Comparative spatial proteomics as a tool to understand difficult-to-express molecules in CHO cell lines

Chinese hamster ovary (CHO) cell line expression systems dominate the biotechnology industry for expressing therapeutic proteins but can still be plagued with unpredictable low production yields, which make for substandard manufacturability of the drug product.

Proteins localise to discrete, subcellular locations within the cell to fulfil their location-specific functions and enable biological processes to occur. Movement between these niches (e.g., organelles) is essential for cellular processes. Modifications, mutations, and other variations that cause deviant protein localisation have been associated with a wide range of diseases, as well as impacting on protein expression. Determining the location of proteins in different cell types and states is essential to understanding the functions and processes involved with their differential localisation.

Here, we have applied the subcellular spatial proteomic method, hyper-plexed localisation of organelle proteins by isotope tagging (hyperLOPIT), to determine differential localisations of proteins between a CHO cell line expressing an monoclonal antibody that is easy to express (ETE) and a cell line expressing a difficult to express protein (DTE). Nearly 5,000 proteins were mapped to 13 organellar compartments with excellent resolution using the hyperLOPIT workflow, serving as a significant resource for anyone utilising CHO cells as an expression platform.

Little is know about the subcellular organisation of proteins specifically within CHO cells, or how this changes with the expression of therapeutic proteins. Therefore a bespoke approach, Bayesian ANalysis of Differential Localisation Experiments (BANDLE), was applied to compute probability of differential localisation of proteins between the the cells that have relative apparent ease or difficulty in expressing therapeutic proteins. Results from this extensive analysis identified many proteins that are differentially localised between cell lines expressing DTE and ETE molecules with an implication in various intracellular processes – particularly the upregulation of proteins involved in endocytosis, autophagy, and phagosome formation in the cell line expressing the DTE protein.

This work provides novel insight into potential gene targets to enhance the manufacturing capability of biologics in CHO cell lines.