US2011158972A1PendingUtilityA1

Compositions and methods for treating collagen-mediated diseases

48
Individually held — no corporate assignee on recordPriority: Jan 30, 2006Filed: Aug 30, 2010Published: Jun 30, 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 43/00A61P 35/00A61P 17/02A61P 17/10A61P 17/00A61P 19/02A61P 19/04A61K 38/4886C12Y 304/24007C12N 9/52A61K 9/19A61K 9/0019A61K 9/08A61K 38/48C12N 9/20
48
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Claims

Abstract

A drug product comprising a combination of highly purified collagenase I and collagenase II from Clostridium 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 - 47 . (canceled) 
     
     
         48 . 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.   
     
     
         49 . The drug product of  claim 48 , wherein the  Clostridium histolyticum  are grown in the presence of porcine-derived proteose peptone. 
     
     
         50 . The 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 hydrophobic interaction chromatography (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.   
     
     
         51 . The drug product of  claim 50 , wherein the  Clostridium histolyticum  are fermented in the presence of porcine-derived proteose peptone. 
     
     
         52 . The drug product of  claim 50 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         53 . The drug product of  claim 52 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         54 . The drug product of  claim 50 , wherein the fermentation step comprises the steps of:
 i) inoculating a non-bovine-derived 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 the mixtures from step (iii) to obtain an aliquot;   v) inoculating the medium in a third stage with aliquots from step (iv) and agitating;   vi) incubating the 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   vii) incubating mixtures from step (vii).   
     
     
         55 . The drug product of  claim 54 , wherein the non-bovine derived medium comprises porcine-derived proteose peptone. 
     
     
         56 . The drug product of  claim 54 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         57 . The drug product of  claim 56 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         58 . 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 hydrophobic interaction chromatography (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.   
     
     
         59 . The process of  claim 58 , wherein the  Clostridium histolyticum  are fermented in the presence of porcine-derived proteose peptone. 
     
     
         60 . The process of  claim 58 , wherein the process further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         61 . The process of  claim 60 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         62 . The process of  claim 58 , wherein the fermentation step comprises the steps of:
 i) inoculating a non-bovine-derived 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 the mixtures from step (iii) to obtain an aliquot;   v) inoculating the medium in a third stage with aliquots from step (iv) and agitating;   vi) incubating the 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   vii) incubating mixtures from step (vii).   
     
     
         63 . The process of  claim 62 , wherein the medium comprises porcine-derived proteose peptone. 
     
     
         64 . The process of  claim 63 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         65 . The process of  claim 64 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
     
     
         66 . 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.   
     
     
         67 . The pharmaceutical formulation of  claim 66 , wherein the  Clostridium histolyticum  is grown in the presence of porcine-derived proteose peptone. 
     
     
         68 . 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 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 media;   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 hydrophobic interaction chromatography (HIC) column; 
 4. adding leupeptin to the filtrate; 
 5. removing the ammonium sulphate; 
 6. filtering the mixture of step (5); and 
 7. separating collagenase I and collagenase II using ion-exchange; and 
   iv. combining the collagenase I and collagenase II purified from step (iii) at a ratio of about 1 to 1.   
     
     
         69 . The pharmaceutical composition of  claim 68 , wherein the  Clostridium histolyticum  are fermented in the presence of porcine-derived proteose peptone. 
     
     
         70 . The pharmaceutical composition of  claim 68 , wherein the process further comprises the step of conducting cell bank preparations in the absence of bovine-derived media. 
     
     
         71 . The pharmaceutical composition of  claim 70 , wherein the cell bank preparations are conducted in the presence of proteose peptone. 
     
     
         72 . 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.   
     
     
         73 . The drug product of  claim 72 , wherein the  Clostridium histolyticum  are grown in the presence of porcine-derived proteose peptone.

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