阅读说明：本技术 一种人尿激酶原料精制过程中去除内毒素的方法 (Method for removing endotoxin in refining process of human urokinase raw material ) 是由 顾京 李争 倪萌 蔡宏兵 唐维 于 2020-07-10 设计创作，主要内容包括：本发明提供了一种人尿激酶原料精制过程中去除内毒素的方法,涉及生物工程及化学工程领域,该方法具体包括以下步骤：(1)样品预处理；(2)层析柱制备与装柱；(3)消毒以及上样处理。该方法通过采用一种新的聚合物阴离子层析介质并优化各项工艺条件,处理过程简单,可以有效去除尿激酶中间产品中的内毒素,同时保证尿激酶活性收率达到75％以上。在该处理过程中,节约了工时与材料,降低了生产成本。(The invention provides a method for removing endotoxin in the refining process of human urokinase raw material, which relates to the field of biological engineering and chemical engineering, and specifically comprises the following steps: (1) sample pretreatment; (2) preparing a chromatographic column and filling the chromatographic column; (3) and (4) sterilizing and loading. The method adopts a new polymer anion chromatography medium and optimizes various process conditions, has simple treatment process, can effectively remove endotoxin in the intermediate product of urokinase, and simultaneously ensures that the activity yield of urokinase reaches more than 75 percent. In the treatment process, the working hours and materials are saved, and the production cost is reduced.)

1. A method for removing endotoxin in the refining process of human urokinase raw material is characterized in that: the method comprises the following steps:

(1) sample pretreatment: adjusting the pH and the conductance of the intermediate solution of the human urokinase raw material;

(2) preparing and packing a chromatographic column: adding water into the polymer anion exchange chromatographic packing to obtain a suspension, and filling the suspension into a column to obtain a chromatographic column;

(3) and (3) disinfection: sterilizing the chromatographic column and the chromatographic system, detecting endotoxin, and obtaining a negative result;

(4) loading treatment: and (2) balancing the chromatographic column by using a balance buffer solution, loading the sample after the ultraviolet, the electric conductivity and the pH value are stabilized, wherein the loaded sample is the intermediate solution of the human urokinase raw material in the step (1), collecting the flow through liquid when the chromatographic system detects that the ultraviolet absorption of the flow through peak is greater than 50mAU, continuously washing the chromatographic column by using the balance buffer solution after the loading is finished until the ultraviolet absorption of the system is less than 50mAU, and stopping collecting, wherein the collection liquid is the urokinase raw material solution for removing the endotoxin.

2. The method of claim 1, wherein: the polymer anion exchange chromatography filler in the step (2) is Chromalite MQ/F weak base anion resin of British Brand resin Co., Ltd, TOYOPEARL ion exchange resin super Q-650M of Tokyo Japan, or SOURCE 30Q high-speed low-back pressure ion exchange medium of American general electric company.

3. The method of claim 2, wherein: the polymer anion exchange chromatography filler in the step (2) is Chromalite MQ/F weak base anion resin of British Brand resin Co.

4. The method of claim 1, wherein: the height of the packed column in the step (2) is 10-30cm, and the diameter of the chromatographic column is 1.6-30 cm.

5. The method of claim 1, wherein: in the step (1), the pH is 6.0-9.0, and the conductance is 1-10 ms/cm.

6. The method of claim 1, wherein: the concrete steps of column packing in the step (2) are as follows: replacing the preserving fluid in the chromatographic packing with water for injection for three times; starting the chromatographic system, draining air in the chromatographic system and the chromatographic column head screen by using water for injection, uniformly mixing chromatographic filler and water, and adding the mixture into the chromatographic column at a uniform speed at one time; installing a chromatographic column head, discharging bubbles in the pipeline, opening an outlet valve of the chromatographic column, and starting to install the column; and (3) as the time is prolonged, the chromatographic packing is settled in the chromatographic column to form a stable column bed, the pump is stopped, the outlet valve is closed, the column head valve at the inlet of the chromatographic column is switched to the exhaust port, the water below the column head is discharged by rotating the column head adjusting device of the chromatographic column, and the column head screen is ensured to be tightly contacted with the chromatographic packing, so that the column packing is completed.

7. The method of claim 1, wherein: the chromatography in the step (2) comprises the following specific steps: continuously washing 3-5 column volumes with NaOH solution; using water for injection, 5-10 column volumes were washed successively.

8. The method of claim 1, wherein: the equilibration buffer in the step (4) comprises phosphate buffer and/or Tris-HCl buffer.

9. The method of claim 1, wherein: the balancing conditions in the step (4) are that the upper and lower deviation of an ultraviolet horizontal line is less than 0.1mAU, the pH value is 6.0-9.0, and the electric conductance is 1-10 ms/cm.

Technical Field

The invention relates to the field of biological engineering and chemical engineering, in particular to a method for removing endotoxin in the refining process of a human urokinase raw material.

Background

Urokinase is a thrombolytic drug extracted from fresh human urine, which can activate plasminogen to convert into active plasmin, which can convert insoluble fibrin into soluble peptides, thereby dissolving thrombus. Therefore, it is clinically used for treating thrombosis, thromboembolism and other diseases. When the urokinase is combined with the 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. Urokinase is used as an injection medicine, the finished product has strict requirements on endotoxin, and the content of the endotoxin in the finished urokinase product in pharmacopoeia has specific requirements (<1EU/1 ten thousand units), so the endotoxin must be effectively removed in the production process of a urokinase raw material. However, most of the techniques have been to separate endotoxin removal and urokinase purification processes, thereby increasing production costs.

Chinese patent CN104031901B discloses a method for purifying urokinase, which comprises the steps of separately processing proteins in 2 main molecular weight regions in a urokinase crude product, further removing impurities, heat sources and cell endotoxins in the crude product by means of pH value adjustment and the like, and obtaining a urokinase refined product. However, the method of the invention is relatively complex, and meanwhile, ultrafiltration is mainly carried out by using an ultrafiltration membrane, and most of the ultrafiltration membranes cannot be subjected to chemical treatment, so that the service life is short, and the cost is increased in the replacement aspect.

Aiming at the problems of complexity, high cost, low urokinase activity yield and the like of a urokinase purification method, a method for removing endotoxin by urokinase with high activity yield and excellent removal effect is urgently needed to be found, so that the endotoxin in a urokinase intermediate product is effectively removed, and the production cost is reduced.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for removing endotoxin in the process of refining human urokinase raw material, which adopts a new polymer anion chromatography medium and optimizes various process conditions, has simple treatment process, can effectively remove the endotoxin in the intermediate product of urokinase, has simple and convenient operation, saves working hours and materials and reduces the production cost.

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

the invention provides a method for removing endotoxin in the refining process of human urokinase raw material, which comprises the following steps:

(1) sample pretreatment: adjusting the pH and the conductance of the intermediate solution of the human urokinase raw material;

(2) preparing and packing a chromatographic column: adding water into the polymer anion exchange chromatographic packing to obtain a suspension, and filling the suspension into a column to obtain a chromatographic column;

(3) and (3) disinfection: sterilizing the chromatographic column and the chromatographic system, detecting endotoxin, and obtaining a negative result;

(4) loading treatment: and (2) balancing the chromatographic column by using a balance buffer solution, loading the sample after the ultraviolet, the electric conductivity and the pH value are stabilized, wherein the loaded sample is the intermediate solution of the human urokinase raw material in the step (1), collecting the flow through liquid when the chromatographic system detects that the ultraviolet absorption of the flow through peak is greater than 50mAU, continuously washing the chromatographic column by using the balance buffer solution after the loading is finished until the ultraviolet absorption of the system is less than 50mAU, and stopping collecting, wherein the collection liquid is the urokinase raw material solution for removing the endotoxin.

Further, in the step (1), the pH is 6.0-9.0, and the conductance is 1-10 ms/cm.

Further, the intermediate solution of human urokinase raw material in the step (1) is an eluent purified by an affinity chromatography step.

Further, the polymeric anion exchange chromatography packing material in the step (2) is a Chromalite MQ/F weak base anion resin of British Brand resin Co., Ltd, a TOYOPEARL ion exchange resin superQ-650M of Tokyo Japan, or a SOURCE 30Q high-speed low-back pressure ion exchange medium of the United states general electric company.

Preferably, the polymeric anion exchange chromatography packing in step (2) is a chromolite MQ/F weak base anion resin from British Brand resins, Inc.

Further, the height of the column in the step (2) is 10-30cm, and the diameter of the chromatographic column is 1.6-30 cm.

Further, the step (2) of column packing comprises the following specific steps: replacing the preserving fluid in the chromatographic packing with water for injection for three times; starting the chromatographic system, draining air in the chromatographic system and the chromatographic column head screen by using water for injection, uniformly mixing chromatographic filler and water, and adding the mixture into the chromatographic column at a uniform speed at one time; installing a chromatographic column head, discharging bubbles in the pipeline, opening an outlet valve of the chromatographic column, and starting to install the column; and (3) as the time is prolonged, the chromatographic packing is settled in the chromatographic column to form a stable column bed, the pump is stopped, the outlet valve is closed, the column head valve at the inlet of the chromatographic column is switched to the exhaust port, the water below the column head is discharged by rotating the column head adjusting device of the chromatographic column, and the column head screen is ensured to be tightly contacted with the chromatographic packing, so that the column packing is completed.

Further, the step (2) of column packing comprises the following specific steps: replacing the preserving fluid in the chromatographic packing with water for injection for three times, and adjusting the ratio of the chromatographic packing to the water to be 1:0.4-1 after the last replacement; starting the chromatographic system, exhausting air of the chromatographic system and the chromatographic column head screen by using water for injection, fully and uniformly mixing chromatographic filler and water, and adding the mixture into the chromatographic column at a uniform speed at one time; installing a chromatographic column head, discharging bubbles in the pipeline, adjusting the flow rate to be 30-60cm/h, opening an outlet valve of the chromatographic column, and starting to install the column; and (3) the chromatographic packing settles into a stable column bed in the chromatographic column along with the prolonging of the time, the pump is stopped, the outlet valve is closed, the column head valve at the inlet of the chromatographic column is switched to the exhaust port, the water below the column head is discharged by rotating the column head adjusting device of the chromatographic column, and the column head screen is ensured to be tightly contacted with the chromatographic packing, so that the column packing is completed.

Further, the chromatography in the step (2) comprises the following specific steps: continuously washing 3-5 column volumes with NaOH solution; using water for injection, 5-10 column volumes were washed successively.

Further, the chromatography in the step (2) comprises the following specific steps: using 0.1-2M NaOH solution, setting the flow rate of a chromatography system to be 30-60cm/h, and continuously washing 3-5 column volumes; the flow rate of the chromatography system is set to be 60-90cm/h by using water for injection, and 5-10 column volumes are continuously washed.

Further, the equilibration buffer in the step (4) comprises phosphate buffer and/or Tris-HCl buffer. Preferably a phosphate buffer.

Further, the balancing conditions in step (4) are that the upper and lower deviation of ultraviolet horizontal line is less than 0.1mAU, pH is 6.0-9.0, and conductance is 1-10 ms/cm.

And further, washing the chromatographic column with a cleaning solution to remove impurities (mainly endotoxin) adsorbed on the chromatographic packing, and washing the chromatographic column with injection water until the pH is neutral and the endotoxin is detected to be negative when the ultraviolet absorption detected by the chromatographic system is zero and stable. Wherein the cleaning solution is 2-6M guanidine hydrochloride, 0.1-2M NaOH solution or 20-50% ethanol, and the cleaning time is 0.5-2 hours.

The technical effects obtained by the invention are as follows:

1. the method of the invention adopts a new polymer anion chromatography medium and optimizes various process conditions, has simple treatment process, can effectively remove endotoxin in the intermediate product of urokinase, has simple and convenient operation, saves working hours and materials and reduces the production cost.

2. The polymer anion chromatography medium used in the invention comprises Chromalite MQ/F weak base anion resin of British Delite resin Co., Ltd, TOYOPEARL ion exchange resin super Q-650M of Tokyo Japan and SOURCE 30Q high-speed low-back pressure ion exchange medium of American general electric company, compared with other media, the three materials have remarkable advantages in the aspect of endotoxin removal, and by combining with the process optimization in the invention, the endotoxin can be effectively removed, and the activity yield of urokinase can be ensured to be more than 75%.

Detailed Description

It should be noted that the polymeric anion exchange chromatography packing used in the present invention is shown in table 1, and the other raw materials are general commercial products, and thus the source thereof is not particularly limited.

TABLE 1 chromatography packing

Chromatography packing	Company/manufacturer
		Chromalite MQ/F	British bleached resin Co., Ltd (Purolite)
TOYOPEARL superQ-650M	Nippon east Cao Co Ltd (TOSOH)
		SOURCE 30Q	General electric company of America (GE)