Method for the purification of albumin conjugates

• US 20080146783A1
• Filed: 10/30/2007
• Published: 06/19/2008
• Est. Priority Date: 04/23/2004
• Status: Abandoned Application

• Current Assignee: ConjuChem LLC (Colabor Group, Inc.)
• Original Assignee: ConjuChem Biotechnologies, Inc. /Old/ (Colabor Group, Inc.)
• Inventors: Quraishi, Omar, Bousquet-Gagnon, Nathalie, Bridon, Dominique P.
• Application Number:
  US11/981,474
• Publication Number:
  US 20080146783A1
• Time in Patent Office: Days
• 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

1-22. -22. (canceled)

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

contacting said solution with a hydrophobic interaction chromatography matrix under conditions wherein the albumin conjugate binds to said matrix and the unconjugated albumin does not bind to said matrix, wherein the albumin conjugate comprises a peptide.

24. The method of claim 23, wherein said hydrophobic interaction chromatography matrix is a column containing a hydrophobic resin.

25. The method of claim 24, 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.

26. The method of claim 25, wherein said hydrophobic resin is a bead-formed agarose-based gel filtration matrix covalently coupled to a butyl group.

27. The method of claim 23, wherein the hydrophobic interaction chromatography matrix is, prior to contact with said solution, equilibrated in aqueous buffer at a salt concentration high enough to promote matrix-protein interactions.

28. The method of claim 27, wherein the said salt concentration is between 500 and 3000 mM.

29. The method of claim 27, wherein said salt is selected from the group consisting of ammonium phosphate, ammonium sulfate, magnesium phosphate.

30. The method of claim 27, wherein said salt is ammonium sulfate.

31. The method of claim 27, wherein the pH of said aqueous buffer is between 3.0 and 9.0.

32. The method of claim 27, wherein the pH of said aqueous buffer is 7.0.

33. The method of claim 27, wherein said aqueous buffer and said hydrophobic interaction chromatography matrix are at a temperature of between 4°

C. and about 25°

C.

34. The method of claim 27, further comprising applying a gradient of decreasing salt concentration to said hydrophobic interaction chromatography matrix following contact with said solution.

35. The method of claim 34, further comprising collecting the eluted albumin conjugate.

36. The method of claim 23, wherein said albumin conjugate consists of a peptide comprising a Michael acceptor covalently bonded to albumin.

37. The method of claim 36, wherein said bond is between said Michael acceptor and cysteine 34 of said albumin.

38. The method of claim 37, wherein said Michael acceptor is maleimide-propionic acid.

39. The method of claim 36, wherein said peptide is covalently bonded to said Michael acceptor, optionally through a linker.

40. The method of claim 39, wherein said peptide is covalently bonded to said Michael acceptor through a linker selected from the group consisting of (2-amino)ethoxy acetic acid (AEA), ethylenediamine (EDA), 2-[2-(2-amino)ethoxy]ethoxy acetic acid (AEEA), amino ethoxy ethyl amino succinic acid (AEEAS), glycine, 3-aminopropionic acid (APA), 8-aminooctanoic acid (AOA), octanoic acid (OA), and 4-aminobenzoic acid (APhA).

41. The method of claim 39, wherein said peptide is selected from the group consisting of glucagon like peptide 1 (GLP-1), glucagon like peptide 2 (GLP-2), atrial natriuretic peptide (ANP), kringle 5 (K5), 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), peptide YY (PYY), and analogs thereof.

42. The method of claim 39, wherein said peptide is GLP-1 (7-36) dAla⁸ Lys³⁷-CONH₂.

43. The method of claim 39, wherein said peptide is Exendin-4 (1-39) Lys⁴⁰-CONH₂.

44. The method of claim 36, wherein said peptide comprising a Michael acceptor 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 Gln⁸ 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-Ile-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) Lys⁷ (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 Lys²⁰ 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).

45. The method of claim 23, wherein said albumin is selected from the group consisting of serum albumin and recombinant albumin.

46. The method of claim 23, wherein said albumin is human serum albumin.

47. A hydrophobic interaction chromatography matrix to which an albumin conjugate is bound, wherein said albumin conjugate comprises a peptide.