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 purified collagenase I having the sequence of  Clostridium histolyticum  collagenase I, wherein the collagenase I is at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         2 . The isolated and purified collagenase I of  claim 1 , having a SRC assay activity of 13,000 to 23,000 fSRC units/mg. 
     
     
         3 . The isolated and purified collagenase I of  claim 1 , wherein the collagenase I contains less than 2% by area aggregated protein. 
     
     
         4 . The isolated and purified collagenase I of  claim 1 , wherein the collagenase I contains less than 1% by area of clostripain. 
     
     
         5 . The isolated and purified collagenase I of  claim 1 , wherein the collagenase I contains less than 1% by area of gelatinase. 
     
     
         6 . The isolated and purified collagenase I of  claim 1 , wherein the collagenase I contains less than 1 ug/mg (w/w) of leupeptin. 
     
     
         7 . The isolated and purified collagenase I of  claim 1 , wherein the collagenase I has a bioburden less than 1 cfu/ml, and wherein the drug product is sterilized. 
     
     
         8 . The isolated and purified collagenase I of  claim 7 , wherein the collagenase I contains less than 10 EU/ml of endotoxin. 
     
     
         9 . The isolated and purified collagenase I of  claim 7 , wherein the collagenase I contains less than 5 EU/mg of endotoxin. 
     
     
         10 . The isolated and purified collagenase I of  claim 1 , wherein the collagenase I is at least 97% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         11 . The isolated and purified collagenase I of  claim 1 , wherein the collagenase I is at least 98% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         12 . Isolated and purified collagenase I having the sequence of  Clostridium histolyticum  collagenase I which is at least 95% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the collagenase I comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase I;   c) purifying collagenase I 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 using ion-exchange chromatography. 
   
     
     
         13 . The isolated and purified collagenase I of  claim 12 , wherein preparation of the collagenase I further comprises the step of conducting cell bank preparations in the presence of phytone peptone or vegetable peptone. 
     
     
         14 . The isolated and purified collagenase I 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 I of  claim 12 , wherein the collagenase I is stored at a temperature of about −70° C. 
     
     
         16 . A process for producing isolated and purified collagenase I having the sequence of Clostridium histolyticum collagenase I, wherein the collagenase I 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 I; and   c) purifying collagenase I 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 using ion-exchange chromatography. 
   
     
     
         17 . The process of  claim 16 , wherein the collagenase I 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 I is stored at a temperature of about −70° C. 
     
     
         21 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and isolated and purified collagenase I having the sequence of  Clostridium histolyticum  collagenase I, wherein the collagenase I 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 I, 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 I, 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 I having the sequence of  Clostridium histolyticum  collagenase I, wherein the collagenase I is at least 95% pure by area as determined by reverse phase high performance liquid chromatography, wherein the preparation of the drug product comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase I; and   c) purifying collagenase I 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 using ion-exchange chromatography. 
 
   
     
     
         27 . The pharmaceutical composition of  claim 26 , wherein the drug product 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 I, 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 I, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         31 . Isolated and purified collagenase I having the sequence of  Clostridium histolyticum  collagenase I, wherein the collagenase I is at least 97% by area as determined by reverse phase high performance liquid chromatography. 
     
     
         32 . The isolated and purified collagenase I of  claim 31 , having a SRC assay activity of 13,000 to 23,000 fSRC units/mg. 
     
     
         33 . The isolated and purified collagenase I of  claim 31 , wherein the collagenase I contains less than 2% by area aggregated protein. 
     
     
         34 . The isolated and purified collagenase I of  claim 31 , wherein the collagenase I contains less than 1% by area of clostripain. 
     
     
         35 . The isolated and purified collagenase I of  claim 31 , wherein the collagenase I contains less than 1% by area of gelatinase. 
     
     
         36 . The isolated and purified collagenase I of  claim 31 , wherein the collagenase I contains less than 1 ug/mg (w/w) of leupeptin. 
     
     
         37 . The isolated and purified collagenase I of  claim 31 , wherein the collagenase I has a bioburden less than 1 cfu/ml, and wherein the drug product is sterilized. 
     
     
         38 . The isolated and purified collagenase I of  claim 37 , wherein the collagenase I contains less than 10 EU/ml of endotoxin. 
     
     
         39 . The isolated and purified collagenase I of  claim 38 , wherein the collagenase I contains less than 5 EU/mg of endotoxin. 
     
     
         40 . Isolated and purified collagenase I having the sequence of  Clostridium histolyticum  collagenase I which is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the collagenase I comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase I;   c) purifying collagenase I 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 using ion-exchange chromatography. 
   
     
     
         41 . The isolated and purified collagenase I of  claim 40 , wherein preparation of the collagenase I further comprises the step of conducting cell bank preparations in the presence of phytone peptone or vegetable peptone. 
     
     
         42 . The isolated and purified collagenase I of  claim 41 , 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 I of  claim 40 , wherein the collagenase I is stored at a temperature of about −70° C. 
     
     
         44 . A process for producing isolated and purified collagenase I having the sequence of  Clostridium histolyticum  collagenase I, wherein the collagenase I 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 I; and   c) purifying collagenase I 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 using ion-exchange chromatography. 
   
     
     
         45 . The process of  claim 44 , wherein the collagenase I 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 I is stored at a temperature of about −70° C. 
     
     
         49 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and isolated and purified collagenase I having the sequence of  Clostridium histolyticum  collagenase I, wherein the collagenase I 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 I, 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 I, 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 I having the sequence of  Clostridium histolyticum  collagenase I, wherein the collagenase I is at least 95% pure by area as determined by reverse phase high performance liquid chromatography, wherein the preparation of the drug product comprises the steps of:
 a) fermenting  Clostridium histolyticum;      b) harvesting a crude fermentation comprising collagenase I; and   c) purifying collagenase I 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 using ion-exchange chromatography. 
   
     
     
         55 . The pharmaceutical composition of  claim 54 , wherein the drug product 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 I, 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 I, about 12.0 mg of sucrose and about 0.7 mg of Tris.