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Nucleic Acids Research Advance Access originally published online on July 10, 2008
Nucleic Acids Research 2008 36(15):e96; doi:10.1093/nar/gkn423
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Nucleic Acids Research, 2008, Vol. 36, No. 15 e96
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Gaurav Backliwal1, Markus Hildinger2, Sebastien Chenuet1, Sarah Wulhfard1, Maria De Jesus2 and Florian M. Wurm1,2,*

1Ecole Polytechnique Fédérale de Lausanne, Laboratory of Cellular Biotechnology, Institute of Bioengineering, Faculty of Life Sciences, Lausanne, Switzerland and 2ExcellGene SA, Monthey, Switzerland

*To whom correspondence should be addressed. Tel: +41 (0) 21 693 61 41; Fax: +41 (0) 21 693 61 40; Email: florian.wurm{at}epfl.ch

Received December 30, 2007. Revised June 17, 2008. Accepted June 18, 2008.

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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