A mechanism-based kinetic analysis of succinimide-mediated deamidation, racemization, and covalent adduct formation in a model peptide in amorphous lyophiles.

The succinimide intermediate generated during deamidation of asparagine-containing peptides and proteins has been implicated as having a role in the formation of multiple types of degradants in addition to hydrolysis products, including racemization products and, more recently, amide-linked, nonreducible protein and peptide aggregates. The formation of alternative degradants may be particularly important in solid-state formulations. This study quantitatively examines the role of the succinimide intermediate in hydrolysis, racemization, and covalent, amide-linked adduct formation in amorphous lyophiles. The degradation of a model peptide, Gly-Phe-L-Asn-Gly, and its L- or D-succinimide intermediates were examined in lyophiles containing hydroxypropyl methylcellulose and varying amounts of excess Gly-Val. Disappearance of the starting reactants and formation of up to 10 degradants were monitored when lyophiles were exposed to either 27°C/40% relative humidity (RH) or 40°C/75 RH using a stability indicating high-performance liquid chromatography method. Terminal degradant profiles were the same when the starting reactant was either Gly-Phe-L-Asn-Gly or its succinimide intermediate. Nucleophilic attack occurred preferentially at the α-carbonyl of the succinimide intermediate at ratios of approximately 2:1 for both water and the N-terminus of Gly-Val as the attacking nucleophiles. A mechanism-based kinetic model analysis indicates that hydrolysis, racemization, and covalent, amide-linked adduct formation all proceed via the succinimide intermediate.