Divor Kiseljak

A simple high-yielding process for transient gene expression in CHO cells.

Here we describe a simplified method for transient gene expression (TGE) in suspension-adapted Chinese hamster ovary (CHO) cells using polyethylenimine (PEI) for DNA delivery. Both the transfection and production phases of the bioprocess were performed at a density of 4 × 10⁶ cells/mL at 31 °C. In addition, the amounts of both PEI and plasmid DNA were reduced up to 50% on a per cell basis compared to previously published protocols from this laboratory, resulting in higher cell viability after transfection and higher volumetric recombinant protein yields. In batch cultures of up to 14 days, reproducible recombinant antibody yields up to 300 mg/L were achieved at small scale (5 mL) and up to 250 mg/L at large scale (500 mL). The simplicity and improved yields are expected to increase the utility of CHO cells for the rapid production of recombinant proteins at larger scales by TGE.

Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells.

Abstract Transient gene expression (TGE) from mammalian cells is an increasingly important tool for the rapid production of recombinant proteins for research applications in biochemistry, structural biology, and biomedicine. Here we review methods for the transfection of human embryo kidney (HEK-293) and Chinese hamster ovary (CHO) cells in suspension culture using the cationic polymer polyethylenimine (PEI) for gene delivery.

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

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 cells transcription machinery, resulting in a higher level of transgene mRNA. Biotechnol. Bioeng. 2012;109: 2271–2278.

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.

Enhanced plasmid DNA utilization in transiently transfected CHO-DG44 cells in the presence of polar solvents.

Although the protein yields from transient gene expression (TGE) with Chinese hamster ovary (CHO) cells have recently improved, the amount of plasmid DNA (pDNA) needed for transfection remains relatively high. We describe a strategy to reduce the pDNA amount by transfecting CHO–DG44 cells with 0.06 μg pDNA/106 cells (10% of the optimal amount) in the presence of nonspecific (filler) DNA and various polar solvents including dimethylsufoxide, dimethyl formamide, acetonitrile, dimethyl acetamide (DMA), and hexamethyl phosphoramide (HMP). All of the polar solvents with the exception of HMP increased the production of a recombinant antibody in comparison to the untreated control transfection. In the presence of 0.25% DMA, the antibody yield in a 7‐day batch culture was 500 mg/L. This was fourfold higher than the yield from the untreated control transfection. Mechanistic studies revealed that the polar solvents did not affect polyethylenimine‐mediated pDNA delivery into cells or nuclei. The steady‐state transgene mRNA level was elevated in the presence of each of the polar solvents tested, while the transgene mRNA half‐life remained the same. These results indicated that the polar solvents enhanced transgene transcription. When screening a panel of recombinant antibodies and Fc‐fusion proteins for production in the presence of the polar solvents, the highest increase in yield was observed following DMA addition for 11 of the 12 proteins. These results are expected to enhance the applicability of high‐yielding TGE processes with CHO–DG44 cells by decreasing the amount of pDNA required for transfection.

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.

Influence of glutamine on transient and stable recombinant protein production in CHO and HEK-293 cells.

BackgroundGlutamine is an essential component in culture mediafor most of the mammalian cell lines. It is often used asanalternativesourceofenergybycells,alongwithglu-cose. Glutamine metabolism induces ammonia accumu-lation in cell culture. Elevated ammonia concentrationabove 2 mM has been shown to have negative impacton both cell growth and recombinant protein productiv-ity [1-4]. In this study we investigated the effects ofdecreased glutamine concentration in the medium forCHO-DG44 and HEK-293E cells during transient geneexpression (TGE). The rationale was to reduce ammoniaaccumulation in the culture, and consequently, improvecell viability and recombinant protein productivity.Materials and methods

The use of filler DNA for improved transfection and reduced DNA needs in transient gene expression with CHO and HEK cells

Background Transient gene expression (TGE) is a rapid method for the production of recombinant proteins. Protein productivity in TGE has improved significantly over the past decade, reaching 300 mg/L and 1 g/L in CHO DG44 (CHO) and HEK 293E (HEK) cells, respectively [1,2]. However, the amount of plasmid DNA needed for transfection remains relatively high, contributing significantly to the overall cost of the TGE process. In order to reduce the amount of plasmid DNA in TGE, we examined the possibility of partially replacing it with herring sperm DNA (non-coding “filler” DNA) in transfections of CHO and HEK cells.

Transgene mRNA Levels and Stability are Key Factors to Enhance Transient Gene Expression in CHO DG44 Cells

The aim of this work was to identify some of the limiting factors in transient gene expression (TGE) in CHO cells and to propose strategies to overcome them. Increasing the amount of plasmid DNA in the transfection did not increase recombinant protein yields, and it had a negative impact on transgene mRNA levels. Therefore, two other strategies aimed at increasing transgene mRNA levels were investigated. The first involved hypothermic treatment of transfected cells and the second the addition of valproic acid (VPA) after transfection. Both strategies resulted in recombinant antibody yields of 40–60 mg/L, whereas the untreated control transfections produced only 5–10 mg/L. 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. The two strategies proposed here are cost-effective and scalable making large-scale TGE in CHO cells a feasible alternative for rapid production of gram amounts of recombinant protein.

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.