JPS582672B2 - Urokina Zeno Bunri Seiseihouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for directly and easily separating and purifying urokinase, which is a plasminogen activating enzyme, from a solution containing urokinase.

Urokinase is an enzyme that exists in trace amounts in human urine, blood, and organs, and preparations made by separating and purifying it are known to be effective as thrombolytic agents, anticancer effect enhancers, etc. when injected intravenously.

Therefore, there is a strong industrial need for a method for efficiently obtaining urokinase from human urine, blood, and organ tissue culture fluid.

The conventional method is the porous silica adsorption method (U.S. Patent No. 3).
755083) and lysine-bonded agarose adsorption method (see JP-A-49-125584), etc. are known.

However, as a result of the study of the present invention, the former method requires a complicated elution operation, and the latter method tends to cause the adsorbent itself to become mixed, making it difficult to precisely control the flow rate, which is the key to elution conditions. It became clear that this method still needs improvement.

Furthermore, since all conventional methods require the addition of acid or alkali to adjust the hydrogen ion concentration of the solution containing urokinase before adsorption, there is no way to directly and easily separate urokinase without pre-treating the solution. requested.

From this point of view, the present inventors have conducted extensive research to overcome the shortcomings of these known methods, and as a result have discovered a simpler method for separating urokinase that is superior to conventional methods.

That is, the method of the present invention combines an adsorbent in which an amino acid selected from the group consisting of phenylalanine, tyrosine, leucine, isoleucine, parine, glutamic acid, and aspartic acid is bound to a water-insoluble carrier with an aqueous solution containing urokinase. This is a method for separating and purifying urokinase, which is characterized by adsorbing urokinase in the solution by contacting the solution, and then eluting the adsorbed urokinase.

Next, aspects of the present invention will be explained.

The adsorbent used in the method of the present invention is one in which amino acids are adsorbed onto a water-insoluble carrier, and the amino acids include:
It is at least one selected from the group consisting of phenylalanine, tyrosine, leucine, inleucine, parine, glutamic acid, and aspartic acid, and among these, phenylalanine and valine are particularly preferred because of their high affinity.

The water-insoluble carrier may be either organic or inorganic, as long as it is insoluble in water and can bind to the amino acid.

For example, alumina (At203), silica (S10
2), titania (Tt02), zirconia (ZrO2), nickel oxide (NiO)
Examples of the inorganic carrier include glass, columnite, kaolin, shirasu, diatomaceous earth, and clay.

These inorganic carriers may have any shape, but shapes with a large surface area such as fine particles, porous, and fibrous shapes are preferred.

In addition, polystyrene-based weakly acidic cation exchange resins (product names such as Amberlite CG-50 and Amberlite I RC-5)
0, DOWEX CCR-2), acrylic weakly acidic cation exchange resin (trade name, e.g. DIAION WK-1
0, WK-11, Duolite CC-3, CC-4
Examples of organic carriers include weakly acidic ion-exchange resins having carboxyl groups in their side chains, such as .

Next, in order to prepare the adsorbent used in the method of the present invention, any chemical method for binding amino acids to the above-mentioned carrier may be used. For example, (1) in the case of an inorganic carrier such as alumina or silica, activated A method in which an amino group is subsequently introduced using a silane coubling agent, a carboxyl group is further introduced using succinic anhydride, and finally condensation is performed using water-soluble polyprodiimide [Biotech.Bioeng.] 1 2 ,3
(2) In the case of inorganic supports such as titania and zirconia, the method of Weetall et al. (Biotech. Bioeng. 15, 455)
(1973)), but (3) in the case of inorganic carriers such as nickel oxide, the method of Wea r ing et al.
Biotech. Bioeng, 14. 975( 1
972)) can be applied.

In the case of a weakly acidic ion exchanger, direct condensation with water-soluble carbodiimide may be used.

In order to increase the specific activity (purity) of the obtained urokinase, it is necessary to introduce amino groups and carboxyl groups when an inorganic carrier is used as a carrier, and to introduce exchange groups when a weakly acidic ion exchanger is used as a carrier. Preferably, the carboxyl group is completely replaced by an amino acid.

When carrying out the method of the present invention, the adsorbent may be brought into contact with an aqueous solution containing urokinase to adsorb urokinase in the solution.The adsorbent may be added to the solution in a batch manner, or the adsorbent may be adsorbed in a column. The solution may be passed through a packed column.

When using a carrier with relatively large particles such as glass, Diaion, Amberly 1, etc., a simple method such as filtration or declination may be used to separate the solution from the carrier.

As long as the aqueous solution containing urokinase used in the method of the present invention has a pH of 3 to 8, there is no need to particularly adjust pi{, and urine, serum, organ tissue culture, P.I.
[ is usually used as is.

Of course, the solution may be not only such a body fluid but also a urokinase solution obtained from a body fluid by other methods or by the method of the present invention.

If the salt concentration in the solution is high, adsorption to the adsorbent will be inhibited, so in order to lower the salt concentration before contact, the solution is diluted with water or dialyzed to reduce the conductivity of the solution to 10 m.
It is preferable to lower it to below U/m.

In addition, if the solution contains an inert precipitate or the like, it is preferable to remove the precipitate by an operation such as filtration or centrifugation before contacting.

In order to elute the adsorbed urokinase by the method of the present invention, after washing to completely remove the unadsorbed solution from the adsorbent that has been brought into contact with the solution, an aqueous solution with a high ion concentration,
For example, it is preferable to wash with 4% ammonia water, IM sodium chloride solution, or IM ammonium sulfate solution.

Since urokinase is thus recovered in the eluate, it can then be obtained by conventional enzyme separation methods such as ammonium sulfate salting out, alcohol precipitation, ultraconcentration, and lyophilization.

In the method of the present invention, the adsorbent itself can be economically advantageously prepared;
Moreover, it has the advantage of being suitable for large-scale operations in terms of performance.

During repeated use of basic amino acid-bound polysaccharides, the bound basic amino acids are detached and the adsorption capacity decreases, or they disintegrate into fine particles, which gradually reduces the flow rate of the solution and decreases the gain and recovery rate per unit time. Moreover, since it is decomposed by microorganisms, it has the disadvantage that polysaccharide fragments are eluted and mixed into the product.

The properties of the adsorbent used in the method of the present invention that are suitable for large-scale operation include stability with no change in adsorption capacity even after repeated use, mechanical strength important for continuous force ram operation, and volume when compressed. It is superior to conventional adsorbents in terms of change rate, pressure loss during solution flow, and surface liquid drainage.

Next, the method of the present invention will be explained in detail with reference to Examples, but the method is not necessarily limited thereto.

In addition, the activity units of urokinase in the examples are those measured by the fibrin plate method.

Example 1 5 g of Amberlite IRC-50 (manufactured by Organo), which is a polystyrene-based weakly acidic cation exchange resin, was taken,
It was activated according to a conventional method and used as type H.

And this is 0. 1 M IJ acid buffer pH 7.
After that, water-soluble carbodiimide (manufactured by Protein Research Association) was added and mixed well with stirring.

Next, 5g1 of L-phenylalanine was added and reacted at room temperature for 5 hours.

After the reaction, the insoluble carrier was collected by filtration, thoroughly washed with water, and used as an adsorbent.

Urine 40 was obtained by diluting the adsorbent 2,9 thus prepared 4 times.
The mixture was dispersed in 0ml/l' and stirred at room temperature for 30 minutes.

Thereafter, the insoluble carrier adsorbed with urokinase was collected by filtration, and 4% ammonia water was poured into the funnel using 20 ml of 4% ammonia water. l. Urokinase was eluted by gradually adding .

Recovery rate from raw urine: 76% Specific activity: 140 units/In
9 (protein amount) of urokinase was obtained.

S Example 2 A sample 11' (manufactured by Hanui Chemical Co., Ltd.) was taken and washed with an organic solvent (acetone or alcohol).

Then 0. It was dispersed in 100 ml of IN-NaOH and washed thoroughly.

It was filtered through filter paper, and washed on a funnel with distilled water until the washing liquid became medium S.

Thereafter, the mixture was dispersed in 100rrLl of IN-HCA and stirred well.

It was filtered again using filter paper, washed on a funnel with distilled water until the washing liquid became neutral, and then dried under reduced pressure.

Take 8g of diatomaceous earth converted into gME in this way, and make 80JW
Ll was dispersed in toluene.

Here is Aminopipil I- IJ Engineering 1 xysilane 12
．． 8 g was added, a reflux condenser was attached, and the silicon bath temperature was adjusted to around 180° C. while heating and reacting for over 7 hours.

After the reaction, collect and thoroughly wash with acetone or methanol.
After drying, disperse in cold water, add succinic anhydride 89, add ice-cooled 2N-NaO'H while cooling with ice, and adjust the pH to 6.0.
It was adjusted to

The reaction was allowed to proceed overnight with stirring, and the reaction product was collected by filtration.

After thorough washing with water, the next operation was carried out. The thus obtained diatomaceous material was dissolved in 0.1M phosphate buffer pH 7. 0, 3
1 mg of water-soluble carbodiimide (described above) was added and stirred well.

2 g of L-valine was added to this, and stirring was continued at room temperature for 5 hours.

After the reaction, the insoluble carrier was collected by filtration, thoroughly washed with water, and then used as an adsorbent.

Urokinase was recovered from urine by the same procedure as in Example 1.

The recovery rate from raw urine was 87%, and the specific activity was 260 units/~(
protein content).

Example 3 Diaion W, an acrylic weakly acidic cation exchanger
2 g of K-11 (manufactured by Mitsubishi Kasei) was activated according to a conventional method and used as Type H.

Then, add this to 0.1M phosphate buffer i&pH 7. 0
, after dispersing at 30 mA, water-soluble, carbodiimide 1
rrLl was added and stirred well.

Next, 2 g of L-phenylalanine was added and stirring was continued for 5 hours at room temperature.

After the reaction, the insoluble carrier was collected, thoroughly washed with water, and then used as an adsorbent.

The adsorbent thus prepared was packed into a column of ICrrL x 2CrrL, and urine diluted 4 times was allowed to flow down from above.

After completely washing away, add 20rIL of 4% ammonia water.
was allowed to flow slowly and the urokinase was eluted.

The urokinase preparation thus obtained had an activity of 160 units/Tl1y protein amount, and a recovery rate of 65%.

Example 4 An adsorbent 2y prepared in the same manner as in Example 2 using Shirasu balloon (manufactured by Sun Kogyo) in place of the diatomaceous earth carrier and L-phenylalanine in place of L-valine was used. , was dispersed in 400 rrLl of urine diluted 4 times, and urokinase was allowed to adsorb at room temperature for 30 minutes.

The carrier adsorbed with urokinase was collected on the funnel by filtration, and 4% aqueous ammonia was gradually added on the funnel to allow the urokinase to flow out.

The urokinase preparation thus obtained had a recovery rate of 65% and a specific activity (4270 units/~(protein amount)).

Example 5 By the same operation as in Example 3, an adsorbent was prepared by binding L-valine to diamond ions.

Using the thus obtained adsorbent, a recovery rate of 86% was obtained using the same procedure as in Example 3. f3 sex 190 units/■
(Protein amount) of urokinase preparation was obtained.

Comparative Example 1 Urokinase was adsorbed and eluted in the same manner as in Example 2 using Diatomycin itself which had not been converted into an amino acid. Table 1 shows the results.

An example using a diatomaceous compound bound to glutamic acid or valine is also shown, and it can be seen that both the activity recovery rate and the specific activity are higher in this case, and the effect of the bound amino acid is remarkable.

Comparative Example 2 A case where urokinase was adsorbed and eluted only with Diaion WK-10 (manufactured by Mitsubishi Kasei) and a case where urokinase was adsorbed and eluted with Diaion WK-10 (manufactured by Mitsubishi Kasei).
Table 2 shows a comparison when urokinase was adsorbed and eluted using a carrier to which valine was bound.

When valine is bound, the specific activity increases by about 1.4 times and the activity recovery rate increases by about 4 times, which clearly shows the effect of valine binding.

Claims (1)

[Claims] 1. An adsorbent in which an amino acid selected from the group consisting of phenylalanine, tyrosine, leucine, isoleucine, parine, glutamic acid, and aspartic acid is bound to a water-insoluble carrier is brought into contact with an aqueous solution containing urokinase, and the solution is A method for separating and purifying urokinase, which comprises adsorbing urokinase therein and then eluating the adsorbed urokinase.