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Glycoprotein multi-charge isomer N-glycan structure evaluation method

A technology of charge isomerization and glycoprotein, applied in the field of protein medicine and bioengineering, can solve the problems of decreased cytotoxic activity, affecting cytotoxicity, reducing affinity, etc., and achieve the effect of improving clinical application, good reference significance, and convenient operation

Inactive 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

For example, for monoclonal antibodies or fusion proteins, high mannose glycoforms and lower sialic acid levels at the end will affect the pharmacokinetics (pK), resulting in rapid clearance in the body and shortened half-life; core fucose can reduce the interaction with receptors affinity, leading to a decrease in antibody-dependent cell-mediated cytotoxicity (ADCC) activity; terminal galactose mainly affects complement-dependent cytotoxicity (CDC) activity

Method used

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  • Glycoprotein multi-charge isomer N-glycan structure evaluation method
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  • Glycoprotein multi-charge isomer N-glycan structure evaluation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Structural analysis of N-sugar chains of charge variants of recombinant human VEGFR-Fc fusion protein

[0029] (1) IEF separation charge heterogeneity

[0030] 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.

[0031] Table 1 IEF electrophoresis voltage settings

[0032]

[0033] 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 that, the gel was washed 3 times with ultrapure water, and an appropri...

Embodiment 2

[0069] Example 2, High pI recombinant human VEGFR-Fc fusion protein charge variant N-sugar chain structure analysis

[0070] (1) IEF separation charge heterogeneity

[0071] 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.

[0072] Table 1 IEF electrophoresis voltage settings

[0073]

[0074] 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 an appropria...

Embodiment 3

[0103] Example 3, Low pI Recombinant Human VEGFR-Fc Fusion Protein Charge Isomer N Sugar Chain Structure Analysis

[0104] (1) IEF separation charge heterogeneity

[0105] Take 1 mg of the low pI recombinant human VEGFR-Fc fusion protein, use a 10kD, 0.5ml ultrafiltration tube to replace the medium by ultrafiltration into 100 μl 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.

[0106] Table 1 IEF electrophoresis voltage settings

[0107]

[0108] 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 an ...

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Abstract

The invention discloses a glycoprotein multi-charge isomer N-glycan structure evaluation method, which comprises the following steps of charge isomer separation, charge isomer collection, in-gel digestion, glycan enrichment, glycan labeling, glycan purification and mass spectrometry. Isoelectric focusing electrophoresis (IEF) separates the multiple charge isomers, the in-gel digestion means is used for analyzing the N-glycan structure, the difficulty in analyzing the N-glycan structure by the multiple charge isomers can be overcome, an N-glycan structure of single-charge glycoprotein can be accurately analyzed, the multi-charge isomer N-glycan structure is analyzed more accurately, and the method is applicable to analysis and evaluation of the glycoprotein multi-charge isomer N-glycan structure.

Description

technical field [0001] The invention relates to the field of protein medicine bioengineering and technology, and relates to a method for evaluating the N sugar chain structure of multiple charge isomers of glycoproteins. In particular, it relates to a method for evaluating the N-sugar chain structure of various charge isomers of recombinant human vascular endothelial cell growth factor receptor Fc (VEGFR-Fc) fusion protein. Background technique [0002] Glycosylation is one of the most common protein post-translational modifications. According to different connection methods, it can be divided into N-glycosylation and O-glycosylation. N-glycosylation is the combination of sugar chains and encoded protein amino acid sequences Asn- -NH on the Asn residue in X-Ser / Thr (X is an amino acid other than Pro) 2 Linked, O-glycosylation is the linking of sugar chains to the -OH on the Ser / Thr residues of the amino acid sequence of the encoded protein. At present, most therapeutic pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/72
Inventor 何景昌陈志鑫李维王鑫刘利波
Owner BEIJING TIDE PHARMA
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