Method of producing collagenase
The present invention relates to the fields of collagenase production and collagenase products, and particularly to improving the reproducibility, purity, and stability of collagenase I and collagenase II compositions, where the compositions are pure to at least 95% by area as measured by reverse phase high pressure liquid chromatography (RP-HPLC) and essentially free of neutral protease.
1. An isolated and purified collagenase I and collagenase II composition obtained from Clostridium histolyticum , wherein the collagenase I and collagenase II have a mass ratio of about 1 to 1, and the composition is at least 95% by area pure as determined by reverse phase high performance liquid chromatography, wherein the composition is essentially free of delta toxin, and wherein the composition is made by a process comprising the steps of:
a. fermenting Clostridium histolyticum;
b. harvesting a crude fermentation comprising collagenase I and collagenase II; and
c. purifying collagenase I and collagenase II from the crude harvest via filtration and column chromatography comprising treating the collagenase I and collagenase II with an ammonium sulfate solution having a zinc level of less than about 80 ppm and a nickel level less than about 1.2 ppm.
2. The composition of claim 1 , wherein the process is performed in the presence of low levels of a metal selected from the group consisting of nickel, zinc, aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium, and lead.
3. The composition of claim 1 , wherein the nickel is present in an amount less than about 0.5 ppm.
4. The composition of claim 1 , wherein the nickel is present in an amount selected from the group consisting of less than about 0.9 ppm, less than about 0.8 ppm, less than about 0.7 ppm, less than about 0.6 ppm, less than about 0.5 ppm, less than about 0.4 ppm, less than about 0.3 ppm, less than about 0.2 ppm, less than about 0.1 ppm, less than about 0.09 ppm, less than about 0.08 ppm, less than about 0.07 ppm, less than about 0.06 ppm, less than about 0.05 ppm, less than about 0.04 ppm, less than about 0.03 ppm, less than about 0.02 ppm, and less than about 0.01 ppm.
5. The composition of claim 1 , wherein the zinc is present in an amount of less than about 1 ppm.
6. The composition of claim 1 , wherein the process further comprises a delta toxin elimination step comprising passing a collagenase filtrate through a separation column or filter.