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Monoclonal Antibody Disulfide Bond Pairing Analysis Method

An analysis method and technology of disulfide bonds, applied in the field of antibody disulfide bond pairing analysis, can solve problems such as inconvenience, and achieve the effect of fewer enzyme digestion methods and abundant fragment ions

Active Publication Date: 2020-11-10
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the disulfide bond pairing analysis of Golimumab mostly uses several specific enzymes for combined enzymatic hydrolysis for analysis and confirmation, which is very inconvenient

Method used

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  • Monoclonal Antibody Disulfide Bond Pairing Analysis Method
  • Monoclonal Antibody Disulfide Bond Pairing Analysis Method
  • Monoclonal Antibody Disulfide Bond Pairing Analysis Method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Antibody disulfide bond pairing analysis (trypsin combined with Lys-C digestion)

[0046] Monoclonal antibody disulfide bond pairing analysis steps:

[0047] (1) Protein denaturation treatment

[0048] Take 0.5 mg of the protein sample and add it to 380 μl of denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5M Tris, 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.

[0049] (2) Enzymolysis

[0050] Take 100 μg of the protein in (1), add trypsin at 1:25 (wt / wt), add Lys-C at 1:200, combine with enzymatic hydrolysis monoclonal antibody, and incubate at 37°C for 4 hours.

[0051] (3) Reduction and termination of enzymatic hydrolysis

[0052] After the enzymatic hydrolysis of the sample in (2) is completed, take half of the sample and add formic acid FA to a final volume concentration of 0.1%; add 1 μL of 0.1M DTT to the other half of the sample vo...

Embodiment 2

[0068] Example 2: Monoclonal Antibody Disulfide Bond Pairing Analysis (Trypsin Digestion)

[0069] Monoclonal antibody disulfide bond pairing analysis steps:

[0070] (1) Protein denaturation treatment

[0071] Take 0.5 mg of the protein sample and add it to 380 μl of denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5M Tris, 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.

[0072] (2) Enzymolysis

[0073] Take 100 μg of the protein in (2), add trypsin-digested monoclonal antibody at 1:25 (wt / wt), and incubate at 37°C for 4 hours.

[0074] (3) Reduction and termination of enzymatic hydrolysis

[0075] After the enzymatic hydrolysis of the sample in (2) is completed, take half of the sample and add formic acid FA to a final volume concentration of 0.1%; add 1 μL of 0.1M DTT to the other half of the sample, and add formic acid FA to a final volume concentration o...

Embodiment 3

[0080] Example 3: Monoclonal Antibody Disulfide Bond Pairing Analysis (Lys-C Digestion)

[0081] Monoclonal antibody disulfide bond pairing analysis steps:

[0082] (1) Protein denaturation treatment

[0083] Take 0.5 mg of the protein sample and add it to 380 μl of denaturation buffer (8M guanidine hydrochloride+5mM EDTA+0.5M Tris, 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.

[0084] (2) Enzymolysis

[0085] Take 100 μg of the protein in (1), add Lys-C enzymatic monoclonal antibody at 1:200 (wt / wt), and incubate at 37°C for 4 hours.

[0086] (3) Reduction and termination of enzymatic hydrolysis

[0087] After the enzymatic hydrolysis of the sample in (2) is completed, take half of the sample and add formic acid FA to a final volume concentration of 0.1%; add 1 μL of 0.1M DTT to the other half of the sample, and add formic acid FA to a final volume concentration of ...

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Abstract

The invention discloses an analysis method for disulfide bond pairing of a monoclonal antibody. The method comprises the following steps: 1) subjecting protein to denaturation; 2) subjecting the protein to enzymatic hydrolysis with a specific enzyme under non-reduction conditions; 3) dividing the protein having undergone enzymatic hydrolysis into two samples, subjecting one of the samples to reduction while the other not to reduction and terminating enzymatic hydrolysis of the two samples; and 4) after termination of enzymatic hydrolysis, subjecting the samples to peptide mass fingerprinting. The method provided by the invention overcomes problems of a tedious digestion treatment manner using a plurality of enzymes, employs one enzyme for enzymatic hydrolysis of the protein under non-reduction conditions, and can eliminate steric hindrance action of special amino acid sequences of the protein on enzymatic hydrolysis of the protein and accurately confirm and locate a disulfide bond peptide fragment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an 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 and reduced...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
Inventor 吕锋华环民霞李赛谋刘周阳谭青乔
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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