High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI

Authors

  • Gaurav Backliwal,

    1. Ecole Polytechnique Fédérale de Lausanne, Laboratory of Cellular Biotechnology, Institute of Bioengineering, Faculty of Life Sciences, Lausanne, Switzerland; telephone: 41-21-693-61-41; fax: 41-21-693-61-40
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  • Markus Hildinger,

    1. ExcellGene SA, Monthey, Switzerland
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  • Vivek Hasija,

    1. Ecole Polytechnique Fédérale de Lausanne, Laboratory of Cellular Biotechnology, Institute of Bioengineering, Faculty of Life Sciences, Lausanne, Switzerland; telephone: 41-21-693-61-41; fax: 41-21-693-61-40
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  • Florian M. Wurm

    Corresponding author
    1. Ecole Polytechnique Fédérale de Lausanne, Laboratory of Cellular Biotechnology, Institute of Bioengineering, Faculty of Life Sciences, Lausanne, Switzerland; telephone: 41-21-693-61-41; fax: 41-21-693-61-40
    2. ExcellGene SA, Monthey, Switzerland
    • Ecole Polytechnique Fédérale de Lausanne, Laboratory of Cellular Biotechnology, Institute of Bioengineering, Faculty of Life Sciences, Lausanne, Switzerland; telephone: 41-21-693-61-41; fax: 41-21-693-61-40.
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  • G. Backliwal and M. Hildinger contributed equally to this study.

Abstract

Recombinant proteins are of great commercial and scientific interest. Yet, most production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility; yet, depending on the transfection protocol, transient transfection faces some bottlenecks such as a priori complex formation, limitations in terms of transfection and production media used and the need for medium exchange prior to and/or after transfection. Published protocols for transfection of suspension-adapted HEK-293 cells with polyethyleneimine have shown great promise in overcoming some of these bottlenecks, but still require a priori complex formation for optimal yields and limit the choice of transfection and production media. Here, we report successful in situ transfection of suspension-adapted HEK-293 cells with 25-kDa linear polyethyleneimine at densities up to 20 × 106 cells/mL in complex media followed by production at lower cell densities (1 × 106 cells/mL). After concentrating cells to such high densities, transfection of HEK-293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Furthermore, there is no need to make transfection complexes a priori, a step that prevents inline sterile filtration of the DNA bulk for transfection, an important consideration when scaling processes up to 100 or 1,000 L. Finally, transfecting HEK-293 cells at high density in complex media is superior to existing transfection protocols and doubles yields of recombinant protein obtainable by transient gene expression. Biotechnol. Bioeng. 2008;99: 721–727. © 2007 Wiley Periodicals, Inc.

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