Patent ID: 7128913

Claim:
A process of making a conjugate that comprises an erythropoietin glycoprotein having an N-terminal α-amino group and one poly(ethylene glycol), said erythropoietin glycoprotein being selected from the group consisting of human erythropoietin, analogs thereof that have from 1 to 6 additional sites for glycosylation, and human erythropoietin having at least one glycosylation site that is rearranged, and being covalently linked to one poly(ethylene glycol) group of the formula —CO—(CH 2 ) x —(OCH 2 CH 2 ) m —OR, wherein the —CO of the poly(ethylene glycol) group forms an amide bond with the N-terminal α-amino group of the erythropoietin glycoprotein; R is lower alkyl; x is 2 or 3; and m is from about 450 to about 1350; said process comprising: a) expressing and fermenting a recombinant EPO protein that has an N-terminal peptidic extension that includes a proteolytic cleavage sequence, b) protecting the ε-amino groups, c) proteolytically cleaving the N-terminal peptidic extension, d) pegylating the N-terminal α-amino group, and e) deprotecting the ε-amino groups of the EPO glycoprotein.