ORIGINAL RESEARCH article
Front. Bioeng. Biotechnol.
Sec. Bioprocess Engineering
Volume 13 - 2025 | doi: 10.3389/fbioe.2025.1615263
This article is part of the Research TopicAdvancing Protein Engineering for Biotechnological and Therapeutic ApplicationsView all articles
Dual Formation Mechanisms of Acidic Variants in Cysteine-Engineered Antibodies and Strategies for Their Reduction
Provisionally accepted
- Shanghai AsymBio Biotechnology Co., Shanghai, China
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Cysteine-engineered antibodies (THIOMABs) are pivotal in the site-specific conjugation for ADC therapeutics. In this study, THIOMABs were expressed in CHO cell culture and showed a significantly higher proportion of acidic variants compared to traditional antibodies. A dual formation mechanism for acidic species was identified: glutathione (GSH) capping at engineered cysteine sites and traditional post-translational modifications (PTMs) during cell culture. Moreover, it was found that these two mechanisms exhibited overlapping effects on the formation of acidic species, and their simultaneous elimination was required for significant reduction of acidic variants. Consequently, by modulating temperature and pH to reduce PTMs and supplementing with L-cysteine to displace GSH, the proportion of acidic variants was successfully reduced. This study uncovers dual formation mechanisms of acidic species in THIOMABs and provides a practical cell culture approach, effectively reducing acidic variants and enhancing the quality attributes of THIOMABs.
Keywords: THIOMABs, CHO cell culture, Acidic variants, GSH capping, traditional PTM, dual formation mechanism
Received: 21 Apr 2025; Accepted: 23 Sep 2025.
Copyright: © 2025 Zhou, Cao, Yin, Xu, Gao and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Zhenshou Wang, wangzhenshou@asymchem.com.cn
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