Optimizing signal peptides for expression of recombinant antibodies in HEK293 cells

Abstract: Monoclonal antibodies are well-established as a therapeutic in the biopharmaceutical market, targeting a variety of diseases and with 79 approved products by the United States Food and Drug Administration in December 2019. Therapeutic monoclonal antibodies are commonly produced as recombinant proteins in mammalian cell lines, due to their capacity of post-translational modifications, most notably glycosylation. Furthermore, an identified bottleneck within the production of recombinant proteins is the translocation of nascent proteins from the cytosol into the lumen of the endoplasmic reticulum. The signal peptide, which is located at the N-terminal of nascent proteins, plays a central role in the process of protein secretion. Several studies have shown that optimization of signal peptides is a crucial step for attempting to achieve increased expression of recombinant antibodies in mammalian systems. The aim of this study was to evaluate the expression of three human recombinant antibodies in Human Embryonic Kidney 293 (HEK293) cells by evaluating 16 different signal peptide combinations, consisting of eight heavy chain (HC) and two light chain (LC) signal peptides. The impact goal was an efficient secretion of recombinant antibodies, and thus lower production cost of recombinant antibodies in HEK293 cells. First, 16 HC and LC signal peptide plasmid constructs were generated for each of the three recombinant antibodies. Thereafter, transient gene expression in HEK293 cells were performed at three independent experiments. Finally, the antibody titers were quantified using Biacore concentration analysis. The produced antibody titers for the three studied recombinant antibodies were highly dependent on the used signal peptides. Interestingly, the evaluated HC and LC signal peptide combinations resulted in 3 times higher and 2 times higher antibody titers compared to the original signal peptides used by the Drug Discovery and Development platform at Science for Life Laboratory, for two of the studied antibodies respectively. The results presented in this report further demonstrates the necessity to evaluate signal peptides in order to achieve increased expression of recombinant antibodies in mammalian systems.