Localizing the Conjugation Sites of Cystine-Conjugated Antibody Drug Conjugates by Improved LC-MS Subunit Analysis for ADC Positional Isomer Identification

Subunit analysis of antibody drug conjugates (ADC’s) with LC-MS methods provides insightful structural information, such as conjugation isoforms and their modifications (e.g. oxidation, or water losses). Cysteine proteases (e.g. IdeS or SpeB) are often used to cleave the hinge region to generate the LC, Fc/2 and Fd subunits because of their high specificity for mAb sequences. However, the cleavage for ADC’s was highly dependent upon the degree of conjugation, presumably due to steric hinderance. This application note presents a study to address this cleavage efficiency issue by evaluating Ides, SpecB and Lys-C for the localization of conjugation sites and confirmation of positional isomers. Experiments were conducted using the Acquity UPLC H Class Bio and the Xevo G2S QTof MS or Xevo G2-XS QTof MS system.