Applications of a Novel Cho Glycosylation Mutant

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.