Proteomic and Ubiquitinated Proteome Insights Into ER Stress Responses in Chinese Hamster Ovary Cells Under Mild Hypothermic Conditions.

Chinese hamster ovary (CHO) cells, widely utilised in biopharmaceutical production, experience various stressors during cell culture that can affect protein expression and folding, particularly within the endoplasmic reticulum (ER). Mild hypothermia is widely employed in CHO cell bioproduction to improve recombinant protein yield and quality; however, its impact on ER-associated pathways, particularly those governing protein folding and stress responses, remains insufficiently characterised. Mass spectrometry-based proteomics allows for the identification and relative quantification of proteins, enabling detailed insights into protein expression, modifications, and functional networks. This study investigates the impact of mild hypothermic conditions (31°C) on the whole cell proteome and ubiquitinated proteome of CHO cells, with a specific focus on ER proteins and ER stress. Using high-resolution mass spectrometry, we conducted a comprehensive proteomic and ubiquitinated proteomic analysis to quantify changes in protein abundance and ubiquitinated peptides under mild hypothermia. The downregulation of several proteins involved in the glycosylation of nascent polypeptides at 31°C, including DDOST, P4HB, PRKSCH and LMAN1, in all cell lines studied suggests that mild hypothermic shock disrupts the cell's normal ability to fold new proteins, leading to ER stress as the misfolded proteins build up. When this is coupled with the maintained cell viability and increased productivity at 31°C, it indicates the ER stress response can mitigate the build-up of misfolded proteins. The differential regulation of the transcription factor eIF2α, downregulated in non-producer cells but upregulated in producer cells at 31°C, suggests that recombinant protein-producing CHO cells possess a more adaptive ER stress response, enabling more efficient function under hypothermic culture conditions. Enhanced ubiquitination of misfolded protein substrates highlights an increased reliance on ER-associated degradation (ERAD) pathways to alleviate proteotoxic stress, as well as the wide range of biological processes that are regulated by ubiquitination as part of the hypothermic stress response. These findings provide new insights into the cellular adaptation mechanisms of CHO cells to mild hypothermia, with implications for optimising bioproduction strategies to improve yield and quality of therapeutic proteins. Our study highlights the importance of understanding the more complex aspects of the proteome and how this additional layer of detail can open new avenues for CHO cell engineering.