JPS59187779A - Adsorbent for purifying enzyme - Google Patents

Abstract

PURPOSE:To obtain a novel adsorbent for affinity chromatography capable of purifying urokinase in high purity and yield, by bonding specific two groups within a specific molar ratio range chemically to a water-insoluble carrier. CONSTITUTION:An adsorbent obtained by bonding groups expressed by formula I and groups expressed by formula II within (75:25)-(35:65) molar ratio range chemically to a water-insoluble carrier, e.g. agarose, crosslinked dextran or agar. In the groups expressed by formula II, R is amidino or guanidino group. The adsorbent is packed in a column, and a solution containing crude urokinase is passed through the column to adsorb the crude urokinase therein. The adsorbed urokinase is then washed with a neutral aqueous solution and then eluted with an aqueous solution of an acid salt to give the aimed urokinase having 80,000- 130,000 international units/mg protein specific activity containing no pyrogenic material in >=80% yield.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel adsorbent for 7-infinity chromatography for purifying enzymes, particularly urokinase, to high purity and high yield.

Urokinase is an enzyme that exists in trace amounts in human urine, and when it is isolated and highly purified, urokinase shows an excellent thrombolytic promoting effect when administered to humans, and its anticancer effect can be enhanced when used in combination with anticancer drugs. It is widely used as a medicine due to its unique effects, such as significantly enhancing the

Many adsorbents for purifying urokinase have been reported, including inorganic carriers such as calcium phosphate gel and silica gel, Amberly l-OG50, and DEAB.
-Sephadex, OM-Sephadex, and other ion exchangers are widely used.

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

However, in these conventional methods, pyrogens are removed in one step from crude urokinase, which has a specific activity of about 10,000 international units/flea-white or less, and high-purity hemp with a specific activity of about 100,000 international units/flea-white is removed. Urokinase cannot be obtained.

The present inventors have investigated various methods for easily and economically obtaining highly purified urokinase in high yield, and have succeeded in creating a novel adsorbent for affinity chromatography that has a significantly high purification effect.

That is, the present invention provides: ``(1) A water-insoluble carrier is provided with a group represented by formula (A) and a group represented by formula (B) in a molar ratio of (A) group = (B) group of 75:25. An adsorbent for enzyme purification that is chemically bonded to have a ratio of 35:65 to 35:65.

-NU(OH) 0OOH...(A) (However, R in the formula
represents an amidis or guanidino group) (2) The group represented by formula (A) and the group represented by formula (B) are added to a water-insoluble carrier at a molar ratio of (A) group:(B) group of 75. A method for purifying urokinase, which comprises adsorbing urokinase to an adsorbent for enzyme purification which is chemically bonded so that the ratio is within the range of :25 to 35:65, and eluting the adsorbed urokinase.

-NH(0H2), 0OOH...(A)-NH(0H
2),0ONH○)11. ...(B) (However, in the formula, R represents an amidis group or a guanidino group)
It concerns J.

An adsorbent with 6-aminocaproic acid as a spacer and para-aminobenzamidine as a ligand and a method for purifying urokinase using the adsorbent were already published in Japanese Patent Publication No. 13266/1983.
Publication and Biochimica et Biophy
sicaActa445, 215-222 (1976)
, is known from .

However, in the adsorbents described in these publications, 6-aminocaproic acid is bound to an insoluble carrier as a spacer, and there is no group in which para-aminobenzamidine is bound to all of the terminal carboxy groups.

The present inventors have created a product in which 6-aminocaproic acid is bound to an insoluble carrier, and a ligand is bound to the carboxy group at the end of the carrier, and the relationship between the binding ratio and the purification effect of the original urokinase has been determined. As a result of various studies on the relationship, we found that an adsorbent in which 25 to 65, preferably 25 to 45, carboxy groups are bound to a ligand exhibits the unexpected effect of producing a very pronounced A'N production effect on urokinase. I found out.

According to the present invention, an adsorbent is packed in a column, a crude urokinase-containing solution is passed through the column to adsorb urokinase, and after washing the adsorbed urokinase with a neutral aqueous solution, an aqueous salt solution of the inner throat is washed. When eluted with 20%, it is possible to obtain pyrogen-free urokinase with a specific activity of 5000 to 130,000 international units/flea white in a yield of 8 m% or more. Crude urokinase is obtained from human urine using a conventional method with a specific activity of 500 to 1000 international units/'/flea, and contains 0.2 to 0.4 M of sodium chloride.
4000 in 1 M phosphofermentation buffer (]'II = 6-8)
It is dissolved in a concentration of 0 to 100,000 international points/mA and adsorbed through a column. The washing bath usually uses the same buffer used to dissolve crude urokinase. The salt concentration in the buffer is 0.05
~0.5M, with a good range of 0.2~0.4M. Urokinase bath solution is salt concentration 1 & 0.05-0.5M,
Preferably, a buffer solution containing 0.2 to 0.4 M acetic acid (H=3-5) is preferred. The uninvented adsorbent can be easily produced by appropriately combining reactions known per se. That is, in order to bond the group represented by the formula (A) to a water-insoluble carrier, CDI (
N,N'-carbonino di "midazole" activation method,
Conventional methods such as cyanogen bromide activation can be used.

6-Also, in order to condense a ligand to the terminal of the group of formula (,A,) and convert it with the group (of formula (B)), OMO(1-cyclohexyl-3-(2-mol 7-olinoethyl)calfosiimito. ED]
Conventional condensing agents such as OC1-ethyl-.3-(3-dimethylaminepropyl)carbodiimide] can be used.

The water-insoluble carrier may be one that can bond the group represented by the formula (A), such as agarose, crosslinked dextrin, cellulose, etc. Class i,
Polymers such as polyacrylamide are used.

Next, the effects of the present invention will be demonstrated through trials and measurements.

Test Example Sepharose 0L-6B (manufactured by Pharmacia Fine Chemicals) was activated to bind 776 μmole of 6-aminocazuronic acid per gel, and adsorbents were prepared by condensing various ratios of paraaminobenzamidine to this. Fill each column with 6 ml of gelatin adsorbent and add phase urokinase (specific activity 3400 international units/''/flea white).
was adsorbed onto the gel, washed with a 0.1M phosphoric acid solution containing 0.4M NaCl (PIT = 7.0), and then washed with a 0.1M phosphate solution containing 0.4M NaCl (PIT = 7.0). It was eluted with IM acetic acid buffer (PTi=4), and the eluted urokinase was tested for specific activity, yield, and pyrogen.

The results of this test are shown in Table 1.

As is clear from the test results in Table 1, as the binding ratio of para-aminobenzamidine increases, the specific activity of urokinase in the eluate decreases, and pyrogenic substances also become more difficult to remove. On the other hand, when the binding ratio of paraaminobenzamidine was less than 25, the yield of urokinase was extremely reduced.

On the other hand, as shown in A9 of barley 1, the amount of condensation of baraminobenzamidine was 163 μmol/la1. Ge
It has been discovered that even if the bonding ratio is less than 1, a yield of 8 m% or more can be obtained as long as the bonding ratio is within the range of 25 to 65.

In addition, the binding ratio of paraaminobenzamidine is 45 to 65%.
In the range of , pyrogen testing may become mediastinal.

It is also clear that this is not due to the amount of bound para-aminobenzamidine but to the binding ratio.

In the present invention, the activity of urokinase is determined by fibrin plate method [: BlocMm, Biophys, Act
A 24, 27B (1957), ), and the amount of spacer and ligand bound to the gel was calculated from the quantitative value of nitrogen by the Kermer method.

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

Example 1 Sepharose 0L-6B (7 Almacia ψ Fine Chemicals Co., Ltd.%) 20a/! was activated with cyanogen bromide 3y at pH 11 and 6-aminocaproic acid 25
Sepharose OL-6 was reacted with y at 20°C for 10 hours.
A B-aminocaproic acid conjugate was synthesized.

In this reaction, 80 μmole of 6-aminocaproic acid was bound per liter of gel. This gel is 20 resistant with CMO32
was added, 180 M1I of para-aminobenzamidine was added, and the mixture was reacted for 12 minutes at pH 4.2 to 46 to condense the para-aminobenzamidine. The bound para-aminobenzamidine was 24 μmoles L/mA of the gel, so the binding rate to the spacer was 30.

5 ml of this adsorbent was packed into a column, equilibrated with 0.1M phosphate buffer (PH72) containing 0.4M NaCl, and crude urokinase with a positive pyrogen test of 5,100,000 international years (specific activity 3500 international single international year f flea white) 8011
+7! After dissolving in the same buffer solution and removing insoluble matter, it was passed through a column. Column containing 0.4 M NaCl containing 0.1 M
It was thoroughly washed with a phosphate buffer (PH7.0) until the absorbance value at 280 nrn of the washing solution became 0.02 or less. After washing, add 0.1M acetate buffer containing 0.4M salt (PH4,0
) was used to elute the adsorbed urokinase. The thus obtained urokinase had a specific activity of 115,000 yen, a yield of 85 g, and a pyrogen test of 15.0 g.
000 international rank/Kf rabbit weight was negative.

Example 2 Sepharose OT, -4B-aminocaproic acid conjugate was synthesized in the same manner as in Example 1 by comparing the activities of 20 ml of Sepharose OI, -4B with cyanogen bromide 32 for 10 minutes. Gel is 6 per l me
-Aminocaproic acid was bound to 70 μm ole.

Konoge # 20 me K After adding 350 q of BDO, add 250 q of paraaminone enyl guanidine A-,
P)]: Stirred at room temperature for 5 hours while maintaining the temperature at 4.5 to 4.7. Takeru (ha)l me! 117.5
17 μm of varaminone enylguanidine was bound. Then, the binding rate of the ligand to spacer is calculated by h25tc.

This adsorbent 611/ was packed in a column, equilibrated with 0.1 M phosphate buffer containing 0.3 M sodium chloride (pH 7-2), and crude urokinase 2,700, which had a positive pyrogen test, was
000 international year (specific activity 600 international year q flea white) 5
After dissolving in a 0.3M salt-containing phosphorus Q solution (PH7.2) to remove insoluble matter, it was passed through a column to adsorb urokinase, and then the washing solution was washed with the same buffer solution at 280 nm.
The column was washed with a trench in which the absorbance (+&) was 0.01 or less. After washing, the adsorbed urokinase was eluted with 0.1 M acetate buffer (PH 4,0) containing 0.4 M* salt.

The hexaurokinase thus obtained had a specific activity of 80,000 IU/Kti, a yield of 83T, and a pyrogenic substance test result of 15,000I/Kti (rabbit weight), which was negative.

Patent applicant Wakamoto Pharmaceutical Co., Ltd. 13-

Claims (2)

[Claims] (1) The group represented by formula (A) and the formula (
The group represented by B) has a molar ratio of (A) group=(B) group of 7.
An adsorbent for enzyme purification that is chemically bonded to have a ratio of 5:25 to 35:65. -NH (0H2), 0OOH song...(A) (However, 7
, where R represents an amidino group or a guanidino group. ) (2) The group represented by formula (A) and the group represented by formula (B) are added to a water-insoluble carrier at a molar ratio of (A) group:(B) group in the range of 75:25 to 35:65. 1. A method for purifying urokinase, which comprises adsorbing r+ kinase onto an adsorbent for enzyme purification, which is chemically bonded to the adsorbent, and eluting the adsorbed urokinase. -NH(OT() 0OOH... (A) 6 (However, in the formula, n represents an amidino group or a guanidino group.)