US2011243920A1PendingUtilityA1

Compositions and methods for treating collagen-mediated diseases

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Individually held — no corporate assignee on recordPriority: Jan 30, 2006Filed: Apr 12, 2011Published: Oct 6, 2011
Est. expiryJan 30, 2026(expired)· nominal 20-yr term from priority
Inventors:Gregory L. SabatinoBenjamin J. Del Tito, Jr.Phillip J. BassettHazel A. ThariaAntony G. Hitchcock
A61P 35/00A61P 43/00A61P 19/02A61P 17/10A61P 17/00A61P 17/02A61P 19/04A61K 9/19C12N 9/52C12Y 304/24007A61K 38/4886A61K 9/0019A61K 9/08C12N 9/20A61K 38/48
49
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Claims

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.

Claims

exact text as granted — not AI-modified
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; and   b. subjecting the crude harvest to column chromatography in the presence of leupeptin.   
     
     
         2 . The method of  claim 1 , wherein the column chromatography comprises ion exchange chromatography. 
     
     
         3 . The method of  claim 1 , wherein the method comprises 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 prior to step (b), and wherein the column chromatography of step (b) comprises ion exchange chromatography. 
     
     
         4 . 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. 
     
     
         5 . 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. 
     
     
         6 . The method of  claim 7 , wherein the purified collagenase II 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 I is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         8 . 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. 
     
     
         9 . The method of  claim 7 , wherein the purified collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         10 . The method of  claim 1 , wherein step (a) comprises conducting cell bank preparations in the presence of phytone peptone or vegetable peptone. 
     
     
         11 . The method of  claim 1 , wherein step (a) comprises the steps of:
 i) inoculating the medium in a first stage with  Clostridium histolyticum  and agitating the mixture;   ii) incubating the mixture from step (i) to obtain an aliquot;   iii) inoculating the medium in a second stage with aliquots resulting from step (ii) and agitating the mixture;   iv) incubating mixtures from step (iii) to obtain an aliquot;   v) inoculating the medium in a third stage with aliquots resulting from step (iv) and agitating;   vi) incubating mixtures from step (v) to obtain an aliquot;   vii) inoculating the medium in a fourth stage with an aliquot resulting from step   (vi) and agitating; and   viii) incubating mixtures from step (vii).   
     
     
         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 II 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 II 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 16 , wherein the collagenase II contains less than about 1% by area of clostripain as determined by reverse phase high performance liquid chromatography. 
     
     
         20 . A process for producing a drug product consisting of isolated and purified collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, wherein the process comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase I and collagenase II;   c) purifying collagenase I and collagenase II from the crude harvest via filtration and column chromatography comprising the steps of:
 i) filtering the crude harvest through an anion exchange filter; 
 ii) adding ammonium sulphate; 
 iii) subjecting the harvest through a HIC column; 
 iv) adding leupeptin to the filtrate; 
 v) removing the ammonium sulfate; 
 vi) filtering the mixture of step (v); and 
 vii) separating collagenase I and collagenase II using ion-exchange; 
 d) combining the collagenase I and collagenase II purified from step (c) at a 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 1 , 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 , wherein the collagenase I and collagenase II are present at a mass ratio of about 1 to 1. 
     
     
         23 . 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 20 , wherein the collagenase I and collagenase II have a mass ratio of about 1 to 1. 
     
     
         24 . 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 20 , wherein the collagenase I and collagenase II are present at a mass ratio of about 1 to 1.

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