JPS606191A - Separation of low-molecular urokinase and high- molecular urokinase - Google Patents

Abstract

PURPOSE:To separate the titled substance useful as a thrombolytic agent, etc. in high purity, by adsorbing a crude urokinase in an adsorbent containing aminocaproic acid group bonded chemically to a carrier, selectively eluting the low- molecular urokinase therefrom, and eluting the high-molecular urokinase. CONSTITUTION:Crude urokinase containing high-molecular urokinase and low- molecular urokinase is adsorbed in a neutral buffer solution containig >=0.8mol of salts to an adsorbent containing the aminocaproic acid group of formula I and the group of formula II (R is amidino or guanidino) bonded chemically to a water-insoluble carrier (e.g. agarose). The adsorbed low-molecular urokinase is eluted selectively with an acidic buffer solution containing >=0.8mol of salts, and then the high-molecular urokinase is eluted to achieve the separation of both components.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating low molecular weight urokinase and high molecular weight urokinase.

Urokinase is an enzyme that exists in trace amounts in human urine, and a highly purified preparation of this enzyme has an excellent thrombolysis-promoting effect when administered to humans.

In addition, it is widely used as a drug due to its unique effects, such as its anticancer effect being significantly enhanced when used in combination with anticancer drugs.

Many methods for purifying urokinase have been reported, such as calcium phosphate gel.

Methods using adsorbents such as silica gel, and methods using amber
CG50. Methods using ion exchangers such as DEAR-Sephadex and CM-Sephadex are known.

In addition, in recent years, many methods for purifying urokinase using affinity stomatography have been reported2, for example,
Methods using arginine, lysine, benzamidine, benzguanidine, or the like as a ligand are known.

However, these conventional purification methods are aimed at improving the purity of urokinase, and are not necessarily sufficient as a method for efficiently separating and collecting polymeric urokinase useful as a pharmaceutical. That is, the urokinase contained in fresh urine is a high-molecular urokinase, but during the purification process, a portion of it decomposes and transfers to low-molecular urokinase, which has poor adsorption to fibrin. It was confirmed that its in vivo activity is inferior to that of polymeric urokinase.

High molecular weight urokinase was desired as a pharmaceutical product, and the present invention was completed after conducting two types of research in order to establish an industrially advantageous method for removing the kinase and selectively separating and collecting high molecular weight urokinase. . That is, 9 the present invention is based on the formula (A) -NH(Cz)5cOO) (... Nakayama
... Kawa, a polymeric urokinase and a low molecular weight urokinase were applied to an adsorbent formed by chemically bonding a group represented by (A) and a group represented by (in the formula, R represents an amidino group or a guanidino group) to a water-insoluble carrier. Crude urokinase containing molecular urokinase is adsorbed in a neutral buffer containing 0.8 mol or more of salt, and the adsorbed low molecular urokinase is reduced to 0.8 mol or more.
A method for separating low-molecular-weight urokinase and high-molecular-weight urokinase, which comprises selectively eluating the high-molecular-weight urokinase with an acidic buffer containing 8 mol or more of salt. ”.

According to the present invention, low-molecular-weight urokinase can be separated and removed from crude!17r nearkinase with a specific activity of about 5 (10 to 10,000 international units/da protein) in one step, resulting in a high-purity polymer that can be used for pharmaceutical purposes. Urokinase can be industrially produced very advantageously.

The adsorbent used in the present invention is produced by reacting 6-aminocaproic acid with a water-insoluble carrier and bonding the group represented by (A) above by appropriately combining reactions known per se. 1 part, preferably 25-6
It can be prepared by condensing 5% of aminobenzamidine or aminobenzguanidine to convert it into the group shown in (B) above. That is, in order to bond the group represented by the above formula (A) to a water-insoluble carrier, CDI(N,
Conventional methods such as N'-carbonyldiimidazole activation method and cyanogen bromide activation method can be used.

In addition, in order to convert the group of formula (B) by condensing a ligand to the terminal of the group of formula (9), -P-)luenesulfonic acid], EDCCI-ethyl-3-(3-dimethylaminopropyl)carbodiimide] and the like can be used.

Examples of water-insoluble carriers include those capable of bonding the group represented by formula (A), such as agarose, crosslinked dextran, and cellulose.

Polysaccharides such as agar and polymers such as polyacrylamide are used.

When carrying out the present invention, the adsorbent is packed in a column and pre-adsorbed with a neutral buffer solution (pH 6 to 8) containing salts of 0.8 mol or more, preferably 0.8 to 3.0 mol a degree. Equilibrate. As the salts added to the buffer solution, salts having a salting-out effect, such as ammonium sulfate di-salt and sodium formate, are used.

The crude urokinase used as a raw material has been prepurified by a conventional method and has a specific activity of approximately 5 (10 to 100 QO international units/■
Protein is suitable.

Crude urokinase is typically 4'0000 to I00000 International Units/I in the same buffer used to equilibrate the column.
It is dissolved to a concentration of - and adsorbed through the column.

Wash with the same buffer solution and continue washing until the absorbance value of the eluate at 280 nm becomes 0.02F or less. Thereafter, while maintaining the salt concentration of the same buffer at 0.8 mol or more, the pH is adjusted to an acidic range (preferably 1-:4. By continuing to wash at a lower temperature of 0 to 45, it is possible to leave the high molecular weight urokinase in the column and selectively elute only the low molecular weight urokinase.

After eluting low-molecular-weight urokinase, if the pH of the buffer solution is kept in the acidic range and elution is continued by lowering the salt concentration, a specific activity of 100,000 international units/m9 protein containing no pyrogen a or antigenic substance can be obtained. It is possible to elute high-purity high-molecular urokinase before and after with a yield of 80% or more.

In the present invention, the activity of urokinase is determined by the fibrin plate method [Biocl+em, 13iophs, AcLa
24. 278 (1957)).

The present invention will be explained in detail below with reference to Examples.

Example J Sepharose CL-6B (manufactured by Pharmacia Fine Chemicals) 20 mA was mixed with cyanogen bromide 3 at pH 11.
2 and then 6-aminocaproic acid 19.3F
Sepharose cL-6E3 was reacted at 20°C for 10 hours.
-Aminocaproic acid conjugate was synthesized. In this reaction, 617 μmol of 6-aminocaproic acid was bound per ml of gel. After adding 3 CMC to 20ml of this gel,
Add 170η of para-aminobenzamidine, I) H4
, 2 to 46 for 12 hours to condense para-aminobenzamidine. The para-aminobenzamidine bound was 22.2 moles per gel.

15 ml of the gel adsorbent obtained in this way was packed into a column, and a solution containing 1M sodium chloride (1,1M phosphate buffer (17.0 >
Equilibrated with.

Crude urokinase 8340 X10 with positive pyrogen test
3 international units (specific activity: 7200 international units/my protein, polymeric urokinase content: 93 units) was dissolved in the same 100% buffer solution to remove insoluble matter, and then passed through a column. After thoroughly washing the column with the same buffer solution until the absorbance value at 28 nm of the δL solution became 0.02 or less, low-molecular-weight urokinase was eluted when eluted with 0.1M sodium phosphate (pH 41) containing 1M sodium chloride. . The eluted low molecular weight urokinase has a specific activity of 508 x 10' international units (specific activity 1.
420 (10 international units/+++y tacus), which corresponds to 87 units of low molecular weight urokinase contained in crude roginase.

After eluating the low molecular weight urokinase, dilute the eluate with 4 plls.
．． If the salt concentration is kept at 1 and the elution is continued by continuously decreasing the salt concentration, the salt concentration will be 6,60 between 0.55 and 0.02 mol.
A high molecular weight urokinase with 0 x 10' international units (specific activity 127,000 international units/~protein) was eluted. Yield of polymeric urokinase: 85. Pyrogenic substance test results,
This polymeric urokinase tested negative in the international unit iKy rabbit test of 150.

Example 2゜Similar to Example 1, Sepharose CL-681nI7!
An adsorbent was prepared in which 664 moles of 6-aminocaproic acid groups were bound per unit, and 18.6 μmol of para-amino guanidine was condensed to the terminal carboxyl group.

Seven of these adsorbents were packed into a column, and 2M ammonium sulfate containing 0.1
Equilibration was performed with M phosphate buffer (pH 7,0). Crude urokinase with positive pyrogen test4. 103 international units of oso Then, urokinase was adsorbed. After thoroughly washing the column with the same buffer solution until the absorbance value at 280 nm of the washing solution becomes 0.02 or less, add 0.1M ammonium sulfate containing 2M ammonium sulfate.
Low molecular weight urokinase was eluted with 50ml of sodium phosphate (1) (114.2).

The eluted low molecular weight urokinase was 230 x 10' international units, corresponding to 56 units of the total urokinase activity in the adsorbent.

After eluting the low molecular weight urokinase, the high molecular weight urokinase in the column was eluted by a gradient elution method. That is, 2M ammonium sulfate-containing 0.0.
When elution is continued by continuously lowering only the ammonium sulfate concentration of 1M sodium phosphate (pH 4,2) from 2M, the ammonium sulfate concentration is 10.
The heavy molecule urokinase of 3.35 + l × 10” international units was eluted between ~0.3 mol.
Kinase corresponds to the 82nd part of the total urokinase activity of the raw material. J, activated); J: ]J5. +] tlO International
(Position/Ir! 9 I (I white 2 pyrogenic substance a test) ga・ka result, J5.11 den) International junior high school f3, / '9 family rabbit body 7
I with N (I finished with λ).

1 axis person for patent Wakamoto Pharmaceutical Co., Ltd.

Claims (1)

[Claims] Formula (A) -Nl((CH2)s C0OH...
・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ The group represented by the formula (A) and the group represented by the formula (B) (in the formula, R represents an amidino group or a guanidino group) are chemically applied to a water-insoluble carrier. Crude urokinase containing high molecular weight urokinase and low molecular weight urokinase is adsorbed onto the bound adsorbent in a neutral buffer containing 0.8 mol or more of salt, and the adsorbed low molecular weight urokinase is reduced to 0.
．． A method for separating low-molecular-weight urokinase and high-molecular-weight urokinase, which comprises selectively eluating the high-molecular-weight urokinase with an acidic buffer containing 8 mol or more of salt.