Chemical Degradation

The major known chemical degradation pathways for peptides and proteins are deami-dation, racemization, isomerization, hydrolysis, disulfide formation/exchange, P-elimina-tion, and oxidation.

5.1.1.1. Deamidation

Asparagine residues in peptides and proteins undergo deamidation via cyclic imide formation followed by subsequent hydrolysis to form the corresponding aspartic and wo-aspartic acid peptides. This mechanism occurs primarily under neutral-to-basic pH conditions. Deamidation of an asparagine residue to the corresponding aspartic acid residue may also occur via a mechanism that does not involve cyclic imide formation, as shown in Scheme 80. Glutamine residues also undergo deamidation, but at much slower rates.

Adrenocorticotropic hormone (ACTH), which has 38 amino acid residues, exhibited pseudo-first-order deamidation in the neutral-to-alkaline pH region. The deamidation rate increased with increasing pH and buffer concentration. Deamidation via the cyclic imide of an asparagine residue was suggested since both the aspartic acid and /so-aspartic acid peptides were detected as deamidation products. As shown in Table 13, the rate of disappearance of ACTH showed good mass balance with the rates of appearance of deamidated ACTH and ammonia, indicating that the rate-determining step for the deamidation is not degradation of the cyclic imide but its formation.789 A model hexapeptide with an asparagine residue (Asn-hexapeptide) exhibited a similar deamidation reaction. The deamidation rate was higher for asparagine residues having a smaller amino acid at the C-terminal side ofthe residue, as shown in Table 14, indicating that steric factors may influence cyclic imide formation.790 791

Deamidation of ACTH under acidic pH conditions is considered to be direct deamidation to the aspartic acid peptide since the wo-aspartic acid peptide was not observed as a

Scheme 80. Scheme showing the deamidation, isomerization, and racemization of peptides having asparagine or aspartic acid residues.

Table 13. The Effect of Glycine Buffer Concentration on the Deamidation of ACTH at pH 9.6

Apparent rate constant (h - 1 ) at pH 9.6, 37°C and glycine buffer concentration of:

10mM

50mM

100mM

Disappearance of ACTH

6.6 x 10-2

1.4 x 10-1

2.3 x 10-1

Appearance of deamidated ACTH

4.7 x 10-2

1.4 x 10-1

2.6 x 10-1

Appearance of ammonia

6.5 x 10-2

1.6 x 10-1

2.9 x 10-1

a Reference 789.

a Reference 789.

degradation product.789 Similar direct deamidation of the model Asn-hexapeptide resulted in 100% formation of the aspartic acid peptide.790 791

Insulin has two asparagine residues that undergo deamidation. At acidic pH values, Asn A-21 undergoes deamidation, whereas at neutral pH and in suspensions, deamidation at residue Asn B-3 predominates.792 Deamidation of insulin at pH 2 and 3 was also enhanced by self-association.793

5.1.1.2. Isomerization and Racemization

Peptides and proteins having an aspartic acid residue undergo hydrolysis, isomerization, and racemization via cyclic imide formation. As shown in Scheme 80, L-aspartic acid peptide can isomerize to L-iso -aspartic acid peptide via its L-cyclic imide. The L-cyclic imide intermediate is capable of undergoing racemization to the D-cyclic imide and thus forms the D-aspartic acid peptide and the D-iso-aspartic acid peptide on hydrolysis.

Following storage of a secretin solution, aspartoyl3 secretin (cyclic imide) and P-aspar-tyl3 secretin (isomer) were detected in the solution by reversed-phase HPLC, indicating that isomerization occurred via the cyclic imide.794 795 An Asp-hexapeptide also exhibited isomerization via cyclic imide formation at pH values above 4.796 The rate of formation of the cyclic imide was affected by the size of the amino acid on the C-terminal side of the aspartic acid residue.797 A cyclic imide was also detected as a major degradation product of basic fibroblast growth factor at pH 5.798

Table 14. The Effect of the Amino Acid Residue on the C-terminal Side of Asn on the Deamidation of Asn-Hexapeptidesa

Asn-hexapeptide

t50 (days)

Val-Tyr-Pro- Asn-Gly-Ala

1.89 (pH 7.5)

Val-Tyr-Pro- Asn-Ser- Ala

5.55 (pH 7.5)

Val-Tyr-Pro- Asn- Ala- Ala

20.2 (pH 7.4)

Val-Tyr-Pro-Asn-Val- Ala

106 (pH 7.5)

Val-Tyr-Pro- Asn-Pro-Ala

70 (pH 7.4)

Val-Tyr-Pro- Asn-Pro- Ala

106 (pH 7.4)

a Reference 791.

a Reference 791.

Racemization has also been observed with many peptides and proteins. Casein exhibits racemization at aspartic acid, phenylalanine, glutamic acid, and alanine residues.799 Racemization of serine and histidine residues has been reported for histrelin (a nonapeptide)800 and a decapeptide,801 agonists of luteinizing hormone-releasing hormone (LH-RH). As shown in Fig. 200, the main degradation pathway of decapeptide (an antagonist of LH-RH) above PH 7 was epimerization.802

5.1.1.3. Hydrolysis

Hydrolysis is a pathway often observed during peptide and protein degradation. As shown in Scheme 81, aspartic acid residues in particular are susceptible to hydrolysis in the acidic pH range. Secretin, apart from undergoing isomerization, also undergoes degradation by hydrolysis of its aspartic acid residues at position-3 and position-15.794795 Hydrolysis of aspartic acid residues under acidic conditions has also been observed with recombinant human macrophage colony-stimulating factor,803 recombinant human interleukin- 1 1 ,804 and a hexapeptide.796 Hydrolysis may also occur at serine and histidine residues.799-802

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

Oxidation of cysteine residues of peptide and protein molecules yields intra- and intermolecular disulfide bonds (Scheme 82), leading to changes in tertiary structure. Also, normal disulfide bonds in peptide and protein molecules can undergo thiol-catalyzed intra-and intermolecular exchange reactions, leading to changes in secondary and tertiary structures (Scheme 83). Furthermore, the disulfide bond itself is susceptible to cleavage via ^-elimination and forms dehydroalanine residues and persulfides (Scheme 84). These

1er3

Images Deamidation Pathways

Figure 200. pH-rate profiles for deamidation (•), epimerization (i,), and hydrolysis (■) of a decapeptide LH-RH antagonist at 80°C. (Reproduced from Ref. 802 with permission.)

Figure 200. pH-rate profiles for deamidation (•), epimerization (i,), and hydrolysis (■) of a decapeptide LH-RH antagonist at 80°C. (Reproduced from Ref. 802 with permission.)

Images Deamidation Pathways
Scheme 81. Pathways proposed for the hydrolysis of peptides at aspartic acid residues.

products may also participate further in disulfide exchange reactions, resulting in formation of new cross-linkages.

Lysozyme exhibits deamidation at pH 6 and deamidation and hydrolysis at pH 4, whereas cleavage of disulfide residues and formation of new disulfide bonds were observed at pH 8.805 The half-lives for reaction at the disulfide residues due to ^-elimination were similar for 14 proteins, including insulin, indicating that cleavage of disulfide bonds is relatively independent of both primary and higher structures.806 Intermolecular formation of new disulfide bonds leads to aggregation of peptides and proteins. For example, lyophilized bovine serum albumin and insulin undergo aggregation via intermolecular disulfide bond formation at a rate dependent on the water content of the lyophile.807-810 Lyophilized

HCCHjSH —> HCCHsSSCHjCH Scheme 82. Formation of a disulfide bond through oxidation of cysteine residues.

Scheme 83. Disulfide exchange reactions.

P-galactosidase also exhibited aggregation via disulfide bond formation at relatively low water content. The participation of disulfide bond formation in this case was confirmed by size-exclusion chromatography. The formed aggregate was not dissociated by guanidine hydrochloride but was dissociated by dithiothreitol, a disulfide bond reductant.811

Covalent bond formation, other than disulfide bond formation, is also involved in other intermolecular cross-linkages. The covalent linkages in the aggregates of freeze-dried ribonuclease A appeared to result from the participation of lysine, asparagine, and glutamine residues as suggested by amino acid analysis of the aggregates.812813 Lyophylized formulations of recombinant tumor necrosis factor-a formed dimers and oligomers that were nonreducible.814 Upon storage, insulin formulations yielded covalent dimers; the extent of dimerization was highly dependent on the formulation.815 The deamidated A-21 asparagine of one insulin molecule and the B-1 phenylalanine residue of another were found to be involved in the formation of the dimeric species.816 The aggregates of basic fibroblast growth factor have been characterized by a systematic approach using UV spectroscopy, size-exclusion HPLC, and reversed-phase chromatography.817

5.1.1.5. Oxidation

Formation of disulfide bonds from cysteine residues is an oxidation reaction. A cysteine residue in a-amylase is oxidized at pH 8.0.818 Methionine and histidine residues are also susceptible to oxidation. Oxidation of methionine residues has been observed during storage of parathyroid hormone819 and relaxin.820 Degradation of freeze-dried ribonuclease A was ascribed to oxidation because molecular oxygen was involved in the degradation process.821

Oxidation of methionine to methionine sulfoxide in small peptides was catalyzed by Fe3+ and promoted by ascorbic acid. Intramolecular catalysis by a histidine residue was involved in this oxidation, and its effect was maximal when the histidine and methionine residues were separated by one residue.822

NH NH I I HCCHjSSCHsCH ->

CO CO

HCCHjSS" + CH2=C

Scheme 84. ß-Elimination at a disulfide bond.

Lyophilized porcine pancreatic elastase exhibited denaturation during storage at 40°C and 75% relative humidity in which oxidation of tryptophyl groups was probably involved.823 Reducing sugar impurities in mannitol used as an excipient824 induced solid-state oxidative degradation of a cyclic heptapeptide.

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