JPH0143550B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for recovering urokinase, and aims to provide an industrially advantageous method for recovering urokinase with high yield and high purity from human urine or a liquid containing urokinase. A substance that degrades fibrin exists in human urine, and Williams (1951), Sobel (1952) and others named this substance ``urokinase'' because it is an enzyme that activates plasminogen to plasmin. Since then, urokinase has been used as a dissolving therapeutic agent for thrombosis and embolism in the blood circulation system, such as peripheral arteriovenous thrombosis, cerebrovascular atresia, and myocardial infarction. Furthermore, recently, the usefulness of urokinase in combination with anticancer drugs has been recognized, and the importance of urokinase has further increased. Urokinase isolated from human urine has no side effects such as antigen-antibody reactions and is currently widely used. Conventionally, methods have been known to use barium sulfate, silica gel, Hyflo Super Sale, bentonite, hydroxylapatite, zeolite, cellulose phosphate, Bonnell, and other ion exchangers as adsorbents to obtain urokinase from human urine. There is. The present inventors conducted research to develop an even better method than these known methods, and found that perlite, which has never been used as an adsorbent for urokinase, is effective and can be obtained with high yield and comparatively high yield. The present invention was completed after discovering that a method for recovering highly active urokinase is highly economical even on an industrial scale. The pearlite used in the present invention is made by rapidly heating glassy volcanic rock powder at a temperature of around 1000°C to expand it into a fine cell-like cell structure, which is then crushed and classified to remove a few percent or less of bound moisture. It is a mineral substance whose main component is silicate. The outline of the present invention is to use human urine or a urokinase-containing liquid, such as urine concentrated by a foaming method, to
Adjust the pH to 8.5-9.5, remove the resulting precipitate, and then adjust the pH again to 5.0-7.0. Further water is added to adjust the electrical conductivity (hereinafter abbreviated as EC) to 10 to 20 millimoh/cm. This is brought into contact with perlite that has been previously treated with IN=hydrochloric acid to adsorb urokinase. Approximately 400 to 1600 mg of perlite per raw urine is usually appropriate. The adsorption operation can be carried out either by a column method or by a batch method. The perlite adsorbed with urokinase is washed with 5 times the volume of water of the adsorbent, and then washed with a 0.1-0.5M aqueous salt solution to remove impurities. The appropriate amount of washing liquid with the salt solution is 20 times the volume of the adsorbent. By this washing, most proteins other than urokinase are eluted and removed. Next, the adsorbent is eluted with an alkaline aqueous solution with a pH of 9.5 to 12.0 or a 4% ammonia aqueous solution to which 0.1 to 0.5M salts have been added to obtain a highly pure urokinase eluate containing 5000 to 10000 units/mg protein. can do. The relationship between EC and the adsorption rate of urokinase to perlite when human urine (original urine) or urine concentrated by a foaming method is used is as follows. Table 1 shows human urine concentrated approximately 13 times by the foaming method, and water or salt added to bring the EC to 10-25.
This figure shows the adsorption rate of urokinase to pearlite in millimos/cm. The amount of perlite used was 2W/V% based on concentrated urine.

[Table] This result shows that if the EC of the urokinase-containing solution is adsorbed at 20 mmol/cm or less, 90% or more adsorption is possible. Table 2 shows the relationship between the amount of pearlite and the adsorption rate of urokinase when the urokinase-containing solution has an EC of 13 mmol/cm and a pH of 6.5.

[Table] The results show that an adsorption rate of 90% or more can be achieved by using approximately 400 mg of perlite per 1 portion of urine. In addition, when using 0.5W/V% of urine concentrated approximately 13 times by the foaming method, the adsorption rate was 90%.
% or more. Next, the present invention will be explained in more detail with reference to Examples. The urokinase activity was measured using the fibrin plate method of Ploug et al. [J. Ploug: Biochim.
Biophys.Acta 24 , 278 (1957)]
The activity indication is the international unit (IU) defined by Johnson et al. [A. Johnson: Thrombos. Diathes
haemorrh (Stuttg) 21 , 259, (1969)].
In addition, protein was quantified using the Folin-Lowry method. Example 1 Concentrated liquid 2.0 of human urine 25 using the foaming method
Adjust the pH to 8.5, remove the resulting precipitate by centrifugation, and then adjust the pH to 6.5. Add water, EC = 12 ~
13 mmol/cm, 40 g of perlite which had been previously treated with IN hydrochloric acid and washed with water was added and stirred for 1 hour to adsorb urokinase. Separate the perlite that has adsorbed urokinase, wash it with 200 ml of distilled water, disperse it in 300 ml of water, and apply it to a column (diameter 9 x 3 cm).
Added to. After the water had flowed down, it was further washed with 500 ml of distilled water, and then the contaminant proteins were washed out and removed with 800 ml of a 0.5M NaCl aqueous solution. Finally, the column was eluted with 0.2% ammonia aqueous solution containing 0.5M NaCl.
A colorless and transparent urokinase eluate was obtained. Adsorption rate 95
%, elution rate was 9.8%, and specific activity was 9505 IU/mg protein. Example 2 2600 human urine was processed in the same manner as in Example 1 and concentrated urine
Got 160. This concentrated solution was adjusted to have an EC of 14.5 mmol/cm and a pH of 6.5. To the solution 320, 3.5 kg of perlite that had been previously treated with IN-Hcl and washed with water was added and stirred for 1 hour to adsorb urokinase. Pour this pearlite into a column (diameter 40 x 16 cm),
First, distilled water 100%, then 0.5M−NaCl aqueous solution.
65, and elution was performed with a 0.2% ammonia aqueous solution containing 0.5M NaCl to obtain a colorless and transparent urokinase eluate. Adsorption rate 90%, elution rate 83%, specific activity
The yield of urokinase per ton of human urine was 6580 IU/mg protein and 5.06 million IU/T. Example 3 A column with a diameter of 9 cm was filled with 40 g of perlite that had been treated with IN-Hcl and washed with water (diameter 9 x 3).
cm). Human urine 25.0 is adjusted to pH 8.5, and after removing the resulting precipitate, the pH is adjusted to 6.5. Add water to this liquid
After adjusting the EC to 12-13 mmo/cm, it was passed through the column. After the entire amount had passed through the column, the column was washed with 1 portion of distilled water to remove non-adsorbed substances. Furthermore, the column was washed with 800 ml of 0.5M NaCl aqueous solution to elute contaminant proteins. Finally, elution was performed with a 0.2% ammonia aqueous solution containing 0.5M NaCl to obtain a colorless and transparent urokinase eluate. Adsorption rate 91.5%, elution rate 95%, specific activity:
It was 10158 IU/mg protein. Example 4 A column with a diameter of 15 cm was filled with 40 g of perlite that had been previously treated with IN-HCl and washed with water. Crude urokinase (240IU/mg, specific activity 542IU/mg protein)
3 million IU was made into an aqueous solution of 2000 IU/ml, adjusted to pH 6.5, and then EC=10 mm/cm was passed through the column. After the entire volume has passed through the column, distilled water 8
Wash the column with
Contaminant proteins were washed away with 0.5M NaCl aqueous solution 7. Finally, urokinase was eluted and recovered with a 0.2% ammonia aqueous solution containing 0.1M NaCl. Adsorption rate
The dissolution rate was 90%, the elution rate was 96%, and the specific activity was 8761 IU/mg protein.

Claims (1)

[Scope of Claims] 1. A method for obtaining urokinase, which comprises bringing human urine or a urokinase-containing solution into contact with perlite to adsorb urokinase, and eluting urokinase from the perlite. 2. The method for obtaining urokinase according to claim 1, wherein the amount of perlite used is 400 mg or more and 1600 mg or less per 1 solution converted to human urine. 3. The method for obtaining urokinase according to claim 1, wherein the urokinase is washed with a 0.1-0.5M salt solution before being eluted from the pearlite. 4. The method for obtaining urokinase according to claim 1, wherein the electrical conductivity of human urine or a urokinase-containing liquid is adjusted to 10 to 20 mmo/cm prior to adsorption.