Systems Biology of Recombinant 2G12 and 353/11 mAb Production in CHO-K1 Cell Lines at Phosphoproteome Level

Chinese hamster ovary (CHO) cells are extensively used in the pharmaceutical industry for producing complex proteins, primarily because of their ability to perform human-like post-translational modifications. However, the efficiency of high-quality protein production can vary significantly for mAb producing cell lines, within the CHO host cell lines or by extrinsic factors. To investigate the complex cellular mechanisms underlying this variability, a comprehensive phosphoproteomics analysis was performed using label-free quantitative liquid chromatography after a phospho-peptide enrichment of recombinant CHO-cells producing two different antibodies and a tunicamycin treatment experiment. Using MaxQuant and Perseus for data analysis, we identified 2109 proteins and quantified 4059 phosphosites. Significant phosphorylation dynamics were observed in nuclear proteins of cells producing the difficult-to-produce 2G12 mAb. It suggests that the expression of 2G12 regulates nuclear pathways based on up- and down-regulation of phosphorylation sites. Furthermore, a substantial number of changes in the phosphorylation pattern related to tunicamycin treatment has been detected.TM treatment affects, among other phosphoproteins, the eukaryotic elongation factor 2 kinase (Eef2k). It alters the phosphorylation landscape of key proteins involved in cellular processes including DNA methylation, highlighting the mechanisms behind stress-induced cellular responses.