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

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Abstract

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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Acknowledgments

We thank Dr. Antje Taliana, Dr. Yu Feng, and Marieke Svoboda of Invitrogen Co. for kindly providing EPO, Factor IX clones, vectors, and technical assistance.

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Authors and Affiliations

  1. Research and Development, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, 92008, USA

    Chaoting Liu, Brian Dalby, Weixing Chen, Jennifer M. Kilzer & Henry C. Chiou

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  1. Chaoting Liu
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Correspondence to Chaoting Liu or Henry C. Chiou.

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Liu, C., Dalby, B., Chen, W. et al. Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells. Mol Biotechnol 39, 141–153 (2008). https://doi.org/10.1007/s12033-008-9051-x

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