Serum-free large-scale transient transfection of CHO cells.
To date, methods for large-scale transient gene expression (TGE) in cultivated mammalian cells have focused on two transfection vehicles: polyethylenimine (PEI) and calcium phosphate (CaPi). Both have been shown to result in high transfection efficiencies at scales beyond 10 L. Unfortunately, both approaches yield higher levels of recombinant protein (r-protein) in the presence of serum than in its absence. Since serum is a major cost factor and an obstacle to protein purification, our goal was to develop a large-scale TGE process for Chinese hamster ovary (CHO) cells in the absence of serum. CHO-DG44 cells were cultivated and transfected in a chemically defined medium using linear 25 kDa PEI as a transfection vehicle. Parameters that were optimized included the DNA amount, the DNA-to-PEI ratio, the timing and solution conditions for complex formation, the transfection medium, and the cell density at the time of transfection. The highest levels of r-protein expression were observed when cultures at a density of 2.0 x 10(6) cells/ml were transfected with 2.5 microg/ml DNA in RPMI 1640 medium containing 25 mM HEPES at pH 7.1. The transfection complex was formed at a DNA:PEI ratio of 1:2 (w/w) in 150 mM NaCl with a 10-min incubation at room temperature prior to addition to the culture. The procedure was scaled up for a 20-L bioreactor, yielding expression levels of 10
Laboratory of Cellular Biotechnology, IGBB, Faculty of Life Science, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland.
Madiha
M
Derouazi
Philippe
P
Girard
Frédéric
F
Van Tilborgh
Keyvan
K
Iglesias
Natalie
N
Muller
Martin
M
Bertschinger
Florian M
FM
Wurm
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free / Q000378:metabolism*; D004247:DNA / Q000008:administration & dosage; D016503:Drug Delivery Systems / Q000379:methods*; D014677:Pharmaceutical Vehicles / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection / Q000379:methods*
D016895:Culture Media, Serum-Free; D014677:Pharmaceutical Vehicles; D011994:Recombinant Proteins; D011094:Polyethyleneimine; D004247:DNA