Method for de novo sequencing of a monoclonal antibody and use thereof

By optimizing the enzymatic digestion and mass spectrometry detection process through monoclonal antibody de novo sequencing, the problem of insufficient accuracy in protein identification of deep learning de novo sequencing methods has been solved, enabling rapid and accurate antibody quality evaluation and detection, and promoting the development of antibody product quality evaluation systems.

CN117607307BActive Publication Date: 2026-07-24SHENZHEN INSTITUTE FOR DRUG CONTROL (SHENZHEN TESTING CENTER OF MEDICAL DEVICES)
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Patent Information

Application Number
CN202311663435.X
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2023-12-06
Publication Date
2026-07-24
Estimated Expiration
2043-12-06

AI Technical Summary

Technical Problem

Existing deep learning-based de novo sequencing methods have limitations in protein identification accuracy and cannot fully utilize the information in tandem mass spectrometry, thus limiting the identification accuracy of protein de novo sequencing.

Method used

A monoclonal antibody de novo sequencing method was used to prepare peptide samples through denaturation, reduction, alkylation, and multiple enzymatic digestion steps. High-performance liquid chromatography and mass spectrometry were combined with de novo sequencing software for analysis, and peptide sequences were reassembled. The enzymatic digestion process was optimized to improve detection efficiency and accuracy.

Benefits of technology

It significantly shortens mass spectrometry detection time, increases the detection rate of mutant antibodies from 90% to 99%, simplifies the quality evaluation and testing of antibody reagents, shortens the time, and lays the foundation for the quality evaluation system of antibody and antigen products.

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Abstract

The application provides a method for sequencing a monoclonal antibody from scratch and an application thereof, and the method comprises the following steps: (1) sequentially subjecting a monoclonal antibody sample to denaturation reduction and alkylation to obtain an alkylation reaction solution, dividing the alkylation reaction solution into five parts, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, mixing the five enzymolysis solutions for reaction, and obtaining a polypeptide sample to be detected; (2) using high performance liquid chromatography separation and mass spectrometry to detect the polypeptide sample to be detected, obtaining mass spectrometry data of the polypeptide sample, and using a sequencing software from scratch to analyze and reassemble peptide sequences into a monoclonal antibody sequence. The method can quickly evaluate the quality of the corresponding antibody, accurately detect the mutation site of the antibody, greatly simplifies the operation of quality evaluation and detection of the antibody reagent, and shortens the time of antibody quality evaluation.
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