Since the early days of CHO cell proteomics, quantification methods have steadily evolved ( Bachi and Bonaldi, 2008 Bakalarski and Kirkpatrick, 2016). 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. 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., 2019, 2020 Lakshmanan et al., 2019). 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).

While most of these studies focused on the effects of one factor at a time, more systemic approaches such as quality by design and design of experiments have been used to study the link between applied parameters whilst further pushing the limits of recombinant protein production ( Looby et al., 2011 Nagashima et al., 2013 Weng et al., 2020).

However, it was also shown that an improved energy metabolism under hyperosmotic conditions favors specific productivity of CHO cells ( Lee et al., 2003). Various studies have shown that a temperature shift from 37 to 32☌ drastically increases recombinant protein production, potentially due to improved cell viability ( Yoon et al., 2004 Ahn et al., 2008). Frequently used approaches include shifts in cell culture parameters like temperature and pH as well as the addition of certain media supplements or the use of controlled feeding strategies ( Trummer et al., 2006 Fan et al., 2015 Vergara et al., 2018). Various strategies have been applied to enable optimized CHO cell bioprocessing, aiming not only for an increased specific productivity but also for a higher cell viability while reducing the amount of undesired by-products ( Kunert and Reinhart, 2016). Consequently, CHO cells have been actively engineered over the last decade in order to express high levels of a wide range of mAbs and other therapeutic biomolecules. With an increasing number of approvals every year, the biopharmaceutical market is dominated by monoclonal antibodies (mAbs) which are most commonly produced in Chinese hamster ovary (CHO) cells ( Kaplon and Reichert, 2018 Walsh, 2018). Taken together, the present study allows the conclusion that, in anti-IL8 IgG1 producing CHO DP-12 cells, an improved energy metabolism achieved by lowering the cell culture pH is favorable when aiming towards high antibody production rates while maintaining product quality. Moreover, in-depth product characterization by means of charge variant analysis, peptide mapping, as well as structural and functional analysis, revealed posttranslational and structural changes in the expressed drug substance. Additionally, activation of ERK5 and TNFR1 signaling suggested an affected cell cycle. Pathway analysis indicated that applied bioprocessing conditions resulted in differential activation of oxidative phosphorylation. Furthermore, analysis of the cells using two-dimensional liquid chromatography-mass spectrometry employing tandem mass tag based isotopic quantitation and synchronous precursor selection-MS 3 detection revealed substantial changes in the protein expression profiles of CHO cells. Applying shifts in temperature, pH and dissolved oxygen concentration, respectively, resulted in altered productivity and product quality. Therefore, in this study the effects of commonly applied changes in bioprocessing parameters on an anti-IL8 IgG1 producing CHO DP-12 cell line were investigated on the level of host cell proteome expression combined with product quality assessment of the expressed IgG1 monoclonal antibody. To enable further improvements in manufacturing processes, detailed information about how certain parameters affect cellular mechanisms in the production cells, and thereby also the expressed drug substance, is required. Intense cell engineering, in combination with optimization of various process parameters results in increasing product titers. The biopharmaceutical market is dominated by monoclonal antibodies, the majority of which are produced in Chinese hamster ovary (CHO) cell lines.

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