectors for HPLC 4 25 Pumps 4 26 Detectors 5 27 Theory of HPLC 5

3 BIACORE 2000 10

31 SPR 10 32 Biosensor chip 11 33 Optical system 11 34 IFC - integrated micro-fluidic cartridge 12 35 Sensorgram 12 36 Theory 12

4 CALORIMETRY 15

41 Thermodynamics and kinetics 15 42 The thermopile heat-conduction principle 15 43 The heat flow signal and the heat quantity 16

5 METHODS 17

51 HPLC 17 52 Biacore 2000 18

6 RESULTS 19

61 HPLC 19 62 Biacore 2000 30

7 DISCUSSION 32 8 REFERENCES 33

1

1 Introduction Proteins are molecules consisting of long chains of amino acids For a certain protein the chain is folded in a certain way and therefore has a unique structure The pharmaceutical industry is interested in these different structures because the information in the structures can give them clues how the protein works In this way a drug can be constructed which interact with the protein in a good way Interaction of a drug with and competition for the binding site on a plasma protein might strongly affect its distribution elimination as well as pharmacodynamics and toxic properties Competition between two drugs for their binding to a plasma protein can strongly affect the drug disposition of both drugs with possible serious physiological consequences One problem is that many potential drug molecules tightly bind into the protein that they loose their efficiency in the human body By knowing how different substances binds to proteins and how strong the bindings are the pharmaceutical industry is able to construct drugs with higher effect and optimal compability with the body and other drugs [10] Human serum albumin (HSA) is an interesting protein from that approach

11 Human Serum Albumin HSA One of the most important drug transport protein in human plasma is human serum albumin (HSA) This transport protein is a monomeric protein made up only of amino acids Albumin plays a fundamental role in the transport of drugs metabolites and endogenous ligands HSA contributes to osmotic pressure in the blood stream and provides most of the acidbase buffering action of the plasma proteins In general acidic drugs bind mainly to albumin while basic drugs such as most beta-blocking agents have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma) [4]

12 Chiral drugs Chiral compounds contain at least one asymmetric carbon atom or are molecularly asymmetric They exist in two enantiomorphic forms related as object and mirror images like left and right hand This type of molecules have the same physical and chemical properties and differ only in their interaction with plane-polarised light such as dextrorotatory (+) and the other laevorotatory (-) There are a number of conventions for indicating the spatial configuration as opposed to optical properties of enantiomers The classical D and L system for monosaccharide and amino acids can not be applied easily to other structures and the Cahn-Ingold-Prelog systems which assign R (rectus) or S (sinister) configurations to an enantiomer are of more general use Until recently it has not been possible to resolve mixtures of enantiomers and this has created problems for the pharmaceutical industry in its development and clinical use of drugs many of them chiral Although enantiomers have identical chemical and physical properties they are distinguishable biologically Thus they differ in their ability to interact with the receptors involved in a range of physiological responses and they are often metabolised and excreted at different rates Chromatographic techniques have now been developed that allow mixtures of enantiomers to be dissolved [14]

2

13 Adsorption chromatography In 1903 M Twett first applied adsorption chromatography to the separation of plant pigments using a hydrocarbon solvent and inulin powder (a carbohydrate) as stationary phase [15] The separation of coloured bands led to the name chromatography from the Greek word chromatos meaning ldquocolourrdquo Adsorption chromatography operates on the basis of polarity Compounds that possess functional groups capable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compounds Thus less polar compounds will elute from the column faster than compounds that are highly polar The classic chromatography builds on the principal that one phase is stationary while the other one moves past it Figure 11 shows a solution containing solutes A and B placed on top of a column packed with solid particles and filled with solvent When the outlet opens solutes A and B flow down into the column Fresh solvent applies to the top of the column and the mixture washes down the column by continuous solvent flow If solute A more strongly adsorbs than solute B on the solid particles the solute A spends a smaller fraction of time free in the solution Solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components The mobile phase (the solvent moving down the column) in chromatography is either a liquid or a gas The stationary phase is most commonly a viscous liquid coated on the inside of a capillary tube or on the surface of solid particles packed into the column Alternatively as in fig11 the solid particles themselves may be the stationary phase Columns are either packed or open tubular A packed column is filled with particles containing stationary phase as in fig11 An open tubular column is a narrow hollow capillary with stationary phase coated on the inside walls

Fig 11 A solution containing solutes A (light grey band) and B (dark grey band) flows trough a column Here solute A adsorbs more strongly to the solid particles than solute B Therefore solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components

3

2 HPLC ndash High Performance Liquid Chromatography At present high performance liquid chromatography HPLC is the most important separation method and analytical technique The wide applicability speed and high sensitivity of HPLC has made this the most popular form of chromatography and virtually all types of biological molecules have been assayed or purified using this technique Modern HPLC has many applications including separation identification purification and quantification of various compounds HPLC is widely considered to be a technique mainly for biotechnological biomedical and biochemical research as well as for the pharmaceutical industry Currently HPLC is used by a variety of fields including cosmetics energy food and environmental industries

21 The history of HPLC HPLC has undergone a period of rapid development Until the 1970 only gas chromatography could provide the same high performance as HPLC does today High-pressure liquid chromatography was developed in the mid-1970 and quickly improved with the development of column packing materials and the additional convenience of on-line detectors In the 1980s HPLC began to be commonly used for the separation of chemical compounds New techniques improved separation identification purification and quantification far above the previous techniques Computers and automation added to the convenience of HPLC Improvements in type of columns and thus reproducibility were made as such terms as micro-column affinity columns and Fast HPLC began to immerge [2]

22 Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied into the column or stationary phase The mobile phase acts as a carrier for the sample solution A sample solution is injected into the mobile phase of an assay through the injector port As the sample solution flows through a column with the mobile phase the components of that solution migrate according to the non-covalent interactions of the compound with the column The chemical interactions of the mobile phase and sample determine the degree of migration and separation of components contained in the sample A sample that bounds stronger to the mobile phase than with the stationary phase elutes faster from the column This gives a shorter retention time [12] The range of solvents that can be used in HPLC is wide Water and aqueous buffer solutions are common and some non-aqueous solvents of low viscosity are also used High viscosity solvents are avoided because they require longer time to pass through the column which results in peak broadening and poorer resolution This also requires a higher pressure to force the solvent through the column Solvents for use in HPLC must be free of particles and dissolved air To ensure that sample is free from particles it is usually passed through a Millipore filter Degassing the sample is made in a water bath with UV-sound [16]

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

1

1 Introduction Proteins are molecules consisting of long chains of amino acids For a certain protein the chain is folded in a certain way and therefore has a unique structure The pharmaceutical industry is interested in these different structures because the information in the structures can give them clues how the protein works In this way a drug can be constructed which interact with the protein in a good way Interaction of a drug with and competition for the binding site on a plasma protein might strongly affect its distribution elimination as well as pharmacodynamics and toxic properties Competition between two drugs for their binding to a plasma protein can strongly affect the drug disposition of both drugs with possible serious physiological consequences One problem is that many potential drug molecules tightly bind into the protein that they loose their efficiency in the human body By knowing how different substances binds to proteins and how strong the bindings are the pharmaceutical industry is able to construct drugs with higher effect and optimal compability with the body and other drugs [10] Human serum albumin (HSA) is an interesting protein from that approach

11 Human Serum Albumin HSA One of the most important drug transport protein in human plasma is human serum albumin (HSA) This transport protein is a monomeric protein made up only of amino acids Albumin plays a fundamental role in the transport of drugs metabolites and endogenous ligands HSA contributes to osmotic pressure in the blood stream and provides most of the acidbase buffering action of the plasma proteins In general acidic drugs bind mainly to albumin while basic drugs such as most beta-blocking agents have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma) [4]

12 Chiral drugs Chiral compounds contain at least one asymmetric carbon atom or are molecularly asymmetric They exist in two enantiomorphic forms related as object and mirror images like left and right hand This type of molecules have the same physical and chemical properties and differ only in their interaction with plane-polarised light such as dextrorotatory (+) and the other laevorotatory (-) There are a number of conventions for indicating the spatial configuration as opposed to optical properties of enantiomers The classical D and L system for monosaccharide and amino acids can not be applied easily to other structures and the Cahn-Ingold-Prelog systems which assign R (rectus) or S (sinister) configurations to an enantiomer are of more general use Until recently it has not been possible to resolve mixtures of enantiomers and this has created problems for the pharmaceutical industry in its development and clinical use of drugs many of them chiral Although enantiomers have identical chemical and physical properties they are distinguishable biologically Thus they differ in their ability to interact with the receptors involved in a range of physiological responses and they are often metabolised and excreted at different rates Chromatographic techniques have now been developed that allow mixtures of enantiomers to be dissolved [14]

2

13 Adsorption chromatography In 1903 M Twett first applied adsorption chromatography to the separation of plant pigments using a hydrocarbon solvent and inulin powder (a carbohydrate) as stationary phase [15] The separation of coloured bands led to the name chromatography from the Greek word chromatos meaning ldquocolourrdquo Adsorption chromatography operates on the basis of polarity Compounds that possess functional groups capable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compounds Thus less polar compounds will elute from the column faster than compounds that are highly polar The classic chromatography builds on the principal that one phase is stationary while the other one moves past it Figure 11 shows a solution containing solutes A and B placed on top of a column packed with solid particles and filled with solvent When the outlet opens solutes A and B flow down into the column Fresh solvent applies to the top of the column and the mixture washes down the column by continuous solvent flow If solute A more strongly adsorbs than solute B on the solid particles the solute A spends a smaller fraction of time free in the solution Solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components The mobile phase (the solvent moving down the column) in chromatography is either a liquid or a gas The stationary phase is most commonly a viscous liquid coated on the inside of a capillary tube or on the surface of solid particles packed into the column Alternatively as in fig11 the solid particles themselves may be the stationary phase Columns are either packed or open tubular A packed column is filled with particles containing stationary phase as in fig11 An open tubular column is a narrow hollow capillary with stationary phase coated on the inside walls

Fig 11 A solution containing solutes A (light grey band) and B (dark grey band) flows trough a column Here solute A adsorbs more strongly to the solid particles than solute B Therefore solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components

3

2 HPLC ndash High Performance Liquid Chromatography At present high performance liquid chromatography HPLC is the most important separation method and analytical technique The wide applicability speed and high sensitivity of HPLC has made this the most popular form of chromatography and virtually all types of biological molecules have been assayed or purified using this technique Modern HPLC has many applications including separation identification purification and quantification of various compounds HPLC is widely considered to be a technique mainly for biotechnological biomedical and biochemical research as well as for the pharmaceutical industry Currently HPLC is used by a variety of fields including cosmetics energy food and environmental industries

21 The history of HPLC HPLC has undergone a period of rapid development Until the 1970 only gas chromatography could provide the same high performance as HPLC does today High-pressure liquid chromatography was developed in the mid-1970 and quickly improved with the development of column packing materials and the additional convenience of on-line detectors In the 1980s HPLC began to be commonly used for the separation of chemical compounds New techniques improved separation identification purification and quantification far above the previous techniques Computers and automation added to the convenience of HPLC Improvements in type of columns and thus reproducibility were made as such terms as micro-column affinity columns and Fast HPLC began to immerge [2]

22 Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied into the column or stationary phase The mobile phase acts as a carrier for the sample solution A sample solution is injected into the mobile phase of an assay through the injector port As the sample solution flows through a column with the mobile phase the components of that solution migrate according to the non-covalent interactions of the compound with the column The chemical interactions of the mobile phase and sample determine the degree of migration and separation of components contained in the sample A sample that bounds stronger to the mobile phase than with the stationary phase elutes faster from the column This gives a shorter retention time [12] The range of solvents that can be used in HPLC is wide Water and aqueous buffer solutions are common and some non-aqueous solvents of low viscosity are also used High viscosity solvents are avoided because they require longer time to pass through the column which results in peak broadening and poorer resolution This also requires a higher pressure to force the solvent through the column Solvents for use in HPLC must be free of particles and dissolved air To ensure that sample is free from particles it is usually passed through a Millipore filter Degassing the sample is made in a water bath with UV-sound [16]

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

2

13 Adsorption chromatography In 1903 M Twett first applied adsorption chromatography to the separation of plant pigments using a hydrocarbon solvent and inulin powder (a carbohydrate) as stationary phase [15] The separation of coloured bands led to the name chromatography from the Greek word chromatos meaning ldquocolourrdquo Adsorption chromatography operates on the basis of polarity Compounds that possess functional groups capable of strong hydrogen bonding will adhere more tightly to the stationary phase than less polar compounds Thus less polar compounds will elute from the column faster than compounds that are highly polar The classic chromatography builds on the principal that one phase is stationary while the other one moves past it Figure 11 shows a solution containing solutes A and B placed on top of a column packed with solid particles and filled with solvent When the outlet opens solutes A and B flow down into the column Fresh solvent applies to the top of the column and the mixture washes down the column by continuous solvent flow If solute A more strongly adsorbs than solute B on the solid particles the solute A spends a smaller fraction of time free in the solution Solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components The mobile phase (the solvent moving down the column) in chromatography is either a liquid or a gas The stationary phase is most commonly a viscous liquid coated on the inside of a capillary tube or on the surface of solid particles packed into the column Alternatively as in fig11 the solid particles themselves may be the stationary phase Columns are either packed or open tubular A packed column is filled with particles containing stationary phase as in fig11 An open tubular column is a narrow hollow capillary with stationary phase coated on the inside walls

Fig 11 A solution containing solutes A (light grey band) and B (dark grey band) flows trough a column Here solute A adsorbs more strongly to the solid particles than solute B Therefore solute A moves down the column more slowly than solution B This leads to a separation of the mixture into its components

3

2 HPLC ndash High Performance Liquid Chromatography At present high performance liquid chromatography HPLC is the most important separation method and analytical technique The wide applicability speed and high sensitivity of HPLC has made this the most popular form of chromatography and virtually all types of biological molecules have been assayed or purified using this technique Modern HPLC has many applications including separation identification purification and quantification of various compounds HPLC is widely considered to be a technique mainly for biotechnological biomedical and biochemical research as well as for the pharmaceutical industry Currently HPLC is used by a variety of fields including cosmetics energy food and environmental industries

21 The history of HPLC HPLC has undergone a period of rapid development Until the 1970 only gas chromatography could provide the same high performance as HPLC does today High-pressure liquid chromatography was developed in the mid-1970 and quickly improved with the development of column packing materials and the additional convenience of on-line detectors In the 1980s HPLC began to be commonly used for the separation of chemical compounds New techniques improved separation identification purification and quantification far above the previous techniques Computers and automation added to the convenience of HPLC Improvements in type of columns and thus reproducibility were made as such terms as micro-column affinity columns and Fast HPLC began to immerge [2]

22 Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied into the column or stationary phase The mobile phase acts as a carrier for the sample solution A sample solution is injected into the mobile phase of an assay through the injector port As the sample solution flows through a column with the mobile phase the components of that solution migrate according to the non-covalent interactions of the compound with the column The chemical interactions of the mobile phase and sample determine the degree of migration and separation of components contained in the sample A sample that bounds stronger to the mobile phase than with the stationary phase elutes faster from the column This gives a shorter retention time [12] The range of solvents that can be used in HPLC is wide Water and aqueous buffer solutions are common and some non-aqueous solvents of low viscosity are also used High viscosity solvents are avoided because they require longer time to pass through the column which results in peak broadening and poorer resolution This also requires a higher pressure to force the solvent through the column Solvents for use in HPLC must be free of particles and dissolved air To ensure that sample is free from particles it is usually passed through a Millipore filter Degassing the sample is made in a water bath with UV-sound [16]

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

3

2 HPLC ndash High Performance Liquid Chromatography At present high performance liquid chromatography HPLC is the most important separation method and analytical technique The wide applicability speed and high sensitivity of HPLC has made this the most popular form of chromatography and virtually all types of biological molecules have been assayed or purified using this technique Modern HPLC has many applications including separation identification purification and quantification of various compounds HPLC is widely considered to be a technique mainly for biotechnological biomedical and biochemical research as well as for the pharmaceutical industry Currently HPLC is used by a variety of fields including cosmetics energy food and environmental industries

21 The history of HPLC HPLC has undergone a period of rapid development Until the 1970 only gas chromatography could provide the same high performance as HPLC does today High-pressure liquid chromatography was developed in the mid-1970 and quickly improved with the development of column packing materials and the additional convenience of on-line detectors In the 1980s HPLC began to be commonly used for the separation of chemical compounds New techniques improved separation identification purification and quantification far above the previous techniques Computers and automation added to the convenience of HPLC Improvements in type of columns and thus reproducibility were made as such terms as micro-column affinity columns and Fast HPLC began to immerge [2]

22 Mobile Phase The mobile phase in HPLC refers to the solvent being continuously applied into the column or stationary phase The mobile phase acts as a carrier for the sample solution A sample solution is injected into the mobile phase of an assay through the injector port As the sample solution flows through a column with the mobile phase the components of that solution migrate according to the non-covalent interactions of the compound with the column The chemical interactions of the mobile phase and sample determine the degree of migration and separation of components contained in the sample A sample that bounds stronger to the mobile phase than with the stationary phase elutes faster from the column This gives a shorter retention time [12] The range of solvents that can be used in HPLC is wide Water and aqueous buffer solutions are common and some non-aqueous solvents of low viscosity are also used High viscosity solvents are avoided because they require longer time to pass through the column which results in peak broadening and poorer resolution This also requires a higher pressure to force the solvent through the column Solvents for use in HPLC must be free of particles and dissolved air To ensure that sample is free from particles it is usually passed through a Millipore filter Degassing the sample is made in a water bath with UV-sound [16]

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

4

23 Stationary Phase Columns are commonly 10 to 30 cm in length and with a 3-10 mm inner diameter Most columns for HPLC are made of 316-grade stainless steel resistant to the usual HPLC pressure and also relatively inert to chemical corrosion The high pressure employed requires a very hard packing material If high efficiency is to be obtained uniform packing under high and constant pressure must be achieved These requirements usually result in packing being made from silica or alumina consisting of round particles Porous packing materials are used in all forms of liquid chromatography The particle parameter of the packing material has a considerable influence on the separating efficiency of the column Packing materials for columns are diverse since there are many modes of HPLC They are available in different sizes diameters pore sizes or they can have special materials attached (such as an antigen or antibody for immunoaffinity chromatography) Packing available range from those needed for specific applications (affinity immunoaffinity chiral biological etc) to those for all-purpose applications The packing material is attached to the internal column hull by resins or supports which include oxides polymers carbon hydroxyapatite beads agarose or silica which is the most common type [13]

24 Injectors for HPLC Samples injected into the HPLC via an injection port The injection port of an HPLC commonly consists of an injection valve and the sample loop The sample dissolves in the mobile phase before injection into the sample loop and is then drawn into a syringe and injected into the loop via the injection valve A rotation of the valve rotor closes the valve and opens the loop in order to inject the sample into the stream of the mobile phase Loop volumes can range between 10 microl to over 500 microl Modern HPLC systems has automated sample injections [13]

25 Pumps The pump is one of the major components in a HPLC Two major types the reciprocating pump and the positive displacement pump will be discussed Reciprocating pumps These pumps have the advantages of being able to pump unlimited quantities of solvent Therefore are they often used during long separations or preparative situations exist A disadvantage is that it produces a pulsating pressure and this affects the detector Using two pistons reduces the pulsating effect one compressing while the other is releasing Positive displacement pump (Syringe pump) The advantages with this type of pump is the produce a constant volume through the column no cavation no pulsation and changing solvents is easy A motor-driven screw forces the piston forward This forces a constant volume through the column regardless of changes in the pressure This permits the retention time to stay the same form one run to another This pump has the disadvantage that only a single charge of solvents can be delivered hopefully enough to complete the analysis

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

5

26 Detectors The detector for an HPLC is the component that emits a response due to the eluting sample compound and subsequently signals a peak on the chromatogram The detector positioned immediately posterior to the stationary phase has the mission to detect the compounds as they elute from the column Many different types of detectors are used with HPLC Some of the more common detectors include Refractive Index (RI) Ultra-Violet (UV) Fluorescent Radiochemical Electrochemical Near-Infra Red (Near-IR) Mass Spectroscopy (MS) Nuclear Magnetic Resonance (NMR) and Light Scattering (LS)

27 Theory of HPLC In fig 11 A and B become slightly separated because B showed a greater affinity for the stationary phase and moved more slowly than A Eventually A elutes distinct from and followed by B ndashthis separation is the goal of the chromatography A chromatogram is a two dimensional plot of molecules eluting from a column measurement of absorbance or some other defining property is plotted against elution of time or volume as seen in Fig 21

Fig 21 Basic Liquid Chromatography Parameter Basic HPLC and CE of biomolecules [8] VM is the permeation volume or mobile phase volume and represent the volume of the mobile phase in the column This volume is the sum of the liquid outside the matrix (V0) and the liquid inside the matrix (Vi) Importantly VM is the volume of fluid required for a completely unretained peak to pass through the column and is a major reference point for the chromatographic separation VM is also called dead volume The dead time (t0) is the time required for the dead volume to pass through the column [8] VM = 05 middot L middot dc

2 Where L is the column length and dc is the diameter both in cm T0 calculate the dead time the dead volume in ml must be divided by the flow rate (F) in mlmin T0=VMF

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

6

The total column volume (VT) is the sum of the dead volume and the volume occupied by the matrix or packing material it is therefore the volume of the empty cylinder VT=π middot r2 middot L where r and L are the radius and the length of the column respectively The point at which a sample is injected into a column is sometimes indicated by a characteristic spike and occurs in the baseline (usually downward) The detector or the result of a transient pressure surge that occurs during the injection of the sample may mark the spike not confused with t0 that occurs later in the run Retention volume VR is the volume of the solvent that passes through the column from the time of sample injection to the detection of the peak measured at its apex as shown in Fig 21 Flow rates are generally kept constant during a run permitting a straightforward interconversion between time and volume of the eluent as plotted on the x-axis in the chromatogram VR for a particular component is thus associated with a distinct time called the retention time tR which is related through the flow rate VR=F middot tR where F is the volumetric flow rate usually in mlmin Concept Molecules which are effectively separated by any particular chromatographic system may actually have somewhat different retention times from run to run which can be caused by operational variance of the flow rate mobile phase or other parameters Retention time can be normalized to remove this variance by defining the capacity factor or retention factor (k) which is the number of dead volumes or mobile phase volumes required to elute the molecule or peak of interest Until recently capacity factor was symbolised kacute Most frequently k is expressed into terms of retention time (tR) because time is the usual unit of measurement k = (VR-VM)VM = (tR-T0)t0 The goal of chromatography is separation of multiple peaks The resolution (Rs) of two peaks can be defined quantitatively as Rs=2(t2-t1)(w2+w1) Where w2 and w1 are the peak widths at there respectively bases in the same time units as the retention times Retention In a chromatographic process molecules travel in a mobile phase except when they are interacting with a stationary phase The differential interactions of molecules result in their separation In HPLC the mobile phase is a liquid and the stationary phase is usually a coating bounded at a small solid particle which is contained in a metal cylinder or column A molecule is retained if it partitions into the stationary phase rather than merely travelling with the mobile phase The capacity factor or retention factor k is thus related to these phases

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

7

k = K middot (VSVM) where VS and VM are the volumes of the stationary and mobile phases respectively and K is the distribution coefficient for the solute in the two phases Selectivity The ability of the stationary phase to discriminate between two molecules is an essential aspect of chromatography Selectivity (α) is defined as the ratio of the capacity factor of the most highly retained compound to that of the less retained α = k2k1 Selectivity is dependent on the physical and chemical structures of the analytes the stationary and the mobile phase Adjusting the flow rate or the column dimensions cannot change selectivity It is possible to change selectivity by replacing the mobile phase with one with different properties or to change the column to achieve enough selectivity to resolve two peaks Selectivity is the most important tool of successful chromatography Efficiency The peak width is an integral component of resolution whose basis must be understood in order to achieve an acceptable separation The concept ldquoTheoretical Platesrdquo was developed to explain such variations in peak width and the factors responsible Efficiency is a measure of the easy with which a compound moves through a column and to the extent that it measures transition in and out of the mobile and stationary phases it is a kinetic parameter Efficiency is also a statistical calculation of the standard deviation of the peak in unit time and is measured in theoretical plates (N) N=(tRσ)2

where σ is the standard deviation of the band in times units which can be calculated by using the peak width generally at the base line (w) or at half height (w05) Both the retention time and peak width must be measured in the same units usually in length N=16 middot (tRw)2 = 554 middot (tR w05)2 The height equivalent of the theoretical plate (H) relates the plate number (N) to the column length (L) H = LN and L is usually measured in mm The typical plate heights for HPLC are usually 001 or 01 mm An efficient process allows the sample molecules to migrate in a narrow band in a short period of time with minimal dispersion The plate count (N) therefore gives an indication of the sharpness of the peak

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

8

Interrelationship of factors The three components of a separation ndash speed resolution and retention ndash are interrelated therefore improving one will sometimes compromise the other Optimisation of a separation lies therefore in compromise and is usually achieved most efficiently by a stepwise examination of each parameter without alteration of the others Resolution is a function of the three factors which can individually improve or destroy a separation

1 Retention ndash the ability for molecules to interact with the stationary phase or the affinity of the molecule for the matrix

2 Selectivity ndash the discriminating power of the matrix which produces differential interaction for at least two compounds

3 Efficiency ndash the ease of movement of the solute through the column The distribution coefficient K The relative distribution coefficient of a solute between two immiscible phases can be described by the magnitude of the distribution coefficient which in turn can be defined mathematically by the equation KA = cS(A)cM(A) where KA = distribution coefficient of solute A between stationary and mobile phase cS(A) = concentration of solute A in stationary phase under equilibrium condition cM(A) = concentration of solute A in mobile phase under equilibrium condition The thermodynamic theory of retention Solute retention and selectivity are directly related to the distribution coefficient K Thus the classical thermodynamic expression relating the distribution coefficient with the excess free energy can be employed to describe the control of retention in thermodynamic terms [7] RT middot lnK = -∆G (1) where R = gas constant T = absolute temperature [K˚] ∆G = free energy bearing in mind that ∆G = ∆H ndash T middot ∆S (2) where ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] Substituting for ∆G from Eq 2 into Eq 1 ln K = -(∆H RT- ∆S R) (3)

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

9

Equation 3 gives a direct relationship between the distribution coefficient K (and thus retention) and the thermodynamic function free enthalpy and free entropy By determining the corrected retention volume of a solute in a given phase over a range of specific values the magnitude of ∆H and ∆S can be determined The respective values of the distribution coefficient at each temperature can be calculated from KA = (VM + KA middot VS)VS=VacuteR(A) VS where VM and VS is the total volume of mobile and stationary phase respectively In liquid-solid chromatography the stationary phase has a limited number of possible interaction sites Therefore the ratio between the concentrations in the stationary and mobile phase of the analyte will successively decrease with increasing concentration and it becomes non-linear An adsorption isotherm relates those concentrations at constant temperature [8] The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites [9] The adsorption isotherm q = (a middot cM )(1+ b middot cM ) = cSmax middot b middot cM )(1+ b middot cM ) a = the equilibrium coefficient at infinite dilutionthe initial slope of the isotherm b = the binding constant related to the energy of adsorption = kakd (association constantdissociation constant) cM = the mobile phase concentration of the analyte [mM] cSmax = ab is the maximum concentration of the stationary phase [mM]

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

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5

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30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

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05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

10

3 Biacore 2000 Biacore 2000 is an SPR-based biosensor technology Surface plasmon resonance (SPR) allows sensitive detection of molecular interactions in real time without the use of labels [5] The Real-time BIA technology rests on three cornerstones

bull Surface plasmon resonance (SPR) which detects the mass concentrations at the surface

bull Sensor chip which provide the surface conditions for SPR for attaching molecules of interest

bull A liquid handling system with precision pumps and an integrated micro-fluidic cartridge (IFC) for controlled transport of samples to the sensor surface

Other essential components of a BIACORE analytical system are The optical system is responsible for generation and detection of the SPR signal and the sensorgram that present the results in real-time on a computer screen

31 SPR Surface plasmon resonance (SPR) arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index In Biacore systems the media are the sample and the glass of the sensor chip and the conducting film is a thin layer of gold on the chip surface SPR causes a reduction in the intensity of reflected light at a specific angle of reflection This angle varies with the refractive index close to the surface on the side opposite from the reflected light (the sample side in Biacore) When molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction This plot is called a sensorgram What Biacore actually measures is the angle of minimum reflected light intensity The light absorbs not by the sample instead the light energy dissipates through SPR in the gold film Therefore the light used to detect interaction processes never enters the sample SPR re- sponse values expresses in resonance units (RU) One RU represents a change of 00001deg in the angle of the intensity minimum For most proteins 1000 RU is equivalent to a change in concentration of about 1 ngmm2 on the sensor surface or about 6 mgml in bulk protein con- centration The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the change in concentration [6]

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

11

Fig 31 The SPR functionfrom the Biacore homepage wwwbiacorecom Light is reflected from a conducting film at the interface between two media of different refractive index Molecules in the sample bind to the sensor surface the concentration and therefore the refractive index at the surface changes and an SPR response is detected Then the response against time is plotted in a sensorgram

32 Biosensor chip The biosensor chip CM5 is a glass slide coated with a thin layer of gold to which a matrix of carboxymethylated dextran is covalently attached The dextran matrix allows covalent immobilisation of bio molecules using a well-characterised chemistry and provides a hydrophilic environment suitable for a widely variety of protein interactions Exposing the sensor chip to conditions that dissociate the surface-bound complex without damaging the covalent attached interactant can regenerate the bio specific surface This makes that each sensor chip can be re-used for a repeated series of measurements The precious metal gold is required for generation of the surface plasmon resonance (SPR) response because it is stable and hard to oxidise Figure 31 above shows the SPR function Dextran ndash a polysaccharide consisting of a α-1-6-linked glucose unit For matrix use it is cross-linked with epichlorohydrin but is less stable to cid hydrolysis than cellulose matrices It is stable up to pH 12 and is hydrophilic

33 Optical system The optical system uses a fixed array of diode detectors to monitor the position of the resonance angle The resonance angle or signal expresses in resonance units (RU) A response (ie a change in resonance signal) of 1000 RU corresponds to a change in surface concentration on the sensor chip of about 1 ngmm2 for proteins The specific response is determined by the specific refractive index increment for the analyte and similar for most other bio molecules

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

12

34 IFC - integrated micro-fluidic cartridge The IFC (integrated micro-fluidic cartridge) contains flow channels sample loops and pneumatic valves and controls delivery of sample and eluent to the sensor chip surface Flow cells for detection are formed by pressing the IFC against the sensor chip thus keeping the dead volume between the sample loops and sensor chip surface to a minimum The flow cells designed for minimal sample dispersion and efficient mass transport of bio molecules to the sensor chip surface Among the significant advantages of continuous flow technology with micro fluidics are the rapid exchange of sample and buffer at the sensor surface and the ability to control the contact time of sample with the surface accurately and reproducibly The sensor surface in Biacore forms one wall of a flow cell and measurements are made under conditions of continuous liquid flow over the surface Samples and reagents are delivered to the surface through a micro fluidic system that ensures precise control over the time-point and duration of sample delivery Valves in the micro fluidic system switch between buffer and sample with high precision so that the sample is delivered as a defined liquid segment with minimum dispersion between sample and buffer Simpler systems for biosensors based on SPR or similar technologies deliver the sample to a small cuvette A major advantage of Biacorersquos continuous flow system over the cuvette approach is that sample concentration and flow conditions are exactly defined for the duration of sample contact with the surface improving the precision and robustness of quantitative analyses

35 Sensorgram Results from real-time BIA represents as a sensorgram which is a plot of changes in the resonance signal as a function of time The response is measured in resonance units RU and is proportional to the molecule mass on the surface For an interactant with a given mass the response is therefore proportional to the number of molecules at the surface Observing the change in resonance signal follows the progress of the interaction and kinetic data extracts from the rate of change of the signal

36 Theory When molecules interactions are studied in Biacore one of the interactants is immobilised on the sensor surface while the other is passed over that surface in solution The term ligand refers to the immobilised component The interactant present in the sample injected over the surface is referred to as the analyte [5] The response is measured in resonance units (RU) and is proportional to the molecule mass on the surface The response against the time is showed in a sensorgram on a computer screen during the analysis

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

13

There are three major steps in a Biacore assay bull Immobilisation the process by which the ligand is attached to the sensor chip

surface bull Interaction analysis the analyte is injected over the sensor chip surface and the

interaction between the analyte and the immobilised ligand is monitored bull Regeneration the process of removing bound analyte from the ligand of the surface

Fig 33 Immobilisation sensorgram Figure 33 shows seven steps (a-g) in a sensorgram [6]

a) Baseline b) EDCNHS activation of the dextran matrix c) Baseline after activation d) Attraction and covalent binding of the ligand to the dextran matrix e) Buffer washes away loosely associated ligand The response level at this point gives a

first indication of the immobilised amount f) Deactivation and further washing away of loosely associated ligand g) The difference in response between points a and g reflects the amount immobilised

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

14

The response can be calculated from the formula R = (Rmax K c )( 1+ K c) c = the mobile phase concentration of the analyte [mM] K = distribution constant (distribution of an uncharged compound to an organic phase) or the binding constant calculated from kakd (association ratedissociation rate) The binding capacity of the surface depends on the immobilisation level of the ligand The maximum response Rmax describes the binding capacity of the surface in terms of the response at saturation A theoretical Rmax can be calculated using the formula Rmax = (analyte molar weightligand molar weight) immobilised amount stochiometric ratio The theoretically calculated Rmax is often higher than the experimentally derived Rmax There are many potential reasons for this for example the ligand are not fully active of some reason From the article Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage [1] we get the relationship ln K = ∆H(RT) - ∆SR ∆G= -RT lnK= ∆H ndash T ∆S

∆S = (∆H - ∆G)T [calmol K] where R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol]

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

15

4 Calorimetry

41 Thermodynamics and kinetics

Thermodynamics is the study of transformation of energy in a system Heat can be looked on upon as the most elementary form of energy because any kind of energy can be quantitatively converted into heat Q The energetic of a system can therefore be described in terms of the heat content The first law of thermodynamics states that energy cannot be created or destroyed ie the energy of an isolated system is constant For a system of constant pressure the enthalpy is a measure of the heat content and approximately equal to the energy change except for gases that expand substantially on heating Thermo chemistry is the study of energy changes that occur during chemical reactions If heat is absorbed the reaction is endothermic and if heat is produced the reaction is exothermic The enthalpy change ∆H by itself will not determine the spontaneity of a reaction Another important quantity is the entropy change ∆S A positive entropy change describes an increased disorder for the system The second law of the thermodynamic states that any process that occurs in a system will lead to an increase in the system Reactions are spontaneous in the direction of a decreasing Gibbs function ie Gibbs free energy change ∆G which depends on the enthalpy change and the entropy change together with the absolute temperature T ∆G = ∆H ndash T ∆S Calorimeters measures heat changes They have been constructed for a range of different applications The history of calorimetry dates back to the eighteenth century Around 1760 the physicist and chemist J Black (1728-1799) made for the first time the distinction between the concepts of temperature and heat In this simple rdquophase-transition calorimeterrdquo he observed that the input of heat into a quantity of melting ice did not cause a temperature change until all the ice had melted The first instrument to be called a micro calorimeter due to its high resolution was built by E Calvert in 1948 Micro calorimeters have been developed to allow small quantities to be measured with an improved sensitivity [3]

42 The thermopile heat-conduction principle

Each thermocouple monitors the heat that is conducted across it because the temperature difference between the two sides is proportional to the voltage un and to the heat flow dqdtn For an ideal heat-conduction calorimeter the total heat is conducted across the individual thermocouples according to the equation U = sum(u1+u2+) = c sum (dqdt1+dqdt2+) = c dQdt where U is the total thermopile voltage dQdt is the total heat flow through the thermopile and c is a proportionality constant If the measured reaction rate is constant or very slow the heat flow signal is directly proportional to the output voltage

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

16

dQdt = ε U The heat flow dQdt is obtained in microW (microJs) and ε is a constant obtained by calibration If it is a fast reaction the inertia of the calorimeter will create a time lag The heat equation is then described by the Tian equation dQdt = ε [U+τ1(dUdt)] where τ1 is the time constant and dUdt is the time derivative of the output voltage

43 The heat flow signal and the heat quantity

The signal monitored by the micro calorimeter which will be referred to as the heat flow signal dQdt is proportional to the rate of heat change (the thermal activity) from the reaction in the sample vessel The level of monitored heat flow signal for a decomposition reaction of a drug D is proportional to the reaction rate at that temperature dDdt where D is proportional to the number of moles of the drug and the proportionality constant ∆H is the enthalpy change of the reaction in kJmol dQdt = ∆H dDdt The formula ∆G = -RT lnK = ∆H ndash T ∆S is well known and we also used it in the HPLC and the Biacore system R = gas constant T = absolute temperature [K] ∆H = free enthalpy [kcalmol] ∆S = free entropy [calmol K] ∆G = free energy [kcalmol] K = binding constant calculated from kakd (association ratedissociation rate)

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

17

5 Methods

51 HPLC

Apparatus HP 1100 Series Pumping Systems HP 1100 Series Auto samplers HP 1100 Series Diode-Array Detector Column Nucleosil Diol 500 Aring Particle size 7 microm Length100 mm Inner diameter 2 mm Serial Batch no 180204701101 Chemicals and solutions Mobile phases PBS 50 mM pH 74 (138 mM NaCl 27 mM KCl) NaCl Lot no K31900304 MW 5844 gmol KCl Lot no TA659136009 MW 7455 gmol NaP 50 mM pH 69 (13 acetonitrile 4mM octanoic acid) Acetonitrile Lot no 0413327 MW 4105 gmol (Acros Organics) MeOH Lot no ME0315 HPLC grade (Scharlau) Isopropanol Lot no K26165234 MW6010 gmol (Merck) Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Alprenolol Lot no 68H0478 MW 2858 gmol (Sigma) Propranolol Lot no PRP584 MW 2958 gmol (Sigma) Methylmandelate Lot no 4233881 MW 16618 gmol (Fluka) Immobilisation HAS Lot no 120K76031 (Sigma) NaIO4 Lot no AO13869901 MW 21389 gmol (Acros Organics) NaBH3CN Lot No S17381-263 1 mgml (Aldrich) Ethanolamine Lot no 1048-2131919 1M pH 85 (Biacore) Immobilised protein Human Serum Albumin

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

18

52 Biacore 2000

Chemicals and solutions Running buffer PBS 50mM NaP pH 74 + 138 mM NaCl + 27 mM KCl Injections Ibuprofen Lot no 063K1117 MW 20629 gmol (Sigma) Immobilisation 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide esters (EDC) N-hydroxysuccinimide (NHS) Running buffer NaAc 10 mM pH 43 Immobilised protein Human Serum Albumin Apparatus Biacore 2000

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

19

6 Results One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The Langmuir isotherm is the relationship between the concentration in the mobile and stationary phase with limited number of adsorption sites We can se that adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c) (1+ b c) R = (Rmax K c)(1+ K c) As we can se from the formulas above cSmax = Rmax and K = b = kakd (association ratedissociation rate) This gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G is calculated in the same way in HPLC micro calorimetry and the Biacore system ∆G = -RT ln K therefore these three techniques can be used as complement to each other

61 HPLC

HSA immobilisation The first step was to immobilise the human serum albumin in the column To make sure that the column was clean before the immobilisation the column was rinsed with isopropanol and after that with mQ-H2O We worked with an immobilisation protocol from the article Chromatographia 2001 54 December (No 1112) The article used a 100 46 column and the protein Glucoamylase G2 from Asp Niger With the molar weight 63 kDa the protein concentration 2 mgml and immobilised 100 mg (50 ml) The molar weight for albumin is 66 kDa which is almost the same as the molar weight for Glucoamylase The superficial area for our column was π 12 and the article column superficial area was π 23 2 Therefore we chose R = 53 mm2 and we used 5053 = 94 ml of HSA 2 mgml solution To make an oxidation of diol moieties to aldehydes we used 100 times the volume of the column The mobile phase consisted of 60 mM NaIO4 solved in four parts H2O and one part of MeOH (41) The column volume was π 012 10 = 0314 100 = 314 ml A solution with NaAc 50 mM pH 40 was made From this we mixed 133 ml HAc with 5 ml NaAc 1mM diluted with mQH2O to a total volume of 100 ml Another solution was made 60 mM NaIO4 in H2OMeOH 41

The flow rate we used was 05 mlmin and with 314 ml it were enough to flow the system in 63 min during the pressure on 97 bar To clean the system it was washed with mQH2O

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

20

A volume of 94 ml human serum albumin HSA 2 mgml was mixed with NaBH3CN 1 mgml in NaAc 50 mM pH 4 With the flow rate 01 mlmin 94 ml was enough to flow the system in 94 min and the pressure was 12 bar After 200 min still no break-through appeared Therefore we prepared another HSANaBH3CN solution After six hours we still did not have any break-through It depends probably on problems with the activation of the protein or wrong pH pH 4 lt pH HSA To change the pH we changed the immobilisation buffer to NaP 50 mM pH 60 Another change we did was to use a lower flow rate 005 mlmin and therefore a lower pressure on 7 bar We also collected the elution volumes to measure the weight With this changes a break-through (Fig 61) appeared at approximately 66 minutes and the system was rinsed with NaP buffer 50 mM pH 6 with a flow rate on 02 mlmin and the pressure on 28 bar

0

05

1

0 20 40 60 80 100

[Min]

[V]

Because the sorption of aldehydes proceeds gradually the concentration of sorbate registered in the effluent as a function of volume of passed-through fluid is called break-through curve Although the change in voltage is rather sudden the front of the break-through curve is distinct due to dispersion phenomena [11] After the break-through we blocked the remaining aldehydes groups in 37 min with a solution made of 01 mM ethanolamine pH 73 and 1mgml NBH3CN The flow rate was 02 mlmin and the pressure was 27 bar The used volume was 74 ml The column was then rinsed with NaP 50 mM pH 6 and equilibrated with isopropanol 5 in Mq-H2O

Fig 61 A break-through appeared after approximately 66 minutes

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

21

Control of the column function To see if the immobilisation worked and we used a 50 mM phosphate buffer NaP with the pH 69 The mobile phase was made of 87 50 mM NaP 13 acetonitrile and 4 mM octanoic acid The injection 01 mM ibuprofen was solved in the mobile phase The enantiomers in the ibuprofen are separated and the column seems to work properly

-5

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 Min

mAU

Fig 62 Test of the column function with Ibuprofen injected in 50 mM phosphate buffer

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

22

To see how the curves differ with different buffer concentration we diluted the mobile phase five times to 10 mM NaP and injected 01 Ibuprofen 01 mM ibuprofen was also injected in 50 mM NaP The enantiomers were separated in both buffers When using the diluted mobile phase with lower ion strength (10 mM) the curve moves to the right and therefore gets longer retention times Higher ion strength in the running buffer gives shorter retention times because the protein-drug binding is weaker

-05

0

05

1

15

2

25

3

35

4

0 10 20 30 40 50 60

Min

mAU

50 mM NaP10 mM NaP

Fig 63 Injections of 01 mM ibuprofen in 10 Mm and 50 mM NaP

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

23

Control of physiological conditions Next step was to investigate if the column separated the enantiomers in ibuprofen propranolol and alprenolol The system was flowed with a new buffer PBS (a phosphate buffer) 50mM NaP pH 74 (containing 138 mM NaCl + 27 mM KCl) The enantiomers separate but the curves do not look as they should During the lower concentrations the curves should move to the right and therefore get longer retention times Now happens the opposite

-05

0

05

1

15

2

25

3

35

4

12 17 22 27 32

01 mM

mM

0001mM

001 mM

Fig 64 Three injections ibuprofen with different concentrations 01 001 and 0001 mM in PBS

Min

mAU

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

24

We did the same injections with propranolol in PBS and the enantiomers in the propranolol do not separate at all probably because the albumin binds more easily to acids like ibuprofen

-5

0

5

10

15

20

25

30

0 5 10 15 20Min

mAU

01 mM001 mM0001 mM

Fig 65 Three different concentrations of propranolol 01 001 0001 mM in PBS

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

25

Here 01 001 and 0001 mM alprenolol was injected in PBS The enantiomers in the alprenolol do not separate here either and the curves look strange We should have two peaks with similar area The column or the system is maybe unstable In general acidic drugs (ibuprofen) bind mainly to albumin while basic drugs (propranolol and alprenolol) have higher affinity for α1-acid glycoprotein α-AGP (another transport protein in the plasma)

-15

-1

-05

0

05

1

15

2

25

3

0 5 10 15 20 25

Min

mAU

01 mM001 mM0001 mM

Fig 66 Three injections of 01 001 and 0001 mM alprenolol in PBS

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

26

01 mM ibuprofen was injected two weeks later (compare with fig 23) A new mobile phase PBS (phosphate buffer) 50 mM pH 74 was used The area under the first peak should have the same area as peak number two This gives us indications that something is wrong or unstable

-05

0

05

1

15

2

25

3

35

0 10 20 30 40

Min

mAU

Fig 67 01 mM ibuprofen injected in PBS two weeks later

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

27

Stability test of the HSA column To check the stability in the column and to see if the protein still is active we tested the HSA column at three different dates 153 183 and 223 01 mM Ibuprofen was injected and the same mobile phase was used during the three experiments (50 mM NaP 13 acetonitrile and 4mM octanoic acid The results show that both peaks and retention times differ from each other The temperature is constant at 25˚C but the pressure differs from day to day The pressure the 15th and 22th is 34 bar and the 18th the pressure is 32 bar All those factors mean that both the system and the column may be unstable The peaks should have the same retention times and same heights independent of the date the experiment were made

-10

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6

153183223

mAU

Min

Fig 68 Stability test of the HSA column

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

28

Stability test of the system with a C-18 column We wanted to know if the pump system worked properly during the low flow rates that we used We did not know if it was the column or the system was unstable Therefore we did repeated tests with a C-18 column in 02 and 05 microlmin The C-18 column does not contain living proteins and are therefore more stable than the HSA column The mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile with this column during the low flow rate 02 mlmin

-2

0

2

4

6

8

10

12

14

0 5 10 15Min

mAU

Fig 69 Stability test with a C-18 column flow rate 02 mlmin three injections with the same concentration plotted in black medium gray and light gray

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

29

This time the mobile phase we used was MeOHmQ-H2O (5050) and 01 mM metylmandelate was injected three times The curves seem to be stabile here as well with this column during the low flow rate 05 mlmin The pump and the system seem to be working when using the C-18 column and 05 mlmin The HSA column is not working properly and makes the system unstable The experiment is interrupted here because the bad results and lapping of time With the HPLC system the immobilisation of HSA was successful and separation of enantiomers was made with ibuprofen in PBS with modifier and worked properly When we tried to separate the enantiomers in ibuprofen in a buffer without modifiers the curves did not look as they should The separation of the enantiomers in alprenolol and propranolol did not work at all We also tested the stability in the system and in the column and the tests showed that the HSA column was unstable It probably depends on many factors one for example the protein was denaturised

-2

0

2

4

6

8

10

12

0 1 2 3 4 5 6

Min

mAU

Fig 610 Stability test of a C18-column flow rate 05 mlmin three injections with the same concentration plotted in black medium gray and light gray

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

30

62 Biacore 2000

Immobilisation of the ligand Human Serum Albumin We used amine coupling which is the most widely approach for covalently attaching bio molecules to the sensor chip surface First the dextran matrix on the sensor chip surface was activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide esters (EDC) and N-hydroxysuccinimide (NHS) to get reactive succinimide esters After that our ligand (HSA) was passed over the surface to make the esters react spontaneously with the amino groups (or other nucleophilic groups) in the HSA This process linked the albumin covalently to the dextran After the injection of albumin ethanolamine was passed over the sensor surface to deactivate the remaining active esters Interaction analysis The aim of this part of the experiment was to inject drugs with small molecules and chiral properties The reason was to measure and determining the bindings between the protein and the drugs and this measurement is very important in pharmaceutical industry The drugs we were supposed to inject were ibuprofen alprenolol and propranolol We hade problems with the drifting in the baseline and we are therefore just presenting a sensorgram made with ten injections of ibuprofen see Fig 37 The running buffer is PBS and the injected concentrations of ibuprofen are 16 (the curve with the highest RU) 14 12 10 8 5 1 05 01 and 005 mM

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

31

Fig 611 The Biacore sensorgram shows ten injections of ibuprofen The experiments with HPLC and Biacore were made parallel This study ends here because the HPLC systemcolumn was unstable and therefore interrupted All experiments were very time demanding both with Biacore and HPLC In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen and propranolol were made Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

32

7 Discussion One of the aims with this work was to find and compare the relationships between HPLC Biacore and micro calorimetric techniques The adsorption isotherm q for chromatography and the response R in the Biacore system can be related to each other Chromatography Biacore q = (cSmax b c)(1+ b c) R = (Rmax K c)(1 + K c) The formulas above shows that cSmax = Rmax and K = b = kakd (association ratedissociation rate) and gives us a useful relationship between HPLC and the Biacore system The isotherm function ∆G (∆G = -RT ln K) is calculated in the same way in HPLC Biacore and micro calorimetric techniques Therefore the three systems can be used as complement to each other In the HPLC system the immobilisation of HSA was successful and separation of the enantiomers in ibuprofen in PBS with modifier worked properly Separation of the enantiomers in alprenolol and propranolol did not work at all The reason for this is that albumin more easily binds to acids (ibuprofen) than to basis (alprenolol and propranolol ) Stability tests showed that the system was stable and the HSA column was unstable The reason for this was probably because denaturised protein and therefore we did not get the results we wanted In the Biacore system the protein was immobilised on the microchip CM5 and injections with ibuprofen was made and successful Unfortunately we were not able to make the same comparative injections in both Biacore and HPLC because of lack of time

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co

33

8 References

[1] Abdiche Yasmina N Myszka David G Direct comparison of bonding equilibrium thermodynamic and rate constants on the Biacore homepage Center for University of Utah Salt Lake City USA wwwbiacorese last visited March 15 2004

[2] A users guide of High Performance Liquid Chromatography (HPLC) University of

Kentucky College of Pharmacy wwwpharmukyeduASRGHPLChplcmytryhtml last visited April 3 2004 [3] Angberg Maria Evaluation of isothermal heat-conduction micro calorimetry in

pharmaceutical stability studies Department of Pharmaceutics Uppsala University 1992 [4] Bertucci Carlo Domenici Enrico Reversible and covalent binding of drugs to human

serum albumin methodological approaches and physiological relevance Current Medicinal Chemistry 2002 no 9 1463-1481

[5] Biacore 2000 getting started Ed September 2002 Biacore [6] Biacore 2000 handbook Ed September 2002 Biacore [7] Brown Phyllis R Hartwick Richard A High performance liquid chromatography 1989

Chemical analysis Vol 98 John Wiley amp Sons [8] Cunico Robert L Gooding Karen M Wehr Tim Basic HPLC and CE of Biomolecules

Bay Bio analytical Laboratory Inc 1998 [9] Goumltmar Gustaf Heterogeneous adsorption in chiral LC 2002 Uppsala University [10] Harris C Daniel Quantitative chemical analysis 5th ed Freeman and Co 1998 ISBN 0-

7167-2881-8 [11] Macaacutesek Fedor Navratil James D Separation chemistry 1992 Ellis Horwood Ltd p 320 [12] Meloan Clifton E Chemical separations 1999 John Wiley amp Sons Inc p183-210 [13] Meyer Veronika R Practical high performance liquid chromatography 1994 John Wiley

amp Sons Inc [14] Oravcovaacute Jana Boumlhs Barbara Lindner Wolfgang Drug-protein binding studies ndash new

trends in analytical and experimental methodology Journal of Chromatography B 1996 no 677 1-28

[15] Snyder LR Stadalius MA Quarry MA Analytical chemistry 1983 Vol 55 pp

1412-30 [16] Willard H Merritt L Dean JA Settle Jr FA Instrumental methods of analysis 1988

7th ed Wadsworth Publishing Co