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)


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) 



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


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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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