Method for recovering urokinase from urine containing the same

Claim 2

The method of claim 1 wherein the adsorbant is silicon dioxide, titanium dioxide or a naturally occurring South Carolina hard clay having a surface area of about 200 m2/gm.

Claim 3

The process of claim 1 wherein the urokinase-containing adsorbant of step (b) is resuspended to form a slurry and the slurry washed one or more times with water.

Claim 4

The process of claim 4 wherein the recovery step (c) is carried out by extracting the urokinase-containing adsorbant with tris buffer to which sodium chloride, a sodium phosphate or ammonium sulfate salt has been added at a level of about 10-30% by weight of the slurry, centrifuging the slurry and recovering the supernatant liquid containing urokinase dissolved therein.

Claim 5

The process of claim 5 wherein the recovery step (d) is carried out by precipitating the acid-insoluble proteins present in the urokinase solution with an additional quantity of salt and recovering the urokinase precipitate.

Claim 6

The process of claim 6 wherein the urokinase precipitate is redissolved, the acid-insoluble proteins present in the urokinase solution are precipitated by the addition of a suitable acid precipitant, the supernatant solution containing urokinase is recovered, the urokinase present in the solution is precipitated with a suitable base and the urokinase precipitate is recovered.

Claim 7

The process of claim 7 wherein the urokinase precipitate recovered therein is further purified by the steps of a. redissolving the urokinase precipitate; b. dialyzing the urokinase solution against tris buffer solution; c. adsorbing the urokinase upon an anionic polysaccharidal exchanger; d. recovering the urokinase from the anionic polysaccharidal exchanger/urokinase adsorbate in the form of an aqueous solution of urokinase. e. recovering the urokinase from the solution; f. redissolving the urokinase; g. dialyzing the urokinase against tris buffer solution; h. re-adsorbing the urokinase upon the anionic polysaccharidal exchanger; i. recovering the urokinase from the anionic polysaccharidal exchanger/urokinase adsorbant in the form of an aqueous solution of urokinase; j. purifying the urokinase present in the aqueous solution by column fractionation; k. dialyzing the purified urokinase solution against phosphate buffer. l. heating the dialyzed urokinase solution at a temperature and for a period of time sufficient to inactivate any viral organisms present therein; m. dialyzing the urokinase solution against a solution of a salt of ethylenediaminetetraacetic acid. n. depyrogenating the urokinase solution by ultrafiltration; and o. recovering the urokinase from the solution by lyophilization.