Compositions and Methods for Treating Collagen-Mediated Diseases

Abstract

A drug product comprising a combination of highly purified collagenase I and collagenase II from Colostridium histolyticum is disclosed. The drug product includes collagenase I and collagenase II in a ratio of about 1 to 1, with a purity of greater than at least 95%. The invention further disclosed improved fermentation and purification processes for preparing the said drug product.

1 . A method of purifying collagenase I and collagenase II from  Clostridium histolyticum,  comprising the steps of:
 a. fermenting  Clostridium histolyticum  to obtain a crude harvest comprising collagenase I and collagenase II;   b. subjecting the crude harvest to hydrophobic interaction chromatography (HIC) as a product capture step under conditions suitable for collagenase I and collagenase II binding to the HIC column; and   c. eluting collagenase I and II from the HIC column.   
     
     
         2 . The method of  claim 1 , wherein ammonium sulphate is added to the crude harvest prior to the HIC product capture step and wherein the collagenase I and collagenase II are eluted with a solution containing no ammonium sulphate. 
     
     
         3 . The method of  claim 1 , wherein the crude harvest is filtered through an anion exchange filter prior to step (b). 
     
     
         4 . The method of  claim 1 , wherein leupeptin is added to the eluate from the HIC column. 
     
     
         5 . The method of  claim 1 , comprising the steps of:
 i) filtering the crude harvest obtained in step (a) through an anion exchange filter;   ii) adding ammonium sulphate;   iii) subjecting the harvest obtained from step (ii) to the HIC column;   v) conducting the eluting step (c) with a solution containing no ammonium sulfate;   vi) adding leupeptin to the eluate obtained from step (v); and   vii) separating collagenase I and collagenase II using ion-exchange chromatography;   vii) combining the collagenase I and collagenase II purified from step (vii) at a ratio of about 1 to 1.   
     
     
         6 . The method of  claim 1 , wherein the purified collagenase I is at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         7 . The method of  claim 1 , wherein the purified collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         8 . The method of  claim 6 , wherein the purified collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         9 . The method of  claim 1 , wherein the purified collagenase I is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         10 . The method of  claim 1 , wherein the purified collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         11 . The method of  claim 9 , wherein the purified collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         12 . Isolated and purified collagenase I purified by the method of  claim 1 . 
     
     
         13 . The isolated and purified collagenase I of  claim 12 , wherein the collagenase I is at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         14 . The isolated and purified collagenase I of  claim 12 , wherein the collagenase I is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         15 . The isolated and purified collagenase I of  claim 12 , wherein the collagenase I contains less than about 1% by area of clostripain as determined by reverse phase high performance liquid chromatography. 
     
     
         16 . Isolated and purified collagenase II purified by the method of  claim 1 . 
     
     
         17 . The isolated and purified collagenase II of  claim 16 , wherein the collagenase I is at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         18 . The isolated and purified collagenase II of  claim 16 , wherein the collagenase I is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         19 . The isolated and purified collagenase II of  claim 17 , wherein the collagenase I contains less than about 1% by area of clostripain as determined by reverse phase high performance liquid chromatography. 
     
     
         20 . A drug product comprising isolated and purified collagenase I and collagenase II, wherein the collagenase I and collagenase II are purified according to the method of  claim 1 , and wherein the collagenase I and collagenase II have a mass ratio of about 1 to 1. 
     
     
         21 . A drug product comprising purified collagenase I and collagenase II, wherein the collagenase I and collagenase II are purified according to the method of  claim 5 , and wherein the collagenase I and collagenase II have a mass ratio of about 1 to 1. 
     
     
         22 . A drug product consisting of purified collagenase I and collagenase II, wherein the collagenase I and collagenase II are purified according to the method of  claim 1 , and wherein the collagenase I and collagenase II are present at a mass ratio of about 1 to 1. 
     
     
         23 . A drug product consisting of purified collagenase I and collagenase II, wherein the collagenase I and collagenase II are purified according to the method of  claim 5 , and wherein the collagenase I and collagenase II are present at a mass ratio of about 1 to 1.