KR810000446B1 - Process for production of urokinase - Google Patents

Abstract

Urokinase in human urine was absorbed onto a carboxy-alkylated cellulose absorbent and eluted from this absorbent a soln. contg. cationic surfactants such as lauryltrimethyl ammonium salts, lauryldimethylbenzyl ammonium salts, cethyltrimethyl ammonium salts, stearyltrimethyl ammonium salts, cethyl pyridium salts, lauryl pyridium salts or benzetonium salts to give highly purified urokinase(I). I is used as a thromolysis agent.

Description

Manufacturing method of urokanease

FIG. 1 Elution pattern.

The present invention relates to a method for preparing and purifying urokinase, a human enzyme substance, from urine.

Urokinase is an enzyme in human urine. It is known to promote the production of fibrin soluble protease, which is important for dissolving blood clots. Therefore, urocanase is a known substance known to play an important role in the treatment of thrombolysis.

The amount of urokanease present in human urine is extremely small and therefore its isolation is particularly difficult and expensive.

At present, urocainese is manufactured industrially by contacting urine of healthy people with all kinds of adsorbents and eluting the adsorbed urocaine with alkaline solution. However, according to this known method, proteins other than urokanease are adsorbed to the adsorbent together with urokanease as impurities. These impurities contain substances such as thromboplastin, which are undesirable for the production of urokanases. When eluting with an alkaline solution (pH 11 or higher), such as ammonia water, the yield of urocaneise is small (less than 70), and the yield of urokanease is less than 70. Many proteins, such as boplastin, are eluted. Therefore, only urocaine with small specificity is obtained. In such a case, the urocanase cannot be eluted with a weak alkaline solution (pH 10 or less), so that the eluate containing urocanase cannot be adequately obtained.

In order to invent an eluent capable of eluting urokanease adsorbed on the adsorbent in high yield, the present inventors contacted the urokanease adsorbed on the adsorbent with a cationic surfactant solution as the eluent, thereby eluting the urokanease in high yield. Highly purified and highly active urokanease can be obtained.

Furthermore, according to the present invention, since the content of a substance such as thromboplastin in the purified urocanase is very low, the urocanase purified according to the present invention can dissolve blood clots without causing blood coagulation. Therefore, highly purified, stable and highly active urokinase can be obtained without side effects. Based on these results, the present invention can be completed. The adsorbent used in the present invention can adsorb urokanease. Examples of such adsorbents are cation exchange resins, carboxyalkylcelluloses such as carboxy alkylated cellulose fibers such as cotton and gauze with adsorptive properties, carboxy methyl sefadex, polyacrylonitrile resin fibers, silica gel-bentonite, di Ethylaminoethylcellulose, diethylaminoethyl sefadex and phosphosecellulose.

Carboxyalkylated cellulose fibers such as cotton and gauze having adsorption are particularly preferred adsorbents used in the present invention. Since this adsorbent is a novel substance, it will be explained as follows. Examples of carboxyalkylated cerulosic fibers are cotton, which are fibers, ply yarns, knitted fabrics or cotton fabrics which are all or part of naturally regenerated cellulose. Cottons and gauzes with adsorptive properties are preferred as easily obtainable cotton.

Carboxyalkylation, such as carboxyalkylated cellulose fibers, can be applied with known carboxyalkylation methods. For example, such cellulose fibers are esters or acids of fatty acids substituted with α-halogens such as monochloroacetic acid, monobromoacetic acid and monochlorobutyl acid or alkali metal salts thereof and fatty acids substituted with α-halogens mentioned above. The halide is contacted in the presence of an alkaline substance such as sodium hydroxide according to conventional methods.

Such cellulose fibers, as they are or finely chopped, can be carboxyalkylated according to conventional methods and the degree of substitution of carboxyalkyl groups in the product is preferably about 0.20 to 0.08 for carboxyalkylation. For example, about 0.1 to 1 mole of α- per mole of alkaline solution used in this solution is swelled by dipping and swelling the aforementioned cellulose fiber in a material such as 5.5 to 16 moles of sodium hydroxide or aqueous potassium hydroxide solution per unit of anhydrous glucose. Fatty acid substituted by halogen or derivative thereof is added. The obtained solution is reacted at 30 to 90 ° C. for about 1 to 10 hours. This gives a product with such degree of substitution of carboxyalkyl groups.

The degree of substitution of the carboxyalkyl group in the reaction product can be freely changed by increasing or decreasing the amount and concentration of the alkali substance and the fatty acid substituted with α-halogen. However, in view of the degree of reaction and the rate of reaction, it is preferable to react with the above-mentioned alkali substance in the amount of 2-3 equivalents per fatty acid or derivative thereof substituted with α-halogen.

The above-mentioned carboxyalkylated product having a substitution degree of carboxyalkyl group of 0.21 or more cannot be used as the adsorbent of the present invention because it is difficult to pass the solution (human urine).

On the other hand, the above-mentioned carboxyalkylated product having a degree of substitution of carboxyalkyl groups of 0.07 or less is not preferable to the present invention because it is difficult to adsorb urocanase. Carboxyalkylated products having a degree of adsorption of carboxyalkyl groups between 0.1 and 0.18 are preferred in practice in view of the nature of the passage of the solution, adsorption of urocanise and adsorption amount of urokanase per unit. Carboxymethylated products are particularly preferred as carboxyalkylated products in view of their low cost, ease of production and adsorptivity and use of urokanases.

In contacting human urine or crude urokanease solution obtained from human urine with an adsorbent adsorbing the urokase according to the present invention, for example, a solution of human urine or crude urokanease product may be easily absorbed by the above-mentioned adsorbents. Contact with For example, industrially, a solution of human urine or crude urokinase product is passed through a column with the adsorbents mentioned above or an adsorbent is added to a solution of human urine or crude urokinase product.

The human urine is characterized in that the urine obtained by diluting the human urine obtained by dilution with water at a ratio of 0.5 to 12 g of water per 1 g of human urine originally adsorbed to the adsorbent than the originally obtained human urine itself. It is much preferable to contact with the adsorbent in order to adsorb a lot.

A 4.1 cm × 10 cm column is filled with 3.5 g (40 ml) of carboxy methylated adsorptive cotton (substitution degree of carboxymethyl group = 0.168, see No. 24 human urine, which will be discussed later in this specification). Cotton with carboxymethylated adsorption is washed with 0.1N hydrochloric acid solution, washed with water and deposited in 0.1N sodium chloride solution. The carboxymethylated adsorbent cotton thus obtained is used as the adsorbent. Fresh urine from a healthy person (specific gravity: at 1.016,33 ° C) is adjusted to pH 5.60 with concentrated hydrochloric acid and diluted with demineralized purified water to the mark. In this way, human urine having various dilutions is produced. The ratio of the adsorbed urokanease to the urokanease contained after passing the human urine thus obtained through the column filled with the adsorbent as described above is calculated by the following formula.

The activity of urokinase in the present specification is based on the WHO international urokinase standard method according to the standard method using plasminogen and fibrinogen of the human body, which is the original physiological substrate presented by the Thrombin Soluble Commission (NIH, USA), that is, the international unit ( IU) is calculated by the method. Urokinase must be dialyzed prior to measurement and thereafter the activity measurement calculated.

The adsorbed urocanase is eluted by depositing an adsorbent adsorbed with urocanase in a buffer solution of pH 8.0 containing 0.03% benzalkonium chloride and 0.5 M sodium chloride. The eluted urocanase solution was adjusted to pH 7.0 with hydrochloric acid solution and the activity was calculated for the urocanase thus obtained. The total activity of urokinase recovered from human urine is calculated.

The results thus obtained are shown in the following table (1).

TABLE 1

Human urine obtained by diluting the originally obtained human urine with water at a ratio of 0.5 to 12 g of water (dilution degree of human urine, 1.5 to 13) per 1 g of human urine originally obtained as shown in the above table is urokinase in the present invention. It is preferable to make contact with the adsorbent according to the present invention having a good adsorption force of.

Carboxyalkylated cerulosic fibers are practically useful as adsorbents of the present invention because they have a good ability to pass human urine and can adsorb almost all urokanease. In the case of the carboxyalkylated gauze obtained in Example 1 to be mentioned next, 100 g of carboxymethylated product has a stable capacity of about 1000 ml (density can be changed freely by pressure) and at least 36 liters per hour. When passing, more than 300 liters of human urine can be passed without reducing the ability to pass through the solution. Therefore, a large amount of human urine can be easily processed in a short time by carboxyalkylated cerulose fibers such as cotton and gauze having adsorption properties.

Experimental Example 1

In a beaker, 50 g of commercial gauze (0.309 mole per unit anhydrous glucose), 16 to 184 g of sodium hydroxide and 0.5 to 1.1 l of water are mixed and stirred thoroughly. The mixture is left at room temperature for 2 hours. 60 to 181 g of monochloroacetic acid crystals are added to the mixed solution, and the mixed solution is left to stir at 70 DEG C for 4 hours with occasional stirring. A portion of the product is washed with 2 L of methanol and 1 L of acetone and then dried in a drier under vacuum. The other part of this product itself is used as the adsorbent of the present invention without being washed.

Products with all kinds of degrees of substitution of the carboxymethylated groups will yield all kinds of doses or concentrations of sodium hydroxide and monochloro acetic acid used in the reaction. The obtained results are shown in the following table (2).

TABLE 2

The total exchange capacity (corresponding to 1 g) obtained according to the method for measuring the total exchange capacity in a cation exchange resin which is a weak acid (Ion Exchange Resin Manual (1), issued by Mitsubishi Kasei Kogyo Co., Ltd.) is a composition of cellulose. The degree of substitution is determined by conversion to chemical equivalents per unit of phosphorus anhydrous glucose.

Experimental Example 2

In a 1-liter beaker, 50 g of adsorbent commercial cotton (0.309 mole per unit of anhydrous glucose), sodium hydroxide and 800 ml of water are mixed and stirred thoroughly. The mixture is left at room temperature for 2 hours. Monochloroacetic acid crystals are added to this mixture. Reaction and treatment of the obtained reaction solution yield a dried product by the same method as in Experimental Example 1.

Products having all kinds of substitution degrees of the carboxymethyl group are obtained by using all kinds of doses or concentrations of sodium hydroxide and monochloroacetic acid used in the above reactions corresponding to the degrees of substitution.

The results are shown in the following table (3).

TABLE 3

As the cationic surfactant material of the cationic surfactant solution which is the eluent of the present invention, for example, tetraalkylammonium salt, cetyltrimethylammonium salt, stearyl-trimethyl-ammonium salt, higher alkyl-alalkyl quaternary ammonium salt, higher alkyl-dimethyl Benzyl-ammonium salt, dimethyl-benzyl-lauryl ammonium salt, higher alkyl aryl, quaternary ammonium salt, higher alkyl-dimethyl-phenyl ammonium salt, aryl-alalkyl-alkyl quaternary ammonium salt, dimethylphenyl-benzyl ammonium salt, higher alkylpyridinium salt, Lauryl pyridinium salts and polyalkyl-naphthalene methylpyridinium salts. The alkyl group is lower alkyl having 1 to 6 carbons or higher alkyl group having about 11 to 20 carbon atoms and the aryl group mentioned is phenyl.

Specific examples of such cationic surfactant include lauryltrimethyl ammonium salt, lauryl-dimethyl-benzyl ammonium salt, cetyltrimethyl ammonium salt, stearyl-trimethyl ammonium salt, tetradecyldimethyl-benzyl ammonium salt (benzalkonium salt), N, There are products obtained by condensing N-diethylethylene diamine with fatty acids such as oleic acid and quaternizing the condensation product with alkylating agents such as benzene chloride, dimethyl sulfuric acid (sapamine, BCH, sapamine MS).

Cetyl pyridinium salt, stearamide-methylpyridinium salt, lauryl pyridinium salt, cetyl quinolinium salt, lauryl aminopropionic acid ethyl ester salt, lauryl aminopropionic acid metal salt, lauryl dimethyl betaine, stearyl dimethyl beta Phosphorus, lauryl dihydroxy ethyl betaine and benzetoninium salts.

Aryl is normally a phenyl group. Quaternary ammonium salts that are commercially useful and particularly used in the present invention are as follows:

The eluent used in the present invention has a pH of 6-9 and particularly preferably 7-8.

Increasing the pH of the eluent (greater than 10) decreases the yield and purity of the eluting urocanase.

The concentration of cationic surfactant in the aqueous solution which is the eluent of the present invention is about 0.000001 to 0.5% (W / V). The concentration of the surfactant as an eluent should not be high enough to cause denaturation of urokanase and other proteins.

The eluent, which is a low concentration of cationic surfactant, is not preferred in the present invention because the elution effect of urocaine is very low.

The range of suitable concentrations will vary depending on the type of cationic surfactant used.

For example, it is 0.001 to 0.1% (W / V) for the benzylconium salt, 0.00001 to 0.001% (W / V) for the benzenetoninium salt, 0.001 to 0.1% for the pyridinium salt and the stearyl-trimethylammonium salt. In this case, 0.0005 to 0.5% (W / V) is preferable.

The dissolution method is in accordance with the usual dissolution means or method.

The eluted urokanase solution obtained by the present invention contains such cationic surfactants, but these surfactants can be easily removed by conventional methods using molecular sieves such as dialysis or membrane filters or dextran gels.

In this specification, the quantification of protein content is based on Lowry's method using a folin reagent. The sample must be dialyzed before the measurement, depending on the sample and purpose.

The standard for this measurement is albumin in purified bovine serum. In order to ensure certainty, the content of these proteins may be quantified by the Burrat method.

In the present specification, the content of a substance such as thrombo plastin is determined by measuring the shortening of the remineralization time of the plasma by a conventional plasma-coagulation-controlling method (using plasma of a person added with lyophilized citrate manufactured by Ortho Company). Quantify Samples should be dialyzed against physiological saline prior to measurement, depending on the purpose of sampling. A standard with thromboplastin activity is Ortho Brain ThromboPlastin (freeze-dried thromboplastin in rabbit brains manufactured by Oroto Company).

Coagulation time is measured by the Thromboelastograph (Helig Corporation) because the detection of blood clot production in plasma is difficult in the high-performance state of urocanase by the usual method.

According to the fibrinolytic action on the blood clot of plasma measured by the thrombus elasticity descriptive method, the urokinase obtained in the present invention has a high thrombus decay action.

The following examples are illustrative rather than limiting on the invention.

Example 1

3 g of carboxymethyl cellulose powder (available from Ciba) is packed into a 9 ml column. In the same way, six columns filled with this carboxymethyl cellulose powder were prepared. 100 ml of urine with 560 I.U activity diluted in urine of healthy adults are passed through each column at 5 ° C. The urocane adsorbed on the carboxymethyl cellulose powder adsorbent per column is 510 I.U.

The column was washed with 0.1 M sodium chloride solution, and the following six eluents (25 ml) were passed through each of six columns at 5 ° C. to elute the adsorbed urocanase. Human urine concentrations with activity and specificity as starting materials are 5.6 units / ml and 40 units / 1 mg of protein. The results are shown in the following table (4).

As shown in Table (4), urocanase cannot be eluted at 5 ° C. with an aqueous ammonia solution of pH 8.0, but all of the adsorbed urokanases were used as the eluent under the same pH conditions. Can be eluted. Such cationic surfactants increase the urocanize activity, and thus the recovery rate of the urocanize activity is 100% or more. Considering the fact that the specific concentration of cationic surfactant used increases the urocanase activity by 1.2 to 1.5 times, the recovery rate of the urocanase molecule is greater than 90%. The specificity of the obtained urocanise is 11,000 to 15,000 I.U for 1 mg of protein, so that the urocanise is purified about 300 times.

In the same manner, when urocanise was eluted at 5 ° C. using an aqueous ammonia solution of pH 11.8 as eluent, the activity of urocanase was restored to 55% and the specificity of the eluted urocanase solution was 440 I.U for 1 mg of protein. to be.

TABLE 4

* Purification Ratio: Specificity of purified uro kinase to the specificity of natural urokanease.

Example 2

50 ml of healthy adult urine is passed through a column packed with 1 g of polyacrylonitrile synthetic fiber, Bonnel, at 5 ° C. The column is washed with water. Then 15 ml of 0.01 M Na ₂ HPO ₄ -NaH ₂ HPO ₄ 0.5 M sodium chloride buffer (containing pH = 7.2 at 5 ° C.) containing 0.003% benzalkonium chloride (produced by Takeda Pharmaceutical Co., Ltd.) at 5 ° C. The column is passed through and recovered by elution of 120 I.U of urocanase.

The total activity of urocanease in the initial human urine and the activity of urokanease adsorbed on the adsorbent is 105 I.U. 114% of the adsorbed urokanease is eluted.

After the adsorption of urocaine in the test, 0.01M Na ₂ HPO ₄ -NaH ₂ PO ₄ 0.5M sodium chloride buffer solution containing no benzalkonium chloride was allowed to elute the urocaine when passed through the column. Can't.

The experiment is repeated using Xran, such as polyacrylonitrile synthetic fibers instead of Bonnell. The same result as above is obtained.

Example 3

0.15 g (0.5 ml) of diethylaminoethyl cellulose (available from Brown Co., Ltd.) is charged to the column.

The column is passed 5 ml of Zourokinase (2200 I.U for 1 mg of protein) obtained from human urine having an activity of 10,000 I.U. The activity of urokinase adsorbed on the column is 5,500 I.U.

Pass ₂ ml of 0.01 M Na ₂ HPO ₄ -NaH ₂ PO ₄ 0.3 M sodium chloride buffer (pH = 6.0 at 5 ° C.) with eluent to this column containing 0.02% (W / V) benzalkonium chloride. .

The recovery rate of urokanease is 125%.

The specificity of the obtained urocanize is 92,000 I.U for 1 mg of protein.

In the above experiment, the adsorption of urocaine was followed by a column of 0.01 M Na ₂ HPO ₄ -NaH ₂ PO ₄ 0.3 M sodium chloride buffer solution (pH = 7.2 at 5 ° C.) without benzalkonium chloride. When passed through, the activity of urokanease is 300 I.U, and the recovery rate of urokanease is 5%.

As a result, urocanease can be eluted more advantageously by using the benzalconium salt according to the present invention.

Example 4

Carboxymethylated cotton (Product No. 24, Degree of Substitution of Carboxymethyl Group = 0.168) obtained in 50 g (500 ml) of Experimental Example 2 was packed into a column (x106 mm x 57 mm). 100 L human urine obtained by diluting 50 L urine of 50 L healthy adult man with 50 L water is passed through this column at 20 DEG C under 36 SV. The flow rate of the solution during passage of human urine does not change at all.

The ratio of adsorbed urokanease to urokanese initially applied is greater than 95%. The activity of adsorbed urokinase on column capacity is 275,000 I.U per ml. The activity of adsorbed urocanase per gram of dried carboxymethylated cotton is 5,500 I.U. The efficiency for treating human urine is 36 RV per hour. This experiment for treatment takes 5.5 hours.

Immediately after this treatment, 1250 ml of 0.01 M Na ₂ HPO ₄ -NaH ₂ PO ₄ , 0.5 M sodium chloride, 0.02% (W / V) benzalkonium chloride buffer solution (with pH = 9.0 at 5 ° C.) was brought to 5 ° C. Pass through the column and then elute the adsorbed material.

Immediately, the eluate containing urokanease is neutralized with hydrochloric acid solution. This eluate yields urokanase having an activity of 385,000 I.U.

The recovery rate of urocane activity in all operations (adsorption and elution of urokanease) is 140%.

The above Example 4 was repeated with respect to the carboxymethylated cotton obtained in Experimental Example 2 ((product number 21, degree of substitution of carboxymethyl group = 0.06) and product number 39, degree of substitution of carboxymethyl group = 0.22)]. . In the former case (product number 21), up to 30% of urocaine is adsorbed. In the latter case (product number 39), human urine does not pass and also loses the activity of urokinase in the urine.

The following experiment is conducted to compare with the present invention. 150 g (500 ml) of commercially available carboxymethyl cellulose powder (available from Ciba, Inc.) is packed into a column (106 mm x 57 mm). 100 L human urine obtained by diluting 50 L urine of 50 L healthy adult male with 50 L water is passed through this column at 20 ° C.

In this operation, the flow rate of urine added from the beginning is lowered. Pass at a much lower speed. Pass 1.5 liters of urine for the first hour and 3 liters of urine for the first six hours. After 6 hours, urine no longer passes.

The operation for recovering urokanease is as described above.

Only 3 liters of human urine (total activity of urokanease: 15,000 I.U) cannot be processed, so this operation is not industrially available. However, the treatment for analysis is also performed on such 3 liters of urine.

90% of the urocanise is adsorbed to the adsorbent in the column.

This carboxymethyl cellulose is divided in two similar ways.

One portion of this carboxymethyl cellulose was mixed with 750 ml of aqueous ammonia solution at pH 11.8 at 5 ° C. and the other portion of carboxymethyl cellulose was mixed with 0.01 M Na ₂ HPO ₄ -NaH ₂ PO ₄ , 0.5 M sodium chloride, 0.02% ( W / V) Mix with 750 ml of Benzalconium chloride buffer (pH = 9.0 at 5 ° C.). Thereafter, the eluent containing urocaine is centrifuged to separate it from the carboxymethyl cellulose powder.

In the former case, the activity of urocanase recovered by this elution method was 5,400 I.U (recovery rate: 36%), and in the latter case, the activity of urokanase recovered by this elution method was 10,800 I.U. (Recovery rate: 71%) This is thought to cause the loss of the activity of urocanize in carboxymethyl cellulose before dissolution.

Only the passed human urine is particularly excellent as seen from the above comparison.

The test was repeated for each commercially available carboxymethyl cellulose powder (manufactured by Brown Co., Ltd.), but the results were unsatisfactory as described above.

Example 5

0.7 g of carboxymethyl cellulose powder (CM-32, manufactured by Whatman Co., Ltd.) for chromatography was packed in a column (2 ml, 1 / d = 5).

Thromboplastin activity corresponding to 100 ml of crude urocaine solution (100 I.U / ml, specificity = 2,000 IU / 1 mg protein, 1 mg rabbit brain thromboplastin) obtained from human urine with an activity of 10,000 IU on this column. The ratio of thromboplastin / urocaine = 0.1 μg / 1I.U) is passed at 5 ° C. to adsorb the urocaine to the adsorbent. The carboxymethyl cellulose column thus obtained is washed with water. A small amount of aqueous ammonia solution was added to an aqueous solution of 1 M sodium chloride and 0.01 (W / V) benzalkonium chloride at 5 ° C. to adjust the pH to 9.0 to obtain an eluent. 25 ml of the eluent thus obtained is passed through the column at a rate of 5 ml / hr at 5 ° C. to collect the eluate into a fraction collector.

Urokinase with an activity of 11,000 I.U is recovered in the 0-2RV (0-4ml) fraction from the beginning. In this fraction, 110% of the adsorbed urokanease is recovered.

The specificity of urocanize of this fraction is 41.000 I.U for 1 mg of protein. (100 μg of thromboplastin activity) (20.5 fold purified; ratio of thromboplastin / urocaine = 0.01 μg / 1I.U) This ratio is reduced by this operation. Thus, highly purified good urokanases containing trace amounts of thromboplastin are recovered in high yield.

450 μg of thromboplastin activity (recovery rate = 45%) was recovered from a fraction of 8-10 RV (16-10 ml) of the eluate. No activity of urokanease is found in this fraction. Thus, 55% of the original dissolved thromboplastin is recovered from the total eluate.

As shown in the examples, the eluting urocaine is eluted with the eluent according to the present invention, so that highly purified urokanease can be recovered in high yield, and it is difficult to remove from the urocaine product by various known methods. It can be seen that it is easy to remove such substances.

The dissolution pattern is shown in FIG.

Tromboplastin activity is calculated in the same manner as above.

Claims (1)

Human urine or a urocanise solution obtained from human urine is contacted with a carboxyalkyl cellulose adsorbent of alkyl groups having 1 to 4 carbon atoms capable of adsorbing urocanises, and the adsorbed urocanase is lauryl trimethylammonium salt, lauryl-dimethyl -Eluting with a solution of cationic surfactants such as benzyl ammonium salt, cetyltrimethyl ammonium salt, stearyl-trimethyl ammonium salt, tetradecyl dimethyl-benzyl ammonium salt, cetylpyridinium salt, laurylpyridinium salt, benzethonium salt Method of manufacturing kinase.

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