Method for purification of albumin conjugates

• US 7,307,148 B2
• Filed: 04/22/2005
• Issued: 12/11/2007
• Est. Priority Date: 04/23/2004
• Status: Expired due to Fees

• Current Assignee: ConjuChem LLC (Colabor Group, Inc.)
• Original Assignee: ConjuChem Biotechnologies, Inc. /Old/ (Colabor Group, Inc.)
• Inventors: Bridon, Dominique P., Quraishi, Omar, Bousquet-Gagnon, Nathalie
• Primary Examiner(s): Carlson; Karen Cochrane
• Assistant Examiner(s): Rooke; Agnes B.
• Application Number:
  US11/112,277
• Publication Number:
  US 20050267293A1
• Time in Patent Office: 963 Days
• Field of Search: 514/12, 530/350
• US Class Current: 530/364
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: B01D 15/166 Fluid composition conditioning, e.g. gradient
• CPC Class Codes: B01D 15/327 with hydrophobic interaction
• CPC Class Codes: B01D 15/426 Specific type of solvent
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 1/303 by salting out
• CPC Class Codes: C07K 14/76 Albumins
• CPC Class Codes: C07K 14/765 Serum albumin, e.g. HSA

Abstract

The present invention relates to a method for separating albumin conjugate from unconjugated albumin in a solution comprising albumin conjugate and unconjugated albumin by loading the solution onto a hydrophobic support equilibrated in aqueous buffer having a high salt content; applying to the support a gradient of decreasing salt concentration; and collecting the eluted albumin conjugate.

Claims

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) dAla⁸ Lys³⁷ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

1),GRF (1-29) dAla² Gin⁸ Ala¹⁵ Leu²⁷ Lys³⁰ (ε

-MPA) CONH₂ (SEQ ID NO;

2), Ac-K5 Lys⁸ (ε

-MPA)-NH₂ (SEQ ID NO;

3), Insulin B1-MPA (SEQ ID NO;

4), Insulin A1-MPA (SEQ ID NO;

5), MPA-AEEA-C34-CONH₂ (SEQ ID NO;

6), C34 (1-34) Lys³⁵ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

7), C34 (1-34) Lys¹³ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

8), GLP-1 (7-36) Lys³⁷ (ε

-MPA)-NH₂ (SEQ ID NO;

9), GLP-1 (7-36) dAla⁸ Lys³⁷ (ε

-MPA)-NH₂ (SEQ ID NO;

10), GLP-1 (7-36) Lys²⁶ (ε

-AEEA-AEEA-MPA) (SEQ ID NO;

11), GLP-1 (7-36) Lys³⁴ (ε

-AEEA-AEEA-MPA) (SEQ ID NO;

12), Exendin-4-(1-39) Lys⁴⁰ (ε

-MPA)-NH₂ (SEQ ID NO;

13), Exendin-4 (9-39) Lys⁴⁰ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

14), Dyn A (1-13) (MPA)-NH₂ (SEQ ID NO;

15), MPA-AEEA-ANP (99-126)-CONH₂ (SEQ ID NO;

16), Dyn A (7-13) Lys¹³ (ε

-MPA)-CONH₂ (SEQ ID NO;

17), acetyl-Phe-His-cyclohexylstatyl-lle-Lys (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

18), GLP-1 (7-36) Lys²³ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

19), GLP-1 (7-36) Lys¹⁸ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

20), GLP-1 (7-36) Lys²⁶ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

21), GLP-1 (7-37) Lys²⁷ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

22), GLP-1 (7-36) Lys³⁷ (ε

-AEEA-AEEA-MPA)-CONH₂ (SEQ ID NO;

23), GLP-1 (7-36) Lys³⁷ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

24), Exendin-4-(1-39) Lys⁴⁰ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

25), GLP-1 (7-36) Lys³⁴ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

26), Insulin B1-OA-MPA (SEQ ID NO;

27), Insulin B29-MPA (SEQ ID NO;

28), GRF (1-29) Lys³⁰ (ε

-MPA)-CONH₂ (SEQ ID NO;

29), GRF (1-29) dAla² Gln⁸ dArg¹¹ Ala¹⁵ Leu²⁷ Lys³⁰ (ε

-MPA)-CONH₂ (SEQ ID NO;

30), GRF (1-29) dAla² Lys³⁰ (ε

-MPA)-CONH₂ (SEQ ID NO;

31), GLP-1 (9-36) Lys³⁷ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

32), Ac-T20 (1-36) Lys³⁷ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

33), Ac-T1249 (1-39) Lys⁴⁰ (ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

34), 3′

,4′

-didehydro-4′

-deoxy-C′

-norvincaleukoblastine -AEEA-MPA, C34 (1-34) Lys¹³ (ε

-MPA)-CONH₂ (SEQ ID NO;

36), C34 (1-34) Lys³⁵ (ε

-MPA)-CONH₂ (SEQ ID NO;

37), MPA-C34 (1-34)-CONH₂ (SEQ ID NO;

38), Ac-C34 (1-34) Glu² Lys⁶ Lys⁷ Glu⁹ Glu¹⁰ Lys¹³ Lys¹⁴ Glu¹⁶ Glu¹⁷ Lys²⁰ Lys²¹ Glu²³ Glu²⁴ Lys²⁷ Glu³¹ Lys³⁴ Lys³⁵ Lys³⁶(ε

-AEEA-MPA)-CONH₂ (SEQ ID NO;

39), MPA-AEEA-C34 (1-34) Glu² Lys⁶ Lys⁷ Glu⁹ Glu¹⁰ Lys¹³ Lys¹⁴ Glu¹⁶ Glu¹⁷ Lys²¹ Glu²³ Glu²⁴ Lys²⁷ Glu³¹ Lys³⁴ Lys³⁵ CONH₂ (SEQ ID NO;

40), PYY (3-36) Lys⁴ (ε

-OA-MPA)-CONH₂ (SEQ ID NO;

41), MPA-OA-PYY (3-36)-CONH₂ (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)-CONH₂ (SEQ ID NO;

46), PYY (3-36) Lys³⁷ (ε

-MPA)-CONH₂ (SEQ ID NO;

47), MPA-PYY (22-36)-CONH₂ (SEQ ID NO;

48), Acetyl-PYY (22-36) Lys³⁷ (ε

-MPA)-CONH₂ (SEQ ID NO;

49), MPA-ANP (99-126)-CONH₂ (SEQ ID NO;

50), MPA-EEEEP-ANP (99-126) (SEQ ID NO;

51), and GLP-2 (1-33) Gly² Lys³⁴ (ε

-MPA)-CONH₂ (SEQ ID NO;

52).

26. The method of claim 24, wherein said peptide is GLP-1 (7-36) dAla⁸ Lys³⁷.

27. The method of claim 24, wherein said peptide is Exendin-4 (1-39) Lys⁴⁰.

28. The method of claim 2, 3, or 4, wherein said molecule is GLP-1 (7-36) dAla⁸ Lys³⁷ (ε

-AEEA-MPA)-CONH₂.