Bottom–Up Protein Analysis Workflow for Simultaneous Characterization of Payloads, Disulfide Bonds, and Free Thiols in Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs) are biopharmaceutical agents comprising cytotoxic payloads covalently linked to monoclonal antibodies via chemical linkers. Their structural heterogeneity presents analytical challenges for therapeutic development as conventional methods cannot simultaneously characterize conjugation sites and disulfide linkages. To address this limitation, we have developed a bottom–up liquid chromatography–mass spectrometry (LC–MS) workflow integrated with an online disulfide-ene reaction. This method significantly increases the analytical throughput by providing payload-conjugation sites, disulfide linkages, and free-thiol quantitation from a single analysis pipeline. Using trastuzumab emtansine as a model system, our workflow enables the identification of 46 payload-conjugated sites and 100% disulfide coverage, comparable to results from a conventional bottom–up analysis using two separate LC–MS pipelines. For cantuzumab ravtansine, which contains a spatially hindered disulfide bond in the linker, we have achieved comprehensive mapping of both payload-conjugated sites and disulfide bonds. This workflow has been further applied to quantify free thiol at cysteine residues and linker–payload disulfide bonds for model ADCs under reductive stress, revealing the distinct stability of the disulfide bonds in different structural regions.