SERUM-FREE PRODUCTION OF RECOMBINANT PROTEINS AND ADENOVIRAL VECTORS BY 293SF-3F6 CELLS

This article describes the step-wise approach undertaken to select a s erum-free medium (SFM) for the efficient production of a recombinant a denoviral vectors expressing p-galactosidase (Ad5 CMV-LacZ), in the co mplementing human embryonic kidney 293S cells. In the first step, a 29 3S-derived transfectoma, secreting a soluble epidermal growth factor r eceptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant pro tein as compared to serum-containing medium. Assays showed that only o ne among six commercial serum-free formulations could support both sEG Fr production and cell growth in static or suspension culture. In comm ercially available calcium-containing serum-free formulations, the cel l 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 a bility to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregate s. The 293SF-3F6 clone, first adapted to and then cloned in the select ed 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 tes ted. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreac tor maintained the specific productivities of both p-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspensio n 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 req uired for gene therapy studies. (C) 1998 John Wiley & Sons, Inc. Biote chnol Bioeng 59: 567-575, 1998.