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 . Isolated and pure collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         2 . The isolated and purified collagenase II of  claim 1 , wherein the collagenase II has a GPA assay activity for collagenase II of 200,000 to 380,000 fGPA units/mg. 
     
     
         3 . The isolated and purified collagenase II of  claim 1 , wherein the collagenase II contains less than 2% by area aggregated protein. 
     
     
         4 . The isolated and purified collagenase II of  claim 1 , wherein the collagenase II contains less than 1% by area of clostripain. 
     
     
         5 . The isolated and purified collagenase II of  claim 1 , wherein the collagenase II contains less than 1% by area of gelatinase. 
     
     
         6 . The isolated and purified collagenase II of  claim 1 , wherein the collagenase II contains less than 1 ug/mg (w/w) of leupeptin. 
     
     
         7 . The isolated and purified collagenase II of  claim 1 , wherein the collagenase II has a bioburden less than 1 cfu/ml, and wherein the collagenase II sterilized. 
     
     
         8 . The isolated and purified collagenase II of  claim 7 , wherein the collagenase II contains less than 10 EU/ml of endotoxin. 
     
     
         9 . The isolated and purified collagenase II of  claim 7 , wherein the collagenase II contains less than 5 EU/mg of endotoxin. 
     
     
         10 . The isolated and purified collagenase II of  claim 1 , wherein the collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         11 . The isolated and purified collagenase II of  claim 1 , wherein the collagenase II is at least a least 98% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         12 . Isolated and purified collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the collagenase II comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase II;   c) purifying 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 II using ion-exchange chromatography. 
   
     
     
         13 . The isolated and purified collagenase II of  claim 12 , wherein preparation of the collagenase II further comprises the step of conducting cell bank preparations in the presence of phytone peptone or vegetable peptone. 
     
     
         14 . The isolated and purified collagenase II of  claim 12 , wherein the fermentation step 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).   
     
     
         15 . The isolated and purified collagenase II of  claim 12 , wherein the collagenase II is stored at a temperature of about −70° C. 
     
     
         16 . A process for producing isolated and purified collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography, comprising the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase II; and   c) purifying 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 II using ion-exchange chromatography. 
   
     
     
         17 . The process of  claim 16 , wherein the collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         18 . The process of  claim 16 , further comprising the step of conducting cell bank preparations in the presence of phytone peptone or vegetable peptone. 
     
     
         19 . The process of  claim 16 , wherein the fermentation step 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).   
     
     
         20 . The process of  claim 16 , wherein the collagenase II is stored at a temperature of about −70° C. 
     
     
         21 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 95% pure by area as determined by reverse phase high performance liquid chromatography. 
     
     
         22 . The pharmaceutical formulation of  claim 21 , wherein the formulation is a sterile lyophilized powder and is stored at a temperature of about 5° C. 
     
     
         23 . The pharmaceutical formulation of  claim 21 , wherein the formulation is a lyophilized injectable composition formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         24 . The pharmaceutical formulation of  claim 23 , wherein the formulation is a lyophilized injectable formulation comprising about 0.9 mg of the collagenase II, about 18.5 mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vial fill volume is about 0.9 mL. 
     
     
         25 . The pharmaceutical formulation of  claim 23 , wherein the formulation is a lyophilized injectable formulation comprising about 0.58 mg of the collagenase II, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         26 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and isolated and purified collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the collagenase II comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase II; and   c) purifying 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 II using ion-exchange chromatography. 
   
     
     
         27 . The pharmaceutical composition of  claim 26 , wherein the composition is a sterile lyophilized powder. 
     
     
         28 . The pharmaceutical composition of  claim 26 , wherein the composition is a lyophilized injectable composition formulated with sucrose, Tris and with a pH level of about 8.0. 
     
     
         29 . The pharmaceutical composition of  claim 28 , wherein the composition is a lyophilized injectable composition comprising about 0.9 mg of the collagenase II, about 18.5 mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vial fill volume is about 0.9 mL. 
     
     
         30 . The pharmaceutical composition of  claim 28 , wherein the composition is a lyophilized injectable composition comprising about 0.58 mg of the collagenase II, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         31 . Collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         32 . The isolated and purified collagenase II of  claim 31 , wherein the collagenase II has a GPA assay activity for collagenase II of 200,000 to 380,000 fGPA units/mg. 
     
     
         33 . The isolated and purified collagenase II of  claim 31 , wherein the collagenase II contains less than 2% by area aggregated protein. 
     
     
         34 . The isolated and purified collagenase II of  claim 31 , wherein the collagenase II contains less than 1% by area of clostripain. 
     
     
         35 . The isolated and purified collagenase II of  claim 31 , wherein the collagenase II contains less than 1% by area of gelatinase. 
     
     
         36 . The isolated and purified collagenase II of  claim 31 , wherein the collagenase II contains less than 1 ug/mg (w/w) of leupeptin. 
     
     
         37 . The isolated and purified collagenase II of  claim 31 , wherein the collagenase II has a bioburden less than 1 cfu/ml, and wherein the collagenase II sterilized. 
     
     
         38 . The isolated and purified collagenase II of  claim 37 , wherein the collagenase II contains less than 10 EU/ml of endotoxin. 
     
     
         39 . The isolated and purified collagenase II of  claim 37 , wherein the collagenase II contains less than 5 EU/mg of endotoxin. 
     
     
         40 . Isolated and purified collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the collagenase II comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase II;   c) purifying 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 II using ion-exchange chromatography. 
   
     
     
         41 . The isolated and purified collagenase II of  claim 40 , wherein preparation of the collagenase II further comprises the step of conducting cell bank preparations in the presence of phytone peptone or vegetable peptone. 
     
     
         42 . The isolated and purified collagenase II of  claim 40 , wherein the fermentation step 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).   
     
     
         43 . The isolated and purified collagenase II of  claim 40 , wherein the collagenase II is stored at a temperature of about −70° C. 
     
     
         44 . A process for producing isolated and purified collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, comprising the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase II; and   c) purifying 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 II using ion-exchange chromatography. 
   
     
     
         45 . The process of  claim 44 , wherein the collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         46 . The process of  claim 44 , further comprising the step of conducting cell bank preparations in the presence of phytone peptone or vegetable peptone. 
     
     
         47 . The process of  claim 44 , wherein the fermentation step 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).   
     
     
         48 . The process of  claim 44 , wherein the collagenase II is stored at a temperature of about −70° C. 
     
     
         49 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 97% pure by area as determined by reverse phase high performance liquid chromatography. 
     
     
         50 . The pharmaceutical formulation of  claim 49 , wherein the formulation is a sterile lyophilized powder and is stored at a temperature of about 5° C. 
     
     
         51 . The pharmaceutical formulation of  claim 49 , wherein the formulation is a lyophilized injectable composition formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         52 . The pharmaceutical formulation of  claim 51 , wherein the formulation is a lyophilized injectable formulation comprising about 0.9 mg of the collagenase II, about 18.5 mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vial fill volume is about 0.9 mL. 
     
     
         53 . The pharmaceutical formulation of  claim 51 , wherein the formulation is a lyophilized injectable formulation comprising about 0.58 mg of the collagenase II, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         54 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and isolated and purified collagenase II having the sequence of  Clostridium histolyticum  collagenase II, wherein the collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the collagenase II comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase II; and   c) purifying 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 II using ion-exchange chromatography. 
   
     
     
         55 . The pharmaceutical composition of  claim 54 , wherein the composition is a sterile lyophilized powder. 
     
     
         56 . The pharmaceutical composition of  claim 54 , wherein the composition is a lyophilized injectable composition formulated with sucrose, Tris and with a pH level of about 8.0. 
     
     
         57 . The pharmaceutical composition of  claim 56 , wherein the composition is a lyophilized injectable composition comprising about 0.9 mg of the collagenase II, about 18.5 mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vial fill volume is about 0.9 mL. 
     
     
         58 . The pharmaceutical composition of  claim 56 , wherein the composition is a lyophilized injectable composition comprising about 0.58 mg of the collagenase II, about 12.0 mg of sucrose and about 0.7 mg of Tris.