Process-related impurities and degradants that may occur in peptide drug products. Reproduced from ref. 2 with permission from Elsevier, Copyright 2017.
| Impurity . | Mechanism . |
|---|---|
| Denaturation | Modification of the peptide structure that changes its physical, chemical and biological properties; occurs in the presence of denaturing agents |
| Proteolysis | Upon exposure to harsh conditions, extreme pH, high temperature or enzymes |
| Aggregation and precipitation | Association of hydrophobic amino acid residues to form aggregates; precipitation occurs if on a macroscopic scale |
| Deamination | Hydrolysis of the side-chain amide linkage of an amino acid residue with formation of a free carboxylic acid |
| Oxidation and reduction | Induced by temperature, pH, metal ions and buffers during synthesis and/or storage |
| Disulfide exchange | Change in conformation due to reaction between disulfides within a peptide chain |
| Diastereoisomerization (racemization) | Alteration of l-amino acids to d,l mixtures, with formation of peptide bonds sensitive to proteolytic enzymes |
| β-Elimination | Proceeds through a carbanion intermediate; susceptible residues under alkaline conditions are Cys, Lys, Phe, Ser and Thr |
| Deletion (incomplete coupling) | Incomplete coupling due to incomplete removal of the protecting group of the last coupled amino acid or insufficient activation of the incoming amino acid |
| Truncation | Missing one or more amino acid residues at either the N- or C-terminal end due to precipitation of resin beads during synthesis |
| Amino acid insertion | Insertion of an additional amino acid into the peptide sequence due to excess of 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and improper washing |
| Dimers | Self-association of hydrophobic amino acids residues in peptides to form aggregates |
| By-products generated by incomplete deprotection | Results in protecting groups covalently attached to peptide sequences |
| Impurity . | Mechanism . |
|---|---|
| Denaturation | Modification of the peptide structure that changes its physical, chemical and biological properties; occurs in the presence of denaturing agents |
| Proteolysis | Upon exposure to harsh conditions, extreme pH, high temperature or enzymes |
| Aggregation and precipitation | Association of hydrophobic amino acid residues to form aggregates; precipitation occurs if on a macroscopic scale |
| Deamination | Hydrolysis of the side-chain amide linkage of an amino acid residue with formation of a free carboxylic acid |
| Oxidation and reduction | Induced by temperature, pH, metal ions and buffers during synthesis and/or storage |
| Disulfide exchange | Change in conformation due to reaction between disulfides within a peptide chain |
| Diastereoisomerization (racemization) | Alteration of l-amino acids to d,l mixtures, with formation of peptide bonds sensitive to proteolytic enzymes |
| β-Elimination | Proceeds through a carbanion intermediate; susceptible residues under alkaline conditions are Cys, Lys, Phe, Ser and Thr |
| Deletion (incomplete coupling) | Incomplete coupling due to incomplete removal of the protecting group of the last coupled amino acid or insufficient activation of the incoming amino acid |
| Truncation | Missing one or more amino acid residues at either the N- or C-terminal end due to precipitation of resin beads during synthesis |
| Amino acid insertion | Insertion of an additional amino acid into the peptide sequence due to excess of 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and improper washing |
| Dimers | Self-association of hydrophobic amino acids residues in peptides to form aggregates |
| By-products generated by incomplete deprotection | Results in protecting groups covalently attached to peptide sequences |