Method of purifying therapeutic proteins

Abstract

The present invention relates generally to a method of reducing the level of at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease(s) in a solution comprising at least one protein selected from the group consisting of fibrinogen, Factor VIII and von Willebrand factor (VWF), the method comprising:(i) passing a feedstock comprising at least one protein selected from the group consisting of fibrinogen, Factor VIII and VWF through a hydrophobic charge-induction chromatographic resin under conditions selected such that at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease(s) present in the feedstock is bound to the resin; and(ii) recovering a solution comprising the at least one protein selected from the group consisting of fibrinogen, Factor VIII and VWF which passes through the resin, wherein the concentration of the at least one protein selected from the group consisting of plasminogen, tissue plasminogen activator and other protease(s) in the solution is reduced by at least 50% compared to the feedstock.Also provided are solutions and pharmaceutical formulations comprising the fibrinogen and/or Factor VIII and/or VWF recovered by such methods, and uses thereof.

1 .- 48 . (canceled) 
     
     
         49 . A method for purifying fibrinogen, the method comprising the steps of:
 (i) passing a solution comprising fibrinogen through an anion exchange chromatographic resin under conditions selected such that fibrinogen monomer is bound to the resin,   (ii) eluting the fibrinogen monomer from the anion exchange chromatographic resin with an elution buffer, and   (iii) filtering the fibrinogen monomer eluted from step (ii) through a filter having a pore size in the range of from about 15 nm to about 35 nm.   
     
     
         50 . The method of  claim 49 , wherein the anion exchange chromatographic resin is a strong anion exchange resin. 
     
     
         51 . The method of  claim 50 , wherein the strong anion exchange resin comprises a quaternary amine functional ligand. 
     
     
         52 . The method of  claim 49 , wherein the fibrinogen is eluted from the anion exchange chromatographic resin with an elution buffer comprising from about 150 mM to about 300 mM NaCl. 
     
     
         53 . The method of  claim 49 , wherein the fibrinogen is eluted from the anion exchange chromatographic resin with an elution buffer comprising from about 150 mM to about 270 mM NaCl. 
     
     
         54 . The method of  claim 49 , wherein the elution buffer has a conductivity in the range of from about 18 mS/cm to about 32 mS/cm. 
     
     
         55 . The method of  claim 49 , wherein the elution buffer has a conductivity in the range of from about 19 mS/cm to about 24 mS/cm. 
     
     
         56 . The method of  claim 49 , wherein the filter is a polyvinylidene fluoride (PVDF) filter. 
     
     
         57 . The method of  claim 49 , wherein step (iii) is carried out in the presence of an amino acid. 
     
     
         58 . The method of  claim 57 , wherein the amino acid comprises arginine, glycine, or a combination thereof. 
     
     
         59 . The method of  claim 57 , wherein the amino acid comprises arginine. 
     
     
         60 . The method of  claim 57 , wherein the concentration of the amino acid is about 1% to about 6% (w/w).