	US 12,448,615 B2
	Method of purifying and isolating collagenase
Christine A Sheaffer, Doylestown, PA (US); Michael Berbaum, Pottstown, PA (US); John Hanna, East Norriton, PA (US); Jason Dziadosz, Quakertown, PA (US); and Daniel Shanafelt, Red Hill, PA (US)
Assigned to Endo Operations Limited, Dublin (IE)
Filed by Endo Operations Limited, Dublin (IE)
Filed on Dec. 22, 2021, as Appl. No. 17/559,782.
Application 17/559,782 is a division of application No. 15/939,231, filed on Mar. 28, 2018, granted, now 11,473,074.
Claims priority of provisional application 62/477,846, filed on Mar. 28, 2017.
Prior Publication US 2022/0119791 A1, Apr. 21, 2022
Int. Cl. C12N 9/52 (2006.01); A61K 31/00 (2006.01); A61K 38/00 (2006.01); G01N 33/569 (2006.01)

CPC C12N 9/52 (2013.01) [C12Y 304/24003 (2013.01); A61K 31/00 (2013.01); A61K 38/00 (2013.01); G01N 33/56911 (2013.01)]

12 Claims

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.