Serum-free large-scale transient transfection of CHO cells

Authors

  • Madiha Derouazi,

    1. Laboratory of Cellular Biotechnology, IGBB, Faculty of Life Science, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland; fax: 41 21 693 61 40
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  • Philippe Girard,

    1. Laboratory of Cellular Biotechnology, IGBB, Faculty of Life Science, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland; fax: 41 21 693 61 40
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  • Frédéric Van Tilborgh,

    1. Laboratory of Cellular Biotechnology, IGBB, Faculty of Life Science, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland; fax: 41 21 693 61 40
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  • Keyvan Iglesias,

    1. Laboratory of Cellular Biotechnology, IGBB, Faculty of Life Science, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland; fax: 41 21 693 61 40
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  • Natalie Muller,

    1. Laboratory of Cellular Biotechnology, IGBB, Faculty of Life Science, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland; fax: 41 21 693 61 40
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  • Martin Bertschinger,

    1. Laboratory of Cellular Biotechnology, IGBB, Faculty of Life Science, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland; fax: 41 21 693 61 40
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  • Florian M. Wurm

    1. Laboratory of Cellular Biotechnology, IGBB, Faculty of Life Science, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland; fax: 41 21 693 61 40
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Abstract

To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a large-scale TGE process for Chinese hamster ovary (CHO) cells in the absence of serum. CHO-DG44 cells were cultivated and transfected in a chemically defined medium using linear 25 kDa PEI as a transfection vehicle. Parameters that were optimized included the DNA amount, the DNA-to-PEI ratio, the timing and solution conditions for complex formation, the transfection medium, and the cell density at the time of transfection. The highest levels of r-protein expression were observed when cultures at a density of 2.0 × 106 cells/ml were transfected with 2.5 μg/ml DNA in RPMI 1640 medium containing 25 mM HEPES at pH 7.1. The transfection complex was formed at a DNA:PEI ratio of 1:2 (w/w) in 150 mM NaCl with a 10-min incubation at room temperature prior to addition to the culture. The procedure was scaled up for a 20-L bioreactor, yielding expression levels of 10 mg/l for an intracellular protein and 8 mg/l for a secreted antibody. © 2004 Wiley Periodicals, Inc.

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