Table of ContentsApplying Enantioselective CE in thePharmaceutical Industry ............................................................................................................................. WHY USE CE?..................................................................................................................................... METHOD DEVELOPMENT..................................................................................................................................... EXAMPLES......................................................................................................................................................... CONCLUSIONS............................................................................................................................... REFERENCES ..................................................................................................................................................................................................................................

In-Process Analysis of Plasmid Copy Numberfor Fermentation Control ........................................................................................................................................... INTRODUCTION ............................................................................................................................................................ EXPERIMENTAL............................................................................................................................................................ RESULTS ................................................................................................................................................................................................... CONCLUSIONS ............................................................................................................................................................ REFERENCES ............................................................................................................................................................

How to Bee a Protein Chemist ........................................................................................................................................... INTRODUCTION.............................................................................................................................. EXPERIMENTAL.................................................................................................................................... RESULTS AND DISCUSSION.............................................................................................................................. CONCLUSIONS..............................................................................................................................Quality Control Of Drugs by Capillary Electrophoresis:A Practical Approach ............................................................................................................................................ OBJECTIVE ............................................................................................................................................................ PUBLIC CONCERNED ..................................................................................................................... LOCATION AND DATE ..................................................................................................................... PROGRAMME ............................................................................................................................................................

ICES-2001: An Electrophoretic OdysseyICES-2001: An Electrophoretic Odyssey..........................................................................................................................................

Dr. Schmerr Wins 2001 “Putting CE to Work” Award ..........................................................................

Optimizing Sample Introduction Conditions in CE-MS/MS....................................................................................................... EFFECT OF SHEATH GAS ..................................................................................................................... EFFECT OF SPRAY VOLTAGE..................................................................................................................... OUR RECOMMENDATION ..................................................................................................................... EFFECT OF SHEATH LIQUID .....................................................................................................................

Highly Sulfated Cyclodextrins ............................................................................................................................................................

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The worldwide newsletterfor capil lary electrophoresis

Volume 5, Issue 1 • April 2001

PATRIK PETERSSON, KARIN

JOHANSSON, AND CARIN LINDE

ASTRAZENECA R&D LUND,SWEDEN

WHY USE CE?

Enantioselective CE has, over thelast 10 to 15 years proved to bean efficient tool for

chiral separation(1). GenerallyCE offers high efficiency, shorttime of analysis, and minimalsolvent/reagent consumptionin comparison with otherseparation techniques.Although most chiral selectorscan be used, it is primarilycyclodextrins and cyclodextrinderivatives that are best suitedfor CE. Other importantselectors like macrocyclicantibiotics and proteins can beused but, due to their UV-absorbance, are easier to employin LC methods where they areimmobilized on the column. It is,however, our impression thatmost analytical scale chiralseparations can be accomplishedusing CE with a limited numberof cyclodextrin derivatives.

METHOD DEVELOPMENTIn our strategy for method develop-

ment, we screen eight chiral selectorsusing two types of buffers: 50 mM sodi-um dihydrogenphosphate, pH 2.5, and20 mM sodium tetraborate, pH 9.3. The

choice of buffer pH depends on thetype of analyte and chiral selector.Bases are analyzed at low pH. Acids areanalyzed at high pH or, alternatively, atlow pH using highly sulfated cyclo-dextrins. Neutrals are analyzed atlow/high pH using highly sulfatedcyclodextrins or, alternatively, at highpH using carboxymethyl β-cyclodex-trin. The selectors that we are

screening are the following (usually at20 kV normal polarity, bare fused-silica,20 cm effective length, 50 mm i.d.,20°C, and a selector concentration of20 mM):

• α-, β- and γ-cyclodextrin (α-CD,β-CD, and γ-CD, 15 mM)

• 2,3,6-trimethyl-β-cyclodextrin(TM-β-CD)

• 2,6-dimethyl-β-cyclodextrin(DM-β-CD)

• (2-hydroxy)propyl-β-cyclodextrin(HP-β-CD)

• highly sulfated α-, β-, and γ-cyclo-dextrin (HS-α-CD, HS-β-CD, andHS-γ-CD, reversed polarity,i.e., anode at detector, and 15 kV)

• carboxymethyl-β-cyclodextrin(CM-β-CD)

In our screening procedure, typeof selector is first evaluated. In orderto speed up the method develop-ment, arrays of 2 mL vials containingdifferent chiral selectors and buffer

Applying Enantioselective CE in thePharmaceutical Industry

2

combinations can be kept in thefreezer. For those selectors thatdisplay changes in peak shape, theinfluence of CD concentration isevaluated at high (50 mM) and lowlevels (2 mM). Finally, for the mostpromising selector(s) the CD concen-tration is fine tuned together with an

optimization of temperature,capillary length, and field strength. Ifnecessary, the addition of a modifier(e.g., methanol) is evaluated.

For basic drugs, peak shapeimprovements may be obtained byaltering the composition of thepH 2.5 buffer. A buffer based ontriethanolamine and phosphoric acidshould result in mobility matchingand minimized wall interactions for

bases(2,3). For the analysis of neutralcompounds, systems combining anegatively charged CD (HS-β-CD orCM-β-CD) to create electrophoreticmobility and a neutral CD to createchiral selectivity are an additionalpossibility(4). For primary amines,we also consider the use of a crownether, 18-crown-6-tetracarboxylicacid, as chiral selector(1,5).

Figure 1. (A-B) An example of selectivity and retention dif-ferences between sulfated γ-CD batches from two differentmanufacturers. According to the manufacturers, the degreeof substitution should be approximately 12-13 sulfate groupsper CD. (B-D) A comparison of three different batches of”raw” HS-γ-CD from one manufacturer. (E-G) Three batchesof the HS-γ-CD commercially available from BeckmanCoulter, Inc.These lots are formulated by blending the raw,synthetic material to meet a tight specification.

Figure 2. The enantiomers of (A) terbutaline, (B) salbutamol (short end injection mode), and (C) bambuterol separated withthe same system based on HS-β-CD.

Volume 5, Issue 1 • April 2001

3

If more than one selector gives anacceptable result, batch-to-batchreproducibility, number of manufac-turers, and cost should be taken intoaccount. Batch-to-batch variations area potential problem for derivatizedCDs. Thus it is advisable to alwaysinvestigate the ruggedness of a newmethod by evaluating different batch-es and manufacturers of the chiralselector. Different manufacturers mayhave products with different degreesand/or positions of substitution. Con-sequently, there can be very largeselectivity and retention differencesas illustrated in Figure 1A-B for sulfat-ed γ-CD from two differentmanufacturers. It should, however, bestressed that derivatized CDs can alsobe produced in a reproducible wayas shown in the same figure(Figure 1B-G) for our evaluation ofsix batches of HS-γ-CD from BeckmanCoulter, Inc. More data on thecharacterization of HS-CDs and thecomparison with other types ofsulfated CDs can be found inreferences 6 and 7.

In order to meet the requirementfrom drug regulatory authorities tobe able to determine related impuri-ties at 0.05% level, both good resolu-tion and a thorough optimization ofthe signal-to-noise ratio is needed.Parameters to optimize are injected

amount and volume, sample focusing,capillary dimensions, as well as detec-tor wavelength, bandwidth, samplingfrequency, and filter settings. Itshould also be noted that a sufficientsignal-to-noise ratio often is difficultto obtain for chiral separations basedon techniques like GC and LC due topoorly separated and/or tailing peaks.

EXAMPLESAs we are working with

pharmaceutical compounds, most ofour analytes are bases. For thesetypes of compounds, it is our experi-ence that HS-CDs and HS-β-CD in par-ticular provide the broadest selectivi-ty and the fastest separations of theCDs listed above. Figure 2 illustratesone example where the enantiomersof three amino alcohols, terbutaline,salbutamol and bambuterol, are sepa-rated with the same CE system basedon HS-β-CD. The enantiomers ofterbutaline and bambuterol have pre-viously been separated using otherCDs(8). To our knowledge, however,only HS-β-CD can separate all of themunder the same conditions. Anotherexample is shown in Figure 3 for aproprietary amine with anenantiomeric purity of 99.3%. For thisamine, HS-γ-CD gave a better selectivi-ty than 17 other chiral selectorsincluding the CDs listed above,proteins, macrocyclic antibiotics, andderivatized cellulose. Similar resultshave also been obtained for othertypes of amines.

CONCLUSIONSIt is our impression that enantios-

elective CE is an important tool forpharmaceutical analysis—perhapsthe most important tool for analyt-ical-scale chiral separations. Amongthe chiral selectors that are availablefor CE today, HS-CDs seem to providenot only the most general selectivitybut also the fastest separations. Theseparation of enantiomers for more

than 140 pharmaceutical compoundshave been reported(6-7,9-12). An addition-al advantage is that bases, acids, andneutrals can be analyzed using thesame systems.

REFERENCES1. Verleysen, K., Sandra, P.

Electrophoresis, 19, 2798-2833(1998)

2. Sänger-van de Griend, C. E.,Gröningsson, K., Westerlund, D.Chromatographia, 42, 263-268(1996)

3. Williams, R. L., Vigh, G. J.Chromatogr., 730, 273-278 (1996)

4. Fillet, M., Hubert, P., Crommen, J.Electrophoresis, 19, 2834-2840(1998)

5. Nishi, H., Nakamura, K., Nakai, H.,Sato, T. J. Chromatogr. A, 757,225-235 (1997)

6. Chen, F.-T. A., Evangelista, R. A.J. Chinese Chem. Soc., 46, 847-860(1999)

7. Chen, F.-T. A., Shen, G., Evangelista,R. A. Submitted to Anal. Chem.

8. Palmarsdottir, S., Edholm, L.-E.J. Chromatogr. A, 666, 337-350(1994)

9. Verleysen, K., Van den Bosch, T.,Sandra, P. Electrophoresis, 20,2650-2656 (1999)

10.Verleysen, K., Sabah, S., Scriba, G.,Chen, A., Sandra, P. J. Chromatogr.A, 824, 91-97 (1998)

11.Lurie, I. S., Odeneal II, N. G.,McKibben, T. D., Casale, J. F.Electrophoresis, 19, 2918-2925(1998)

12.www.beckmacoulter.com/chiral38

Figure 3. Another example, a proprietary amine forwhich HS-γ-CD gave a better selectivity than 17 otherselectors including macrocyclic antibiotics, proteins, andderivatized cellulose.

4

TORSTEN SCHMIDT1, KARL FRIEHS2,MARTIN SCHLEEF1 , CARSTEN

VOSS2, AND ERWIN FLASCHEL2

1PLASMIDFACTORY GMBH & CO.KG, BIELEFELD, GERMANY,WWW.PLASMIDFACTORY.COM2CHAIR OF FERMENTATION

ENGINEERING, UNIVERSITY OF

BIELEFELD, BIELEFELD, GERMANY

INTRODUCTION

In biotechnology, theplasmid copy number,defined as the number of

plasmids per cell, has a highimpact on the productivityof recombinant micro-organisms. A high plasmidcopy number (i.e., a highgene dosage) usually leadsto high expression of plas-mid-encoded genes and,therefore, overproduction ofrecombinant proteins. Sinceplasmid DNA became a phar-maceutical product itself as avector for therapeutic genesin gene therapy or DNA vac-cination trials(1), the micro-bial production of plasmidDNA in Escherichia coli isof great interest. In thesecultivation processes, theplasmid copy number is animportant control parameterfor product concentrationand productivity of theprocess. The quantitation ofplasmid copy number during cultivationneeds a quick DNA isolation in combi-nation with a fast and reliable analysismethod offering in-process controls inless than 30 minutes. In the literature,however, numerous direct or indirectmethods for determination of plasmidcopy number are described, like

measuring the activity of the plasmid-encoded product(2) or dot blotting(3), aswell as quantitation by UV absorbance(4),HPLC(5), or gel electrophoresis(6) afterisolating plasmid DNA from the cells.All these methods have the disadvan-tage of being too time-consuming tobe useful for plasmid copy numberdetermination during a bacterial culti-vation. For this application, the proce-dures for both the DNA isolation and

its quantitative analysis has to be accel-erated compared to common proce-dures. The quick isolation method forplasmid DNA is of primary importancebecause the fast analytical methodshould not be slowed down by timeconsuming isolation. On the other

hand, the completeness of plasmid iso-lation is of interest for getting reliableresults as well.

A novel method, based on the fastquantitation of plasmid DNA withcapillary gel electrophoresis after amodified rapid DNA isolation, allowsdetermination of plasmid copynumber for in-process control in few-er than 30 minutes.

EXPERIMENTALAnalyses were per-

formed using a P/ACE™

2050 CE instrument fromBeckman Coulter equippedwith a LIF detector(488/520 nm). Coatedcapillaries (DB-17; J&W Sci-entific, Folsom, CA, USA)with a length of 30 cm tothe detector window,100 µm I.D., and a coatingthickness of 0.1 µm wereused for separation. Thecapillary was flushed withrun buffer consisting of89 mM Tris, 89 mM boricacid, 2 mM EDTA (pH 8.4),and 0.1% (w/w) hydroxy-propylmethylcellulose(HPMC, Sigma, Deisen-hofen, Germany). Just priorto analysis, the intercalatingdye YOYO (MolecularProbes, Eugene, OR, USA)was added to the runbuffer (1 µL YOYO in15 mL run buffer). After

DNA pre-staining with YOYO, thesamples were injected hydrody-namically for two seconds. Electro-phoresis was carried out at 300 V/cmand 30°C. Plasmid DNA concentra-tion was determined by the correc-ted peak area (peak area migrationtime ratio) using a calibration curve

In-Process Analysis of Plasmid Copy Numberfor Fermentation Control

Volume 5, Issue 1 • April 2001

5

of a reference plasmid standard (Plas-midFactory, Bielefeld, Germany) withconcentrations from 3 to 2000 pg µL-1.

For quantitation, small amounts ofplasmid DNA were quickly isolatedfrom bacteria culture in 20 minutesusing commercially available kitsbased on silica technology, likeNucleoSpin from Macherey-Nagel(Düren, Germany) or QIAprep Spinfrom QIAGEN (Hilden, Germany).However, this kit technology is notsufficient for purification of plasmidDNA to be used in pre-clinical orclinical applications but in quanti-tation it is helpful for separation ofplasmid DNA from genomic DNA,RNA, and proteins.

RESULTSThe quantitation of plasmid DNA

with capillary gel electrophoresiscan be performed in fewer thansix minutes (Figure 1). In contrast toour report in the P/ACE Setter ofOctober 2000 (Volume 4, Issue 2—“Assessing the Homogeneity ofPlasmid DNA: An Important Steptoward Gene Therapy”; see alsowww.CGEservice.com), the separa-tion of different plasmid structuresresulting in longer analysis time isnot necessary for the determinationof plasmid copy number because, inthis case, the quantitation of the totalplasmid amount is required. The elec-tropherogram shows that the plasmidpurity reached with the quick isola-tion method is sufficient. Only onepeak is obtained, representing theplasmid DNA. Other nucleic acid con-

taminants, like RNA or chromosomalDNA, do not disturb the measurements.Approximately 20 minutes for plas-mid sample preparation plus 10 min-utes for CGE analysis results in a totalof a 30-minute method for determin-ing the plasmid copy number. The com-pleteness of plasmid isolation couldbe demonstrated by comparing withother plasmid purification methods(7).

Figure 2 shows the calibrationcurve of corrected peak area (peakarea migration time ratio) versus DNA

concentration of a highly purifiedpBR322 plasmid standard. The linearrange extended over three orders ofmagnitude from 3 to 2000 pg µL-1,respectively. Fortunately, the use of thecalibration curve is independent fromthe size of the quantified plasmids.

The in-process control of plasmidcopy number during a high-cell-density fed-batch cultivation for phar-maceutical, industrial-scale manufac-turing of plasmid DNA performed byPlasmidFactory is demonstrated in

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Figure 2. Calibration curve of corrected peak area vs. DNA concentration of the reference plasmid standard.

Figure 1. Electropherogram of the reference plasmidstandard for quantitation.

6

Figure 3. The fed-batch cultivationprocess enables high biomass yieldsand, thus, high product yields by con-trolling the nutrient level in the culti-vation medium. At the beginning ofthe cultivation, the formation of bio-mass and production of the 7.6 kbpplasmid pUK21CMVβ in the E. colicells does not follow synchronously.The plasmid copy number, which iscalculated from plasmid concentra-tion, plasmid size, and number ofcells, varies from 10 to 250 duringcultivation. Maximum plasmid copynumbers are reached at the end ofthe batch mode (at 15 h cultivationtime) and at the beginning of the sta-tionery phase, indicating the opti-mum point of time for harvesting thecells to get a maximum product yield.

CONCLUSIONSCapillary gel electrophoresis offers

fast quantitation of quickly isolatedplasmid DNA resulting in an in-timemethod for the determination of plas-mid copy number, which is an impor-tant parameter for in-process control ofbacterial cultivations. The rapid deter-mination of plasmid copy number infewer than 30 minutes makes itpossible to control process productivi-ty and plasmid stability and to indicatethe point of time for cell harvest.

REFERENCES1.Schleef, M. Plasmids for Therapy

and Vaccination. Weinheim, NewYork: Wiley-VCH, 2001.

2.Miller, et al. Molec. Microbiol. 9,695-702, 1993

3.Hinnebusch, et al. J. Bacteriology174, 5251-5257 (1992)

4.Sambrook, et al. Molecular Cloning:A Laboratory Manual. Cold Spring,Harbor, New York: 1989.

5.Coppella, et al. Biotechnol. Bioeng.29, 646-647 (1987)

6.Projan, et al. Plasmid 9, 182-190(1983)

7.Schmidt, et al. J. Biotechnol. 49,219-229 (1996)

HARRY WHATLEY

BECKMAN COULTER, INC.

INTRODUCTION

Complex natural proteinmixtures provide a difficultanalytical challenge. Capillary

electrophoresis–tandem mass spectro-metry (CE-MS/MS) provides a power-ful tool with which to attack thisproblem and offers new capabilitiesto the scientist engaged in proteinresearch. The identification of theprotein constituents in a mixture canbe performed rapidly without time-consuming protein purification steps.

In this approach, components areidentified by CE-MS/MS analysis of pep-tides produced by partial enzymaticdigestion of the entire mixture. Thehigh resolving power of the CE systemseparates the peptides in as few as 20minutes. Automated ion selection andfragmentation coupled with computeranalysis of the fragment ion patternprovides user-friendly identification ofcomponents in the mixture. The speedand low reagent consumption of thismethod facilitates the testing ofmultiple buffer chemistries. Data fromseveral different systems is presentedhere; each provides a different perspec-tive on this complex mixture.

Animal venoms provide interest-ing model systems for studying theeffectiveness of this approach. Thesevenoms consist of multiple proteinswith a variety of biological activitiesmixed with inactive proteins andnon-protein components. The tech-nique described here is applicable toother natural protein mixtures as wellas to protein mixtures created bybiotechnology. This report willdescribe studies with the venom ofthe honeybee, Apis mellifera.

The honeybeehas been valued forcenturies as asource of honeyand beeswax. Beeproducts have longbeen used in folkmedicine. In recent years, the venomof these insects has been studied as apotential source of therapeutics for thetreatment of a variety of conditions,including rheumatoid arthritis, multiplesclerosis, and local inflammation.Whether or not bee venom is effectiveagainst disease, the separation scientistfinds bee venom to be an easily ob-tained mixture of bioactive substancesthat serves well as a model for theanalysis of other protein mixtures.

The mixture of components inbee venom, shown in Table 1, providesome challenges to the analyst. Melittincomposes 65-80% of a typical beevenom preparation. The remainingcomponents are relatively minor.Melittin and some of the other com-ponents have significant hydrophobicproperties and tend to form multimers.Many of the components of bee venomhave very high pI values. The largerproteins are compactly folded and notvery accessible to digestive enzymes.

How to Bee a Protein Chemist

Table 1: Major Componentsof Bee Venom

Component MW

Apamin 2089Melittin 2848Secapin 2867Mast Cell Degranulating

Protein 3354Phospholipase A2 15239Hyaluronidase 40837

Serotonin, dopamine, nor-epinephrine,and histamine are also present.

Volume 5, Issue 1 • April 2001

EXPERIMENTALDried bee venom (Sigma, St. Louis,

MO) was dissolved in water and clari-fied by centrifugation. Preliminary analy-sis of the crude venom was performed

on a P/ACE™ MDQ capillary electrophore-sis system (Beckman Coulter, Fullerton,CA) with PDA detection or coupled toan LCQ DUO MS/MS system (Thermo-Finnigan, San Jose, CA). See the figures

for details of the separations. To gener-ate fragments, crude venom was dis-solved in ammonium carbonate bufferat pH 8.3 and digested with 1% w/wtrypsin without prior reduction. After

7

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NL:1.42E9TIC MS Crude-am-13

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Figure 1. CE-MS of undigested bee venom; Amine coated capillary 50 µm x 80cm Ld/Lt 85:15; 1N HOAc:Acetonitrile; 30 kV, cathode at outlet; total ion current; ion at m/z of 949.78 istriply charged.

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Figure 2. Analysis of tryptic digest of bee venom. Labeled components are peptides from Phospholipase A2 (1 and 3), Melittin (2), and Secapin (4). Automated CE-MS/MS; PEO capillary50 µm x 80 cm Ld=Lt; 1N HOAc; 30 kV, cathode at outlet; components identified by TurboSequest.

8

digestion, the mixture was reacted withexcess Tris (carboxyethyl) phosphineto reduce disulfide bonds, then adjustedto pH <2 with acetic acid to stop thereactions. Peptide identification waspreformed with the TurboSequest soft-ware from ThermoFinnigan. CE separa-tion conditions are shown in the figures.

RESULTS AND DISCUSSIONUsing this technique, we were

able to verify the presence of theprotein components listed in Table 1.Melittin is by far the dominantcomponent and could be identifiedwithout digestion. It is known toform dimers, tetramers, and higher-order aggregates. Figure 1 indicatesthat it may be possible to separatethese multimers by CE. In the MS,they dissociate into the monomer.

Using CE-MS/MS on the trypticdigest of bee venom results in a fairlycomplex profile. By using an amine-

coated capillary, the complete analysisof the sample is possible in 20 min-utes. The Xcalibur software allows theMS to be programmed in “data depen-dent triple-play” mode. In this mode,individual ion species in a peak areautomatically isolated and fragmented,so that even unresolved componentscan be isolated. The triple-play datacan be read by the TurboSequest pro-gram (ThermoFinnigan, Inc.) and thepeptide fragmentation patternmatched to known proteins in asequence database. Figure 2 showsdata from such an experiment.

In addition to the known compo-nents, a peak was found in the crudevenom with an m/z of 1003. This wasdetermined to be a triply chargedspecies. The mass of the parent mole-cule can be calculated to be about3010 which does not correspond toany known venom component. Workis under way to identify this protein.

CONCLUSIONSIn this study, most of the known

components of crude bee venomwere identified with relatively simpletechniques and without sample puri-fication. A previously unreported pro-tein of mass 3010 was found in thissample. CE-MS/MS combined withenzymatic fragmentation provides asimple and rapid method for theidentification of proteins in complexmixtures. The technique describedhere does not require the purificationof individual proteins prior to analysis.CE is a “protein friendly” separationtechnique, particularly with samplesthat tend to aggregate or bind to sur-faces. The speed and low bufferrequirements of CE simplify methoddevelopment and optimization. TheP/ACE™ MDQ–LCQ DUO combina-tion provides a turnkey systemapproach to the analysis of complexprotein mixtures.

Volume 5, Issue 1 • April 2001

MAY 14-17, 2001FACULTY OF PHARMACY

UNIVERSITY OF MONTPELLIER

OBJECTIVEThis training course aims to show

through lectures, laboratory sessions,and computer simulations how it is pos-sible to optimise the criteria required forvalidating an assay method for an activedrug and its degradation products.Particular attention is paid to the quanti-tative aspects of the technique.

PUBLIC CONCERNEDTechnical executives and

technical staff of Development andQuality Control laboratories frompharmaceutical, chemical, agro-chem-ical, and cosmetic industries;

laboratories involved in the quantita-tive aspects of the CE; those wishingto acquire training in specialised ana-lytical techniques for quality control.

LOCATION AND DATEFaculté de Pharmacie, Montpellier,

France, from 2pm Monday, 14th May,2001, through 5pm Thursday,17th May, 2001.

PROGRAMMEThe programme includes:

• Principles (CZE, MECC);instrumentation; backgroundreview of applications(impurities, assay, identification,chiral separations and small ions)

• Optimisation of selectivity,repeatability, and sensitivity

• Quantitative procedures

• Method validation, method transfer• Chiral separations• Analysis of small ions• Non-aqueous CE• Microemulsion CE• Electrochromatography

Teaching will be given as lectures,laboratory sessions, and computersimulations by Dr. K. D. Altria (GLAXOWELLCOME; Ware, UK).

To register, or for more informationon this course, please contact:

Prof. H. Fabre or Dr. M. D. BlanchinLaboratoire de Chimie Analytique -Faculté de Pharmacie - F-34060MONTPELLIER CEDEX 2 FRANCE

Tél. (33) 4 67 54 45 20 - Fax (33) 4 67 52 89 15

E-mail : hfabre@pharma.univ-montp1.fr

TRAINING COURSE

Quality Control Of Drugs by Capillary Electrophoresis:A Practical Approach

9

Dear Friends and Colleagues,

In fact Dear “Electrophoreticists”(let me use this neologism so that wecan have our niche in SeparationScience, just like those working in chro-matography, who call themselves “Chro-matographers”), on behalf of ICES (theInternational Council of ElectrophoresisSocieties), I have the pleasure ofinviting you to our 10th InternationalCongress, to be held in Verona duringJune 10-14, 2001. Our Congress, as youare aware, takes place every other year,the last three having been held in Paris(1995), Seattle (1999) and Tokyo(1999). It was in Tokyo that ICES select-ed the University of Verona as the sitefor the Congress of the third Millen-nium, a selection which, although beinga big honour for our University, filledme with “Fear of Flying”, i.e. of beingunable to carry out such a task of greatresponsibility. We are doing our best forassembling a great meeting, but certain-ly the main contribution will comefrom all of you, the future participants,the Electrophoreticists who want againto meet each other, to renew oldacquaintances and to make new ones,and to positively contribute to thesuccess of this meeting by presentingyour newest data in the field.

A great contribution to the presentICES meeting has already come fromGlaxoWellcome who generously donat-ed their congress facilities, a unique set-

ting where the ICES-2001 will take place(you can have a glimpse at these facili-ties by browsing into our web site,although the pictures there do not ren-der justice to the elegance and beautyof the place). Companies dealing withelectrophoretic products are alsogenerously contributing, but, again, theultimate contribution will be your par-ticipation, which we hope will be ingreat numbers.

Thus, I hope you will not miss thisunique opportunity of being “electro-phoresed” into the third Millennium bycoming to a meeting offering the mostadvanced topics in the field and someof the top speakers available today inthe scientific arena. But let me also fin-ish with a note of caution: if youdecide to attend this meeting, be anearly planner! Due to severe limitationsin accommodations in town, we arestrictly forced to limit attendance to400 participants, which represents themaximum capacity of our CongressCenter as well as the maximum numberof rooms we could reserve intown. I hope what happenedrecently at the third Siena meet-ing on “Proteome Analysis”(a classic in the field by now)will not happen to you.Although attendance there hadalso been limited to 400, in theend, approximately 800 applica-tions poured in and 400

scientists had to be left out (believeme, it was a fierce battle for thelatecomers to try to win a seat in theparterre!). In addition, quite a numberof the latecomers, due to hotel roomlimitations, had to find accommoda-tions as far as 30 Km out of town. Iwould not want this to happen to you.Remember that the participants at ourmeeting will be a tiny minority amongthe visitors storming the town to enjoythis magnificent setting and to strollalong the banks of the Adige river (yes,June is a magnificent month, with theglorious light of the summer and thelong, long days due to the daylight sav-ing time schedule). Plan your trip wellin advance and reserve your hotel veryearly in the game. I would not wantyou to have to spend your nightsunder the bridges of the Adige river(romantic, yes, but hardly pleasant).

With my best wishes,

Prof. Pier Giorgio Righetti

ICES—THE INTERNATIONAL COUNCIL OF ELECTROPHORESIS SOCIETIES

INVITES YOU TO ATTEND THE MEETING

ICES-2001: An Electrophoretic OdysseyGLAXOWELLCOME CONGRESS CENTER

10-14 JUNE 2001UNDER THE AUSPICES OF THE UNIVERSITY OF VERONA (ITALY)

ORGANIZING SECRETARIAT:EMMEZETA CONGRESSI

VIA C. FARINI 81, MILANO 20159, ITALY

TEL. +39 02 66802323 – FAX +39 02 6686699WEB SITE : WWW.MZCONGRESSI.COM/ICES2001

1010

Dr. Mary-Jo SchmerrNational Animal Disease Center

Transmissible spongiformencephalopathies are a group offatal neurodegenerative diseases

in animals and humans. The mostfamous member of this family isbovine spongiform encephalopathy or“mad cow disease.” The oldest knownmember of these diseases is sheepscrapie. The causative agent of thesediseases is an altered form of a normalhost glycoprotein (prion protein). Mostof the diagnostic tests are post-mortemtests. Tissue biopsies performed in liveanimals are risky, expensive, andpainful. Since blood is readily availableand easily obtained, a blood assay isthe “Holy Grail” for testing for TSEs.

The electropherogram shownrepresents a competition immunoassayfor the presence of the abnormal prionprotein in a blood sample from ascrapie-infected sheep. We used 0.2 M

Tricine, pH 8.0, containing theadditives 0.1% N-octyl glucoside and0.1% BSA as our buffer. The additivesprevented the sample from binding tothe capillary walls. Uncoated glass cap-illaries, 20 µm I.D. x 20 cm to thedetector, were used for the analysis.The voltage was 26 kV with a currentof ~12 µA. Samples were stored at 7°Cuntil they were run. The runningtemperature of the capillary was 20°C.The analysis was done on a P/ACE™

MDQ using LIF for detection.

A peptide from prion protein,amino acid positions 218-232 waslabeled during synthesis through agamma butyric acid bridge withfluorescein. Theoretically, each peptidewas labeled with the dye. Rabbitantibodies to this peptide werepurified over a column that had thecorresponding peptide linked to thematrix. A single peak was obtainedwhen the peptide was run alone. Theaffinity purified antibody was titrated

with the peptide so that the ratio ofthe peaks of the bound peptide tothe free peptide would be ~1. Thepresence of the prion protein woulddisplace the fluorescein–peptide fromthe antibody resulting in a lower signalfor the immunocomplex peak and anincrease in the free peptide peak. Aftertesting several normal animals, the“cutoff value” was determined as 70%of the antibody control. The ratio ofthe peaks on the electropherogram is0.47 and, thus, a positive sample. Thissheep was clinically normal when thetest was performed but died of clinicalscrapie approximately 14 months later.We have determined that the amountof abnormal prion protein in 10 mL ofblood ranges from 2-25 pg. This com-bination of the extraction protocol forthe abnormal prion protein and thesensitivity of CE-LIF system makes atest using blood possible for the TSEs.Thus, the “Holy Grail” is within reach.

Dr. Schmerr Wins 2001 “Putting CE to Work” AwardSEARCHING FOR THE “HOLY GRAIL”

The “Putting CE to Work” Award celebrates innovation in CE analysis, with the recipient being chosen by scientific peerson the merits of applying CE technology to solve key analytical challenges—essentially Putting CE to Work. This year,entries were submitted from around the world with the selection and presentation made during the 2001 P/ACE Userscelebration, held in conjunction with HPCE Symposium in Boston, MA. The winning entry was submitted by Dr. Mary-JoSchmerr from the National Animal Disease Center, Ames, IA, and was titled, “Searching for the Holy Grail.”We extendour congratulations to Dr. Schmerr and invite you to consider planning your entry for next year’s award.

1.5 2.0 2.5

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0.02

0.04

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0.08

Labeled Peptide bound to antibody

Labeled Free Peptide

Rel

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time (minutes)

Volume 5, Issue 1 • April 2001

11

Sample introduction into the sep-aration capillary for CE-MS/MS isperformed by positive pressure

displacement or by electrokinesis. Inmost CE-MS systems, sample is intro-duced while the MS system is in a“standby” mode, with the start of MSdata acquisition being triggered bythe start of the CE separation step.

It is the “standby” status of the MSsystem that concerns us here. Inmany systems, the spray voltage,sheath gas, and sheath liquid are lefton during the sample introductionstep, unfortunately resulting in poorreproducibility. The impact of thiseffect may range from unexpectedlyincreased or decreased quantitiesloaded to the complete loss ofsample from the capillary.

EFFECT OF SHEATH GASConsider the sheath gas. Depend-

ing on the capillary position in thespray needle and the velocity of thesheath gas, a venturi effect can devel-op. The moving gas stream creates anarea of low pressure at the capillarytip. Because pressure injection dependson a pressure difference between thetwo ends of the capillary, a diminishedpressure at the outlet end will causethe sample volume introduced to begreater than expected. In extremecases, the shape of the sample plugmay be distorted by laminar flow, orair may be drawn into the capillary asthe inlet end is moved from vial to vial.

EFFECT OF SPRAY VOLTAGESpray voltage is somewhat more

complex. This is the voltage appliedbetween the sprayer tip and the heat-ed capillary in the MS. Because theCE and the MS are at a commonground potential, the spray voltageand the separation voltage caninfluence each other. The capillaryvoltage is approximately the algebraicsum of the applied capillary voltageand the spray voltage. This can beverified by noting the change in theCE capillary current as the spray volt-age is changed. In separation mode,the applied capillary voltage is usuallymuch greater than the spray voltage.During sample introduction, however,the presence of voltage on the sprayertip can result in a significant currentflow down the CE capillary. Depend-ing on the nature of the sample, thiscurrent can either reduce or enhancethe amount of sample loaded. Underthe right conditions, the sample mayeven back-migrate out the end of thecapillary during the few secondswhen no pressure or voltage is beingapplied to the CE capillary.

OUR RECOMMENDATIONTurn off the spray voltage and the

sheath gas during the sample intro-duction step. Fortunately, the LCQseries mass spectrometers can beprogrammed to turn off the offend-ing elements until the CE separationhas begun. This is accomplished byusing multiple tune files in the acqui-sition method. First, the MS system istuned as usual to maximize sensitivityto the ions of interest. After savingthe tune file, it is modified. Spray volt-age and/or sheath gas are set to zero.

The file is saved with a new name.The Xcalibur software allows theuser to program multiple segmentsfor MS acquisition, and each segmentcan have a different tune file. Thespray conditions during the “standby”phase will be those defined in thetune file of Segment 1. By creating ashort Segment 1 with our “zero volts-zero gas” tune file, those factors willnot be present during sample intro-duction. A length of 15 to 30 secondsis sufficient for this segment, whichonly needs to be longer than thetime programmed for the ramp-up ofthe separation voltage (usually tenseconds). No ions will be producedin this segment, but no sample isexpected to travel the length of thecapillary in so short a time.

EFFECT OF SHEATH LIQUIDSheath liquid flow is not likely

to create a problem with sample intro-duction. And since the sheath liquidprovides the electrical connection forthe outlet end of the CE capillary, weadvise you to keep the sheath liquidflowing at a low rate (2-5 µL/min)during sample introduction.

Optimizing Sample IntroductionConditions in CE-MS/MS

HYPHENATIONS

HARRY WHATLEY

Developing innovative solutions in genetic analysis, drug discovery, and instrument systems.

Beckman Coulter, Inc.• 4300 N.Harbor Boulevard,Box 3100 • Fullerton,California 92834-3100Sales:1-800-742-2345 • Service:1-800-551-1150 • Telex:678413 • Fax:1-800-643-4366 • www.beckmancoulter.com

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All trademarks are the property of their respective owners.

The HSCDs are a proprietary distribution of cyclodextrinswith an average number of 12 sulfates for the b-CD, 11 forthe a-CD and 13 for the g-CD. These distributions were

preferentially selected over single isomers to provide increasedresolution under defined separation conditions. These productsperform well for the analysis of many neutral, basic and weaklyacidic compounds of pharmaceutical and biological interest.To ensure batch-to-batch consistency, Beckman Coulter has

developed a processwhich generates thefinal product as a20% w/v solution ofthe HSCDs. Ourprocesses incombination with avery rigorous qualitycontrol programensure consistentmatching of all lots toa Gold standard.

Highly Sulfated Cyclodextrins

Volume 5, Issue 1 • April 2001

Submit Manuscripts—Exchange Ideas

Providing a forum for the exchange of ideas, weinvite you to submit articles or manuscriptsdescribing novel uses of capillary electrophoresis

in your laboratory.

Content: You decide. If the subject is of interest toyou, it most likely is interesting to others as well.

Length: Please keep your article to less than 2,000 words.

Use appropriate referencing where copyright and trade-marks are involved.

Beckman Coulter reserves the right to editfor length/clarity.

Send a hardcopy version of your article and electronicversion on disk to:

Beckman Coulter, Inc.P/ACE Setter Review Group, M/S D-31-E4300 N. Harbor Blvd./P.O. Box 3100Fullerton, CA 92834-3100 U.S.A.

or send your article via email to:pacesetter@beckmancoulter.com

P/N DESCRIPTION

149869 Highly Sulfated Cyclodextrin 20% w/v Trial KitConsists of HS α Cyclodextrin (5 mL) 20% w/v, HS βCyclodextrin (5 mL) 20% w/v, HS γ Cyclodextrin (5 mL)Test Mix, PTS Marker (1 mL @ 10 mMolar), CapillaryConditioning Solution and Phosphate Buffer, pH 2.5 (50 mL).

713348 HS α Cyclodextrin (5 mL) 20% w/v713349 HS α Cyclodextrin (100 mL) 20% w/v713331 HS β Cyclodextrin (5 mL) 20% w/v713347 HS β Cyclodextrin (100 mL) 20% w/v713350 HS γ Cyclodextrin (5 mL) 20% w/v713351 HS γ Cyclodextrin (100 mL) 20% w/v

713330 HS Cyclodextrin Test MixIncludes:• pseudoephedrine enantiomers• glutethimide enantiomers

713328 PTS Marker (1 mL @ 10 mMolar)713333 Capillary Conditioning Solution (10 mL)477422 Phosphate Buffer, pH 2.5 (50 mL)