Development of a Novel\nLabel-Free Subunit HILIC-MS\nMethod for Domain-Specific Free Thiol Identification and Quantitation\nin Therapeutic Monoclonal Antibodies

Cysteine residues are crucial for the formation of conserved\ndisulfide\nbonds in therapeutic monoclonal antibodies (mAbs), which are essential\nfor their folding and structural stability. The presence of free thiols\nin mAbs can indicate incomplete disulfide bond formation, potentially\nimpacting the molecule’s conformational stability. Free thiol\nquantitation has been achieved using labeling-based strategies such\nas maleimide and haloalkyl derivatives at both intact and peptide\nlevels. However, intact-level measurement only provides total free\nthiol levels, while peptide-level measurement is time-consuming and\nmore prone to assay-induced artifacts. In this study, we present a\nnovel label-free HILIC-MS method that separates free thiol species\nat the subunit level, followed by free thiol localization by the MS2\nfragmentation pattern. This allows for facile identification and quantitation\nof intrachain free thiols at domain-specific resolution. Compared\nto bottom-up approaches, this subunit HILIC-MS method excels in simpler\nsample preparation and higher throughput and enables chain-specific\nfree thiol analysis for bispecific mAbs. This method can be readily\napplied for screening mAb candidates with elevated levels of free\nthiols in early-stage developability assessment and facilitating an\neffective comparability evaluation of mAb samples during process development.