Iodoacetyl Tandem Mass Tag-Based Site-Specific Free Thiol Analysis (TMT-SiFTA) of Monoclonal Antibodies

Monoclonal antibodies (mAbs) consist of four polypeptide chains that are covalently linked by disulfide bonds formed between cysteine residues. However, low levels of free thiols derived from under-processed/reduced cysteines are commonly present. Because free thiols may increase the risk of undesired immunogenicity in mAb therapeutics, monitoring free thiol occupancies during drug development is crucial to ensure drug quality and safety. Multiple assays have been established to identify and quantify the free thiols of proteins; these assays generally involve spectroscopy-based or mass spectrometry (MS)-based methods. Spectroscopy-based methods may be limited by their sensitivity and the lack of site-specific information. In contrast, MS-based workflows have been developed to address these problems; however, free thiol quantitation mostly relies on peak integration at the full MS level, which is vulnerable to signal variability and suppression. Herein, we describe an iodoacetyl tandem mass tag-based site-specific free thiol analysis (TMT-SiFTA) method incorporating downstream parallel reaction monitoring analysis, thus enabling sensitive and robust characterization of low-abundance free thiols. TMT-SiFTA was validated with the mAb standard NISTmAb and achieved the detection and quantitation of free thiols as low as ∼0.1% for individual cysteine residues. The developed workflow was also successfully applied to one in-house mAb and seven commercialized mAbs. The findings revealed consistent patterns across various molecules: cysteine residues participating in interchain disulfide bonds displayed low free thiol percentages (below 0.6%), whereas those involved in intrachain disulfide bonds exhibited higher percentages (up to 11%). TMT-SiFTA provides a powerful tool for free thiol analysis to support the development of therapeutic mAbs development.