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Dr. Heni Rachmawati
School of Pharmacy Institut Teknologi Bandung
Biologically active peptides and proteins are becoming an increasingly important class of
BACKGROUND
m g g y mp fdrugs.
Their use for human and animal treatment is problematic, however, because some of these drugs are generally ineffective when taken orally
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PREFORMULATION STUDY
Protein/peptide formulation is quite different from the formulation of conventional drugs because of protein formulation of conventional drugs, because of protein structure (1,2,3,4), in which they are degraded not through one step reaction, and the degradation product can not be detected with only 1 analytic method
Factors must be concerned during development of protein formulation:
Protein structureFactors influencing both chemical and physical degradationsTechniques used to improve protein stabilization
Preformulation study:Study on the physicochemical properties both protein and excipientStudy how to evaluate the proteinStudy how the protein stability and factors influencing
Understanding on the solubility, stability, isoelectric point will determine how to formulate protein properly
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PROTEIN
Macromolecul compound (BM 5500 –220.000 dalton or 50 – 2000 amino acids)
Composed by amino acids(BUILDING BLOCK)
Peptide bonding connects the amino acids formingpolypeptide (protein)
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ESSENTIAL AMINO ACIDS
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PROTEIN BIOSYNTHESIS
The protein synthesis is coded by certain gen(genetic code)
Each protein has a unique amino acid sequencederived from nucleotide sequence coding the protein(genetic code)Genetic code is a sequence containing 3 nucleotides named CODONEach codon will code 1 amino acid
PROTEIN STRUCTURE
Primer: Sequence from amino acids chain
Sekunder:If the amino acid sequence connected by hydrogen boundconnected by hydrogen bound
TersierIf any interaction between alpha helix and beta sheet. The structure is stabilized by hydrogen bound, salt bridge, disulphide and hydrophobic core
QuarternerProtein composed by more than 1 protein molecule: dimer, trimer, olygomer, either homomer or heteromer.
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CHEMICAL BONDING IN PROTEIN FOR STABILIZATION OF PROTEIN
Metabolisme: enzyme, hormone
PROTEIN FUNCTION(endogenous protein)
y ,Immunity: antibody, cytokine
Growth: hormone, growth factorTransport and storage: hemoglobin
Etc
Potential as a drug
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Protein characteristicsProtein characteristics
1. Protein is a complex macromolecule2. Its biological activity is influenced strongly by
its structure and conformation (primer, sekunder, tersier, and quarterner)
3 Very potent drug (the dose is very low)3. Very potent drug (the dose is very low)4. Sensitive by many factors
Some endogenous proteins are produced constitutively (at normal condition):
PRODUCTION OF ENDOGENOUS PROTEIN
constitutively (at normal condition) hormone, enzyme, albuminSome endogenous proteins are producedinductively (at stimulated condition):antibody, cytokine, growth factor, enzyme Some proteins are produced both and inductively: albumin hormone enzymeand inductively: albumin, hormone, enzyme
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PROBLEM USING ENDOGENOUS PROTEIN AS A DRUG
The amount of produced of endogenous protein is not sufficientThe protein production is lowered under certain conditionhe prote n product on s lowered under certa n cond t on
Required protein from outside the body (exogenous protein)
EXOGENOUS PROTEIN MUST BE IDOLATED FROM LIVING ORGANISM
Donor limit or ethical reason
PROBLEM USING ENDOGENOUS PROTEIN AS A DRUG
Rejection response (species different)
RECOMBINANT PROTEIN
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VECTOR FOR PRODUCTION OF RECOMBINANT PROTEIN
BACTERIAYEAST
PLANTMAMMALIA CELLS
Glycosilated( i i id )
nonglycosilated( i id )(containing sugar residue) (not contain sugar residue)
ACTIVITY?STABILITY?
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PROTEIN DEGRADATION
CHEMISTRY
PHYSICS
BIOLOGY
MECHANIC
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PROTEIN DEGRADATION IS OCCURRED DURING:
ISOLATIONISOLATIONPURIFICATIONSTIRAGEPFURTHER HANDLINGUSE
GOOD PREPARATION OF REC PROTEIN
PROTEIN DEGRADATION WILL REDUCE OR LOSS PROTEIN ACTIVITY
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PROTEIN DEGRADATION BY CHEMICAL FACTORS
CHEMICAL REACTION BY:
WaterImproper pHExcipients in formulationCosolventsTemperatureSaltsMetal ions
( h ) Aggitation (mechanic) High concentration of protein
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Protein is composed by polypeptide forming a unique 3p y p yp p g qdimensional structureProtein structure is stabilized by combination ofelectrostatic and hydrophobic interactions with molecule flexibility at the inner part of protein
In solution, the structure of protein can be destroyed if the environment changes including alteration in protein size.
Protein stability in solution is difficult to keep
TYPE OF CHEMICAL REACTIONS DESTABILIZED PROTEIN
DeamidationHydrolisisHydrolisisIsomerizationDeglycosilationOksidationReaksi Maillard
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PROTEIN DEGRADATION
Type of degradation protein condition
Cleavage bFGF very acidD id ti hEGF t l bDeamidation hEGF neutral – basaDeamidation bFGF neutral – basa Deamidation insulin pH < 5Deamidation RNase A high pHOksidation rhPTH pH 10Succinimidation bFGF pH 4 - 5
(DENATURATION)
The change in global folding of protein (disturbance of The change in global folding of protein (disturbance of the highest structure of protein: tersier)
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Agregation and precipitation
AGGREGATION
The main physical destabilization process. h ( k
non-native self association form of protein in solution and can not be visualized with naked eyes
At certain condition, the protein structures (sekunder,tersier and quarterner) changes leading to aggregation
Activities solubility immunogenicity
PRECIPITATION
A makroscopic process resulting in visible alteration (increased viscosity or turbidity of solution)
Physical factors cause the aggregation and precipitation
Temperatur (increase or decrease)Ionic strenght
Polymerization (covalen) and aggregation (noncovalen) contribute to insoluble precipitate formation
Ionic strenghtMechanic (Vortex)pH Organic solvent, surfactants
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Improper temperature will denaturate protein:
Weaken the hydrogen boundStrenghten hydrophobic boundNoncovalen force decreasesProtein is more flexible leading to partial unfoldingunfo ngCoalition of protein increases leading to proteinpolymerization
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pH is the most important parameter determining protein structure.
At low pH the stability of protein is less due to At low pH, the stability of protein is less due to electrostatic interaction induced by increased in the positive charge of protein.
At high pH, the stability of protein is less due to refusal interaction of negative charge of protein.
Most proteins show maximum stability at or close to isoelectric point when the charge of protein is 0 (neutral)
Most proteins show unfolded at low pH (2-3)
Reversibility of aggregat protein
Reversible Soluble in reductor/renature agentsg
Irreversible Insoluble in reductor/renature agents
The physical characteristic of reversible andi sibl t s is si ilirreversible aggregates is similar
In reversible aggregat, the structure is more stablemore ordered and looser compared to irreversibleaggreagate
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EXCIPIENT IN PROTEIN FORMULATION
1. BUFFER SYSTEMThe buffers for protein formulation:p
Phosphate (pH 6,2-8,2)Acetate (pH 3,8 – 5,8)Citrate (pH 2,1 – 6,2; pK 3,15 and 6,4)Succinate (pH 3,2 – 6,6; pK 4,2 and 5,6)Hystidin (pK 1,8;6 and 9)Glysin (pK 2,35 and 9,8)y (p )Arginine (pK 2,18 and 9,1)Triethanolamine (pH 7-9)Tris-hydroksimethylaminomethane (pK 8,1)Maleate
Function of buffer in formulation:
Stabilize the protein and keep the bioactivityInfluence the solubility of protein in addition toionic strenght
Protein charge ~ isoelectric point
0 at pH on isoelectric point+ at pH below isoelectric point+ at pH below isoelectric point- at pH above isoelectric point
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2. PROTEIN SOLUBILITY
Protein solubility: very soluble, slightly soluble,insoluble, depends on the amino acid sequence and theconformationconformationSolubility of amino acid inverse propotional with thesize and polarityProtein solubility is determined by ability of polargroup interacting with waterGenerally, the protein solubility is minimum at electricpoint because at neutral charge, interaction betweenproteins is maximum
Influenced by:pHExcipientsTemperature
3. PRESERVATION OF PROTEIN
Preparation containing protein is sensitive to i is t i ti s th t s timicroorganism contamination so that preservative
agent is important component moreover formultiple dose preparationPreservative selection is a critical factor as addingwrong preservative agent can influence the physicalstability of protein (precipitation or turbidity ofsolution)solution)
Example: NaHSO3 used in the insulin injection caused insulin degradation at pH 4-7
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TYPE OF EXCIPIENTS
1.Albumin (human serum albumin, HSA)Protein (BM 66,4 kDa) functions in bindingwith various compoundsBecause of superiority in solubility and stability, albumin is oftently used as anexcipient (stabilizer and prevent adsorptionof peptide or other proteins on various surfaces)Albumin prevents surface adsorption of otherprotein through preferentially adsorptionmechanism
Disadvantages of albumin as an excipient:
Interfere the analisis of other proteinsProbably contamination with pathogens is isolatedProbably contamination with pathogens is isolatedfrom blood (alternative: recombinant HSA)
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2. Amino acid
Functions:Reduce surface adsorptionReduce surface adsorptionInhibits aggregationStabilize protein against high temperatureImprove the protein stabilityExamples: glysin, arginine, alanine
3. CarbohydratePolisacharide fuctions as protein stabilizer against stress conditions: heat, lyophilizationProtein stabilization by polysaccharide is basedon its effect on water structure Prevent protein aggregation induced by moisture
Examples: sucrose, maltose, lactose, trehalose- Sucrose,
Improve the surface tension of water leadingto protein stabilization Th t ff ti b t t t t The most effective substance to protect
hemoglobin from spontaneous oxidation into methemoglobin during lyophilization and storage
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4. Chelating agent
Some anion and cation can bind to protein leading to decrease protein solubilityp yThe functions of chelating agent:
Remove ions and keep the protein solubilityInhibits oxidation of sulphidrile from protein catalyzed by metalImprove the protein stability againts aggregation induced by heataggregation induced by heatExample: EDTA
5. Cyclodextrin
A carbohydrate compound but has a unique stabilization mechanism than other carbohydratesFunctions:
Improve protein stabilityStabilize protein (prevent aggregation)Carrier in drug delivery
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Cyclodextrine structure
Ring structure of cyclodextrine makes it property of its inner partCavity size is 5-10 ÅThe most used of cyclodextrine is β-cyclodextrine although its solubility is low
Aromatic amino acid interacts forming a complex Aromatic amino acid interacts forming a complex with cyclodextrine
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6. Polyhidric alcoholExamples: glycerol, erythritol, arabitol, xylitol, sorbitol, manitol
Function: Stabilizer (prevent aggregation)7. Polyethelene glycol
Functions:Reduce surface adsorption of proteinImprove protein stability (physic + chemistry)Improve protein stability (physic + chemistry)Improve protein solubility
Covalent bounding
8. Salts
Improve protein solubility at low ion strenght (salting in) but at high strenght (salting in) but at high concentration, there is a competition between ion and protein to water molecules leading to decreased in protein solubility (salting out)
Salts concentration is crucial factor
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9. Surfactan
Stabilize proteinThe most use of surfactant is tween 80The peroxide contamination in tween 80 canThe peroxide contamination in tween 80 canspeed up the protein degradationProtein stabilization by surfactant is throughpreferrential adsorption
no surfactant dimmerization aggregasi
+ surfactant, no aggregasi
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Market demand of recombinant
Preformulation consideration and Screening of formulable molecules
Required the successful of proteinformulation
m f mprotein is fast growing
Stabilizer to extend the life of protein formulation
Technique to determine the stability(monitoring of increase of contamination)
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Structure ModificationSubstitution of amino acid enhances the thermostabilityGlycosilation increases pharmacokinetic profile, proteolytic
i t d th t bilitresistance and thermostabilityDisulphide formation will protect protein from denaturation
Pegylation will improve solubility, decrease immunogenicity, enhance t1/2 and reduce toxicity
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Cryoprotectants : inhibit crystal growth during storage of protein at -20C
Excipients
pCryoprotectant: polysaccharides (non-reducing sugar), polyol,
Inhibitor protease to inhibit proteolytic degradation
Antimicrobe (natrium azide, NaN3) at final concentration of0.02-0.05% (w/v) or timerosal at final concentration 0.01%
Chelating agent (EDTA) at final concentration of 1-5 mM toChelating agent (EDTA) at final concentration of 1-5 mM toinhibit metal-catalyzed oxidation
Reductor (DTT, ditiotritiol and mercaptoethanol, 2-ME) at final concentration of 1-5 mM to maintain protein from oxidative degradation (i.e. cys).
Polymers such as albumin and cyclodextrin: protect proteinfrom surface adsorption
Amino acids such as hystidine and methyonine: antioxidant
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Formulation of recombinant protein
liquid solid
To control particle size and particle distribution of protein in liquid preparation is not easy
Solid preparation is preferred
Spray-drying Freeze drying/lyophilization
Supercritical drying
Protein is more stable during process and storage
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Proses Spry freeze drying/lyophilization of protein
Atomization of protein + carrierinto liquid nitrogen
Course dispersion
Transformation of solid dispersionto precooled freeze dryer
Drying process
Stable protein:StructureActivity
reconsitutable
Drying process
Lyophilisate of protein
Water content < 3%
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CRUCIAL FACTOR DURING LYOPHILIZATION
1. Type and concentration of cryoprotectant. ype and concentrat on of cryoprotectant2.Concentration of protein3.Concentration of bulking agent
Concentration of protein vs activity
Lyophilization vs protein stability : aktivitas
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Concentration of cryoprotectant vs activity
Cont’d
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Concentration of protein vs activity
AAPS PharmsciTech 2005
Lyophilization vs protein stability: aggregation
J.Pharm.Sci., 2001, 90
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Type and concentration of cryoprotectant vs aggregation
J.Pharm.Sci., 2001, 90
Conc protein vs aggregation
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Lyophilization vs reconstitution
Protein conc
Conc protein vs reconstitution time
10 min after reconstituion
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Protein lyophilisateProtein lyophilisate
Freeze dryer
Delivery system
The integral part in the development of protein formulation
Play a role not only to improve the life of protein but also the pharmacological activityThe most important issue in the development of protein f m l ti n:formulation:
High patient complianceEffectiveEffisien
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Common route for peptides/proteins nowadays
PARENTERAL ROUTE
Low patient compliance Invasive route
Sterility assurance
PROBLEM
Sterility assurancecostly and limited used
1. Pulmonary delivery: insulin2. Oral, especially for long-term use: MOST PREFERRED
Carrier selection to improve GI stability
ALTERNATIVE ROUTES FOR DELIVERY OF PROTEIN
p yStrategy to improve oral absorption
3. Nasal 4. Transdermal
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THE AIM OF FORMULATING OF PROTEIN
In general similar to other conventional drugs:
1 b l1. Improve patient acceptability2. Improve stability and efficacy3. Improve protein performance4. Improve easiness in therapy