Site-Specific Conjugation Quantitation of a Cysteine-Conjugated Antibody–Drug Conjugate Using Stable Isotope Labeling Peptide Mapping LC–MS/MS Analysis

Drug-load (DL) characterization of antibody–drug conjugates (ADCs) is an important analytical task due to its designation as a critical quality attribute (CQA) affecting potency and stability. Intact and subunit liquid chromatography–mass spectrometry (LC–MS) analyses can determine global drug-to-antibody ratios (DARs) that correlate well with other orthogonal analytical methods; however, peptide mapping liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis has struggled to provide complementary site-specific quantitation of drug conjugation sites. The peptide mapping method described herein utilizes stable isotope labeling to accurately quantitate the site-specific conjugation levels of a cysteine-conjugated ADC to provide “bottom-up” DAR characterization in parallel with protein sequence and post-translational modification (PTM) characterization in one multi-attribute analytical method (MAM).