title: Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants.
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.
authors.affiliation: Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
authors.forename: John S Y
authors.initials: JS
authors.lastname: Goh
authors.forename: Kah Fai
authors.initials: KF
authors.lastname: Chan
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Song
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D003412:Cricetulus; D004921:Erythropoietin / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics*; D006031:Glycosylation; D006801:Humans; D009154:Mutation / Q000235:genetics*; D037121:Plant Lectins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
chemical_list: D006023:Glycoproteins; D037121:Plant Lectins; D011994:Recombinant Proteins; C017707:Ricinus communis agglutinin-1; D004921:Erythropoietin; D013762:Tetrahydrofolate Dehydrogenase