Process Development for Transient Gene Expression in Mammalian Cells at The 3 L Scale: 10–50 MG of R-Protein in Days

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.