title: Highly sialylated recombinant human erythropoietin production in large-scale perfusion bioreactor utilizing CHO-gmt4 (JW152) with restored GnT I function.
abstract: Therapeutic glycoprotein drugs require a high degree of sialylation of their N-glycans for a better circulatory half-life that results in greater efficacy. It has been demonstrated that Chinese hamster ovary (CHO) glycosylation mutants lacking N-acetylglucosaminyltransferase I (GnT I), when restored by introduction of a functional GnT I, produced highly sialylated erythropoietin (EPO). We have now further engineered one of such mutants, JW152, by inactivating the dihydrofolate reductase (DHFR) gene to allow for the amplification of the EPO gene with methotrexate (MTX). Several MTX-amplified clones maintained the ability to produce highly sialylated EPO and one was selected for culture in a perfusion bioreactor that is used in an existing industrial EPO-production bioprocess. Extensive characterization of the EPO produced was performed using total sialic quantification, HPAEC-PAD and MALDI-TOF MS analyses. Our results demonstrated that the EPO produced by the mutant line exhibits superior sialylation compared to the commercially used EPO-producing CHO clone cultured under the same conditions. Therefore, this mutant has the industrial potential for producing highly sialylated recombinant EPO and potentially other recombinant glycoprotein therapeutics.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: John S Y
authors.initials: JS
authors.lastname: Goh
authors.forename: Yingwei
authors.initials: Y
authors.lastname: Liu
authors.forename: Haifeng
authors.initials: H
authors.lastname: Liu
authors.forename: Kah Fai
authors.initials: KF
authors.lastname: Chan
authors.forename: Corrine
authors.initials: C
authors.lastname: Wan
authors.forename: Gavin
authors.initials: G
authors.lastname: Teo
authors.forename: Xiangshan
authors.initials: X
authors.lastname: Zhou
authors.forename: Fusheng
authors.initials: F
authors.lastname: Xie
authors.forename: Peiqing
authors.initials: P
authors.lastname: Zhang
authors.forename: Yuanxing
authors.initials: Y
authors.lastname: Zhang
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Song
mesh_terms: D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D060846:Cell Engineering; D006224:Cricetinae; D003412:Cricetulus; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005784:Gene Amplification / Q000187:drug effects; D006031:Glycosylation; D006207:Half-Life; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin; D013762:Tetrahydrofolate Dehydrogenase; D017351:N-Acetylglucosaminyltransferases; D019158:N-Acetylneuraminic Acid; D008727:Methotrexate
keywords: CHO cells; Erythropoietin (EPO); Glycosylation; N-Acetylglucosaminyltransferase-I (GnT I); Sialylation