Compositions and Methods for Treating Collagen-Mediated Diseases
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 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;
b. 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; and
c. eluting collagenase I and II from the HIC column.
2 . The method of claim 1 , wherein ammonium sulphate is added to the crude harvest prior to the HIC product capture step and wherein the collagenase I and collagenase II are eluted with a solution containing no ammonium sulphate.
3 . The method of claim 1 , wherein the crude harvest is filtered through an anion exchange filter prior to step (b).
4 . The method of claim 1 , wherein leupeptin is added to the eluate from the HIC column.
5 . The method of claim 1 , comprising the steps of:
i) filtering the crude harvest obtained in step (a) through an anion exchange filter;
ii) adding ammonium sulphate;
iii) subjecting the harvest obtained from step (ii) to the HIC column;
v) conducting the eluting step (c) with a solution containing no ammonium sulfate;
vi) adding leupeptin to the eluate obtained from step (v); and
vii) separating collagenase I and collagenase II using ion-exchange chromatography;
vii) combining the collagenase I and collagenase II purified from step (vii) at a ratio of about 1 to 1.
6 . 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.
7 . 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.
8 . The method of claim 6 , wherein the purified collagenase II is at least 95% by area pure as determined by reverse phase high performance liquid chromatography.
9 . 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.
10 . 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.
11 . The method of claim 9 , wherein the purified collagenase II is at least 97% by area pure as determined by reverse phase high performance liquid chromatography.
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 I 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 I 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 17 , wherein the collagenase I contains less than about 1% by area of clostripain as determined by reverse phase high performance liquid chromatography.
20 . A drug product comprising isolated and purified collagenase I and collagenase II, wherein the collagenase I and collagenase II are purified according to the method of claim 1 , and wherein the collagenase I and collagenase II have a mass 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 5 , and 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 , and wherein the collagenase I and collagenase II are present at a mass ratio of about 1 to 1.
23 . 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 5 , and wherein the collagenase I and collagenase II are present at a mass ratio of about 1 to 1.