On-column Thiol Protection Strategy to Control Charge and Stability of ThiomAb

Background Cysteine-engineered monoclonal antibodies (mAbs), or ThiomAb, enable site-specific conjugation and form the basis of several next-generation antibody-drug conjugates (ADCs). However, the presence of intentionally unpaired cysteine residues introduces reactive free thiols that pose challenges for manufacturing consistency, including variable capping by cysteine/glutathione (Cys/GSH) or uncapped free thiol during cell culture. This increases the charge heterogeneity of protein and may compromise stability due to increased risk of oxidation, disulfide scrambling, or aggregation. Methods To address these risks, we developed an on-column capping strategy, in which the antibody is immobilized on chromatography resin during the capture step and subjected to selective thiol masking under optimized redox conditions. This in-process approach allows for early and efficient stabilization of engineered thiol sites prior to polishing and formulation. High-throughput condition screening and analytical techniques—including mass spectrometry and charge variant profiling—were employed to fine-tune buffer conditions and understand on-column redox chemistry. Results The optimized thiol protection conditions substantially reduce the population of free thiols, resulting in more consistent charge profiles and improved stability during downstream processing and storage. Conclusion This strategy offers a scalable and platform-adaptable solution for enhancing the manufacturability and alternative control strategy of ThiomAb intermediates used in site-specific ADC processes.