Identifying novel engineering targets from evolved host populations using proteomics

Chinese Hamster Ovary (CHO) cells are the cell type of choice for the production of recombinant biopharmaceuticals, specifically antibodies. Thus, there have been many efforts to increase recombinant protein productivity and product quality. Directed evolution of CHO cells has been shown to improve their functionality, in terms of higher antibody expression or product quality. In this process, host cells are subjected to a physical or chemical stress and cells that survive these conditions are able to persist and form a resistant population. Here, we have compared the proteome landscape of several evolved hosts to identify differentially regulated pathways that may explain phenotypic differences observed between the hosts.

Expressing and null transfectant pools of seven evolved hosts were analysed and compared to the parental host. Using a multiplexing strategy, we found that both groups, expressing and null, showed distinct protein profiles. This indicates that the proteomes of expressing and null cell lines diverge when cells are expressing the antibody. This analysis revealed several proteins commonly downregulated in the antibody expressing pools, including proteins involved in degradative autophagy, endoplasmic-reticulum-associated protein degradation (ERAD) pathways and response to endoplasmic reticulum stress. Additionally, proteins related to organelle assembly and cellular localization were upregulated in all of the antibody expressing pools.

Moreover, our results demonstrate that the evolved hosts with higher antibody expression and specific productivity (qP) undergo similar changes in their proteome profiles, and importantly, this response was different to the hosts with lower expression and qP. Particularly, proteins related to Golgi apparatus, cell cycle, endosome or nucleolus were found to be upregulated among these evolved hosts. These results suggest that the evolved hosts with higher antibody expression undergo similar mechanisms that are mostly associated with pathways involved in protein secretion.

Finally, given that changes in the distribution of proteins between organelles may be fundamental in the regulation of the secretory pathway, we conducted a LOPIT-DC approach to identify changes in the protein subcellular location of a subset of pools which exhibited more changes in the total protein expression levels.

Altogether, the work presented provides an insight into the fundamental proteomic changes underlying phenotypic differences in these manufacturing host cell lines.