Compositions and methods for treating collagen-mediated diseases

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.

1 .- 61 . (canceled) 
     
     
         62 . A drug product comprising as the sole active ingredients 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 are mixed in a mass ratio of about 1 to 1, and both the collagenase I and collagenase II are at least 95% by area pure as determined by reverse phase high performance liquid chromatography wherein the drug product contains less than 10 parts per million residual host protein. 
     
     
         63 . The drug product of  claim 62 , wherein the drug product contains less than about 2% by area aggregated protein as determined by reverse phase high performance liquid chromatography. 
     
     
         64 . The drug product of  claim 62 , wherein the drug product contains less than about 1% by area of clostripain as determined by reverse phase high performance liquid chromatography. 
     
     
         65 . The drug product of  claim 62 , wherein the drug product contains less than about 1% by area of gelatinase as determined by anion exchange chromatography. 
     
     
         66 . The drug product of  claim 62 , wherein the drug product contains less than about 1 ug/mg (w/w) of leupeptin. 
     
     
         67 . The drug product of  claim 62 , wherein the drug product has a bioburden less than 1 cfu/ml, and wherein the drug product is sterile. 
     
     
         68 . The drug product of  claim 67 , wherein the drug product contains less than 10 EU/ml of endotoxin. 
     
     
         69 . The drug product of  claim 62 , further comprising a pharmaceutically acceptable carrier or excipient selected from the group consisting of a sugar; a starch; cellulose and its derivatives; powdered tragacanth; malt; gelatin; talc; a glycol; propylene glycol; an ester; ethyl oleate; ethyl laurate; agar; a buffering agent; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; sodium lauryl sulfate; magnesium stearate; a coloring agent; a releasing agent; a coating agent; a perfuming agent; a preservative; an antioxidant; and any combination of the foregoing. 
     
     
         70 . A drug product comprising as the sole active ingredients 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 both collagenase I and collagenase II are at least 95% 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;      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.   
     
     
         71 . The drug product of  claim 70 , wherein collagenase I and collagenase II are both at least 96% by area pure as determined by reverse phase high performance liquid chromatography wherein the drug product contains less than 10 parts per million residual host protein. 
     
     
         72 . The drug product of  claim 70 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the presence of phytone peptone or vegetable peptone. 
     
     
         73 . The drug product of  claim 70 , 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).   
     
     
         74 . The drug product of  claim 70 , wherein the drug product is stored at a temperature of about −70° C. 
     
     
         75 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and a drug product comprising as the sole active ingredients 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 collagenase I and collagenase II are both at least 95% by area pure as determined by reverse phase high performance liquid chromatography wherein the formulation contains less than 10 parts per million residual host protein. 
     
     
         76 . The pharmaceutical formulation of  claim 75 , wherein the drug product is a sterile lyophilized powder and is stored at a temperature of about 5° C. 
     
     
         77 . The pharmaceutical formulation of  claim 75 , wherein the formulation is a lyophilized injectable formulation formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         78 . The pharmaceutical formulation of  claim 77 , wherein the formulation is a lyophilized injectable formulation comprising about 0.9 mg of the said drug product, 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. 
     
     
         79 . The pharmaceutical formulation of  claim 77 , wherein the formulation is a lyophilized injectable formulation comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         80 . The pharmaceutical formulation of  claim 75 , wherein the preparation of the drug product 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.   
     
     
         81 . The pharmaceutical formulation of  claim 80 , wherein the formulation is a sterile lyophilized powder. 
     
     
         82 . The pharmaceutical formulation of  claim 81 , wherein the formulation is a lyophilized injectable composition formulation formulated with sucrose, Tris and with a pH level of about 8.0. 
     
     
         83 . The pharmaceutical formulation of  claim 82 , wherein the formulation is a lyophilized injectable formulation comprising about 0.9 mg of the said drug product, 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. 
     
     
         84 . The pharmaceutical formulation of  claim 82 , wherein the formulation is a lyophilized injectable composition comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         85 . The pharmaceutical formulation of  claim 75 , wherein collagenase I and collagenase II are both at least 96% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         86 . The pharmaceutical formulation of  claim 85 , wherein the formulation is a sterile lyophilized powder and is stored at a temperature of about 5° C. 
     
     
         87 . The pharmaceutical formulation of  claim 85 , wherein the formulation is a lyophilized injectable formulation formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         88 . The pharmaceutical formulation of  claim 87 , wherein the formulation is a lyophilized injectable formulation comprising about 0.9 mg of the said drug product, 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. 
     
     
         89 . The pharmaceutical formulation of  claim 87 , wherein the formulation is a lyophilized injectable formulation comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         90 . The pharmaceutical formulation of  claim 85 , wherein the preparation of the drug product 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.   
     
     
         91 . The pharmaceutical formulation of  claim 90 , wherein the formulation is a sterile lyophilized powder. 
     
     
         92 . The pharmaceutical formulation of  claim 90 , wherein the formulation is a lyophilized injectable formulation formulated with sucrose, Tris and with a pH level of about 8.0. 
     
     
         93 . The pharmaceutical formulation of  claim 92 , wherein the formulation is a lyophilized injectable composition comprising about 0.9 mg of the said drug product, 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. 
     
     
         94 . The pharmaceutical formulation of  claim 92 , wherein the formulation is a lyophilized injectable formulation comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         95 . A drug product for a collagen-mediated disease comprising as the sole active ingredients  Clostridium histolyticum  collagenase I and collagenase II, wherein the collagenase I and collagenase II are mixed in a mass ratio of about 1 to 1, and wherein the collagenase I and collagenase II both are at least 95% by area pure as determined by reverse phase high performance liquid chromatography wherein the drug product contains less than 10 parts per million residual host protein. 
     
     
         96 . A drug product for a collagen-mediated disease comprising  Clostridium histolyticum  collagenase I and collagenase II, wherein the collagenase I and collagenase II are mixed in a mass ratio of about 1 to 1, wherein and collagenase I and collagenase II both are at least 95% by area pure as determined by reverse phase high performance liquid chromatography, and wherein the drug product has a potency of between about 1700 and about 3500 SRC units per milligram of collagenase I and collagenase II. 
     
     
         97 . A drug product for a collagen-mediated disease comprising  Clostridium histolyticum  collagenase I and collagenase II, wherein the collagenase I and collagenase II are mixed in a mass ratio of about 1 to 1, wherein and collagenase I and collagenase II both are at least 95% by area pure as determined by reverse phase high performance liquid chromatography, and wherein the drug product has a potency of between about 50,000 and about 90,000 GPA units per milligram of collagenase I and collagenase II. 
     
     
         98 . A drug product for a collagen-mediated disease comprising  Clostridium histolyticum  collagenase I and collagenase II, wherein the collagenase I and collagenase II are mixed in a mass ratio of about 1 to 1, wherein and collagenase I and collagenase II both are at least 95% by area pure as determined by reverse phase high performance liquid chromatography, and wherein the collagenase I has a potency of between about 1700 and about 3500 SRC units per milligram of collagenase I, and collagenase II has a potency of between about 79,000 and about 170,000 GPA units per milligram of collagenase II. 
     
     
         99 . A drug product for a collagen-mediated disease comprising  Clostridium histolyticum  collagenase I and collagenase II, wherein the collagenase I and collagenase II are mixed in a mass ratio of about 1 to 1, wherein and collagenase I and collagenase II both are at least 95% by area pure as determined by reverse phase high performance liquid chromatography, and wherein the drug product has no detectable deterioration of collagenase I or collagenase II for at least 10 days when tested by SDS-PAGE gel electrophoresis.