Capillary electrophoresis detection method for analyzing impurities of recombinant human prourokinase for injection and application of capillary electrophoresis detection method

A technology of capillary electrophoresis and detection method, applied in the field of medicine, can solve the problems of affecting migration, broadening the band, reducing efficiency, etc., and achieves the effects of high repeatability, simple method, and clear and easy-to-distinguish map.

Pending Publication Date: 2022-07-01
TASLY BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the current passes through the conductor, Joule heat will be generated. Joule heat can cause inhomogeneity of temperature, viscosity and separation speed inside the sieving medium, affect migration, reduce efficiency, and widen the zone
The biggest limitation of traditional slab gel electrophoresis is that it cannot overcome the negative effects of Joule heat brought about by the high voltage at both ends
Since this negative effect is proportional to the electric field strength, it greatly limits the introduction of high voltage and it is difficult to increase the electrophoresis speed.

Method used

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  • Capillary electrophoresis detection method for analyzing impurities of recombinant human prourokinase for injection and application of capillary electrophoresis detection method
  • Capillary electrophoresis detection method for analyzing impurities of recombinant human prourokinase for injection and application of capillary electrophoresis detection method
  • Capillary electrophoresis detection method for analyzing impurities of recombinant human prourokinase for injection and application of capillary electrophoresis detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] A kind of detection of real sample (reduced CE-SDS) is provided in the present embodiment, and the concrete steps are as follows:

[0122] 1. Preparation of reduced sample: Dilute the sample to 1 mg / mL with SDS sample buffer (pH 9.0). Take 95 μL of 1 mg / mL sample and add 5 μL of β-mercaptoethanol, seal well and mix well.

[0123] Heat in a 70°C water bath for 5 minutes.

[0124] After cooling to room temperature, centrifuge at 6000 r / min for 1 min, transfer 75 μL to the sample tube, and close the lid.

[0125] 2. For other experimental operations, refer to the experimental operation steps in the content of the invention.

[0126] The test results are: the purity of reduced CE-SDS is 92.1%;

[0127] figure 1 It is a capillary electrophoresis pattern; the main peak appears at about 20 minutes, and the main peak has multiple shoulder peaks, which is presumed to be caused by various glycosylation modifications. Glycosylation is a very important post-translational modif...

Embodiment 2

[0129] A kind of detection of real sample (non-reduced CE-SDS) is provided in the present embodiment, and the concrete steps are as follows:

[0130] 1. Preparation of non-reducing samples: Dilute the sample to 1 mg / mL with SDS sample buffer (pH 9.0). Take 95 μL of 1 mg / mL sample, add 5 μL of 800 mM iodoacetamide, and mix thoroughly after sealing.

[0131] Heat in a 70°C water bath for 5 minutes.

[0132] After cooling to room temperature, centrifuge at 6000 rpm for 1 min, transfer 75 μL to the sample tube, and close the lid.

[0133] 2. For other experimental operations, refer to the experimental operation steps in the content of the invention.

[0134] The test results are: the purity of non-reduced CE-SDS is 99.7%; figure 2 It is a capillary electrophoresis pattern, and the main peak appears at about 20.5 min. The main peak has multiple shoulders, presumably caused by multiple glycosylation modifications. Glycosylation is a very important post-translational modificati...

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Abstract

The invention discloses a capillary electrophoresis detection method for analyzing impurities of recombinant human pro-urokinase for injection and application of the capillary electrophoresis detection method. The capillary electrophoresis detection method comprises the following steps: (1) diluting a sample which comprises a non-reducing sample or a reducing sample by using a sample buffer solution, then adding a reducing agent into the reducing sample or adding an alkylating reagent into the non-reducing sample, and mixing; (2) heating a sample obtained by mixing in the step (1), cooling, centrifuging, and transferring into a loading tube; and (3) placing the sample loading tube on a capillary electrophoresis apparatus, operating the capillary electrophoresis apparatus, starting to inject the sample, separating the sample to obtain a capillary electrophoresis detection map, and analyzing the map to obtain a detection result. According to the method, impurities in the recombinant human pro-urokinase can be effectively separated, the reproducibility of multiple batches of samples is good, and the accuracy is high.

Description

technical field [0001] The invention belongs to the field of medicine, in particular to a capillary electrophoresis detection method for analyzing the impurities of recombinant human urokinase for injection. Background technique [0002] Prourokinase is the precursor of urokinase, a glycoprotein composed of 411 amino acids, which is inactive by itself, and is activated by fibrinolytic enzyme and kininase to open the peptide chain between Lys158-Ile159 Only after the formation of urokinase can it exert its thrombolytic function, it is a specific thrombolytic drug. At present, recombinant human prourokinase is mainly expressed in Chinese hamster ovary cells (CHO cells) constructed by genetic engineering, and is mainly used for thrombolytic therapy of acute ST-segment elevation myocardial infarction. [0003] Capillary electrophoresis is electrophoresis performed by moving the gel on the plate to a capillary tube as a support. The gel is porous and acts like a molecular sieve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447
CPCG01N27/447
Inventor 韩君李凤智黄瑞晶王莎莎贾国勇李静吕明启李剑
Owner TASLY BIOPHARMACEUTICALS CO LTD
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