JPS5858073B2 - Purification method of urokinase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention uses a strongly acidic cation exchange resin having a sulfonic acid group (NH4+ type) as an adsorbent, and a solution containing crude urokinase derived from human urine as a raw material. The present invention relates to a method for obtaining purified urokinase with high yield and high purity on a large scale.

Urokinase is an enzyme that generates plasmin, a fibrinolytic enzyme, from plasminogen, and is not only effective against various thromboses, but has also been found to significantly increase the efficacy of anticancer drugs when used in combination with anticancer drugs. There is.

Urokinase isolated from human urine has no broad effects such as antigen-antibody reactions and is currently widely used.

Conventionally, urokinase was purified by extracting crude urokinase from human urine using adsorbents such as barium sulfate, hydroxyapatite, silicates, and acrylonitrile fibers, and then using various weakly acidic cation exchangers and weakly basic anions to extract crude urokinase from human urine. A method of purification using an exchanger or the like is used.

However, none of these methods is necessarily superior in terms of yield and purity of the obtained urokinase, and more advantageous methods are currently being pursued.

As a result of intensive research into an advantageous purification method for urokinase, the present inventors found that urokinase was adsorbed by contacting a strongly acidic cation exchange resin with a sulfonic acid group prepared in the NH4+ form with a crude urokinase-containing solution, and an alkaline solution The present inventors have discovered the novel finding that urokinase with high specific activity can be obtained at a high recovery rate by elution with a salt solution, etc., and have completed the present invention.

As a technique related to the present invention, Kurt Bergs-
trom report (Arkiv Fer kemi
, vol. 21, p. 535, 1963).

According to this report, 0.02M ammonium acetate buffer of crude urokinase was added to DOWEX 50W (manufactured by Dow Chemical Co., USA) with a degree of crosslinking of 2-12% equilibrated with 0.02M ammonium acetate buffer pH 10. pH10
After contacting the solution, urokinase was eluted with 1M aqueous ammonia, and the crude urokinase was compared to 2.8
~8.1 times purified product was obtained, and the recovery rate of activity was 1
It is 8-32%.

The present inventors prepared DOWEX 50W in 0.02M ammonium acetate buffer pH 10 as shown in this report.
In other words, N)L, + substituted with the W ion of the exchange group on the ion exchange resin without following equilibration with
Without adjusting the ion ratio to pH 10,
All exchange groups on the strongly acidic cation exchange resin are NlH4+
The pH of the crude urokinase-containing solution to be prepared and brought into contact with the ion exchange resin was adjusted to 4 to 9. They succeeded in dramatically improving this and completed the present invention.

In order to prepare the ion exchange resin into the NH4+ type, a commonly used method is used: bringing an ammonium chloride solution into contact with the H+ type ion exchange resin, and measuring the pH to confirm that all H+ ions have been replaced with NH4+ ions. After checking, wash with distilled water (this can be done).

The dramatic improvement in activity recovery rate and purification rate in the present invention is due to
A strongly acidic cation exchange resin having a sulfonic acid group is prepared in the NH4+ form without equilibration with not only the pH 10 buffer described by Bergstrom but also various buffers (this is the first time this has been possible). be.

The method for purifying urokinase according to the present invention is as follows.

A strongly acidic cation exchange resin having a sulfonic acid group prepared in the NH4+ type is preliminarily adjusted to pH 4 to 9, preferably pH 6.
A crude urokinase-containing solution adjusted to a concentration of ~8 is brought into contact with the ion exchange resin to adsorb urokinase onto the ion exchange resin.

The pH is adjusted using a buffer such as a phosphate buffer.

Urokinase adsorbed on the ion exchange resin is specifically eluted (this can be carried out using an alkaline solution, a highly concentrated salt solution, or a combination thereof, taking into account the properties of urokinase).

As the alkaline solution, a buffer solution or dilute ammonia water having a pH of 10 to 12 can be used, and as the salt solution, an ammonium chloride solution can be used, and an alkaline solution with a pH of 8 to 12 containing salts such as ammonium chloride can also be used.

After adsorbing urokinase to the ion exchange resin, it may be eluted immediately with the eluent described above, but if it is washed with water or a low concentration salt solution in advance, most of the impurities other than urokinase are removed and the ion exchange resin is further eluted. This is advantageous because it can enhance the purification effect.

In this way, urokinase that is 10 times more purified than crude urokinase can be obtained at a high recovery rate of 60 to 70%.

The used ion exchange resin is washed and regenerated with a sulfur hydroxide solution and hydrochloric acid, and further prepared into NI (4+ type) with an ammonium chloride solution, so that it can be repeatedly used without reducing the adsorption ability of urokinase. It is economical and can be used in many ways.

The strongly acidic cation exchange resins having sulfonic acid groups used in the present invention include those into which sulfonic acid groups are introduced, such as polystyrene resins with a three-dimensional network structure and phenol-formalin resins, such as those manufactured by Dow Chemical Company, USA. Dowex 50W1 Amberlite I R-120B, CG-120, manufactured by Rohm & Barth, USA, Duolite C- manufactured by Diamond Jamrock, USA
20, etc., all of which are widely commercially available, but the use of DOWEX 50WX8 (200 to 400 mesh) gives the best results.

EXAMPLE The present invention will be specifically explained using the following example, but it goes without saying that the present invention is not limited to this example.

The urokinase activity was measured using the prolag fibrin plate method (Ploug et al.: Bicchem, Bioph
ys, Acta, vol. 24, p. 278, 1957).

Example l Dowex 50WX8 (200
~400 mesh) column (2,8X25crIl)
0.1M phosphate buffer containing 0.1M salt pH
Specific activity 648 international units (hereinafter referred to as I) dialyzed against 6,5
(abbreviated as U)/■Protein crude urokinase 343,000 IU
After washing the resin with 830 ml of distilled water, 150 ml of 4% ammonia water was passed through the column to elute urokinase and separate the fraction having urokinase activity. .

Here (the obtained urokinase is 246,000 I
U1 specific activity is 101,501 U/■ protein, which is 15.7 times more purified than crude urokinase, and the recovery rate is 71.7%.
Met.

Example 2 Duolite C-20 column prepared in NH4+ type (
2,8X 25 crrL) 190,000 IU of crude urokinase with a specific activity of 716 IU of protein, dialyzed against 0.1 M IJ acid buffer containing 0.1 M NaCl.
48 TL of a solution containing U was passed through to adsorb urokinase.

Next, after washing the column with 750M distilled water, 150ml
Urokinase was eluted with 4% ammonia water, and a fraction having urokinase activity was separated.

Urokinase obtained here (1 total activity 120000 I
The specific activity was 8190 IU/7'n9 protein, which was 11.4 times more purified than crude urokinase, and the recovery rate of activity was 63.2%.

Example 3 DOWEX 50WX, 8 (200
~400 mesh) packed column (2, OX 16
CrIL) #This, specific activity 869 IU dialyzed against 0.1 M phosphate buffer pH 6.5 containing 0.1 M sodium chloride
40 TL of a solution containing 159,000 IU of protein crude urokinase was passed through to adsorb urokinase.

Then, after washing the column with 260T/l distilled water, the
M ammonium chloride solution pH 9.6 (pH 9.6 with aqueous ammonia)
Urokinase was eluted using (adjusted H) and fractions having urokinase activity were collected.

The total amount of urokinase obtained here was 107,000 IU.
It has a specific activity of 10,800 IU/■ protein, which is 12.4 times more purified than crude urokinase, and the recovery rate of activity is 67.
It was 1%.

Claims (1)

[Claims] 1. Contact the crude urokinase-containing solution with a strongly acidic cation exchange resin containing sulfonic acid groups prepared in the N H,+ form to adsorb urokinase, and then add an alkaline solution, a highly concentrated salt solution, or a combination thereof (this A method for purifying urokinase, which comprises eluting urokinase using a solution according to the present invention.