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Title: Applications of a Novel Cho Glycosylation Mutant
Keywords: Recombinant glycoproteins, CHO glycosylation mutants, CHO-gmt4, sialylation, erythropoietin, N-acetylglucosaminyltransferase I,
Issue Date: 22-Jan-2014
Source: JOHN GOH SOO YANG (2014-01-22). Applications of a Novel Cho Glycosylation Mutant. ScholarBank@NUS Repository.
Abstract: Recombinant 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.
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