A method for separating and purifying urokinase

Abstract

The application provides a separation and purification method of urokinase, and relates to the technical field of separation and extraction. The separation and purification method comprises the following steps: S1: male urine, adjusting pH to 8.5, standing, and taking supernatant; S2: mixing the supernatant with adsorption material, eluting with an eluent, and obtaining a crude urokinase solution; the adsorption material is a core-shell silica gel polymer, Fe3O4@SiO2-NH2 is used as the core, and polystyrene is used as the shell; the eluent is 0.5-0.15M sodium citrate buffer, 1wt%-5wt% acetonitrile and 0.5-1.5mM EDTA; S3: the crude urokinase solution is subjected to anion exchange column chromatography, eluted with the eluent, and precipitated, and the urokinase is obtained. The urokinase prepared by the method has high content of high-molecular urokinase, high recovery rate and high activity yield.

Description

Technical Field

[0001] This invention relates to the field of separation and extraction technology, specifically to a method for the separation and purification of urokinase. Background Technology

[0002] Urokinase, abbreviated as UK, is an enzyme protein isolated from the urine of healthy individuals or obtained from human kidney tissue culture. Urokinase catalyzes the conversion of plasminogen into plasmin. Clinically, urokinase is mainly used to treat acute myocardial infarction, acute cerebral thrombosis, cerebral vascular embolism, thrombotic occlusive diseases, pulmonary embolism, and central retinal vein thrombosis. Moreover, because urokinase is derived from human urine or kidney cell tissue culture medium, it has advantages over fibrinolytic enzymes, such as being non-antigenic, pyrogen-free, and having fewer toxic side effects.

[0003] Human urinary urokinase mainly exists in two forms: natural high molecular weight urokinase (HUK, 54,000 Dalc) and low molecular weight urokinase (LUK, 33,000 Dalc). Although the enzymatic activities of HUK and LUK are almost identical in the presence of plasma in vitro, HUK has a higher affinity for plasminogen activator in protozoa and a longer half-life in blood, resulting in a thrombolytic effect 2-3 times greater than LUK. Therefore, the activity of HUK is considered more important than that of LUK. Thus, from a production process perspective, the yield and purity of high molecular weight urokinase are crucial indicators for evaluating the quality of the process.

[0004] Existing technologies have disclosed several methods for purifying urokinase. For example, Chinese patent application CN111647585A discloses a method for purifying urokinase. This method includes the following steps: taking a urokinase intermediate solution, loading it onto a gel chromatography column, eluting with a equilibration buffer, collecting the mid-elution buffer, and obtaining a purified urokinase solution; the gel chromatography packing material includes at least one of Superdex 75pg / GE, Superdex 200pg / GE, SephacrylS-100HR / GE, Chromadex 75PG / BESTCHROM, and Chromadex 200PG / BESTCHROM. However, it only discloses a purity of 95%-99%, without specifying whether it is HUK, LUK, or a mixture of both, and the activity yield is only 72%-83%.

[0005] Chinese patent application CN104031901A discloses a method for purifying urokinase, which separates the proteins in two major molecular weight regions of crude urokinase and further removes impurities, pyrogens and intracellular toxins from the crude product by means of adjusting pH value, etc., to obtain refined urokinase. The relative content of high molecular weight urokinase in the refined urokinase is 98.5%, but the activity recovery rate is only 80%.

[0006] A novel extraction method for urokinase from human urine (Li Yan, Food and Fermentation Technology, Vol. 49, No. 3) discloses a new extraction method using 3 mmol / L CuSO4 to precipitate urokinase from human urine. The pH is then adjusted to approximately 9.0 using ammonia, the precipitate is dissolved with EDTA, and finally, the urokinase is purified using 60% ethanol to obtain high-purity urokinase. The urokinase recovery rate is over 90%. However, the purity of the high molecular weight urokinase is not disclosed.

[0007] Therefore, developing a new method for the isolation and purification of urokinase, while improving the purity, yield, and activity yield of high molecular weight urokinase, is a key research focus for researchers in this field. Summary of the Invention

[0008] To address the above-mentioned problems, this invention provides a method for the isolation and purification of urokinase.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] On one hand, the present invention provides a method for isolating and purifying urokinase, comprising the following steps:

[0011] S1: Male urine, adjust pH to 8.5, let stand, and collect the supernatant;

[0012] S2: The supernatant is mixed with the adsorbent material and eluted with elution buffer to obtain crude urokinase solution;

[0013] The adsorbent material is a core-shell silica polymer with Fe3O4@SiO2-NH2 as the core and polystyrene as the shell;

[0014] The elution buffer is 0.05-0.15M sodium citrate buffer, 1wt%-5wt% acetonitrile, and 0.5-1.5mM EDTA;

[0015] S3: The crude urokinase solution is obtained by anion exchange column chromatography, elution with eluent, precipitation.

[0016] Preferably, step S1 is performed at a temperature of 5-15°C.

[0017] Preferably, in step S2, the preparation method of the core-shell silica polymer is as follows:

[0018] (1) Synthesis of Fe3O4 nanoparticles by solvothermal method;

[0019] (2) Fe3O4 nanoparticles were mixed with ethanol solution, the pH was adjusted to 9-10, tetraethyl orthosilicate (TEOS) was added to react, and then aminopropyltriethoxysilane (APTES) was added to modify it to obtain Fe3O4@SiO2-NH2;

[0020] (3) Mix Fe3O4@SiO2-NH2, water and emulsifier, add styrene, crosslinking agent and initiator, and react to obtain the product.

[0021] Preferably, step (1) specifically involves mixing FeCl3·6H2O, ethylene glycol, sodium acetate, and polyethylene glycol, and reacting them at 150-180℃ for 6-10 hours to obtain Fe3O4 nanoparticles.

[0022] Preferably, the molar ratio of FeCl3·6H2O, ethylene glycol, sodium acetate, and polyethylene glycol is 1:40-60:6-10:0.05-0.1;

[0023] More preferably, the molar ratio of FeCl3·6H2O, ethylene glycol, sodium acetate and polyethylene glycol is 1:50:8:0.08.

[0024] Preferably, in step (2), the mass ratio of the Fe3O4 nanoparticles, tetraethyl orthosilicate and aminopropyltriethoxysilane is 1:2-4:2-4.

[0025] More preferably, in step (2), the mass ratio of the Fe3O4 nanoparticles, tetraethyl orthosilicate and aminopropyltriethoxysilane is 1:3:2.

[0026] Preferably, in step (3), the emulsifier is sodium dodecyl sulfate;

[0027] Preferably, in step (3), the crosslinking agent is divinylbenzene;

[0028] Preferably, in step (3), the initiator is ammonium persulfate;

[0029] Preferably, in step (3), the emulsifier is 1-2 wt% of the mass of water;

[0030] Preferably, in step (3), the mass ratio of Fe3O4@SiO2-NH2, emulsifier, styrene, crosslinking agent and initiator is 1:2-8:20-50:0.5-3:0.5-1.

[0031] More preferably, in step (3), the mass ratio of Fe3O4@SiO2-NH2, emulsifier, styrene, crosslinking agent and initiator is 1:6:30:1:0.5.

[0032] Preferably, in step S2, the elution solution is 0.1M sodium citrate buffer, 3wt% acetonitrile and 1mM EDTA.

[0033] Preferably, in step S3, the anion exchange column uses Fractogel EMD TMAE as the medium.

[0034] Preferably, in step S3, the elution solution is ammonium butyrate buffer.

[0035] Preferably, in step S3, the precipitant used for precipitation is a mixed solution of ethanol and acetone; the volume ratio of ethanol to acetone is 8-10:1.

[0036] Preferably, in step S3, the pH of the precipitate is 5-5.5, the temperature is 1-5℃, and the time is 8-16h.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] This invention provides a method for the isolation and purification of urokinase. By optimizing the adsorption material and elution solution, the prepared urokinase has a high content of high molecular weight urokinase, a high recovery rate, and a high activity yield. Detailed Implementation

[0039] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0040] Example 1

[0041] A method for isolating and purifying urokinase, comprising the following steps:

[0042] S1: Male urine, adjusted to pH 8.5, allowed to stand at 10℃, and the supernatant was collected;

[0043] S2: The supernatant was mixed with the adsorbent material and eluted with an elution buffer consisting of 0.1 M sodium citrate buffer, 3 wt% acetonitrile and 1 mM EDTA to obtain a crude urokinase solution.

[0044] The adsorbent is a core-shell silica polymer with Fe3O4@SiO2-NH2 as the core and polystyrene as the shell; the preparation method is as follows:

[0045] (1) FeCl3·6H2O, ethylene glycol, sodium acetate and polyethylene glycol were mixed in a molar ratio of 1:50:8:0.08 and Fe3O4 nanoparticles were synthesized by solvothermal method;

[0046] (2) Fe3O4 nanoparticles were mixed with ethanol solution, the pH was adjusted to 9-10, tetraethyl orthosilicate (TEOS) was added to react, and then aminopropyltriethoxysilane (APTES) was added to modify the mixture to obtain Fe3O4@SiO2-NH2; wherein the mass ratio of Fe3O4 nanoparticles, tetraethyl orthosilicate and aminopropyltriethoxysilane was 1:3:2.

[0047] (3) Mix Fe3O4@SiO2-NH2, water and sodium dodecyl sulfate, the mass of sodium dodecyl sulfate being 1.5% of the mass of water, add styrene, divinylbenzene and ammonium persulfate, and react. The mass ratio of Fe3O4@SiO2-NH2, sodium dodecyl sulfate, styrene, divinylbenzene and ammonium persulfate is 1:6:30:1:0.5, and the mixture is obtained.

[0048] S3: The crude urokinase solution was chromatographically passed through an anion exchange column (using Fractogel EMD TMAE as the medium), eluted with ammonium butyrate buffer, and precipitated. The precipitant was a mixed solution of ethanol and acetone; the volume ratio of ethanol to acetone was 9:1, the pH was 5.5, the temperature was 5℃, and the time was 14h.

[0049] Example 2

[0050] A method for isolating and purifying urokinase, comprising the following steps:

[0051] S1: Male urine, adjusted to pH 8.5, allowed to stand at 5°C, and the supernatant was collected;

[0052] S2: The supernatant was mixed with the adsorbent material and eluted with an elution buffer consisting of 0.05 M sodium citrate buffer, 1 wt% acetonitrile and 0.5 mM EDTA to obtain a crude urokinase solution.

[0053] The adsorbent is a core-shell silica polymer with Fe3O4@SiO2-NH2 as the core and polystyrene as the shell; the preparation method is as follows:

[0054] (1) FeCl3·6H2O, ethylene glycol, sodium acetate and polyethylene glycol were mixed in a molar ratio of 1:40:6:0.05 and Fe3O4 nanoparticles were synthesized by solvothermal method.

[0055] (2) Fe3O4 nanoparticles were mixed with ethanol solution, the pH was adjusted to 9-10, tetraethyl orthosilicate (TEOS) was added to react, and then aminopropyltriethoxysilane (APTES) was added to modify the mixture to obtain Fe3O4@SiO2-NH2; wherein the mass ratio of Fe3O4 nanoparticles, tetraethyl orthosilicate and aminopropyltriethoxysilane was 1:2:4.

[0056] (3) Mix Fe3O4@SiO2-NH2, water and sodium dodecyl sulfate, the mass of sodium dodecyl sulfate being 1.0% of the mass of water, add styrene, divinylbenzene and ammonium persulfate, and react. The mass ratio of Fe3O4@SiO2-NH2, sodium dodecyl sulfate, styrene, divinylbenzene and ammonium persulfate is 1:8:50:3:1, and the mixture is obtained.

[0057] S3: The crude urokinase solution was chromatographically passed through an anion exchange column (using Fractogel EMD TMAE as the medium), eluted with ammonium butyrate buffer, and precipitated. The precipitant was a mixed solution of ethanol and acetone; the volume ratio of ethanol to acetone was 8:1, the pH was 5, the temperature was 1℃, and the time was 16h.

[0058] Example 3

[0059] A method for isolating and purifying urokinase, comprising the following steps:

[0060] S1: Male urine, adjusted to pH 8.5, allowed to stand at 15℃, and the supernatant was collected;

[0061] S2: The supernatant was mixed with the adsorbent material and eluted with an elution buffer consisting of 0.15 M sodium citrate buffer, 5 wt% acetonitrile and 1.5 mM EDTA to obtain a crude urokinase solution.

[0062] The adsorbent is a core-shell silica polymer with Fe3O4@SiO2-NH2 as the core and polystyrene as the shell; the preparation method is as follows:

[0063] (1) FeCl3·6H2O, ethylene glycol, sodium acetate and polyethylene glycol were mixed in a molar ratio of 1:60:10:0.1 and Fe3O4 nanoparticles were synthesized by solvothermal method;

[0064] (2) Fe3O4 nanoparticles were mixed with ethanol solution, the pH was adjusted to 9-10, tetraethyl orthosilicate (TEOS) was added to react, and then aminopropyltriethoxysilane (APTES) was added to modify the mixture to obtain Fe3O4@SiO2-NH2; wherein the mass ratio of Fe3O4 nanoparticles, tetraethyl orthosilicate and aminopropyltriethoxysilane was 1:4:2.

[0065] (3) Mix Fe3O4@SiO2-NH2, water and sodium dodecyl sulfate, the mass of sodium dodecyl sulfate being 2% of the mass of water, add styrene, divinylbenzene and ammonium persulfate, and react. The mass ratio of Fe3O4@SiO2-NH2, sodium dodecyl sulfate, styrene, divinylbenzene and ammonium persulfate is 1:2:20:0.5:0.8, and the mixture is obtained.

[0066] S3: The crude urokinase solution was chromatographically passed through an anion exchange column (using Fractogel EMD TMAE as the medium), eluted with ammonium butyrate buffer, and precipitated. The precipitant was a mixed solution of ethanol and acetone; the volume ratio of ethanol to acetone was 10:1, the pH was 5.5, the temperature was 2℃, and the time was 8h.

[0067] Comparative Example 1

[0068] A method for isolating and purifying urokinase, which differs from Example 1 in that the adsorbent material is replaced by nano-iron oxide and silica gel as described in Example 1 of Chinese Patent CN 115558656 A, with a mass ratio of nano-iron oxide to silica gel of 0.6:1.

[0069] Includes the following steps:

[0070] A method for isolating and purifying urokinase, comprising the following steps:

[0071] S1: Male urine, adjusted to pH 8.5, allowed to stand at 10℃, and the supernatant was collected;

[0072] S2: The supernatant was mixed with the adsorbent material and eluted with an elution buffer consisting of 0.1 M sodium citrate buffer, 3 wt% acetonitrile and 1 mM EDTA to obtain a crude urokinase solution.

[0073] The adsorbent materials are nano-ferric oxide and silica gel, with a mass ratio of nano-ferric oxide to silica gel of 0.6:1.

[0074] S3: The crude urokinase solution was chromatographically passed through an anion exchange column (using Fractogel EMD TMAE as the medium), eluted with ammonium butyrate buffer, and precipitated. The precipitant was a mixed solution of ethanol and acetone; the volume ratio of ethanol to acetone was 9:1, the pH was 5.5, the temperature was 5℃, and the time was 14h.

[0075] Comparative Example 2

[0076] A method for isolating and purifying urokinase, which differs from Example 1 in that the elution buffer in step S2 is replaced with a buffer solution containing 0.3 mol / L sodium chloride and 0.15 mol / L phosphate, while the rest is the same as in Example 1.

[0077] Comparative Example 3

[0078] A method for isolating and purifying urokinase, which differs from Example 1 in that the packing material of the anion exchange column in step S3 is replaced with Whatman DE-52, while the rest is the same as in Example 1.

[0079] Test Example 1

[0080] The relative content of high molecular weight urokinase in urokinase was calculated in Examples 1-3 and Comparative Examples 1-3, and the activity yield and specific activity of each urokinase were calculated according to the methods described in Part I of the main text of the 2020 edition of the Chinese Pharmacopoeia. The test results are shown in Table 1 below:

[0081] Table 1. Test Results

[0082]

[0083] As shown in Table 1, compared with the comparative example, the specific urokinase separation and purification method of the present invention, through optimization of adsorption materials, elution solutions, etc., produces urokinase with high content of high molecular weight urokinase, high recovery rate, and high activity yield.

[0084] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for isolating and purifying urokinase, characterized in that, Includes the following steps: S1: Male urine, adjust pH to 8.5, let stand, and collect the supernatant; S2: The supernatant is mixed with the adsorbent material and eluted with an eluent consisting of 0.05-0.15M sodium citrate buffer, 1wt%-5wt% acetonitrile, and 0.5-1.5mM EDTA to obtain a crude urokinase solution. The adsorbent material is a core-shell silica polymer with Fe3O4@SiO2-NH2 as the core and polystyrene as the shell; The preparation method of the core-shell silica polymer is as follows: (1) Solvothermal synthesis of Fe3O4 nanoparticles: FeCl3·6H2O, ethylene glycol, sodium acetate and polyethylene glycol are mixed and reacted at 150-180℃ for 6-10h to obtain Fe3O4 nanoparticles; the molar ratio of FeCl3·6H2O, ethylene glycol, sodium acetate and polyethylene glycol is 1:40-60:6-10:0.05-0.1; (2) Fe3O4 nanoparticles were mixed with ethanol solution, pH was adjusted to 9-10, tetraethyl orthosilicate was added to react, and then aminopropyltriethoxysilane was added to modify it to obtain Fe3O4@SiO2-NH2; the mass ratio of Fe3O4 nanoparticles, tetraethyl orthosilicate and aminopropyltriethoxysilane was 1:2-4:2-4. (3) Mix Fe3O4@SiO2-NH2, water and emulsifier, add styrene, crosslinking agent and initiator, react to obtain the product; the emulsifier is sodium dodecyl sulfate; the crosslinking agent is divinylbenzene; the initiator is ammonium persulfate; the emulsifier is 1-2 wt% of water; the mass ratio of Fe3O4@SiO2-NH2, emulsifier, styrene, crosslinking agent and initiator is 1:2-8:20-50:0.5-3:0.5-1; S3: The crude urokinase solution was subjected to anion exchange column chromatography using Fractogel EMDTMAE as the medium and eluted with ammonium butyrate buffer. The precipitant was a mixed solution of ethanol and acetone with a volume ratio of 8-10:

1. The pH of the precipitate was 5-5.5, the temperature was 1-5℃, and the precipitation time was 8-16h.

2. The separation and purification method according to claim 1, characterized in that, Step S1 is performed at a temperature of 5-15℃.

3. The separation and purification method according to claim 1, characterized in that, In step S2, the elution buffer is 0.1M sodium citrate buffer, 3wt% acetonitrile and 1mM EDTA.

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