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 . 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 solid support equilibrated in aqueous buffer having a high salt content;   b) applying to said support a gradient of decreasing salt content; and   c) collecting eluted albumin conjugate.   
     
     
         2 . The method of  claim 1 , wherein said albumin conjugate consists of a molecule having a Michael acceptor covalently coupled thereto which bonds to albumin. 
     
     
         3 . The method of  claim 2 , wherein said bond is between said Michael acceptor and cysteine 34 of said albumin. 
     
     
         4 . The method of  claim 2 , wherein said Michael acceptor is a maleimide group. 
     
     
         5 . The method of  claim 4 , wherein said maleimide group is maleimid-propionic acid (MPA). 
     
     
         6 . The method of  claim 1 , wherein said albumin is selected from the group consisting of serum albumin, recombinant albumin and albumin from a genomic source. 
     
     
         7 . 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. 
     
     
         8 . The method of  claim 1 , wherein said albumin is human serum albumin. 
     
     
         9 . The method of  claim 1 , wherein said albumin is modified with at least one selected from the group consisting of fatty acids, metal ions, small molecules having high affinity to albumin, and sugars. 
     
     
         10 . The method of  claim 9 , wherein said sugars are selected from the group consisting of glucose, lactose and mannose. 
     
     
         11 . The method of  claim 2 , wherein said molecule is selected from the group consisting of a peptide, DNA, RNA, small organic molecule and a combination thereof. 
     
     
         12 . The method of  claim 11 , wherein said peptide has a molecular weight of at least 57 daltons. 
     
     
         13 . The method of  claim 11 , wherein said peptide is selected from the group consisting of GLP-1, ANP, K5, dynorphin, GRF, insulin, natriuretic peptides, T-20, T-1249, C-34, SC-35, PYY and analogs thereof. 
     
     
         14 . The method of  claim 11 , wherein said small organic molecule is selected from the group consisting of vinorelbine, gemcitabine and paclitaxel. 
     
     
         15 . The method of  claim 11 , 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. 
     
     
         16 . The method of  claim 1 , wherein said hydrophobic solid support is a column containing a hydrophobic resin. 
     
     
         17 . The method of  claim 16 , wherein said hydrophobic resin is selected from the group consisting of octyl sepharose, phenyl sepharose and butyl sepharose. 
     
     
         18 . The method of  claim 16 , wherein said hydrophobic resin is butyl sepharose. 
     
     
         19 . The method of  claim 1 , wherein said salt has a sufficent salting out effect. 
     
     
         20 . The method of  claim 1 , wherein said salt is selected from the group consisting of ammonium phosphate, ammonium sulfate and magnesium phosphate. 
     
     
         21 - 22 . (canceled)