(750b) Early Activation and Late Resolution of the Unfolded Protein Response in Fed-Batch Culture Associated with Highly Productive CHO Cells

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 can lead to an imbalance in ER homeostasis. Accumulation of improperly folded proteins is a particularly challenging bottleneck in cell line engineering and initiates the unfolded protein response (UPR). This research aims to demonstrate higher specific productivity results in unavoidable ER stress by measuring the UPR in cell lines engineered for high-levels of recombinant protein production. In fed-batch culture, we achieve high specific productivity with two distinct CHO cell lines, one producing immunoglobulin G (IgG) and one producing erythropoietin (EPO-Fc), respectively. Western blot and quantitative polymerase chain reaction (qPCR) were used to investigate the UPR time course, correlated to product titer. In comparison to the parental cell line controls, both CHO cell lines exhibited early activation of the IRE1 and PERK pathways. By Day 3 of fed-batch culture, spliced XBP1 mRNA expression increased up to 2⁶ fold, and both ATF4 and CHOP mRNA expression increased up to 2³ fold. Transcriptomics and subsequent differential gene expression analysis revealed other protein processing genes were activated in addition to the UPR. Furthermore, genes associated with other signaling transduction pathways, including calcium signaling and glutathione synthesis, were found to be differentially expressed in both protein-producing cell lines. To determine if highly productive lines have been selected for the ability to cope with ER stress, the UPR was monitored in fed-batch cultures of CHO cell pools with reduced IgG and EPO production via shRNA interference. Lastly, we explore the impacts of engineering the protein-producing cell lines using targets identified in our transcriptomic analysis. It is anticipated this work will lead to advancements in CHO cell engineering and protein production.