A simple high-yielding process for transient gene expression in CHO cells.

Here we describe a simplified method for transient gene expression (TGE) in suspension-adapted Chinese hamster ovary (CHO) cells using polyethylenimine (PEI) for DNA delivery. Both the transfection and production phases of the bioprocess were performed at a density of 4 × 10⁶ cells/mL at 31 °C. In addition, the amounts of both PEI and plasmid DNA were reduced up to 50% on a per cell basis compared to previously published protocols from this laboratory, resulting in higher cell viability after transfection and higher volumetric recombinant protein yields. In batch cultures of up to 14 days, reproducible recombinant antibody yields up to 300 mg/L were achieved at small scale (5 mL) and up to 250 mg/L at large scale (500 mL). The simplicity and improved yields are expected to increase the utility of CHO cells for the rapid production of recombinant proteins at larger scales by TGE.

[1]  S. Geisse.  Reflections on more than 10 years of TGE approaches. , 2009, Protein expression and purification.

[2]  F. Wurm,et al.  Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions , 2008, Nucleic acids research.

[3]  F. Wurm,et al.  Novel Orbital Shake Bioreactors for Transient Production of CHO Derived IgGs , 2007, Biotechnology progress.

[4]  C. Fux,et al.  Recombinant protein production by transient gene transfer into Mammalian cells. , 2009, Methods in enzymology.

[5]  F. Wurm,et al.  High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. , 2008, Biotechnology and bioengineering.

[6]  F. Wurm.  Production of recombinant protein therapeutics in cultivated mammalian cells , 2004, Nature Biotechnology.

[7]  Amine Kamen,et al.  Large-Scale transfection of mammalian cells for the fast production of recombinant protein , 2006, Molecular biotechnology.

[8]  Y. Durocher,et al.  Development of a scalable process for high‐yield lentiviral vector production by transient transfection of HEK293 suspension cultures , 2009, The journal of gene medicine.

[9]  F. Wurm,et al.  Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. , 2008, Biotechnology and bioengineering.

[10]  D. James,et al.  Control of Culture Environment for Improved Polyethylenimine‐Mediated Transient Production of Recombinant Monoclonal Antibodies by CHO Cells , 2006, Biotechnology progress.

[11]  F. Wurm,et al.  Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells , 2008, Biotechnology progress.

[12]  P. Chatellard,et al.  Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. , 2001, Biotechnology and bioengineering.

[13]  F. Wurm,et al.  Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection , 2006, Biotechnology Letters.

[14]  Lucia Baldi,et al.  Valproic acid enhances recombinant mRNA and protein levels in transiently transfected Chinese hamster ovary cells. , 2010, Journal of biotechnology.

[15]  Natalie Muller,et al.  Orbital shaker technology for the cultivation of mammalian cells in suspension. , 2005, Biotechnology and bioengineering.

[16]  Florian M. Wurm,et al.  Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives , 2007, Biotechnology Letters.

[17]  L. Chasin,et al.  Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[18]  Jianxin Ye,et al.  High‐level protein expression in scalable CHO transient transfection , 2009, Biotechnology and bioengineering.