Taku Tsukidate, , , Zhenshu Wang, , , Andrew Hsieh, , , Patricia Rose, , and , Xuanwen Li*,
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引用次数: 0
Abstract
Monoclonal antibodies (mAbs) are crucial biotherapeutics in the face of increasing global demand, but their production can be impacted by the reduction of disulfide bonds. This study presents a chemical proteomics workflow aimed at elucidating the mechanisms underlying disulfide bond reduction in mAbs produced from Chinese hamster ovary (CHO) cells. We employed iodoacetamide-desthiobiotin (IA-DTB) and the parallel accumulation and serial fragmentation combined with data-independent acquisition (diaPASEF) methodology for cysteine reactivity profiling and successfully quantified approximately 4,500 cysteine-containing peptides from harvested cell culture fluids (HCCF). Our findings reveal that various protein disulfide oxidoreductases were active in reducing HCCF, offering critical insights into the redox environment affecting mAb stability. Notably, we quantified specific cysteine residues in enzymes such as glutaredoxin and thioredoxin domain–containing protein 12, suggesting potential links between their activity and disulfide bond dynamics. This workflow not only complements conventional abundance proteomics but also enhances our understanding of functional enzyme states in bioprocessing. Ultimately, our approach provides a promising strategy for assessing enzymes contributing to disulfide bond reduction, paving the way for improved manufacturing processes of mAbs.
期刊介绍:
The Journal of the American Society for Mass Spectrometry presents research papers covering all aspects of mass spectrometry, incorporating coverage of fields of scientific inquiry in which mass spectrometry can play a role.
Comprehensive in scope, the journal publishes papers on both fundamentals and applications of mass spectrometry. Fundamental subjects include instrumentation principles, design, and demonstration, structures and chemical properties of gas-phase ions, studies of thermodynamic properties, ion spectroscopy, chemical kinetics, mechanisms of ionization, theories of ion fragmentation, cluster ions, and potential energy surfaces. In addition to full papers, the journal offers Communications, Application Notes, and Accounts and Perspectives