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 and low specific productivity with two 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 XBP-1 mRNA expression increased up to 64 fold, and both ATF4 and CHOP mRNA expression increased up to 8 fold. Transcriptomic analysis was conducted to monitor UPR targets and identify additional signaling transduction pathways activated during fed-batch culture. To determine if highly productive lines have been selected for the ability to cope better with ER stress, the UPR was monitored in fed-batch cultures of CHO cell pools with reduced IgG production via shRNA interference. Lastly, we explore the impacts of combining chemicals known to enhance productivity or reduce ER stress on cell productivity, titer and the UPR.

Early activation and late resolution of the unfolded protein response in fed-batch culture associated with high productivity CHO cells

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.