In chinese hamster ovary cells specific signaling pathways are largely unknown. Often, optimization approaches of monoclonal antibody production process with CHO cells are untargeted and therefore partially ineffective. To overcome this, signaling effects of cell growth enhancing agents are analysed on molecular level via quantitative MS approaches.
CHO cultivation was performed in chemical defined medium. For SILAC experiments cells were cultivated with l/m/h lysine and arginine. Bottom-up proteomic MS sample preparation, measurement and data analysis was performed. Phospho-targets were evaluated by Western blots of label-free cultivated cells.
Insulin-like growth factor stimulation induced rapid changes in signaling directly linked to cellular metabolism and in parallel, long-term differences in growth and viability was observed . Next to this, media supplementation with the non-essential amino acid L-glutamine resulted in increased growth of mAb-producing CHO cells, but in also in a remarkebly reduced IGF-induced upregulation of phosphorylation sites.
To elucidate this, the proteomic composition of CHO cells growing on two different media is analyzed via triple SILAC-MS (including label-swap conditions). Significantly differential expressed proteins calculated for both label conditions were compared and validated via two quantification workflows, Proteome Discoverer and MaxQuant.
As expected, in glutamine containing media the glutamine synthesis and also parts of glucose metabolism pathways were found to be downregulated. However, also MAPK interacting proteins, like GSTP1, were downregulated and protein phosphatases as well as cytoskeletal proteins were upregulated. Based on this, the cellular reportoire contributing to decreased signaling intensity is presented on a new designed signaling network by expression data mapping.
The combination of validated Western blot-, SILAC- and LFQ-MS-based data led to a first characterization of a specific proteome, which contributes to a shift in IGF-signaling in CHO in media with or without glutamine. With this knowledge, a more targeted process design can be realized and will improve mAb-production efficiency.