CN117607307A - Method for de novo sequencing of monoclonal antibody and application thereof - Google Patents

Abstract

The invention provides a method for de novo sequencing of monoclonal antibodies and application thereof, wherein the method comprises the following steps: (1) Carrying out denaturation reduction and alkylation on a monoclonal antibody sample in sequence to obtain an alkylation reaction solution, dividing the alkylation reaction solution into 5 parts averagely, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, and mixing and reacting 5 parts of enzymolysis solution to obtain a polypeptide sample to be detected; (2) Detecting the polypeptide sample to be detected by using high performance liquid chromatography and mass spectrometry to obtain mass spectrum data of the polypeptide sample; the peptide sequences were reassembled into monoclonal antibody sequences using de novo sequencing software for analysis. The method can rapidly perform quality evaluation on the corresponding antibody, accurately detect the mutation site of the antibody, greatly simplify the quality evaluation and detection operation of the antibody reagent and shorten the quality evaluation time of the antibody.

Description

Method for de novo sequencing of monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of amino acid sequencing, and particularly relates to a method for de novo sequencing of a monoclonal antibody and application thereof.

Background

In recent years, with the rapid development of molecular biology and mass spectrometry technology, research on proteomics has received more and more attention, and has been widely used in many fields. As a key technology of proteomics, the de novo sequencing method can directly identify the amino acid sequence of the obtained peptide fragment according to tandem mass spectrometry, and has the advantage of irreplaceability of other protein identification methods.

As one of the core contents of proteomics research, the basic task of protein identification is to identify the amino acid sequence corresponding to the peptide fragment of the protein, which is the basis and key of research on protein structure, function, interaction among proteins and the like. As one of the most commonly used techniques in proteomics research at present, mass spectrometry is mainly used in the aspects of biological macromolecular mass measurement, protein structure analysis, protein peptide fragment sequence identification and the like, and has the advantages of high processing speed and high result accuracy, so that the mass spectrometry has been paid attention to in the field of biological research. The basic principle of mass spectrometry is: protein molecules are split into a series of charged fragment ions by a mass spectrometer under vacuum conditions, and the information of the fragment ions is sequentially analyzed and recorded according to the mass-to-charge ratio m/z (i.e. the ratio of mass m to charge z) of the fragment ions, so as to form a mass spectrogram.

In general, a mass spectrum obtained by analysis using only one mass spectrometer is referred to as a primary mass spectrum in which each ion peak corresponds to one peptide fragment of a protein. However, because of the complexity of protein samples, the primary mass spectrum contains less information, and the requirements of related researches such as protein identification cannot be well met. To extract more abundant information contained in proteins, further improving the accuracy of the results, tandem mass spectrometry (MS/MS, tandem mass spectrometry) techniques have been proposed. In short, tandem mass spectrometry refers to a process of connecting two or more mass spectrometers in series, selecting a specific parent ion (i.e. a specific peptide of a protein) in a primary mass spectrum, performing collision and fragmentation to generate a series of ion, and analyzing the child ion by a mass analyzer in the tandem mass spectrometer to form a secondary mass spectrum. The mass spectrum produced by tandem mass spectrometry techniques is referred to as a tandem mass spectrum. The relationship between parent ion and child ion is essentially the relationship between a protein peptide fragment and the amino acids constituting the peptide fragment.

With the appearance and continuous development of tandem mass spectrometry technology, a protein identification method based on tandem mass spectrometry rapidly stands out with the characteristics of high accuracy, high sensitivity, high reliability and the like, and is a main method in the current large-scale protein molecular identification and is widely applied. The protein de novo sequencing method is independent of protein database information, can directly obtain the amino acid sequence of a protein peptide fragment according to tandem mass spectrometry analysis, can be used for identifying new proteins which do not exist in a database, can provide related information of protein post-translational modification, and is one of the most commonly used protein identification methods at present.

However, the accuracy of identification from the head sequencing method is affected to some extent due to the complexity of tandem mass spectrometry data, the drawbacks of the identification method itself, and the like. At present, a number of de novo sequencing methods based on deep learning have been proposed, which can greatly improve the accuracy of de novo sequencing compared to the traditional de novo sequencing of proteins, but at the same time have some disadvantages. Therefore, how to utilize deep learning to learn the information contained in the tandem mass spectrum more fully, design a more accurate and reliable protein identification method, further improve the identification accuracy of protein sequencing from the head, and still be the key content in the related research of proteomics at present, and also be one of the research difficulties.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a method for sequencing monoclonal antibodies from scratch and application thereof. The invention uses the established monoclonal antibody de novo sequencing method to evaluate the quality of the original antibody and the mutant antibody, sequences the mutant antibody and the original antibody to obtain the corresponding amino acid sequence, and performs BLAST comparison between the amino acid sequence obtained by sequencing and the amino acid sequence translated by the nucleic acid sequence, thus being capable of accurately detecting the position of the mutant amino acid in the CDR3 region of the variable region. The method for sequencing the monoclonal antibody from the head can quickly evaluate the quality of the corresponding antibody, accurately detect the mutation site of the antibody, greatly simplify the quality evaluation and detection operation of the antibody reagent and shorten the quality evaluation time of the antibody.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in a first aspect, the invention provides a method for de novo sequencing of monoclonal antibodies, the method comprising:

(1) Carrying out denaturation reduction and alkylation on a monoclonal antibody sample in sequence to obtain an alkylation reaction solution, dividing the alkylation reaction solution into 5 parts averagely, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, and mixing and reacting 5 parts of enzymolysis solution to obtain a polypeptide sample to be detected;

(2) Detecting the polypeptide sample to be detected by using high performance liquid chromatography and mass spectrometry to obtain mass spectrum data of the polypeptide sample; the peptide sequences were reassembled into monoclonal antibody sequences using de novo sequencing software for analysis.

The method for sequencing the monoclonal antibody from the head can quickly evaluate the quality of the corresponding antibody, accurately detect the mutation site of the antibody, greatly simplify the quality evaluation and detection operation of the antibody reagent and shorten the quality evaluation time of the antibody. Meanwhile, the method of sequencing monoclonal antibodies from scratch is used as a starting point and breakthrough, and the relevant quality evaluation technology of antibodies and relevant quality evaluation technology of antigens are developed in an important way, so that a foundation is laid for establishing a scientific, complete research and supervision system for evaluating the quality of antigens and antibody products, and the important role in the development of IVD raw material industry is played.

Compared with the prior art, the method for sequencing the monoclonal antibody from the head has the advantages that the mass spectrum detection time is shortened from 120 minutes to 40 minutes, and the detection rate of the mutant antibody is improved from 90% to 99%, so that the operation of evaluating and detecting the quality of the antibody reagent is simplified, and the time of evaluating the quality of the antibody is shortened. Meanwhile, the method of sequencing monoclonal antibodies from scratch is used as a starting point and breakthrough, and the relevant quality evaluation technology of antibodies and relevant quality evaluation technology of antigens are developed in an important way, so that a foundation is laid for establishing a scientific, complete research and supervision system for evaluating the quality of antigens and antibody products, and the important role in the development of IVD raw material industry is played.

In the invention, the enzymolysis step is optimized, trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme are selected to carry out enzymolysis on the antibody respectively, and the enzyme in the specific selection 5 can fully decompose the antibody into small fragments, so that the coverage rate is high, the subsequent detection is facilitated, and the influence among enzymes can be avoided by adding the enzymes separately, the mutual decomposition of the enzymes is avoided, and the enzymolysis efficiency is reduced. In the invention, the enzymolysis efficiency of the antibody by the combination of different enzymes is examined, and the result shows that the combination of the 5 enzymes has excellent enzymolysis efficiency, can meet the requirement of cost control, and can meet the result of subsequent mass spectrum detection.

Preferably, the polypeptide sample is prepared by a method comprising the steps of:

(A) Denaturation reduction: mixing and reacting the monoclonal antibody sample with urea and dithiothreitol to obtain a denatured and reduced monoclonal antibody;

(B) Alkylation: carrying out alkylation reaction on the denatured and reduced monoclonal antibody and iodoacetamide solution to obtain an alkylation reaction solution;

(C) Enzymolysis: dividing the alkylation reaction solution into 5 parts averagely, and respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis; and stopping the enzymolysis reaction by adopting formic acid to obtain the polypeptide sample to be detected.

Preferably, in the step (A), the concentration of the monoclonal antibody sample in the reaction solution of the mixing reaction is 0.1-0.5mg/mL, for example, 0.1mg/mL, 0.3mg/mL, 0.5mg/mL, or the like.

Preferably, in the step (a), the concentration of urea in the reaction solution of the mixing reaction is 2 to 4M, and may be, for example, 2M, 3M, 4M, or the like.

In the invention, when the concentration of urea is 2-4M, the urea has better denaturation and reduction effects; urea concentration is too low, denaturation and reduction are incomplete, and urea concentration is too high, so that subsequent enzymolysis reaction can be influenced.

Preferably, in the step (A), the concentration of dithiothreitol in the reaction solution of the mixed reaction is 4 to 10mM, and may be, for example, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM or the like.

In the invention, the dithiothreitol has the function of opening disulfide bonds in the antibody, and the dithiothreitol has better action effect when the concentration of the dithiothreitol is 4-10 mM. The concentration of dithiothreitol is too low, disulfide bonds are incompletely opened, and the subsequent enzymolysis result is affected.

Preferably, in the step (A), the temperature of the mixing reaction is 35-65deg.C, for example, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ or 65 ℃, etc., and the time is 20-60 minutes, for example, 20, 30, 40, 50 or 60, etc.

In the present invention, the reaction time can be properly prolonged when the temperature is low during the mixing reaction, and the denaturation reduction can be completed in a shorter time when the temperature is high.

Preferably, in the step (B), the concentration of iodoacetamide in the reaction liquid of the alkylation reaction is 10-20mM, for example, 10mM, 15mM, 20mM, etc.

In the invention, the iodoacetamide is used as an alkylating reagent, so that the protein sample can be completely denatured and kept in a reduced state, the oxidization of sulfhydryl into disulfide bond is prevented, and the reduced environment is maintained.

Preferably, in the step (B), the alkylation reaction is carried out for 20-60 minutes, such as 20, 30, 40, 50 or 60, etc., under the condition of light-shielding reaction.

In the invention, the alkylation reaction is carried out at room temperature, the time of the light-shielding reaction is controlled to be 20-60 minutes, and the incomplete alkylation can be caused by too short time.

Preferably, in the step (C), the final concentration of trypsin in the reaction solution of the enzymolysis reaction is 0.005-0.05mg/mL (for example, 0.005mg/mL, 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, etc.); the final concentration of chymotrypsin is 0.005-0.05mg/mL (e.g., may be 0.005mg/mL, 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, etc.); the final concentration of pepsin is 0.005-0.05mg/mL (e.g., 0.005mg/mL, 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, etc.); the final concentration of elastase is 0.005-0.05mg/mL (e.g., may be 0.005mg/mL, 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, etc.); and the final concentration of Glu-C enzyme is 0.005-0.05mg/mL (e.g., may be 0.005mg/mL, 0.01mg/mL, 0.02mg/mL, 0.03mg/mL, 0.04mg/mL, 0.05mg/mL, etc.).

In the invention, the enzyme concentration cannot be too low, the enzyme digestion efficiency is affected by too low, the number of effective peptide fragments for enzyme digestion is reduced, and the coverage rate of detection results is reduced; the enzyme concentration cannot be too high, the enzyme digestion of the enzyme digestion peptide fragments can be too small due to too high concentration, the liquid phase separation is not facilitated, the coverage rate of the detection results is reduced, meanwhile, the enzyme can be subjected to enzymolysis, and the accuracy of the detection results can be influenced by the fragments which are too high in solubility and are digested by the enzyme digestion enzyme.

Preferably, in the step (C), the condition of the enzymolysis reaction is 34-37 ℃ (for example, 34 ℃, 35 ℃, 36 ℃ or 37 ℃ and the like) and the enzymolysis is carried out for 9-18 hours (for example, 9, 12, 15 or 18 and the like).

In the invention, the enzymolysis reaction time cannot be too short, otherwise, the enzymolysis is incomplete, the measured result is inaccurate, the enzyme is influenced by the result exceeding 18 hours, impurities are brought, and the subsequent detection is interfered.

Preferably, in step (C), the reaction is stopped by adjusting the pH to acidic with formic acid.

In the present invention, the acidity for adjusting the pH to acidity is not particularly limited as long as it is acidic.

Preferably, in step (2), the chromatographic column used for the high performance liquid chromatography separation is C18.

Preferably, in the step (2), the mobile phase adopted by the high performance liquid chromatography separation consists of a mobile phase A and a mobile phase B; the mobile phase A is 0.08-0.12% (for example, 0.08%, 0.1% or 0.12% etc.) formic acid aqueous solution, and the mobile phase B is formic acid aqueous-acetonitrile solution.

Preferably, the concentration of the formic acid aqueous solution in the formic acid aqueous solution-acetonitrile solution is 0.08-0.12%, for example, 0.08%, 0.1% or 0.12% and the like.

Preferably, the volume ratio of the formic acid aqueous solution to the acetonitrile solution in the formic acid aqueous solution-acetonitrile solution is (18-22): (78-82), and can be 18:82, 20:80 or 22:78, for example.

Preferably, the flow rate of the mobile phase is 280-320nL/min, and can be 280nL/min, 300nL/min, 320nL/min, or the like.

Preferably, in step (2), the high performance liquid chromatography separation employs gradient elution, and the gradient elution is performed according to the following procedure:

in the invention, the elution mode has the advantages of short elution time, greatly shortened detection time, high separation efficiency, and capability of effectively separating out the detection peptide fragments, so that the coverage rate and the accuracy of the detection result are improved.

Preferably, in step (2), the mass spectrometry detection is performed using an ultra-high resolution mass spectrometer.

Preferably, in the step (2), the spraying voltage in the mass spectrum detection is 3.8-4.0kV, the collection is carried out in a positive ion mode, the capillary temperature is 300-320 ℃, and the S lens RF level is 20-40; parent ion scan range: 350-1500m/z; the secondary mass spectrum selects parent ions with the intensity of ten in the first time to dissociate under the data dependent mode, and the normalized collision energy is 20-40%.

As a preferred embodiment of the present invention, the method includes:

(1) Sequentially carrying out denaturation reduction, alkylation and enzymolysis on a monoclonal antibody sample to prepare a polypeptide sample;

(A) Denaturation reduction: mixing a monoclonal antibody sample with urea and dithiothreitol at 35-65 ℃ for reaction for 20-60 minutes, wherein the concentration of the monoclonal antibody sample in the reaction solution is 0.1-0.5mg/mL, the concentration of urea is 2-4M, and the concentration of dithiothreitol is 4-10mM, so as to obtain a denatured and reduced monoclonal antibody;

(B) Alkylation: carrying out light-shielding reaction on the denatured and reduced monoclonal antibody and iodoacetamide solution for 20-60 minutes, wherein the concentration of iodoacetamide in the reaction solution of the alkylation reaction is 10-20mM, so as to obtain an alkylation reaction solution;

(C) Enzymolysis: dividing the alkylation reaction solution into 5 parts, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, and carrying out enzymolysis for 9-18 hours at 34-37 ℃; the final concentration of trypsin in the reaction liquid of the enzymolysis reaction is 0.005-0.05mg/mL; the final concentration of chymotrypsin is 0.005-0.05mg/mL; the final concentration of pepsin is 0.005-0.05mg/mL; the final concentration of elastase is 0.005-0.05mg/mL; and the final concentration of Glu-C enzyme is 0.005-0.05mg/mL;

(2) Detecting the polypeptide sample to be detected by using high performance liquid chromatography and mass spectrometry to obtain mass spectrum data of the polypeptide sample; analyzing by using de novo sequencing software, and re-assembling the peptide fragment sequences into monoclonal antibody sequences;

the chromatographic column adopted by the high performance liquid chromatography separation is C18; the adopted mobile phase consists of a mobile phase A and a mobile phase B; the mobile phase A is 0.08-0.12% formic acid aqueous solution, and the mobile phase B is formic acid aqueous-acetonitrile solution; the concentration of the formic acid aqueous solution is 0.08-0.12%, and the volume ratio of the formic acid aqueous solution to the acetonitrile solution is (18-22) (78-82); the flow rate of the mobile phase is 280-320nL/min; gradient elution is adopted, and the gradient elution comprises the following steps:

performing mass spectrum detection by adopting an ultra-high resolution mass spectrometer; the spraying voltage in the mass spectrum detection is 3.8-4.0kV, the collection is carried out in a positive ion mode, the capillary temperature is 300-320 ℃, and the S lens RF level is 20-40; parent ion scan range: 350-1500m/z; the secondary mass spectrum selects parent ions with the intensity of ten in the first time to dissociate under the data dependent mode, and the normalized collision energy is 20-40%.

In a second aspect, the invention provides the use of a method for de novo sequencing of monoclonal antibodies according to the first aspect in monoclonal antibody sequencing.

The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention uses five enzymes to carry out enzyme cutting sequencing, can cover the sequence of the antibody by 100 percent, accurately detect the amino acid sequence of the antibody, and has simple detection method, wide operability and controllable cost. The mass spectrum detection time is shortened from 120 minutes to 40 minutes, and the detection rate of the mutant antibody is improved from 90% to 99%, so that the operation of evaluating and detecting the quality of the antibody reagent is simplified, and the time of evaluating the quality of the antibody is shortened.

(2) The invention uses the method of monoclonal antibody de novo sequencing as the starting point and breakthrough, and focuses on developing the antibody related quality evaluation technology and antigen related quality evaluation technology, which lays the foundation for establishing a scientific, complete research and supervision system for antigen and antibody product quality evaluation, and plays a significant role in the development of in vitro diagnosis test raw material industry.

(3) The invention can rapidly carry out quality evaluation and detection operation on the corresponding antibody in-vitro diagnostic reagent by using the antibody sequencing technology method, can identify whether mutation is generated or not and whether activity is generated or not, and greatly shortens the time for registering the in-vitro diagnostic test related reagent and the accuracy of the performance thereof.

Drawings

FIG. 1 is the result of the experiment of comparative example 1.

FIG. 2 shows the experimental results of example 1.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.

Example 1

The present embodiment provides a method for de novo sequencing of monoclonal antibodies, the method comprising:

(1) Sequentially carrying out denaturation reduction, alkylation and enzymolysis on a monoclonal antibody sample to prepare a polypeptide sample;

(A) Denaturation reduction: mixing a monoclonal antibody sample with urea and dithiothreitol at 35 ℃ for reaction for 60 minutes, wherein the concentration of the monoclonal antibody sample in the reaction solution is 0.3mg/mL, the concentration of urea is 3M, and the concentration of dithiothreitol is 6mM, so as to obtain a denatured and reduced monoclonal antibody;

(B) Alkylation: carrying out light-shielding reaction on the denatured and reduced monoclonal antibody and an iodoacetamide solution for 25 minutes, wherein the concentration of iodoacetamide in the reaction solution of the alkylation reaction is 10mM, so as to obtain an alkylation reaction solution;

(C) Enzymolysis: dividing the alkylation reaction solution into 5 parts averagely, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, and carrying out enzymolysis for 9 hours at 37 ℃; the final concentration of trypsin in the reaction liquid of the enzymolysis reaction is 0.02mg/mL; the final concentration of chymotrypsin is 0.02mg/mL; the final concentration of pepsin is 0.02mg/mL; the final concentration of elastase was 0.02mg/mL; and the final concentration of Glu-C enzyme is 0.02mg/mL; regulating the pH value to be acidic by adopting formic acid to terminate the reaction, so as to obtain a polypeptide sample to be detected;

(2) Detecting the polypeptide sample to be detected by using high performance liquid chromatography and mass spectrometry to obtain mass spectrum data of the polypeptide sample; analyzing by using de novo sequencing software, and re-assembling the peptide fragment sequences into monoclonal antibody sequences;

the chromatographic column adopted by the high performance liquid chromatography separation is C18; the adopted mobile phase consists of a mobile phase A and a mobile phase B; the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is formic acid aqueous-acetonitrile solution; the concentration of the formic acid aqueous solution is 0.1%, and the volume ratio of the formic acid aqueous solution to the acetonitrile solution is 20:80; the flow rate of the mobile phase is 300nL/min; gradient elution is adopted, and the gradient elution comprises the following steps:

performing mass spectrum detection by adopting an ultra-high resolution mass spectrometer; in the mass spectrum detection, the spraying voltage is 3.8kV, the acquisition is carried out in a positive ion mode, the capillary temperature is 320 ℃, and the S lens RF level is 40; parent ion scan range: 350-1500m/z; the secondary mass spectrum selects parent ions with the intensity of ten in the first time to dissociate under the data dependent mode, and the normalized collision energy is 20%.

Example 2

The present embodiment provides a method for de novo sequencing of monoclonal antibodies, the method comprising:

(1) Sequentially carrying out denaturation reduction, alkylation and enzymolysis on a monoclonal antibody sample to prepare a polypeptide sample;

(A) Denaturation reduction: mixing a monoclonal antibody sample with urea and dithiothreitol at 65 ℃ for reaction for 20 minutes, wherein the concentration of the monoclonal antibody sample in the reaction solution is 0.1mg/mL, the concentration of urea is 2M, and the concentration of dithiothreitol is 4mM, so as to obtain a denatured and reduced monoclonal antibody;

(B) Alkylation: carrying out light-shielding reaction on the denatured and reduced monoclonal antibody and an iodoacetamide solution for 35 minutes, wherein the concentration of iodoacetamide in the reaction solution of the alkylation reaction is 15mM, so as to obtain an alkylation reaction solution;

(C) Enzymolysis: dividing the alkylation reaction solution into 5 parts averagely, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, and carrying out enzymolysis for 9 hours at 37 ℃; the final concentration of trypsin in the reaction liquid of the enzymolysis reaction is 0.05mg/mL; the final concentration of chymotrypsin is 0.05mg/mL; the final concentration of pepsin is 0.05mg/mL; the final concentration of elastase was 0.05mg/mL; and the final concentration of Glu-C enzyme is 0.05mg/mL; regulating the pH value to be acidic by adopting formic acid to terminate the reaction, so as to obtain a polypeptide sample to be detected;

(2) Detecting the polypeptide sample to be detected by using high performance liquid chromatography and mass spectrometry to obtain mass spectrum data of the polypeptide sample; analyzing by using de novo sequencing software, and re-assembling the peptide fragment sequences into monoclonal antibody sequences;

the chromatographic column adopted by the high performance liquid chromatography separation is C18; the adopted mobile phase consists of a mobile phase A and a mobile phase B; the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is formic acid aqueous-acetonitrile solution; the concentration of the formic acid aqueous solution is 0.1%, and the volume ratio of the formic acid aqueous solution to the acetonitrile solution is 20:80; the flow rate of the mobile phase is 300nL/min; gradient elution is adopted, and the gradient elution comprises the following steps:

performing mass spectrum detection by adopting an ultra-high resolution mass spectrometer; in the mass spectrum detection, the spraying voltage is 3.8kV, the acquisition is carried out in a positive ion mode, the capillary temperature is 320 ℃, and the S lens RF level is 40; parent ion scan range: 350-1500m/z; the secondary mass spectrum selects parent ions with the intensity of ten in the first time to dissociate under the data dependent mode, and the normalized collision energy is 20%.

Example 3

The present embodiment provides a method for de novo sequencing of monoclonal antibodies, the method comprising:

(A) Denaturation reduction: mixing a monoclonal antibody sample with urea and dithiothreitol at 45 ℃ for reaction for 40 minutes, wherein the concentration of the monoclonal antibody sample in the reaction solution is 0.5mg/mL, the concentration of urea is 4M, and the concentration of dithiothreitol is 10mM, so as to obtain a denatured and reduced monoclonal antibody;

(B) Alkylation: carrying out light-shielding reaction on the denatured and reduced monoclonal antibody and an iodoacetamide solution for 35 minutes, wherein the concentration of iodoacetamide in the reaction solution of the alkylation reaction is 10mM, so as to obtain an alkylation reaction solution;

(C) Enzymolysis: dividing the alkylation reaction solution into 5 parts averagely, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, and carrying out enzymolysis for 9 hours at 37 ℃; the final concentration of trypsin in the reaction liquid of the enzymolysis reaction is 0.005mg/mL; the final concentration of chymotrypsin is 0.005mg/mL; the final concentration of pepsin is 0.005mg/mL; the final concentration of elastase is 0.005mg/mL; and the final concentration of Glu-C enzyme is 0.005mg/mL; regulating the pH value to be acidic by adopting formic acid to terminate the reaction, so as to obtain a polypeptide sample to be detected;

the chromatographic column adopted by the high performance liquid chromatography separation is C18; the adopted mobile phase consists of a mobile phase A and a mobile phase B; the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is formic acid aqueous-acetonitrile solution; the concentration of the formic acid aqueous solution is 0.1%, and the volume ratio of the formic acid aqueous solution to the acetonitrile solution is 20:80; the flow rate of the mobile phase is 300nL/min; gradient elution is adopted, and the gradient elution comprises the following steps:

performing mass spectrum detection by adopting an ultra-high resolution mass spectrometer; in the mass spectrum detection, the spraying voltage is 3.8kV, the acquisition is carried out in a positive ion mode, the capillary temperature is 320 ℃, and the S lens RF level is 40; parent ion scan range: 350-1500m/z; the secondary mass spectrum selects parent ions with the intensity of ten in the first time to dissociate under the data dependent mode, and the normalized collision energy is 20%.

Example 4

This example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in step (C), the final concentration of trypsin in the reaction solution of the enzymatic hydrolysis reaction is 0.2mg/mL; the final concentration of chymotrypsin is 0.2mg/mL; the final concentration of pepsin is 0.2mg/mL; the final concentration of elastase was 0.2mg/mL; and the final concentration of Glu-C enzyme is 0.2mg/mL. The remaining steps are described in example 1.

Example 5

This example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in step (C), the final concentration of trypsin in the reaction solution of the enzymatic hydrolysis reaction is 0.001mg/mL; the final concentration of chymotrypsin is 0.001mg/mL; the final concentration of pepsin is 0.001mg/mL; the final concentration of elastase was 0.001mg/mL; and the final concentration of Glu-C enzyme is 0.001mg/mL. The remaining steps are described in example 1.

Example 6

This example provides a method for de novo sequencing of monoclonal antibodies which differs from example 1 only in that in step (C) the time of enzymatic hydrolysis is 6 hours; the remaining steps are described in example 1.

Example 7

This example provides a method for de novo sequencing of monoclonal antibodies which differs from example 1 only in that in step (C) the time of enzymatic hydrolysis is 20 hours; the remaining steps are described in example 1.

Comparative example 1

This comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the step (C) enzymatic hydrolysis step, trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme are simultaneously enzymatically hydrolyzed in one system; the remaining steps are described in example 1.

Comparative example 2

This comparative example provides a method for de novo sequencing of monoclonal antibodies which differs from example 1 only in that in the enzymatic hydrolysis step of step (C) the enzyme used is trypsin; the remaining steps are described in example 1.

Comparative example 3

This comparative example provides a method for de novo sequencing of monoclonal antibodies which differs from example 1 only in that in the enzymatic hydrolysis step of step (C) the enzyme employed is chymotrypsin; the remaining steps are described in example 1.

Comparative example 4

This comparative example provides a method for de novo sequencing of monoclonal antibodies which differs from example 1 only in that in the enzymatic hydrolysis step of step (C) the enzyme used is pepsin; the remaining steps are described in example 1.

Comparative example 5

This comparative example provides a method for de novo sequencing of monoclonal antibodies which differs from example 1 only in that in the enzymatic hydrolysis step of step (C) the enzyme used is elastase; the remaining steps are described in example 1.

Comparative example 6

This comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the enzymatic hydrolysis step of step (C), the enzyme employed is Glu-C; the remaining steps are described in example 1.

Comparative example 7

This comparative example provides a method for de novo sequencing of monoclonal antibodies which differs from example 1 only in that in the enzymatic hydrolysis step of step (C) the enzyme used is Lys-C enzyme; the remaining steps are described in example 1.

Comparative example 8

This comparative example provides a method for de novo sequencing of monoclonal antibodies which differs from example 1 only in that in the enzymatic hydrolysis step of step (C) the enzyme employed is Asp-N enzyme; the remaining steps are described in example 1.

Comparative example 9

The present comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the enzymatic hydrolysis step of step (C), the enzymes used are trypsin, chymotrypsin and Glu-C enzyme, and trypsin, chymotrypsin and Glu-C enzyme are used for enzymatic hydrolysis, respectively; the remaining steps are described in example 1.

Comparative example 10

This comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the enzymatic hydrolysis step of step (C), the enzymes used are trypsin, chymotrypsin and pepsin, and the enzymes are trypsin, chymotrypsin and pepsin, respectively; the remaining steps are described in example 1.

Comparative example 11

This comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the enzymatic hydrolysis step of step (C), the enzymes used are trypsin, chymotrypsin and Lys-C, and trypsin, chymotrypsin and Lys-C are used for enzymatic hydrolysis, respectively; the remaining steps are described in example 1.

Comparative example 12

The present comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the enzymatic hydrolysis step of step (C), the enzymes used are trypsin, chymotrypsin, pepsin and Glu-C enzyme, and the enzymes are trypsin, chymotrypsin, pepsin and Glu-C enzyme, respectively, are used for enzymatic hydrolysis; the remaining steps are described in example 1.

Comparative example 13

The present comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the enzymatic hydrolysis step of step (C), enzymes selected from trypsin, chymotrypsin, pepsin and elastase are used, and trypsin, chymotrypsin, pepsin and elastase are used for enzymatic hydrolysis, respectively; the remaining steps are described in example 1.

Comparative example 14

This comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the enzymatic hydrolysis step of step (C), the enzymes used are trypsin, chymotrypsin, pepsin, elastase and Lys-C enzyme, and trypsin, chymotrypsin, pepsin, elastase and Lys-C enzyme are used for enzymatic hydrolysis, respectively; the remaining steps are described in example 1.

Comparative example 15

This comparative example provides a method for de novo sequencing of monoclonal antibodies, which differs from example 1 only in that in the enzymatic hydrolysis step of step (C), the enzymes used are trypsin, chymotrypsin, pepsin, elastase, glu-C enzyme and Lys-C enzyme, and the enzymatic hydrolysis is performed with trypsin, chymotrypsin, pepsin, elastase, glu-C enzyme and Lys-C enzyme, respectively; the remaining steps are described in example 1.

Comparative example 16

The comparative example provides a method for sequencing monoclonal antibodies, which differs from example 1 in that the specific steps of the sequencing method are as follows:

step S1, 100. Mu.g of a monoclonal antibody sample was taken and added with 6M guanidine hydrochloride buffer and 1M dithiothreitol to a final concentration of 10mM, and the reaction was carried out at 56℃for 30 minutes to effect denaturation reduction, with the addition of Dithiothreitol (DTT) to a final concentration of 10mM.

Step S2, cooling the denatured and reduced monoclonal antibody sample to room temperature, adding 1M iodoacetamide to the final concentration of iodoacetamide of 20mM, and carrying out alkylation by light-shielding reaction at room temperature for 30 minutes.

Step S3, desalting the monoclonal antibody sample after alkylation by using a zeba desalting column.

Step S4, adding trypsin into the desalted monoclonal antibody sample, and performing enzymolysis for 10-18 hours at room temperature to obtain a monoclonal antibody: the mass ratio of trypsin is 50:1, and the pH value of the solution is adjusted to be acidic to terminate enzymolysis, so that the polypeptide sample is obtained.

And S5, separating peptide fragments of the polypeptide sample obtained in the step S4 by using reverse phase chromatography, and then carrying out mass spectrometry detection.

The chromatographic conditions are as follows, the chromatographic column is C18, the column temperature is 55 ℃, the temperature of a sample chamber is 5 ℃, the loading volume is 2 mu L, the mobile phase A is 0.1% FA aqueous solution, the mobile phase B is 0.1% FA acetonitrile solution, the flow rate is 0.3mL/min, and the gradient elution is carried out:

the mass spectrum conditions were as follows, spray voltage was 3.8kV, acquisition was performed in positive ion mode, capillary temperature was 320 ℃, S-lens RF level was 50; parent ion scan range: 300-2000m/z; the secondary mass spectrum selects the parent ion with the first ten intensities for dissociation in the data dependent mode (DDA), and the Normalized Collision Energy (NCE) is 27%.

The raw Data obtained were analyzed using commercial de novo sequencing software peak AB, data refine select Correct precursor-Mass only (correct for parent ions only); enzymatic mode selection Specified by each sample (specified by each sample); the immobilization modification was chosen from carbamidomethyl (+ 57.0215 Da) (formamide methylation); the maximum number of variable modifications allowed per peptide is 3; and performing de-novo sequencing of the peptide fragment by using the accurate mass number of the primary parent ion and various fragment ions rich in the secondary spectrogram, and then re-assembling the peptide fragment sequence into a monoclonal antibody sequence.

Test example 1

In this test example, three monoclonal antibodies were tested using examples 1-7 and comparative examples 1-16, respectively, each monoclonal antibody was subjected to three parallel enzymatic steps, each sample was tested in one needle, and the test results are shown in Table 1. Wherein the sequences of the three monoclonal antibodies to be detected are as follows:

antibody 1: heavy chain (SEQ ID No. 1):

EVQLEESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVALIRSKSNNYETNYADSVKDRFSISRDDSENMLYLQMNNLKSEDSAMYYCVRHGAVVEGAWFPYWGQGTLVTVSAASTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK。

antibody 1: light chain (SEQ ID No. 2):

DIVMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGNSPQLLVYAATNLADGVPSRFSGSVSGTQYSLKINSLQSEDFGIYYCQHFWGSPPTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

antibody 2: heavy chain (SEQ ID No. 3):

EVQLEQSGPELVKPGASVKLSCKASGYSFTAYYIHWVKQSHGNILDWIGYIYPYNGLSNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREKTTVEGTWFAYWGQGTLVTVSAASTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK。

antibody 2: light chain (SEQ ID No. 4):

DIVMTQTTSSLSASLGDRVTISCSASQGIHNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKPPYTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

antibody 3: heavy chain (SEQ ID No. 5):

QVQLQQSGAELVRPGTSVRVSCKASGYAFTDYFIEWVKQRPGQGLEWIGVINPGSGGSNSYEKFKGQATLTADKASSTAYMQITSLTSEDSAVYFCARSLRLHRYFDYWGQGTAITVSSASTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK。

antibody 3: light chain (SEQ ID No. 6):

DIVLTQSPASLAVSLGQRATISCRASQSVSSSGYSYIHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYSCQHSWELPWTFGGGTKLDIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

TABLE 1

As can be seen from a comparison of example 1 with examples 4 and 5, the concentration of the enzyme in the reaction solution of the enzymatic hydrolysis reaction affects the final fragment of the antibody, and the appropriate enzyme concentration can sufficiently degrade the antibody, facilitating subsequent mass spectrometric detection.

As can be seen from a comparison of examples 1 and examples 6-7, the time of the enzymolysis reaction cannot be too short, otherwise the enzymolysis is incomplete, the measured result is inaccurate and exceeds 18 hours, and the enzyme has influence on itself, so that impurities can be brought to interfere with subsequent detection.

As can be seen from the comparison of the example 1 and the comparative example 1, the separate enzymolysis of different enzymes can be used for enzymolysis of the monoclonal antibody into proper peptide fragments, which is more beneficial to the subsequent mass spectrum detection. The mixed enzymes are detected simultaneously, and mutual interference can occur.

As is clear from the comparison of example 1 and comparative examples 2 to 16, the detection effect of the combination of different enzymes is different, and the combination of partial enzymes has better detection effect on all three antibodies; however, for the combination of partial enzymes, which has no versatility, only a good detection effect on partial antibodies, the combination of enzymes in example 1 has better versatility and higher detection coverage than other combinations; the comparison example 15 adopts the combination of 6 enzymes to prepare a sample to be tested, and the result shows that the enzyme type is further increased, the increase of the detection effect is not facilitated, and the detection cost is increased to a certain extent.

Test example 2

The comparison of the experimental results was performed on the antibody sample (antibody 1) under the same equipment conditions using the sequencing methods in comparative example 16 and example 1.

After assembly by software sequencing, a map of the coverage of the sequence of the mab (fig. 1 and 2) was obtained, each amino acid in the sequence was reliably sequenced, and the de novo score was greater than 85%, wherein the experimental result of comparative example 16 is shown in fig. 1, the coverage of the heavy chain of mab was 98%, the coverage of the light chain was 100%, and the experimental result of example 1 is shown in fig. 2, the coverage of the heavy chain of mab was 100%, and the coverage of the light chain was 100%.

In conclusion, the invention can rapidly carry out quality evaluation on the corresponding antibody by using the established antibody sequencing quality evaluation platform, accurately detect the mutation site of the antibody, greatly simplify the quality evaluation and detection operation of the antibody reagent and shorten the quality evaluation time of the antibody. Meanwhile, the antibody sequencing quality evaluation platform is taken as a starting point and breakthrough, and the antibody related quality evaluation technology and the antigen related quality evaluation technology are developed in an emphasized manner, so that a foundation is laid for establishing a scientific, complete research and supervision system for evaluating the quality of antigens and antibody products, and the important role in the development of IVD raw material industry is played.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims (10)

1. A method of de novo sequencing a monoclonal antibody, the method comprising:

(1) Carrying out denaturation reduction and alkylation on a monoclonal antibody sample in sequence to obtain an alkylation reaction solution, dividing the alkylation reaction solution into 5 parts averagely, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, and mixing and reacting 5 parts of enzymolysis solution to obtain a polypeptide sample to be detected;

(2) Detecting the polypeptide sample to be detected by using high performance liquid chromatography and mass spectrometry to obtain mass spectrum data of the polypeptide sample; the peptide sequences were reassembled into monoclonal antibody sequences using de novo sequencing software for analysis.

2. The method of claim 1, wherein the polypeptide sample is prepared by a method comprising the steps of:

(A) Denaturation reduction: mixing and reacting the monoclonal antibody sample with urea and dithiothreitol to obtain a denatured and reduced monoclonal antibody;

(B) Alkylation: carrying out alkylation reaction on the denatured and reduced monoclonal antibody and iodoacetamide solution to obtain an alkylation reaction solution;

(C) Enzymolysis: dividing the alkylation reaction solution into 5 parts averagely, and respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis; and stopping the enzymolysis reaction by adopting formic acid to obtain the polypeptide sample to be detected.

3. The method of de novo sequencing of monoclonal antibodies according to claim 2, wherein in step (a), the concentration of the monoclonal antibody sample in the reaction solution of the mixing reaction is 0.1-0.5mg/mL;

preferably, in the step (A), the concentration of the urea in the reaction solution of the mixed reaction is 2-4M;

preferably, in the step (A), the concentration of dithiothreitol in the reaction solution of the mixed reaction is 4-10mM;

preferably, in step (A), the temperature of the mixing reaction is 35-65℃and the time is 20-60 minutes.

4. A method of de novo sequencing of monoclonal antibodies according to claim 2 or 3, wherein in step (B) the concentration of iodoacetamide in the reaction solution of the alkylation reaction is 10-20mM;

preferably, in step (B), the alkylation reaction conditions are light-shielding reaction for 20-60 minutes.

5. The method for de novo sequencing of monoclonal antibodies according to any of claims 2-4, wherein in step (C) the final concentration of trypsin in the reaction solution of the enzymatic hydrolysis reaction is 0.005-0.05mg/mL; the final concentration of chymotrypsin is 0.005-0.05mg/mL; the final concentration of pepsin is 0.005-0.05mg/mL; the final concentration of elastase is 0.005-0.05mg/mL; and the final concentration of Glu-C enzyme is 0.005-0.05mg/mL;

preferably, in the step (C), the enzymolysis reaction condition is 34-37 ℃ enzymolysis for 9-18 hours;

preferably, in step (C), the reaction is stopped by adjusting the pH to acidic with formic acid.

6. The method of de novo sequencing of monoclonal antibodies according to any of claims 1-5, wherein in step (2) the high performance liquid chromatography separation employs a chromatography column of C18;

preferably, in the step (2), the mobile phase adopted by the high performance liquid chromatography separation consists of a mobile phase A and a mobile phase B; the mobile phase A is 0.08-0.12% formic acid aqueous solution, and the mobile phase B is formic acid aqueous-acetonitrile solution;

preferably, the concentration of the formic acid aqueous solution in the formic acid aqueous solution-acetonitrile solution is 0.08-0.12%;

preferably, the volume ratio of the formic acid aqueous solution to the acetonitrile solution in the formic acid aqueous solution-acetonitrile solution is (18-22): 78-82;

preferably, the flow rate of the mobile phase is 280-320nL/min.

7. The method of de novo sequencing of monoclonal antibodies according to claim 6, wherein in step (2) the high performance liquid chromatography separation employs a gradient elution with the procedure of:

8. the method of de novo sequencing of monoclonal antibodies according to any of claims 1-7, wherein in step (2) the mass spectrometric detection is performed using an ultra-high resolution mass spectrometer;

preferably, in the step (2), the spraying voltage in the mass spectrum detection is 3.8-4.0kV, the collection is carried out in a positive ion mode, the capillary temperature is 300-320 ℃, and the S lens RF level is 20-40; parent ion scan range: 350-1500m/z; the secondary mass spectrum selects parent ions with the intensity of ten in the first time to dissociate under the data dependent mode, and the normalized collision energy is 20-40%.

9. The method of de novo sequencing of monoclonal antibodies according to any of claims 1-8, wherein the method comprises:

(1) Sequentially carrying out denaturation reduction, alkylation and enzymolysis on a monoclonal antibody sample to prepare a polypeptide sample;

(A) Denaturation reduction: mixing a monoclonal antibody sample with urea and dithiothreitol at 35-65 ℃ for reaction for 20-60 minutes, wherein the concentration of the monoclonal antibody sample in the reaction solution is 0.1-0.5mg/mL, the concentration of urea is 2-4M, and the concentration of dithiothreitol is 4-10mM, so as to obtain a denatured and reduced monoclonal antibody;

(B) Alkylation: carrying out light-shielding reaction on the denatured and reduced monoclonal antibody and iodoacetamide solution for 20-60 minutes, wherein the concentration of iodoacetamide in the reaction solution of the alkylation reaction is 10-20mM, so as to obtain an alkylation reaction solution;

(C) Enzymolysis: dividing the alkylation reaction solution into 5 parts, respectively adding trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme for enzymolysis, and carrying out enzymolysis for 9-18 hours at 34-37 ℃; the final concentration of trypsin in the reaction liquid of the enzymolysis reaction is 0.005-0.05mg/mL; the final concentration of chymotrypsin is 0.005-0.05mg/mL; the final concentration of pepsin is 0.005-0.05mg/mL; the final concentration of elastase is 0.005-0.05mg/mL; and the final concentration of Glu-C enzyme is 0.005-0.05mg/mL; regulating the pH value to be acidic by adopting formic acid to terminate the reaction, so as to obtain a polypeptide sample to be detected;

(2) Detecting the polypeptide sample to be detected by using high performance liquid chromatography and mass spectrometry to obtain mass spectrum data of the polypeptide sample; analyzing by using de novo sequencing software, and re-assembling the peptide fragment sequences into monoclonal antibody sequences;

the chromatographic column adopted by the high performance liquid chromatography separation is C18; the adopted mobile phase consists of a mobile phase A and a mobile phase B; the mobile phase A is 0.08-0.12% formic acid aqueous solution, and the mobile phase B is formic acid aqueous-acetonitrile solution; the concentration of the formic acid aqueous solution is 0.08-0.12%, and the volume ratio of the formic acid aqueous solution to the acetonitrile solution is (18-22) (78-82); the flow rate of the mobile phase is 280-320nL/min; gradient elution is adopted, and the gradient elution comprises the following steps:

performing mass spectrum detection by adopting an ultra-high resolution mass spectrometer; the spraying voltage in the mass spectrum detection is 3.8-4.0kV, the collection is carried out in a positive ion mode, the capillary temperature is 300-320 ℃, and the S lens RF level is 20-40; parent ion scan range: 350-1500m/z; the secondary mass spectrum selects parent ions with the intensity of ten in the first time to dissociate under the data dependent mode, and the normalized collision energy is 20-40%.

10. Use of the method of de novo sequencing of monoclonal antibodies according to any of claims 1-9 in monoclonal antibody sequencing.