Specific purified high-molecular urokinase chromatography media as well as preparation method and application thereof

A chromatographic medium, urokinase technology, applied in biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of loss, limited actual effect, lack of resolution, etc., and achieve the effect of mild reaction conditions and simple preparation process.

Active Publication Date: 2010-04-14
江西浩然生物制药有限公司
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  • Summary
  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

The above-mentioned improvement and exploration of urokinase purification medium and process only focused on the final recovery rate and purification multiple of urokinase biological activity, but it was difficult to effectively separate high-molecular-weight urokinase and low-molecular-weight urokinase, so the actual effect was limited
The main reason is that traditional affinity chromatography is based on the "pocket" advanced structure composed of the active site (His204, Asp255 and Ser356) of the β-chain C-terminal serine protease catalytic active domain of the urokinase molecule, while the polymer urokinase an

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0014] (1) Synthesis of 1L polymer urokinase-specific chromatography medium:

[0015] Take the purchased Separose-CL4B wet glue (bare glue) 1L to remove the protective agent, wash it once with water, then drain it, add water to 2000mL, keep warm in an ice-water bath (0°C) in advance, and resuspend with magnetic stirring. Activate with 100mL of 0.5g / mL cyanogen bromide solution, and adjust the pH value to 10.0-11.0 with NaOH solution while adding cyanogen bromide solution dropwise. After adding the cyanogen bromide solution, use NaOH solution to maintain the pH from 11.0 to about 15 minutes. After the reaction is completed, suction filter to dryness with a pre-cooled sand filter funnel, and place excessive solid ferrous sulfate powder in the suction filter bottle to remove unreacted cyanogen bromide. A volume of 10 L was washed with pre-cooled dilute HCl solution (3 mmol / L HCl). Add about 1000ml of drained activated agarose gel Separose-CL4B into 2000ml of 1mol / L 5-aminovaler...

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Abstract

A specific purified high-molecular urokinase chromatography media is characterized by having a structure as follows: HOOC-(CH2)4(NH)C-O-R-O-C(NH)NH(CH2)4-CO-X,X=NHC6H5C(NH)NH2, wherein R represents cross-linked agarose substrate. The specific purified high-molecular urokinase chromatography media has the advantages of being simple in preparation process and mild for reaction conditions, being applicable to high-efficiency separation of high-molecular urokinase media and low-molecular urokinase media, and being capable of implementing industrial production of high-purification and high-molecular urokinase.

Description

technical field [0001] The invention relates to a high molecular urokinase chromatographic medium and a preparation method, in particular to a specific purification high molecular urokinase chromatographic medium, a preparation method and an application. Background technique [0002] According to the forecast of Datamonitor, a market research organization, by 2015, the total market value created by global novel antithrombotic drugs will reach about 10 billion US dollars. Among them, the thrombolytic agent plasminogen activators (PAs) class of drugs reached 1.5 billion US dollars. The main plasminogen activators include streptokinase (Streptokinase), urokinase (Urokinase), tissue plasminogen activator (tPA) and so on. With the aging of the world's population, the demand for such drugs will inevitably increase further. As one of the thrombolytic drugs, urokinase can directly promote the inactive plasminogen into active plasmin, and hydrolyze the fibrin that makes up the thro...

Claims

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Application Information

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IPC IPC(8): C08B37/12C12N9/72
Inventor 杨华英王启要万偲刘志勇胡小光
Owner 江西浩然生物制药有限公司
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