Abstract
This is the first report of two successful 100 l scale transienttransfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression(LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one encoding for the heavychain of a human recombinant immunoglobulin, the other for the corresponding light chain and a third one for the green fluorescent protein (GFP, 2–4% of DNA in transfection cocktail)were co-transfected. The GFP vector was chosen to monitor transfection efficiency. Expression of GFP could be registered asearly as 20 h after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the100 l scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. Akey advantage of LS-TGE resides in its speed. In the presentedcases, the entire production process for the synthesis of halfa gram of a recombinant antibody, including DNA preparationand necessary expansion of cells prior to transfection, wasexecuted in less than a month. Having an established transfection/expression process allows to run productioncampaigns for any given protein, within one facility, with onesingle host cell line and therefore only one single seed train. Without any need to create and maintain stable cell lines, expression of new r-proteins is not only faster and more economical but also more flexible.
Keywords: bioreactor, calcium phosphate co-precipitation, GFP, HEK 293, human antibody, large-scale gene expression, transient transfection
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References
- Aunins J (2001) Optimizing the Process of Process Transfer into Manufacturing Facilities. Presented at: The 17th ESACT Meeting From Target to Market Tylösand, Sweden.
- Girard P. Rapid Protein Expression in Mammalian Cells: Large-Scale Transient Transfection. Lausanne: Swiss Federal Institute of Technology Lausanne; 2001. [Google Scholar]
- Gorman CM, Howard BH, Reeves R. Expression of recombinant plasmids in mammalian-cells is enhanced by sodium-butyrate. Nucleic Acids Res. 1983;11:7631–7648. doi: 10.1093/nar/11.21.7631. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Graham FL, vd Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973;52:456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
- Jordan M. Transient Gene Expression in Mammalian Cells Based on the Calcium Phosphate Transfection Method. In: Al-Rubeai M, editor. Cell Engineering: Transient Transfection. Dordrecht: Kluwer Academic Publishers; 2000. pp. 56–77. [Google Scholar]
- Jordan M, Fraboulet D, Fourmestraux G, Wurm FM, Freitag R. Sandwich Elisa Today: Limits for Sensitivity, Speed, Precision and Throughput. In: Bernard A, Griffiths B, Noé W, Wurm F, editors. Animal Cell Technology: Products from Cells, Cells as Products. Dordrecht: Kluwer Academic Publishers; 1999. pp. 483–485. [Google Scholar]
- Jordan M, Köhne C, Köhne FMW. Calcium-phosphate mediated DNA transfer into Hek-293 cells in suspension: Control of physicochemical parameters allows transfection in stirred media transfection and protein in expression in mammalian cells. Cytotechnology. 1998;26:39–47. doi: 10.1023/A:1007917318181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jordan M, Schallhorn A, Wurm FM. Transfecting mammalian cells: Optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 1996;24:596–601. doi: 10.1093/nar/24.4.596. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kruh J. Effects of sodium butyrate, a new pharmacological agent, on cells in culture. Mol Cell Biochem. 1982;42:65–82. doi: 10.1007/BF00222695. [DOI] [PubMed] [Google Scholar]
- Meissner P, Girard P, Kulangara A, Tsao MC, Jordan M, Wurm FM. Process developement for transient gene expression in mammalian cells at the 3 l scale: 10–50 Mg of R-protein in days. In: Bernard A., Griffiths B., Noé W., Wurm F., editors. Animal Cell Technology: Products from Cells, Cells as Products. Dordrecht: Kluwer Academic Publishers; 1999. pp. 351–357. [Google Scholar]
- Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FM. Transient gene expression: Recombinant protein production with suspension-adapted Hek293-Ebna cells. Biotechnol Bioeng. 2001;75:197–203. doi: 10.1002/bit.1179. [DOI] [PubMed] [Google Scholar]
- Miescher S, Zahn-Zabal M, De Jesus M, Moudry R, Fisch I, Vogel M, Kobr M, Imboden MA, Kragten E, Bichler J, et al. CHO expression of a novel human recombinant IgG1 anti-Rhd antibody isolated by phage display. Br J Haematol. 2000;111:157–166. doi: 10.1046/j.1365-2141.2000.02322.x. [DOI] [PubMed] [Google Scholar]
- Parham JH, Iannone MA, Overton LK, Hutchins JT. Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation. Cytotechnology. 1998;28:147–155. doi: 10.1023/A:1008046101653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peshwa MV, Kyung Y-S, McClure DB, Hu W-S. Cultivation of mammalian cells as aggregates in bioreactors: Effect of calcium concentration on spatial distribution of viability. Biotechnol Bioeng. 1993;41:179–187. doi: 10.1002/bit.260410203. [DOI] [PubMed] [Google Scholar]
- Yates JL, Warren N, Sugden B. Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature. 1985;313:812–815. doi: 10.1038/313812a0. [DOI] [PubMed] [Google Scholar]