Process development of transient gene expression in mammalian cells for high biopharmaceutical production

Abstract

Transient gene expression (TGE)-based recombinant protein production in biotechnologically important mammalian cells such as human embryonic kidney 293 (HEK293) cells and Chinese hamster ovary (CHO) cells has been a valuable technology for the past several years. Remarkable development of the TGE system was accomplished with the recombinant therapeutic protein yield increasing from mg/L to g/L due to huge demand for pre-clinical research and development purposes. This progression of TGE-based recombinant protein production is attributed to many factors: optimized efficient transfection reagents, an expression vector with strong gene expression, suitable cell lines, cell line-specific serum-free media, and an optimized cell culture process for suspension cells. Therefore, to improve the production of recombinant protein, this study focused on the cell engineering of mammalian cell lines such as HEK293 and CHO cells, expression vector engineering, and optimization of culture process for TGE-based recombinant protein production in mammalian cells. In an attempt to determine the relationship between the Epstein Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (qp), EBNA-1-amplified HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. qp was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R2 = 0.7407). The Fc-fusion protein production and qp in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in qp by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in HEK293 cell-based TGE systems. Despite the relatively low transfection efficiency and low qp of CHO cell-based transient gene expression systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, EBNA-1/polyoma virus large T-antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dhfr/methotrexate (MTX)-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems. Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus of swine with high morbidity and mortality in new born piglets. The S1 domain of the spike protein of PEDV is responsible for virus binding and fusion to the cellular receptor inducing a number of neutralizing antibodies. Therefore, we expressed recombinant S1 protein in HEK293 cell-based TGE systems for the development of efficient vaccines against PEDV. The recombinant S1 protein with human Fc region (S1-Fc) was successfully expressed in a secretory form in HEK293 cells. Affinity purified recombinant S1-Fc protein was analyzed by SDS-PAGE, Western blot, and size-exclusion chromatography. The human-derived seven signal peptides were then evaluated for their impacts on the production of recombinant S1-Fc protein in HEK293 cell-based TGE systems. Also, tagging of recombinant S1-Fc protein with oligomerization domain such as GCN4 and foldon was assessed for potential formation of trimeric structure. It was observed that the maximum recombinant S1-Fc protein production with thrombopoietin isoform 1 precursor (THPO) was 1.5-fold of higher than that in native signal peptide of S1 protein. We also found that the ability of vaccination with the recombinant S1 protein against PEDV. The purified recombinant S1-THPO-Fc protein with GCN4 was shown to mediate highly potent antibody responses in PEDV whole virus antigen. These results show that recombinant S1 protein expressed in HEK293 cell-based TGE systems will be promising method for an efficient subunit vaccine for PEDV prevention. For high-level production of heterologous therapeutic proteins, the mostly widely used mammalian expression systems in biopharmaceutical industry is the dhfr/MTX-mediated gene amplification system. In particular, dhfr/MTX-mediated gene amplification procedure is a based on the use of dhfr-deficient CHO cell lines such as CHO-DG44 and CHO-DXB11. However, the use of gene amplification approach has been restricted due to the limitation of mammalian cell lines that lack the amplifiable genes. In an attempt to develop the dhfr-deficient HEK293 cell lines, expression vector containing short hairpin RNA (shRNA) that targeting the 3′-untranslated region (3′-UTR) of endogenous dhfr was applied for amplifying the foreign gene in wild-type HEK293 cell lines. shRNAs were designed to target the 3′-UTR of endogenous dhfr specifically, and then efficiently down-regulated dhfr expression of HEK293 cells. The shRNA-2 decreased the mRNA expression level of endogenous dhfr by 52%. The protein expression level of endogenous dhfr also reduced by the action of shRNA-2. The EBNA-1 expression levels of shRNA/EBNA-1-amplified pools were successfully amplified by stepwise increments of MTX concentration. As a result, compared with a Null, the qp increased by more than 2.0 times in shRNA/EBNA-1-amplified pool at 100 nM MTX. Taken together, combining shRNA by downregulation of endogenous target gene and gene amplification approach could be potentially applied for amplifying the target gene in wild-type cell lines.