Method for purifying FSH

Abstract

The present invention provides a method for purifying follicle stimulating hormone (FSH) from biological samples, for example, from human pituitary glands or human postmenopausal urine, wherein the FSH is contaminated with other proteins, by use of dye-ligand affinity chromatography (DAC). Depending on the starting material used and the initial purity of FSH in the starting material, additional purification steps may be employed. These steps preferably involve the use of hydrophobic interaction chromatography. This process may be used to generate affinity pure FSH suitable for therapeutic applications. The methods of the invention provide high purity FSH with high overall yield. A further advantage is the ability to easily regenerate the chromatography media for re-use, thus providing added economy to the purification process.

What is claimed is:  
     
         1 . A method for purification of FSH from an FSH-containing sample comprising the steps of: 
 (a) applying the sample in a first buffer to a dye affinity chromatography matrix comprising a dye ligand;    (b) washing out contaminants from the chromatography matrix with a second buffer;    (c) and eluting the FSH with a third buffer comprising less than about 0.8 M NaCl;    wherein the FSH is selected from a group consisting of human recombinant FSH, human FSH secreted from gonadotropes maintained in cell culture, genetically altered forms of human FSH, bovine FSH, equine FSH, porcine FSH, ovine FSH, canine FSH, rat FSH, feline FSH, mouse FSH, and monkey FSH.    
     
     
         2 . The method of  claim 1  wherein step (c) comprises a step-wise increase in ionic strength.  
     
     
         3 . The method of  claim 1  wherein step (c) comprises the use of a linear gradient.  
     
     
         4 . The method of  claim 1  wherein step (c) comprises a step-wise increase in ionic strength and the FSH group further consists of human urinary FSH.  
     
     
         5 . A method for purification of human pituitary FSH from an FSH-containing sample comprising the steps of: 
 (a) applying the sample in a first buffer to a dye affinity chromatography matrix comprising a dye ligand;    (b) washing out contaminants from the chromatography matrix with a second buffer;    (c) and eluting the FSH with a step-wise increase in ionic strength with a third buffer comprising less than about 1.0 M NaCl.    
     
     
         6 . A method for purification of FSH from a sample comprising the steps of: 
 (a) applying the sample in a first buffer to a dye affinity chromatography matrix comprising a dye ligand;    (b) washing out contaminants from the chromatography matrix with a second buffer;    (c) and eluting the FSH with a third buffer comprising a pH of greater than or equal to about 8.0.    
     
     
         7 . A method for purification of FSH from an FSH-containing sample comprising the steps of: 
 (a) applying the sample in a first buffer to a dye affinity chromatography matrix comprising a dye ligand;    (b) washing out contaminants from the chromatography matrix with a second buffer;    (c) and eluting the FSH with a third buffer comprising a competitor of FSH binding to the dye ligand.    
     
     
         8 . A method as in one of claims  1 - 7  wherein the first buffer comprises a pH of less than about 6 and a conductivity of less than about 1 mS.  
     
     
         9 . A method as in one of claims  1 - 7  wherein the dye ligand is Orange 1, Orange 2, Yellow 2, or Green 1.  
     
     
         10 . A method as in one of claims  1 - 7  wherein the dye affinity chromatography matrix further comprises cross linked agarose triazine coupled to Orange 1.  
     
     
         11 . A method as in one of claims  1 - 7  wherein the second buffer comprises a salt concentration of about less than about 50 mM and a pH of less than about 8.  
     
     
         12 . A method as in one of claims  1 - 7  further comprising the step of purifying the FSH by chromatography on a hydrophobic solid phase.