JPS5937071B2 - Purification method of urokinase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying urokinase, and more specifically to a method for purifying urokinase.
-Using as an adsorbent a bead-shaped gel of a polysaccharide containing sulfate groups in the molecular structure whose main components are galactose and 3,6-anhydro-D-galactose or more than IOW/W, human urine or crude derived therefrom The present invention relates to an industrially advantageous method for obtaining highly purified urokinase from urokinase.

Urokinase has the function of activating plasminogen contained in serum to produce plasmin, which has fibrinolytic ability, so it is useful as an activator of the fibrinolytic system.
It is widely used clinically to treat peripheral arterial thrombosis and myocardial stiffness.

In addition, in recent years, its use as an enhancer for anticancer drugs has been developed.
Its demand is rapidly increasing.

Methods for purifying urokinase include methods using ion exchangers such as cellulose phosphate, Amberlite IRC-50, carboxymethyl textolan, and carboxymethyl cellulose, silica gel, glass beads, porous glass,
Carbowax-treated porous glass, method using physicochemical adsorbents such as magnesium aluminum silicate, barium sulfate, diatomaceous earth, activated diatomaceous earth, hydroxyapatite, bentonite, acrylonitrile synthetic fiber, cyanoalkylated cellulose, polyamide containing polyethyleneimine Methods using specific adsorbents such as fibers and agarose;
Various methods are known.

The present inventors have repeatedly conducted various studies on purification methods for urokinase. As a result, the present inventors have found that a molecular structure consisting mainly of D-galactose and 3,6-anhydro-D-galactose, which has never been used for the purification of urokinase, has been found. IOW sulfate group
It has been discovered that a bead-shaped polysaccharide gel containing at least /W% has a specific adsorption effect on urokinase, and based on this knowledge, an industrially advantageous method for obtaining highly pure urokinase has been developed. It has been established.

That is, the present invention combines human urine or crude urokinase derived therefrom with D-galactose and 3,6-anhydro-
A process for preparing urokinase, which involves adsorbing urokinase by contacting it with a bead-shaped gel of a polysaccharide containing LOW/W% or more of sulfate groups in the molecular structure mainly composed of D-galactose, and then eluting the adsorbed urokinase. It is a purification method.

Examples of the polysaccharide according to the present invention include carrageenan, farcellan, and the like.

Carrageenan is a red algae (Rodophyceae).
igartinac-eae and 5olieriacea
Sulfate group 20-30W extracted and purified from seaweed belonging to e.
/W% containing polysaccharides, such as Genyugel W
G (trade name manufactured by Copenhagen Pectin Factory), Kenyugel CWG (same as above), Genyuvisco J
(same as above).

In addition, farcellan is a red algae (Rodophycea
Furcellaria fastigia, a species of e)
It is a polysaccharide containing 12 to 16 w/w% of sulfate groups extracted and purified from ta, and for example, Farcellan manufactured by Nortex, Denmark, is an example.

In the method of the present invention, such a polysaccharide is formed into a bead-like gel (
A product molded into a bead-shaped polysaccharide gel (hereinafter referred to as a bead-shaped polysaccharide gel) is used.

The method for preparing the bead-shaped polysaccharide gel is summarized as follows.

First, add l
~IOW/W% of the above polysaccharide aqueous solution is added and emulsified and dispersed.

The emulsion thus obtained was then pressed down for 10 to 5 minutes.
℃, or by adding ions such as ammonium ions and potassium ions to the emulsion, or by adding an alkylene diamine such as nonamethylene diamine to the emulsion.
A bead-shaped polysaccharide gel of about μ size is obtained.

Examples of the emulsifier used in preparing the bead-shaped polysaccharide gel include higher fatty acid esters of sorbitan such as sorbitan monooleate and sorbitan monostearate.

Further, examples of the organic solvent include a toluene/carbon tetrachloride mixed solvent, a cyclohexane/chloroform mixed solvent, and the like.

The method of the present invention will be specifically explained below.

First, human urine or a crude urokinase solution derived therefrom is allowed to flow down a column filled with bead-shaped polysaccharide gel, and urokinase is adsorbed onto the bead-shaped polysaccharide gel.

This adsorption operation is preferably carried out at a pH of 3 to 9, and the electrical conductivity of the human urine or crude urokinase solution flowing down the column is preferably adjusted to 20 to 100 m mho/Cm.

Under these conditions, urokinase is adsorbed on the beaded polysaccharide gel, and some impurities flow out without being adsorbed.

The column is then washed with a dilute salt solution having a pH of 3 to 9 (for example, a phosphate buffer with an electrical conductivity of 20 to 100 mmho/CIrL).

The amount of this washing solution is preferably 2 to 3 times the column capacity.

Although impurities are completely removed by this washing operation, urokinase remains adsorbed.

To elute the adsorbed urokinase, add p to the column.
Concentrated salt solution of H3-9 (e.g. electrical conductivity 100 m
This can be easily carried out by flowing down a phosphate buffer solution (saline solution, saline solution) of m11 o/crIL. In addition, when elution is performed using a saline solution, a concentration gradient method in which the concentration of the saline bath solution is increased stepwise is used. In some cases, impurities can be removed more completely.

The urokinase eluate thus obtained is subjected to salting out with ammonium sulfate in a conventional manner, followed by dialysis and lyophilization to isolate urokinase.

Incidentally, the adsorption operation and elution operation of the present invention can be carried out not only by the above-mentioned column method but also by a batch method.

The method of the present invention as described above has the advantage that highly pure urokinase can be obtained using a small amount of bead-shaped polysaccharide gel, and the bead-shaped gel can be used repeatedly.

The method of the present invention will be explained below with reference to Reference Examples and Examples, but the present invention is not limited to these examples in any way.

In addition, the titer of urokinase in the Examples was determined by the fibrin plate method of Ploug et al.
phys, Acta, 24, 278 (1957)], and the titer is expressed in the international unit determined by Johnson et al. [A, J-Johnson et al.: Thr om
bos.

Diathes, haernorrh(Stutt
g), 21, 259 (1969)].

Reference example l Niracol TO106 (sorbitan monooleate) 3
g to toluene-carbon tetrachloride mixture (3:lV/V) 4
Dissolve 8 oml/lc and heat at 50°C.

To this solution, 240 rILl of a 5% Genyugel WG (carrageenan manufactured by Copenhagen Pectin Factory) aqueous solution kept at 50°C in advance is added and emulsified and dispersed.

When this emulsion is cooled to 4°C while stirring, a bead-shaped gel is obtained.

By thoroughly washing this gel with acetone and a 2% potassium chloride aqueous solution, 160 g (wet weight) of bead-shaped carrageenan gel with a diameter of 40 to 250 μm is obtained.

Reference example 2 Niracol TS106 (sorbitan monostearate)
19 to toluene-carbon tetrachloride mixture (3:IV/V) 12
Dissolve in 0 ml and warm to 60°C.

Add to this solution 60 rIL of a 4% Faseleran (manufactured by Noritex, Denmark) aqueous solution kept at 60°C in advance.
1 and emulsify and disperse.

When this emulsion is stirred and cooled to 0° C., a bead-shaped gel is obtained.

This gel was thoroughly washed with ether and a 2% potassium chloride aqueous solution to form a bead-shaped furcerelan gel with a diameter of 40 to 250 μm.25. N wet polydispersion is obtained.

Example 1 Beaded carrageenan gel prepared in Reference Example 1 1Qml
was packed into a column (1,32 x 7.3 cm) and 0.1 M
0.01M phosphate buffer containing potassium chloride (pH 8.0
) for buffering.

This column was filled with a crude urokinase solution extracted from fresh human urine (50,000 units, 5.500 units/0TL).
ml protein, 0.1M potassium chloride content, electrical conductivity 2
0 mmh O/Cr1L9p) (s, 0) is allowed to flow down.

The column was washed with 20 ml of 0.01 M phosphate buffer (pH 8,0) containing 0.1 M potassium chloride, and then washed with 20 ml of 0.01 M phosphate buffer (pH 8,0) containing 0.1 M potassium chloride.
0.01M phosphate buffer containing 1M potassium chloride (pH 8)
20 ml of a 0.4M salt solution dissolved in 0.0,0) is poured down to remove impurities.

The column was then filled with 0.01 M potassium chloride.
Urokinase is eluted by flowing down 50 rrL of 0.5M saline solution dissolved in M phosphate buffer (pH 8.0).

The specific activity of the urokinase solution obtained in this way was 3
2,000 units/■ protein, and the activity yield from the crude urokinase solution was 90%.

Example 2 Bead-shaped farselan gel prepared in Reference Example 2 1Or
Using ul, 10 d of crude urokinase solution (50,00
0 units, 5,500 units/~ protein, 0.1M potassium chloride content, electrical conductivity 20mmho/CrfL,
pH 8.0) was treated in the same manner as in Example 1.

The specific activity of the urokinase solution obtained in this way was 3
1,000 units/q protein, and the activity yield from the crude urokinase solution was 80 units.

Example 3 100m of beaded carrageenan gel prepared in Reference Example 1
1 is buffered with 0.01M phosphate buffer (pH 8.0) containing 0.1M canalum chloride.

On the other hand, potassium chloride was added to 5 tons of fresh urine collected from an adult male to a concentration of 0.1M.

The electrical conductivity at this time was 30 m mho/cIIL.

Add the entire amount of the beaded carrageenan gel to this,
When stirred at 4°C for 10 minutes, all of the urinary urokinase is adsorbed to the gel.

The gel was collected by mouth filtration and diluted with 0.0
．． 200ml of 0.1M phosphate buffer (pH 8.0) followed by 200ml of 0.4M saline solution dissolved in o.oiM phosphate buffer (pH 8.0) containing 0.1M potassium chloride.
Wash at

The gel was then dissolved in 0.5M saline solution 5 in 0.01M phosphate buffer (pH 8.0) containing 0.1M potassium chloride.
After suspending the gel in 00ml and stirring for 10 minutes to elute the urokinase, the gel is removed by mouth.

The total activity of the urokinase solution thus obtained was 3
2,000 units, and the specific activity is 14,000 units/~
It was protein.

Claims (1)

[Claims] 1. Human urine or crude urokinase derived therefrom, D
- Urokinase is adsorbed by contacting with a polysaccharide bead-like gel containing sulfate groups in IOW/W% or more in the molecular structure mainly composed of galactose and 3,6-anhydro-D-galactose, and then adsorbed. A method for purifying urokinase, which comprises eluating urokinase. 2. The purification method according to claim 1, wherein the polysaccharide bead-like gel is a bead-like carrageenan gel or a bead-like furcellane gel.