Combined metabolomics and proteomics reveals hypoxia as a cause of lower productivity on scale‐up to a 5000‐liter CHO bioprocess

Abstract

Large-scale bioprocessing is key to the successful manufacturing of a biopharmaceutical. However, cell viability and productivity are often lower in the scale-up from laboratory to production. In this study, we analyzed CHO cells, which showed lower percent viabilities and productivity in a 5-KL production scale bioreactor compared to a 20-L bench-top scale under seemingly identical process parameters. An increase in copper concentration in the media from 0.02 µM to 0.4 µM led to a doubling of percent viability in the production scale albeit still at a lower level than the bench-top scale. Combined metabolomics and proteomics revealed the increased copper reduced the presence of reactive oxygen species (ROS) in the 5-KL scale process. The reduction in oxidative stress was supported by the increased level of glutathione peroxidase in the lower copper level condition. The excess ROS was shown to be due to hypoxia (intermittent), as evidenced by the reduction in fibronectin with increased copper. The 20-L scale showed much less hypoxia and thus less excess ROS generation, resulting in little to no impact to productivity with the increased copper in the media. The study illustrates the power of 'Omics in aiding in the understanding of biological processes in biopharmaceutical production.

Proteomics in biomanufacturing control: Protein dynamics of CHO‐K1 cells and conditioned media during apoptosis and necrosis

“…LC‐MS based “omics”‐approaches are increasingly used to gain advanced understanding of biomanufacturing processes such as to study host cell proteins (HCPs), productivity during process scale‐up or cellular response to altered media composition (Farrell et al, ; Gao et al, ; Liu et al, ). The recent elucidation of the CHO cellular and secreted proteome further facilitates research in this area (Baycin‐Hizal et al, ; Kumar et al, ).…”

Hypoxia and transforming growth factor‐beta1 pathway activation promote Chinese Hamster Ovary cell aggregation

“…Bench‐top bioreactors generally have better culture mixing, CO 2 stripping, and pH maintenance than large‐scale bioreactors. More prominent DO gradients and hypoxic stress were reported in 5,000‐L commercial production bioreactors (Gao et al, ; Xing, Kenty, Li, & Lee, ). It has been observed in some of our bioprocesses that shake flask cultures can behave similarly to the large‐scale bioreactors.…”

“…Subsequent multi‐omics analysis showed an increased formation of reactive oxygen species at 5,000‐L scale and demonstrated that this increase was caused by hypoxic stress even though the online DO reading was maintained within a normal range. (Gao et al, ). In one of our other studies, DO conditions were controlled at 60% (normoxic) and 15% (hypoxic) in 50‐L bioreactors (Qian et al, ) and the cells tended to be more aggregative under 15% DO condition (Supplementary data, Figure S4) although the correlation between hypoxic condition and cell aggregation was not studied in detail at that time.…”

Quantitative intracellular flux modeling and applications in biotherapeutic development and production using CHO cell cultures

“…The researchers now have access to perform in silico metabolic flux simulation and analysis at a system level based on genome‐scale CHO cell metabolic network (Hefzi et al, ; Yusufi et al, ). Furthermore, increasing volumes of omics data in CHO cells are becoming publicly available with the help of advanced analytical techniques (Gao et al, ; Lewis et al, ), and it's believed that genome‐scale constraint‐based metabolic models are a reliable framework to fully utilize these multi‐omics data. However, constraint‐based approaches have several limitations.…”