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Capillary gel electrophoresis detection kit containing reducing agent

A detection kit and gel electrophoresis technology, applied in the field of capillary gel electrophoresis detection kits containing reducing agents, can solve problems such as high hazards, and achieve the effect of reducing pollution and safety problems

Pending Publication Date: 2021-07-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The actual technical problem to be solved in the present invention is to provide a capillary gel electrophoresis detection kit containing a reducing agent for the problem that the existing reduced CE-SDS detection method is more harmful. The sample buffer contains a reducing agent, and the kit can reduce the hazards of reduced CE-SDS detection to experimenters and the environment while ensuring the accuracy of the results of antibody molecular size isomers

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  • Capillary gel electrophoresis detection kit containing reducing agent
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  • Capillary gel electrophoresis detection kit containing reducing agent

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Example 1 Different concentrations of mercaptoethanol affect the purity of the reduced CE-SDS of recombinant anti-ricin humanized monoclonal antibody (mAb1) in a buffer solution of pH 9.0

[0060] Weigh 0.606g of Tris, 0.5g of SDS, dissolve with 42mL of deionized water until clear, add 2.5mL of mercaptoethanol stock solution (14M), then adjust the pH to 9.0 with 3M hydrochloric acid, then dilute to 50mL with deionized water to obtain 700 mM mercaptoethanol in sample buffer. Then use the sample buffer solution without reducing agent and the sample buffer solution containing 700mM mercaptoethanol to sequentially dilute to obtain the sample buffer solution containing 70mM, 7mM, 0.7mM and 0.07mM mercaptoethanol. The recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg / mL with the prepared sample buffer, then denatured in a water bath, and analyzed by capillary gel electrophoresis.

[0061] Subsequently, the effects of different concentrations of ...

Embodiment 2

[0064] Example 2 Effect of different concentrations of 11-mercapto-1-undecanol on the purity of the reduced CE-SDS of recombinant anti-ricin humanized monoclonal antibody (mAb1) in a buffer solution of pH 9.0

[0065] Weigh 0.606g of Tris, 0.5g of SDS, 0.7153g of 11-mercapto-1-undecyl alcohol (MW: 204.37), dissolve with 42mL of deionized water until clear, then adjust the pH to 9.0 with 3M hydrochloric acid, and then use deionized water to Dilute to 50 mL to obtain a sample buffer solution containing 70 mM 11-mercapto-1-undecanol. 700 mM buffer was not available due to solubility limitations. Then use the sample buffer without reducing agent and the sample buffer containing 70mM 11-mercapto-1-undecanol, and sequentially dilute to obtain the sample buffer containing 7mM, 0.7mM and 0.07mM 11-mercapto-1-undecanol liquid. The recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg / mL with the prepared sample buffer, then denatured in a water bath, and ana...

Embodiment 3

[0069] Example 3 Effects of Different Concentrations of Mercaptosuccinic Acid on the Reduced CE-SDS Purity of Recombinant Anti-ricin Humanized Monoclonal Antibody (mAb1) in the Buffer Solution of pH 9.0

[0070] Weigh 0.606g of Tris, 0.5g of SDS, 5.255g of mercaptosuccinic acid (MW: 150.15), dissolve with 42mL of deionized water until clear, then adjust the pH to 9.0 with 3M hydrochloric acid, then dilute to 50mL with deionized water, A sample buffer containing 700 mM mercaptosuccinic acid was obtained. Then use the sample buffer without reducing agent and the sample buffer containing 700mM mercaptosuccinic acid to sequentially dilute to obtain the sample buffer containing 70mM, 7mM, 0.7mM and 0.07mM mercaptosuccinic acid. The recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg / mL with the prepared sample buffer, then denatured in a water bath, and analyzed by capillary gel electrophoresis.

[0071] Subsequently, the effects of different concentrat...

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Abstract

The invention relates to a capillary gel electrophoresis detection kit containing a reducing agent. The reducing agent is selected from a monothiol compound and a dithiol compound, the monothiol compound is selected from 11-mercapto-1-undecanol, and the dithiol compound is selected from 2, 2'-(1, 2-ethanedioxy) bis-ethanethiol and ethylene glycol dithioglycolate. The kit disclosed by the invention can be used for reducing environmental pollution and harm to a human body in the process of analyzing biopharmacy such as monoclonal antibodies through reduced capillary gel electrophoresis, and ensuring accuracy of purity under a reduction condition at the same time; the biological drugs comprise a recombinant anti-ricin humanized monoclonal antibody, a recombinant anti-HER2 humanized monoclonal antibody, T-DM1 and a recombinant human type II tumor necrosis factor receptor-antibody fusion protein for injection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the analysis of protein by reduced capillary gel electrophoresis, in particular to a capillary gel electrophoresis detection kit containing a reducing agent that can accurately detect the purity of biopharmaceuticals such as antibodies. Background technique [0002] With the continuous development of the field of biopharmaceuticals, more and more techniques are applied to the heterogeneity characterization of therapeutic proteins. Among them, capillary gel electrophoresis (CGE), as a technology developed from traditional slab gel electrophoresis, has gradually become the standard for the purity of many biopharmaceuticals, especially antibodies, due to its advantages of automation, rapidity, high resolution and on-line quantification. One of the characterization tools. Taking monoclonal antibody as an example, in the reduction analysis, the protein molecule will be reduced by the reduc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/447G01N33/68
CPCG01N33/561G01N33/6803
Inventor 方伟杰沈斌彬张中伟
Owner ZHEJIANG UNIV
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