Stability Of Peptides And Proteins

1. STABILITY PROBLEMS AND
PREVENTION IN PROTEINS
AND PEPTIDES DRUG
DELIVERY SYSTEM
B.THILAK CHANDRA
M.PHARMACY (PHARMACEUTICS)
VAAGDEVI INSTITUTE OF PHARMACEUTICAL SCIENCES

2. Contents
• INTRODUCTION
• PEPTIDE AND PROTEIN STRUCTURE
• PROPERTIES AFFECTED BY UNSTABILITY
• TYPES OF STABILITY PROBLEMS
1. PHYSICAL STABILITY
2. CHEMICAL STABILTY
• CONCLUSION
• REFERNCES

3. INTRODUCTION
 Proteins are biochemical compounds consisting of one
or more polypeptides typically folded into a globular or
fibrous form, facilitating a biological function.
 Peptides - 20 amino acids. proteins - 50 or more
amino acids. Polypeptides- 20 to 50 amino acids.
 Peptide chains in peptides and proteins are seldom
linear and adapt a variety of specific folded three
dimensional patterns and conformations.
 Conformation in a peptide chain is determined by the
covalently bonded amino acids sequence, by disulfide
bridges between cysteine residues and by total
conformational energy

4. PEPTIDE AND PROTEIN
STRUCTURE
1. Primary structure
2. Secondary
structure
3. Tertiary structure
4. Quaternary
structure

5. EXAMPLES
Insulin Interferon γ
Interferon β
TPA
5

6. PROPERTIES AFFECTED
BY UNSTABILITY
 PHYSICAL PROPERTIES solubility, spectral
properties such as circular dichorism.
 CHEMICAL PROPERTIES alteration of stabilized
reactive group or group sterically shield from the reagents.
 BIOLOGICAL PROPERTIES 3-D structures place
catalytic groups into proper orientation for enzymatic
activity or place backbone and side-chain groups into
proper orientation for hormone receptor interaction.
 Stability to enzymatic cleavage since some of the amide
groups susceptible to proteolysis are deterred due to
sterical peptide chain orientation.

7. TYPES OF STABILITY
PROBLEMS

8. PHYSICAL STABILITY
 Physical instability involves transformations in the
secondary, tertiary, or quaternary structure of the molecule.
1. DENATURATION
• Any nonproteolytic modification of the unique structure of a
native protein that effects definite changes in physical,
chemical, and biological properties.
• Peptides and proteins are comprised of both polar amino
resides and nonpolar amino acid residues.

9. FACTORS THAT FAVOUR
THE DENATURATION
 When solvent changes from
an aqueous to organic
solvents or to a mixed
solvent.
 On unfolding hydrophobic
and hydrogen bonds are
broken.

10. FACTORS THAT FAVOUR THE
DENATURATION
 pH changes –alters the ionization of the carboxylic acid
and amino acids and there by the charges carried by the
molecules.
 Alteration in the ionic strength.
 Temperature rise.

11.  Denaturation may be reversible or irreversible
 Denaturation may lead to decrease in in solubility,
alteration in surface tension, loss of crystallizing ability,
changes in constituent group reactivity and molecular
profile, vulnerability to enzymic degradation, loss or
alteration of antigencity and loss of specific biological
activity.
DENATURING AGENTS
category mechanism examples
Polar and protic Disrupt H-bonds Urea, guanidine HCL, alcohol,
acetic acid
chemicals
surfactants Hydrophobic disruption Sodium dodecyl sulphate,
and charge group polyethylene gltcol, dodecyl
separation ammonium chloride

12. METHODS TO PREVENT
DENATURATION
 Denaturated protein is restored on removal of denaturants.
 Maintaining pH.
 Maintaining ionic strength.
 Maintaining Temperature .

13. ADSORPTION
 Peptides and proteins are amphiphilic in nature, hence
they tend to adsorb at interfaces such as air-water and air-
solid. Example-Insulin
 Polar – hydrophilic , nonpolar – hydrophobic
 Conformational rearrangement leading to denaturation can
be induced by their interfacial adsorption.
 After adsorption, they form some short-range bonds (van
der Waals, hydrophobic, electrostatic, hydrogen, ion-pair
bonds) with the surface resulting into further denaturation
of polypeptide moieties.

14. ADSORPTION

15.  Adsorption of peptides and proteins at the interfaces are
rapid, but the rates of conformational changes are
relatively slower.
 On adsorption there may be a loss or change in biological
activity as the molecular structure is rearranged.
 If peptide and protein drug entities are adsorbed at
interfaces there may be a reduction in the concentration of
drug available to elicit its function.
 Such loss of proteinaceous drug(s) may occur during
purification, formulation, storage and/or delivery.

16. METHODS TO PREVENT
ADSORPTION
 Insulin adsorption may be minimized by the addition of
0.1% to 1% albumin.
 Excess agitation should prevented during production.
 The headspace within the confines of the container should
be small.
 Use of surfactants to reduce adsorption.
 Smooth glass walls best to reduce adsorption or
precipitation

17. Aggregation and
Precipitation
 The denatured, unfolded protein may rearrange in such a
manner that hydrophobic amino acid residue of various
molecules associate together to form the aggregates.

18.  If the aggregation is on a macroscopic scale, precipitation
occurs.
 Interfacial adsorption may be followed by aggregation and
precipitation.
 The extent to which aggregation and precipitation occurs is
defined by the relative hydrophilicity of the surfaces in
contact with the polypeptide/protein solution.

19. CAUSES OF AGGREGATION
AND PRECIPITATION
 The presence of large air-water interface generally
accelerates this process.
 Presence of large headspace within the confines of the
container also accelerates the course of precipitation.
 Insulin forms finely divided precipitates on the walls of the
containers, referred to as frosting. The presence of large
air-water interface generally accelerates this process.

20. CAUSES OF AGGREGATION
AND PRECIPITATION
 Increase in thermal motion of the molecules due to
agitation.
 Solvent composition,
 solvent dielectric profile,
 ionic strength
 pH

21. METHODS TO PREVENT
AGGREGATION AND
PRECIPITATION
 Organic solvent such as10-15% propylene glycol can
suppress the formation of peptide liquid crystals.
 Excess agitation should prevented during production.
 The headspace within the confines of the container should
be small.
 The ionic strength, solvent composition, solvent dielectric
profile and ph should be carefully controlled at every step
in production.
 Use of surfactants to reduce aggregation.

22. Chemical instability
 Involves alteration in the molecular
structure producing a new
chemical entity, by bond formation
or cleavage.
 The stability of peptide and
proteins against a chemical
reagent is decided by temperature,
length of exposure, and the amino
acid composition, sequence and
conformation of the
peptide/protein.

23. DEAMIDATION
 This reaction involves the hydrolysis of the side chain
amide linkage of an amino acid residue leading to the
formation of a free carboxylic acid.
 Asparagine glutamine
 leading to conversion of a neutral residue to a negatively
charged residue and primary sequence isomerization.
 In vivo deamidation is observed with human growth
hormone, bovine growth hormone, prolactin, adreno-
corticotropic hormone , insulin, lysozyme and secretin.

24. Factors that favour the rate
of deamidation
 pH
 temperature
 ionic strength
 The deamidation of Asn residues is accelerated at neutral
and alkaline pH
 The tertiary structure of the protein also affects its stability,
as observed with trypsin in which the tertiary structure
prevents deamidation.

25. METHODS TO PREVENT
DEAMIDATION
 The use of genetic engineering and by recombinant DNA
technology.
 The Asparagine residues can be selectively eliminated and
replaced by other residues, provided conformations and
bioactivity of protein can be maintained.

26. Oxidation and Reduction
 Major degradation pathways
 Oxidation commonly occurs during isolation, synthesis and
storage of proteins

27. Factors that favour the
Oxidation and Reduction
 The oxidative degradation reactions can even occur in
atmospheric oxygen under mild conditions (autoxidation).
 Temperature, pH, trace amounts of metal ions and buffers
influence these reactions.
 Oxidation may take place involving side chains of histidine
(His), lysine (Lys), tryptophan (Trp), and thyronine (Tye)
residues in proteins.
 The thioether group of methionine (Met) is particularly
susceptible to oxidation.
 Under acidic conditions Met residues can be oxidized by
atmospheric oxygen.

28. Factors that favour the
Oxidation and Reduction(cont…)
 Oxidizing agents like hydrogen peroxide,
dimethylsulphoxide and iodine can oxidize Met-to-Met
sulphoxides.
 Thethiol group of cysteine can be oxidized to sulphonic
acid; oxidation by iodine and hydrogen peroxide is
catalyzed by metal ions and may occur spontaneously by
atmospheric oxygen.
 Usually the oxidation of amino acid residues is followed by
a significant loss of biological activity as observed after
oxidation of Met residues in calcitonin, corticotrophin and
gastrin. Glucagon is an exception as it retains biological
activity even after oxidation.


29. METHODS TO PREVENT
OXIDATION AND REDUCTION
 Oxidation scavengers may block these acid or base
catalyzed oxidations.
Example phenolic compounds, propyl gallate.
 Reducing agents –methionine, ascorbic acid, sodium
sulphate, thioglycerol and thioglycolic acid.
 Chelating agents –EDTA, Citric Acid
 Nitrogen flush, refrigeration, protection from light and
adjustment of ph.

30. METHODS TO PREVENT
OXIDATION AND REDUCTION
 Avoiding vigorous stirring and exclusion of air by degassing
solvents can prevent air initiated oxidation.

31. PROTEOLYSIS
 The hydrolysis of peptide bonds within the polypeptide or
protein destroys or at least reduces its activity.
 The vulnerability of peptide bonds to cleavage is
dependent on the other residues involved.
 In comparison to other residues, Asn residues are unstable
and in particular the Asn-Proline bond

32. FACTORS THAT FAVOUR THE
PROTEOLYSIS AND PREVENTION
 Proteolysis may occur on exposing the proteins to harsh
conditions, such as prolonged exposure to extremes of pH
or high temperature or proteolytic enzymes.
 Bacterial contamination is the most common source of
proteases. This can be avoided by storing the protein in the
cold under sterile conditions.
 Proteases may also gain access during the isolation,
purification and recovery of recombinant proteins from cell
extracts or culture fluid.

33. FACTORS THAT FAVOUR THE
PROTEOLYSIS AND PREVENTION
 This problem can be minimized by the manipulation of the
solution conditions during the stage of purification and/or
by addition of protease inhibitors.
 Some proteins even have autoproteolytic activity. This
property aids in controlling the level or function of protein in
vivo .

34. DISULPHIDE EXCHANGE
 Thiol-disulfide exchange showing the linear intermediate in
which the charge is shared among the three sulfur atoms.
The thiolate group (shown in red) attacks a sulfur atom
(shown in blue) of the disulfide bond, displacing the other
sulfur atom (shown in green) and forming a new disulfide
bond.
Cystine
 Disulphide bonds may break and reform with incorrect
pairings. This results in an alteration in the three-
dimensional structure followed by a resultant change in
biological activity.


35.  A peptide chain with more than one disulphide can enter
into disulphide exchange reactions, leading to scrambling
of disulphide bridges and thereby a change in
conformation.
 By analogous reactions, trimers and dimers can be formed.
 The reaction is concentration dependent, particularly for
oligomer formation.
 These oligomers appear at low Rf value on TLC and are
readily removed by gel filtration.

36. METHODS TO PREVENT
DISULPHIDE EXCHANGE
 BY THIOL SCAVENGERS SUCH AS
P-MERCURIBENZOATE
N-EHYLMALEIMIDE
COPPER IONS

37. RACEMIZATION
 Racemization is the alteration of L-amino acids to D,L-
mixtures.
 With the exception of Gly, all the mammalian amino acids
are chiral at the carbon bearing chain and are susceptible
to base-catalyzed racemization.
 Racemization may form peptide bonds that are sensitive to
proteolytic enzymes.
 This reaction can be catalyzed in neutral and alkaline
media by thiols, which may arise as a result of hydrolytic
cleavage of disulphides.

38. METHODS TO PREVENT
RACEMIZATION
 The thiolated ions carry out nucleophilic attack on a
sulphur atom of the disulphide.
 Addition of thiol scavengers such as p-mercuribenzoate, N-
thylmaleimide and copper ions, may prevent susceptible
sulphur and disulphide.

39. BETA-ELIMINATION
 The mechanism involved in the beta-elimination is similar
to the racemization, i.e. it proceeds through a carbanion
intermediate.
 Higher elimination rate prevails under alkaline conditions
which ultimately lead to loss of biological activity.
 Protein residues susceptible to beta-elimination under
alkaline conditions include Cys, Lys, Phe, Ser.

40.  Stabilize proteins against thiol-disulfide exchange by
chemically block the thiol group(s) involved in the process.
For example, S-alkylating the Cys-34 of albumin stabilizes
the protein not only during high temperature and high
humidity storage, but also when loaded within a polymeric
matrix.

41. CONCLUSION
 Therapeutic peptides and proteins can degrade by
several physical and chemical pathways.
 In most cases, more than one pathway of physical or
chemical instability is responsible for the degradation of
peptides and proteins.
 The primary structure will often reveal potential sites of
chemical degradation.
 Physical instability is more difficult to predict from
primary structure.

42. CONCLUSION
 However, if most residues are hydrophobic amino
acids, it does suggest a strong tendency toward
adsorption and aggregation.
 For proteins, the secondary and tertiary structures may be
more useful predictors of physical stability.
 Comparability protocols for well-characterized biologics will
allow the introduction of biogenerics into the market.

43. REFERENCES
 CONTROLLED DRUG DELIVERY S.P.VYAS AND ROOP K.KHAR
 Therapeutic Peptides and Proteins Ajay K. Banga, Ph.D.
 Biochemistry U.Satyanarayana
 http://en.wikipedia.org