Claims for Patent: 5,332,503

Summary for Patent: 5,332,503

Title:	Process for purifying collagenase
Abstract:	A process for purifying crude collagenase is disclosed. The collagenase purification process includes providing a stabilized crude collagenase solution containing collagenase, pigment, toxins, bacterial materials, and proteolytic enzyme impurities including clostripain, trypsin, and caseinase. The stabilized collagenase solution is applied to hydroxylapatite packing. pigment and caseinase are eluted with a first solution comprising about 0.05 M to about 0.3 M phosphate buffer, and then collagenase, trypsin, and clostripain are eluted with a second solution comprising about 0.35 M to about 0.5 M phosphate buffer to provide a first collected solution. The first collected solution is then applied to gel filtration packing and collagenase and clostripain are eluted with a neutral pH buffer solution, to provide a second collected solution. The second collected solution is then applied to Reactive Red 120-Agarose packing and collagenase is eluted with a neutral pH buffer solution to provide purified collagenase. The process provides extremely pure collagenase in high yield with reduced consumption of eluting solutions and avoids unpredictable gradient eluting techniques.
Inventor(s):	Lee; Catherine (Laguna Hills, CA), Hornacek; Cynthia (Trabuco Cyn., CA), Dinh; Tan T. (Garden Grove, CA)
Assignee:	Baxter International Inc. (Deerfield, IL)
Application Number:	08/073,272
Patent Claims:	1. A process for purifying crude collagenase compositions containing collagenase, pigment, toxins, bacterial material, and proteolytic enzyme impurities including clostripain, trypsin, and caseinase, said process comprising the steps of: applying a stabilized crude collagenase solution to a column containing hydroxylapatite packing; eluting pigment and caseinase from the hydroxylapatite packing with a first solution comprising about 0.05 M to about 0.3 M phosphate, buffered to about pH 6 to about pH 8, and a nonionic surfactant; eluting collagenase, clostripain, and trypsin with a second solution comprising about 0.35 M to about 0.5 M phosphate, buffered to about pH 6 to about pH 8, and a nonionic surfactant, to provide a first collected solution; applying said first collected solution to a column containing gel filtration packing; eluting collagenase and clostripain with a third solution comprising a neutral pH buffer, to provide a second collected solution; applying said second collected solution to a column containing Reactive Red 120-Agarose packing; and eluting collagenase with a fourth solution comprising a neutral pH buffer, to provide a solution comprising purified collagenase. 2. The process of claim 1 wherein said stabilized crude collagenase solution comprises collagenase, CaCl.sub.2, and arginine. 3. The process of claim 1 further comprising the step of adding deoxyribonuclease to said stabilized crude collagenase solution in an amount sufficient to provide a deoxyribonuclease activity of about 40 u/mL. 4. The process of claim 1 further comprising the steps of: dialyzing said stabilized crude collagenase solution against a solution comprising about 0.15 M potassium phosphate, about 5 mM arginine, and about 1 mM CaCl.sub.2, buffered to about pH 6.7, to provide a first dialysate; and centrifuging said first dialysate to provide a supernatant comprising dialyzed and stabilized crude collagenase solution. 5. The process of claim 4 further comprising the steps of: concentrating said first collected solution; dialyzing the concentrated first collected solution with a solution comprising NaCl, Tricine, CaCl.sub.2, and arginine, buffered to about pH 7.5, to provide a second dialysate; and centrifuging said second dialysate to provide a supernatant comprising dialyzed first collected solution. 6. The process of claim 1 wherein said first solution comprises about 0.15 M potassium phosphate, about 0.01 wt. % nonionic surfactant, and about 1 mM CaCl.sub.2, buffered to about pH 6.7. 7. The process of claim 1 wherein said second solution comprises about 0.4 M potassium phosphate, about 0.01 wt. % nonionic surfactant, and about 1 mM CaCl.sub.2, buffered to about pH 6.7. 8. The process of claim 1 wherein said third solution comprises about 1.5 M NaCl, about 5mM CaCl.sub.2, and about 5 mM Tricine, buffered to about pH 7.5. 9. The process of claim 1 wherein said fourth solution comprises about 1.5 M NaCl, about 5mM CaCl.sub.2, and about 5 mM Tricine, buffered to about pH 7.5. 10. An process for purifying collagenase compositions comprising the steps of: digesting a crude collagenase solution containing collagenase, pigment, toxins, bacterial materials, and proteolytic enzyme impurities, including clostripain, trypsin, and caseinase, in a solution comprising magnesium chloride, potassium phosphate, and deoxyribonuclease; dialyzing the digested crude collagenase solution against a solution comprising about 0.15 M potassium phosphate, about 5 mM arginine, and about 1 mM CaCl.sub.2, buffered to about pH 6.7, to provide a dialysate; centrifuging said dialysate to provide a supernatant; applying said supernatant to a hydroxylapatite packed chromatography column; eluting pigment with a first solution comprising about 0.15 M potassium phosphate, about 0.01 wt. % nonionic surfactant, and about 1 mM CaCl.sub.2, buffered to about pH 6.7; eluting proteolytic enzyme impurities and collagenase with a second solution comprising about 0.4 M potassium phosphate, about 0.01 wt. % nonionic surfactant, and about 1 mM CaCl.sub.2, buffered to about pH 6.7, to provide a first collected solution; applying said first collected solution to a gel filtration packed chromatography column; eluting collagenase and clostripain with a third solution comprising about 1.5 M NaCl, about 5 mM CaCl.sub.2, and about 5mM Tricine, buffered to about pH 7.5, to provide a second collected solution; applying said second collected solution to a Reactive Red 120-Agarose packed chromatography column; and eluting purified collagenase with a fourth solution comprising about 1.5 M NaCl, about 5mM CaCl.sub.2, and about 5mM Tricine, buffered to about pH 7.5. 11. The process of claim 10 further comprising the step of regenerating said hydroxylapatite packing with sodium hydroxide. 12. The process of claim 10 further comprising the step of regenerating said gel filtration packing with sodium hydroxide. 13. The process of claim 10 further comprising the steps of: regenerating said Reactive Red 120-Agarose packing first with sodium chloride and then with urea. 14. The process of claim 10 further comprising the steps of: collecting fractions eluted from hydroxylapatite; assaying said fractions for collagenase, trypsin, and clostripain activity; and pooling collected fractions having a collagenase activity greater than 15 .mu.kat/L and a ratio of clostripain activity to collagenase activity less than or equal to 0.25, to provide said first collected solution. 15. The process of claim 10 further comprising the steps of: collecting fractions eluted from gel filtration packing; assaying said fractions for collagenase, trypsin, and clostripain activity; and pooling collected fractions having a collagenase activity greater than 1.240 .mu.kat/L or a ratio of clostripain activity to collagenase activity less than or equal to 2.0 and trypsin activity less than 1.170 .mu.kat/L, to provide said second collected solution. 16. The process of claim 10 further comprising the steps of: collecting fractions eluted from Reactive Red 120-Agarose packing; assaying said fractions for collagenase, trypsin, and clostripain activity; and pooling collected fractions having a collagenase activity greater than 1.24 .mu.kat/L.

Details for Patent 5,332,503

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2039-03-29