Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells.
This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.
Côté
J
J
Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montréal, Québec, Canada, H4P 2R2.
Garnier
A
A
Massie
B
B
Kamen
A
A
D000256:Adenoviridae / Q000235:genetics*; D019149:Bioreactors; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D066246:ErbB Receptors; D001616:beta-Galactosidase; D005947:Glucose