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.

Development of high-titer transient gene expression processes for manufacturing recombinant proteins

Gaurav Backliwal

The market for recombinant therapeutic proteins (including antibodies) is estimated to be greater than $50-billion and has a compounded annual growth rate of around 20%. More than 50% of all approved processes for manufacturing recombinant proteins use mammalian cells as expression hosts due to their ability to carry out complex assembly and processing, human-like glycosylation and secretion of proteins. This percentage is not expected to change and should even increase with time. Classically, these manufacturing processes use stable cell lines for protein expression, wherein the plasmid coding for the protein of interest is stably integrated into the host cell chromosomes (Stable Gene Expression, SGE). An alternative method for expressing recombinant proteins is Transient Gene Expression (TGE). Herein, the expression of the protein of interest takes place from plasmids which are transfected into the cells and are maintained extra-chromosomally. TGE has become a rapid and easy method for obtaining proteins for structural, biochemical or pre-clinical studies, where only small amounts of one or more proteins might be required. As yet, TGE has not been used for manufacturing recombinant proteins for clinical testing or approved clinical use due to the requirement of large amounts of protein of consistent quality. Even though TGE has been scaled-up to the 100-liter scale and is being attempted at the 1000-liter scale, due to low specific productivity, low volumetric yields and the high cost of DNA, TGE processes are not able to economically produce sufficient amounts of proteins for such purposes. Therefore, the goal of this thesis was to improve TGE in order to create high-titer processes, which would be economically more feasible for manufacturing large-amounts of recombinant proteins. This was achieved by: Improving and simplifying gene delivery – by the combination of high cell density at time of transfection and in-situ complex formation (as apposed to a-priori complex formation), we could enhance protein expression levels and gene delivery. This made implementation more robust and easy, with greater choice of media which could be used and cell density which could be maintained. Improving gene expression – by using a combination of inhibitors of histone deacetylases like valproic acid, and growth factors like acidic fibroblast growth factor, we could enhance specific productivity by ∼20-fold. Enhancing the cell densities at which cells could be maintained after transfection – by the combination of cell cycle regulators like human p18 and p21 and treatment with inhibitors of histone deacetylases, we blocked cell growth which allowed cells to be maintained at significantly higher cell densities, allowing us to enhance volumetric productivity by ∼100-fold. Overall these improvements allowed antibody titers in excess of 1 g/l. The gram per liter range of volumetric productivity has previously only been reported for optimized SGE based manufacturing processes. We therefore improved the economic feasibility of TGE for manufacturing recombinant proteins and reduced the difference in manufacturing costs between TGE and SGE, for a given amount of protein, by an estimated 50-fold. With some further development work and study of related issues like protein quality, TGE based processes can be expected to become part of mainstream recombinant protein manufacturing in the future.

EPFL2008

Transient gene expression for rapid protein production

Natalie Muller

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

Development of plasmid-based expression technology for rapid and sustained production of recombinant proteins in insect cells

Xiao Shen

Due to the high demand for heterogeneous protein production in insect cells, we have established efficient methods for plasmid-based transient gene expression (TGE) in various insect cell lines, including commonly used Spodoptera cell lines Sf9 and HighFive™ (H5) and the Diptera cell line Schneider S2 from Drosophila melanogaster, as a high-yield protein production platform. The cells were grown in suspension in orbitally shaken containers at working volumes of 5-300 mL. Rapid cell growth and high cell densities were obtained. Among the various transfection agents tested, polyethylenimine supported high transfection efficiencies in all three cell lines. Intensive optimizations of TGE processes for the three cell lines were executed. For example, various promoter and enhancer elements were compared. Using an optimal TGE process, transfection efficiencies (percentage of transfected cells) of over 85% were obtained for all three cell lines. Yields of the secreted protein tumor necrosis factor receptor-Fc fusion (TNFR-Fc) ranged from 100 – 200 mg/L for the three cell lines using a gene optimized for expression in insect cells. The molecular weight of the Sf9-derived dimeric TNFR-Fc was about 6 kDa less than the equivalent product synthesized by Chinese hamster ovary cells due to differences in glycosylation. In addition, several difficult-to-express proteins, that failed to be delivered at sufficient quantity using other expression systems, were successfully produced in S2 cells by TGE. A human serotonin receptor was expressed at 20-60 mg/L; empty and peptideloaded MHC II tetramer complexes were expressed at up to 150 mg/L; neutrophil serine proteases were expressed at levels over 50 mg/L; and protein tyrosine kinases were expressed at levels over 2 mg/L. These yields were obtained within 5 days of transfection at volumetric scales up to 300 mL. We also developed methods for the generation of stable polyclonal pools of recombinant S2 cells based on plasmid cotransfection in the context of the Piggybac transposon. After two weeks of genetic selection of recombinant cells, the recombinant pools remained stable for at least 100 days in the absence of selection with volumetric TNFR-Fc productivity over 40 mg/L in a 4-day batch culture. Our data highlight the use of plasmid-based expression systems in insect cells to meet the demand for rapid and sustained production of proteins in sufficient quantities for biomedical research and for the biotechnology industry.

EPFL2014