US2010330065A1PendingUtilityA1

Compositions and methods for treating collagen-mediated diseases

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Individually held — no corporate assignee on recordPriority: Jan 30, 2006Filed: Jul 16, 2010Published: Dec 30, 2010
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. HitchcockBo YuThomas L. Wegman
A61P 35/00A61P 43/00A61P 17/00A61P 19/04A61P 19/02A61P 17/02A61P 17/10C12N 9/52A61K 38/4886A61K 9/19A61K 9/0019C12Y 304/24007A61K 9/08A61K 38/48C12N 9/20
51
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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 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 collagenase I and collagenase II:
 a) are obtained from  Clostridium histolyticum  grown in the absence of bovine-derived media; and   b) have a mass ratio of about 1 to 1;   and the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.   
     
     
         2 . The drug product of  claim 1 , wherein the  Clostridium histolyticum  are grown in the presence of porcine-derived proteose peptone. 
     
     
         3 . 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 collagenase I and collagenase II have a mass ratio of about 1 to 1 and the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the drug product comprises the steps of:
 a) fermenting  Clostridium histolyticum  in the absence of bovine-derived media;   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 sulphate; 
 vi. filtering the mixture of step (v); and 
 vii. separating collagenase I and collagenase II using ion-exchange; and 
   d) combining the collagenase I and collagenase II purified from step (c) at a ratio of about 1 to 1.   
     
     
         4 . The drug product of  claim 3 , wherein the  Clostridium histolyticum  are fermented in the presence of porcine-derived proteose peptone. 
     
     
         5 . The drug product of  claim 3 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         6 . The drug product of  claim 5 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         7 . The drug product of  claim 3 , wherein the fermentation step comprises the steps of:
 a) inoculating a non-bovine-derived medium in a first stage with  Clostridium histolyticum  and agitating the mixture;   b) incubating the mixture from step (a) to obtain an aliquot;   c) inoculating the medium in a second stage with aliquots resulting from step (b) and agitating the mixture;   d) incubating mixtures from step (c) to obtain an aliquot;   e) inoculating the medium in a third stage with aliquots resulting from step (d) and agitating;   f) incubating mixtures from step (e) to obtain an aliquot;   g) inoculating the medium in a fourth stage with an aliquot resulting from step (f) and agitating; and   h) incubating mixtures from step (g).   
     
     
         8 . The drug product of  claim 7 , wherein the non-bovine-derived medium comprises porcine-derived proteose peptone. 
     
     
         9 . The drug product of  claim 7 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         10 . The drug product of  claim 9 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         11 . 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 collagenase I and collagenase II have a mass ratio of about 1 to 1 and the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, comprising the steps of:
 a) fermenting  Clostridium histolyticum  in the absence of bovine-derived media;   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 sulphate; 
 vi. filtering the mixture of step (v); and 
 vii. separating collagenase I and collagenase II using ion-exchange; and 
   d) combining the collagenase I and collagenase II purified from step (c) at a ratio of about 1 to 1.   
     
     
         12 . The process of  claim 11 , wherein the  Clostridium histolyticum  are fermented in the presence of porcine-derived proteose peptone. 
     
     
         13 . The process of  claim 11 , wherein the process further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         14 . The process of  claim 13 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         15 . The process of  claim 11 , wherein the fermentation step comprises the steps of
 a) inoculating a non-bovine-derived medium in a first stage with  Clostridium histolyticum  and agitating the mixture;   b) incubating the mixture from step (a) to obtain an aliquot;   c) inoculating the medium in a second stage with aliquots resulting from step (b) and agitating the mixture;   d) incubating mixtures from step (c) to obtain an aliquot;   e) inoculating the medium in a third stage with aliquots resulting from step (d) and agitating;   f) incubating mixtures from step (e) to obtain an aliquot;   g) inoculating the medium in a fourth stage with an aliquot resulting from step (f) and agitating; and   h) incubating mixtures from step (g).   
     
     
         16 . The process of  claim 15 , wherein the medium comprises porcine-derived proteose peptone. 
     
     
         17 . The process of  claim 15 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         18 . The process of  claim 17 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         19 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and 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 collagenase I and collagenase II:
 a) are obtained from  Clostridium histolyticum  grown in the absence of bovine-derived media; and   b) have a mass ratio of about 1 to 1; and   the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.   
     
     
         20 . The pharmaceutical formulation of  claim 19 , wherein the  Clostridium histolyticum  is grown in the presence of porcine-derived proteose peptone. 
     
     
         21 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and 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 collagenase I and collagenase II:
 a) are obtained from  Clostridium histolyticum  grown in the absence of bovine-derived media; and   b) have a mass ratio of about 1 to 1; and   the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the drug product comprises the steps of:
 i. fermenting  Clostridium histolyticum  in the absence of bovine-derived medium; 
 ii. harvesting a crude fermentation comprising collagenase I and collagenase II; 
 iii. purifying collagenase I and collagenase II from the crude harvest via filtration and column chromatography comprising the steps of:
 1. filtering the crude harvest through an anion exchange filter; 
 2. adding ammonium sulphate; 
 3. subjecting the harvest through a HIC column; 
 4. adding leupeptin to the filtrate; 
 5. removing the ammonium sulphate; 
 6. filtering the mixture of step (v); and 
 7. separating collagenase I and collagenase II using ion-exchange; and 
 
 iv. combining the collagenase I and collagenase II purified from step (c) at a ratio of about 1 to 1. 
   
     
     
         22 . The pharmaceutical composition of  claim 21 , wherein the  Clostridium histolyticum  are fermented in the presence of porcine-derived proteose peptone. 
     
     
         23 . The pharmaceutical composition of  claim 21 , wherein the process further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         24 . The pharmaceutical composition of  claim 23 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         25 . A drug product consisting of collagenase I and collagenase II, wherein the collagenase I and collagenase II are isolated and purified from  Clostridium histolyticum  and wherein the collagenase I and collagenase II;
 a) are obtained from  Clostridium histolyticum  grown in the absence of bovine-derived media; and   b) have a mass ratio of about 1 to 1; and   the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.   
     
     
         26 . The drug product of  claim 23 , wherein the  Clostridium histolyticum  are grown in the presence of porcine-derived proteose peptone.

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