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 μg/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 at 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 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 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, 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 stored at 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. 
   
   
       48 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and a drug product consisting of 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 characterized by a purity of at least 97% by area as determined by reverse phase high performance liquid chromatography. 
   
   
       49 . The pharmaceutical formulation of  claim 48 , wherein the drug product is a sterile lyophilized powder and is stored at a temperature of about 5° C. 
   
   
       50 . The pharmaceutical formulation of  claim 48 , wherein the formulation is a lyophilized injectable composition formulated with Sucrose, Tris and with a pH level of about 8.0. 
   
   
       51 . The pharmaceutical formulation of  claim 50 , wherein the formulation 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 vial fill volume is about 0.9 mL. 
   
   
       52 . The pharmaceutical formulation of  claim 50 , wherein the formulation 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. 
   
   
       53 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a drug product consisting of 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 characterized by a purity of at least 97% 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 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.   
   
   
       54 . The pharmaceutical formulation of  claim 53 , wherein the drug product is a sterile lyophilized powder. 
   
   
       55 . The pharmaceutical formulation of  claim 54 , wherein the formulation is a lyophilized injectable composition formulated with sucrose, Tris and with a pH level of about 8.0. 
   
   
       56 . The pharmaceutical formulation of  claim 55 , wherein the formulation 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 vial fill volume is about 0.9 mL. 
   
   
       57 . The pharmaceutical formulation of  claim 55 , 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. 
   
   
       58 . 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 have a mass ratio of about 1 to 1 and the drug product is characterized by a purity of at least 97% by area as determined by reverse phase high performance liquid chromatography. 
   
   
       59 . The process of  claim 33 , wherein the purification step comprises the steps of:
 a) filtering the crude harvest through an anion exchange filter;   b) capturing proteins using a HIC column;   c) subjecting the filtrate obtained in step (b) to tangential flow filtration; and   d) separating collagenase I and collagenase II using anion exchange chromatography and tangential flow filtration.   
   
   
       60 . The process of  claim 33  wherein cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
   
   
       61 . The process of  claim 33  wherein cell bank preparations are conducted in the absence of bovine-derived media. 
   
   
       62 . A drug product consisting of 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 characterized by a purity of at least 97% by area as determined by reverse phase high performance liquid chromatography. 
   
   
       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 μg/mg (w/w) of leupeptin. 
   
   
       67 . The drug product of  claim 62 , wherein having a bioburden less than 1 cfu/ml, and wherein the drug product is sterile. 
   
   
       68 . The drug product of  claim 67 , containing less than 10 EU/ml of endotoxin. 
   
   
       69 . The drug product of  claim 67 , containing less than 5 EU/mg of endotoxin. 
   
   
       70 . A drug product consisting of 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 characterized by a purity of at least 97% 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 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.   
   
   
       71 . The drug product of  claim 70 , wherein the purity is at least 98% by area as determined by reverse phase high performance liquid chromatography. 
   
   
       72 . The drug product of  claim 70 , wherein cell bank preparations are conducted in the presence of phytone peptone or vegetable peptone. 
   
   
       73 . The drug product of  claim 70 , 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.   
   
   
       74 . The drug product of  claim 70 , wherein the purification step comprises the steps of:
 a) filtering the crude harvest through an anion exchange filter a Mustang Q column;   b) adding ammonium sulphate;   c) subjecting the harvest through a HIC column;   d) adding leupeptin to the filtrate;   e) removing the ammonium sulfate and maintaining leupeptin for correct binding of collagenase I and collagenase II with buffer exchange by tangential flow filtration (TFF);   f) filtering the mixture of step (e);   g) separating collagenase I and collagenase II using Q-Sepharose HP   h) preparing TFF concentration and formulation for collagenase I and collagenase II separately; and   i) filtering through a 0.2 um filtration system.   
   
   
       75 . The drug product of  claim 70 , wherein the drug product is stored at a temperature of about −70° C. 
   
   
       76 . A process for producing a drug product consisting of 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 characterized by a purity of at least 97% by area 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 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.   
   
   
       77 . The process of  claim 76 , wherein the drug product has a purity of at least 98% by area as determined by reverse phase high performance liquid chromatography. 
   
   
       78 . The process of  claim 76 , wherein cell bank preparations are conducted in the presence of phytone peptone or vegetable peptone. 
   
   
       79 . The process of  claim 76 , 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.   
   
   
       80 . The process of  claim 76 , wherein the purification step comprises the steps of:
 a) filtering the crude harvest through an anion exchange filter;   b) adding ammonium sulphate;   c) subjecting the harvest through a HIC column;   d) adding leupeptin to the filtrate;   e) removing the ammonium sulfate and maintaining leupeptin for correct binding of collagenase I and collagenase II with buffer exchange by tangential flow filtration (TFF);   f) filtering the mixture of step (e);   g) separating collagenase I and collagenase II using Q-Sepharose HP   h) preparing TFF concentration and formulation for collagenase I and collagenase II separately; and   i) filtering through a 0.2 um filtration system.   
   
   
       81 . An injectable drug product consisting essentially of collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, wherein said drug product is prepared by a method 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 which can be easily and efficiently determined and controlled. 
   
   
       82 . The drug product of  claim 81 , wherein the collagenase I has a purity of at least 95% by area. 
   
   
       83 . The drug product of  claim 81 , wherein the collagenase I has a purity of at least 97% by area. 
   
   
       84 . The drug product of  claim 81 , wherein the collagenase I has a purity of at least 98% by area. 
   
   
       85 . The drug product of  claim 81 , wherein the collagenase II has a purity of at least 95% by area. 
   
   
       86 . The drug product of  claim 81 , wherein the collagenase II has a purity of at least 97% by area. 
   
   
       87 . The drug product of  claim 81 , wherein the collagenase II has a purity of at least 98% by area. 
   
   
       88 . The drug product of  claim 81 , wherein the collagenase I and the collagenase II have a purity of at least 95% by area. 
   
   
       89 . The drug product of  claim 81 , wherein the collagenase I and the collagenase II have a purity of at least 97% by area. 
   
   
       90 . The drug product of  claim 81 , wherein the collagenase I and the collagenase II have a purity of at least 98% by area. 
   
   
       91 . The drug product of  claim 81 , wherein the collagenase I and collagenase II are combined at an optimized fixed mass ratio which maximizes synergistic activity provided by said collagenase I and collagenase II. 
   
   
       92 . An injectable drug product consisting essentially of collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, wherein said collagenase I and collagenase II have a mass ratio of about 1 to 1 and are characterized by a purity of at least 95%. 
   
   
       93 . An injectable drug product comprising collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, wherein said collagenase I and collagenase II are combined at a ratio which can be easily and efficiently determined and controlled and are characterized by a purity of at least 95%, and wherein said drug product is substantially free of non-collagenase enzymes. 
   
   
       94 . An injectable drug product consisting essentially of collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, wherein said drug product is prepared by a method 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 to a purity of at least 95%; and d) combining the collagenase I and collagenase II purified from step (c) at a ratio which can be easily and efficiently determined and controlled. 
   
   
       95 . An injectable drug product comprising collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, wherein said drug product is prepared by a method 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 to a purity of at least 95%; and d) combining the collagenase I and collagenase II purified from step (c) at a ratio which can be easily and efficiently determined and controlled and wherein said drug product is substantially free of non-collagenase enzymes. 
   
   
       96 . The drug product of  claim 93 , wherein the collagenase I has a purity of at least 95%. 
   
   
       97 . The drug product of  claim 96 , wherein the collagenase I has a purity of at least 97%. 
   
   
       98 . The drug product of  claim 97 , wherein the collagenase I has a purity of at least 98%. 
   
   
       99 . The drug product of  claim 93 , wherein the collagenase II has a purity of at least 95%. 
   
   
       100 . The drug product of  claim 99 , wherein the collagenase II has a purity of at least 97%. 
   
   
       101 . The drug product of  claim 100 , wherein the collagenase II has a purity of at least 98%. 
   
   
       102 . The drug product of  claim 93 , wherein the collagenase I and the collagenase II have a purity of at least 95%. 
   
   
       103 . The drug product of  claim 102 , wherein the collagenase I and the collagenase II have a purity of at least 97%. 
   
   
       104 . The drug product of  claim 103 , wherein the collagenase I and the collagenase II have a purity of at least 98%. 
   
   
       105 . The drug product of  claim 93 , wherein the strain of  Clostridium histolyticum  produces relatively fewer non-collagenase proteins compared to collagenase I and collagenase II proteins. 
   
   
       106 . The drug product of  claim 105 , wherein the strain is “013”. 
   
   
       107 . The drug product of  claim 94 , wherein the harvesting is performed after the peak cell density is reached. 
   
   
       108 . The drug product of  claim 107 , wherein the harvesting is performed 5 to 10 hours after peak cell density is reached. 
   
   
       109 . The drug product of  claim 93 , wherein the drug product is suitable for injection as solution within a lactose carrier medium, and wherein the ratio by mass of drug product to lactose is 1:1.9 for the solution. 
   
   
       110 . The drug product of  claim 93 , wherein the collagenase I and the collagenase II have a purity of at least 98% by SDS PAGE. 
   
   
       111 . The drug product of  claim 93 , wherein the drug product has an SRC assay activity for collagenase I of about 13,000 to about 23,000 FSRC units/mg. 
   
   
       112 . The drug product of  claim 93 , wherein the drug product has a GPA assay activity for collagenase II of about 200,000 to about 380,000 fGPA units/mg. 
   
   
       113 . The process of  claim 33  wherein the fermentation is conducted in the presence of porcine-derived proteose peptone. 
   
   
       114 . The process of  claim 33  wherein the fermentation is conducted in the absence of bovine-derived media. 
   
   
       115 . The process of  claim 76 , wherein the drug product stored at temperature of about −70° C. 
   
   
       116 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and a drug product consisting of 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 characterized by a purity of at least 97% by area as determined by reverse phase high performance liquid chromatography. 
   
   
       117 . The pharmaceutical formulation of  claim 116 , wherein the drug product is a sterile lyophilized powder and is stored at a temperature of about 5° C. 
   
   
       118 . The pharmaceutical formulation of  claim 116 , wherein the formulation is a lyophilized injectable composition formulated with Sucrose, Tris and with a pH level of about 8.0. 
   
   
       119 . The pharmaceutical formulation of  claim 118 , wherein the formulation 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 vial fill volume is about 0.9 mL. 
   
   
       120 . The pharmaceutical formulation of  claim 118 , wherein the formulation 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. 
   
   
       121 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a drug product consisting of 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 characterized by a purity of at least 97% 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 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.   
   
   
       122 . The pharmaceutical formulation of  claim 121 , wherein the drug product is a sterile lyophilized powder. 
   
   
       123 . The pharmaceutical formulation of  claim 122 , wherein the formulation is a lyophilized injectable composition formulated with sucrose, Tris and with a pH level of about 8.0. 
   
   
       124 . The pharmaceutical formulation of  claim 123 , wherein the formulation 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 vial fill volume is about 0.9 mL. 
   
   
       125 . The pharmaceutical formulation of  claim 123 , 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. 
   
   
       126 . 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 have a mass ratio of about 1 to 1 and the drug product is characterized by a purity of at least 97% by area as determined by reverse phase high performance liquid chromatography. 
   
   
       127 . The process of  claim 76 , wherein the purification step comprises the steps of:
 a) filtering the crude harvest through an anion exchange filter;   b) capturing proteins using a HIC column;   c) subjecting the filtrate obtained in step (b) to tangential flow filtration; and   d) separating collagenase I and collagenase II using anion exchange chromatography and tangential flow filtration.   
   
   
       128 . The process of  claim 76  wherein cell bank preparations are conducted in the presence of porcine-derived proteose peptone. 
   
   
       129 . The process of  claim 76  wherein cell bank preparations are conducted in the absence of bovine-derived media.