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

Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants
Title:
Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants
Other Titles:
Methods in Molecular Biology
Keywords:
Publication Date:
07 May 2021
Citation:
Goh JS, Chan KF, Song Z. Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants. Methods Mol Biol. 2015;1321:323‐333. doi:10.1007/978-1-4939-2760-9_22
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.
License type:
PublisherCopyrights
Funding Info:
This project is supported by the A*STAR, under A*STAR/BMRC’s Strategic Positioning Fund (SPF Ref No. SPF 2013/001)
Description:
This is a post-peer-review, pre-copyedit version of an article published in Methods in Molecular Biology. The final authenticated version is available online at: https://doi.org/10.1007/978-1-4939-2760-9_22
ISBN:
978-1-4939-2759-3
978-1-4939-2760-9
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