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 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.