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
View PDF11 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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
Comparison scheme
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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): G01N30/02G01N30/06
Inventor 陈志鑫何景昌李维王鑫刘利波
Owner BEIJING TIDE PHARMA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap