Cysteine in cell culture media induces acidic IgG1 species by disrupting the disulfide bond network

• Published in: Biotechnology and bioengineering Vol. 118; no. 3; pp. 1091 - 1104
• Main Authors: Prade, Elke, Zeck, Anne, Stiefel, Fabian, Unsoeld, Andreas, Mentrup, David, Arango Gutierrez, Erik, Gorr, Ingo H.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-03-2021; John Wiley and Sons Inc
• Subjects: acidic charge variants; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - isolation & purification; bioprocess optimization; Bioprocessing; Cell culture; Cell Culture Techniques; Chemical bonds; CHO Cells; Chromatography, Liquid; Cricetulus; Culture media; Cysteine; Cysteine - chemistry; Disulfide bonds; Disulfides - chemistry; Fermentation; Heterogeneity; Immunoglobulin G; Immunoglobulin G - biosynthesis; Immunoglobulin G - chemistry; Immunoglobulin G - isolation & purification; Impurities; Incubation; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Monoclonal antibodies; product quality; Redox potential; redox‐sensitive modifications; Reducing agents; Species; redox-sensitive modifications; acidic charge variants; product quality; bioprocess optimization; cysteine
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.27628

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.