Two strategies to improve transient gene expression in HEK293E and/or CHO-DG44 cells

Transient gene expression (TGE) is based on episomal DNA expression. This technique is used for the production of recombinant proteins that can be used as therapeutics. TGE is for example extremely useful for shortening the time between candidate proteins screening and their production. Understanding and optimizing TGE is a major concern in the field of recombinant proteins. Thus, we selected here two different approaches to further improve TGE. First, we studied effect of signal peptide (SP) modification on TNFR-Fc protein in transiently transfected HEK293E and CHO-DG44 cells. Secondly, we studied effect of Valproic acid on the production of TNFR-Fc protein in HEK293E cells and on its influence on plasmid methylation. Modification of TNFR-Fc was done with eighteen others SPs. Artificial SPs exhibiting different scores using a prediction method and SPs coming from naturally secreted proteins were tested. None of them could induce a better secretion than the native SP. Nevertheless, they all showed comparable secretion abilities, except one that could not induce protein secretion. We showed here that SP modification could not increase protein secretion in HEK293E or in CHO-DG44. But also that without a valid SP, protein secretion is inhibited. Addition of VPA in HEK293E cells transfected with pXLG-A3 plasmid increased IgG productivity by 11 fold. Nevertheless, we showed here that VPA did not increase TNFR-Fc productivity to the same extent (1.2 fold) even by optimization of its concentration. Furthermore, we investigated the effect of plasmid methylation (TNFRFc and IgG) on TGE using VPA. We found that IgG expression was less influenced by this modification than TNFR-Fc. In fact, TNFR-Fc plasmid methylation drastically decreased specific productivity (0.2 fold). And VPA could almost entirely compensate this effect (0.8 fold). We therefore hypothesized that VPA has an impact on pDNA methylation in the context of TGE.

Transient recombinant protein expression in mammalian cells

Sarah Wulhfard

Transient gene expression (TGE) is a rapid method for generating recombinant proteins in mammalian cells, but the volumetric productivities for secreted proteins in transiently transfected CHO DG44 cells are typically more than an order of magnitude lower than the yields achieved with recombinant CHO-derived cell lines. The goals of the thesis are to identify the limitations to higher TGE yields in CHO DG44 cells and to find possible solutions to overcome the problems. Initially an attempt was made to enhance TGE production by increasing the amount of transfected plasmid DNA. However, this approach did not result in increased recombinant protein levels; on the contrary, transfection with an excess of plasmid DNA (> 1.25 μg/ml) had a negative impact on transgene mRNA levels and protein production. Moreover, it was also observed that recombinant protein yield was strongly dependent on the mRNA level. Therefore, three strategies aimed at increasing the amount of transgene mRNA were investigated. For the first approach, transfected cells were exposed to hypothermic conditions during the production phase. It was already known that lower temperatures increase protein production several fold in recombinant CHO DG44-derived cell lines. The second strategy involved the treatment of transfected cells with valproic acid, a histone deacetylase inhibitor that reduces the effects of gene silencing. The third approach aimed to increase transgene mRNA levels by overexpressing transcription factors and growth factors. With the first two strategies recombinant antibody yields of 60-80 mg/L were achieved whereas the untreated control transfections produced only 5-10 mg/L. Combination of the two strategies led to the production of 90 mg/L of antibody. Moreover, in the treated cultures, the steady-state level of transgene mRNA was 3-5 times higher than in the untreated cultures and remained stable up to 6 days post-transfection. Using the third approach, the increase in recombinant protein production was moderate and transgene mRNA amounts were only 2-fold higher in treated samples compared to the control. When specific proteins such as c-fos, c-jun, NF-kB, and acidic fibroblast growth factor (aFGF) were overexpressed the recombinant antibody production was 20 mg/L compared to 5 mg/L for the control transfection. Overexpression of either a transcription factor or a growth factor in combination with treatment with valproic acid allowed the recombinant protein yield to reach 90 mg/L. However, the benefit of the overexpressed factors was minimal compared to the effect of valproic acid alone. In conclusion, it was demonstrated that the level and stability of transgene mRNA are important factors for increasing volumetric yields in transiently transfected CHO DG44 cells. Furthermore, three approaches aimed to increase mRNA amounts were tested. Exposure to hypothermic conditions and treatment with valproic acid were the two best strategies tested. Both are simple, cost-effective, and scalable making transient gene expression in CHO DG44 cells a feasible alternative for rapid production of gram amounts of recombinant protein.

EPFL2009

Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells

Klaus Friedrich, Horst Pick, Florian Maria Wurm

Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.

2001

Transient recombinant protein expression in mammalian cells

Sarah Wulhfard

EPFL2009