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Transient gene expression: Recombinant protein production with suspension‐adapted HEK293‐EBNA cells
Author(s) -
Meissner Petra,
Pick Horst,
Kulangara Alexandra,
Chatellard Philippe,
Friedrich Kirstin,
Wurm Florian M.
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.1179
Subject(s) - recombinant dna , transfection , green fluorescent protein , hek 293 cells , microbiology and biotechnology , cell culture , chinese hamster ovary cell , expression vector , biology , plasmid , chemistry , gene , biochemistry , genetics
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 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 × 10 6 molecules/cell. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 197–203, 2001.