Quantification of in vivo site-specific Asp isomerization and Asn deamidation of mAbs in animal serum using IP-LC–MS

ABSTRACT Background: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. Results: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC–MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. Conclusion: IP-LC–MS can be applied to simultaneously quantify...