Using Digestion by IdeS Protease to Improve Quantification of Degradants in Monoclonal Antibodies by Non-Reducing Capillary Gel Electrophoresis

作者：Andrew McClain, Yiting Zhang, Yan Yin, Qi Wang, Lih‐Yueh Hwang, Yan Gu, Jeff Beckman, Richard Ludwig · 发表于：Analytical Chemistry · 年份：2022 · DOI：10.1021/acs.analchem.2c02630 · 被引用次数：7 · 研究领域：Protein purification and stability、Viral Infectious Diseases and Gene Expression in Insects、Monoclonal and Polyclonal Antibodies Research

Monoclonal antibodies (mAbs) have become predominant therapeutics by providing highly specific mechanisms of action enabling treatment of complex diseases. However, mAbs themselves are highly complex and require thorough testing and characterization to ensure efficacy and patient safety. In this regard, fragmentation is a degradation product of concern. The biotechnology industry uses capillary gel electrophoresis (CGE) to quantify fragmentation by electrophoretically resolving size variants, such as products resulting from partial reduction of interchain disulfides. However, standard CGE methods may not adequately separate less typical fragments, particularly when there is minimal size difference to the parent molecule. For mAb-1, a degradant only ∼11 kDa smaller than the intact mAb (∼149 kDa) was unable to be resolved under typical non-reducing conditions, preventing an accurate purity assessment and precluding tracking of product purity within stability studies. To address these deficiencies, a subunit-based non-reducing CGE method was developed to employ IdeS protease to produce F(ab')2 and Fc fragments, which resulted in baseline resolution of the clipped subunit species from its parent species. This enabled more accurate trending of purity throughout stability studies. Method characterization ensured that this subunit method monitored expected impurities observed by intact non-reducing CGE and thus could suitably replace non-reducing CGE in the release and stability tes...