Cytotechnology

, Volume 38, Issue 1–3, pp 15–21 | Cite as

100-liter transient transfection

  • Philippe Girard
  • Madiha Derouazi
  • Gwendoline Baumgartner
  • Michaela Bourgeois
  • Martin Jordan
  • Barbara Jacko
  • Florian M. Wurm
Article

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.

bioreactor calcium phosphate co-precipitation GFP HEK 293 human antibody large-scale gene expression transient transfection 

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Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Philippe Girard
    • 1
  • Madiha Derouazi
    • 1
  • Gwendoline Baumgartner
    • 1
  • Michaela Bourgeois
    • 1
  • Martin Jordan
    • 1
  • Barbara Jacko
    • 2
  • Florian M. Wurm
    • 1
  1. 1.LBTC, Center of Biotechnology, EPFLLausanneSwitzerland
  2. 2.BioWhittaker Inc.WalkersvilleUSA

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