title: 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.
abstract: 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.
authors.affiliation: Ecole Polytechnique Fédérale de Lausanne, Laboratory of Cellular Biotechnology, Institute of Bioengineering, Faculty of Life Sciences, Lausanne, Switzerland.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Backliwal
authors.forename: Markus
authors.initials: M
authors.lastname: Hildinger
authors.forename: Sebastien
authors.initials: S
authors.lastname: Chenuet
authors.forename: Sarah
authors.initials: S
authors.lastname: Wulhfard
authors.forename: Maria
authors.initials: M
authors.lastname: De Jesus
authors.forename: Florian M
authors.initials: FM
authors.lastname: Wurm
mesh_terms: D000906:Antibodies / Q000235:genetics; D000917:Antibody Formation / Q000235:genetics*; D002460:Cell Line; D016895:Culture Media, Serum-Free; D005822:Genetic Vectors; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection / Q000379:methods*; D019076:Transgenes
chemical_list: D000906:Antibodies; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins