For example, deamidation of an Asn-Gly site in hemoglobin alters its affinity for oxygen, while the same modification alters the proteolytic cleavage of human growth hormone (hGH). As deamidation changes the peptide/protein structure and conformation, it can significantly affect the function and stability of proteins. Deamidation occurs at a much faster rate (up to 70 times) when an unhindered amino acid residue such as glycine is on the C-terminal side of an asparagine in the primary sequence (XXX-Asn-Gly-XXX), but its rate is also affected by other conditions and characteristics such as temperature, pH, and protein structure. This reaction is spontaneous and non-enzymatic, where asparagine residues undergo formation of a five-membered succinimide ring intermediate from an intramolecular attack, and subsequently hydrolyze under physiological conditions to form either aspartyl or isoaspartyl peptides, which can be found in both the D and L configurations (Figure 1). Īlthough little is known about the effects that deamidation of asparagine have on protein function, it is known that deamidation is involved in protein degradation and development.
The separation and quantitation of peptides that have these modifications is of paramount importance in protein biotherapeutics because the modifications can contribute to a loss of stability or activity. These modifications can include the oxidation of methionine and the deamidation of asparagine, among others. Many of the chemical modifications that accumulate in biotherapeutic agents during bioprocessing, purification, storage, or other stages increase the hydrophilicity of the amino acid side chain on the altered residue(s).