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Method for fast and high throughput detection of N-linked glycans in glycoprotein based on membrane separation

A technology for linking sugars and glycoproteins, applied in the biological field, can solve the problems of long time, complex steps, low throughput, etc., and achieve the effects of shortening time, simplifying steps, and improving throughput

Inactive Publication Date: 2017-03-22
SHANGHAI HAOZHE INFORMATION TECH LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method takes a long time, has complicated steps, and low throughput, which cannot meet the requirements of rapid analysis and detection

Method used

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  • Method for fast and high throughput detection of N-linked glycans in glycoprotein based on membrane separation
  • Method for fast and high throughput detection of N-linked glycans in glycoprotein based on membrane separation
  • Method for fast and high throughput detection of N-linked glycans in glycoprotein based on membrane separation

Examples

Experimental program
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Effect test

preparation example Construction

[0042] (1) Preparation of reference substance solution: get standard substance, dissolve, obtain reference substance solution;

[0043] (2) Preparation of the test solution: take the glycoprotein sample and pre-treat to obtain the test solution containing N-linked sugar chains;

[0044] (3) measure, adopt UPLC to detect respectively reference substance solution and need testing solution.

[0045]In step (1), the standard is selected from: G0 (NGA2), G0F (NGA2F), Man-5, G1, (G0F+GN), G1Fa, G1Fb, G1F+GN, Man-6, G2 (NA2 ), G2F(NA2F), G1F+SA, or G2F+SA.

[0046] In the step (1), the preparation method of the reference substance solution includes: accurately weighing an appropriate amount of the standard substance respectively, adding water to dissolve, and obtaining the standard substance solution as the reference substance solution. The water is preferably HPLC grade water.

[0047] In step (2), the pretreatment includes: glycoprotein denaturation, sugar chain release, sugar c...

Embodiment 1

[0061] Detection of N-linked sugar chains in purified Herceptin in Example 1

[0062] 1. Preparation of standard solution:

[0063] Standards: G0(NGA2), G0F(NGA2F), Man-5, G1 and (G0F+GN), G1Fa, G1Fb, G1F+GN, Man-6, G2(NA2), G2F(NA2F), G1F+SA , G2F+SA were purchased from Waters.

[0064] Dissolve 228pmol of each of the above standard substances in 100uL of HPLC grade water to obtain a standard solution. The resulting standard solution was stored at -20°C.

[0065] 2. Preparation of the test solution

[0066] (1) Cleaning of the porous plate with hydrophobic filter membrane: Centrifuge and rinse the 96-well plate with PVDF membrane with 200 μL of 70% alcohol and ultrapure water for one time, and the centrifugation condition is 500×g for 1 minute;

[0067] (2) Glycoprotein denaturation

[0068] Add 50 μL of glycoprotein sample (purified Herceptin) with a concentration of 2 mg / ml, 150 μl of guanidine hydrochloride with a concentration of 8 M, and 4 μL of TCEP with a concentr...

Embodiment 2

[0090] Example 2 Detection of N-linked sugar chains in purified Herceptin

[0091] 1. Preparation of standard solution:

[0092] Standards: G0(NGA2), G0F(NGA2F), Man-5, G1 and (G0F+GN), G1Fa, G1Fb, G1F+GN, Man-6, G2(NA2), G2F(NA2F), G1F+SA , G2F+SA were purchased from Waters.

[0093] Dissolve 228pmol of each of the above standard substances in 100uL of HPLC grade water to obtain a standard solution. The resulting standard solution was stored at -20°C.

[0094] 2. Preparation of the test solution

[0095] (1) Cleaning of the porous plate with hydrophobic filter membrane: Centrifuge and rinse the 96-well plate with PVDF membrane with 200 μL of 70% alcohol and ultrapure water for one time, and the centrifugation condition is 500×g for 1 minute;

[0096] (2) Glycoprotein denaturation

[0097] Add 50 μL of glycoprotein sample (purified Herceptin) with a concentration of 1 mg / ml, 150 μL of guanidine hydrochloride with a concentration of 8 M, and 4 μL of TCEP with a concentrati...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method for fast and high throughput detection of N-linked glycans in a glycoprotein sample based on membrane separation. A hydrophobic membrane porous plate and a hydrophilic membrane porous plate are used to process the glycoprotein sample, the glycoprotein sample processing flux is improved, and steps are simplified. In addition, by changing the reaction conditions of PNGase F enzyme, and the sample processing time is successfully shortened. The detection time of the N-linked glycans in the glycoprotein sample can be shortened from 2 days of a traditional method to within 4-6 hours. In addition, the method successfully solves the problem that in traditional experiments only a purified glycoprotein sample can be used to carry out the experiments, and by use of the method, cell culture supernatant can be directly used for experiments. A strong support is provided for fast qualitative analysis of the N-linked glycans in the glycoprotein for screening of early cell lines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for fast and high-throughput detection of N-linked sugar chains of glycoprotein samples based on membrane separation. Background technique [0002] As we all know, antibody drugs have become a hot field in the pharmaceutical industry at home and abroad in recent years. Antibody drugs are complex macromolecular glycoproteins, which undergo a series of post-translational modifications during their expression, among which protein glycosylation is an important modification for proteins to exert their biological functions. Glycoproteins have both N-linked sugar chains and O-linked sugar chains, and more attention is paid to N-linked sugar chains in antibody drug research. The relationship between antibody drug function and glycosylation has been studied in depth at present: the effect of glycosylation on affinity, the effect of glycosylation on cytotoxicity, the effe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/00
CPCG01N30/00
Inventor 孙奕扬贺芸芬殷家骏
Owner SHANGHAI HAOZHE INFORMATION TECH LTD
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