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Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants

  • John S. Y. Goh
  • Kah Fai Chan
  • Zhiwei Song
Part of the Methods in Molecular Biology book series (MIMB, volume 1321)

Abstract

The degree of sialylation of therapeutic glycoproteins affects its circulatory half-life and efficacy because incompletely sialylated glycoproteins are cleared from circulation by asialoglycoprotein receptors present in the liver cells. Mammalian expression systems, often employed in the production of these glycoprotein drugs, produce heterogeneously sialylated products. Here, we describe how to produce highly sialylated glycoproteins using a Chinese hamster ovary (CHO) cell glycosylation mutant called CHO-gmt4 with human erythropoietin (EPO) as a model glycoprotein. The protocol describes how to isolate and characterize the CHO glycosylation mutants and how to assess the sialylation of the recombinant protein using isoelectric focusing (IEF). It further describes how to inactivate the dihydrofolate reductase (DHFR) gene in these cells using zinc finger nuclease (ZFN) technology to enable gene amplification and the generation of stable cell lines producing highly sialylated EPO.

Key words

Chinese hamster ovary (CHO) cells Glycosylation mutant Sialylation Glycoprotein Erythropoietin (EPO) Ricinus communis agglutinin-I (RCA-I) 

Notes

Acknowledgment

This work was funded by the Agency for Science, Technology and Research (A*STAR).

References

  1. 1.
    Morell AG, Gregoriadis G, Scheinberg IH et al (1971) The role of sialic acid in determining the survival of glycoproteins in the circulation. J Biol Chem 246:1461–1467PubMedGoogle Scholar
  2. 2.
    Grabenhorst E, Schlenke P, Pohl S et al (1999) Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj J 16:81–97PubMedCrossRefGoogle Scholar
  3. 3.
    Hossler P, Khattak SF, Li ZJ (2009) Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 19:936–949PubMedCrossRefGoogle Scholar
  4. 4.
    Goh JS, Zhang P, Chan KF et al (2010) RCA-I-resistant CHO mutant cells have dysfunctional GnT I and expression of normal GnT I in these mutants enhances sialylation of recombinant erythropoietin. Metab Eng 12:360–368PubMedCrossRefGoogle Scholar
  5. 5.
    Goh JS, Liu Y, Liu H et al (2013) Highly sialylated recombinant human erythropoietin production in large-scale perfusion bioreactor utilizing CHO-gmt4 (JW152) with restored GnT I function. Biotechnol J 9:100–109PubMedCrossRefGoogle Scholar
  6. 6.
    Rees S, Coote J, Stables J et al (1996) Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein. Biotechniques 20:102–104, 106, 108–110PubMedGoogle Scholar
  7. 7.
    Ho SC, Bardor M, Feng H et al (2012) IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol 157:130–139PubMedCrossRefGoogle Scholar
  8. 8.
    Schriebl K, Trummer E, Weik R et al (2006) A novel strategy for quantitative isoform detection directly performed from culture supernatant. J Pharm Biomed Anal 42:322–327PubMedCrossRefGoogle Scholar
  9. 9.
    Santiago Y, Chan E, Liu PQ et al (2008) Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A 105:5809–5814PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Doyon Y, Vo TD, Mendel MC et al (2010) Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods 8:74–79PubMedCrossRefGoogle Scholar
  11. 11.
    Urnov FD, Miller JC, Lee YL et al (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646–651PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • John S. Y. Goh
    • 1
  • Kah Fai Chan
    • 1
  • Zhiwei Song
    • 1
  1. 1.Bioprocessing Technology Institute (BTI)Agency for Science, Technology and Research (A*STAR)SingaporeSingapore

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