Gaurav Backliwal

Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.

Valproic acid: A viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures

Various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) were screened for their ability to enhance transient gene expression (TGE) in Human Embryonic Kidney 293-EBNA (HEK293E) cells. The effects in HEK293E cells were compared to those in Chinese Hamster Ovary DG44 (CHO-DG44) cells. The iDNMTs and iHDACs were chosen based on their different cellular activities and mechanisms of action. For each inhibitor tested, the optimum concentration was determined for both cell lines, and these conditions were used to evaluate the effect of each compound using a recombinant monoclonal antibody as a reporter protein. All the iHDACs increased transient antibody yield at least 4-fold in HEK293E and at least 1.5-fold in CHO-DG44. By comparison, the iDNMTs increased antibody yields by a maximum of approximately 2-fold. Pairwise combinations of iDNMTs and iHDACs had a linearly additive effect on TGE in CHO-DG44 but not in HEK293E. With valproic acid (VPA), volumetric and specific productivities of 200 mg/L and 20 pg/cell/day, respectively, were achieved in HEK293E cells with a 10-day process. As VPA is both FDA-approved and 5-fold less expensive than sodium butyrate (NaBut), we recommend it as a cost-effective alternative to this widely used enhancer of recombinant protein production from mammalian cells.

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells.

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells.

Recombinant Antibody Yield Over 2 g/L by Transient Transfection of HEK 293 EBNA Cells in a Fed-Batch Process

Transient gene expression (TGE) is an established technology for the rapid production of recombinant proteins in mammalian cells. We developed a high-density PEI-mediated transfection protocol for HEK 293E cells using an IgG antibody as a model protein. Under previously optimized conditions in a batch culture, cell viability decreased rapidly soon after a relatively short period of exponential growth because of a complete depletion of glucose and glutamine from the medium. In this report we present results of feeding optimization. Glucose and protein hydrolysates had the biggest impact on cell viability and protein production. We compared the effect of five different plant-based protein hydrolysates on product yield and cell viability. We optimized the time of feeding, as well as the amounts of glucose and hydrolysate added to the medium. The best results were obtained when glucose and hydrolysate were added to final concentrations of 3 and 4 g/L, respectively, on day 3 and day 6 post-transfection. Under these conditions IgG titers over 2 g/L within 10 days after transfection were reached.

High Cell Density Transient Gene Expression in HEK 293 EBNA Cells

Transient gene expression (TGE) is a simple method for the production of recombinant proteins in a few days. In this report we present results obtained by optimization of a PEI-mediated high-density transient transfection protocol in HEK-293E cells with emphasis on reducing the cost of DNA needed for transfection. First, we constructed different vectors having the expression cassettes for both the IgG heavy chain and light chain genes on a single plasmid. This allowed us to reduce the DNA cost by 30–50% for large-scale transfections. With these vectors we optimized several transfection parameters including the cell density and the DNA and PEI amounts. Under optimized conditions we reproducibly obtained IgG titers over 700 mg/L in only five days of cultivation. This optimization allowed us furthermore to reduce cost of DNA for 40% by decreasing amount of DNA needed for transfection.

A Transient Gene Expression Process with Recombinant Antibody Titer of 0.5 g/L in CHO Cells

Here we describe our work towards an optimized transfection method for Chinese hamster ovary (CHO) cells that allows the transient production of recombinant antibody at titers approaching 0.5 g/L by two weeks post-transfection. This was achieved, in part, by transfecting at high cell density and by direct addition of polyethylenimine (PEI) and DNA to the culture followed by incubation of the cells in mild hypothermic conditions. In addition, by systematic screening and optimization of various medium additives such as glucose, amino acids, lipids, and peptones in the context of a serum free suspension medium, the integral viable cell density (IVCD) of the culture was improved.

Optimization and Comparison of Different DNA Methyl Transferase and Histone Deacetylase Inhibitors for Enhancing Transient Protein Expression

We have evaluated various DNA methyl transferase inhibitors (iDNMTs) and histone deacetylase inhibitors (iHDACs) for their effect on transient gene expression in mammalian cells. The inhibitors were selected on the basis of differences in their reported mechanism of action or cellular activities. The optimal concentrations of the inhibitors were determined and their activities were compared in HEK293E and CHO-DG44 cells transiently expressing a recombinant antibody. Their effects on transient gene expression from different vectors were also evaluated.

A Serum-Free, Transient Transfection System for Enhancing Production of Recombinant Antibodies in Mammalian Cells

Here, we present an optimized transfection system for mammalian cells that allows the serum-free production of recombinant antibodies with titers considerably higher than any currently published protocol. This was achieved and reproducibly shown by combining multi-plasmid transfection of suspension-adapted HEK293E cells with 25-kd linear polyethyleneimine (PEI) in serum-free medium with an improved protocol, encompassing optimized culture conditions and by manipulation of apoptotic and cell cycle pathways with HEK 293-adapted and optimized expression vectors.

Release of Plasmid-DNC from PEI/DNA Particles

The cationic polymer polyethylenimine (PEI) spontaneously forms complexes with DNA that efficiently transfect mammalian cells. To study the reversibility of the DNA/PEI interaction in vitro, preformed complexes were diluted with varying amounts of plasmid DNA and RNA. The released plasmid DNA was visualized by gel electrophoresis. With this simple assay we determined the kinetics of the release of plasmid DNA. Even at very low concentrations RNA instantaneously replaced most of the plasmid DNA in the complex, whereas DNA-mediated release takes up to one day. The results of these in vitro experiments are important for understanding the transport and release of plasmid DNA from PEI/DNA particles after transfection of cells.