Method for purifying recombinant fsh

Abstract

The present invention relates to a method for purifying a recombinant follicle stimulating hormone (FSH) or recombinant FSH variant. The method comprises the steps of subjecting a liquid containing a recombinant FSH or recombinant FSH variant to an anion exchange chromatography, to a hydrophobic interaction chromatography, and to a dye affinity chromatography, wherein these chromatographies may be performed in any order, and wherein the method neither comprises a weak anion exchange chromatography nor a reverse phase chromatography. The method of purification results in a high yield of recombinant FSH having a desired degree of purity. The obtained FSH is especially useful for the prophylaxis and treatment of disorders and medical indications where FSH preparations are considered as useful remedies.

1 . A method of purifying a recombinant FSH or recombinant FSH variant, comprising the steps of subjecting a liquid containing said FSH or FSH variant to:
 an anion exchange chromatography,   a hydrophobic interaction chromatography, and   a dye affinity chromatography,   which are performed in any order,   wherein the method neither comprises a weak anion exchange chromatography nor a reverse phase chromatography.   
     
     
         2 . The method of  claim 1 , wherein the steps are performed in the following order:
 a) an anion exchange chromatography   b) a hydrophobic interaction chromatography, and   c) a dye affinity chromatography.   
     
     
         3 . The method of  claim 1 , wherein the anion exchange chromatography is performed using a strong anion exchange resin having —N + (CH 3 ) 3  functional groups, or a resin having similar characteristics. 
     
     
         4 . The method of  claim 1 , wherein the hydrophobic interaction chromatography is performed using a resin consisting of cross-linked agarose beads derivatized with phenyl or butyl groups, or a resin having similar characteristics. 
     
     
         5 . The method of  claim 1 , wherein the dye affinity chromatography is performed with Cibacron Blue 3G as the ligand, covalently coupled to any matrix. 
     
     
         6 . The method of  claim 1 , wherein the chromatography is performed using a Tris-HCl/sodium chloride buffer as eluent at a pH in the range between 7.0 and 9.0. 
     
     
         7 . The method of  claim 1 , the method further comprising a cation exchange chromatography. 
     
     
         8 . The method of  claim 7 , wherein the membrane cation exchange chromatography is performed with a strong acidic cation exchanger sulfonic acid fixed on a membrane, or an exchanger having similar characteristics. 
     
     
         9 . The method of  claim 7 , wherein the steps are performed in the following order:
 a) an anion exchange chromatography,   b) a hydrophobic interaction chromatography,   c) a dye affinity chromatography, and   d) a cation exchange chromatography.   
     
     
         10 . The method of  claim 1 , further comprising a further anion exchange chromatography. 
     
     
         11 . The method of  claim 10 , wherein the anion exchange chromatography is performed using a strong anion exchange resin having —N + (CH 3 ) 3  functional groups, or a resin having similar characteristics. 
     
     
         12 . The method of  claim 10 , wherein the anion exchange chromatography is performed using a Tris-HCl/sodium chloride buffer as eluent at a pH in the range between 7.0 and 9.0. 
     
     
         13 . The method of  claim 10 , wherein the steps are performed in the following order:
 a) a first anion exchange chromatography,   b) a hydrophobic interaction chromatography,   c) a dye affinity chromatography,   d) an optional cation exchange chromatography, and   e) a second anion exchange chromatography.   
     
     
         14 . The method of  claim 1 , the method further comprising a size exclusion chromatography. 
     
     
         15 . The method of  claim 14 , wherein the size exclusion chromatography is performed using a matrix of spherical composite of cross-linked agarose and dextran. 
     
     
         16 . The method of  claim 14 , wherein the steps are performed in the following order:
 a) a first anion exchange chromatography,   b) a hydrophobic interaction chromatography,   c) a dye affinity chromatography,   d) an optional cation exchange chromatography,   e) an optional second anion exchange chromatography, and   f) a size exclusion chromatography.   
     
     
         17 . The method of  claim 1 , comprising the following steps in the following order:
 a) a first anion exchange chromatography,   b) a hydrophobic interaction chromatography,   c) a dye affinity chromatography,   d) a membrane cation exchange,   e) a second anion exchange chromatography, and   f) a size exclusion chromatography.   
     
     
         18 . The method of  claim 1 , further comprising one or more ultrafiltration and/or nanofiltration steps. 
     
     
         19 . The method of  claim 1 , wherein no metal ion affinity chromatography is performed. 
     
     
         20 . The method of  claim 1 , wherein no immunoaffinity chromatography is performed. 
     
     
         21 .- 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the FSH has an α-subunit according to SEQ ID No. 1 and a β-subunit according to SEQ ID No. 2. 
     
     
         25 . An FSH or FSH variant obtained by the method according to  claim 1 . 
     
     
         26 . A pharmaceutical composition comprising the FSH or FSH variant according to claim  22  as well as a pharmaceutically acceptable excipient. 
     
     
         27 . Use of the FSH or FSH variant according to claim  22  for the treatment of fertility disorders. 
     
     
         25 . Use of the pharmaceutical composition according to claim  23  for the treatment of fertility disorders.