Using Matrix Attachment Regions to Improve Recombinant Protein Production

  • Niamh Harraghy
  • Montserrat Buceta
  • Alexandre Regamey
  • Pierre-Alain Girod
  • Nicolas Mermod
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 801)

Abstract

Chinese hamster ovary (CHO) cells are the system of choice for the production of complex molecules, such as monoclonal antibodies. Despite significant progress in improving the yield from these cells, the process to the selection, identification, and maintenance of high-producing cell lines remains cumbersome, time consuming, and often of uncertain outcome. Matrix attachment regions (MARs) are DNA sequences that help generate and maintain an open chromatin domain that is favourable to transcription and may also facilitate the integration of several copies of the transgene. By incorporating MARs into expression vectors, an increase in the proportion of high-producer cells as well as an increase in protein production are seen, thereby reducing the number of clones to be screened and time to production by as much as 9 months. In this chapter, we describe how MARs can be used to increase transgene expression and provide protocols for the transfection of CHO cells in suspension and detection of high-producing antibody cell clones.

Key words

Matrix attachment region Recombinant protein production Chinese hamster ovary cells Antibody IgG ELISA 

References

  1. 1.
    Walsh, G. (2006) Biopharmaceutical benchmarks 2006. Nat Biotechnol, 24, 769–776.PubMedCrossRefGoogle Scholar
  2. 2.
    Wurm, F.M. (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol, 22, 1393–1398.PubMedCrossRefGoogle Scholar
  3. 3.
    Kaufman, R.J. (1990) Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol, 185, 537–566.PubMedCrossRefGoogle Scholar
  4. 4.
    Kim, N., Byun, T. and Lee, G. (2001) Key determinants in the occurrence of clonal variation in humanized antibody expression of CHO cells during dihydrofolate reductase mediated gene amplification. Biotechnol Prog, 17, 69–75.PubMedCrossRefGoogle Scholar
  5. 5.
    Kim, S., Kim, N., Ryu, C., Hong, H. and Lee, G. (1998) Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol Bioeng, 58, 73–84.PubMedCrossRefGoogle Scholar
  6. 6.
    Chusainow, J., Yang, Y.S., Yeo, J.H., Toh, P.C., Asvadi, P., Wong, N.S. and Yap, M.G. (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng, 102, 1182–1196.PubMedCrossRefGoogle Scholar
  7. 7.
    Pilbrough, W., Munro, T.P. and Gray, P. (2009) Intraclonal protein expression heterogeneity in recombinant CHO cells. PLoS One, 4, e8432.PubMedCrossRefGoogle Scholar
  8. 8.
    Raj, A., Peskin, C., Tranchina, D., Vargas, D. and Tyagi, S. (2006) Stochastic mRNA synthesis in mammalian cells. PLoS Biol, 4, e309.PubMedCrossRefGoogle Scholar
  9. 9.
    Gorman, C., Arope, S., Grandjean, M., Girod, P. and Mermod, N. (2009) Use of MAR elements to increase the production of recombinant proteins. Cell Engineering, 6, 1–32.CrossRefGoogle Scholar
  10. 10.
    Yang, Y., Mariati, Chusainow, J. and Yap, M.G. (2010) DNA methylation contributes to loss in productivity of monoclonal antibody-producing CHO cell lines. J Biotechnol, 147, 180–185.Google Scholar
  11. 11.
    Ferrai, C., Xie, S.Q., Luraghi, P., Munari, D., Ramirez, F., Branco, M.R., Pombo, A. and Crippa, M.P. (2010) Poised transcription factories prime silent uPA gene prior to activation. PLoS Biol, 8, e1000270.PubMedCrossRefGoogle Scholar
  12. 12.
    Galbete, J.L., Buceta, M. and Mermod, N. (2009) MAR elements regulate the probability of epigenetic switching between active and inactive gene expression. Mol Biosyst, 5, 143–150.PubMedCrossRefGoogle Scholar
  13. 13.
    Kwaks, T.H. and Otte, A.P. (2006) Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells. Trends Biotechnol, 24, 137–142.PubMedCrossRefGoogle Scholar
  14. 14.
    Girod, P.A., Zahn-Zabal, M. and Mermod, N. (2005) Use of the chicken lysozyme 5′ matrix attachment region to generate high producer CHO cell lines. Biotechnol Bioeng, 91, 1–11.PubMedCrossRefGoogle Scholar
  15. 15.
    Zahn-Zabal, M., Kobr, M., Girod, P.A., Imhof, M., Chatellard, P., de Jesus, M., Wurm, F. and Mermod, N. (2001) Development of stable cell lines for production or regulated expression using matrix attachment regions. J Biotechnol, 87, 29–42.PubMedCrossRefGoogle Scholar
  16. 16.
    Phi-Van, L., von Kries, J.P., Ostertag, W. and Stratling, W.H. (1990) The chicken lysozyme 5′ matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol, 10, 2302–2307.PubMedGoogle Scholar
  17. 17.
    Girod, P.A., Nguyen, D.Q., Calabrese, D., Puttini, S., Grandjean, M., Martinet, D., Regamey, A., Saugy, D., Beckmann, J.S., Bucher, P. et al. (2007) Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat Methods, 4, 747–753.PubMedCrossRefGoogle Scholar
  18. 18.
    Dang, Q., Auten, J. and Plavec, I. (2000) Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviral vector. J Virol, 74, 2671–2678.PubMedCrossRefGoogle Scholar
  19. 19.
    Liebich, I., Bode, J., Frisch, M. and Wingender, E. (2002) S/MARt DB: a database on scaffold/matrix attached regions. Nucleic Acids Res, 30, 372–374.PubMedCrossRefGoogle Scholar
  20. 20.
    Harraghy, N., Gaussin, A. and Mermod, N. (2008) Sustained transgene expression using MAR elements. Curr Gene Ther, 8, 353–366.PubMedCrossRefGoogle Scholar
  21. 21.
    Kim, J.-M., Kim, J.-S., Park, D.-H., Kang, H., Yoon, J., Baek, K. and Yoon, Y. (2004) Improved recombinant gene expression in CHO cells using matrix attachment regions. J Biotechnol, 107, 95–105.PubMedCrossRefGoogle Scholar
  22. 22.
    Kim, J., Yoon, Y., Hwang, H.-Y., Park, J., Yu, S., Lee, J., Baek, K. and Yoon, J. (2005) Efficient selection of stable Chinese hamster ovary (CHO) cell lines for expression of recombinant proteins by using human interferon b SAR element. Biotechnol Prog, 21, 933–937.PubMedCrossRefGoogle Scholar
  23. 23.
    Varghese, J., Alves, W., Brill, B., Wallace, M., Calabrese, D., Regamey, A. and Girod, P. (2008) Rapid development of high-performance, stable mammalian cell lines for improved clinical development. Bioprocess J, 7, 30–36.Google Scholar
  24. 24.
    Evans, K., Ott, S., Hansen, A., Koentges, G. and Wernisch, L. (2007) A comparative study of S/MAR prediction tools. BMC Bioinformatics, 8, 71.PubMedCrossRefGoogle Scholar
  25. 25.
    Singh, G.B., Kramer, J.A. and Krawetz, S.A. (1997) Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucleic Acids Res, 25, 1419–1425.PubMedCrossRefGoogle Scholar
  26. 26.
    Frisch, M., Frech, K., Klingenhoff, A., Cartharius, K., Liebich, I. and Werner, T. (2002) In silico prediction of scaffold/matrix attachment regions in large genomic sequences. Genome Res, 12, 349–354.PubMedCrossRefGoogle Scholar
  27. 27.
    Jenke, A.C., Stehle, I.M., Herrmann, F., Eisenberger, T., Baiker, A., Bode, J., Fackelmayer, F.O. and Lipps, H.J. (2004) Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome. Proc Natl Acad Sci USA, 101, 11322–11327.PubMedCrossRefGoogle Scholar
  28. 28.
    Piechaczek, C., Fetzer, C., Baiker, A., Bode, J. and Lipps, H.J. (1999) A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic Acids Res, 27, 426–428.PubMedCrossRefGoogle Scholar
  29. 29.
    Stehle, I.M., Postberg, J., Rupprecht, S., Cremer, T., Jackson, D.A. and Lipps, H.J. (2007) Establishment and mitotic stability of an extra-chromosomal mammalian replicon. BMC Cell Biol, 8, 33.PubMedCrossRefGoogle Scholar
  30. 30.
    Giannakopoulos, A., Stavrou, E.F., Zarkadis, I., Zoumbos, N., Thrasher, A.J. and Athanassiadou, A. (2009) The functional role of S/MARs in episomal vectors as defined by the stress-induced destabilization profile of the vector sequences. J Mol Biol, 387, 1239–1249.PubMedCrossRefGoogle Scholar
  31. 31.
    Rosser, M.P., Xia, W., Hartsell, S., McCaman, M., Zhu, Y., Wang, S., Harvey, S., Bringmann, P. and Cobb, R.R. (2005) Transient transfection of CHO-K1-S using serum-free medium in suspension: a rapid mammalian protein expression system. Protein Expr Purif, 40, 237–243.PubMedCrossRefGoogle Scholar
  32. 32.
    Albano, C.R., Randers-Eichhorn, L., Bentley, W.E. and Rao, G. (1998) Green fluorescent protein as a real time quantitative reporter of heterologous protein production. Biotechnol Prog, 14, 351–354.PubMedCrossRefGoogle Scholar
  33. 33.
    Meng, Y.G., Liang, J., Wong, W.L. and Chisholm, V. (2000) Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene, 242, 201–207.PubMedCrossRefGoogle Scholar
  34. 34.
    Pick, H.M., Meissner, P., Preuss, A.K., Tromba, P., Vogel, H. and Wurm, F.M. (2002) Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D IgG1 synthesis. Biotechnol Bioeng, 79, 595–601.PubMedCrossRefGoogle Scholar
  35. 35.
    Brezinsky, S.C., Chiang, G.G., Szilvasi, A., Mohan, S., Shapiro, R.I., MacLean, A., Sisk, W. and Thill, G. (2003) A simple method for enriching populations of transfected CHO cells for cells of higher specific productivity. J Immunol Methods, 277, 141–155.PubMedCrossRefGoogle Scholar
  36. 36.
    Bergman, L.W., Harris, E. and Kuehl, W.M. (1981) Glycosylation causes an apparent block in translation of immunoglobulin heavy chain. J Biol Chem, 256, 701–706.PubMedGoogle Scholar
  37. 37.
    Bibila, T. and Flickinger, M.C. (1991) A structured model for monoclonal antibody synthesis in exponentially growing and stationary phase hybridoma cells. Biotechnol Bioeng, 37, 210–226.PubMedCrossRefGoogle Scholar
  38. 38.
    Schlatter, S., Stansfield, S.H., Dinnis, D.M., Racher, A.J., Birch, J.R. and James, D.C. (2005) On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol Prog, 21, 122–133.PubMedCrossRefGoogle Scholar
  39. 39.
    English, C., Merson, S. and Keer, J. (2006) Use of elemental analysis to determine comparative performance of established DNA quantification methods. Anal Chem, 78, 4630–4633.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Niamh Harraghy
    • 1
  • Montserrat Buceta
    • 2
  • Alexandre Regamey
    • 2
  • Pierre-Alain Girod
    • 2
  • Nicolas Mermod
    • 1
  1. 1.Laboratory of Molecular BiotechnologyUniversity of LausanneLausanneSwitzerland
  2. 2.Selexis SAPlan-les-OuatesSwitzerland

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