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 . A drug product consisting of collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, having a mass ratio of about 1 to 1 with a purity of at least 95% by area. 
     
     
         2 . The drug product of  claim 1 , wherein the drug product has a SRC assay activity for collagenase I of about 13,000 to about 23,000 fSRC units/mg, and a GPA assay activity for collagenase II of about 200,000 to about 380,000 fGPA units/mg, when the drug product is in 10 mM Tris buffer and 60 mM sucrose at a pH of about 8. 
     
     
         3 . The drug product of  claim 1 , wherein the drug product contains less than about 2% by area aggregated protein. 
     
     
         4 . The drug product of  claim 3 , wherein the drug product contains less than about 1% by area of clostripain. 
     
     
         5 . The drug product of  claim 4 , wherein the drug product contains less than about 1% by area of gelatinase. 
     
     
         6 . The drug product of  claim 5 , wherein the drug product contains less than about 1 ug/mg (w/w) of leupeptin. 
     
     
         7 . The drug product of  claim 6 , wherein having a bioburden less than 1 cfu/ml, and wherein the drug product sterilized. 
     
     
         8 . The drug product of  claim 7 , containing less than 10 EU/ml of endotoxin. 
     
     
         9 . The drug product of  claim 7 , containing less than 5 EU/mg of endotoxin. 
     
     
         10 . The drug product of  claim 1 , wherein the collagenase I and II have a purity of a least about 97% by area. 
     
     
         11 . The drug product of  claim 1 , further comprising a pharmaceutically acceptable excipient. 
     
     
         12 . The drug product of  claim 1 , wherein the drug product is a sterile lyophilized powder is stored at a temperature of about 5° C. 
     
     
         13 . The drug product of  claim 11 , wherein the drug product is a lyophilized injectable composition formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         14 . The drug product of  claim 13 , wherein the drug product is a lyophilized injectable composition 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 a vial fill volume is about 0.9 mL. 
     
     
         15 . The drug product of  claim 13 , wherein the drug product is a lyophilized injectable composition formulation comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         16 . A kit comprising a vial for providing the drug product according to  claim 11  and instructions explaining how to deliver said drug product with said device. 
     
     
         17 . The drug product of  claim 1 , wherein the drug product is used to treat a subject suffering from a collagen-mediated disease. 
     
     
         18 . A drug product consisting of collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, having a mass ratio of about 1 to 1 with a purity of at least 95% by area, wherein the preparation of the drug product comprising 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; and   d) combining the collagenase I and collagenase II purified from step (c) at a ratio of about 1 to 1.   
     
     
         19 . The drug product of  claim 18 , wherein the drug product has a SRC assay activity for collagenase I of about 13,000 to about 23,000 fSRC units/mg, and a GPA assay activity for collagenase II of about 200,000 to about 380,000 fGPA units/mg. 
     
     
         20 . The drug product of  claim 18 , wherein the purity is at least 97% by area. 
     
     
         21 . The drug product of  claim 18 , wherein the purity is at least 98% by area. 
     
     
         22 . The drug product of  claim 18 , wherein cell bank preparations are conducted in the presence of phytone peptone or vegetable peptone. 
     
     
         23 . The drug product of  claim 18 , wherein the fermentation step comprises the steps of:
 a) inoculating the 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);   e) inoculating the medium in a third stage with aliquots resulting from step (d) and agitating;   f) incubating mixtures from step (e);   g) inoculating the medium in a fourth stage with an aliquot resulting from step (f) and agitating;   h) incubating mixtures from step (g); and   i) harvesting culture resulting from step (h) by filtration.   
     
     
         24 . The drug product of  claim 18 , wherein the purification step comprises the steps of:
 a) filtering the crude harvest through a Mustang Q column;   b) adding ammonium sulphate;   c) filtering the crude harvest;   d) subjecting the filtrate through a HIC column;   e) adding leupeptin to the filtrate;   f) removing the ammonium sulfate and maintaining leupeptin for correct binding of collagenase I and collagenase II with buffer exchange by TFF;   g) filtering the mixture of step (f);   h) separating collagenase I and collagenase II using Q-Sepharose HP;   i) preparing TFF concentration and formulation for collagenase I and collagenase II separately; and   j) filtering through a 0.2 μm filtration system.   
     
     
         25 . The drug product of  claim 18 , wherein the drug product is stored at a temperature of about −70° C. 
     
     
         26 . The drug product of  claim 18 , further comprising a pharmaceutically acceptable excipient. 
     
     
         27 . The drug product of  claim 18 , wherein the drug product is a sterile lyophilized powder and is stored at a temperature of about 5° C. 
     
     
         28 . The drug product of  claim 26 , wherein the drug product is a lyophilized injectable composition formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         29 . The drug product of  claim 28 , wherein the drug product is a lyophilized injectable composition 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 a vial fill volume is about 0.9 mL. 
     
     
         30 . The drug product of  claim 28 , wherein the drug product is a lyophilized injectable composition formulation comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         31 . A kit comprising a vial for providing the drug product according to  claim 26  and instructions explaining how to deliver said drug product with said device. 
     
     
         32 . The drug product of  claim 18 , wherein the drug product is used to treat a subject suffering from a collagen-mediated disease. 
     
     
         33 . A process for producing a drug product according to  claim 1 , comprising 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; and   d) combining the collagenase I and collagenase II purified from step (c) at a ratio of about 1 to 1.   
     
     
         34 . The process of  claim 33 , wherein the drug product has a SRC assay activity for collagenase I of about 13,000 to about 23,000 fSRC units/mg, and a GPA assay activity for collagenase II of about 200,000 to about 380,000 fGPA units/mg. 
     
     
         35 . The drug product of  claim 33 , wherein the drug product has a purity of at least 97% by area. 
     
     
         36 . The drug product of  claim 33 , wherein the drug product has a purity of at least 98% by area. 
     
     
         37 . The process of  claim 33 , wherein cell bank preparations are conducted in the presence of phytone peptone or vegetable peptone. 
     
     
         38 . The process of  claim 33 , wherein the fermentation step comprises the steps of:
 a) inoculating the 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);   e) inoculating the medium in a third stage with aliquots resulting from step (d) and agitating;   f) incubating mixtures from step (e);   g) inoculating the medium in a fourth stage with an aliquot resulting from step (f) and agitating;   h) incubating mixtures from step (g); and   i) harvesting culture resulting from step (h) by filtration.   
     
     
         39 . The process of  claim 33 , wherein the purification step comprises the steps of:
 a) filtering the crude harvest through a Mustang Q column;   b) adding ammonium sulphate;   c) filtering the crude harvest;   d) subjecting the filtrate through a HIC column;   e) adding leupeptin to the filtrate;   f) removing the ammonium sulfate and maintaining leupeptin for correct binding of collagenase I and collagenase II with buffer exchange by TFF;   g) filtering the mixture of step (f);   h) separating collagenase I and collagenase II using Q-Sepharose HP;   i) preparing TFF concentration and formulation for collagenase I and collagenase II separately; and   j) filtering through a 0.2 μm filtration system.   
     
     
         40 . The process of  claim 33 , wherein the drug product is stored at a temperature of about −70° C. 
     
     
         41 . The drug product of  claim 33 , further comprising a pharmaceutically acceptable excipient. 
     
     
         42 . The drug product of  claim 33 , wherein the drug product is a sterile lyophilized powder is stored at a temperature of about 5° C. 
     
     
         43 . The drug product of  claim 33 , wherein the drug product is a lyophilized injectable composition formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         44 . The drug product of  claim 43 , wherein the drug product is a lyophilized injectable composition 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 a vial fill volume is about 0.9 mL. 
     
     
         45 . The drug product of  claim 43 , wherein the drug product is a lyophilized injectable composition formulation comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         46 . A kit comprising a vial for providing the drug product according to  claim 41  and instructions explaining how to deliver said drug product with said device. 
     
     
         47 . The process of  claim 33 , wherein the drug product is used to treat a subject suffering from a collagen-mediated disease.