Charged-tagging ion-exchange chromatography enables rapid, accurate and comprehensive (RAC) profiling of free thiols and charge heterogeneity in mAbs

作者：Tao Liu, S X Xu, Chenjing Xu, Jinhao Li, Jinhao Li, Anqi Zhou, Yu Wei, Na Li, Weitao Zhang, Weitao Zhang, Qiaoshuang Chen, Xinyu Zhang, Mengjiao Xu, Chunguang Zheng, Chaoxin Zhou, Chaoxin Zhou, Wenqiang Fan, Congrong Wang, Qingcheng Guo, Xiuling Wang, Xin Zhang, Xin Zhang, Lankun Song, Ziqiao Sun, Xuekun Wang, Dapeng Zhang, Jianxin Dai, Hao Wang, Jing Li, Jin Xu, Huaizu Guo, Jin Xu, Huaizu Guo · 发表于：Journal of Pharmaceutical Analysis · 年份：2026 · DOI：10.1016/j.jpha.2026.101740 · 研究领域：Sulfur Compounds in Biology、Analytical Chemistry and Chromatography、Redox biology and oxidative stress

Free thiol content and charge heterogeneity are critical quality attributes for therapeutic antibodies, impacting stability, activity, and batch consistency. Currently, no single analytical method can simultaneously profile both attributes in a rapid, accurate, and comprehensive manner, especially with the ability to distinguish between exposed and buried free thiols. Here, we present a novel charged-tagging strategy using N-2-aminoethyl maleimide, a thiol-specific derivatizing agent that introduces a defined positive charge upon conjugation. This enables clear separation by ion-exchange chromatography while preserving the inherent capability to resolve charge heterogeneity. For exposed free thiols, direct labeling provides excellent resolution and quantitative recovery. For buried or partially buried thiols, a mild structural-unfolding step enables quantitative derivatization while preserving native charge profiles. Implemented on a standard high performance liquid chromatography-ultraviolet detector (HPLC-UV) system, this platform delivers the first integrated, rapid, accurate and comprehensive-compatible approach for simultaneous free thiol and charge heterogeneity profiling. Our work significantly expands the detection ability of ion-exchange chromatography and offers a practical solution for mAb process development, optimization, and routine quality control.