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Large-Scale Transfection of Mammalian Cells

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Protein Expression in Mammalian Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 801))


The large-scale transfection of mammalian cells allows moderate (milligram to gram) amounts of recombinant proteins (r-proteins) to be obtained for fundamental or clinical research. In this article, we describe a one-liter transfection using polyethyleneimine (PEI) for DNA delivery into human embryonic kidney (HEK-293) cells cultivated in serum-free suspension to produce a recombinant human monoclonal antibody that yields up to about 1 g/L in a 10-day process. The method is based on a DNA delivery step performed at high cell density (20×106 cells/mL) by direct addition of DNA and PEI to the culture. Subsequently, the cells are diluted 20-fold for the 10-day production phase in the presence of valproic acid (VPA), a histone deacetylase inhibitor. The methods for plasmid purification, antibody quantification by enzyme-linked immunosorbent assay (ELISA), and affinity purification with protein A are also described.

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  1. Wurm, F. M. (2004) Production of recombinant protein therapeutics in cultivated mammalian cells, Nat Biotechnol 22, 1393–1398.

    Google Scholar 

  2. Pham, P. L., Kamen, A., and Durocher, Y. (2006) Large-scale transfection of mammalian cells for the fast production of recombinant protein, Mol Biotechnol 34, 225–237.

    Google Scholar 

  3. Baldi, L., Hacker, D. L., Adam, M., and Wurm, F. M. (2007) Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives, Biotechnol Lett 29, 677–684.

    Google Scholar 

  4. Baldi, L., Muller, N., Picasso, S., Jacquet, R., Girard, P., Thanh, H. P., Derow, E., and Wurm, F. M. (2005) Transient gene expression in suspension HEK-293 cells: application to large-scale protein production, Biotechnol Prog 21, 148–153.

    Google Scholar 

  5. Backliwal, G., Hildinger, M., Hasija, V., and Wurm, F. M. (2008) High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI, Biotechnol Bioeng 99, 721–727.

    Google Scholar 

  6. Backliwal, G., Hildinger, M., Kuettel, I., Delegrange, F., Hacker, D. L., and Wurm, F. M. (2008) Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures, Biotechnol Bioeng 101, 182–189.

    Google Scholar 

  7. Gottlicher, M., Minucci, S., Zhu, P., Kramer, O. H., Schimpf, A., Giavara, S., Sleeman, J. P., Lo Coco, F., Nervi, C., Pelicci, P. G., and Heinzel, T. (2001) Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells, EMBO J 20, 6969–6978.

    Google Scholar 

  8. Muller, N., Girard, P., Hacker, D. L., Jordan, M., and Wurm, F. M. (2005) Orbital shaker technology for the cultivation of mammalian cells in suspension, Biotechnol Bioeng 89, 400–406.

    Google Scholar 

  9. Stettler, M. (2007) Bioreactor processes based on disposable materials for the production of recombinant proteins from mammalian cells, EPFL Thesis no. 3947, Lausanne, Switzerland,

  10. Backliwal, G., Hildinger, M., Chenuet, S., Wulhfard, S., De Jesus, M., and Wurm, F. M. (2008) 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, Nucleic Acids Res 36, e96.

    Google Scholar 

  11. Pick, H. M., Meissner, P., Preuss, A. K., Tromba, P., Vogel, H., and Wurm, F. M. (2002) Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D IgG1 synthesis, Biotechnol Bioeng 79, 595–601.

    Google Scholar 

  12. Meissner, P., Pick, H., Kulangara, A., Chatellard, P., Friedrich, K., and Wurm, F. M. (2001) Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells, Biotechnol Bioeng 75, 197–203.

    Google Scholar 

  13. Zhang, G., Gurtu, V., and Kain, S. R. (1996) An enhanced green fluorescent protein allows sensitive detection of gene transfer in mammalian cells, Biochem Biophys Res Commun 227, 707–711.

    Google Scholar 

  14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2 ed., Cold Spring Harbor Laboratory.

    Google Scholar 

  15. Eastman, E. M., and Durland, R. H. (1998) Manufacturing and quality control of plasmid-based gene expression systems, Adv Drug Deliv Rev 30, 33–48.

    Google Scholar 

  16. Pham, P. L., Perret, S., Cass, B., Carpentier, E., St-Laurent, G., Bisson, L., Kamen, A., and Durocher, Y. (2005) Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis, Biotechnol Bioeng 90, 332–344.

    Google Scholar 

  17. Tuvesson, O., Uhe, C., Rozkov, A., and Lullau, E. (2008) Development of a generic transient transfection process at 100 L scale, Cytotechnology 56, 123–136.

    Google Scholar 

  18. Schlaeger, E. J., and Christensen, K. (1999) Transient gene expression in mammalian cells grown in serum-free suspension culture, Cytotechnology 30, 71–83.

    Google Scholar 

  19. Schlatter, S., Stansfield, S. H., Dinnis, D. M., Racher, A. J., Birch, J. R., and James, D. C. (2005) On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells, Biotechnol Prog 21, 122–133.

    Google Scholar 

  20. Bentley, K. J., Gewert, R., and Harris, W. J. (1998) Differential efficiency of expression of humanized antibodies in transient transfected mammalian cells, Hybridoma 17, 559–567.

    Google Scholar 

  21. Bertschinger, M., Schertenleib, A., Cevey, J., Hacker, D. L., and Wurm, F. M. (2008) The kinetics of polyethyleneimine-mediated transfection in suspension cultures of Chinese hamster ovary cells, Mol Biotechnol 40, 136–143.

    Google Scholar 

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Correspondence to Lucia Baldi .

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Baldi, L., Hacker, D.L., Meerschman, C., Wurm, F.M. (2012). Large-Scale Transfection of Mammalian Cells. In: Hartley, J. (eds) Protein Expression in Mammalian Cells. Methods in Molecular Biology, vol 801. Humana Press.

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-351-6

  • Online ISBN: 978-1-61779-352-3

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