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.
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Acknowledgment
This work was funded by the Agency for Science, Technology and Research (A*STAR).
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Goh, J.S.Y., Chan, K.F., Song, Z. (2015). Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants. In: Castilho, A. (eds) Glyco-Engineering. Methods in Molecular Biology, vol 1321. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2760-9_22
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DOI: https://doi.org/10.1007/978-1-4939-2760-9_22
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2759-3
Online ISBN: 978-1-4939-2760-9
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