1

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

2

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

3

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)

4

ESSENTIAL AMINO ACIDS

5

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.

6

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

7

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

8

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

9

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?

10

PROTEIN DEGRADATION

CHEMISTRY

PHYSICS

BIOLOGY

MECHANIC

11

PROTEIN DEGRADATION IS OCCURRED DURING:

ISOLATIONISOLATIONPURIFICATIONSTIRAGEPFURTHER HANDLINGUSE

GOOD PREPARATION OF REC PROTEIN

PROTEIN DEGRADATION WILL REDUCE OR LOSS PROTEIN ACTIVITY

12

PROTEIN DEGRADATION BY CHEMICAL FACTORS

CHEMICAL REACTION BY:

WaterImproper pHExcipients in formulationCosolventsTemperatureSaltsMetal ions

( h ) Aggitation (mechanic) High concentration of protein

13

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

14

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)

15

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

16

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

17

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

18

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

19

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

20

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)

21

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

22

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

23

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

24

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

25

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

26

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)

g

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

27

28

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

29

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

30

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%

31

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

32

Concentration of cryoprotectant vs activity

Cont’d

33

Concentration of protein vs activity

AAPS PharmsciTech 2005

Lyophilization vs protein stability: aggregation

J.Pharm.Sci., 2001, 90

34

Type and concentration of cryoprotectant vs aggregation

J.Pharm.Sci., 2001, 90

Conc protein vs aggregation

35

Lyophilization vs reconstitution

Protein conc

Conc protein vs reconstitution time

10 min after reconstituion

36

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

37

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

38

39

40

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