(127f) Analyzing ER Stress and UPR Activation in Highly Producing Chinese Hamster Ovary (CHO) Cell Lines

Chinese hamster ovary (CHO) cells are common protein production platforms due to efficient post-translational modification machinery and endoplasmic reticulum (ER) quality control; however , secretion levels needed for industrial cell lines likely leads to an imbalance in ER homeostasis, resulting in increased cellular stress. As a result, accumulation of improperly folded proteins is a particularly challenging bottleneck in cell line engineering. Misfolded or unfolded proteins induce ER stress, which initiates the unfolded protein response (UPR) to alleviate this stress. In mammalian cells, the UPR is activated via three pathways, namely IRE1, PERK, and ATF6. Activation of these pathways leads to a downstream cascade of changes in gene expression and protein abundance of XBP-1, CHOP, and ATF6α as indicative of IRE1, PERK, and ATF6 pathway activation, respectively. In order to measure ER stress in cell lines engineered for high-levels of recombinant protein production, this research aims to demonstrate the presence of ER stress associated with immunoglobulin G (IgG) antibody production, elucidate the progression of UPR in response to highly producing recombinant protein, and determine the effect of UPR on product quality. Current work is focused on measuring the UPR, product titer, and product quality in a CHO cell line engineered for high productivity of IgG. ELISA assays are used to investigate IgG titer, followed by glycan analysis to measure changes in product quality. Western blot and quantitative polymerase chain reaction (qPCR) assays are used to investigate the UPR, which is then correlated to product titer and decline in product quality. Connections between amino acid and energy metabolism and the UPR are also investigated, as these processes are intertwined and can be influence by media condition. It is expected research in this area will lead to improved engineering strategies for developing cell lines for higher titers of desired recombinant proteins, including relevant therapeutic protein products.