Method for the purification of albumin conjugates

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.

1 - 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) dAla 8  Lys 37 -CONH 2 . 
     
     
         43 . The method of  claim 39 , wherein said peptide is Exendin-4 (1-39) Lys 40 -CONH 2 . 
     
     
         44 . The method of  claim 36 , wherein said peptide comprising a Michael acceptor 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  Gln 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-Ile-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 7  (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 20  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). 
     
     
         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.