Vector engineering strategies for protein production in CHO cells

Cost-effective, rapid transient gene expression (TGE) systems are essential for producing recombinant proteins for both research and early drug development. Monoclonal antibodies (mAbs) continually dominate the market, with thousands requiring manufacture. The development of a generic manufacturing process capable of consistently enhancing the expression of any chosen mAb is a worthwhile objective, aiming to ensure sufficient production levels whilst minimising the costly resource requirements associated with repeated production runs. Chinese hamster ovary (CHO) cells are the preferred hosts for biopharmaceutical production, and over the past decade, TGE processes have been developed that can achieve g/L yields. However, these developments have historically focused on host cell and process engineering, whilst the expression vector is often overlooked; despite playing a key role in controlling expression dynamics, such as transcription, translation, and mRNA stability. Presented here is a generic CHO cell TGE vector that combines synthetic biology and vector-based cell engineering strategies to consistently outperform an existing industry-standard vector and is suitable for use with a range of mAbs. First, to address the current limitation on transcription, highly-active synthetic promoters were designed using existing knowledge of CHO cell transcription factor regulatory elements (TFRE). Two libraries of novel synthetic promoters were created, which successfully exceeded expression of the cytomegalovirus (CMV) promoter up to 2.2-fold using an easy-to-express (ETE) model mAb. At the time of writing, this is the first demonstration of CHO cell synthetic promoters driving the expression of an industrially relevant mAb. Secondly, additional commercially available elements (introns, 3’ UTR, and polyadenylation elements), and synthetic variations thereof, were evaluated for their impact on mAb expression. Combinations of efficacious elements were examined for their generic effect on three representative IgG1 mAbs, leading to broad improvements in expression. Thirdly, two well-known cell engineering strategies were employed to address widely reported limitations in TGE processes, (i) The overexpression of the transcription factor, spliced X-box binding protein (XBP1s) to expand the cell’s endoplasmic reticulum (ER) capacity, whilst its cognate TFRE (Unfolded response element; UPRE) was incorporated into the mAb vectors to further enhance transcriptional output; and (ii) the OriP/EBNA-based episomal vector system was employed to overcome plasmid dilution. Both strategies facilitated improvements in mAb titre, up to 2.2-fold and 3.5-fold, respectively, although XBP1s co-expression exhibited product-specific effects. The final vector configuration comprising of a synthetic promoter, intron, and polyadenylation element, and the OriP/EBNA system, increased titre up to 8-fold compared to the original industrial vector for the highest expressing mAb, thus providing a potentially generic vector for substantially enhanced TGE recombinant protein production.