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Analysis method for disulfide bond pairing of monoclonal antibody

An analysis method, disulfide bond technology, applied in the field of antibody disulfide bond pairing analysis, can solve the inconvenience and other problems, and achieve the effect of fewer enzyme cleavage methods and rich fragment ions

Active Publication Date: 2016-11-23
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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  • Analysis method for disulfide bond pairing of monoclonal antibody
  • Analysis method for disulfide bond pairing of monoclonal antibody
  • Analysis method for disulfide bond pairing of monoclonal antibody

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 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.

[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 volume, and a...

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 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.

[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 of 0.1 after...

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 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.

[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 0.1 after b...

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Abstract

The invention discloses a monoclonal antibody disulfide bond pairing analysis method. The method comprises the following steps: 1), denaturing the protein; 2), using specific enzymes to enzymolyze the protein under non-reducing conditions; 3), The protein after enzymolysis was divided into two samples, one of the samples was reduced, and the other sample was not reduced, and the enzymolysis was terminated for both samples; 4), the mass peptide spectrum was performed on the sample after the end of enzymolysis graph analysis. The method of the present invention can overcome the tedious and various enzymatic cleavage treatment methods, and adopts one enzymatic cleavage method to enzymolyze the protein white under non-reducing conditions, which can eliminate the steric hindrance effect of the special amino acid sequence of the protein on the enzymatic protein hydrolysis, Can accurately confirm and locate disulfide bonded peptides.

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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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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