PURIFICATION OF PEPTIDE CONJUGATES BY HYDROPHOBIC INTERACTION CHROMATOGRAPHY
The present invention provides polypeptides that include an O-linked glycosylation site that is not present in the wild-type peptide. The polypeptides of the invention include glycoconjugates in which a species such as a water-soluble polymer, a therapeutic agent of a biomolecule is covalently linked through an intact O-linked glycosyl residue to the polypeptide. Also provided are methods of making a glycoconjugate, methods of isolating a glycoconjugate from a reaction mixture, pharmaceutical compositions containing a glycoconjugate, and methods of treating, ameliorating or preventing diseased in mammals by administering an amount of a glycoconjugate sufficient to achieve the desired response.
1 . A method of separating a covalent conjugate between a peptide and a modifying group from a reaction mixture, said method comprising:
(a) contacting said reaction mixture with a hydrophobic interaction chromatography medium; and
(b) collecting said covalent conjugate from said hydrophobic interaction chromatography medium, thereby separating said covalent conjugate from said reaction mixture,
wherein said modifying group is covalently attached to said peptide at a glycosyl or amino acid residue of said peptide via an intact glycosyl linking group covalently attached to said glycosyl or amino acid residue by a glycosyltransferase.
2 . The method according to claim 1 , wherein said modifying group is poly(ethylene glycol).
3 . The method of claim 1 , wherein said peptide is Factor VIII.
4 . The method of claim 2 , wherein said peptide is Factor VIII.
5 . A covalent conjugate between a peptide and a modifying group, wherein said covalent conjugate is obtained by a method comprising:
(a) contacting a reaction mixture comprising said covalent conjugate with a hydrophobic interaction chromatography medium; and
(b) collecting said covalent conjugate, thereby separating said covalent conjugate from said reaction mixture, and
wherein said modifying group is covalently attached to said peptide at a glycosyl or amino acid residue of said peptide via an intact glycosyl linking group covalently attached to said glycosyl or amino acid residue by a glycosyltransferase.
6 . The covalent conjugate according to claim 5 , wherein said modifying group is poly(ethylene glycol).
7 . The covalent conjugate of claim 5 , wherein said peptide is Factor VIII.
8 . The covalent conjugate of claim 6 , wherein said peptide is Factor VIII.