Patent ID: 8716240

Claim:
An isolated covalent conjugate between an erythropoietin peptide and a water-soluble polymer, wherein the covalent conjugate is prepared by a cell-free, in vitro method comprising: (a) contacting an asialo erythropoietin peptide with a first glycosyltransferase and a first modified sugar donor under conditions suitable for said first glycosyltransferase to covalently attach a sugar moiety of said first modified sugar donor to a preselected first glycosyl residue of said erythropoietin peptide via a first intact glycosyl linking group, wherein said sugar moiety of said first modified sugar donor comprises one modifying group which is a first water-soluble polymer, (b) contacting said erythropoietin peptide with a second glycosyltransferase and a second modified sugar donor under conditions suitable for said second glycosyltransferase to covalently attach a sugar moiety of said second modified sugar donor to a preselected second glycosyl residue of said erythropoietin peptide via a second intact glycosyl linking group, wherein said sugar moiety of said second modified sugar donor comprises one modifying group which is a second water-soluble polymer, thereby forming the covalent conjugate, wherein (i) said first glycosyltransferase and said second glycosyltransferase are independently selected from the group consisting of sialyltransferase, galactosyltransferase, and GalNAc transferase, and (ii) said first water-soluble polymer and said second water-soluble polymer are selected from the group consisting of poly(ethers), poly(amines), and poly(carboxylic acids), and (c) isolating the covalent conjugate, thereby forming an isolated covalent conjugate, with the proviso that three or less glycosyl residues of said isolated covalent conjugate comprise a modifying group covalently attached thereto.