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High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
Published on: December 28, 2015
High-density transient gene expression in suspension-adapted 293 EBNA1 cells
Xiangming Sun1, Hui Ching Hia, Peen Ern Goh
1Bioprocessing Technology Institute, 20 Biopolis Way, Centros 138668, Singapore. sun_xiang_ming@bti.a-star.edu.sg
Biotechnology and Bioengineering
|July 17, 2007
Summary
Transient gene expression (TGE) in mammalian cells was optimized for high cell density, significantly increasing recombinant protein yields. This advancement enables scalable protein production for pre-clinical research using improved transfection methods.
Area of Science:
- Biotechnology
- Mammalian cell culture
- Protein expression
Background:
- Large-scale transient gene expression (TGE) in mammalian cells is crucial for rapid recombinant protein production for pre-clinical studies.
- Current TGE methods yield low protein concentrations (approx. 20 mg/L) due to low cell densities (approx. 5 x 10^5 cells/mL).
Purpose of the Study:
- To enhance TGE yields by developing a high-density cell culture and transfection strategy.
- To establish an economical plasmid preparation protocol for TGE.
Main Methods:
- Established high-density perfusion culture of 293 EBNA1 cells in a 2-L bioreactor.
- Optimized transfection at 1 x 10^7 cells/mL using polyethylenimine (PEI) with specific DNA/PEI ratios and amounts.
- Developed an anion-exchange based plasmid preparation method.
Main Results:
- Achieved 80.8 mg/L of erythropoietin (EPO) in a fed-batch mode post-transfection.
- Produced 227 mg of EPO in 10.7 L using perfusion culture with enriched medium, demonstrating scalable production.
- Successfully performed transient transfections at cell densities up to 1 x 10^7 cells/mL.
Conclusions:
- High-density TGE significantly improves recombinant protein production yields in mammalian cell cultures.
- The developed methods enable the production of hundreds of milligrams of protein in a 2 L bioreactor.
- This study presents the first report of high-density TGE at 1 x 10^7 cells/mL.
