US 5,332,503 A · 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.

Claims16

Dependent claims

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.

5 more elements

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.

9 more elements

Dependencies are inferred from the claim text.

Process for purifying collagenase

Life of this patent

Process for purifying collagenase

Life of this patent

Abstract

Claims16