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A method for determining the electrophoretic purity of recombinant human prourokinase for injection

A technique for prourokinase and injection, which is applied in the field of medicine and can solve the problems of low accuracy of the assay method, unsatisfactory separation of test samples, inaccurate test results and the like

Active Publication Date: 2020-06-02
TASLY BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results of the current detection method are inaccurate, and the separation of the test product is not ideal. The sample of pro-urokinase is usually treated at a high temperature of 100°C, which causes protein damage, so that the accuracy of the detection method is not high, and it is not suitable for industrial production.

Method used

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  • A method for determining the electrophoretic purity of recombinant human prourokinase for injection
  • A method for determining the electrophoretic purity of recombinant human prourokinase for injection
  • A method for determining the electrophoretic purity of recombinant human prourokinase for injection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Testing of real samples:

[0084] 1. Operating procedures

[0085] 1.1 Reagents and solutions required for inspection

[0086] 1.1.1 30% gel stock solution: pure for electrophoresis, store in the dark at 2-8°C.

[0087] 1.1.2 1.5mol / L Tris-HCl buffer solution (pH8.8): Weigh 18.2g of trishydroxyaminomethane (Tris, pure by electrophoresis), add an appropriate amount of MilliQ water for injection to dissolve, adjust the pH to 8.8 with dilute hydrochloric acid, and constant volume To 100ml, store at 2-8°C.

[0088] 1.1.3 1.0mol / L Tris-HCl buffer (pH6.8): Weigh 12.1g of trishydroxyaminomethane (Tris, pure by electrophoresis), add an appropriate amount of MilliQ water for injection to dissolve, adjust the pH to 6.8 with dilute hydrochloric acid, and constant volume To 100ml, store at 2-8°C.

[0089] 1.1.4 10% Sodium Dodecyl Sulfate (SDS): Weigh 10 g of Sodium Dodecyl Sulfate (SDS, pure by electrophoresis), add 100 ml of MilliQ water for injection to dissolve, and store at...

Embodiment 2

[0170] The non-reducing sample buffer, electrophoresis buffer, 15% separating gel solution 5, and % stacking gel solution described in Example 1 are replaced as follows:

[0171] Non-reducing sample buffer refers to weighing 0.2g of trishydroxyaminomethane, 0.5g of sodium lauryl sulfate, and 0.001g of bromophenol blue, measuring 2ml of glycerol, and 0.05ml of hydrochloric acid, adding MilliQ water for injection to dissolve and constant volume. Can.

[0172] Electrophoresis buffer: Weigh 10g of trishydroxyaminomethane, 65g of glycine, 4g of sodium lauryl sulfate, add MilliQ water for injection to dissolve, adjust the pH to 7.5-9.0 with hydrochloric acid, and continue to dilute with milliQ water to obtain the product.

[0173]15% separating gel solution includes MilliQ water for injection 1ml, 30% gel stock solution 1.5ml, Tris-HCl buffer (PH8.8, 1.5M) 1ml, 10% SDS 0.02ml, 10% AP 0.01ml, TEMED 0.001ml ;

[0174] The 5% concentrated gel solution includes MilliQ water for inject...

Embodiment 3

[0176] The non-reducing sample buffer, electrophoresis buffer, 15% separating gel solution 5, and % stacking gel solution described in Example 1 are replaced as follows:

[0177] Non-reducing sample buffer refers to weighing 0.5g of trishydroxyaminomethane, 1g of sodium lauryl sulfate, and 0.003g of bromophenol blue, measuring 6ml of glycerol, and 1ml of hydrochloric acid, adding MilliQ water for injection to dissolve and constant volume.

[0178] Electrophoresis buffer: Weigh 20g of trishydroxyaminomethane, 85g of glycine, 8g of sodium lauryl sulfate, add MilliQ water for injection to dissolve, adjust the pH to 7.5-9.0 with hydrochloric acid, and continue to dilute with milliQ water to obtain the product.

[0179] Described 15% separation gel solution comprises MilliQ water for injection 2.5ml, 30% gel stock solution 3.5ml, Tris-HCl buffer (PH8.8, 1.5M) 2.5ml, 10%SDS0.02-0.1 part, 10 %AP 0.01-0.05 part, TEMED0.005ml; The 5% concentrated gel solution includes MilliQ water for ...

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Abstract

The invention relates to a method for electrophoresis determination of purity of recombinant human prourokinase for injection, wherein the method includes the following steps: (1) preparation of a test sample solution: taking the recombinant human prourokinase for injection on ice, thawing, diluting with MilliQ water, adding a non reductive sample buffer solution, placing at the temperature of 55-65 DEG C and heating for 1-5 minutes, immediately placing in an ice bath to obtain the non reductive test sample solution; (2) preparation of a Marker solution: taking a Marker solution, thawing, placing in an 80-100 DEG C water bath for 1-5 minutes, and immediately placing in an ice bath for standby application; and (3) determination method: adopting a vertical plate gel electrophoresis system, and detecting the test sample solution and the Marker solution, wherein the sample loading amount of the two solutions is 10-20 [mu]L, and the instrument initial voltage is 50-60 V; and adjusting the voltage to 100-110 V when entering a separation gel, when a bromophenol blue band runs to the lower edge of the gel, finishing the electrophoresis, after electrophoresis, dyeing and decoloring to obtain an atlas, and calculating the purity of the test sample solution according to the atlas.

Description

technical field [0001] The invention belongs to the field of medicine, in particular to a method for determining the electrophoretic purity of recombinant human urokinase for injection. Background technique [0002] Prourokinase is the precursor of urokinase. It is a glycoprotein consisting of 411 amino acids. It is inactive by itself. It is activated by fibrinolytic enzyme and kininase to open the peptide chain between Lys158-Ile159 The formation of urokinase can exert its thrombolytic effect. It is a specific thrombolytic drug. [0003] Recombinant human prourokinase produced by Shanghai Tasly is obtained through the expression of Chinese hamster ovary cells (CHO cells) constructed by genetic engineering methods. It is mainly used for thrombolytic therapy of acute ST-segment elevation myocardial infarction. A class of new drugs was successfully launched. [0004] Polyacrylamide gel vertical plate electrophoresis technology is a kind of zone electrophoresis technology ba...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
Inventor 陶铜静陈维维袁婀娜韩进
Owner TASLY BIOPHARMACEUTICALS CO LTD
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