Rapid and in-depth proteomic analysis of CHO cell bioprocesses

Abstract

Chinese hamster ovary cells (CHO) are the main platform for monoclonal antibody production in the growing biopharmaceutical market. Compared to other production platforms, CHO cells, being mammalian cells, have the major benefit of providing appropriate protein folding and glycosylation patterns. However, further knowledge on the effect of protein burden and optimisations aimed at improving the viability and culture longevity for higher protein production are required. Programmed cell death is the predominant form of response occurring from biochemical stresses in bioreactor culturing. Cell longevity, yield and product quality are thus negatively affected. However, cell death is not yet comprehensively understood. Proteomics - the investigation of the whole set of proteins and their associated changes in any cell, tissue or organism, allows for global investigation of CHO cell bioprocesses and identification of pathways or markers of desired phenotype - targets for cell process improvement. Due to high sample number per experiment collected, a requirement for developing a high throughput methodology emerged, which could be used reliably in both industrial scale experiments and in other proteomic spheres such as large-sample clinical cohorts. Utilisation of shorter nano liquid chromatography columns such as µPAC Neo High Throughput HPLC column and EASY-Spray Pepmap 15 cm column, coupled with a 7-minute chromatography gradient and data independent acquisition, allowed for reproducible proteomic analysis within a tractable timescale. The developed high throughput methodologies were compared to a standard 90 minute data dependent acquisition method, established in the author’s laboratory, in terms of number of detected peptide and protein groups, carryover and differentially abundant proteins in CHO cells upon stress induction. This allowed for the global proteomic investigation of different CHO cells under shake flask and bioreactor conditions to assess the molecular stresses, which they undergo. Data generated provided insights into the mechanisms of execution and protection of cell death – ferroptosis, in CHO cell lines. Comparative proteomic analysis of CHO cell lines expressing distinct monoclonal antibodies identified differences in molecular pathways associated with productivity and bioprocess performance. Additional comparison of a bioreactor-adapted producer cell line and a non-adapted non-producer cell line confirmed the mechanisms through which cells adapt to the stress caused by change in the environment and the cell death mechanisms occurring when culturing a non-adapted line in bioreactor conditions. Proteomic analysis revealed pathways associated with induction of ferroptosis and shed light into the mechanisms that are activated when ROS scavenging chemicals are utilised. Enzymatic inhibition indeed provided the best results for ferroptosis inhibition, indicating that targeting the redox metabolism and preventing its dysregulation, provides better results, than attempting to reduce the consequences of its action – ROS. Finally, this research reports the generation of a ferroptosis inducer resistant CHO cell line through directed selection, that has not been previously reported in literature. Molecular biology strategies exploiting the enzymatic targets discovered are currently in progress. In summary, this work provided insights into CHO cell bioprocesses through the utilisation of a high throughput liquid chromatography mass spectrometry proteomics technique. Cellular stresses including a not fully investigated cell death type were highlighted in the proteomic analysis of a producer cell line in fed-batch bioreactor conditions. Targeting a ferroptosis inducing enzyme (found overabundant during the death phase of culturing) through chemical inhibition resulted in higher culture viability, which led to the development of a new ferroptosis inducer resistant producer cell line. This served as a groundwork for further genetic engineering to improve CHO cell culture longevity and thus productivity.