Novel cell engineering of the Unfolded Protein Response to achieve efficient therapeutic protein production cell line

Abstract

Bioproduction of therapeutic proteins such as monoclonal antibodies (mAb) continues to be a fast growing sector of advanced manufacturing. An ever increasing repertoire of therapeutic proteins coupled with the emergence of biosimilars has led to increasing global demand for higher yielding, faster, and more cost-effective manufacturing process. Central to any good production platform is the capacity of the production cell line. A suitable production cell line exhibits physiological traits such as high specific productivity (qp), short doubling time, high peak cell density and efficient metabolism. The emergence of a suitable production cell line with all the aforementioned traits is an extremely rare event, as it requires that all facets of cellular transcription, translation, secretion and metabolic efficiency are both individually optimized and collectively synchronized into a system capable of high level protein expression. Cellular translation and secretion capacity is thought to be one of the major cellular bottlenecks limiting protein production in mammalian cells. The over-expression of complex recombinant proteins such as mAbs driven by strong viral promoters exerts considerable burden on the Endoplasmic Reticulum (ER) and Golgi apparatus. Increased ER stress triggers the Unfolded Protein Response (UPR), which may lead to cell apoptosis and thus elimination of cells with high expression of the Gene of Interest (GOI). In this thesis, we adopted a host cell engineering approach to expand cellular translation and secretion capacity prior to expressing the GOI, in order to increase the probability of isolating high producers. Through screening of a panel of mAb producing clonal cell lines using qPCR, the XBP1 mRNA splice ratio was identified as a suitable target to augment host cell translation and secretion machinery. A high ratio of spliced over unspliced XBP1 mRNA is required to overcome the negative regulatory effect of unspliced XBP1 protein. Spliced XBP1 mRNA translates into a potent transcription factor that up-regulates a wide range of folding, secretory and quality control proteins, which forms the cytoprotective branch of the UPR. A stable CHO host cell line engineered with a high splice ratio of XBP1 mRNA exhibited increased expression of various chaperone and secretary vesicle proteins without increasing UPR associated apoptotic markers. This engineered host cell line was compared against the parental cell line in transient mAb production studies. The XBP1 cell line displayed a 7.5 fold increase in qp over the control cell line and more than 4 fold increase in final volumetric productivity. Moreover, a 3-fold increase in the percentage of high producers was observed in the cell distributions post-transfection when using the XBP1 host cell line. mAb producing cells derived from the XBP1 host cell line also demonstrated competency in conventional stirred-tank bioreactors, showing promise of a commercially viable production platform. In conclusion, XBP1 mRNA splice ratio engineered host cells have expanded translation and secretion capacity to alleviate the ER stress experienced from mAb production. The augmented capacity enabled multi-fold improvements in qp and volumetric productivity and increased the frequency of high producers in the transformed pool. The engineered host cell line showed great promise to become a commercially viable production platform cell line to significantly reduce the time and resources associated with cell line development.