JPS5820191A - Preparation of urokinase - Google Patents

Abstract

PURPOSE:To elute the titled substance and utilize the substance widely as a medicine, etc., by bringing an adsorbent adsorbing the titled substance into contact with a nonionic surfactant in an aqueous solution thereof in a given concentration. CONSTITUTION:An adsorbent adsorbing urokinase, e.g. aluminum hydroxide gel, barium sulfate or weakly acidic or medium acidic ion exchange resin,is brought into contact with a nonionic surfactant. A polyoxyethylene type is preferred as the nonionic surfactant, and used in an aqueous solution in a concentration of usually 0.01-10% (W/V). The salt concentration adjusted to 0.1-2M is effective for enhancing the eluting effect of the urokinase. A neutral salt, e.g. common salt, magnesium chloride or potassium chloride, is preferred as the salt to be used.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for eluating urokinase adsorbed to an adsorbent from a crude p-kinase aqueous solution derived from human urine or human urine and a tissue culture fluid of renal cells in a stable state and with high yield. It relates to a method of elution.

Urokinase is an enzyme that exists in trace amounts in human urine and tissue culture fluid of kidney cells, and has the function of activating plasminogen contained in human plasma to generate plasmin that has fibrinolytic ability.

For this reason, urokinase is collected and purified from human urine, etc.
It is widely used clinically to treat various types of thrombosis and myocardial infarction. In addition, it has recently been known to improve the efficacy of anticancer drugs.

Moreover, since urokinase is extracted and purified from human urine or kidney cell tissue culture fluid, it does not contain any antigenicity, so it is widely praised for its lack of side effects caused by antigen-antibody reactions.

By the way, as a method for producing urokinase from a solution containing urokinase, it is purified by adsorption using an adsorbent tube such as silica gel, barium sulfate, kaolin, zeolite, magnesium aluminate silicate, hydroxyamethite, etc. It has been known. However, in methods using these adsorbents, a large amount of urokinase enters the interior of the adsorbent, resulting in a significantly low elution rate under normal elution conditions. Furthermore, aqueous ammonia is used to elute urokinase from these adsorbents, but in this case the pH is extremely high, which worsens the stability of urokinase and reduces the yield. First, it has the disadvantage that impurities other than urokinase are also eluted.

In order to overcome these drawbacks, the present inventors conducted research on a method for eluting urokinase adsorbed to an adsorbent in a high yield under mild conditions. We found that highly purified T44 kinases can be eluted in high yields when contacted with a surfactant.

The present invention was completed based on the above findings, and is a method for producing urokinase, which is characterized in that urokinase is eluted by bringing the adsorbent adsorbed with urokinase into full contact with a nonionic surfactant.

The adsorbent that has adsorbed urokinase, which is the starting material in the present invention, may be any adsorbent that has urokinase adsorbed thereto.

Therefore, any adsorbent for adsorbing urokinase may be used as long as it can adsorb urokinase.
Specifically, adsorbents such as aluminum hydroxide gel, barium sulfate, magnesium sulfate, diatomaceous earth, bentonite, acid clay, hydroxyapatite, calcium phosphate glue, glass peas, magnesium aluminium monate compound, weakly acidic to moderately acidic ion exchange resins, Weakly acidic to strongly acidic ion-exchanged cellulose, weakly acidic ion-exchanged Sephadex, weakly acidic ion-exchanged agarase, cellulose materials with carboxyl groups introduced, weakly basic ion-exchange resins, weakly basic ion-exchanged cellulose, Examples include ion exchangers such as weakly basic ion-exchange adsorbents, weakly basic ion-exchange agarose, agarose, polyacrylonitrile resin powder, and other adsorbents such as fiber dye-immobilized adsorbents. .

The method of the present invention is carried out by contacting an adsorbent adsorbing urokinase with a nonionic surfactant as an eluent to elute urokinase.

The pH during elution is neutral to alkaline, specifically pH 7 to 1O. As the nonionic surfactant, preferably a polyoxyethylene surfactant is used, specifically sorbitan monomer. Alkyl ester type (for example, various Tween'), alkyl ester type (for example, various Trito-n), alkyl ester type (for example, various types of Trito-n),
For example, various polyethylene glycols), polyoxyethylene polyoxypropylene copolymers (such as various P-1uronie), etc.0 Nonionic surfactants are usually used in the form of an aqueous solution, and their concentration is generally 0.01 to 10% (W/ma),
Preferably it is O, 1 to 5 inches (W/ma). This is preferred because when urokinase is eluted using a cationic surfactant such as lauryltrimethylammonium, a concentration of α5- or higher causes irreversible denaturation of proteins, especially urokinase.

This is largely different from the description in Japanese Patent Application Publication No. 55-20675, which states that there is no such thing.

At this time, in order to enhance the elution effect of urokinase, it is more effective to adjust the salt concentration to 0.1 M to 2 M, especially around 1 M. Examples of the salt used here include common salt, magnesium chloride, chloride Neutral salts such as potassium are preferred.

The eluate of urokinase is desalted, concentrated, and highly purified using conventional methods to obtain urokinase that can be used as a pharmaceutical product. According to the present invention, when urokinase is eluted from an adsorbent that has adsorbed urokinase, nonionic surface activity is used. Since the eluate containing all the agents is used, urokinase can be eluted in a stable state with good yield, improving the recovery rate of urokinase and suppressing the loss of activity during the work. This method is extremely advantageous for producing urokinase on a large scale.

Next, the present invention will be specifically explained with reference to examples.Example 1 10Gt fresh silver t'pI (9
, remove the precipitate formed in step 5, and rinse the supernatant with t-6N hydrochloric acid.
Adjusted to H7.0. Prepare 200 Alt-200 Alt suspensions in diluted hydrochloric acid in advance, add this to fresh silver supernatant to adsorb urokinase, and add polyoxyethylene polyoxy at a final concentration of 2% w/m to a 0015 molar ammonia sulfate solution. Propylene copolymer (product name: Pluronic)
The eluate was adjusted to 7'hpH7.5, and the bentonite adsorbed with urokinase was immersed in this eluate, and the eluent was neutralized with 6N sulfuric acid to remove ammonium sulfate. 60 - saturated 0th order saturated liquid 4
After leaving it overnight at °C, collect the precipitate # by centrifugation.
The mixture was dissolved in u-1 oom of distilled water and dialyzed against 4°C water, and the dialysate was lyophilized to obtain purified 44-kinase. If the raw material for p-kinase obtained in this way is urine, the recovery rate is 2.
Example 2 100 tons of fresh silver collected from a large number of healthy people pH 9.5
After removing the precipitate formed, the supernatant was diluted with 6N hydrochloric acid to pH 7.
Adjusted to 0. Bentonite 20 suspended in dilute hydrochloric acid in advance
0 #t- was prepared and added to the supernatant of fresh silver to adsorb urokinase onto bentonite. An eluent solution of pH 8.0 was prepared by adding polyoxyethylene polyoxypropylene Pruck polymer with a final concentration of 2% W/V to 1 molar sodium chloride solution, and the eluate was adjusted to 3 tf, and the bentonite adsorbed with urokinase was immersed in this eluate. urokinase was eluted.

This eluate was neutralized with 6N sulfuric acid to saturate it with ammonium sulfate by 60°. Next, the saturated solution was allowed to stand overnight at 4 DEG C., and then the precipitate was collected by centrifugation, and the precipitate was dissolved in t-100 ml of distilled water to obtain a urokinase solution. This p-kinase solution is further purified by ion exchange chromatography or gel filtration to a high purity (
Clokinase with a specific activity of 70,000 international units/da) can be obtained.

Example 3 My of 5 million national units partially purified by the method of Example 1
Tauronase solution (ao o o international units/■) to pH
Dialyze against 7,0,0,005M phosphate buffer through Puisking dialysis tubing.

100 meshes of polishing gas beads 4KP manufactured by Toshiba Corporation are equilibrated with p) i7.0.0.005M phosphate buffer, and packed into a column. When the dialyzed crude urokinase solution is allowed to flow down this column, impurities pass through and p-kinase is adsorbed onto the glass beads.

Thereafter, impurities adhering to the glass beads are eluted with sodium chloride at pH 9, 0, 0, and 1M.

The urokinase is then eluted using IM sodium chloride solution, PH9t- containing 2'4 w/v polyoxyethylene polyglupyrene block polymer.

The obtained urokinase solution contains 875Gt of urokinase in the starting material (urine), and its purity is 5o, o o
o International units/Da or higher.

Example 4
B - Crude urokinase solution of 300,000 international units extracted from human urine and partially purified (15,000 international units/liter)
Dialyze against H7,5,0,0725M phosphate buffer through Puisking dialysis tubing.

Equilibrate with and fill the column. When a dialyzed urokinase solution is allowed to flow down this column, urokinase is adsorbed and impurities pass through. Thereafter, the equilibration buffer is sufficiently poured down to remove as much impurities as possible.

Next, 1.5 M at pH 9 containing Z% (W/ma) of polyoxyethylene polyoxybu tetrapyrene block polymer
Elute with urokinase using potassium chloride solution.

The obtained urokinase solution contains 88- of the urokinase in the starting material, and its purity is so,o.

0 International Ranking/Wisdom or higher.

Example 5 Extracted from human urine, the electrical conductivity was 4 to 5 mha/m
/ Crude urokinase solution adjusted to 25°C 50 - (Kame 50
0 international single international rate s specific activity 350 international units/da protein),
A 15M amount of sodium chloride was dissolved and the pH was adjusted to 6.8.

The column was packed with 25 d (force 2 μm volume) of phenyl octyl Sepharose and 3.5 d in 0.05 M phosphate solution.
M amount of sodium chloride was dissolved and equilibrated by passing through a liquid tube whose pH was adjusted to 6.8.

The p-kinase solution prepared previously was passed through this to adsorb urokinase, and then washed with an equilibration solution.

Then, a pH 8 solution containing 1% (ma/ma) polyoxyethylene-based sorbitan fatty acid ester nonionic surfactant (trade name, Tween 8) was passed through.
Kinase was eluted.

After removing all the surfactant from the eluate and purifying it with a weakly acidic cation exchanger, the specific activity was so and oo.

Urokinase of international unit 7w9 protein was obtained with a recovery rate of 80%.

Example 6 Extracted from human urine, electrical conductivity 4-5 m mhV-72
100 d of partially purified crude urokinase solution adjusted to 5°C (
20,000 international modulus d, specific activity 7.000 international units/IIv protein), 2.0M amount of potassium chloride was added and the pH was adjusted to 6.8.

It was packed into an 80 m (column volume) octyl agarose tube column, and equilibrated by passing a solution in which 2.0 M potassium chloride was dissolved in a 0.05 M phosphate solution and adjusted to pH' (H6.8). .

The urokinase solution that had been applied previously was passed through this to adsorb urokinase, and then washed with an equilibration solution.

Then, 2% (ma/ma) of a polyethylene glycol alkyl ether type nonionic surfactant (trade name, T
Urokinase was eluted by passing through a pH 8 solution containing riton X-100)i. The surfactant was removed from the eluate, and purified urokinase with a recovery rate of 60,000 international units/da protein was obtained with a recovery rate of 84. Ta.

Example 7 Crude urokinase solution of Example 6 100 dK2. Dissolve OM amount of potassium chloride, 1) H? Adjusted to 6.8.

A column was filled with 80 t7 (column volume) of Octyl Sepharose, and a solution prepared by dissolving 20 M potassium chloride in a 0.05 M phosphate solution and adjusting the pH'i to 6.8 was passed through the column for equilibration.

A nine-urokinase solution prepared in advance was passed through this to adsorb urokinase, and then washed with an equilibration solution.

Thereafter, 2% (vr/ma) of a polyoxyethylene ether type nonionic surfactant (poo to i
1. Urokinase was eluted by passing a pH 9 solution containing 0.0, 0.0, 0).

The surfactant was removed from the eluate and the specific activity was 72. .

It was obtained at an international rate of 0.00/a recovery rate of purified urokinase 'i9.1 of Da Protein.

Claims (2)

[Claims] (1) Urokinase is eluted by contacting the adsorbed adsorbent with a nonionic surfactant tube.
Characteristic method for producing Tsushikinase. (2) Nonionic surfactant t-'! Il! When contacting, ■ Nonionic field activator tαO1~1os (W/Ma)
The method for producing urokinase according to claim 1, characterized in that: (1) the salt concentration is made 0.1 to 2M with a neutral inorganic salt.