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

• Published in: Bioanalysis Vol. 8; no. 15; pp. 1611 - 1622
• Main Authors: Mehl, John T, Sleczka, Bogdan G, Ciccimaro, Eugene F, Kozhich, Alexander T, Gilbertson, Deb G, Vuppugalla, Ragini, Huang, Christine S, Stevens, Brenda, Mo, Jingjie, Deyanova, Ekaterina G, Wang, Yun, Huang, Richard Yc, Chen, Guodong, Olah, Timothy V
• Format: Journal Article
• Language: English
• Published: England 01.08.2016
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - blood; Antibodies, Monoclonal - chemistry; Asparagine - analysis; Aspartic Acid - analysis; Chromatography, Liquid - methods; Humans; Immunoprecipitation - methods; Isomerism; Macaca fascicularis; Male; Tandem Mass Spectrometry - methods; CDR; in vivo deamidation; MS; in vivo isomerization; LC–MS; immunoprecipitation; asparagine deamidation; mAb therapeutic; monoclonal antibody; therapeutic protein quantification; aspartic acid isomerization
• ISSN: 1757-6180; 1757-6199
• DOI: 10.4155/bio-2016-0035

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. 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. IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.