Method of purifying and isolating 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. A method of purifying collagenase I and collagenase II, the method comprising:
treating the collagenase I and collagenase II with a salt selected from ammonium sulfate, sodium chloride, potassium chloride, sodium sulfate, and mixtures thereof and forming a collagenase I and collagenase II solution, wherein the salt comprises less than about 1.2 ppm of nickel and less than about 84.4 ppm of zinc;
fractionating the collagenase I and collagenase II solution using chromatography; and
separating a plurality of highly pure fractions of the collagenase I and a plurality of highly pure fractions of collagenase II from fractions of neutral protease, and
mixing the highly pure fractions of the collagenase I and the highly pure fractions of the collagenase II, wherein the mixture has less than 50 ng neutral protease per mg of the collagenase mixture, to thereby purify the collagenase.
2. The method of claim 1 , wherein the salt further comprises low levels of metals selected from aluminum, arsenic, calcium, cadmium, chromium, copper, iron, magnesium, and lead.
3. The method of claim 1 , wherein the salt comprises nickel in an amount of less than about 1.0 ppm, 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, or less than about 0.01 ppm.
4. The method of claim 1 , wherein the salt comprises zinc in an amount of less than about 80 ppm, less than about 50 ppm, less than about 25 ppm, less than about 10 ppm, less than about 5 ppm, less than about 3 ppm, or less than about 1 ppm.
5. The method of claim 2 , wherein the salt comprises less than about 0.75 ppm aluminum, less than about 0.2 ppm arsenic, less than about 10 ppm calcium, less than about 1 ppm cadmium, less than about 0.75 ppm chromium, less than about 1 ppm copper, less than about 2 ppm iron, less than about 5 ppm magnesium, or less than about 2 ppm lead.
6. The method of claim 1 , wherein the separating step comprises testing each fraction of the collagenase on an SDS-PAGE gel and pooling only fractions of the collagenase that are free from detectable amounts of neutral protease on the gel.
7. The method of claim 1 , wherein the purified collagenase I and collagenase II is free from detectable amounts of neutral protease.
8. The method of claim 1 , wherein the salt is ammonium sulfate.
9. The method of claim 8 , wherein the ammonium sulfate has a concentration of about 0.8 M to about 1.2 M.
10. The method of claim 9 , wherein prior to the treating, the method comprises the steps of:
a. harvesting a crude fermentation comprising the collagenase; and
b. filtering the crude fermentation over an anion exchange filter.
11. The method of claim 10 , wherein the chromatography comprises hydrophobic interaction chromatography, ion-exchange chromatography, or a combination thereof.
12. The method of claim 1 , wherein the collagenase is derived from Clostridium histolyticum.