Methods for characterizing disulfide bonds

A technology of disulphide bonds and misconnection of disulfide bonds, which can be used in the preparation of test samples, labels and instruments used in chemical analysis, etc., can solve time-consuming and labor-intensive problems

Pending Publication Date: 2021-08-17
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Current methods for analyzing mAb disulfide bonds are time-consuming and laborious

Method used

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  • Methods for characterizing disulfide bonds
  • Methods for characterizing disulfide bonds
  • Methods for characterizing disulfide bonds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Synthesis of Parallel and Crossed Hinge Peptides

[0073] method :

[0074] cross-linking

[0075] Purchase cysteine-containing peptides from commercial suppliers. By reacting with 1 mM Cu as an oxidizing agent in the presence of air 2+ Incubation results in cross-linking of the peptides. Peptide and Cu 2+ The molar ratio is 5:1.

[0076] N-terminal analysis / Edman degradation

[0077] The cross-linked peptides were suspended in water and placed in a protein sequencer. Peptides were exposed to phenylisothiocyanate (PITC). PITC is coupled to the N-terminal residue to form a PTC polypeptide. Upon addition of trifluoroacetic acid to the reaction, the PTC N-terminal residue undergoes acid cleavage, resulting in the release of the labile ATZ-amino acid. Separate the ATZ-amino acid from the peptide solution into a transformation flask with ethyl acetate. ATZ-amino acids were converted to stable PTH-amino acids with 25% TFA in water (v / v). The PTH-amino ac...

Embodiment 2

[0081] Example 2: Analysis of the hinge regions of two mAbs

[0082] method

[0083] Hinge disulfide bond characterization

[0084] Antibodies are first digested into peptides. IgGl antibodies were subjected to double enzyme digestion using proteinase K followed by trypsin. IgG4 antibodies were subjected to FabRICATOR followed by trypsin digestion. Hinge peptide standards were prepared as described above using the hinge region sequences of the IgGl and IgG4 antibodies used to prepare the peptides. The digested peptide mixture was subjected to LC-MS analysis. Hinge peptide standards were also subjected to LC-MA analysis. Retention time analysis was performed to compare the retention time of the antibody peptide to that of the hinge peptide standard.

[0085] result

[0086] IgG1 mAb1 was subjected to digestion into peptides, and the resulting peptides were subjected to LC / MS analysis. The hinge peptide standard above was also subjected to LC / MS analysis. Such as ...

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Abstract

Compositions and methods for analyzing disulfide bonds are provided. An exemplary method includes preparing peptide standards having no disulfide bonds, scrambled disulfide bond peptide standards, and native disulfide bond peptide standards according to the sequence of the region of the protein drug product that includes the disulfide bond, digesting a sample of protein drug product into peptides, separating the protein drug product peptides, analyzing the protein drug product peptides and the peptide standards, identifying scrambled and native disulfide bond peptides by retention time, and quantifying the level of scrambled disulfide bond peptides.

Description

technical field [0001] The present invention generally relates to systems and methods for characterizing antibodies, particularly disulfide bonds. [0002] Cross references to related patent applications [0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 792,994, filed January 16, 2019, which is incorporated by reference in its entirety. Background technique [0004] During the development of a monoclonal antibody (mAb) from a drug candidate to a marketed product, issues related to stability, post-translational modifications, or other changes to the antibody may arise. Changes in antibody structure and function can lead to problems such as poor shelf life or even poor immunogenicity in patients. Therefore, it is important to properly characterize antibody structures and monitor them throughout the production process. Antibody quality control and quality assurance are critical to the purity and safety of mAb products....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/6848G01N33/6815G01N33/6854G01N1/28G01N30/7233G01N2030/027G01N2030/8831G01N2458/15A61K2039/505A61K39/395
Inventor 王顺海
Owner REGENERON PHARM INC
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