1/20/2016 1DEPARTMENT OF PHARMACEUTICS

STABILITY STUDIES OF PROTEINS AND PEPTIDES

Presented by-

Sulabh Singhania

M.Pharm – 1st SEMEnrollment No-201504100410019

Introduction

• Degradation observed with peptide/protein pharmaceuticalsis classified into chemical and physical mechanisms

• Chemical degradation:- changes in covalent bond– Deamidation

– Racemization

– Hydrolysis

– Disulfide formation/ exchange

– β-elimination

– Oxidation

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Contd..

• Physical degradation:- changes in non-covalent interaction– Hydrophobic bonding and or associations

– Denaturation

– Precipitation

– Aggregation

– Adsorption

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1.Chemical Degradation

1.1.Deamidation:• Asparagine residues in peptides and proteins undergo

deamidation via cyclic imide formation followed bysubsequent hydrolysis to form the corresponding aspartic andiso-aspartic acid peptides is favored within neutral-to-basicpH.

• Glutamine residues also under the same but at a slower rate.

• ExampleAdrenocorticotrophic hormone(ACTH) - 38 amino residues undergoespseudo first order deamidation.

The deamidation rate increased with increasing pH and bufferconcentration.

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• The deamidation rate was higher for asparagine residueshaving a smaller amino acid at the C-terminal side of theresidue, as shown in Table 14, indicating that steric factorsmay influence cyclic imide formation.

• Deamidation of ACTH under acidic pH conditions isconsidered to be direct deamidation to the aspartic acidpeptide since the iso-aspartic acid peptide was not observedas a degradation product.

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Scheme showing deamidation isomerization and racemization

1.2.Racemization and isomerization

• Peptides and proteins having an aspartic acid residue undergohydrolysis, isomerization, and racemization via cyclic imideformation as shown in scheme L-aspartic acid peptide canisomerize to L-iso-aspartic acid peptide via its L-cyclic imide.

• The L-cyclic imide intermediate is capable of undergoingracemization to the D-cyclic imide and thus forms the D-aspartic acid peptide and the D-iso-aspartic acid peptide onhydrolysis.

• Racemization has also been observed with many peptides andproteins. Casein exhibits racemization at aspartic acid,phenylalanine, glutamic acid, and alanine residues

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1.3.Hydrolysis

• As shown in the following scheme aspartic acid residues inparticular are susceptible to hydrolysis in the acidic pH range.

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1.4. Cross-Linking through Disulfide Bond Formation and Other Covalent Interactions

• Oxidation of cysteine residues of peptide and proteinmolecules yields intra- and intermolecular disulfide bondsleading to changes in tertiary structure.

• normal disulfide bonds in peptide and protein molecules canundergo thiol-catalyzed intra and intermolecular exchangereactions, leading to changes in secondary and tertiarystructures.

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• Furthermore, the disulfide bond itself is susceptible tocleavage via β -elimination and forms dehydroalanine residuesand persulfides.

• Lysozyme exhibits deamidation at pH 6 and deamidation andhydrolysis at pH 4,whereas cleavage of disulfide residues andformation of new disulfide bonds were observed at pH 8.

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1.5.Oxidation

• Formation of disulfide bonds from cysteine residues is anoxidation reaction. A cysteine residue in α -amylase is oxidizedat pH 8.0

• Methionine and histidine residues are also susceptible tooxidation. Oxidation of methionine residues has beenobserved during storage of parathyroid hormone819 andrelaxin.

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2.Physical Degradation

• Larger peptides and proteins are susceptible to non-covalentor physical changes (so called physical degradation.) inaddition to chemical degradation.

• Physical degradation includes denaturation, aggregation,adsorption, and precipitation.

• Denaturation, that is, an alteration of tertiary (and/orquaternary) structure, generally results in loss of bioactivity.

• Furthermore, exposure of hydrophobic groups upondenaturation often leads to adsorption onto surfaces,aggregation, and precipitation.

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• Denaturation may also prompt chemical degradationpathways often not seen with the native or natural tertiary(and/or quaternary) structure. Therefore, there isconsiderable interest in preventing denaturation whileformulating protein drugs.

• Cross-linkages via disulfide bond formation cause aggregationof peptides and proteins.

• Hydrophobic bond formation, on the other hand, causesaggregation without covalent changes.

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3.Degradation in Peptide and Protein Formulations• Degradation of peptides and proteins in formulations is

complex because various factors may be involved in thedegradation.

• Analytical methods such as electrophoresis and gelpermeation chromatography are useful in assessing theirstability and elucidating the degradation mechanisms.

• Peptide mapping– degradation of monoclonal antibody

• Temperature gradient gel electrophoresis– for studying irreversible and reversible denaturation.

• Quasi elastic light scattering– Determining changes in size distribution upon aggregation.

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4. Factors Affecting the Degradation of Peptide and Protein Drugs

4.1 Moisture Content and Molecular Mobility.

• Freeze-dried ribonuclease A with a higher water contentexhibited a greater extent of aggregation.

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• Moisture absorption often decreases storage stability oflyophilized proteins; however, extremely low moisturecontent can also decrease storage stability.

• Storage of freeze-dried insulin at high relative humidity causesas expected, a higher moisture absorption, which thenresulted in increased aggregation, as shown

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• Aggregation of lyophilized β -galactosidase and bovine serumalbumin in solution also increased with increasing watercontent

• Destabilization caused by moisture absorption can be ascribedto the plasticizing effect of water, which increases themolecular mobility of Iyophiles.

• Plasticization of lyophilized β -galactosidase and bovine serumalbumin due to moisture absorption was accompanied by anincrease in the mobility of water molecules as measured bythe spin-spin lattice relaxation time of H2

17O.

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4.2 Role of Excipients

• Freeze-dried proteins are most stable in viscous glassy states, and their stability is increased by interactions such as hydrogen bonding between proteins and surrounding molecules, therefore, excipients that contribute to the maintenance of a glassy state or interact with the proteins can stabilize the proteins.

• Excipients such as sugars and HP-β –CD improves the stability of alkaline phosphates.

• Denaturation of urease and IL-2 is inhibited by non-ionic surfactants like polxamers.

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5.Degradation Kinetics of Peptide andProtein Pharmaceuticals

5.1 Quantitative Description of Peptide and ProteinDegradation

• chemical degradation of small peptides in aqueous solutionsgenerally conforms to simple first-order kinetics. For example,first-order kinetics have been reported for the hydrolysis inaqueous solution of secretin, which has 27 amino acidresidues

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• Apparent first order kinetics

5.2 Temperature Dependence of theDegradation Rate of Peptide andProtein Drugs

• Stability prediction for peptide and protein drugs underaccelerated testing conditions is possible if the temperaturedependence of the degradation rate is determined and foundto be well behaved.

• The temperature dependence can often be represented by the Arrhenius equation, as was seen with small-molecule drugs. Linear Arrhenius plots and the values of apparent activation energy calculated from the slopes have been reported for chemical degradation of various peptides in aqueous solutions.

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• Even when mechanisms and pathways are unknown, it issometimes possible to use Arrhenius or empirical equationsto determine apparent rate constants at other temperatures.

• Apparent first-order rate constants for α -chymotrypsin andbromelain inactivation exhibited linear Arrhenius plots.

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• Linear Arrhenius ’plots suggest that prediction ofdenaturation rate by accelerated testing is possible if thedenaturation mechanism does not change in the temperaturerange in question.

• denaturation at lower temperature may occur viamechanisms different from those at higher temperature. Thismakes it difficult to predict the stability of peptide and proteindrugs by accelerated testing.

• But in cases where only thermal denaturation occurs, however, prediction of denaturation rate by accelerated testing may be possible.

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References

• Sumie Yoshioka and Valentino J Stella’s “Stability studies of drugs and dosage forms.

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THANK YOU.

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