阅读说明：本技术 一种尿激酶的精制方法 (Method for refining urokinase ) 是由 顾京 李争 熊心磊 郭芬 张茜 于 2020-07-08 设计创作，主要内容包括：本发明公开了一种尿激酶的精制方法,属于尿激酶纯化技术领域。该方法包括以下步骤：取尿激酶中间体溶液,上凝胶色谱柱,用平衡缓冲液洗脱,收集中段洗脱液,得到精制的尿激酶溶液；其中,所述凝胶色谱的填料包括Superdex 75pg/GE、Superdex 200pg/GE、Sephacryl S-100HR/GE、Chromadex 75PG/BESTCHROM、Chromadex 200PG/BESTCHROM中的至少一种。该方法简便、安全、环保,纯度高、回收率好,适用范围广,通过优化各项工艺参数,仅需要通过一次凝胶色谱柱,满足量产经济效益要求。(The invention discloses a method for refining urokinase, belonging to the technical field of urokinase purification. The method comprises the following steps: taking the urokinase intermediate solution, loading the urokinase intermediate solution on a gel chromatographic column, eluting the urokinase intermediate solution by using an equilibrium buffer solution, and collecting middle-section eluent to obtain a refined urokinase solution; wherein the filler of the gel chromatography comprises at least one of Superdex 75PG/GE, Superdex 200PG/GE, Sephacryl S-100HR/GE, Chromadex 75PG/BESTCHROM and Chromadex200 PG/BESTCHROM. The method is simple, safe, environment-friendly, high in purity, good in recovery rate and wide in application range, and can meet the requirement of mass production economic benefit by optimizing various process parameters and only needing to pass through a gel chromatographic column once.)

1. A method for purifying urokinase, which is characterized by comprising the following steps:

taking the urokinase intermediate solution, loading the urokinase intermediate solution on a gel chromatographic column, eluting the urokinase intermediate solution by using an equilibrium buffer solution, and collecting middle-section eluent to obtain a refined urokinase solution;

wherein the filler of the gel chromatographic column comprises at least one of Superdex 75PG/GE, Superdex 200PG/GE, Sephacryl S-100HR/GE, Chromadex 75PG/BESTCHROM and Chromadex200 PG/BESTCHROM.

2. The method of claim 1, wherein the packing of the gel chromatography column is Superdex 75 pg/GE.

3. The method of claim 1, wherein the protein concentration of the upper gel chromatography column is 2-20 mg/mL.

4. The method of claim 1, wherein the protein concentration of the upper gel chromatography column is 10 mg/mL.

5. The method of claim 1, wherein the upper gel chromatography column has an upper column volume of 2-8% of the column volume.

6. The method of claim 1, wherein the equilibration buffer is at least one of an acetate buffer, a citrate buffer, a phosphate buffer, and a Tris-HCl buffer.

7. The method of claim 1, wherein the equilibration buffer is a citrate buffer.

8. The method of claim 1, wherein the equilibration buffer has a ph of 4.5-8.0.

9. The method according to claim 1, wherein the elution is carried out at a conductance value of 15 to 90 ms/cm.

10. The method of claim 1, wherein the mid-stream eluent is a liquid with a retention time of 40-70 min.

Technical Field

The invention belongs to the technical field of urokinase purification, and particularly relates to a urokinase refining method.

Background

Urokinase is a thrombolytic drug extracted from fresh human urine, is a serine protease produced by human renal tubular epithelial cells, is an alkaline protein, has an isoelectric point of about pH8.7, is white amorphous powder, and is easily soluble in water. The dilute solution is unstable in property, needs to be used fresh, and needs not to be diluted by an acid solution, and the freeze-dried state can be stable for years. Urokinase is a very specific proteolytic enzyme. The activity of the synthetic substrate is similar to that of trypsin and plasmin, and the synthetic substrate also has esterase activity, no antigenicity and no in vivo antibody production. The half-life period in vivo is (14 +/-6) min.

Urokinase activates plasminogen to active plasmin, which converts insoluble fibrin to soluble peptides, thereby dissolving the thrombus. Therefore, it is clinically used for treating thrombosis, thromboembolism and other diseases. When urokinase is combined with an anticancer agent, the urokinase can dissolve fibrin around cancer cells, so that the anticancer agent can penetrate into the cancer cells more effectively, thereby improving the capability of the anticancer agent in killing the cancer cells. Therefore, urokinase is also a good cancer adjuvant, and it has no problem of antigenicity and can be used for a long time.

D-160 adsorption resin is adopted in the past for refining urokinase, and the adsorption resin has the main problems of complicated treatment steps, large use of strong acid and strong base, high environmental protection pressure and great harm to working environment and people due to the use of large-volume strong acid.

Chinese patent application 200410018835.6 discloses a purification method of recombinant human prourokinase, comprising the following steps: A. recovering recombinant human prourokinase from the supernatant of the engineering cell culture by cation exchange Streamml ine-SP expanded bed chromatography; B. further purifying the crude urokinase product collected from A by Sephacryl S-200 gel chromatography; C. removing urokinase from the crude product by affinity chromatography using para aminobenzamidine-Sepharose Fast Flow; D. and (3) removing DNA of residual cells in the recombinant human prourokinase by using DEAE-Sepharose fast Flow anion exchange chromatography, and obtaining the recombinant human prourokinase meeting the SFDA requirement by adopting the method, wherein the total recovery rate is more than 70 percent, and the purity is more than 99 percent.

Chinese patent application 201711260186.4 discloses a four-step purification process of a new thrombolytic drug, recombinant human prourokinase, which comprises four steps of carboxymethyl radial ion exchange chromatography (CM-RIEC), microporous glass bead adsorption chromatography (MPG), dextran-polyacrylamide high performance gel chromatography (Sephacryl S-200HR) and formamidine affinity chromatography (PABZ), and is characterized in that the CM-radial ion exchange chromatography is matched with the combination of other three methods. After purification, the specific activity of prourokinase is increased by 280 times, the impurity protein can be removed by more than 98%, and the total recovery rate is up to more than 50%.

However, the methods all need the combination of gel chromatography and other chromatographic columns, and different chromatographic columns are used for multiple times, so that the complexity of the purification process is greatly improved.

Chinese patent application 201710311962.2 discloses a method for separating and purifying urokinase, which comprises the following steps: 1) preparing a crude urokinase product; 2) purifying urokinase; the step 2) comprises the following steps: purifying the clear liquid obtained by dissolving the crude urokinase prepared in the step 1) by a dextran microsphere column and/or an agarose microsphere column. The separation and purification method provided by the application can simultaneously improve the specific activity of the urokinase to more than 2 ten thousand IU/mg protein, the yield reaches 75-87%, and the proportion of the large and small molecular urokinase is more than 85%. The crude product prepared by the method is prepared by sequentially passing through a diatomite column and a CM-C column, the purity is improved to a certain extent, and the same effect cannot be obtained when the crude product is directly used for purifying a finished urokinase product in a purification step.

In view of the above, the invention provides a simple, safe and environment-friendly urokinase refining method, which has high purity, good recovery rate and wide application range, and meets the economic benefit requirement of mass production by optimizing various process parameters and only needing to pass through a gel chromatographic column once.

Disclosure of Invention

The invention aims to simplify the purification process of urokinase by adopting high-resolution gel chromatography and combining the optimization of other process conditions on the premise of ensuring the purity and recovery rate of urokinase, meet the requirement of mass production on economic benefit, simultaneously do not need strong acid, and are safe and environment-friendly.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for purifying urokinase, comprising the following steps:

and (3) taking the urokinase intermediate solution, loading the urokinase intermediate solution on a gel chromatographic column, eluting the urokinase intermediate solution by using an equilibrium buffer solution, and collecting middle-section eluent to obtain a refined urokinase solution.

Wherein, the filler of the gel chromatographic column comprises at least one of Superdex 75PG/GE, Superdex 200PG/GE, Sephacryl S-100HR/GE, Chromadex 75PG/BESTCHROM and Chromadex200PG/BESTCHROM, preferably Superdex 75 PG.

Preferably, the urokinase intermediate solution is a human urokinase intermediate solution, and the purity of the urokinase intermediate solution is preferably 65-85%, and more preferably 70%.

Preferably, the protein concentration of the upper gel chromatographic column is 2-20mg/mL, and more preferably 10 mg/mL.

Preferably, the upper column volume of the upper gel chromatography column is 2 to 8%, more preferably 5% of the column volume.

Preferably, the equilibrium buffer is at least one of an acetate buffer, a citrate buffer, a phosphate buffer and a Tris-HCl buffer, and preferably is a citrate buffer; the buffer solution contains sodium chloride, and the content of the sodium chloride in the buffer solution is 150-500 mM.

Preferably, the pH of the equilibration buffer is 4.5-8.0, more preferably 6.

Preferably, the elution is carried out at a conductivity value of 15 to 90ms/cm, more preferably 50 ms/cm.

Preferably, the mid-stream eluent is a liquid with a retention time of 40-70min, and more preferably 55 min.

Preferably, after obtaining the refined urokinase solution, the method further comprises a step of washing; the cleaning step is preferably: washing the column with a washing solution, and then washing with injection water to neutrality; further preferably, the cleaning solution is 0.1-2M NaOH solution, and the cleaning time is 0.5-2 hours.

Compared with the prior art, the invention has the beneficial effects that:

under the condition of ensuring good activity yield, the gel chromatography process is used, so that the method is simple and convenient to operate, high in purity, good in recovery rate and wide in application range; strong acid is not used, the environmental protection pressure is low, the working environment and the personnel safety are guaranteed, and the economic benefit meets the requirement of the mass production of enterprises.

Detailed Description

The present invention will be further explained with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easy to understand, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The urokinase intermediate solutions used in the following examples were made by the company under lots 20190426, 20190518.