GTC Biotherapeutics (fka GTCB)

[DewDiligence]

Apropos to this thread…

http://tinyurl.com/23ucjl

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Transient Transfection Factors for Recombinant Protein Production in Suspension Cultured Mammalian Cells

Mol Biotechnol. 2008 Mar 8 [Epub ahead of print]

Liu C, Dalby B, Chen W, Kilzer JM, Chiou HC.

Research and Development, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, 92008, USA, [e-mail].

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX™ to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.
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