Transient gene expression: Recombinant protein production with suspension-adapted HEK293-EBNA cells

Transient 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 IgG1‐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 × 106 molecules/cell. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 197–203, 2001.