Abstract
Objective
Chinese hamster ovary (CHO) cells are the dominant platform for monoclonal antibody (mAb) production. Despite their widespread use, an understanding of endoplasmic reticulum (ER) stress during recombinant protein production remains limited, often creating bottlenecks that hinder improvements in production titres and product quality. Because many critical regulators of protein folding, processing and quality control are membrane-embedded or membrane-associated, they are frequently underrepresented in whole-cell lysate proteomic analyses. In this study, we applied a membrane proteome enrichment strategy to selectively enhance detection of ER-resident and secretory pathway proteins and thereby improve the resolution of ER stress responses in recombinant CHO cell lines.Methods
Sustained ER stress was induced using tunicamycin for 72 h in two IgG producing CHO cell lines, CHO DP-12 and NISTCHO. Membrane-enriched proteomes were analysed using label-free quantitative LC-MS/MS, followed by orthogonal validation using targeted LC-MS.Results
Membrane enrichment expanded coverage of ER-resident and secretory pathway proteins, revealing minimal overlap with whole-cell lysate analyses. ER stress induced distinct, pathway-level changes in the two cell lines that were more clearly resolved in the membrane fraction. In CHO DP‑12 cells, ER stress resulted in decreased abundance of proteins involved in protein folding, oxidative folding, and glycosylation, together with altered abundance of proteins involved in ER-associated degradation (ERAD) suggesting remodelling of the ER quality-control network and a potential shift towards enhanced clearance of misfolded proteins. In contrast, NISTCHO cells showed more moderate changes, including increased abundance of several ER chaperones and oxidative folding proteins, alongside comparatively limited changes in degradation-related pathways. Targeted LC-MS using parallel reaction monitoring (PRM) confirmed the direction of regulation of a panel of these proteins, providing additional confidence in the observed trends.Conclusions
This study highlights membrane proteomics as an important approach for studying ER-associated processes relevant to CHO cell function under ER stress conditions.