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

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.

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

Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells

Lucia Baldi Unser, Martin Bertschinger, David Hacker, Florian Maria Wurm

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.

2004

The Use of Valproic Acid in Transient Gene Expression with HEK-293E Cells

Divor Kiseljak

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins. Recently, valproic acid (VPA), a well known histone deacetylase inhibitor, has been suggested for the use in cell cultures to enhance recombinant protein expression. However, little was known about protein expression in HEK-293E cells under VPA treatment. Therefore, the first aim of this thesis was to describe the effects of VPA on protein expression by TGE in HEK-293E cells. The addition of VPA induced a dose-dependent increase in protein production. Under the optimal VPA concentration (3.75 mM) IgG volumetric productivity increased over 8-fold compared to the control. This was achieved through improvements in both integral viable cell density (IVCD) and cell specific productivity. The IVCD was improved by the VPA-induced cell growth arrest resulting in longer culture duration and extended protein production phase. The enhancement in specific productivity in VPA-treated cells was a consequence of higher cellular pDNA copy number and improved pDNA utilization that led to higher transgene mRNA levels. The study on limitations governing VPA-based TGE protein expression in HEK-293E cells revealed the bottlenecks at the transcriptional and translational levels, however the limitation did not appear to be at the level of protein folding or assembly for the model IgG antibody studied. In addition, the effect of VPA was tested on the expression of different recombinant monoclonal antibodies and Fc-fusion proteins. The results showed that VPA addition did not always significantly improve protein titers. Since the optimal VPA concentration was dependent on both the recombinant protein being expressed and the amount of coding pDNA used for transfection, we recommend evaluating the suitability of VPA addition and the optimization of VPA concentration in order to find the optimal conditions for TGE protein expression. We believe that this work will contribute to the design of more rational, robust, and successful expression platforms for recombinant protein production in HEK-293E cells. One of the major contributors to the cost of TGE process at large scale is the considerable amount of pDNA required for transfection. Therefore, the second aim of III this thesis was to determine a cost-effective method to reduce the pDNA amount. We showed that a part of the coding pDNA can be replaced with nonspecific (filler) DNA with minimal loss in volumetric productivity. The studies revealed that the presence of filler DNA allowed the coding pDNA to be distributed over a larger number of DNA-PEI complexes. This resulted in a higher percentage of transfected cells and may favor the better utilization of the coding pDNA by the cell’s transcription machinery. The use of filler DNA as a part of defined fed-batch process (based on the addition of glucose and plant derived protein hydrolysates) resulted in 25 fold reduction in coding pDNA amount requirement while maintaining volumetric protein productivity over 1 g/l. The cost reduction of TGE-produced proteins, due to significant decrease in pDNA amount requirement for transfections, will certainly facilitate the development and implementation of large scale TGE as a feasible method for TGE-based protein production.