Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation




Pharmaceutical Sciences

First Advisor

Dr. Bradley D. Anderson


Peptides and proteins are prone to chemical and physical degradation in solutions and solids. One of the most common chemical degradation pathways involves deamidation of asparagine residues through a reactive succinimide intermediate resulting in the formation of aspartyl and isoaspartyl degradation products.

In this work, kinetic studies in aqueous solutions demonstrated that the succinimide intermediate is susceptible to attack by nucleophiles other than water. This may lead to a variety of alternative degradants. In solutions containing high concentrations of ammonia, the succinimide generated in the deamidation of the model peptides Phe-Asn-Gly and Phe-isoAsn-Gly underwent back-reaction with ammonia leading to peptide isomerization. Intramolecular cyclization involving the N-terminus of Phe-Asn-Gly to form a cyclic diketopiperazine was also observed. Experiments employing the succinimide as the starting reactant and kinetic models confirmed that the above pathways proceed exclusively through an imide intermediate.

To maximize their stability, proteins and peptides are formulated as lyophilized solids. However, succinimide-mediated deamidation remains important in solid-state degradation. Accelerated stability studies of the model peptide Gly-Phe-Asn-Gly in the presence of excess Gly-Val in amorphous lyophiles revealed the formation of four deamidation products from the reaction of the succinimide intermediate with water as well as four covalent amide-linked adducts. The amide-linked adducts were identified as diastereomers formed by nucleophilic attack of the Gly-Val N-terminus at either the alpha or beta-carbonyl of the succinimide. A comprehensive kinetic model confirmed that the formation of all four hydrolysis products and the four covalent-adducts proceed exclusively through the succinimide.

The relative ratio of formation of hydrolysis products to covalent-adducts reflects a competition between water and the N-terminus of neighboring peptides for reaction with the succinimide intermediate. This ratio varies with formulation. The sensitivity of the individual reaction steps involved in deamidation and covalent adduct formation to formulation changes (e.g., variation in water content and dilution of reactants by increasing excipient content) were explored. Increasing water content accelerated adduct and hydrolysis degradant formation while increases in concentration of a polymeric excipient (HPMC) appeared to have no significant effect.

The novel mechanism described offers an alternative pathway for peptide and protein aggregation, particularly in amorphous solid formulations.