Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells

✍ Scribed by Johanne Côté; Alain Garnier; Bernard Massie; Amine Kamen

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 132 KB
• Volume: 59
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(19980905)59:5<567::aid-bit6>3.0.co;2-8

✦ Synopsis

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 ␤-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 serumfree 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 ␤-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.