Open AccessRational 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
Author(s) -
Gaurav Backliwal,
Markus Hildinger,
Sébastien Chenuet,
Sarah Wulhfard,
María de Jesús,
Florian M. Wurm
Publication year - 2008
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkn423
Subject(s) - transfection , biology , hek 293 cells , cell culture , recombinant dna , titer , chinese hamster ovary cell , microbiology and biotechnology , antibody , gene , biochemistry , immunology , genetics
Transient transfection allows for fast production of recombinant proteins. However, the current bottle- necks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfec- tion protocol that yields titers exceeding 1g/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 2l, thus delivering `1g 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.