Please use this identifier to cite or link to this item: https://scholarbank.nus.edu.sg/handle/10635/53712
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dc.titleApplications of a Novel Cho Glycosylation Mutant
dc.contributor.authorJOHN GOH SOO YANG
dc.date.accessioned2014-05-31T18:03:01Z
dc.date.available2014-05-31T18:03:01Z
dc.date.issued2014-01-22
dc.identifier.citationJOHN GOH SOO YANG (2014-01-22). Applications of a Novel Cho Glycosylation Mutant. ScholarBank@NUS Repository.
dc.identifier.urihttp://scholarbank.nus.edu.sg/handle/10635/53712
dc.description.abstractRecombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are quickly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are called CHO-gmt4 cells. CHO-gmt4 cells were observed to transiently and stably express erythropoietin (EPO) that was better sialylated than the wild-type CHO-K1 cells when functional GnT I was restored. CHO-gmt4D cells, derived from CHO-gmt4 by knocking out dihydrofolate reductase, were stably transfected with both EPO and GnT I and after gene amplification, a panel of clones that produced EPO with superior sialylation was generated. One of these clones, named CHO-gmt4D-EPO-GnT I was cultured in an industrial perfusion-culture based bioreactor and the resulting superior sialylation of EPO was maintained as shown through isoelectric focusing, HPAEC-PAD, sialic acid quantification and MALDI-TOF analyses. These results demonstrate that the CHO-gmt4 cell line can be applied in the production of better sialylated recombinant EPO and possibly other recombinant therapeutic glycoproteins.
dc.language.isoen
dc.subjectRecombinant glycoproteins, CHO glycosylation mutants, CHO-gmt4, sialylation, erythropoietin, N-acetylglucosaminyltransferase I,
dc.typeThesis
dc.contributor.departmentBIOCHEMISTRY
dc.contributor.supervisorSONG ZHIWEI
dc.contributor.supervisorSHEN HAN MING
dc.description.degreePh.D
dc.description.degreeconferredDOCTOR OF PHILOSOPHY
dc.identifier.isiutNOT_IN_WOS
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