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Method for preparing urokinase and freeze-dried powder of urokinase

A technology of urokinase and freeze-dried powder, which is applied in the preparation method of urokinase and the field of freeze-dried powder, can solve the problems of high production cost, inability to ensure high purity and high activity yield of large molecular weight urokinase, and reduce production cost, reduce side effects, and ensure curative effect

Active Publication Date: 2020-03-20
JIANGSU YOULIKA BIOTECHNOLOYG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is that in the production and purification method of urokinase in the prior art, the production cost is high, and it cannot ensure that the obtained large molecular weight urokinase has technical problems such as high purity and high activity yield at the same time

Method used

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  • Method for preparing urokinase and freeze-dried powder of urokinase
  • Method for preparing urokinase and freeze-dried powder of urokinase

Examples

Experimental program
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Effect test

Embodiment 1

[0035] (1) Preparation of crude urokinase: add fresh male urine to silica gel, stir and absorb, pack the silica gel into a column, rinse the silica gel column with tap water, elute with 3% ammonia water, and collect the eluate. Add solid ammonium sulfate to the eluent and precipitate at 4-8°C. Use food-grade diatomaceous earth as the bottom of the filter layer, and the filter screen is 150 mesh nylon filter cloth. Filter and collect the brown filter residue, which is the crude urokinase.

[0036](2) Preparation of urokinase high-quality goods: take the above-mentioned urokinase crude product, dissolve in water to make a solution with a concentration of 3000iu / ml-5000iu / ml concentration, use food-grade diatomaceous earth as the bottom of the filter layer, and the filter screen is a 150-mesh nylon filter Cloth was filtered to obtain the refined urokinase.

[0037] (3) Purification:

[0038] (a) Take the refined urokinase, dissolve it in water, and obtain the aqueous solution of...

Embodiment 2

[0045] (1) Preparation of crude urokinase: add fresh male urine to silica gel, stir and absorb, pack the silica gel into a column, rinse the silica gel column with tap water, elute with 3% ammonia water, and collect the eluate. Add solid ammonium sulfate to the eluent and precipitate at 4-8°C. Use food-grade diatomaceous earth as the bottom of the filter layer, and the filter screen is 150 mesh nylon filter cloth. Filter and collect the brown filter residue, which is the crude urokinase.

[0046] (2) Preparation of urokinase high-quality goods: take the above-mentioned urokinase crude product, dissolve in water to make a solution with a concentration of 3000iu / ml-5000iu / ml concentration, use food-grade diatomaceous earth as the bottom of the filter layer, and the filter screen is a 150-mesh nylon filter Cloth was filtered to obtain the refined urokinase.

[0047] (3) Purification:

[0048] (a) Take the refined urokinase, dissolve it in water, and obtain the aqueous solution o...

Embodiment 3

[0055] The urokinase preparation process is similar to Example 1, and the difference is only in step (3) purification:

[0056] (a) Take the refined urokinase, dissolve it in water, and obtain the aqueous solution of the refined urokinase, adjust the pH to 7.5 with phosphoric acid solution or sodium hydroxide solution, adjust the conductance to 18-25ms / cm with an equilibrium buffer, and add high-resolution cation exchange chromatography Column (Capto SimpAct, available from GE Company of the U.S.), wash the column with equilibration buffer (acetate buffer containing 150-500mM sodium chloride), until UV280nm<0.1, then wash the column with elution buffer (containing 500-1000mM sodium chloride acetate buffer) to elute the column, and collect 2 liters of the eluate.

[0057] (b) Take the above eluate, concentrate it with an ultrafiltration membrane with a molecular weight cut-off of 10KD, wash and filter to replace the freeze-drying buffer, and collect 500ml of the concentrated so...

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Abstract

The invention relates to a method for preparing high-purity macromolecular urokinase and freeze-dried powder of the urokinase and belongs to the technical field of biochemical engineering. The methodspecifically comprises the steps: (1) preparing a urokinase crude product; (2) preparing a urokinase fine product; and (3) carrying out purifying, wherein in the step (3), the urokinase fine product obtained in the step (2) is dissolved, and then, the urokinase fine product solution is added into a cation exchange chromatography column for purification; and the purified urokinase is subjected to freeze-drying, thereby obtaining the freeze-dried powder of the urokinase. Fillers filled in the cation exchange chromatography column comprise one of Capto S impAct, SP sepharose HP and source 30S. According to the method for preparing the urokinase and the freeze-dried powder of the urokinase, high-resolution ion-exchange chromatography is selected, so that on the premise of guaranteeing purity,activity and yield, the production cost is reduced greatly, and large-scale production of high-purity high-molecular-weight urokinase becomes possible.

Description

technical field [0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a preparation method of urokinase and freeze-dried powder thereof. Background technique [0002] Urokinase is a thrombolytic drug extracted from fresh human urine. It can activate plasminogen and convert it into active plasmin. dissolve. Therefore, it is mostly used clinically to treat thrombosis, thromboembolism and other diseases. When urokinase is used in combination with anticancer agents, because it can dissolve fibrin around cancer cells, anticancer agents can penetrate into cancer cells more effectively, thereby improving the ability of anticancer agents to kill cancer cells. So urokinase is also a good adjuvant therapy for cancer, and it has no antigenicity problem and can be used for a long time. [0003] Urokinase is a serine protease produced by human renal tubular epithelial cells. It is a basic protein with an isoelectric point of aroun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/72
CPCC12N9/6462C12Y304/21073
Inventor 李尔华顾京赵江峰魏文培
Owner JIANGSU YOULIKA BIOTECHNOLOYG CO LTD
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