Method for purification of albumin conjugates

1. A method for separating albumin conjugate from unconjugated albumin in a solution comprising albumin conjugate and unconjugated albumin, the method comprising:

a) loading said solution onto a hydrophobic interaction chromatography matrix equilibrated in aqueous buffer at a salt concentration high enough to promote matrix-protein interactions;

b) applying to said matrix a gradient of decreasing salt concentration; and

c) collecting said albumin conjugate.

2. The method of claim 1 , wherein said albumin conjugate consists of a molecule comprising a Michael acceptor covalently bonded to albumin.

3. The method of claim 2 , wherein said Michael acceptor is a maleimide group.

4. The method of claim 3 , wherein said maleimide group is maleimid-propionic acid.

5. The method of claim 1 , wherein said albumin is selected from the group consisting of serum albumin and recombinant albumin.

6. The method of claim 1 , wherein said albumin is selected from the group consisting of human albumin, rat albumin, mouse albumin, swine albumin, bovine albumin, dog albumin, and rabbit albumin.

7. The method of claim 1 , wherein said albumin is human serum albumin.

8. The method of claim 1 , wherein said albumin is modified with a fatty acid, a metal ion, a sugar, or a combination thereof.

9. The method of claim 8 , wherein said sugar is selected from the group consisting of glucose, lactose, and mannose.

10. The method of claim 2 , wherein said molecule is selected from the group consisting of a peptide, a DNA, an RNA, and a combination thereof, to which said Michael acceptor is covalently bonded, optionally through a linker.

11. The method of claim 10 , wherein said molecule is a peptide having a molecular weight of at least 57 daltons.

12. The method of claim 10 , wherein said peptide is selected from the group consisting of glucagon like peptide 1 (GLP-1), atrial natriuretic peptide (ANP), kringle 5 (KS), dynorphin, exendin-4, growth hormone releasing factor (GRF), insulin, natriuretic peptides, enfuvirtide (T-20), T-1249, C-34, soluble C-35 peptide BK (SC-35), peptide YY (PYY), and analogs thereof.

13. The method of claim 10 , wherein said molecule is selected from the group consisting of:

3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine;

2′-deoxy-2′,2′-difluorocytidine; and

5β,20-epoxy-1 ,2α,4,7β,10β,13α-hexahydroxy-tax-11-en-9-one, 4,10-diacetate 2-benzoate, 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine.

14. The method of claim 10 , wherein said molecule is covalently attached to said albumin through an acid sensitive covalent bond or a peptide sequence susceptible to proteolytic cleavage, thereby allowing the separation of said molecule and albumin and the entry of the molecule into a cell.

15. The method of claim 1 , wherein said hydrophobic interaction chromatography matrix is a column containing a hydrophobic resin.

16. The method of claim 15 , wherein said hydrophobic resin is a bead-formed agarose-based gel filtration matrix covalently coupled to a ligand selected from the group consisting of an octyl group, a phenyl group, and a butyl group.

17. The method of claim 15 , wherein said hydrophobic resin is a bead-formed agarose-based gel filtration matrix covalently coupled to a butyl group.

18. The method of claim 1 , wherein said gradient of decreasing salt concentration has a starting salt concentration of between 500 and 3000 mM.

19. The method of claim 1 , wherein said salt is selected from the group consisting of ammonium phosphate, ammonium sulfate, and magnesium phosphate.

20. The method of claim 1 , wherein said salt is ammonium phosphate or ammonium sulfate.

21. The method of claim 1 , wherein said salt is ammonium sulfate.

22. The method of claim 2 , wherein said bond is between said Michael acceptor and cysteine 34 of said albumin.

23. The method of claim 2 , 3 , or 4 , wherein said molecule is a peptide to which said Michael acceptor is covalently bonded, optionally through a linker.

24. The method of claim 23 , wherein said peptide is an analog of glucagon like peptide 1 (GLP-1), glucagon like peptide 2 (GLP-2), atrial natriuretic peptide (ANP), kringle 5 (KS), dynorphin, exendin-4, growth hormone releasing factor (GRF), insulin, natriuretic peptides, enfuvirtide (T-20), T-1249, C-34, soluble C-35 peptide EK (SC-35), or peptide YY (PYY).

25. The method of claim 2 , 3 , or 4 , wherein said molecule is selected from the group consisting of GLP-1 (7-36) dAla 8 Lys 37 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:1),GRF (1-29) dAla 2 Gin 8 Ala 15 Leu 27 Lys 30 (ε-MPA) CONH 2 (SEQ ID NO:2), Ac-K5 Lys 8 (ε-MPA)-NH 2 (SEQ ID NO:3), Insulin B1-MPA (SEQ ID NO:4), Insulin A1-MPA (SEQ ID NO:5), MPA-AEEA-C34-CONH 2 (SEQ ID NO:6), C34 (1-34) Lys 35 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:7), C34 (1-34) Lys 13 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:8), GLP-1 (7-36) Lys 37 (ε-MPA)-NH 2 (SEQ ID NO:9), GLP-1 (7-36) dAla 8 Lys 37 (ε-MPA)-NH 2 (SEQ ID NO:10), GLP-1 (7-36) Lys 26 (ε-AEEA-AEEA-MPA) (SEQ ID NO:11), GLP-1 (7-36) Lys 34 (ε-AEEA-AEEA-MPA) (SEQ ID NO:12), Exendin-4-(1-39) Lys 40 (ε-MPA)-NH 2 (SEQ ID NO:13), Exendin-4 (9-39) Lys 40 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:14), Dyn A (1-13) (MPA)-NH 2 (SEQ ID NO:15), MPA-AEEA-ANP (99-126)-CONH 2 (SEQ ID NO:16), Dyn A (7-13) Lys 13 (ε-MPA)-CONH 2 (SEQ ID NO:17), acetyl-Phe-His-cyclohexylstatyl-lle-Lys (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:18), GLP-1 (7-36) Lys 23 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:19), GLP-1 (7-36) Lys 18 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:20), GLP-1 (7-36) Lys 26 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:21), GLP-1 (7-37) Lys 27 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:22), GLP-1 (7-36) Lys 37 (ε-AEEA-AEEA-MPA)-CONH 2 (SEQ ID NO:23), GLP-1 (7-36) Lys 37 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:24), Exendin-4-(1-39) Lys 40 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:25), GLP-1 (7-36) Lys 34 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:26), Insulin B1-OA-MPA (SEQ ID NO:27), Insulin B29-MPA (SEQ ID NO:28), GRF (1-29) Lys 30 (ε-MPA)-CONH 2 (SEQ ID NO:29), GRF (1-29) dAla 2 Gln 8 dArg 11 Ala 15 Leu 27 Lys 30 (ε-MPA)-CONH 2 (SEQ ID NO:30), GRF (1-29) dAla 2 Lys 30 (ε-MPA)-CONH 2 (SEQ ID NO:31), GLP-1 (9-36) Lys 37 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:32), Ac-T20 (1-36) Lys 37 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:33), Ac-T1249 (1-39) Lys 40 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:34), 3′,4′-didehydro-4′-deoxy-C′-norvincaleukoblastine -AEEA-MPA, C34 (1-34) Lys 13 (ε-MPA)-CONH 2 (SEQ ID NO:36), C34 (1-34) Lys 35 (ε-MPA)-CONH 2 (SEQ ID NO:37), MPA-C34 (1-34)-CONH 2 (SEQ ID NO:38), Ac-C34 (1-34) Glu 2 Lys 6 Lys 7 Glu 9 Glu 10 Lys 13 Lys 14 Glu 16 Glu 17 Lys 20 Lys 21 Glu 23 Glu 24 Lys 27 Glu 31 Lys 34 Lys 35 Lys 36 (ε-AEEA-MPA)-CONH 2 (SEQ ID NO:39), MPA-AEEA-C34 (1-34) Glu 2 Lys 6 Lys 7 Glu 9 Glu 10 Lys 13 Lys 14 Glu 16 Glu 17 Lys 21 Glu 23 Glu 24 Lys 27 Glu 31 Lys 34 Lys 35 CONH 2 (SEQ ID NO:40), PYY (3-36) Lys 4 (ε-OA-MPA)-CONH 2 (SEQ ID NO:41), MPA-OA-PYY (3-36)-CONH 2 (SEQ ID NO:42), Insulin B29-AEES2-MPA (SEQ ID NO:43), Insulin B1-AEES2-MPA (SEQ ID NO:44), Insulin B29-OA-MPA (SEQ ID NO:45), MPA-PYY (3-36)-CONH 2 (SEQ ID NO:46), PYY (3-36) Lys 37 (ε-MPA)-CONH 2 (SEQ ID NO:47), MPA-PYY (22-36)-CONH 2 (SEQ ID NO:48), Acetyl-PYY (22-36) Lys 37 (ε-MPA)-CONH 2 (SEQ ID NO:49), MPA-ANP (99-126)-CONH 2 (SEQ ID NO:50), MPA-EEEEP-ANP (99-126) (SEQ ID NO:51), and GLP-2 (1-33) Gly 2 Lys 34 (ε-MPA)-CONH 2 (SEQ ID NO:52).

26. The method of claim 24 , wherein said peptide is GLP-1 (7-36) dAla 8 Lys 37 .

27. The method of claim 24 , wherein said peptide is Exendin-4 (1-39) Lys 40 .

28. The method of claim 2 , 3 , or 4 , wherein said molecule is GLP-1 (7-36) dAla 8 Lys 37 (ε-AEEA-MPA)-CONH 2 .