Label-free quantitative proteomics analysis of producer and non-producer Chinese Hamsters Ovary (CHO) cells under ER stress conditions

Abstract

Chinese Hamster Ovary (CHO) cells are the most commonly used host cell line used in the biopharmaceutical industry for the production of biotherapeutics. However, the lack of understanding of endoplasmic reticulum (ER) stress mechanisms involved in protein production, folding and secretion remains a bottleneck preventing improved production titres and product quality. This study aimed to uncover differentially expressed proteins in ER stress pathways in a non-producer (CHO-K1) and two producer (CHO DP-12 and CHO SK15-EPO) cell lines following exposure to the known stress inducer, thapsigargin using label-free quantitative LC-MS/MS proteomic analysis. This resulted in the identification of 607, 322 and 900 differentially expressed proteins in CHO-K1, CHO DP-12 and CHO SK15-EPO cells, respectively, compared to untreated controls. We identified key pathways related to protein folding, the unfolded protein response (UPR), protein processing in the ER, ER-associated degradation (ERAD), RNA transport, aminoacyl-tRNA biosynthesis and amino acid biosynthesis following induction of ER stress conditions, with expression changes observed in several glycosyltransferases, protein disulfide isomerases and molecular chaperones. Proteins targeting to the ubiquitin-ligase complex and ERAD were observed to be more enriched in the producer cells following induction of ER stress. This study extends existing knowledge of proteomic changes that govern the cellular stress response potentially identifying targets that can facilitate targeted cell line engineering to obtain production cell lines that demonstrate a higher stress tolerance and improved productivity.