A high degree of charge heterogeneity is an unfavorable phenomenon commonly observed for therapeutic monoclonal antibodies (mAbs). Removal of these impurities during manufacturing often comes at the cost of impaired step yields. A wide spectrum of posttranslational and chemical modifications is known to modify mAb charge. However, a deeper understanding of underlying mechanisms triggering charged species would be beneficial for the control of mAb charge variants during bioprocessing. In this study, a comprehensive analytical investigation was carried out to define the root causes and mechanisms inducing acidic variants of an immunoglobulin G1‐derived mAb. Characterization of differently charged species by liquid chromatography–mass spectrometry revealed the reduction of disulfide bonds in acidic variants, which is followed by cysteinylation and glutathionylation of cysteines. Importantly, biophysical stability and integrity of the mAb are not affected. By in vitro incubation of the mAb with the reducing agent cysteine, disulfide bond degradation was directly linked to an increase of numerous acidic species. Modifying the concentrations of cysteine during the fermentation of various mAbs illustrated that redox potential is a critical aspect to consider during bioprocess development with respect to charge variant control.

中文翻译：

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细胞培养基中的半胱氨酸通过破坏二硫键网络诱导酸性 IgG1 物种

高度的电荷异质性是治疗性单克隆抗体 (mAb) 常见的不利现象。在制造过程中去除这些杂质通常是以降低步骤产量为代价的。已知广泛的翻译后和化学修饰可以修饰 mAb 电荷。然而，更深入地了解触发带电物质的潜在机制将有利于在生物加工过程中控制 mAb 电荷变体。在这项研究中，进行了一项全面的分析调查，以确定导致免疫球蛋白 G1 衍生 mAb 酸性变体的根本原因和机制。通过液相色谱-质谱法对不同电荷物质的表征揭示了酸性变体中二硫键的减少，随后是半胱氨酸的半胱氨酸化和谷胱甘肽化。重要的是，mAb 的生物物理稳定性和完整性不受影响。通过 mAb 与还原剂半胱氨酸的体外温育，二硫键降解与许多酸性物质的增加直接相关。在各种 mAb 的发酵过程中改变半胱氨酸的浓度表明，氧化还原电位是在生物工艺开发过程中关于电荷变异控制的一个关键方面。