A method for purifying urokinase

Abstract

The invention discloses a method for purifying urokinase, and relates to the technical field of separation and purification. The method comprises: using a re-solution containing EDTA to re-dissolve a crude urokinase enzyme to obtain a crude product solution, after the crude product solution is allowed to stand, filtering the supernatant, heating the filtered solution without insoluble impurities in a water bath, filtering again, performing affinity chromatography on the obtained filtered solution, and obtaining urokinase after elution. The invention alleviates the reduction of enzyme activity in the process of inactivating bacteria by water bath heating by using EDTA, is conducive to improving the retention of urokinase, and improves the enzyme activity yield and urokinase purity of urokinase in the capture chromatography process, and improves the specific activity of urokinase.

Description

Technical Field

[0001] The invention relates to the technical field of separation and purification, in particular to a method for purifying urokinase. Background Art

[0002] Urokinase is an enzyme protein isolated from healthy human urine or obtained from human kidney tissue culture. It mainly exists in the human body in two forms: small molecular weight urokinase and large molecular weight urokinase. Urokinase has the advantages of rapid onset, strong fibrinolytic effect and long-lasting effect on new thrombosis. Therefore, urokinase is used as the first choice drug for the treatment of diseases such as cerebral thrombosis, venous embolism and acute myocardial infarction. However, the existing urokinase purification method has the problem of easily reducing the enzyme activity and urokinase retention when purifying urokinase, and does not consider the problem of improving the enzyme activity yield and specific activity in the urokinase capture stage. In addition, the removal and classification of diatomaceous earth in the crude urokinase product are not considered during the purification process of urokinase.

[0003] Since the 1970s, the preparation of urokinase has been in the ascendant, and many refining methods have been reported. For example, CN105238772A discloses a method for separating and purifying urokinase, wherein urokinase is separated and purified to obtain a urokinase solution with endotoxin removed, specifically, male urine is acidified, adsorbed with a diatomaceous earth column, eluted with ammonia water and sodium chloride solution, and then the eluate is salted out to obtain a crude urokinase product, and then the crude urokinase product is added to ammonia water for dissociation to obtain a urokinase solution.

[0004] CN96104829.8 discloses a four-step purification process for recombinant human prourokinase, which consists of four steps: carboxymethyl radial ion exchange chromatography (CM-RIEC), microporous glass bead adsorption chromatography (MPG), dextran polyacrylamide high-performance gel chromatography (Sephacryl, S-200HR) and p-aminobenzamide affinity chromatography (PABZ), characterized in that CM-radial ion exchange chromatography is combined with the other three methods. After purification, the specific activity of prourokinase is increased by more than 280 times, more than 98% of foreign proteins can be removed, and the total recovery rate reaches more than 50%. Summary of the invention

[0005] The object of the present invention is to provide a method for purifying urokinase to solve the problems raised in the above background technology.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a method for purifying urokinase, the purification method comprising using a re-solution containing EDTA to re-dissolve the crude urokinase enzyme to obtain a crude solution, after the crude solution is allowed to stand, taking the upper clear liquid for filtration separation and affinity chromatography, and obtaining urokinase after elution.

[0007] Preferably, the reconstituted solution for reconstituted crude urokinase enzyme is a crude enzyme reconstituted solution containing 5-20 mM EDTA buffer salt.

[0008] Preferably, during the supernatant affinity chromatography stage, 5-20 mM EDTA is added to the supernatant.

[0009] Preferably, the purification method comprises the following steps:

[0010] S1, dissolving crude urokinase in a crude enzyme reconstitution solution containing 5-20 mM EDTA buffer salt, and adjusting the pH value to obtain a crude urokinase solution, wherein the crude enzyme reconstitution solution is prepared by mixing a buffer balance solution and an EDTA buffer salt, wherein the buffer balance solution is at least one of an acetate buffer, a citrate buffer, a phosphate buffer and a Tris-HCl buffer;

[0011] S2, the crude urokinase solution obtained in S1 is allowed to stand for 30-60 minutes, after which the upper clear solution is taken and filtered through a filter membrane to obtain insoluble impurities and a yellow solution containing no insoluble impurities, wherein the pore size of the filter membrane is 0.22 μm;

[0012] S3, heating the yellow solution 1 obtained in S2 at 60° C. in a water bath for 10 h, and after cooling, filtering the yellow solution 1 through a 0.22 μm filter membrane to obtain a yellow solution 2 free of insoluble impurities;

[0013] S4. Using the yellow solution II obtained in S3 as the supernatant II for affinity chromatography, adjusting the pH value of the supernatant II for affinity chromatography to 7.0-7.5, and performing affinity capture chromatography on the adjusted yellow solution II.

[0014] Preferably, the step S4 further includes the following steps: adding a mixed solution prepared with EDTA and a balancing solution to the second yellow solution, wherein the balancing solution is at least one of a phosphate buffer and a Tris-HCl buffer.

[0015] Preferably, S4 further includes the following steps: after affinity chromatography, eluting the chromatography column with an eluent, wherein the eluent is a saline acetic acid system with a pH of 3.0-5.0 and a conductivity of 0-80 mS / cm.

[0016] Preferably, the crude urokinase solution in S2 is allowed to stand for 45 minutes.

[0017] Preferably, the buffer is a citrate phosphate buffer, which contains sodium chloride, and the sodium chloride content is 150-500 mM.

[0018] Preferably, the balancing solution is a sodium phosphate buffer solution, which contains sodium chloride, and the sodium chloride content is 150-500 mM.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention alleviates the reduction of enzyme activity during the water bath heating and inactivation of bacteria by using EDTA, which is beneficial to improving the retention of urokinase, and improves the enzyme activity yield and urokinase purity of urokinase during the capture chromatography process, thereby improving the specific activity of urokinase.

[0021] 2. The present invention accelerates the rapid precipitation of insoluble impurities in urokinase by adding EDTA to the crude urokinase solution, which is beneficial for separating and removing diatom insoluble impurities in the crude urokinase, thereby improving the purification purity of urokinase.

[0022] 3. The present invention purifies urokinase by dissolving, precipitating, filtering and separating, and affinity chromatography, thereby proposing a simple-to-operate method for purifying urokinase, which is beneficial to the large-scale purification and production of urokinase.

[0023] 4. The present invention uses an eluent containing a saline acetic acid system to perform two-side elution capture during the affinity chromatography of urokinase, which is beneficial to increasing the amount of purified urokinase. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison diagram of the solutions to be subjected to affinity chromatography in the present invention. DETAILED DESCRIPTION

[0025] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0026] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0027] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0028] The present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commonly available products in the technical field.

[0029] The present application provides a method for purifying urokinase, which specifically comprises the following steps: S1, dissolving crude urokinase in a crude enzyme reconstitution solution containing 5-20 mM EDTA buffer salt, and adjusting the pH value to obtain a crude urokinase solution, wherein the crude enzyme reconstitution solution is prepared by mixing a buffer balance solution and an EDTA buffer salt, wherein the buffer balance solution is at least one of an acetate buffer, a citrate buffer, a phosphate buffer and a Tris-HCl buffer;

[0030] S2, the crude urokinase solution obtained in S1 is allowed to stand for 30-60 minutes, after which the upper clear solution is taken and filtered through a filter membrane to obtain insoluble impurities and a yellow solution containing no insoluble impurities, wherein the pore size of the filter membrane is 0.22 μm;

[0031] S3, heating the yellow solution 1 obtained in S2 at 60° C. in a water bath for 10 h, and after cooling, filtering the yellow solution 1 through a 0.22 μm filter membrane to obtain a yellow solution 2 free of insoluble impurities;

[0032] S4. Using the yellow solution 2 obtained in S3 as the supernatant 2 for affinity chromatography, adjusting the pH value of the supernatant 2 for affinity chromatography to 7.0-7.5, and performing affinity chromatography on the adjusted yellow solution 2.

[0033] The preparation method of crude urokinase used in this embodiment is as follows: fresh male urine is added to silica gel for stirring and adsorption, the silica gel is loaded into a column, the silica gel column is rinsed with tap water, and then the silica gel column is eluted with 2.5% by mass ammonia water, the eluate is collected, solid ammonium sulfate is added to the eluate, and precipitation is carried out at a temperature of 4-8°C, food-grade diatomaceous earth is used as the bottom of the filter layer, and 150 mesh nylon filter cloth is used as the filter screen, the eluate after precipitation is filtered, and the brown filter residue is collected by filtration, which is the crude urokinase enzyme powder.

[0034] Embodiment 1: A method for purifying urokinase, comprising the following steps:

[0035] S1, dissolving crude urokinase in a crude enzyme reconstitution solution containing 10 mM EDTA buffer salt, and adjusting the pH value to 7.0 to obtain a crude urokinase solution, wherein the crude enzyme reconstitution solution is prepared by mixing a buffer balance solution and EDTA buffer salt, and the buffer balance solution is a citrate buffer;

[0036] S2, the crude urokinase solution obtained in S1 is allowed to stand for 45 minutes, after which the supernatant is taken and filtered through a filter membrane to obtain insoluble impurities and a yellow solution containing no insoluble impurities, wherein the pore size of the filter membrane is 0.22 μm;

[0037] S3, heating the yellow solution 1 obtained in S2 at 60° C. in a water bath for 10 h, and after cooling, filtering the yellow solution 1 through a 0.22 μm filter membrane to obtain a yellow solution 2 free of insoluble impurities;

[0038] S4. Use the yellow solution II obtained in S3 as the supernatant II for affinity chromatography, and adjust the pH value of the supernatant II for affinity chromatography to 7.0, and perform affinity chromatography on the adjusted yellow solution II. Before affinity chromatography, add a mixed solution prepared with EDTA and a balancing solution to the yellow solution II, wherein the balancing solution is a sodium phosphate buffer solution. After chromatography, capture and elution are performed to obtain urokinase.

[0039] Embodiment 2: A method for purifying urokinase, comprising the following steps:

[0040] S1, dissolving crude urokinase in a crude enzyme reconstitution solution containing 15 mM EDTA buffer salt, and adjusting the pH value to 7.0 to obtain a crude urokinase solution;

[0041] S2, the crude urokinase solution obtained in S1 is allowed to stand for 45 minutes, after which the upper clear solution is taken and filtered through a filter membrane to obtain insoluble impurities and a yellow solution containing no insoluble impurities, wherein the pore size of the filter membrane is 0.22 μm;

[0042] S3, heating the yellow solution 1 obtained in S2 at 60° C. in a water bath for 10 h, and after cooling, filtering the yellow solution 1 through a 0.22 μm filter membrane to obtain a yellow solution 2 free of insoluble impurities;

[0043] S4. Use the yellow solution II obtained in S3 as the supernatant II for affinity chromatography, and adjust the pH value of the supernatant II for affinity chromatography to 7.0, and perform affinity chromatography on the adjusted yellow solution II. Before affinity chromatography, add a mixed solution prepared with EDTA and a balancing solution to the yellow solution II, wherein the balancing solution is a sodium phosphate buffer solution. After chromatography, capture and elution are performed to obtain urokinase.

[0044] Embodiment 3: A method for purifying urokinase, comprising the following steps:

[0045] S1, dissolving crude urokinase in a crude enzyme reconstitution solution containing 19 mM EDTA buffer salt, and adjusting the pH value to 7.0 to obtain a crude urokinase solution;

[0046] S2, the crude urokinase solution obtained in S1 is allowed to stand for 45 minutes, after which the upper clear solution is taken and filtered through a filter membrane to obtain insoluble impurities and a yellow solution containing no insoluble impurities, wherein the pore size of the filter membrane is 0.22 μm;

[0047] S3, heating the yellow solution 1 obtained in S2 at 60° C. in a water bath for 10 h, and after cooling, filtering the yellow solution 1 through a 0.22 μm filter membrane to obtain a yellow solution 2 free of insoluble impurities;

[0048] S4. Use the yellow solution II obtained in S3 as the supernatant II for affinity chromatography, and adjust the pH value of the supernatant II for affinity chromatography to 7.0, and perform affinity chromatography on the adjusted yellow solution II. Before affinity chromatography, add a mixed solution prepared with EDTA and a balancing solution to the yellow solution II, wherein the balancing solution is a sodium phosphate buffer solution. After chromatography, capture and elution are performed to obtain urokinase.

[0049] Comparative Example 1:

[0050] A method for purifying urokinase comprises the following steps:

[0051] S1, dissolving crude urokinase in a crude enzyme reconstitution solution containing 0 mM EDTA buffer salt, and adjusting the pH value to 7.0 to obtain a crude urokinase solution;

[0052] S2, the crude urokinase solution obtained in S1 is allowed to stand for 45 minutes, after which the upper clear solution is taken and filtered through a filter membrane to obtain insoluble impurities and a yellow solution containing no insoluble impurities, wherein the pore size of the filter membrane is 0.22 μm;

[0053] S3, heating the yellow solution 1 obtained in S2 at 60° C. in a water bath for 10 h, and after cooling, filtering the yellow solution 1 through a 0.22 μm filter membrane to obtain a yellow solution 2 free of insoluble impurities;

[0054] S4. Use the yellow solution II obtained in S3 as the supernatant II for affinity chromatography, and adjust the pH value of the supernatant II for affinity chromatography to 7.0, and perform affinity chromatography on the adjusted yellow solution II. Before affinity chromatography, add a mixed solution prepared with EDTA and a balancing solution to the yellow solution II, wherein the balancing solution is a sodium phosphate buffer solution. After chromatography, capture and elution are performed to obtain urokinase.

[0055] Furthermore, a capture chromatography method in the affinity chromatography process of urokinase is proposed, comprising the following steps:

[0056] Example 4: S1. The filler used in the capture chromatography was NM90 Agarase Benzamidine (Nanomicron, China), and a sodium phosphate buffer containing salt was used before loading, and the equilibrium volume was 5CV;

[0057] S2, using the yellow solution 2 in Example 1, Example 2, Example 3 and Comparative Example 1 as crude solutions, respectively, performing affinity chromatography on the crude solutions, and adjusting the pH value to 7.0 before affinity chromatography to obtain a crude urokinase solution for chromatography, and performing affinity chromatography on the crude urokinase solution for chromatography using a capture chromatography column;

[0058] S4, performing a capture elution on the chromatography column, and the capture chromatography eluent is a saline acetic acid system with a pH of 5.0 and a conductivity of 40 mS / cm;

[0059] S5, performing secondary capture elution on the chromatography column after the primary elution, and the secondary capture chromatography eluent is a saline acetic acid system, pH 4.0, conductivity 40mS / cm, and collecting ultraviolet peaks to detect enzyme activity and protein content at this stage;

[0060] Using the capture chromatography method proposed in Example 4, the yellow solution 2 containing urokinase in S4 in Example 1, Example 2, Example 3 and Comparative Example 1 was subjected to affinity chromatography, elution capture, and purification of urokinase.

[0061] The urokinase prepared in Example 1, Example 2 and Comparative Example 1 was taken to prepare a urokinase solution in the same proportion, and the enzyme activity and protein content were detected according to the relevant method of the Chinese Pharmacopoeia 2015 edition, and the specific activity and enzyme activity yield were calculated, wherein the specific activity (U / mg) = enzyme activity per unit volume / protein content per unit volume × 100%, and the enzyme activity yield (%) = total enzyme activity / total protein content × 100%. The results are shown in the following table:

[0062]

[0063] The yellow solution 2 used for affinity chromatography in Example 1, Example 2 and Comparative Example 1 is as shown in the attached manual. Figure 1 The yellow solution 2 described in the comparative example 1, the embodiment 1 and the embodiment 2 are shown from left to right.

[0064] Combine the above table and the instructions Figure 1 It can be seen that the method for purifying urokinase proposed in the present application uses EDTA to accelerate the precipitation of insoluble impurities in the crude urokinase solution during the purification of urokinase, and alleviates the loss of enzyme activity during the process of heating and inactivating viruses, thereby improving the enzyme activity yield of capture chromatography and the purity of urokinase. The operation is simple and is conducive to large-scale production.

[0065] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for purifying urokinase, characterized in that: The purification method comprises the following steps: S1, dissolving crude urokinase in a crude enzyme reconstitution solution containing 5-20 mM EDTA buffer salt, and adjusting the pH value to obtain a crude urokinase solution, wherein the crude enzyme reconstitution solution is prepared by mixing a buffer balance solution and an EDTA buffer salt, wherein the buffer balance solution is at least one of an acetate buffer, a citrate buffer, a phosphate buffer and a Tris-HCl buffer; S2, the crude urokinase solution obtained in S1 is allowed to stand for 30-60 minutes, after which the upper clear solution is taken and filtered through a filter membrane to obtain insoluble impurities and a yellow solution containing no insoluble impurities, wherein the pore size of the filter membrane is 0.22 μm; S3, heating the yellow solution 1 obtained in S2 at 60° C. in a water bath for 10 h, and after cooling, filtering the yellow solution 1 through a 0.22 μm filter membrane to obtain a yellow solution 2 free of insoluble impurities; S4, using the yellow solution 2 obtained in S3 as the supernatant 2 for affinity chromatography, and adjusting the pH value of the supernatant 2 for affinity chromatography to 7.0-7.5, and adding a mixed solution prepared with EDTA and an equilibrium solution to the yellow solution 2 before affinity chromatography, and the equilibrium solution is at least one of a phosphate buffer and a Tris-HCl buffer, and performing affinity capture chromatography on the adjusted yellow solution 2; In S4, the following steps are also included: after the chromatography affinity, the chromatography column is eluted with an eluent, wherein the eluent is a saline acetic acid system, and the pH is 3.0-5.0, and the conductivity is 0-80 mS / cm; The elution times of the chromatography column are twice, the first capture chromatography eluent is a saline acetic acid system with a pH of 5.0, and the second capture chromatography eluent is a saline acetic acid system with a pH of 4.

0.

2. A method for purifying urokinase according to claim 1, characterized in that: The re-dissolving solution for re-dissolving the crude urokinase enzyme is a crude enzyme re-dissolving solution containing 5-20 mM EDTA buffer salt.

3. A method for purifying urokinase according to claim 2, characterized in that: During the affinity chromatography stage of the supernatant, 5-20 mM EDTA is added to the supernatant.

4. A method for purifying urokinase according to claim 1, characterized in that: The crude urokinase solution in S2 was allowed to stand for 45 min.

5. A method for purifying urokinase according to claim 1, characterized in that: The buffer balance solution is a citrate buffer.

6. A method for purifying urokinase according to claim 1, characterized in that: The balancing solution is a sodium phosphate buffer solution, which contains sodium chloride, and the sodium chloride content is 150-500mM.

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