GB1411422A - Methods for purifying urokinase preparations - Google Patents

Abstract

1411422 Purifying urokinase solutions CHOAY SA 22 Sept 1972 [24 Sept 1971 30 June 1972 (2)] 15502/75 Divided out of 1411421 Heading C3H A method of purifying an aqueous solution containing 1.5-3 mg./ml. of urokinase, foreign proteins and pyrogenic substances involves adjusting the pH of the solution to between 3 and 4.5 and its conductivity to between 15,000 and 25,000 micromhos/cm., determining, e.g. with the aid of a graph prepared from experimental results, the concentration of ammonium suplate needed for forming a precipitate of foreign proteins &c. containing only 2-5% of the total activity of the urokinase in the initial solution, adding to the solution the appropriate quantity of ammonium sulphate and collecting the supernatant. The concentration of ammonium sulphate employed should preferably correspond to a degree of saturation of the solution of from 0.25 to 0.35. The urokinase is then precipitated out with a further quantity of ammonium sulphate and the precipitate is dissolved in distilled, sterile apyrogenic water at pH 7. The initial urokinase solution may be obtained from human urine by absorbing the proteins &c. from the urine on a filtration adjuvant, re-extracting the urokinase and other adsorbed proteins &c. therefrom, salting out urokinase from the solution by adding ammonium sulphate, dissolving the resulting precipitate in a glucose solution and dialysing the latter to obtain a concentrated crudeurokinase solution of specified conductivity, two steps of fractionation using diethylaminoethyl cellulose employing in the first step solutions of pH 4-6 and conductivity 15,000- 25,000 micromhos/cm. with 15-40 g. of resin per 14 million CTA units of urokinase, and in the second step solutions of pH 6-7 and conductivity 15,000-18,000 micromhos/cm. with 30-60 g. of resin per 10 million CTA units of the enzyme, the urokinase-containing medium during the second step being diluted with quantities of water sufficient to lower the conductivity by about 1000 micromhos/cm. at each successive dilution until a conductivity of about 9000 micromhos/cm. is reached, and then absorbing the urokinase in the solution obtained by combining the effluent and the washing liquors from the resin used in the second fractionation on either carboxymethyl cellulose at a pH of about 5 and eluting the adsorbed urokinase using a phosphate buffer at pH 6.8-6.9, or kaolin at pH 6.2 and eluting the urokinase retained with a solution of ammonium chloride in 4% ammonia, followed by precipitation with ammonium sulphate and dialysis against a 0.05 M phosphate solution buffered to pH 6.2.