COMPARISON OF RECOMBINANT GLYCOSYLPHOSPHATIDYL INOSITOL ANCHORED AND SECRETED HUMAN RECOMBINANT ERYTHROPOIETIN FROM CHINESE HAMSTER OVARY CELLS

Secreted human recombinant erythropoietin (rEPO) produced from Chinese hamster ovary (CHO) cells show glycosylation heterogeneity with a loss in biological activity. The manuscript describes expression of fully glycosylated human rEPO without any glycosylation heterogeneity when expressed as a glycosylphosphatidyl inositol (GPI) anchored cell surface molecule (rEPO-g) in CHO cells. Homogenous fully glycosylated human rEPO-g is expressed as a homogenous 42kd band from CHO cells. In contrast secreted human rEPO (rEPO-s) is expressed with glycosylation heterogeneity as a smear between 19kd-42kd. PNGase digestion of the rEPO-g showed the presence of a 24kd and 29kd band indicating 1 and 2 intact N-glycosylation sites respectively. Digestion of rEPO-s gave a 18.9kd unglycosylated rEPO-s band. The GPI anchor of the rEPO-g can be removed during the purification process by thrombin digestion. Thrombin digestion of the rEPO-g showed a decrease in 1.9kd kd with a decrease in rEPO-g molecular weight to 40kd indicating complete removal of the His6tag, Factor Xa cleavage site, GPI attachment peptide of 9 amino acids and the GPI anchor. This is the first report for the production of homogenous and completely glycosylated human rEPO at 40kd from CHO cells.