JPS5847490A - Purification of urokinase - Google Patents

Abstract

PURPOSE:To obtain high-purity urokinase in high yield, by using an adsorbing material prepared by immobilizing a specific dye on a water-insoluble carrier. CONSTITUTION:A dye selected from the group consisting of azo dye, phthalocyanine dye, and anthraquinone dye is immobilized on a water-insoluble carrier such as agarose, cellulose, polyacrylamide, etc. to give an adsorbing material. A urokinase-containing aqueous solution is brought into contact with the adsorbing material so that urokinase is adsorbed on the adsorbing material. Urokinase is then eluted with an eluate containing a nonionic surface active agent.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying urokinase using an adsorbent in which a specific dye is immobilized on a water-insoluble carrier.

Rip-kinase 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 has been collected and purified from human urine, and has not only been widely used clinically for the treatment of various thromboses and myocardial infarctions, but also recently has been used in combination with anticancer drugs to enhance the efficacy of anticancer drugs. It is known to improve

Moreover, urokinase is found in human urine or kidney cells.
1 Extraction from culture medium l/lI#! Because it is a urokinase, it does not have antigenicity, so there are no side effects caused by antigen-antibody reactions, and it is widely prized as a sorption method used to recover p-kinase from urine, crude urokinase solutions, etc. Various methods have been reported in the past, but there are still few that are specifically superior and efficient.

The present inventors have conducted various studies on specific adsorbents for urokinase, and found that urokinase is specifically adsorbed to specific dyes, namely azo dyes, 7-talocyanine dyes, and anthraquinone dyes. As a result of further research on adsorption methods, elution methods, etc., we succeeded in obtaining urokinase in high yield and high purity, and completed the present invention 8At. , azo dye,
This is a method for producing urokinase, which is characterized in that a dye selected from phthalocyanine dyes and anthraquinone dyes is adsorbed onto an adsorbent bound to a water-insoluble carrier, and then urokinase is eluted.

Examples of the aqueous urokinase-containing solution to be purified in the present invention include urine, a crude urokinase solution obtained by partially purifying urine, and a crude urokinase aqueous solution such as tissue culture fluid of kidney cells. Regarding the dye used in the present invention. Examples of the azo dyes, anthraquinone dyes and phthalocyanine dyes include those illustrated in Figures 1 to 3. Particularly preferred are prodione-based and cibaculone-based dyes having an azo group, an anthraquinone ring, and azo dyes. Dye can be given.

(Left below) As the water-insoluble carrier, any water-insoluble carrier that can bind with the above dye can be used. Preferred examples include water-insoluble polysaccharides such as Sephadex (Pharmacia Co., Ltd.), polyacrylamide, etc. The water-insoluble carrier is activated by a known method and then combined with a dye to form an adsorbent. Examples of adsorbents currently available on the market include Affi-Gel® (manufactured by Biorad), Blue Sepharose (manufactured by Falicia), and Madrex Blue A (manufactured by Amicon). 0, which can be easily manufactured by the same method or a similar method.
Adsorption in the present invention is performed according to the principle of affinity chromatography.

The adsorption method is as follows, for example: The aqueous solution containing the p-kinase tube is adjusted to pH before adsorption treatment.
about 7-8, preferably about 7.5, salt concentration 0.1-0.
It is preferable to perform a pretreatment such as dialysis against a 3M% buffer, preferably about 0.15M buffer, such as a phosphate buffer.

Next, the adsorbent is fully equilibrated with a buffer solution with a low salt concentration and a pH of approximately 7 to 8 (this may be the same buffer solution for pretreatment of an aqueous solution containing urokinase), and the adsorbent is packed into a column. . An aqueous solution containing urokinase is developed on this column, and the urokinase is adsorbed onto the adsorbent. Subsequently, the buffer used for equilibration of the column is passed through to remove unadsorbed contaminants.
4I! Wash and remove J.

Elution of adsorbed urokinase is generally performed by increasing the salt concentration. Specifically, the elution is, for example, about 1 to 2
M, preferably about 1.5 M, of an inorganic salt (for example, an alkali metal salt of a halogen such as potassium chloride, sodium chloride, etc.) and a pH 7 to 8 buffer solution is flowed down. In addition, in order to specifically elute urokinase in a high yield, approximately 0.1 to 5% (w/v), preferably 2% (v/m) of a polarity-reducing agent was added to the eluate. It is preferable to use As the polarity reducing agent, nonionic surfactants [for example, sorbitan monoalkyl ester type (for example, various types of Two-・n■), alkyl ether type (for example, various types of Tritono), alkyl ester type (for example, various types of polyethylene glycol); , polyoxyethylene polyoxypropylene copolymers (eg, various types of Pluronic O)], etc. are exemplified as preferred examples. In this case, it is more effective to set the salt concentration of the eluate to about 0.1 M to 0.3 M and to maintain the pH at about 7 to 9.

The urokinase thus eluted and purified is then subjected to further treatments such as high concentration, desalting, and precipitation according to conventional methods, followed by sterilization, freeze-drying, etc., and is used as a pharmaceutical product. The urokinase that can be used may be 0 liquid 100a, in which the present invention will be described in detail by way of examples below.
J (4700 international unit ratio 1 l-, specific activity 220 international units/WIg protein) was dialyzed against pH 7.5, O, 15M phosphate buffer. Prodione HE3Bt, which is a prodione dye, is fixed with 80 volumes of agarose (column volume), equilibrated with the above buffer solution, and packed into a column.
After the dialysis, the aqueous solution containing urokinase was developed on the column to adsorb urokinase t, and then the buffer solution was allowed to flow down to wash and remove impurities.

Thereafter, 0.15 M phosphate buffer of pZ (7,5) containing 1.5 M potassium chloride was poured down to elute urokinase.

The obtained urokinase had a specific activity of 9.800 international index/Da, and the amount recovered was 91 qb.

Example 2 100w of the same crude urokinase solution as the starting material of Example 1
Dialysis was performed against Llk pH 7,5,0,15M phosphate buffer. □ A commercially available ClbacromoBlue F3GAi agarose-immobilized gel, 80 d (column volume), was equilibrated with the above buffer and packed into a column. The dialyzed catalytic kinase solution was passed through the column to coat urokinase tli, and the buffer was then allowed to flow down to wash out the contaminants. Then, 1.5M potassium chloride containing, pl(
Urokinase was eluted by flowing down 7,5 0.15 M9 phosphate buffer i[.

The obtained urokinase had a specific activity of 10,200 ap and a recovery rate of 93%.

After that, as a result of performing n-manufacturing by ion exchange chromatography using CM-cellulose, the specific activity was 56,000.
Purified urokinase from Kokusai Kokusai Chichi was obtained with a total recovery rate of 87%.

Example 3 2%' (w/v) polyoxyethylene as eluent.
pH containing polyoxypropylene block polymer nonionic surfactant (trade name, PluronicO)
The procedure of Example 2 was followed except that 1M sodium chloride solution of &5 was used to elute urokinase.

The obtained p-kinase had a specific activity of 21,000 DA and a recovery rate of 87 DA.

Thereafter, affinity chromatography using lysine sepharose was performed, and urokinase with a specific activity of 84,800 international units was obtained with an overall recovery rate of 73%.

Example 4 100 ml of the same crude urokinase solution as the starting material of Example 1
g was dialyzed against pH 7,5,0,15M phosphate buffer.

Cibacron ■ Blue F 3, a Cibacron dye
Equilibrate 80 d (column volume) of G A k-immobilized agarose with the buffer solution, fill the column, pass the dialyzed p-kinase solution through the column to adsorb urokinase, and then allow the buffer solution to flow down, Impurities were washed and removed. After that, 1% (w/v) of a polyoxyethylene-based sorbitan fatty acid ester nonionic surfactant (trade name, Tween 80) with a pH of 8.0 was added.
M potassium chloride solution was allowed to flow down to elute urokinase. The surfactant was removed from the eluate and the product was purified by ion-exchange chromatography using CM-cellulose to obtain urokinase with a specific activity of 58,000 international dp/da protein. The recovery rate was 85.

Claims (3)

[Claims] (1) After adsorbing an aqueous solution containing urokinase onto an adsorbent in which a dye selected from azo dyes, phthalocyanine dyes and anthraquinone dyes is bonded to a water-insoluble carrier, urokinase is eluted. Purification method. (2) The method for purifying urokinase according to claim (1), characterized in that in the elution of urokinase from the adsorbent, the eluate contains a nonionic surfactant. (3) The nonionic surfactant is one selected from the group consisting of polyethylene glycol type, polyoxyethylene type ether type, ester type and ester ether type. (2
) Purification method of urokinase described in section 0