Kinetics and mechanisms of deamidation and covalent amide-linked adduct formation in amorphous lyophiles of a model asparagine-containing Peptide.

ABSTRACT

PURPOSE: Asparagine containing peptides and proteins undergo deamidation via a succinimide intermediate. This study examines the role of the succinimide in the formation of covalent, amide-linked adducts in amorphous peptide formulations. METHODS: Stability studies of a model peptide, Gly-Phe-L-Asn-Gly, were performed in lyophiles containing an excess of Gly-Val at 'pH' 9.5 and 40°C/40% RH. Reactant disappearance and the formation of ten different degradants were monitored by HPLC. Mechanism-based kinetic models were used to generate rate constants from the concentration vs. time profiles. RESULTS: Deamidation of Gly-Phe-L-Asn-Gly in lyophiles resulted in L- and D-aspartyl and isoaspartyl-containing peptides and four amide-linked adducts between the succinimide and Gly-Val. The kinetic analysis demonstrated competition between water and terminal amino groups in Gly-Val for the succinimide. The extent of covalent adduct formation was dependent on dilution effects due to its second order rate law. CONCLUSION: The cyclic imide formed during deamidation of asparagine containing peptides in lyophiles can also lead to covalent adducts due to reaction with other neighboring peptides. A reaction model assuming a central role for the succinimide in the formation both hydrolysis products and covalent adducts was quantitatively consistent with the kinetic data. This mechanism may contribute to the presence of covalent, non-reducible aggregates in lyophilized peptide formulations.