Metabolic understanding of disulfide reduction during monoclonal antibody production
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Metabolic understanding of disulfide reduction during monoclonal antibody production Anthony J. Cura 1 & Xuankuo Xu 1 & Susan Egan 1 & Kathryn Aron 1 & Lauren Jenkins 1 & Tyler Hageman 2 & Yunping Huang 2 & Srinivas Chollangi 1 & Michael Borys 1 & Sanchayita Ghose 1 & Zheng Jian Li 1 Received: 24 June 2020 / Revised: 9 September 2020 / Accepted: 17 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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. Keywords Monoclonal antibody . Disulfide bond reduction . CHO metabolism . Biomanufacturing . GAPDH . HCCF
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10916-1) contains supplementary material, which is available to authorized users. * Xuankuo Xu [email protected] * Kathryn Aron [email protected] 1
Biologics Development, Global Product Development and Supply, Bristol-Myers Squibb Company, 38 Jackson Road, Devens, MA 01434, USA
2
Biophysical Characterization, Global Product Development and Supply, Bristol-Myers Squibb Company, 1 Squibb Drive, New Brunswick, NJ 08901, USA
The cells used to produce mAbs in bioreactors are exposed to many stresses, including accumulation of toxic metabolites (Pereira et al. 2018), ER stress from high protein production rates (Xiao et al. 2014) and oxid
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