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Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

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Abstract

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2–10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time––weeks. With “classical” approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.

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References

  • Baldi L, Muller N, Picasso S et al (2005) Transient gene expression in suspension HEK-293 cells: application to large-scale protein production. Biotechnol Prog 21(1):148–153

    Article  PubMed  CAS  Google Scholar 

  • Barnes D, Sato G (1980) Serum-free cell culture: a unifying approach. Cell 22(3):649–655

    Article  PubMed  CAS  Google Scholar 

  • Batard P, Jordan M, Wurm F (2001) Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection. Gene 270(1–2):61–68

    Article  PubMed  CAS  Google Scholar 

  • Bentley KJ, Gewert R, Harris WJ (1998) Differential efficiency of expression of humanized antibodies in transient transfected mammalian cells. Hybridoma 17(6):559–567

    Article  PubMed  CAS  Google Scholar 

  • Blasey HD, Brethon B, Hovius R et al (2000) Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System. Cytotechnology 32(3):199–208

    Article  CAS  PubMed  Google Scholar 

  • Boussif O, Lezoualc’h F, Zanta MA et al (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 92(16):7297–7301

    Article  PubMed  CAS  Google Scholar 

  • Boyce FM, Bucher NL (1996) Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci USA 93(6):2348–2352

    Article  PubMed  CAS  Google Scholar 

  • Buchman AR, Berg P (1988) Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol 8(10):4395–4405

    PubMed  CAS  Google Scholar 

  • Cho MS, Yee H et al (2002) Establishment of a human somatic hybrid cell line for recombinant protein production. J Biomed Sci 9(6 Pt 2):631–638

    Article  PubMed  CAS  Google Scholar 

  • Cho MS, Yee H, Brown C et al (2003) Versatile expression system for rapid and stable production of recombinant proteins. Biotechnol Prog 19(1):229–232

    Article  PubMed  CAS  Google Scholar 

  • Côté J, Garnier A, Massie B et al (1998) Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells. Biotechnol Bioeng 59(5):567–575

    Article  PubMed  Google Scholar 

  • Dang JM, Leong KW (2006) Natural polymers for gene delivery and tissue engineering. Adv Drug Deliv Rev 58(4):487–499

    Article  PubMed  CAS  Google Scholar 

  • Davies A, Greene A, Lullau E et al (2005) Optimisation and evaluation of a high-throughput mammalian protein expression system. Protein Expr Purif 42(1):111–121

    Article  PubMed  CAS  Google Scholar 

  • Derouazi M, Girard P, Van Tilborgh F et al (2004) Serum-free large-scale transient transfection of CHO cells. Biotechnol Bioeng 87(4):537–545

    Article  PubMed  CAS  Google Scholar 

  • Durocher Y, Perret S, Kamen A (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30(2):E9

    Article  PubMed  Google Scholar 

  • Edwards RH, Selby MJ, Garcia PD et al (1988) Processing of the native nerve growth factor precursor to form biologically active nerve growth factor. J Biol Chem 263(14):6810–6815

    PubMed  CAS  Google Scholar 

  • Falkner FG, Turecek PL, MacGillivray RT et al (1992) High level expression of active human prothrombin in a vaccinia virus expression system. Thromb Haemost 68(2):119–124

    PubMed  CAS  Google Scholar 

  • Fussenegger M, Bailey JE (1998) Molecular regulation of cell-cycle progression and apoptosis in mammalian cells: implications for biotechnology. Biotechnol Prog 14(6):807–833

    Article  PubMed  CAS  Google Scholar 

  • Galbraith DJ, Tait AS, Racher AJ et al (2006) Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells. Biotechnol Prog 22(3):753–762

    Article  PubMed  CAS  Google Scholar 

  • Garnier A, Côté J, Nadeau I et al (1994) Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells. Cytotechnology 15(1–3):145–155

    Article  PubMed  CAS  Google Scholar 

  • Geisse S, Henke M (2005) Large-scale transient transfection of mammalian cells: a newly emerging attractive option for recombinant protein production. J Struct Funct Genomics 6(2–3):165–170

    Article  PubMed  CAS  Google Scholar 

  • Gill DR, Smyth SE, Goddard CA et al (2001) Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Ther 8(20):1539–1546

    Article  PubMed  CAS  Google Scholar 

  • Girard P, Derouazi M, Baumgartner G et al (2002) 100-liter transient transfection. Cytotechnology 38:15–21

    Article  CAS  PubMed  Google Scholar 

  • Graham FL, Smiley J, Russell WC et al (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36(1):59–74

    Article  PubMed  CAS  Google Scholar 

  • Graham FL (1987) Growth of 293 cells in suspension culture. J Gen Virol 68(Pt 3):937–940

    PubMed  Google Scholar 

  • Hacker DL, Bertschinger M, Baldi L et al (2004) Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells. Gene 341:227–234

    Article  PubMed  CAS  Google Scholar 

  • Hassaine G, Wagner R, Kempf J et al (2006) Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells. Protein Expr Purif 45(2):343–351

    Article  PubMed  CAS  Google Scholar 

  • Hofmann C, Sandig V, Jennings G et al (1995) Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci USA 92(22):10099–10103

    Article  PubMed  CAS  Google Scholar 

  • Jarvis DL (2003) Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology 310(1):1–7

    Article  PubMed  CAS  Google Scholar 

  • Jordan M, Schallhorn A, Wurm FM (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4):596–601

    Article  PubMed  CAS  Google Scholar 

  • Kim NS, Lee GM (2002) Response of recombinant Chinese hamster ovary cells to hyperosmotic pressure: effect of Bcl-2 overexpression. J Biotechnol 95(3):237–248

    Article  PubMed  CAS  Google Scholar 

  • Kost TA, Klein JL, Condreay JP (2000) Application of Recombinant Baculoviruses in Biopharmaceutical Research. In: Al-Rubeai M, Fussenegger M (eds) Cell engineering, Kluwer Academic Publishers

  • Lechardeur D, Verkman AS, Lukacs GL (2005) Intracellular routing of plasmid DNA during non-viral gene transfer. Adv Drug Deliv Rev 57(5):755–767

    Article  PubMed  CAS  Google Scholar 

  • Lee CC, MacKay JA, Frechet JM et al (2005) Designing dendrimers for biological applications. Nat Biotechnol 23(12):1517–1526

    Article  PubMed  CAS  Google Scholar 

  • Li LH, Shivakumar R, Feller S et al (2002) Highly efficient, large volume flow electroporation. Technol Cancer Res Treat 1(5):341–350

    PubMed  CAS  Google Scholar 

  • Lindell J, Girard P, Muller N et al (2004) Calfection: a novel gene transfer method for suspension cells. Biochim Biophys Acta 1676(2):155–161

    PubMed  CAS  Google Scholar 

  • Makrides SC (1999) Components of vectors for gene transfer and expression in mammalian cells. Protein Expr Purif 17(2):183–202

    Article  PubMed  CAS  Google Scholar 

  • Mastrangelo AJ, Hardwick JM, Zou S et al (2000) Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults. Biotechnol Bioeng 67(5):555–564

    Article  PubMed  CAS  Google Scholar 

  • Meissner P, Pick H, Kulangara A et al (2001) Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol Bioeng 75(2):197–203

    Article  PubMed  CAS  Google Scholar 

  • Muller N (2005) Transient gene expression for rapid protein production: studies & optimizations under serum-free conditions. Dissertation, Ècole Polytechnique Fédérale de Lausanne

  • Pavirani A, Meulien P, Harrer H et al (1987) Two independent domains of factor VIII co-expressed using recombinant vaccinia viruses have procoagulant activity. Biochem Biophys Res Commun 145(1):234–240

    Article  PubMed  CAS  Google Scholar 

  • Pham PL, Perret S, Doan HC et al (2003) Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng 84(3):332–342

    Article  PubMed  CAS  Google Scholar 

  • Pham PL, Perret S, Cass B et al (2005) Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis. Biotechnol Bioeng 90(3):332–344

    Article  PubMed  CAS  Google Scholar 

  • Rosser MP, Xia W, Hartsell S et al (2005) Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr Purif 40(2):237–243

    Article  PubMed  CAS  Google Scholar 

  • Schlaeger EJ, Christensen K (1999) Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology 30:71–83

    Article  CAS  PubMed  Google Scholar 

  • Seo NS, Hollister JR, Jarvis DL (2001) Mammalian glycosyltransferase expression allows sialoglycoprotein production by baculovirus-infected insect cells. Protein Expr Purif 22(2):234–241

    Article  PubMed  CAS  Google Scholar 

  • Shi C, Shin YO, Hanson J et al (2005) Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells. Biochemistry 44(48):15705–15714

    Article  PubMed  CAS  Google Scholar 

  • Stadler J, Lemmens R, Nyhammar T (2004) Plasmid DNA purification. J Gene Med 6 Supply 1:S54–S66

    Google Scholar 

  • Sutter G, Moss B (1992) Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc Natl Acad Sci USA 89(22):10847–10851

    Article  PubMed  CAS  Google Scholar 

  • Tait AS, Brown CJ, Galbraith DJ et al (2004) Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol Bioeng 88(6):707–721

    Article  PubMed  CAS  Google Scholar 

  • Tey BT, Singh RP, Piredda L et al (2000) Influence of bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotechnol Bioeng 68(1):31–43

    Article  PubMed  CAS  Google Scholar 

  • Thomas M, Klibanov AM (2003) Non-viral gene therapy: polycation-mediated DNA delivery. Appl Microbiol Biotechnol 62(1):27–34

    Article  PubMed  CAS  Google Scholar 

  • Thomas M, Lu JJ, Ge Q et al (2005) Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci USA 102(16):5679–5684

    Article  PubMed  CAS  Google Scholar 

  • Urlaub G, Chasin LA (1980) Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA 77(7):4216–4220

    Article  PubMed  CAS  Google Scholar 

  • Van Craenenbroeck K, Vanhoenacker P, Haegeman G (2000) Episomal vectors for gene expression in mammalian cells. Eur J Biochem 267(18):5665–5678

    Article  PubMed  Google Scholar 

  • Wakelin SJ, Sabroe I, Gregory CD et al (2006) “Dirty little secrets”-Endotoxin contamination of recombinant proteins. Immunol Lett 106(1):1–7

    Article  PubMed  CAS  Google Scholar 

  • Wright JL, Jordan M, Wurm FM (2003) Transfection of partially purified plasmid DNA for high level transient protein expression in HEK293-EBNA cells. J Biotechnol 102(3):211–221

    Article  PubMed  CAS  Google Scholar 

  • Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22(11):1393–1398

    Article  PubMed  CAS  Google Scholar 

  • Young JM, Cheadle C, Foulke JJS et al (1988) Utilization of an Epstein-Barr virus replicon as a eukaryotic expression vector. Gene 62(2):171–185

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors wish to thank Matthieu Stettler, Sarah Wuhlfard and Markus Hildinger for sharing unpublished results. This work was partly supported by the swiss KTI/CTI (Science to Market Fund). We thank A. Kühner AG for providing cell culture incubator shakers.

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Correspondence to Lucia Baldi.

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Baldi, L., Hacker, D.L., Adam, M. et al. Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives. Biotechnol Lett 29, 677–684 (2007). https://doi.org/10.1007/s10529-006-9297-y

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  • DOI: https://doi.org/10.1007/s10529-006-9297-y

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