Claims for Patent: 5,989,888

Summary for Patent: 5,989,888

Title:	Purified mixture of collagenase I, collagenase II and two other proteases
Abstract:	A purified enzyme mixture useful for isolating cells or cell clusters from tissue is disclosed. The mixture includes at least two collagenase enzymes, at least two other proteases, and additional non-protease components. The mixture is purified by removing at least some of the non-protease components. The purified mixture may then be used to isolate cells or cell clusters from tissue. Also disclosed are the essential components of the purified enzyme mixture as well as preferred ranges and ratios of these essential components for isolating cells or cell clusters from tissue. Finally, a dissociation system is disclosed that can be used with the purified enzyme mixture to dissociate tissue and recover cells or cell clusters.
Inventor(s):	Dwulet; Francis E. (Greenwood, IN), Ellis; Bernice B. (Greenfield, IN), Gill; John F. (Indianapolis, IN), Jacobsen; Linda B. (Indianapolis, IN), Smith; Marilyn E. (McCordsville, IN), Waters; David G. (Indianapolis, IN)
Assignee:	Roche Diagnostics Corporation (Indianapolis, IN)
Application Number:	09/042,295
Patent Claims:	1. An enzyme composition prepared by combining defined amounts of proteases, said composition comprising collagenase I having a Wunsch activity of about 0.3 to about 0.7 U/mg from C. histolyticum and collagenase II having a Wunsch activity of about 7.5 to about 10.0 U/mg from C. histolyticum, and two other proteases, wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of the collagenase II in the composition is about 0.3 to about 0.6. 2. The enzyme composition of claim 1 wherein at least one of the two other proteases is a neutral protease. 3. The enzyme composition of claim 2 wherein the neutral protease is selected from the group consisting of thermolysin, dispase and C. histolyticum neutral protease. 4. The enzyme composition of claim 2 wherein the neutral protease is thermolysin. 5. The enzyme composition of claim 4 wherein the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch units of activity of the masses of collagenase I and collagenase II in the composition is about 19:1 to about 70:1. 6. The enzyme composition of claim 2 wherein the neutral protease is dispase. 7. The enzyme composition of claim 6 wherein the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch units of activity of the masses of collagenase I and collagenase II in the composition is about 19:1 to about 62:1. 8. The enzyme composition of claim 2 wherein the neutral protease is dispase or thermolysin and the ratio of total FITC casein activity of the enzyme composition to the total Wunsch units of the masses of collagenase I and collagenase II in the composition is about 41:1. 9. The enzyme composition of claim 2 wherein the two other proteases are a neutral protease and clostripain. 10. The enzyme composition of claim 9 wherein the neutral protease is selected from the group consisting of thermolysin, dispase and C. histolyticum neutral protease. 11. The enzyme composition of claim 9 wherein the neutral protease is thermolysin. 12. The enzyme composition of claim 1 wherein the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch units of activity of the masses of collagenase I and collagenase II in the composition is about 19:1 to about 70:1. 13. The enzyme composition of claim 9 wherein the neutral protease is dispase. 14. The enzyme composition of claim 13 wherein the ratio of the total FITC casein activity of the enzyme composition to the total Wunsch units of activity of the masses of collagenase I and collagenase II in the composition is about 19:1 to about 62:1. 15. The enzyme composition of claim 9 wherein the neutral protease is dispase or thermolysin and the ratio of total FITC casein activity of the enzyme composition to the total Wunsch units of the masses collagenase I and collagenase II in the composition is about 41:1. 16. The composition of claim 2 wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II is about 0.4 to about 0.45. 17. The composition of claim 3 wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II is about 0.4 to about 0.45. 18. The composition of claim 9 wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of collagenase II is about 0.4 to about 0.45. 19. A method for isolating islets cells or islet cell clusters from pancreas, said method comprising the steps of perfusing the pancreas with a solution of an enzyme composition to initiate dissociation of islet cells or islet cell clusters from the pancreas said enzyme composition comprising collagenase I having a Wunsch activity of about 0.3 to about 0.7 U/mg from C. histolyticum and collagenase II having a Wunsch activity of about 7.5 to about 10.0 U/mg from C. histolyticum, and two other proteases, wherein the ratio of the mass of collagenase II to the mass of collagenase I plus the mass of the collagenase II in the composition is about 0.3 to about 0.6; contacting the perfused pancreas with a circulating solution of said enzyme composition; and, isolating the islet cells or islet cell clusters from the dissociation solution. 20. The method of claim 19 wherein at least one of the other two proteases is a neutral protease selected from the group consisting of thermolysin, dispase and C. Histolyticum neutral protease.

Details for Patent 5,989,888

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2016-01-24