Method for purification of proteins

Abstract

The present invention relates to methods for purification of antithrombin-III (AT-III) by precipitation of impurities. The said methods comprise (a) adding, to a solution comprising antithrombin-III, a saccharide and citrate, in an amount sufficient for impurities to precipitate while antithrombin-III essentially remains in solution; (b) allowing impurities to precipitate; and (c) removing the precipitated impurities, thereby obtaining a solution comprising purified antithrombin-III. The invention also relates to pharmaceutical compositions, obtainable by the said methods, comprising purified antithrombin-III, as well as to reconstituted pharmaceutical compositions essentially free from visible particles.

1 . A process for purifying antithrombin-III, comprising the steps of: 
 (a) adding, to a solution comprising antithrombin-III, a saccharide and citrate, in an amount sufficient for impurities to precipitate while antithrombin-III essentially remains in solution;    (b) allowing impurities to precipitate; and    (c) removing the precipitated impurities, thereby obtaining a solution comprising purified antithrombin-III.    
     
     
         2 . The process according to  claim 1 , wherein the saccharide is selected from the group consisting of monosaccharides and disaccharides.  
     
     
         3 . The process according to  claim 2 , wherein the saccharide is a monosaccharide.  
     
     
         4 . The process according to any one of the preceding claims, wherein the concentration of saccharide is from 10 to 30%.  
     
     
         5 . The process according to  claim 4 , wherein the concentration of saccharide is from  15  to 25%.  
     
     
         6 . The process according to any one of the preceding claims, wherein the saccharide is sucrose.  
     
     
         7 . The process according to any one of the preceding claims, wherein the concentration of citrate is from 0.1 to 3 M.  
     
     
         8 . The process according to  claim 7 , wherein the concentration of citrate is from 0.5 to 1.5 M.  
     
     
         9 . The process according to any one of the preceding claims, wherein the source of citrate is sodium citrate.  
     
     
         10 . The process according to any one of  claims 1  to  9 , followed by a pasteurization step of the obtained solution comprising purified antithrombin-III in the presence of the said saccharide and citrate as stabilizing agents.  
     
     
         11 . The process according to any one of  claims 1  to  10 , further comprising a lyophilization step.  
     
     
         12 . A pharmaceutical composition obtainable by the method according to any one of claims  1 - 11 , comprising 
 (i) antithrombin-III; and    (ii) a saccharide and citrate as stabilizing agents.    
     
     
         13 . The pharmaceutical composition according to  claim 12 , wherein the saccharide is a monosaccharide.  
     
     
         14 . The pharmaceutical composition according to  claim 13 , wherein the monosaccharide is sucrose.  
     
     
         15 . The composition according to any one of claims  12 - 14 , wherein the amount of saccharide is from 0.5 to 2.5 parts per part antithrombin-III by weight.  
     
     
         16 . The composition according to  claim 15 , wherein the amount of saccharide is from 1.0 to 1.5 parts per part antithrombin-III by weight.  
     
     
         17 . The composition according to any one of  claims 12  to  16 , wherein the amount of citrate is from 1 to 4 parts per part antithrombin-III by weight.  
     
     
         18 . The composition according to  claim 17 , wherein the amount of citrate is from 2.5 to 3.0 per part antithrombin-III by weight.  
     
     
         19 . The composition according to any one of  claims 12  to  18 , wherein the source of citrate is sodium citrate.  
     
     
         20 . The composition according to any one of  claims 11  to  19 , characterized in that it is free of albumin.  
     
     
         21 . A pharmaceutical composition according to any one of claims  12 - 20  in lyophilized form.  
     
     
         22 . A reconstituted pharmaceutical composition according to  claim 21 , wherein the reconstitution media is a non-ionic surfactant in a concentration of at least from 0.01% (w/w).  
     
     
         23 . A reconstituted pharmaceutical composition according to  claim 22 , wherein the reconstitution media is a polyoxyethylene sorbitan fatty acid ester.  
     
     
         24 . A reconstituted pharmaceutical composition according to  claim 23 , wherein the recoconstitution media is selected from any one of polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85 and polysorbate 120.  
     
     
         25 . A reconstituted pharmaceutical composition according to  claim 24 , wherein the recoconstitution media is polysorbate 80.  
     
     
         26 . A reconstituted pharmaceutical composition according to  claim 22 , wherein the recoconstitution media is a sorbitan fatty acid ester.