Meissner, P.Pick, H.Kulangara, A.Chatellard, P.Friedrich, K.Wurm, F. M.2007-06-052007-06-052007-06-05200110.1002/bit.1179https://infoscience.epfl.ch/handle/20.500.14299/7617WOS:000171118200007156611536142Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.BioreactorsBlottingWesternCell LineCulture MediaDNA-Binding ProteinsElectrophoresisPolyacrylamide GelEpstein-Barr Virus Nuclear Antigens/geneticsEscherichia coli/geneticsGene ExpressionGenesReporterGenetic MarkersGenetic VectorsGreen Fluorescent ProteinsHumansImmunoglobulin G/geneticsLuminescent ProteinsMicroscopyConfocalPlasmidsReceptorsSerotonin/geneticsReceptorsSerotonin5-HT3Recombinant Proteins/*biosynthesis/geneticsTime FactorsTransfection/*methodsReceptorstext::journal::journal article::research article