Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

IgG2 type monoclonal antibody disulfide bond pairing analysis method

An analysis method, disulfide bond technology, applied in the field of monoclonal antibody disulfide bond pairing analysis, can solve inconvenience and other problems, and achieve the effect of enriching fragment ions

Active Publication Date: 2016-11-23
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the existing technical methods use several specific enzymes for combined enzymatic hydrolysis to confirm the disulfide bond pairing form of Denosumab, which is very inconvenient

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
  • IgG2 type monoclonal antibody disulfide bond pairing analysis method
  • IgG2 type monoclonal antibody disulfide bond pairing analysis method
  • IgG2 type monoclonal antibody disulfide bond pairing analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Antibody Protein Disulfide Bond Pairing Analysis (Trypsin Digestion)

[0051] Antibody protein disulfide bond pairing analysis steps:

[0052] (1) Protein denaturation treatment

[0053] Take 0.5 mg protein sample and add it to 380 μl denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5MTris, pH8.3), mix well, and incubate at 37°C for 60 minutes. Ultrafiltration centrifugation or desalting column desalting to 50mM NH 4 HCO 3 (pH8.0) buffer.

[0054] (2) Enzymolysis

[0055] Take 100 μg of the protein in (1), add trypsin to digest the antibody protein at a ratio of 1:10 (wt / wt, enzyme / protein), and incubate at 37°C for 4 hours.

[0056] (3) Reduction and termination of enzymatic hydrolysis

[0057] After the enzymatic hydrolysis of the sample in (2) is completed, take out half of the sample volume and add formic acid FA to a final volume concentration of 0.1%; add 1 μL of 0.1M DTT (dithiothreitol) to the other half of the sample volume. Formic a...

Embodiment 2

[0075] Example 2: Analysis of antibody protein disulfide bond pairing (trypsin 1:25 digestion)

[0076] Antibody protein disulfide bond pairing analysis steps:

[0077] (1) Protein denaturation treatment

[0078] Take 0.5 mg protein sample and add it to 380 μl denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5MTris, pH8.3), mix well, and incubate at 37°C for 60 minutes. Ultrafiltration centrifugation or desalting column desalting to 50mM NH 4 HCO 3 (pH8.0) buffer.

[0079] (2) Enzymolysis

[0080] Take 100 μg of the protein in (1), add trypsin to digest the antibody protein at 1:25 (wt / wt), and incubate at 37°C for 4 hours.

[0081] (3) Reduction and termination of enzymatic hydrolysis

[0082] After the enzymatic hydrolysis of the sample in (2) is completed, take out half of the sample volume and add formic acid FA to a final concentration of 0.1%; add 1 μL of 0.1M DTT to the other half of the sample volume, and add formic acid FA to a final concentration of 0...

Embodiment 3

[0087] Example 3: Analysis of antibody protein disulfide bond pairing (trypsin combined with chymotrypsin digestion)

[0088] Antibody protein disulfide bond pairing analysis steps:

[0089] (1) Protein denaturation treatment

[0090] Take 0.5 mg protein sample and add it to 380 μl denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5MTris, pH8.3), mix well, and incubate at 37°C for 60 minutes. Ultrafiltration centrifugation or desalting column desalting to 50mM NH 4 HCO 3 (pH8.0) buffer.

[0091] (2) Enzymolysis

[0092] Take 100 μg of the protein in (2), add trypsin at 1:25 (wt / wt), add chymotrypsin combined with enzymatic antibody protein at 1:50 (wt / wt), and incubate at 37°C for 4 hours.

[0093] (3) Reduction and termination of enzymatic hydrolysis

[0094] After the enzymatic hydrolysis of the sample in (2) is completed, take out half of the sample volume and add formic acid FA to a final concentration of 0.1%; add 1 μL of 0.1M DTT to the other half of the s...

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 an IgG2 type monoclonal antibody disulfide bond pairing analysis method. The method comprises the steps: 1), carrying out denaturating treatment on a protein; 2), under non reducing conditions, carrying out enzymolysis on the protein after the denaturating treatment by using a specific enzyme; 3), dividing the protein after enzymolysis into two samples, adding DTT to one sample and reducing, allowing the other sample not to be reduced, and making enzymolysis of both of the two samples terminated; and 4), carrying out mass peptide spectrogram analysis of a disulfide bond peptide fragment of the samples having enzymolysis terminated. The method can overcome various complicated enzyme digestion treatment ways, adopts a single enzyme trypsin for enzymolysis of the antibody protein under the non reducing conditions, can exclude the space steric hindrance effect of an antibody protein special amino acid sequence during enzymolysis of the protein, can accurately confirm and position the disulfide bond peptide fragment, and can detect a mismatched disulfide bond peptide fragment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a monoclonal antibody disulfide bond pairing analysis method. Background technique [0002] The disulfide bond is the S-S bond, which is a covalent bond between sulfur atoms in the form of -S-S- formed by the oxidation of two sulfhydryl groups. Cysteine ​​plays a very important role in forming a stable protein spatial structure, maintaining the correct spatial conformation, and regulating biological activity. The correct pairing of disulfide bonds is conducive to the rapid folding of the peptide chain and the formation of a tight and stable spatial structure, forming a local hydrophobic center, which can prevent water molecules from entering the interior of the peptide to destroy hydrogen bonds and form a stable higher-order structure region. [0003] The disulfide bond is only relatively stable, and its covalent bond is easily reduced and broken. When the disulfide bond is broken a...

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
Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
Inventor 吕锋华环民霞刘周阳谭青乔
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com