Method of separating and purifying two active forms of urokinase using affinity chromatography

An affinity chromatography method for the isolation and purification of an active form of urokinase characterized by a molecular weight of approximately 33,400 and a specific activity of approximately 226,000 CTA units/mg protein from a crude urokinase preparation, comprising the steps of:

• a. preparing a solution of the crude urokinase preparation in 0.01 molar sodium phosphate buffer, pH 6.0-9.0;
• b. contacting said crude urokinase solution with an extracting agent comprising agmatine covalently coupled to the surface of a water-insoluble solid support material, whereby urokinase becomes preferentially adsorbed on said extracting agent;
• c. washing said extracting agent with 0.01 molar sodium phosphate buffer, pH 6.0-9.0, so as to remove extraneous material from said extracting agent;
• d. eluting said active form of urokinase from said extracting agent with 0.02 molar sodium phosphate buffer, pH 6.0-9.0, and collecting the resulting eluate in fractions;
• and
• e. recovering said active form of urokinase in a purified state from the high specific activity fractions of said eluate.

An affinity chromatography method for the isolation and purification of an active form of urokinase characterized by a molecular weight of approximately 47,000 and a specific activity of approximately 104,000 CTA units/mg protein from a crude urokinase preparation, comprising the steps of:

• a. preparing a solution of the crude urokinase preparation in 0.01 molar sodium phosphate buffer, pH 6.0-9.0;
• b. contacting said crude urokinase solution with an extracting agent comprising agmatine covalently coupled to the surface of a water-insoluble solid support material, whereby urokinase becomes preferentially adsorbed on said extracting agent;
• c. washing said extracting agent with 0.01 molar sodium phosphate buffer, pH 6.0-9.0, so as to remove extraneous material from said extracting agent;
• d. eluting a portion of the urokinase from said extracting agent with 0.02 molar sodium phosphate buffer, pH 6.0-9.0;
• e. eluting said active form of urokinase from said extracting agent with 0.1 molar sodium phosphate -- 0.4 molar sodium chloride buffer, pH 5.0-8.0, and collecting the resulting eluate in fractions;
• and
• f. recovering said active form of urokinase in a purified state from the eluate resulting from step (e) as a pool of those eluate fractions having an activity of at least 1 .times. 10.sup.4 IU per ml.