阅读说明：本技术 一种尿激酶中间体的纯化方法 (Method for purifying urokinase intermediate ) 是由 顾京 唐维 倪萌 韩庆坤 黄俊杰 于 2020-07-08 设计创作，主要内容包括：本发明提供了一种尿激酶中间体的纯化方法,涉及生物工程、化学工程领域。具体包括以下步骤：(1)取尿液、吸附,洗脱,收集洗脱液；(2)洗脱液进行沉淀,过滤,收集沉淀物,即得人尿激酶粗品；(3)取人尿激酶粗品,溶解,过滤后经葡聚糖凝胶层析,收集洗脱液,得到尿激酶中间体粗品；(4)取尿激酶中间体粗品,溶解后调节pH和电导,然后经过亲和琼脂糖凝胶层析柱,洗脱,收集洗脱液,即得高纯度尿激酶中间体。实施过程中采用亲和琼脂糖凝胶层析,结合其他工艺条件的优化,如平衡缓冲液配方、洗脱缓冲液配方、洗脱方法、pH、电导等,使最终得到的尿激酶的活性收率提高到90％以上,纯度提高到85％以上。(The invention provides a method for purifying a urokinase intermediate, and relates to the fields of biological engineering and chemical engineering. The method specifically comprises the following steps: (1) taking urine, adsorbing, eluting and collecting eluent; (2) precipitating the eluent, filtering, and collecting precipitate to obtain human urokinase crude product; (3) dissolving a human urokinase crude product, filtering, performing sephadex chromatography, and collecting eluent to obtain a urokinase intermediate crude product; (4) and (3) dissolving the crude urokinase intermediate product, adjusting the pH and the conductance, then passing through an affinity agarose gel chromatographic column, eluting, and collecting the eluent to obtain the high-purity urokinase intermediate. In the implementation process, affinity agarose gel chromatography is adopted, and optimization of other process conditions, such as a balance buffer solution formula, an elution method, pH, conductivity and the like, is combined, so that the activity yield of the finally obtained urokinase is improved to more than 90%, and the purity is improved to more than 85%.)

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

(1) adding silica gel into fresh male urine for adsorption, filtering, washing the silica gel with water, then adding ammonia water for elution, and collecting the eluate;

(2) adding ammonium sulfate into the eluent obtained in the step (1) for precipitation, filtering, and collecting precipitates to obtain a human urokinase crude product;

(3) dissolving the human urokinase crude product obtained in the step (2), filtering, performing sephadex chromatography, and collecting eluent to obtain a urokinase intermediate crude product;

(4) and (4) dissolving the crude urokinase intermediate obtained in the step (3), adjusting the pH and the conductance, then passing through an affinity agarose gel chromatography column, eluting, and collecting the eluent to obtain the high-purity urokinase intermediate.

2. The purification process according to claim 1, characterized in that: the content ratio of the silica gel to the urine in the step (1) is 1-5:100 Kg/L; the concentration of the ammonia water is 0.01-0.05%.

3. The purification process according to claim 1, characterized in that: the content ratio of the ammonium sulfate to the eluent in the step (2) is 350-430: 1 g/L; the pH value is 6.0-8.0.

4. The purification process according to claim 1, characterized in that: the step (3) comprises the following steps:

(31) dissolving the crude human urokinase with phosphate buffer solution with pH of 5.0-8.0, and filtering to obtain filtrate;

(32) using buffer solution to balance the sephadex chromatographic column, wherein the pH value of the buffer solution is 6.0-8.0 and the electric conductance is 1-30ms/cm in the balancing process;

(33) and (3) enabling the filtrate obtained in the step (31) to pass through a sephadex chromatographic column at the speed of 0.5-5mL/min, then adding a buffer solution for elution, wherein the pH value of the buffer solution in the elution process is 6.0-8.0, the electric conductivity is 30-90ms/cm, and collecting the eluate to obtain a crude urokinase intermediate product.

5. The purification method according to claim 4, characterized in that: the equilibrium buffer solution in the step (32) is one of acetate, phosphate and Tris-HCl buffer solution;

the equilibrium buffer solution also contains one of 150-1000mM sodium chloride, sodium sulfate, ammonium sulfate or magnesium chloride.

6. The purification method according to claim 4, characterized in that: the elution buffer solution in the step (33) is one of acetate, phosphate and Tris-HCl buffer solution;

the elution buffer also contains 150-1000mM sodium chloride, sodium sulfate, ammonium sulfate or magnesium chloride.

7. The purification process according to claim 1, characterized in that: the step (4) comprises the following steps:

(41) dissolving the crude urokinase intermediate product in a buffer solution with the pH value of 5.0-8.0, and filtering to obtain a filtrate;

(42) balancing affinity agarose gel chromatographic column with buffer solution of pH 6.0-8.0 and conductance 20-60 ms/cm; preferably, the pH value is 7.0-8.0, and the conductance is 30-50 ms/cm; more preferably, the pH is 7.0 and the conductance is 50 ms/cm;

(43) and (4) passing the filtrate obtained in the step (41) through an affinity agarose gel chromatography column at the speed of 0.5-5mL/min, eluting, wherein the pH value of a buffer solution in the elution process is 2.0-4.0, the electric conductivity is 30-90ms/cm, and collecting the eluate to obtain the high-purity urokinase intermediate.

8. The purification process according to claim 7, characterized in that: the equilibrium buffer solution in the step (42) is one of acetate buffer solution, citrate buffer solution, phosphate buffer solution or Tris-HCl buffer solution;

the equilibrium buffer solution also contains one of 150-1000mM sodium chloride, sodium sulfate, ammonium sulfate or magnesium chloride.

9. The purification process according to claim 1, characterized in that: the elution buffer solution in the step (43) is one of Tris-HCl buffer solution, acetic acid buffer solution, glycine buffer solution or hydrochloric acid buffer solution;

the elution buffer also contains 150-1000mM sodium chloride, sodium sulfate, ammonium sulfate or magnesium chloride.

10. The purification process according to claim 1, characterized in that: the filler of the affinity Sepharose chromatography column in the step (4) is one of para aminobenzamidine Sepharose, Benzamidine Sepharose 4FFHS/GE and Benzamidine Sepharose 6B/GE.

Technical Field

The invention relates to the fields of biological engineering and chemical engineering, in particular to a method for purifying a urokinase intermediate.

Background

Urokinase is a thrombolytic drug extracted from fresh human urine. It can activate plasminogen to convert to active plasmin, which can convert insoluble fibrin to soluble peptides, thereby dissolving 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.

Urokinase is a serine protease produced by human renal tubular epithelial cells, is a basic protein, has an isoelectric point of about pH8.7, is white amorphous powder, and is easily soluble in water. The dilute solution has unstable properties, must be used fresh, and must not be diluted with acidic solution. The lyophilized state is 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 activity of esterase is also high. Has no antigenicity, does not generate antibody in vivo, and has an in vivo half-life of (14 +/-6) min.

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

For example, chinese patent application 200910186618.0 discloses a method for preparing urokinase, which is characterized in that D-160 cation resin exchange method, affinity membrane chromatography and affinity chromatography are combined to prepare urokinase with crude urokinase as raw material. The quality of the obtained urokinase is superior to the existing domestic level, the specific activity of the urokinase can reach more than 150,000IU/mg & pr, the relative content of the high molecular urokinase can reach more than 96 percent, and the recovery rate of the biological activity of the urokinase can reach 65 percent. The method adopts D-160 adsorption resin, and the main problems of the adsorption resin are that the treatment steps are complicated, a large amount of strong acid and strong base are used, the environmental protection pressure is high, the use of large-volume strong acid has great harm to the working environment and people, and the content and the purity of the obtained urokinase can not meet the requirements.

For another example, chinese patent application 201611197428.5 discloses a method for purifying urokinase by affinity chromatography. Specifically, fresh urine of healthy adult males is used as a raw material, and high-purity urokinase is prepared by modern protein high-end biochemical separation technologies such as chitin adsorption, adsorption column chromatography, affinity chromatography and the like, so that the total yield can be improved to more than 80%, and the total titer is not lower than 40000 iu/mg.

Therefore, there is an urgent need for a urokinase purification method which can achieve high purity and high activity yield of urokinase intermediates by optimizing various process parameters such as equilibrium buffer formula, elution method, pH, conductivity and the like on the premise of ensuring the activity recovery rate in the urokinase intermediate purification process, and meet the requirements of mass production economic benefits.

Disclosure of Invention

Based on the defects and shortcomings in the prior art, the invention aims to provide a urokinase intermediate purification method, and aims to adopt affinity agarose gel chromatography and combine with the optimization of other process conditions, such as the optimization of conditions of a balance buffer solution formula, an elution method, pH, conductivity and the like, on the premise of ensuring the activity recovery rate in the urokinase intermediate purification process, so that the purity and the activity yield of the urokinase intermediate can be obviously improved, and the requirement of mass production economic benefit is met.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention relates to a method for purifying a urokinase intermediate, which adopts affinity agarose gel to purify the urokinase intermediate, and can improve the purity of the urokinase intermediate to more than 85 percent and the activity yield to more than 90 percent.

A method for purifying a urokinase intermediate specifically comprises the following steps:

(1) adding silica gel into fresh male urine for adsorption, filtering, washing the silica gel with water, then adding ammonia water for elution, and collecting the eluate;

(2) adding ammonium sulfate into the eluent obtained in the step (1) for precipitation, filtering, and collecting precipitates to obtain a human urokinase crude product;

(3) dissolving the human urokinase crude product obtained in the step (2), filtering, performing sephadex chromatography, and collecting eluent to obtain a urokinase intermediate crude product;

(4) and (4) dissolving the crude urokinase intermediate obtained in the step (3), adjusting the pH and the conductance, then passing through an affinity agarose gel chromatography column, eluting, and collecting the eluent to obtain the high-purity urokinase intermediate.

The content ratio of the silica gel to the urine in the step (1) is 1-5:100 (Kg/L); the concentration of the ammonia water is 0.01-0.05%.

The content ratio of the ammonium sulfate to the eluent in the step (2) is 350-430: 1 (g/L); the pH value is 6.0-8.0.

Preferably, the step (3) includes the steps of:

(31) dissolving the crude human urokinase with phosphate buffer solution with pH of 5.0-8.0, and filtering to obtain filtrate;

(32) using buffer solution to balance the sephadex chromatographic column, wherein the pH value of the buffer solution is 6.0-8.0 and the electric conductance is 1-30ms/cm in the balancing process; preferably, the pH value is 7.0-8.0, and the electric conductivity is 10-20 ms/cm; more preferably, the pH is 7.0 and the conductance is 15 ms/cm;

(33) passing the filtrate obtained in the step (31) through a sephadex chromatographic column at the speed of 0.5-5mL/min, then adding a buffer solution for elution, wherein the pH value of the buffer solution is 6.0-8.0 in the elution process, the electric conductivity is 30-90ms/cm, and collecting the eluate to obtain a crude urokinase intermediate product;

preferably, the pH value of the buffer solution in the elution process is 6.5-7.5, and the electric conductance is 50-90 ms/cm; preferably, the pH value of the buffer solution in the elution process is 7.0, and the conductance is 60-80 ms/cm; further preferably, the pH value of the buffer during the elution process is 7.0, and the conductance is 80 ms/cm.

The pH of the filtrate described in step (33) above is the same as that of the conductivity and equilibration buffers.

The equilibrium buffer solution in the step (32) is one of acetate, phosphate and Tris-HCl buffer solution; preferably a phosphate buffer;

in some preferred embodiments, the equilibration buffer further comprises one of 150-1000mM sodium chloride, sodium sulfate, ammonium sulfate, or magnesium chloride, preferably sodium chloride.

The elution buffer solution in the step (33) is one of acetate, phosphate and Tris-HCl buffer solution; preferably a phosphate buffer;

in some preferred embodiments, the elution buffer further comprises 150-; sodium chloride is preferred.

Preferably, the step (4) includes the steps of:

(41) dissolving the crude urokinase intermediate product in a buffer solution with the pH value of 5.0-8.0, and filtering to obtain a filtrate;

(42) balancing affinity agarose gel chromatographic column with buffer solution of pH 6.0-8.0 and conductance 20-60 ms/cm; preferably, the pH value is 7.0-8.0, and the conductance is 30-50 ms/cm; more preferably, the pH is 7.0 and the conductance is 50 ms/cm;

(43) passing the filtrate obtained in step (41) through an affinity agarose gel chromatography column at a speed of 0.5-5mL/min, eluting, wherein the pH value of a buffer solution in the elution process is 2.0-4.0, the electric conductivity is 30-90ms/cm, and collecting the eluate to obtain a high-purity urokinase intermediate;

preferably, the pH value of the buffer solution in the elution process is 3.0-4.0, and the electric conductance is 50-90 ms/cm; preferably, the pH value of the buffer solution in the elution process is 3.0, and the conductance is 60-80 ms/cm; (ii) a Further preferably, the pH value of the buffer solution in the elution process is 3.0, and the electric conductance is 80 ms/cm.

The equilibrium buffer solution in the step (42) is one of an acetate buffer solution, a citrate buffer solution, a phosphate buffer solution or a Tris-HCl buffer solution; preferably Tris-HCl buffer;

in some preferred embodiments, the equilibration buffer further comprises one of 150-1000mM sodium chloride, sodium sulfate, ammonium sulfate, or magnesium chloride, preferably sodium chloride.

The elution buffer solution in the step (43) is one of a Tris-HCl buffer solution, an acetate buffer solution, a glycine buffer solution or a hydrochloric acid buffer solution, and preferably is a Tris-HCl buffer solution and an acetate buffer solution;

in some preferred embodiments, the elution buffer further comprises 150-; sodium chloride is preferred.

The filler of the affinity Sepharose chromatography column in the step (4) is one of p-aminobenzamidine Sepharose, Benzamidine Sepharose 4FF (HS)/GE and Benzamidine Sepharose 6B/GE, and is preferably self-produced p-aminobenzamidine Sepharose.

The elution in the step (4) adopts a gradient elution mode, and the specific operation steps are as follows:

stationary phase: p-aminobenzamidine Sepharose, Benzamidine Sepharose 4FF (HS)/GE or Benzamidine Sepharose 6B/GE;

mobile phase: mobile phase A: Tris-HCl buffer, mobile phase B: acetic acid buffer solution;

gradient elution:

time of day	Mobile phase A	Mobile phase B
			0-15min	100% Tris-HCl buffer	-
15-50min	100% → 0% Tris-HCl buffer	0% → 100% acetic acid buffer

The urokinase intermediate in the invention refers to: the urine is adsorbed, eluted and precipitated by ammonium sulfate, and the solid obtained after diatomite filtration is subjected to first-step chromatography to obtain a target product, wherein the concentration and the purity of the target product are improved to a certain extent, and the target product still needs to be subsequently purified.

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

(1) in the implementation process, the urokinase is purified by using an agarose gel process, so that the activity yield and purity of the urokinase are obviously improved, the method is simple and convenient to operate, the environmental protection pressure is low, strong acid is not used, and the working environment and the safety of personnel are guaranteed;

(2) in the implementation process of the invention, affinity agarose gel chromatography is adopted, and optimization of other process conditions, such as optimization of conditions of a balance buffer solution formula, an elution method (step gradient optimization), pH, conductivity and the like, is combined, so that the purity and the activity yield of the urokinase intermediate can be obviously improved, and the requirement of mass production economic benefit is met;

(3) the purification method provided by the invention can improve the activity yield of the finally obtained urokinase to more than 90% and the purity to more than 85%.

Detailed Description

Basic example 1 preparation of crude urokinase intermediate

(1) Taking 500L of fresh male urine, adding 5g of silica gel for adsorption, filtering, washing the silica gel with water, then adding 0.01% ammonia water for elution, and collecting eluent;

(2) adding 35g of ammonium sulfate into 0.1L of the eluent with the pH value of 6.0 obtained in the step (1) for precipitation, filtering, and collecting precipitates to obtain a crude product of human urokinase;

(3) dissolving the human urokinase crude product obtained in the step (2), filtering, performing sephadex chromatography, and collecting eluent to obtain a urokinase intermediate crude product;

(31) dissolving the crude human urokinase by using a phosphate buffer solution with the pH value of 6.0, and filtering to obtain a filtrate;

(32) equilibrating the sephadex column using a phosphate buffer containing 150mM sodium chloride, the pH of the buffer being 6.0 and the conductance being 5ms/cm during equilibration;

(33) and (3) enabling the filtrate obtained in the step (31) to pass through a sephadex chromatographic column at the speed of 0.5mL/min, then adding a phosphate buffer solution containing 150mM sodium chloride for elution, wherein the pH value of the buffer solution in the elution process is 6.0, the electric conductivity is 30ms/cm, and collecting the eluent to obtain a crude urokinase intermediate product.

The crude product yield is 85.6 percent, the purity is 28.2 percent, and the specific activity is 18 ten thousand IU/mg.

Basic example 2 preparation of crude urokinase intermediate

(1) Taking 500L of fresh male urine, adding 20g of silica gel for adsorption, filtering, washing the silica gel with water, then adding 0.03% ammonia water for elution, and collecting eluent;

(2) adding 40g of ammonium sulfate into 0.1L of the eluent with the pH value of 7.0 obtained in the step (1) for precipitation, filtering, and collecting precipitates to obtain a crude product of human urokinase;

(3) dissolving the human urokinase crude product obtained in the step (2), filtering, performing sephadex chromatography, and collecting eluent to obtain a urokinase intermediate crude product;

(31) dissolving the crude human urokinase by using a phosphate buffer solution with the pH value of 7.0, and filtering to obtain a filtrate;

(32) equilibrating the sephadex column using a phosphate buffer containing 500mM sodium chloride, the pH of the buffer being 7.0 and the conductance being 15ms/cm during equilibration;

(33) and (3) enabling the filtrate obtained in the step (31) to pass through a sephadex chromatographic column at the speed of 3.5mL/min, then adding a phosphate buffer solution containing 500mM sodium chloride for elution, wherein the pH value of the buffer solution in the elution process is 7.0, the electric conductivity is 80ms/cm, and collecting the eluent to obtain a crude urokinase intermediate product.

The crude product yield is 90.5%, the purity is 39.6%, and the specific activity is 20 ten thousand IU/mg.

Basic example 3 preparation of crude urokinase intermediate

(1) Taking 500L of fresh male urine, adding 25g of silica gel for adsorption, filtering, washing the silica gel with water, then adding 0.05% ammonia water for elution, and collecting eluent;

(2) adding 43g of ammonium sulfate into 0.1L of the eluent with the pH value of 7.0 obtained in the step (1) for precipitation, filtering, and collecting precipitates to obtain a crude product of human urokinase;

(3) dissolving the human urokinase crude product obtained in the step (2), filtering, performing sephadex chromatography, and collecting eluent to obtain a urokinase intermediate crude product;

(31) dissolving the human urokinase crude product by using a Tris-HCl buffer solution with the pH value of 8.0, and filtering to obtain a filtrate;

(32) equilibrating the Sephadex column with Tris-HCl buffer containing 1000mM magnesium chloride, pH of the buffer being 8.0 and conductance being 30ms/cm during equilibration;

(33) and (3) enabling the filtrate obtained in the step (31) to pass through a sephadex chromatographic column at the speed of 5mL/min, then adding a Tris-HCl buffer solution containing 1000mM magnesium chloride for elution, wherein the pH value of the buffer solution in the elution process is 8.0, the electric conductivity is 90ms/cm, and collecting the eluent to obtain a crude urokinase intermediate product.

The crude product yield is 82.4%, the purity is 25.8%, and the specific activity is 16 ten thousand IU/mg.

Basic example 4 preparation of crude urokinase intermediate

(1) Taking 500L of fresh male urine, adding 22g of silica gel for adsorption, filtering, washing the silica gel with water, then adding 0.05% ammonia water for elution, and collecting eluent;

(2) adding 38g of ammonium sulfate into 0.1L of the eluent with the pH value of 7.0 obtained in the step (1) for precipitation, filtering, and collecting precipitates to obtain a crude product of human urokinase;

(3) dissolving the human urokinase crude product obtained in the step (2), filtering, performing sephadex chromatography, and collecting eluent to obtain a urokinase intermediate crude product;

(31) dissolving the crude human urokinase with acetate buffer solution with the pH value of 8.0, and filtering to obtain filtrate;

(32) equilibrating the sephadex column using an acetate buffer containing 800mM sodium sulfate, the pH of the buffer being 8.0 and the conductance being 25ms/cm during equilibration;

(33) and (3) enabling the filtrate obtained in the step (31) to pass through a sephadex chromatographic column at the speed of 4mL/min, then adding an acetate buffer solution containing 800mM sodium sulfate for elution, wherein the pH value of the buffer solution in the elution process is 6.5, the electric conductivity is 50ms/cm, and collecting the eluate to obtain a crude urokinase intermediate product.

The crude product yield was 84.9%, the purity was 23.8%, and the specific activity was 17 ten thousand IU/mg.