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Protective-agent-containing capillary gel electrophoresis detection kit

A detection kit and gel electrophoresis technology, applied in measurement devices, material analysis by electromagnetic means, instruments, etc., can solve the problems of increased purity and high fragment content, and achieve the effect of improving accuracy and reducing detection deviation.

Inactive Publication Date: 2016-06-08
HISUN BIORAY PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using iodoacetamide recommended by the manufacturer as an inhibitor can significantly reduce the fragment content, but the fragment content is still significantly high (such as containing 12.5mM iodoacetamide as a protective agent can increase the antibody mAbl purity from 81% to 89.5%, but continue to increase Iodoacetamide concentration to 37.5mM can only increase the purity to 90.6%)

Method used

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  • Protective-agent-containing capillary gel electrophoresis detection kit
  • Protective-agent-containing capillary gel electrophoresis detection kit
  • Protective-agent-containing capillary gel electrophoresis detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of a typical kit of embodiment 1

[0030] Weigh 6.06g of Tris, 5.0g of SDS, and 75g of sucrose, dissolve them in 420mL of deionized water until clear, adjust the pH to 9.0 with 3M hydrochloric acid, and then dilute to 500mL with deionized water to obtain a sample buffer.

[0031] Dissolve 46 mg of iodoacetamide in 1 mL of deionized water to prepare a 250 mM iodoacetamide solution.

[0032]The recombinant anti-HER2 humanized monoclonal antibody (mAb1) was diluted to 1 mg / mL with the prepared sample buffer, and then mixed with the prepared iodoacetamide solution at a ratio of 95 μL: 5 μL.

Embodiment 2

[0033] Example 2 Effect of different types of protective agents on the purity of non-reduced CE-SDS of recombinant anti-HER2 humanized monoclonal antibody (mAb1)

[0034] The effects of different types of protective agents on the purity of non-reduced CE-SDS of recombinant anti-HER2 humanized monoclonal antibody (mAb1) were compared. The results are shown in Table 1 and image 3 . Among them, sucrose and trehalose had the best effect, and the CE-SDS purity increased from 89.5% to 92.1%; the effect of mannitol was next, with a purity of 91.6%; and the effect of glycine was the worst, only 89.7%.

[0035] Table 1. Kit components containing different protective agents and their effects on the CE-SDS main peak content of recombinant anti-HER2 humanized monoclonal antibody (mAb1)

[0036]

[0037]

[0038] Note: #1 is a kit from Beckman, and the rest are kits with various protective agents added on this basis. The concentration of each protective agent is 500mM.

Embodiment 3

[0039] Example 3 Effect of different types of protective agents on the purity of non-reduced CE-SDS of recombinant anti-tumor necrosis factor-alpha human-mouse chimeric monoclonal antibody (mAb2)

[0040] The effects of different types of protective agents on the non-reduced CE-SDS purity of recombinant anti-tumor necrosis factor-α human-mouse chimeric monoclonal antibody (mAb2) were compared. The results are shown in Table 2 and Figure 4 . Among them, glucose, trehalose and sucrose had the best effects, and the CE-SDS purity increased from 88.9% to 91.6%, 91.3% and 91.0% respectively; % and 90.0%; and glycerol had the worst effect, only 89.1%.

[0041] Table 2. Kit components containing different protective agents and their effects on the CE-SDS main peak content of recombinant anti-tumor necrosis factor-α human-mouse chimeric monoclonal antibody (mAb2)

[0042]

[0043]

[0044] Note: #6 is a kit from Beckman, and the rest are kits with various protective agents ad...

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Abstract

The present invention relates to a protective-agent-containing capillary gel electrophoresis detection kit, a protective agent is selected from white sugar, a polyol or an amino acid. The kit can improve the accuracy of capillary electrophoresis detection of the purity of biopharmaceuticals such as antibodies, reduces the detection deviation generated in an actual experimental operation process and can better reflect actual contents of various isomers, especially debris, of antibody molecules contained in a sample itself, and antibodies include recombinant anti-HER2 humanized monoclonal antibodies, recombinant human-mouse chimeric anti-tumor-necrosis-factor-alpha monoclonal antibodies, recombinant anti-interleukin-6 receptor humanized monoclonal antibodies, T-DM1, recombinant anti-tumor-necrosis-factor-alpha fully-humanized monoclonal antibody injections and recombinant anti-CD52 humanized monoclonal antibodies.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a capillary gel electrophoresis detection kit capable of accurately detecting the purity of biopharmaceuticals such as antibodies. Background technique [0002] Capillary electrophoresis (capillary electrophoresis, CE) technology is developed from traditional gel electrophoresis (gel electrophoresis), which is driven by a strong electric field generated by high voltage electricity, and uses a micro-diameter quartz capillary as a separation channel. Compared with traditional gel electrophoresis, CE has the advantages of automation, rapidity, accurate quantification, and high resolution. Many biomolecules, such as proteins, polysaccharides, and nucleic acids, can be analyzed using capillary electrophoresis. Sodium dodecylsulfonate capillary electrophoresis (CE-SDS) technology is to add linear gel to form molecular sieve in capillary separation buffer, add anionic surfactant sodium dode...

Claims

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Application Information

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IPC IPC(8): G01N27/447
Inventor 方伟杰王海彬黄春辉王宗襄张中伟白骅
Owner HISUN BIORAY PHARMA CO LTD
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