Preparative purification process for human furin

Abstract

Recombinant truncated human furin was expressed in CHO cells and concentrated approximately 50-fold by ultrafiltration and diafiltration. The concentrate was purified by column chromatography on Capto-MMC™ resulting in a 30-50 fold purification factor and a yield of at least 60%. The at least 20% pure preparation obtained after Capto-MMC™ chromatography had already a purification degree allowing on-column maturation of pro-VWF. Then an additional Arginine Sepharose chromatography purification was carried out. This two column process for purification of truncated human furin resulted in an almost pure furin preparation with a specific activity of approximately 290,000 U furin/mg protein and a yield of about 50%.

1 - 10 . (canceled) 
     
     
         11 . A furin polypeptide or furin polypeptide derivative having furin biological activity purified from a protein solution by a method comprising:
 (a) binding the furin polypeptide or furin polypeptide derivative to a mixed cation exchange/hydrophobic interaction resin that binds the furin polypeptide or furin polypeptide derivative at pH 6.0, and   (b) recovering the furin polypeptide or furin polypeptide derivative from the resin by elution.   
     
     
         12 . The furin polypeptide or furin polypeptide derivative according to  claim 11  having a specific activity of at least 100,000 U/mg of protein. 
     
     
         13 . A pharmaceutical composition comprising an endoproteolytically active amount of the furin polypeptide or furin polypeptide derivative according to  claim 11  and a pharmaceutically acceptable carrier. 
     
     
         14 - 16 . (canceled) 
     
     
         17 . A furin polypeptide or furin polypeptide derivative having furin biological activity purified from a protein solution by a method comprising:
 (a) binding the furin polypeptide or furin polypeptide derivative to a mixed cation exchange/hydrophobic interaction resin,   (b) eluting the furin polypeptide or furin polypeptide derivative from the mixed cation exchange/hydrophobic interaction resin,   (c) binding the eluted furin polypeptide or furin polypeptide derivative to an Arginine-Sepharose resin, and   (d) eluting the furin polypeptide or furin polypeptide derivative from the Arginine-Sepharose resin.   
     
     
         18 . The furin polypeptide or furin polypeptide derivative according to  claim 17  having a specific activity of at least 290,000 U/mg of protein. 
     
     
         19 . A pharmaceutical composition comprising an endoproteolytically active amount of the furin polypeptide or furin polypeptide derivative according to  claim 18  and a pharmaceutically acceptable carrier. 
     
     
         20 . The furin polypeptide or furin polypeptide derivative of  claim 11 , wherein the furin polypeptide derivative is a truncated furin polypeptide. 
     
     
         21 . The furin polypeptide or the furin polypeptide derivative of  claim 17 , wherein the furin polypeptide derivative is a truncated furin polypeptide.