Development of improved high-performance liquid chromatography conditions for nonisotopic detection of isoaspartic acid to determine the extent of protein deamidation.

A rapid and accurate ion-pairing reversed-phase high-performance liquid chromatography (IP-RP-HPLC) procedure has been developed for nonisotopic detection of isoaspartic acid residues in protein or peptides resulting from deamidation of asparagine residues. The IP-RP-HPLC procedure specifically detects and quantifies S-adenosylhomocysteine (SAH). SAH is a by-product of the reaction between protein isoaspartyl methyltransferase (PIMT), S-adenosylmethionine (SAM), and isoaspartic acid residues. The HPLC conditions described in this paper have been demonstrated to offer significantly better reproducibility compared to earlier studies. The HPLC method allows determination of the extent of protein deamidation without the use of radioisotopes and therefore offers significant advantages for biopharmaceutical development laboratories. Copyright 2000 Academic Press.