Transcriptional and post-transcriptional limitations of high-yielding, PEI-mediated transient transfection with CHO and HEK-293E cells

Transient gene expression (TGE) in human embryonic kidney (HEK-293) and Chinese hamster ovary (CHO) cells is a well-established technology for the rapid generation of recombinant proteins. Although the maximum TGE yields have reached 1 g/L or more, the amount of plasmid DNA (pDNA) required for transfection remains high. Although greater than 103 copies of pDNA are present per transfected cell, protein yields are still lower than those achieved in recombinant cell lines with only one or a few copies of the transgene. This indicates a clear limitation to TGE in terms of the maximum level of recombinant protein production. In this study, we investigated the limitations to high-yielding TGE processes with CHO and HEK-293E cells using a monoclonal antibody as a model protein. For either cell host, both the intracellular and intranuclear pDNA levels increased linearly with the amount of pDNA added to the culture. In contrast, transgene mRNA accumulation reached a plateau as the intranuclear pDNA amount increased, suggesting a limitation in pDNA transcription. A post-transcriptional limitation to TGE yields was revealed by calculating the amount of antibody produced per transgene mRNA (mRNA utilization). For both hosts the transgene mRNA utilization decreased dramatically when transfected pDNA amounts increased beyond the level giving the maximum protein yield. The post-transcriptional limitation did not appear to be due to bottlenecks in antibody assembly or secretion, suggesting that transgene mRNA translation may be limiting. The results show that TGE yields are not limited by pDNA delivery into the nuclei, but in pDNA and transgene mRNA utilization.

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 in HEK-293E Cells for Recombinant Protein Production

Sophie Nallet

The growing demand of biopharmaceutical products is boosting the market for therapeutic recombinant proteins (r-proteins). More than half of the 140 r-proteins that have gained approval for human therapeutic use are manufactured in mammalian cells that make possible complex post-translational modifications, like glycosylation. In the industry, r-proteins are manufactured by stable gene expression (SGE) following Good Manufacturing Practice (GMP) standards. Transient gene expression (TGE) in mammalian cells is an alternative method for the expression of r-proteins whose main advantages are rapidity and flexibility. TGE involves the expression of the protein of interest from plasmids, which are transfected into the cells with a non-viral DNA transfecting agent, usually polyethylenimine (PEI). DNA is transcribed from the plasmids without the latter having been integrated into the host genome. Despite the recent improvements in r-protein titers by TGE and the scale up of this method to the 100-L scale, TGE remains so far a tool for protein production for research purposes. Therefore, to determine if TGE can be used for manufacturing therapeutic r-protein following GMP standards, different features related to reproducibility and product quality from TGE of a recombinant anti-rhesus D immunoglobulin G in HEK-293E cells using linear PEI were characterized. To test the effects of the size distribution and chemical structure of PEI on TGE, different commercially available PEIs were characterized and their impact on transfection and r-protein expression were assessed. The results showed that both the molecular weight distribution and the chemical structure of the PEI had an effect on TGE. However, the small variations in size distribution and structure between the batches of PEI from the same supplier did not affect significantly the transfection and r-protein production by TGE. Then, the fate of PEI and plasmid DNA in cell culture over time and their removal from the final r-protein during purification were determined and measured. Most of the PEI and pDNA remained in the cell culture medium after transfection. However, both PEI and plasmid DNA, which are additional components in TGE compared to processes based on recombinant cell lines, could be reduced to a concentration below the limit of detection of the assays after Protein A affinity purification of the antibody. To test the reproducibility of a TGE process, 10 batches of transfected cells were run in 500-mL orbitally shaken bottles. The cell specific productivity of the antibody was 20.2 ± 2.6 pg.cell-1.day-1 on average for the 10 batches. The purity and size consistency of the recombinant antibody produced in those 10 batches was demonstrated by gel electrophoresis and size exclusion chromatography. Then, the glycosylation profile of the antibody produced by TGE in HEK-293E cells was determined by mass spectrometry and was reproducible over the 10 batches. Moreover, it was similar to the glycosylation profile of the antibody produced by 3 stable HEK-293E cell lines. Finally, as a proof of concept, TGE was applied for the development of a vaccine against the respiratory syncytial virus. The fusion protein of the respiratory syncytial virus was produced by TGE and used as an antigen for the development of a recombinant vaccine. TGE enabled the rapid evaluation of the candidate vaccine in animal trials. Overall, the results of this study suggest that TGE is a reliable and reproducible method for manufacturing r-proteins of a consistent quality, provided that some particular features, such as reagents quality and process parameters, are well defined and controlled. Since TGE makes possible the rapid production of virtually any protein, even toxic, it could be used to provide GMP approved biopharmaceuticals for clinical trials in a short time, reducing development costs of biological therapeutic products.

EPFL2010

Role of non-specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK-293E cells

Lucia Baldi Unser, David Hacker, Divor Kiseljak, Sagar Shashidhar Manoli, Yashas Rajendra, Florian Maria Wurm

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the TGE volumetric productivity has improved significantly over the past decade, the amount of plasmid DNA (pDNA) needed for transfection remains very high. Here, we examined the use of non-specific (filler) DNA to partially replace the transgene-bearing plasmid DNA (coding pDNA) in transfections of Chinese hamster ovary (CHO) and human embryo kidney (HEK-293E) cells. When the optimal amount of coding pDNA for either host was reduced by 67% and replaced with filler DNA, the recombinant protein yield decreased by only 25% relative to the yield in control transfections. Filler DNA did not affect the cellular uptake or intracellular stability of coding pDNA, but its presence lead to increases of the percentage of transfected cells and the steady-state level of transgene mRNA compared to control transfections. Studies of the physicochemical properties of DNA-polyethyleneimine (PEI) complexes with or without filler DNA did not reveal any differences in their size or surface charge. The results suggest that filler DNA allows the coding pDNA to be distributed over a greater number of DNA-PEI complexes, leading to a higher percentage of transfected cells. The co-assembly of filler DNA and coding pDNA within complexes may also allow the latter to be more efficiently utilized by the cell's transcription machinery, resulting in a higher level of transgene mRNA.