COMPOSITIONS AND METHODS FOR TREATING COLLAGEN-MEDIATED DISEASES

1 - 47 . (canceled)

48 . A drug product consisting of 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:

a) are obtained from Clostridium histolyticum grown in the absence of bovine-derived media; and

b) have a mass ratio of about 1 to 1;

and the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.

49 . The drug product of claim 48 , wherein the Clostridium histolyticum are grown in the presence of porcine-derived proteose peptone.

50 . The drug product consisting of 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 the drug product is at least 97% 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 in the absence of bovine-derived media;

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 hydrophobic interaction chromatography (HIC) column;

iv. adding leupeptin to the filtrate;

v. removing the ammonium sulphate;

vi. filtering the mixture of step (v); and

vii. separating collagenase I and collagenase II using ion-exchange; and

d) combining the collagenase I and collagenase II purified from step (c) at a ratio of about 1 to 1.

51 . The drug product of claim 50 , wherein the Clostridium histolyticum are fermented in the presence of porcine-derived proteose peptone.

52 . The drug product of claim 50 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media.

53 . The drug product of claim 52 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone.

54 . The drug product of claim 50 , wherein the fermentation step comprises the steps of:

i) inoculating a non-bovine-derived 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 the mixtures from step (iii) to obtain an aliquot;

v) inoculating the medium in a third stage with aliquots from step (iv) and agitating;

vi) incubating the 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

vii) incubating mixtures from step (vii).

55 . The drug product of claim 54 , wherein the non-bovine derived medium comprises porcine-derived proteose peptone.

56 . The drug product of claim 54 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media.

57 . The drug product of claim 56 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone.

58 . A process for producing a drug product consisting of 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 the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, comprising the steps of:

a) fermenting Clostridium histolyticum in the absence of bovine-derived media;

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 hydrophobic interaction chromatography (HIC) column;

iv. adding leupeptin to the filtrate;

v. removing the ammonium sulphate;

vi. filtering the mixture of step (v); and

vii. separating collagenase I and collagenase II using ion-exchange; and

d) combining the collagenase I and collagenase II purified from step (c) at a ratio of about 1 to 1.

59 . The process of claim 58 , wherein the Clostridium histolyticum are fermented in the presence of porcine-derived proteose peptone.

60 . The process of claim 58 , wherein the process further comprises the step of conducting cell bank preparations in the absence of bovine-derived media.

61 . The process of claim 60 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone.

62 . The process of claim 58 , wherein the fermentation step comprises the steps of:

i) inoculating a non-bovine-derived 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 the mixtures from step (iii) to obtain an aliquot;

v) inoculating the medium in a third stage with aliquots from step (iv) and agitating;

vi) incubating the 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

vii) incubating mixtures from step (vii).

63 . The process of claim 62 , wherein the medium comprises porcine-derived proteose peptone.

64 . The process of claim 63 , wherein preparation of the drug product further comprises the step of conducting cell bank preparations in the absence of bovine-derived media.

65 . The process of claim 64 , wherein the cell bank preparations are conducted in the presence of porcine-derived proteose peptone.

66 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and a drug product consisting of 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:

a) are obtained from Clostridium histolyticum grown in the absence of bovine-derived media; and

b) have a mass ratio of about 1 to 1; and

the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.

67 . The pharmaceutical formulation of claim 66 , wherein the Clostridium histolyticum is grown in the presence of porcine-derived proteose peptone.

68 . A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a drug product consisting of 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 II:

a) are obtained from Clostridium histolyticum grown in the absence of bovine-derived media; and

b) have a mass ratio of about 1 to 1 and;

the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography, wherein the preparation of the drug product comprises the steps of:

i. fermenting Clostridium histolyticum in the absence of bovine-derived media;

ii. harvesting a crude fermentation comprising collagenase I and collagenase II;

iii. purifying collagenase I and collagenase II from the crude harvest via filtration and column chromatography comprising the steps of:

1. filtering the crude harvest through an anion exchange filter;

2. adding ammonium sulphate;

3. subjecting the harvest through a hydrophobic interaction chromatography (HIC) column;

4. adding leupeptin to the filtrate;

5. removing the ammonium sulphate;

6. filtering the mixture of step (5); and

7. separating collagenase I and collagenase II using ion-exchange; and

iv. combining the collagenase I and collagenase II purified from step (iii) at a ratio of about 1 to 1.

69 . The pharmaceutical composition of claim 68 , wherein the Clostridium histolyticum are fermented in the presence of porcine-derived proteose peptone.

70 . The pharmaceutical composition of claim 68 , wherein the process further comprises the step of conducting cell bank preparations in the absence of bovine-derived media.

71 . The pharmaceutical composition of claim 70 , wherein the cell bank preparations are conducted in the presence of proteose peptone.

72 . 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;

a) are obtained from Clostridium histolyticum grown in the absence of bovine-derived media; and

b) have a mass ratio of about 1 to 1; and

the drug product is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.

73 . The drug product of claim 72 , wherein the Clostridium histolyticum are grown in the presence of porcine-derived proteose peptone.