COMPOSITIONS AND METHODS FOR TREATING COLLAGEN-MEDIATED DISEASES

1 . A method of purifying collagenase I and collagenase II from Clostridium histolyticum , comprising the steps of:

a. fermenting Clostridium histolyticum to obtain a crude harvest comprising collagenase I and collagenase II; and

b. subjecting the crude harvest to column chromatography in the presence of leupeptin.

2 . The method of claim 1 , wherein the column chromatography comprises ion exchange chromatography.

3 . The method of claim 1 , wherein the method comprises subjecting the crude harvest to hydrophobic interaction chromatography (HIC) as a product capture step under conditions suitable for collagenase I and collagenase II binding to the HIC column prior to step (b), and wherein the column chromatography of step (b) comprises ion exchange chromatography.

4 . The method of claim 1 , wherein the purified collagenase I is at least 95% by area pure as determined by reverse phase high performance liquid chromatography.

5 . The method of claim 1 , wherein the purified collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography.

6 . The method of claim 7 , wherein the purified collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography.

7 . The method of claim 1 , wherein the purified collagenase I is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.

8 . The method of claim 1 , wherein the purified collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.

9 . The method of claim 7 , wherein the purified collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.

10 . The method of claim 1 , wherein step (a) comprises conducting cell bank preparations in the presence of phytone peptone or vegetable peptone.

11 . The method of claim 1 , wherein step (a) 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).

12 . Isolated and purified collagenase I purified by the method of claim 1 .

13 . The isolated and purified collagenase I of claim 12 wherein the collagenase I is at least 95% by area pure as determined by reverse phase high performance liquid chromatography.

14 . The isolated and purified collagenase I of claim 12 wherein the collagenase I is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.

15 . The isolated and purified collagenase I of claim 12 , wherein the collagenase I contains less than about 1% by area of clostripain as determined by reverse phase high performance liquid chromatography.

16 . Isolated and purified collagenase II purified by the method of claim 1 .

17 . The isolated and purified collagenase II of claim 16 , wherein the collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography.

18 . The isolated and purified collagenase II of claim 16 , wherein the collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.

19 . The isolated and purified collagenase II of claim 16 , wherein the collagenase II contains less than about 1% by area of clostripain as determined by reverse phase high performance liquid chromatography.

20 . 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 process 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.

21 . A drug product comprising purified collagenase I and collagenase II wherein the collagenase I and collagenase II are purified according to the method of claim 1 , wherein the collagenase I and collagenase II have a mass ratio of about 1 to 1.

22 . A drug product consisting of purified collagenase I and collagenase II, wherein the collagenase I and collagenase II are purified according to the method of claim 1 , wherein the collagenase I and collagenase II are present at a mass ratio of about 1 to 1.

23 . A drug product comprising purified collagenase I and collagenase II wherein the collagenase I and collagenase II are purified according to the method of claim 20 , wherein the collagenase I and collagenase II have a mass ratio of about 1 to 1.

24 . A drug product consisting of purified collagenase I and collagenase II, wherein the collagenase I and collagenase II are purified according to the method of claim 20 , wherein the collagenase I and collagenase II are present at a mass ratio of about 1 to 1.