High cysteine concentrations in cell culture media lead to oxidative stress and reduced bioprocess performance of recombinant CHO cells. Issue 11 (10th August 2022)

• Record Type: Journal Article
• Authors: Komuczki, Daniel; Stadermann, Anna; Bentele, Maximilian; Unsoeld, Andreas; Grillari, Johannes; Mueller, Markus M.; Paul, Albert; Fischer, Simon
• Is Part Of: Biotechnology journal. Volume 17:Issue 11(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-08-10
• Subjects: CDM -- CHO -- cysteine -- oxidative stress -- ROS -- transcriptomics -- upstream development; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.202200029
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract: Cysteine is considered an essential amino acid in the cultivation of Chinese hamster ovary (CHO) cells. An optimized cysteine supply during fed‐batch cultivation supports the protein production capacity of recombinant CHO cell lines. However, we observed that CHO production cell lines seeded at low cell densities in chemically defined media enriched with cysteine greater than 2.5 mm resulted in markedly reduced cell growth during passaging, hampering seed train performance and scale‐up. To investigate the underlying mechanism, seeding cell densities and initial cysteine concentrations ranging from low to high cysteine concentrations were varied followed by an analysis of cell culture performance. Additionally, cell cycle analysis, intracellular quantification of reactive oxygen species (ROS) as well as transcriptomic analyses by next‐generation sequencing were carried out. Our results demonstrate that CHO cells seeded at low cell densities at high initial cysteine concentrations encountered increased oxidative stress leading to a p21‐mediated cell cycle arrest in the G1/S phase. The resulting oxidative stress caused redox imbalance in the endoplasmic reticulum and activation of the unfolded protein response as well as the major antioxidant nuclear factor‐like 2 response pathways. Potential signature genes related to oxidative stress and the inhibition of the pentose phosphate pathway were identified in the study. Finally, the study presents that seeding cells at a higher concentration counteract oxidative stress in cysteine‐enriched cell culture media. Graphical Abstract and Lay Summary: The concentration of L‐cysteine in chemically‐defined cell culture media is critical for CHO cell line performance. In this report, the impact of elevated L‐cysteine concentrations have been carefully investigated via transcriptomic studies, which revealed that elevated L‐cysteine concentrations lead to increased formation of reactive oxygen species (ROS) that negatively affect cell culture performance of monoclonal antibody producting CHO cells.