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Glycoprotein multi-charge isomer post-translational modification evaluation method

A technology of post-translational modification and charge isomerization is applied in the field of evaluation of post-translational modification of various charge isoforms of glycoproteins, which can solve the problems of complex modification and achieve the effect of convenient operation and good reference.

Active Publication Date: 2019-06-25
BEIJING TIDE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The protein contains multiple glycosylation modification sites, has a variety of charge isomers, and the post-translational modification is extremely complex. In order to ensure the consistency of each production batch, a stable production process and an effective post-translational modification analysis method are required

Method used

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  • Glycoprotein multi-charge isomer post-translational modification evaluation method
  • Glycoprotein multi-charge isomer post-translational modification evaluation method
  • Glycoprotein multi-charge isomer post-translational modification evaluation method

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1. Structural Analysis of Post-translational Modifications of Charge Variant of Recombinant Human VEGFR-Fc Fusion Protein

[0023] (1) IEF separation charge heterogeneity

[0024] Take 1mg of recombinant human VEGFR-Fc fusion protein, use 10kD, 0.5ml ultrafiltration tube to replace liquid by ultrafiltration into 100μl ultrapure water, and quantify the protein concentration with a microplate reader. Take an appropriate amount of ultrafiltered sample and mix it with 2×sample buffer in an equal volume before loading the sample. The protein loading amount is 100 μg / band, and the gel used is pH3-10 IEF gel. Electrophoresis conditions were as follows.

[0025] Table 1 IEF electrophoresis voltage settings

[0026]

[0027] After electrophoresis, the gel was rinsed twice with ultrapure water, then soaked in 12% trichloroacetic acid fixative solution for fixation, and shaken at room temperature for 30 min. After washing the gel three times with ultrapure water, add...

Embodiment 2

[0052] Example 2, High pI recombinant human VEGFR-Fc fusion protein charge variant post-translational modification structure analysis

[0053] (1) IEF separation charge heterogeneity

[0054] Take 1mg of high pI recombinant human VEGFR-Fc fusion protein, use 10kD, 0.5ml ultrafiltration tube to replace liquid by ultrafiltration into 100μl ultrapure water, and quantify the protein concentration with a microplate reader. Take an appropriate amount of ultrafiltered sample and mix it with 2×sample buffer in an equal volume before loading the sample. The protein loading amount is 100 μg / band, and the gel used is pH3-10 IEF gel. Electrophoresis conditions were as follows.

[0055] Table 1 IEF electrophoresis voltage settings

[0056]

[0057] After electrophoresis, the gel was rinsed twice with ultrapure water, then soaked in 12% trichloroacetic acid fixative solution for fixation, and shaken at room temperature for 30 min. After washing the gel three times with ultrapure water...

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Abstract

The invention discloses a glycoprotein multi-charge isomer post-translational modification evaluation method, which comprises the following steps: 1) multiple charge isomers are separated through isoelectric focusing electrophoresis; 2) a strip after separation of the multiple charge isomers is cut and collected; 3) the multiple collected isomers are subjected to in-gel digestion respectively; 4)peptides after in-gel digestion are extracted; and 5) the extracted peptides are subjected to mass spectrometry to confirm oxidation, deamidation, C-terminal Lys deletion, glycosylation and other post-translational modification. The method overcomes the difficulty of post-translational modification analysis by the multiple charge isomers, multiple kinds of post-translational modification for single-charge glycoprotein can be accurately analyzed, accurate analysis on multiple kinds of post-translational modification related to the multiple charge isomers is more facilitated, and the method is applicable to analysis and evaluation of glycoprotein multi-charge isomer post-translational modification.

Description

technical field [0001] The invention relates to the field of protein medicine bioengineering and technology, and relates to an evaluation method for post-translational modification of multiple charge isoforms of glycoproteins. In particular, it relates to an evaluation method for post-translational modification of various charge variants of recombinant human vascular endothelial cell growth factor receptor Fc (VEGFR-Fc) fusion protein. Background technique [0002] Mature protein drugs not only have a complete amino acid sequence, but also often require a series of post-translational modifications to have specific biological activities. There are various forms of modification and processing, including oxidation, deamidation, deletion of terminal Lys, glycosylation, disulfide bonds, etc. These post-translational modifications (PTMs) affect protein activity and immunogenicity, and are directly related to drug efficacy, half-life, stability and safety in vivo. For example, fo...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 陈志鑫何景昌李维王鑫刘利波
Owner BEIJING TIDE PHARMA
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