| Publication number |
CLPUB00638 |
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| Authors |
Goh J.S.Y. |
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| Title |
Applications of a novel CHO glycosylation mutant. |
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| Citation |
Thesis PhD (2014), National University of Singapore, Singapore |
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| Web pages |
https://scholarbank.nus.edu.sg/handle/10635/53712 |
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| 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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| Cell lines |
CVCL_W354; CHO-gmt4 CVCL_A8SP; CHO-gmt4D CVCL_A8SQ; CHO-gmt4D-EPO-GnT I |
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