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

Abstract

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 β‐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 β‐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. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 567–575, 1998.

Production of Adenoviral Vectors in 293 Cells: A Case Study of the Adaptation of Attached Cells to Grow in Suspension

“…The price comparison was obtained from current prices of a liter of medium for attached and suspension HEK 293 cells (Invitrogen Corp., Carlsbad, CA and Sigma-Aldrich Ltd, UK). These concerns are increased when the final product is to be used as an injectable therapeutic [9].…”

“…This may be due to the fact that infected suspended cells are more fragile than the non-infected cells especially when they are growing in SFM. This correlated with the results reported previously for the 293 cells growing in a different SFM [9]. Similarly to attached cells, the highest adenoviral vector titer was obtained at 48 hpi.…”

“…Likewise, cell culture using commercially available microcarriers has some drawbacks such as limitations in scaling-up, high cost implications, cell damage caused by the collisions between microcarriers and the internals of a stirred bioreactor. In addition, the adenoviral vector yield is not as high as that reported for cells in suspension [3,8,9].…”

Effect of Pluronic F-68, 5% CO, Atmosphere, HEPES, and Antibiotic-Antimycotic on Suspension Adapted 293 Cells

“…In some large-scale protocols for growing 293 cells in suspension the gas is supplied to control the pH during the culture [32][33][34]. In this study, the effect of a 5% CO 2 atmosphere upon the cell density and viability of the suspension adapted 293 cells was evaluated, since previous reports showed the capacity of 293 cells to grow at normal rate in the absence of aeration [21].…”

“…Even though the 293 cells tend to grow in attached form, the line can be adapted to grow in suspension using serum containing medium (SCM) [19] and SFM [20,21]. The objective in this study was to evaluate the influence of different parameters including aeration, surfactant (Pluronic F-68), carbon dioxide (5% CO 2 atmosphere), buffer (HEPES) and a antibiotic-antimycotic on the cell growth of suspension adapted 293 cells.…”