Abstract
Transient gene expression in mammalian cells at scales of 1–100 L may become a powerful tool of producing large quantities of proteins in a very short period of time. In this study, we continued to optimize process parameters for transient transfection of HEK-293 cells with the calcium-phosphate method cultivated in a 3 Liter bioreactor. Two different HEK-293 cell lines (expressing EBNA or T-Antigen) were adapted to suspension culture using a newly developed medium, 293G (BioWhittaker). Process parameters, during and after transfection, were optimized using a vector that expresses green fluorescent protein (GFP) as a reporter. Cells were expanded in standard spinner culture. During the exponential growth phase, cells were recovered by centrifugation, resuspended in DMEM-F12 medium and transferred into a bioreactor at an initial cell density of 3 × 105 cells/ml. The transfection cocktail with 0.5–1.0 mg of supercoiled plasmid DNA was transferred into the reactor by syringe injection, 2 hours after inoculation. The setpoint for pH, initially at pH 7.4, was shifted to pH 7.1 4–6 hours post-transfection. With transfection efficiencies ranging between 70 and 90% — as established with GFP vectors — anti-human RhD IgGI antibody was secreted at concentrations of up to 20 mg/L within a time frame of less than one week.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
Similar content being viewed by others
References
Blasey, H.D., Aubry, J.-P., Mazzei, G.J. and Bernard, A.R. (1996) Large scale transient expression with COS cells, Cytotechnology 18, 138–192.
Jordan, M., Schallhorn, A. and Wurm, F.M. (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation, Nucleic Acid Res 24, 596–601.
Jordan, M., Köhne, C. and Wurm, F.M. (1998) Calcium-phosphate mediated UNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media, Cytotechnology 26, 39–47.
Verma, R., Boleti, E. and George, A.J.T. (1998) Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems, J Immunol Methods 216, 165–181.
Wurm, F.M. and Bernard, A. (1999) Large-scale transient expression in mammalian cells for recombinant protein production, Curr Opin Biotechnol 10, 156–159.
Author information
Authors and Affiliations
Editor information
Rights and permissions
Copyright information
© 1999 Kluwer Academic Publishers
About this chapter
Cite this chapter
Meissner, P., Girard, P., Kulangara, A., Tsao, M., Jordan, M., Wurm, F. (1999). Process Development for Transient Gene Expression in Mammalian Cells at The 3 L Scale: 10–50 MG of R-Protein in Days. In: Bernard, A., Griffiths, B., Noé, W., Wurm, F. (eds) Animal Cell Technology: Products from Cells, Cells as Products. Springer, Dordrecht. https://doi.org/10.1007/0-306-46875-1_76
Download citation
DOI: https://doi.org/10.1007/0-306-46875-1_76
Publisher Name: Springer, Dordrecht
Print ISBN: 978-0-7923-6075-9
Online ISBN: 978-0-306-46875-9
eBook Packages: Springer Book Archive