Metabolic understanding of disulfide reduction during monoclonal antibody production
| Autor: | Lauren Jenkins, Anthony J. Cura, Sanchayita Ghose, Kathryn L. Aron, Yunping Huang, Tyler Hageman, Srinivas Chollangi, Xuankuo Xu, Michael C. Borys, Susan Egan, Zheng Jian Li |
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| Rok vydání: | 2020 |
| Předmět: |
Dehydrogenase
CHO Cells Pentose phosphate pathway Applied Microbiology and Biotechnology 03 medical and health sciences Cricetulus Cricetinae Animals Humans Disulfides Glyceraldehyde 3-phosphate dehydrogenase 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology 030306 microbiology Chemistry Chinese hamster ovary cell Antibodies Monoclonal General Medicine Enzyme assay Enzyme Biochemistry Cell culture Antibody Formation biology.protein GAPDH Gene Biotechnology |
| Zdroj: | Applied microbiology and biotechnology. 104(22) |
| ISSN: | 1432-0614 |
| Popis: | The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract. |
| Databáze: | OpenAIRE |
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