The asparagine (Asn) deamidation and aspartate (Asp) isomerization reactions are nonenzymatic intra-molecular reactions occurring in peptides and proteins that are a source of major stability concern in the formulation of these biomolecules. The mechanisms for the deamidation and isomerization reactions are similar since they both proceed through an intra-molecular cyclic imide (Asu) intermediate. The formation of the Asu intermediate, which involves the attack by nitrogen of the peptide backbone on the carbonyl carbon of the Asn or the Asp side chain, is the rate-limiting step in both the deamidation and the isomerization reactions at physiological pH. In this article, the influence of factors such as formulation conditions, protein primary sequence, and protein structure on the reactivity of Asn and Asp residues in proteins are reviewed. The importance of formulation conditions such as pH and solvent dielectric in influencing deamidation and isomerization reaction rates is addressed. Formulation strategies that could improve the stability of proteins to deamidation and isomerization reactions are described. The review is intended to provide information to formulation scientists, based on protein sequence and structure, to predict potential degradative sites on a protein molecule and to enable formulation scientists to set appropriate formulation conditions to minimize reactivity of Asn and Asp residues in protein therapeutics.
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