SU1325076A1 - Method of recovering and purifying urokinase from biological fluids - Google Patents

Abstract

The invention relates to methods for producing purified enzymes, namely, urokinase, and can be used in the medical industry to produce urokinase preparations. The aim of the invention is to increase the yield and specific activity of the target product. The method involves the use of porous silica or lignin washing the sorbent with 0.05-0.2 M Tris-HC1 as a sorbent during adsorption of the enzyme with a pH 8-10 buffer solution and elution of the enzyme with a 2-4% aqueous solution of ammonia. The culture fluid obtained after growing a transplanted culture of human kidney cells, of 5 liters volume and 30 CTA activity and 1 ml is passed through a cm column filled with porous glass MPS-2000-UHC for 12 hours. Then the column is washed with 500 ml of 0.1 M Tris -NS buffer solution pH 10. Urokinase is eluted from the column after washing, using 4% aqueous ammonia solution as eluent. The elution rate was 6 ml / min. Concentrated and purified urokinase eluted from the column along with ammonia in a volume of 90 ml and has an activity of 1600 CTA / ML. The protein content in the resulting preparation is 0.4 mg / ml. The yield of urokinase is 96%, the specific activity of 4000 STL / mg. Table I Ate

Description

113250762

The invention relates to methods 1 to 7 CTA / ml is passed through a column for the preparation of purified enzymes, namely urokinase, and can be used 12 cm filled with silochrome S for 2 hours. Then the column is washed with 0.05 M Tris-HC buffer, pH 8 (30m g After washing, a 2% ammonia solution is passed through the column. At the ammonia output from the column, a 4 ml fraction containing the concentrated and purified urokinase, as

in the medical industry to obtain urokinae preparations.

The aim of the invention is to increase the yield and specific activity of the target product.

The method consists in the fact that the adsorption of urokinase is carried out on porous silica or lignin, and the removal of the ballast proteins from the sorbent is carried out by washing it with Tris-HC1 buffer pH 8.0-10.0.

The proposed method is carried out as follows.

The biological fluid containing urokinase activity is passed through a column filled with porous glass or lignin. The volume of the column corresponds to 0.1-0.01 of the volume of liquid to be passed Then the column is washed from impurities, passes Tris-HC1 buffer solution, pH 10. Usually 2-5 volumes of the column of such buffer are passed. After removal of the column, the adsorbed urokinase is eluted with a 2–4% aqueous solution of ammonia, passing it at a rate of about I cm / min.

Urokinase is eluted by a narrow zone, usually in a volume of 0.5 column volume, with the result that it is concentrated ten times. The yield of purified and concentrated urokinase is 80-95%.

Example 1. The selection of urokinase from the culture fluid.

The culture fluid obtained after growing a transplanted culture of human kidney cells, of 5 liters volume and 30 CTA / ml activity, passes through a 448 cm column filled with porous glass MPS 2000 GC for 12 hours. Then the column is washed with 500 ml of 0.1 M Tris-HCl buffer solution, pH 10. Urokinase is eluted from the column after washing, using A% A as eluent.

I12 cm, filled with Silochrome C80, for 2 hours. Then the column is washed with 0.05 M Tris-HC buffer, pH 8 (30 ml). g After washing, a 2% ammonium solution is passed through the column. At the ammonia outlet from the column, a 4 ml fraction containing concentrated and purified urokinase is collected, the activity of the fraction is 950 CTA / ml. The output of urokinates 90% The specific activity of the drug is 3300 CTA / mg.

PRI me R 3. The selection of urokinase from urine.

15 A person’s urine, diluted in half with water, 2.5 liter in volume and 9 STA / ML activity, is passed through a column of 1,510 cm, filled with porous glass MPS - 2000-VGH. The flask is washed with 0.05 M Tris-HC1 buffer, pH 9. Urokinase is eluted with ammonia solution. 7 ml of purified and concentrated urokinase are obtained. The output of urokinase 85%,

25 specific activity 2600 cta / mg.

PRI me R 4. The selection of urokinaei on lignin.

Culture fluid., Obtained after growing transplantable

30 cultures of a human kidney with a volume of 500 ml and an activity of 17 CTA / ml are passed through a column filled with lignin (isolated from coniferous trees). The volume of the column is 11 2 cm.

35 Then the column is washed with 0.05 M Tris-HC buffer, pH 10 (30 ml). After washing, a 4% aqueous solution of a is passed through the column; 1miaka. At the outlet of ammonia from the column is collected

40 fraction with a volume of 4 ml containing concentrated and purified urokinase. The activity of the fraction 1250 cta / ml, the protein content of 0.25 mg / ml. The output of urokinase 80%, specific activity

45 drugs 5000 CTA / mg.

EXAMPLE 5 Urokinase extraction was carried out according to Example 4. A 0.05 M aqueous ammonia solution was used to wash the column. The rate of elu- 50 Tris-HC1 buffer, pH 8.0. Elution of urocation was 6 ml / min. Concentrated and carried out with a 2% aqueous solution.

purified urokinase eluted from the column along with ammonia in a volume of 90 ml and has an activity of 1600 CTA / ml. The protein content in the resulting preparation is 0.4 mg / ml. Urokinase yield 96%, specific activity 4000 CTA / mg.

PR and Mer 2, the culture fluid with a volume of 250 ml and activity

1 7 CTA / ml is passed through the column

I12 cm, filled with Silochrome C80, for 2 hours. Then the column is washed with 0.05 M Tris-HC buffer, pH 8 (30 ml). After washing, a 2% ammonium solution is passed through the column. At the outlet of ammonia from the column, a 4 ml fraction is collected containing concentrated and purified urokinase, the activity of the fraction is 950 CTA / ml. The output of urokinates 90%. The specific activity of the drug is 3300 CTA / mg.

PRI me R 3. The selection of urokinase from urine.

Human urine, diluted in half with water, a volume of 2.5 liters and activity of 9 CTA / ML, is passed through a column of 1.510 cm, filled with porous glass MPS - 2000-UGH. The lamination of the column was carried out with 0.05 M Tris-HC1 buffer, pH 9. Urokinase was eluted with ammonia solution. 7 ml of purified and concentrated urokinase are obtained. The output of urokinase 85%,

specific activity 2600 cta / mg.

PRI me R 4. The selection of urokinaei on lignin.

Culture fluid., Obtained after growing transplantable

Cultures of a human kidney with a volume of 500 ml and an activity of 17 CTA / ml are passed through a column filled with lignin (isolated from coniferous trees). The volume of the column is 11 2 cm.

Then the column is washed with 0.05 M Tris-HC buffer, pH 10 (30 ml). After washing, a 4% aqueous solution of a is passed through the column; 1miaka. At the outlet of ammonia from the column is collected

4 ml fraction containing concentrated and purified urokinase. The activity of the fraction 1250 cta / ml, the protein content of 0.25 mg / ml. The output of urokinase 80%, specific activity

drug 5000 CTA / mg.

ammonia. The yield of urokinase is 85% of the specific activity of the drug 4500 cta / mg. il р and Мер 6. The culture fluid with a volume of 1 l and activity of 20 CTA / ML is passed through column 1, cm, filled with macroporous glass MPS-2500-VG X, for 4 hours. Then the column is washed with 0.2 M Tris3132

HCl buffer solution, pH 10. Urokinazu elute with an aqueous solution of ammonia. Concentrated urokinase (volume 9 ml) has a specific activity of 3080 CTA / mg. The yield of urokinase is 98%.

Example. 16000 ml of fresh human urine with 13 CTA / ML activity passed through a column

2,220 cm, filled with a porous stack of urokinase, which does not work with an ISP 1,150-YOGH islam, overnight. Washing of ballast proteins was performed with a 0.1 M solution of Tris-HCl buffer, pH 10. Urokinae was eluted with a 2% aqueous solution of ammonia. 170 ml of a solutionless solution of concentrated purified yi tcnase are obtained at a f5, specific activity 3900 o HL / mg, yield 96%.

The proposed method allows obtaining in one stage with a yield of 80-98% a preparation of urokinase with a specific activity up to 4500 CTA / mg. In order to obtain a highly purified preparation, conventional xpo-S matrix operations are performed.

The sorbents used in the proposed method, provide a fairly complete yield of the enzyme. Previously, these sorbents 30 were not used to obtain urokinase.

The proposed method has significant advantages over the known ones; first of all, it is simple to manufacture, since the isolation and purification of the enzyme occurs in one stage using a single sorbent. Purification is accomplished by washing the column with an experimentally selected buffer solution, after which the enzyme is eluted.

Urokinase has been found to be fairly strongly bound to porous silica and lignin. Consequently, washing is performed with buffer 45 solutions with a sufficiently high pH, which ensures the removal of most impurity proteins. The pH range of the buffer solutions at which impurity proteins are removed, but the enzyme is not washed out, should be in the range of 8-10. Disabling the pH in one direction or the other does not provide the necessary purity and yield of the drug.

The effect of the pH of the washing buffer solution on the purification of urokinase is shown in the table.

using different buffer solutions. A higher percentage of ammonia is not desirable for desorption, since this does not increase the yield of the enzyme, but leads only to an increase in the solubility of the sorbent matrix.

The previous method has a technical character, since it can be easily implemented with sterile conditions of pro-M1, of a large scale. Scaffolds 1u; 1al}, and the liquid and buffer solutions are sterilely fed into a column, which is pre-sterilized and sealed. Process monitoring: - carried out using a flow spectrophotometer. Sorbents are used repeatedly, they do not contain harmful impurities, their sorption properties (they are restored after progtusk-chpi through a column of 4% rasporov l. Tmiaka.

In addition, it is possible to obtain npen-iiites that have a high specific activity when used in the form of a lignin separator lignin. This is due to the fact that lignin is more specifically bound to urokinase in comparison with porous silica.

The proposed method can be tested in analytical and njieiu ipaTiiaHoM variants, it can be used to obtain urokinase in laboratories and medical industry (.The biological indicators of the preparations obtained (using the method type) show their possible use for it; I1TS1PP, 1,

Claims (1)

Invention Formula The method of isolation and purification of urokinase and biological liquids, 55 involves the adsorption of fer; 1e} 1ta on the sorbent, washing the sorbent by placing urokinase with a 2–4% ammonia solution, and so on. TaKiiM, when the pH of the Bbmie 10 clear buffer is observed, the enzyme is washed out, and when the pH is below 8, the content of impurity protein in the preparation rises. The specific eluent of d.p. uroknaey is ammonia. An aqueous 2-4% solution of ammonia can completely elute the urokinase adsorbed on the column, which is not successful when using using different buffer solutions. A higher percentage of ammonia is not desirable for desorption, since this does not increase the yield of the enzyme, but leads only to an increase in the solubility of the sorbent matrix. The previous method has a technical character, since it can be easily implemented with sterile conditions of pro-M1, of a large scale. Scaffolds 1u; 1al}, and the liquid and buffer solutions are sterilely fed into a column, which is pre-sterilized and sealed. Process monitoring: - carried out using a flow spectrophotometer. Sorbents are used repeatedly, they do not contain harmful impurities, their sorption properties (they are restored after progtusk-chpi through a column of 4% rasporov l. Tmiaka. In addition, it is possible to obtain npen-iiites that have a high specific activity when used in the form of a lignin separator lignin. This is due to the fact that lignin is more specifically bound to urokinase in comparison with porous silica. The proposed method can be tested in analytical and njieiu ipaTiiaHoM variants, it can be used to obtain urokinase in laboratories and medical industry (.The biological indicators of the preparations obtained (using the method type) show their possible use for it; I1TS1PP, 1, Invention Formula The method of isolation and purification of urokinase and biological fluids, which involves the adsorption of fer; 1e} 1ta on the sorbent, washing the sorbent by placing urokinase with a 2–4% ammonia solution, and so on. 513250766 that, in order to increase the yield and fhraKy of the sorbent with simultaneous removal of the specific activity of the target product of ballast proteins, it is carried out that 0.05-0.2 M Tris-HC1 buffer pH 8.0 is used as the sorbent , and from 10.0. The content of urocinae in the laundering eluate (9%),% O 0.8 0.4 0.5 0.1 M Tris-HCl. 15 22 0.3 0.3 0.3 0.2 0.2