Multi‐Omics Reveals Impact of Cysteine Feed Concentration and Resulting Redox Imbalance on Cellular Energy Metabolism and Specific Productivity in CHO Cell Bioprocessing

Abstract

Chinese hamster ovary (CHO) cells are currently the primary host cell lines used in biotherapeutic manufacturing of monoclonal antibodies (mAbs) and other biopharmaceuticals. Cellular energy metabolism and endoplasmic reticulum (ER) stress are known to greatly impact cell growth, viability, and specific productivity of a biotherapeutic; but the molecular mechanisms are not fully understood. The authors previously employed multi‐omics profiling to investigate the impact of a reduction in cysteine (Cys) feed concentration in a fed‐batch process and found that disruption of the redox balance led to a substantial decline in cell viability and titer. Here, the multi‐omics findings are expanded, and the impact redox imbalance has on ER stress, mitochondrial homeostasis, and lipid metabolism is explored. The reduced Cys feed activates the amino acid response (AAR), increases mitochondrial stress, and initiates gluconeogenesis. Multi‐omics analysis reveals that together, ER stress and AAR signaling shift the cellular energy metabolism to rely primarily on anaplerotic reactions, consuming amino acids and producing lactate, to maintain energy generation. Furthermore, the pathways are demonstrated in which this shift in metabolism leads to a substantial decline in specific productivity and altered mAb glycosylation. Through this work, meaningful bioprocess markers and targets for genetic engineering are identified.

A Metabolomics Approach to Increasing Chinese Hamster Ovary (CHO) Cell Productivity

“…Cysteine is an important substrate for synthesis of antioxidants, including taurine and glutathione (GSH), which support high rates of oxidative phosphorylation associated with high-qP cells. A recent study found that a depletion of cysteine in CHO cell culture medium negatively impacted VCD, viability, and qP, and attributed these observations to redox imbalance, endoplasmic reticulum stress, and mitochondrial dysfunction [46]. The accumulation we observed in high-qP cells could reflect the ability of these cells to supplement feed cysteine with endogenous production.…”

Insights into the Impact of Rosmarinic Acid on CHO Cell Culture Improvement through Transcriptomics Analysis

“…Recently, Lakshmanan et al (2019) employed transcriptomic and proteomic profiling to systematically link phenotypic differences, including cell growth and glycosylation, with genotypes across different CHO hosts [16]. Ali et al (2019 and2020) conducted omics studies to explore the impact of cysteine feed level on CHO cell metabolism [17,18]. Additionally, omics approaches have been applied to support scale-up activities.…”

Proteomic Profiling of IgG1 Producing CHO Cells Using LC/LC-SPS-MS3: The Effects of Bioprocessing Conditions on Productivity and Product Quality

“…Even though expression was not increased for the chaperone proteins measured, a significant upregulation of BAK1 and Caspase-3 was observed indicating ER stress. While it would need additional experiments to verify our findings on the level of protein expression, it seems that in both cases, lowering the pH has beneficial effects on maintaining redox balance which is ultimately favoring recombinant protein expression (Ali et al, 2019(Ali et al, , 2020. Generally, combining the proteome analysis with additional omics-data, such as metabolomics or phosphoproteomics, would be beneficial to allow for a deeper biological interpretation even though it increases the experimental effort and time.…”

“…Nevertheless, there is still limited knowledge about of how these process alterations affect fundamental biological processes within the production cells and how these alterations ultimately relate to quality attributes such as, e.g., charge variants which could alter product efficacy and safety (Torkashvand and Vaziri, 2017;Papathanasiou and Kontoravdi, 2020). For this reason 'omics techniques have been applied to explore CHO cells under various conditions in order to provide a foundation for knowledge-driven improvements of manufacturing processes (Baycin-Hizal et al, 2012;Farrell et al, 2014;Heffner et al, 2017;Stolfa et al, 2018;Ali et al, 2019Ali et al, , 2020Lakshmanan et al, 2019). More accurate and complete genome references (Rupp et al, 2018) in combination with technological improvements in analytical instrumentation (Williamson et al, 2016) now enable proteomic profiling of CHO cells to a depth and accuracy that was previously not possible.…”