Characterization of glutamine synthetase (GS)/MSX HEK293 expression system mediated by GS gene knockout cells

Abstract

From the first approval of biotherapeutic protein in 1982, the technologies for their production have developed substantially. Although over 70% of biotheriapeutic proteins have been produced in Chinese hamster ovary (CHO) cells, there has been a recent shift toward the use of human cell lines because of their human-derived post-translational modifications (PTMs). Human embryonic kidney (HEK) 293 cells has been extensively used as an expression tool for recombinant proteins in scientific research. Recently, HEK293 cell lines were also used for the stable production of complex biotherapeutic proteins because their human-like PTMs, suspension-free culture, and high transfection efficiency among human cell lines. However, lack of efficient selection system was regarded as limitations for using HEK293 cells as a stable host cell line. Glutamine sytnehtase (GS) ？ mediated gene amplification system is one of stable protein production systems used in industrial field to isolate high-producing clonal cells. In this study, GS-mediated gene amplification system was applied in HEK293 cells for stringent and effective selection for high-producing clonal cell lines. To investigate the feasibility of GS-mediated gene amplification in HEK293 cells for the high-level stable production of therapeutic proteins, HEK293E cells were transfected by the GS expression vector containing antibody genes and were selected at various methionine sulfoximine (MSX) concentrations in 96-well plates. For a comparison, CHOK1 cells were transfected by the same GS expression vector and selected at various MSX concentrations. Unlike CHOK1 cells, HEK293E cells producing high levels of antibodies were not selected at all. For HEK293E cells, the number of wells with the cell pool did not decrease with an increase in the concentration of MSX up to 500 μM MSX. A q-RT-PCR analysis confirmed that the antibody genes in the HEK293E cells, unlike the CHOK1 cells, were not amplified after increasing the MSX concentration. It was found that the GS activity in HEK293E cells was much higher than that in CHOK1 cells (P < 0.05). In a glutamine-free medium, the GS activity of HEK293E cells was approximately 4.8 times higher than that in CHOK1 cells. Accordingly, it is inferred that high GS activity of HEK293E cells results in elevated resistance to MSX and therefore hampers GS-mediated gene amplification by MSX. Thus, in order to apply the GS-mediated gene amplification system to HEK293 cells, the endogenous GS expression level in HEK293 cells needs to be minimized by knock-out or down-regulation methods. Previously, it was inferred that a high GS activity in HEK293E cells results in elevated resistance to MSX and consequently hampers GS-mediated gene amplification and selection by MSX. To overcome this MSX resistance in HEK293E cells, a GS-knockout HEK293E cell line was generated using the CRISPR/Cas9 system to target the endogenous human GS gene. The GS-knockout in the HEK293E cell line (RK8) was confirmed by Western blot analysis of GS and by observation of glutamine-dependent growth. Unlike the wild type HEK293E cells, the RK8 cells were successfully used as host cells to generate a recombinant HEK293E cell line (rHEK293E) producing a monoclonal antibody (mAb). When the RK8 cells were transfected with the GS expression vector containing the mAb gene, selection efficiency was decreased by MSX and rHEK293E cells producing the mAb could be selected in the absence as well as in the presence of MSX. The gene copies and mRNA expression levels of the mAb in rHEK293E cells were also quantified using qRT-PCR. In addition, the stability of rHEK293E clones were sustained in the presence of selection pressure. Taken together, the GS-knockout HEK293E cell line can be used as host cells to generate stable rHEK293E cells producing a mAb through GS-mediated gene selection in the absence as well as in the presence of MSX. In conclusion, the GS/MSX system for isolating high-producing clonal cells successfully applied in HEK293E cells by minimizing the MSX resistance of HEK293 cells, which derived by high-level of endogenous GS expression in HEK293E cells. The GS-HEK293 system can be used for the protein production in human-derived cell line, especially for the production of complex proteins.