JPS6215196B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for purifying urokinase. The aim is to obtain urokinase with high activity and no pyrogenic substances. Urokinase is an enzyme present in human urine.
It is a useful substance in the treatment of peripheral arteriovenous thrombosis and myocardial infarction. This urokinase can be obtained by gel filtration, ion exchange chromatography, affinity chromatography, etc., but these methods do not completely remove impurities, especially pyrogens and inactive substances, so the amount per unit is low. A product with high activity could not be obtained. Moreover, this separation operation is also complicated, and the equipment used for the operation has drawbacks such as difficulty in repeated use. As a result of research to solve the above-mentioned drawbacks, the present inventor has found that when purifying crude urokinase, the activity per unit can be improved by bringing the crude urokinase into contact with ion-exchange polysaccharide granules and then performing affinity chromatography. The present inventors have completed the present invention by discovering that urokinase, which has extremely high pyrogenicity and contains almost no pyrogenic substances, can be obtained in good yield. In the present invention, a solution obtained by contacting a buffer solution of crude urokinase with ion-exchanged polysaccharide granules is contacted with a carrier made of a synthetic urokinase inhibitor bound to polysaccharide granules via carbon chains, and urokinase is adsorbed. This is a purification method for urokinase in which the urokinase is purified by elution. The term "crude urokinase" as used in the present invention refers to urokinase separated from human urine using adsorbents such as barium sulfate, silicic acid and its salts, various ion exchangers, or precipitated substances such as ammonium sulfate. In addition, "ion-exchange polysaccharide granules" refer to polysaccharide granules such as cellulose, cross-linked dextran, agarose, etc., with ion-exchange groups bonded to them. , anion exchange polysaccharide granules such as triethylaminoethylcellulose, aminoethylcellulose, guanidoethylcellulose, carboxymethylcephadex, sulfonylethylscephadex (manufactured by Pharmacia), carboxymethylcellulose, phosphocellulose, phosphonomethylcellulose, sulfonate Examples include cation exchange polysaccharide granules such as ethyl cellulose and sulfonomethyl cellulose. Furthermore, the term "synthetic urokinase inhibitor" refers to a substance that specifically inhibits the action of urokinase, and includes, for example, para-aminobenzamidine, meta-aminobenzamidine, agmatine (aminobutylene guanidine), and the like. A carrier in which this synthetic urokinase inhibitor and polysaccharide granules as described above are bound via a carbon chain spacer is used for affinity chromatography. This carrier can be used, for example, in "Biochimica et Biophysica Acta" No. 445.
It can be easily produced by the method described in Vol. 215-222 (1976). In the method of the present invention, first, a buffered solution of crude urokinase is brought into contact with ion exchange polysaccharide granules. The type and concentration of the buffer used in this case varies depending on the type of ion exchange polysaccharide granules and the state of the enzyme, but for example, when using anion exchange polysaccharide granules, a basic buffer with a pH of about 6 to 8, etc. is used, and the appropriate concentration of the buffer is 0.05 to 0.3 mol. If the crude urokinase to be purified contains a particularly large number of impurities, it is preferable to use a buffer solution in the higher range of the concentration range shown above, that is, a concentration of 0.1 to 0.3 molar. However, if the concentration of the buffer solution exceeds the above range, the removal of pyrogenic substances, which is the main purpose of this purification, will not be achieved, which is undesirable. Other conditions during ion-exchange purification, such as the flow rate of a buffer solution of crude urokinase, can be appropriately selected depending on each case. The urokinase solution obtained by the ion exchange purification treatment is one in which most of the pyrogenic substances contained in the crude urokinase have been removed, and is then brought into contact with an affinity carrier for further purification. First, the urokinase solution is replaced with a buffer solution suitable for the conditions of affinity chromatography by a method such as dialysis. At this time, a buffer solution suitable for the type of affinity chromatography and the state of the enzyme solution is used, such as a neutral high concentration salt solution such as a phosphate buffer solution containing common salt. The urokinase buffer solution prepared as described above is brought into contact with the affinity carrier. Through this treatment, urokinase is adsorbed onto the carrier, and other impurities, mainly proteins, flow out of the chromatographic system, allowing separation of urokinase and impurities. Urokinase adsorbed on the carrier is
For example, it flows out from the column by passing a low-PH, high-concentration salt solution such as an acetate buffer containing sodium chloride. Since the urokinase thus obtained does not contain impurities, particularly pyrogens and inert substances, it has very high activity per unit, and is particularly useful when used in combination with anticancer drugs. Furthermore, since the operating steps are simple, there is no decrease in activity, yield, or enzyme decomposition during purification, making it an extremely excellent method. According to the method of the present invention, it is estimated that extremely effective purification of urokinase can be achieved through the following separation process.

[Table] Affinity Chromato

Claims (1)

[Claims] 1. A buffer solution of crude urokinase is brought into contact with ion-exchanged polysaccharide granules, and the thus treated solution is brought into contact with a carrier made of a synthetic urokinase inhibitor bound to the polysaccharide granules via carbon chains to adsorb urokinase. , and then elution and recovery of the adsorbed urokinase.