Transient gene expression in suspension HEK-293 cells: application to large-scale protein production

Lucia Baldi Unser, Philippe Girard, Natalie Muller, Florian Maria Wurm
2005
Journal paper

Abstract

Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.

Official source

https://infoscience.epfl.ch/record/105025?ln=en

About this result

This page is automatically generated and may contain information that is not correct, complete, up-to-date, or relevant to your search query. The same applies to every other page on this website. Please make sure to verify the information with EPFL's official sources.

Related concepts

Related publications

Lucia Baldi Unser, Philippe Girard, Natalie Muller, Florian Maria Wurm
2005
Journal paper

Abstract

Official source

https://infoscience.epfl.ch/record/105025?ln=en

About this result

Related concepts (22)

Related concepts

Related publications (95)

Related publications

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

Recombinant proteins are important for biomedical research and for the treatment of human disease. Therefore it is necessary to develop reproducible bioprocesses to rapidly produce proteins of adequate quality and quantity. Expression in mammalian cells is preferred if the proteins are to be properly folded and post-translationally modified. For the rapid production of milligram to gram quantities of a protein in mammalian cells, large-scale transient gene expression is an attractive option. The two most important components of this technology are the cell cultivation system and the gene delivery method. For this reason the goal of this thesis was to develop novel cost-efficient cell cultivation systems and gene delivery methods for the large-scale transfection of mammalian cells in chemically defined media free of any animal-derived components including serum. Cultivation of mammalian cells in suspension is essential for large-scale transient gene expression. A superior system for the cultivation of mammalian cells based on the agitation of cells in "square-shaped" glass bottles (square bottles) was developed. It is an inexpensive but efficient means to grow cells to a high density without instrumentation. The growth and viability of cultures of both human embryo kidney (HEK) 293E and Chinese hamster ovary (CHO) DG44 cells in agitated one-liter square bottles exceeded that in spinner flasks, reaching cell densities 1.5 times higher on average. Furthermore, an efficient and reproducible method to transfect cells in agitated square bottles was developed for both HEK 293E and CHO DG44 cells. A novel gene delivery method termed calfection that relies on the addition of DNA and CaCl2 directly to a suspension culture was developed. Calfection has a number of advantages over existing gene delivery methods such as calcium phosphate-DNA coprecipitation in that there are no time-dependent steps. The DNA/CaCl2 solution can be stored for long periods and is filterable. It is suitable for both large-scale transfections in bioreactors and for high-throughput transfections in microtiter plates. One drawback, however, is its dependence on the presence of serum in the culture medium. A second gene delivery method, serum-free transfection with polyethylenimine (polyfection), proved to be highly efficient for the transfection of both HEK 293E and CHO DG44 cells. A reproducible method for polyfection in microtiter plates, 50 ml centrifuge tubes, agitated square and round bottles, spinner flasks, and bioreactors was optimized for these two cell lines. Polyfection proved to be a simple and successful gene transfer method in both instrumented and non-instrumented cultivation systems. Importantly, it could be performed in serum-free chemically defined media. With HEK 293E cells, 80 mg/l of antibody was produced in agitated square bottles and with CHO DG44 cells, more than 2 g of antibody were produced in one week in a 150-liter bioreactor.

EPFL2005

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.

EPFL2013