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Affinity chromatography filler for purifying urokinase and preparation method and application thereof

A technology of urokinase and affinity layer, which is applied in the field of affinity chromatography filler and preparation for purifying urokinase, can solve the problems that the separation and purification methods are not reported in the literature, and the purity and activity cannot meet the demand, and achieve low production cost and separation The effect of short time and high yield

Active Publication Date: 2020-11-20
武汉人福药业有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this application is mainly used to purify polymer urokinase (HUK), and its purity and activity still cannot meet the demand
[0006] There are also many literatures about urokinase affinity chromatography at present, but there are almost no reports on the separation and purification method literature for separating high-molecular-weight urokinase (HUK) and low-molecular-weight urokinase (LUK) from human urine, so it is necessary to develop a method that can separate Separation and purification method for separation of high molecular weight urokinase (HUK) and low molecular weight urokinase (LUK)

Method used

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  • Affinity chromatography filler for purifying urokinase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Basic Example 1 Preparation method of a novel affinity filler

[0056] Specifically include the following steps:

[0057] (1) Preparation of aminomethylbenzoic acid-Sepharose CL-6B

[0058] a. Wash 40mL of Sepharose CL-6B sequentially with 400mL of water, 400mL of 20% dioxane aqueous solution, 400mL of 60% dioxane aqueous solution and 400mL of 100% dioxane to obtain washed Sepharose CL-6B;

[0059] b. Put the washed 40mL Sepharose CL-6B in 30mL100% dioxane to obtain Sepharose CL-6B solution, and add 2.0g N,N'-carbonyldiimidazole (CDI) to the Sepharose CL-6B solution to obtain a suspension ;

[0060] c. Stir the suspension at room temperature for 30 minutes, then wash with 400mL of 100% dioxane, 400mL of 60% dioxane aqueous solution, 400mL of 20% dioxane aqueous solution and 400mL of water to obtain activated Sepharose CL-6B;

[0061] d. At room temperature, react 40mL of activated Sepharose CL-6B with 20g of aminomethylbenzoic acid in water, then wash with 400mL of w...

Embodiment 3

[0073] Basic Example 3 A method for preparing a novel affinity filler, specifically comprising the following steps:

[0074] (1) Preparation of aminomethylbenzoic acid-Sepharose CL-6B

[0075] a. Wash 40mL of Sepharose CL-6B sequentially with 500mL of water, 500mL of 20% dioxane aqueous solution, 500mL of 60% dioxane aqueous solution and 500mL of 100% dioxane to obtain washed Sepharose CL-6B;

[0076]b. Put the washed 40mL Sepharose CL-6B in 40mL100% dioxane to obtain a Sepharose CL-6B solution, and add 2.4g N,N'-carbonyldiimidazole (CDI) to the Sepharose CL-6B solution to obtain a suspension ;

[0077] c. Stir the suspension at room temperature for 30 minutes, then wash with 500 mL of 100% dioxane, 500 mL of 60% dioxane aqueous solution, 500 mL of 20% dioxane aqueous solution and 500 mL of water to obtain activated Sepharose CL-6B;

[0078] d. At room temperature, react activated 40mL Sepharose CL-6B with 21.6g aminotoluic acid in water, then wash with 500mL water, 500mL 0....

Embodiment 2

[0087] Example 2 The method of using Sepharose CL-6B-aminomethylbenzoic acid-p-AB filler to separate and purify urokinase

[0088] Include the following steps:

[0089] A. Weigh 200mg (61182IU / mg) of urokinase raw material, use 0.1M phosphate buffer containing 0.1M NaCl, dissolve at pH=7.0, obtain sample solution, and set aside;

[0090] B. Take the 20mL Sepharose CL-6B-aminotoluic acid-p-AB filler prepared in Basic Example 2, put it into the column, and then use 0.1M phosphate buffer containing 0.1M NaCl to balance at pH=7.0;

[0091] C. Inject the sample solution obtained in step A into the well-balanced column in step B, respectively use 0.1M phosphate buffer containing 0.1M NaCl, pH=7.0 and 0.1M phosphate buffer containing 1M NaCl, Wash the column at pH=7.0 until the absorbance of the permeate at 280nm is less than 5mAU, stop washing;

[0092] D. Use 0.1M phosphate buffer containing 1M NaCl to elute at pH=4.0. Stop collecting when the pH drops to 4.0 and the absorbance a...

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Abstract

The invention provides an affinity chromatography filler for purifying urokinase and a preparation method and application of the affinity chromatography filler. The invention belongs to the field of biochemical purification. In the implementation process of the preparation method, aminomethylbenzoic acid and 4-aminobenzamidine dihydrochloride are used for modifying Sepharose CL-6B, the Sepharose CL-6B-aminomethylbenzoic acid-p-AB filler can better purify and separate macromolecular urokinase and low molecular urokinase, the activity recovery rate is high and is 95% or above, and the recovery effect is good.

Description

technical field [0001] The invention belongs to the field of biochemical purification, and in particular relates to an affinity chromatography filler for purifying urokinase, a preparation method and application thereof. Background technique [0002] Tissue plasminogen activator is the most representative thrombolytic agent in the treatment of acute myocardial infarction. However, the side effects of tissue plasminogen activator are significant, such as bleeding and reocclusion. Studies have shown that combined therapy of tissue plasminogen activator and urokinase can reduce these side effects, therefore, urokinase is also widely used as an effective thrombolytic agent for the treatment of acute myocardial infarction. [0003] Human urinary urokinase mainly includes natural high molecular weight urokinase (HUK, 54000Dal) and low molecular weight urokinase (LUK, 33000Dal). Urokinase (LUK) is thought to be derived from macromolecular urokinase (HUK) by proteolytic degradatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D15/38C12N9/72
CPCB01D15/3804C12N9/6462C12Y304/21073
Inventor 田瀚付贞唐维熊心磊郭芬
Owner 武汉人福药业有限责任公司
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