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

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.

Description

Technical Field

[0001] This invention belongs to the field of amino acid sequencing technology, specifically relating to a method for de novo sequencing of monoclonal antibodies and its application. Background Technology

[0002] In recent years, with the rapid development of molecular biology and mass spectrometry, proteomics research has received increasing attention and has been widely applied in many fields. As a key technology in proteomics, de novo sequencing can directly identify the amino acid sequence of peptides based on tandem mass spectrometry, offering advantages that are irreplaceable by other protein identification methods.

[0003] As a core component of proteomics research, the fundamental task of protein identification is to determine the amino acid sequences corresponding to protein peptides. This is the basis and key to studying protein structure, function, and protein-protein interactions. Mass spectrometry, one of the most commonly used techniques in proteomics research, is primarily used for determining the mass of biological macromolecules, analyzing protein structure, and identifying protein peptide sequences. Its fast processing speed and high accuracy have made it a highly regarded technique in biological research. The basic principle of mass spectrometry is as follows: under vacuum conditions, a mass spectrometer fragments protein molecules into a series of charged fragment ions. These fragment ions are analyzed and recorded sequentially according to their mass-to-charge ratio (m / z), forming a mass spectrum.

[0004] Generally, a mass spectrum obtained using only one mass spectrometer is called a primary mass spectrum, where each ion peak corresponds to a peptide of the protein. However, due to the complexity of protein samples, primary mass spectra contain limited information and cannot adequately meet the requirements of protein identification and related research. To extract richer information from proteins and further improve the accuracy of results, tandem mass spectrometry (MS / MS) was proposed. Simply put, tandem mass spectrometry combines the operations of mass spectrometry in series. Specifically, it involves connecting two or more mass spectrometers in series, selecting a specific precursor ion (i.e., a specific peptide of the protein) from the primary mass spectrum, causing it to collide and fragment, generating a series of daughter ions. The mass analyzer in the tandem mass spectrometer then analyzes these daughter ions to form a secondary mass spectrum. The mass spectrum generated by tandem mass spectrometry is called a tandem mass spectrum. Furthermore, the relationship between the precursor ion and daughter ions is essentially the relationship between the protein peptide and the amino acids that make up the peptide.

[0005] With the emergence and continuous development of tandem mass spectrometry (SMMS) technology, protein identification methods based on SMMS have rapidly emerged due to their high accuracy, high sensitivity, and high reliability, becoming the main method for large-scale protein molecular identification and gaining widespread application. Among these methods, de novo protein sequencing does not rely on protein database information and can directly obtain the amino acid sequence of protein peptides from SMMS analysis. It can not only identify novel proteins not found in databases but also provide information on post-translational modifications, making it one of the most commonly used protein identification methods.

[0006] However, due to the complexity of tandem mass spectrometry data and inherent limitations of the identification methods themselves, the accuracy of de novo sequencing has been affected to some extent. Currently, many deep learning-based de novo sequencing methods have been proposed. Compared to traditional protein de novo sequencing methods, deep learning-based methods can significantly improve the accuracy of de novo sequencing, but they also have some shortcomings. Therefore, how to utilize deep learning to more fully learn the information contained in tandem mass spectrometry, design more accurate and reliable protein identification methods, and further improve the accuracy of protein de novo sequencing remains a key focus and one of the research challenges in proteomics. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for de novo sequencing of monoclonal antibodies and its applications. This invention utilizes the established method for de novo sequencing of monoclonal antibodies to evaluate the quality of both original and mutated antibodies. Sequencing of both antibodies yields the corresponding amino acid sequences. BLAST alignment of the sequenced amino acid sequences with the amino acid sequences translated from the nucleic acid sequences accurately detects the positions of mutated amino acids in the CDR3 variable region. The de novo sequencing method for monoclonal antibodies established by this invention allows for rapid quality evaluation of corresponding antibodies, accurately detecting antibody mutation sites, significantly simplifying the quality evaluation process for antibody reagents, and shortening the antibody quality evaluation time.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for de novo sequencing of monoclonal antibodies, the method comprising:

[0010] (1) The monoclonal antibody sample was denatured, reduced and alkylated in sequence to obtain an alkylation reaction solution. The alkylation reaction solution was divided into 5 equal parts, and trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme were added to each part for enzymatic hydrolysis. The 5 hydrolysates were mixed and reacted to obtain the polypeptide sample to be tested.

[0011] (2) The peptide sample to be tested was separated by high performance liquid chromatography and mass spectrometry to obtain mass spectrometry data of the peptide sample; the peptide sequence was reassembled into a monoclonal antibody sequence using de novo sequencing software.

[0012] The monoclonal antibody de novo sequencing method established in this invention can rapidly evaluate the quality of corresponding antibodies, accurately detect antibody mutation sites, greatly simplify the operation of antibody reagent quality evaluation, and shorten the antibody quality evaluation time. Furthermore, starting with and breaking through the monoclonal antibody de novo sequencing method, this invention focuses on developing antibody-related quality evaluation technologies and antigen-related quality evaluation technologies. This lays the foundation for establishing a scientific and comprehensive research and regulatory system for antigen and antibody product quality evaluation, and will play a crucial role in the development of the IVD raw material industry.

[0013] Compared with existing technologies, the monoclonal antibody de novo sequencing method established in this invention reduces mass spectrometry detection time from 120 minutes to 40 minutes and increases the detection rate of mutant antibodies from 90% to 99%, thereby simplifying the operation of antibody reagent quality evaluation and shortening the antibody quality evaluation time. Furthermore, starting with and breaking through the monoclonal antibody de novo sequencing method, this invention focuses on developing antibody-related quality evaluation technologies and antigen-related quality evaluation technologies. This lays the foundation for establishing a scientific and comprehensive research and regulatory system for antigen and antibody product quality evaluation and will play a crucial role in the development of the IVD raw material industry.

[0014] In this invention, the enzymatic digestion steps were optimized. Trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme were selected to digest the antibody separately. The five enzymes specifically chosen in this invention can effectively break down the antibody into small fragments with high coverage, which is beneficial for subsequent detection. Furthermore, adding each enzyme separately avoids interference between enzymes and prevents mutual decomposition, thus preventing reduced digestion efficiency. This invention also investigated the digestion efficiency of different enzyme combinations for antibodies. The results show that the combination of the above five enzymes has excellent digestion efficiency, meeting cost control requirements while also satisfying the requirements for subsequent mass spectrometry detection.

[0015] Preferably, the polypeptide sample is prepared using a method comprising the following steps:

[0016] (A) Denaturation and Reduction: The monoclonal antibody sample is mixed with urea and dithiothreitol and reacted to obtain the denatured and reduced monoclonal antibody;

[0017] (B) Alkylation: The denatured and reduced monoclonal antibody is subjected to an alkylation reaction with iodoacetamide solution to obtain an alkylation reaction solution;

[0018] (C) Enzymatic hydrolysis: The alkylation reaction solution was divided into 5 equal portions, and trypsin, chymotrypsin, pepsin, elastase and Glu-C enzyme were added to each portion for enzymatic hydrolysis; the enzymatic hydrolysis reaction was terminated with formic acid to obtain the polypeptide sample to be tested.

[0019] Preferably, in step (A), the concentration of the monoclonal antibody sample in the reaction solution of the mixed reaction is 0.1-0.5 mg / mL, for example, it can be 0.1 mg / mL, 0.3 mg / mL or 0.5 mg / mL, etc.

[0020] Preferably, in step (A), the concentration of urea in the reaction solution of the mixed reaction is 2-4M, for example, it can be 2M, 3M or 4M.

[0021] In this invention, a urea concentration of 2-4M has a better denaturation and reduction effect; if the urea concentration is too low, the denaturation and reduction will be incomplete, and if the concentration is too high, it will affect the subsequent enzymatic hydrolysis reaction.

[0022] Preferably, in step (A), the concentration of dithiothreitol in the reaction solution of the mixed reaction is 4-10 mM, for example, it can be 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM.

[0023] In this invention, the role of dithiothreitol is to open the disulfide bonds in the antibody. The effect is best when the concentration of dithiothreitol is between 4-10 mM. If the concentration of dithiothreitol is too low, the disulfide bonds will not open completely, affecting the results of subsequent enzymatic hydrolysis.

[0024] Preferably, in step (A), the temperature of the mixing reaction is 35-65°C, for example, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 65°C, and the time is 20-60 minutes, for example, 20, 30, 40, 50 or 60 minutes.

[0025] In this invention, during the mixed reaction, the reaction time can be appropriately extended when the temperature is low, while at a high temperature, the denaturation and reduction can be completed in a shorter time.

[0026] Preferably, in step (B), the concentration of iodoacetamide in the reaction solution of the alkylation reaction is 10-20 mM, for example, it can be 10 mM, 15 mM or 20 mM.

[0027] In this invention, the iodoacetamide, as an alkylating agent, can ensure complete denaturation of the protein sample and maintain its reduced state, preventing the thiol group from being oxidized into a disulfide bond and maintaining a reducing environment.

[0028] Preferably, in step (B), the alkylation reaction is carried out under light-protected conditions for a reaction time of 20-60 minutes, for example, 20, 30, 40, 50 or 60 minutes.

[0029] In this invention, the alkylation reaction is carried out at room temperature, and the reaction time is controlled to be 20-60 minutes in the dark. Too short a time will result in incomplete alkylation.

[0030] Preferably, in step (C), the final concentration of trypsin in the reaction solution of the enzymatic hydrolysis reaction is 0.005-0.05 mg / mL (e.g., 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, or 0.05 mg / mL, etc.); the final concentration of chymotrypsin is 0.005-0.05 mg / mL (e.g., 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, or 0.05 mg / mL, etc.); and the final concentration of pepsin is 0.005-0.05 mg / mL (e.g., 0.005 mg / mL, etc.). The final concentrations of elastase are 0.005-0.05 mg / mL (e.g., 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, or 0.05 mg / mL, etc.); and the final concentrations of Glu-C enzyme are 0.005-0.05 mg / mL (e.g., 0.005 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, or 0.05 mg / mL, etc.).

[0031] In this invention, the enzyme concentration cannot be too low, as this will affect the enzyme digestion efficiency, reduce the effective peptide fragments digested, and decrease the coverage of the detection results. The enzyme concentration also cannot be too high, as this will cause the enzyme-digested peptide fragments to be too small, which is not conducive to liquid phase separation and will reduce the coverage of the detection results. At the same time, the enzyme itself will also undergo enzymatic hydrolysis, and if the concentration is too high, the fragments digested by the enzyme itself will affect the accuracy of the detection results.

[0032] Preferably, in step (C), the enzymatic hydrolysis reaction is carried out at 34-37°C (e.g., 34°C, 35°C, 36°C, or 37°C) for 9-18 hours (e.g., 9, 12, 15, or 18 hours).

[0033] In this invention, the enzymatic hydrolysis reaction time cannot be too short, otherwise the enzymatic hydrolysis will be incomplete and the measured results will be inaccurate. If it exceeds 18 hours, it will also affect the enzyme itself, introduce impurities, and interfere with subsequent detection.

[0034] Preferably, in step (C), formic acid is used to adjust the pH to acidity to terminate the reaction.

[0035] In this invention, there is no specific range for the pH to be adjusted to acidic; any acidic pH is acceptable.

[0036] Preferably, in step (2), the chromatographic column used for high performance liquid chromatography separation is C18.

[0037] Preferably, in step (2), the mobile phase used in the high performance liquid chromatography separation consists of mobile phase A and mobile phase B; mobile phase A is a 0.08-0.12% (for example, it can be 0.08%, 0.1% or 0.12% etc.) aqueous formic acid solution, and mobile phase B is an aqueous formic acid-acetonitrile solution.

[0038] Preferably, the concentration of the formic acid aqueous solution in the formic acid aqueous solution-acetonitrile solution is 0.08-0.12%, for example, it can be 0.08%, 0.1% or 0.12%, etc.

[0039] Preferably, the volume ratio of formic acid aqueous solution to acetonitrile solution in the formic acid aqueous solution-acetonitrile solution is (18-22):(78-82), for example, it can be 18:82, 20:80 or 22:78, etc.

[0040] Preferably, the flow rate of the mobile phase is 280-320 nL / min, for example, it can be 280 nL / min, 300 nL / min or 320 nL / min.

[0041] Preferably, in step (2), the high-performance liquid chromatography separation employs gradient elution, and the gradient elution procedure is as follows:

[0042]

[0043] In this invention, the elution method has the advantages of short elution time, which greatly shortens the detection time, and high separation efficiency, which can effectively separate the detection peptides and increase the coverage and accuracy of the detection results.

[0044] Preferably, in step (2), the mass spectrometry detection is performed using an ultra-high resolution mass spectrometer.

[0045] Preferably, in step (2), the spray voltage in the mass spectrometry detection is 3.8-4.0 kV, the acquisition is performed in positive ion mode, the capillary temperature is 300-320℃, the S lens RF level is 20-40, the precursor ion scanning range is 350-1500 m / z, and the secondary mass spectrometry selects the top ten precursor ions in intensity in data-dependent mode for dissociation, with a normalized collision energy of 20-40%.

[0046] As a preferred embodiment of the present invention, the method includes:

[0047] (1) Monoclonal antibody samples were sequentially denatured, reduced, alkylated and enzymatically digested to prepare polypeptide samples;

[0048] (A) Denaturation and Reduction: The monoclonal antibody sample is mixed with urea and dithiothreitol and reacted at 35-65℃ for 20-60 minutes. The concentration of the monoclonal antibody sample in the reaction solution is 0.1-0.5 mg / mL, the concentration of urea is 2-4 M, and the concentration of dithiothreitol is 4-10 mM, to obtain the denatured and reduced monoclonal antibody.

[0049] (B) Alkylation: The denatured and reduced monoclonal antibody is reacted with iodoacetamide solution in the dark for 20-60 minutes. The concentration of iodoacetamide in the alkylation reaction solution is 10-20 mM to obtain the alkylation reaction solution.

[0050] (C) Enzymatic hydrolysis: The alkylation reaction solution was divided into 5 equal portions, and trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme were added to each portion for enzymatic hydrolysis at 34-37℃ for 9-18 hours. The final concentrations of trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme in the reaction solution were 0.005-0.05 mg / mL, 0.005-0.05 mg / mL, 0.005-0.05 mg / mL, and 0.005-0.05 mg / mL, respectively.

[0051] (2) The peptide sample to be tested was separated by high performance liquid chromatography and mass spectrometry to obtain mass spectrometry data of the peptide sample; the peptide sequence was reassembled into a monoclonal antibody sequence using de novo sequencing software.

[0052] The high-performance liquid chromatography (HPLC) separation uses a C18 column; the mobile phase consists of mobile phase A and mobile phase B; mobile phase A is a 0.08-0.12% formic acid aqueous solution, and mobile phase B is a formic acid aqueous solution-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 280-320 nL / min; gradient elution is used, and the gradient elution program is as follows:

[0053]

[0054] Mass spectrometry was performed using an ultra-high resolution mass spectrometer. The spray voltage was 3.8-4.0 kV, and data was collected in positive ion mode. The capillary temperature was 300-320℃, and the S-lens RF level was 20-40. The precursor ion scanning range was 350-1500 m / z. For secondary mass spectrometry, the top ten precursor ions by intensity in data-dependent mode were dissociated, with a normalized collision energy of 20-40%.

[0055] Secondly, the present invention provides the application of the method for de novo sequencing of monoclonal antibodies described in the first aspect in monoclonal antibody sequencing.

[0056] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] (1) This invention utilizes five enzymes for enzyme digestion and sequencing, which can 100% cover the antibody sequence and accurately detect the antibody's amino acid sequence. The detection method is simple, widely operable, and cost-controllable. The mass spectrometry detection time is reduced from 120 minutes to 40 minutes, and the detection rate of mutant antibodies is increased from 90% to 99%, thereby simplifying the operation of antibody reagent quality evaluation and shortening the antibody quality evaluation time.

[0059] (2) This invention takes the de novo sequencing method of monoclonal antibodies as its starting point and breakthrough, and focuses on developing antibody-related quality evaluation technology and antigen-related quality evaluation technology. It lays the foundation for establishing a scientific and complete research and supervision system for the quality evaluation of antigen and antibody products, and will also play a pivotal role in the development of the in vitro diagnostic reagent raw materials industry.

[0060] (3) The present invention utilizes antibody sequencing technology to quickly perform quality evaluation and testing on corresponding antibody-based in vitro diagnostic reagents, which can identify whether mutations have occurred and whether they are active, greatly shortening the registration time of in vitro diagnostic reagents and improving the accuracy of their performance. Attached Figure Description

[0061] Figure 1 The results are for Comparative Example 1.

[0062] Figure 2 The results are from Example 1. Detailed Implementation

[0063] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0064] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0065] Example 1

[0066] This embodiment provides a method for de novo sequencing of monoclonal antibodies, the method comprising:

[0067] (1) Monoclonal antibody samples were sequentially denatured, reduced, alkylated and enzymatically digested to prepare polypeptide samples;

[0068] (A) Denaturation and reduction: The monoclonal antibody sample was mixed with urea and dithiothreitol and reacted at 35°C for 60 minutes. The concentration of the monoclonal antibody sample in the reaction solution was 0.3 mg / mL, the concentration of urea was 3 M, and the concentration of dithiothreitol was 6 mM, to obtain the denatured and reduced monoclonal antibody.

[0069] (B) Alkylation: The denatured and reduced monoclonal antibody was reacted with iodoacetamide solution in the dark for 25 minutes. The concentration of iodoacetamide in the alkylation reaction solution was 10 mM, and the alkylation reaction solution was obtained.

[0070] (C) Enzymatic hydrolysis: The alkylation reaction solution was divided into 5 equal portions, and trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme were added to each portion for enzymatic hydrolysis at 37°C for 9 hours. The final concentrations of trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme in the reaction solution were all 0.02 mg / mL. The reaction was terminated by adjusting the pH to acidic using formic acid to obtain the polypeptide sample to be tested.

[0071] (2) The peptide sample to be tested was separated by high performance liquid chromatography and mass spectrometry to obtain mass spectrometry data of the peptide sample; the peptide sequence was reassembled into a monoclonal antibody sequence using de novo sequencing software.

[0072] The high-performance liquid chromatography (HPLC) separation uses a C18 column; the mobile phase consists of mobile phase A and mobile phase B; mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a formic acid aqueous solution-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 300 nL / min; gradient elution is used, and the gradient elution program is as follows:

[0073]

[0074] Mass spectrometry was performed using an ultra-high resolution mass spectrometer. The spray voltage was 3.8 kV, and the data was collected in positive ion mode. The capillary temperature was 320 °C, and the S lens RF level was 40. The precursor ion scanning range was 350-1500 m / z. For secondary mass spectrometry, the top ten precursor ions in intensity were selected for dissociation in data-dependent mode, and the normalized collision energy was 20%.

[0075] Example 2

[0076] This embodiment provides a method for de novo sequencing of monoclonal antibodies, the method comprising:

[0077] (1) Monoclonal antibody samples were sequentially denatured, reduced, alkylated and enzymatically digested to prepare polypeptide samples;

[0078] (A) Denaturation and reduction: The monoclonal antibody sample was mixed with urea and dithiothreitol and reacted at 65°C for 20 minutes. The concentration of the monoclonal antibody sample in the reaction solution was 0.1 mg / mL, the concentration of urea was 2 M, and the concentration of dithiothreitol was 4 mM, to obtain the denatured and reduced monoclonal antibody.

[0079] (B) Alkylation: The denatured and reduced monoclonal antibody was reacted with iodoacetamide solution in the dark for 35 minutes. The concentration of iodoacetamide in the alkylation reaction solution was 15 mM, and the alkylation reaction solution was obtained.

[0080] (C) Enzymatic hydrolysis: The alkylation reaction solution was divided into 5 equal portions, and trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme were added to each portion for enzymatic hydrolysis at 37°C for 9 hours. The final concentrations of trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme in the reaction solution were all 0.05 mg / mL. The reaction was terminated by adjusting the pH to acidic using formic acid to obtain the polypeptide sample to be tested.

[0081] (2) The peptide sample to be tested was separated by high performance liquid chromatography and mass spectrometry to obtain mass spectrometry data of the peptide sample; the peptide sequence was reassembled into a monoclonal antibody sequence using de novo sequencing software.

[0082] The high-performance liquid chromatography (HPLC) separation uses a C18 column; the mobile phase consists of mobile phase A and mobile phase B; mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a formic acid aqueous solution-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 300 nL / min; gradient elution is used, and the gradient elution program is as follows:

[0083]

[0084]

[0085] Mass spectrometry was performed using an ultra-high resolution mass spectrometer. The spray voltage was 3.8 kV, and the data was collected in positive ion mode. The capillary temperature was 320 °C, and the S lens RF level was 40. The precursor ion scanning range was 350-1500 m / z. For secondary mass spectrometry, the top ten precursor ions in intensity were selected for dissociation in data-dependent mode, and the normalized collision energy was 20%.

[0086] Example 3

[0087] This embodiment provides a method for de novo sequencing of monoclonal antibodies, the method comprising:

[0088] (A) Denaturation and reduction: The monoclonal antibody sample was mixed with urea and dithiothreitol and reacted at 45°C for 40 minutes. The concentration of the monoclonal antibody sample in the reaction solution was 0.5 mg / mL, the concentration of urea was 4 M, and the concentration of dithiothreitol was 10 mM, to obtain the denatured and reduced monoclonal antibody.

[0089] (B) Alkylation: The denatured and reduced monoclonal antibody was reacted with iodoacetamide solution in the dark for 35 minutes. The concentration of iodoacetamide in the reaction solution of the alkylation reaction was 10 mM, and the alkylation reaction solution was obtained.

[0090] (C) Enzymatic hydrolysis: The alkylation reaction solution was divided into 5 equal portions, and trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme were added to each portion for enzymatic hydrolysis at 37°C for 9 hours. The final concentrations of trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme in the reaction solution were all 0.005 mg / mL. The reaction was terminated by adjusting the pH to acidic using formic acid to obtain the polypeptide sample to be tested.

[0091] The high-performance liquid chromatography (HPLC) separation uses a C18 column; the mobile phase consists of mobile phase A and mobile phase B; mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is a formic acid aqueous solution-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 300 nL / min; gradient elution is used, and the gradient elution program is as follows:

[0092]

[0093] Mass spectrometry was performed using an ultra-high resolution mass spectrometer. The spray voltage was 3.8 kV, and the data was collected in positive ion mode. The capillary temperature was 320 °C, and the S lens RF level was 40. The precursor ion scanning range was 350-1500 m / z. For secondary mass spectrometry, the top ten precursor ions in intensity were selected for dissociation in data-dependent mode, and the normalized collision energy was 20%.

[0094] Example 4

[0095] This embodiment provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that, in step (C), the final concentrations of trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme in the enzymatic digestion reaction solution are 0.2 mg / mL;

[0096] Example 5

[0097] This embodiment provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that, in step (C), the final concentrations of trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme in the enzymatic digestion reaction solution are 0.001 mg / mL;

[0098] Example 6

[0099] This embodiment provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C), the enzymatic digestion time is 6 hours; the remaining steps are the same as in Example 1.

[0100] Example 7

[0101] This embodiment provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C), the enzymatic digestion time is 20 hours; the remaining steps are the same as in Example 1.

[0102] Comparative Example 1

[0103] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C) enzymatic digestion, trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme are simultaneously digested in one system; the remaining steps are the same as in Example 1.

[0104] Comparative Example 2

[0105] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that the enzyme used in step (C) enzymatic digestion is trypsin; the remaining steps are the same as in Example 1.

[0106] Comparative Example 3

[0107] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that the enzyme used in step (C) enzymatic digestion is chymotrypsin; the remaining steps are the same as in Example 1.

[0108] Comparative Example 4

[0109] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that the enzyme used in step (C) enzymatic digestion is pepsin; the remaining steps are the same as in Example 1.

[0110] Comparative Example 5

[0111] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that the enzyme used in step (C) enzymatic digestion is elastase; the remaining steps are the same as in Example 1.

[0112] Comparative Example 6

[0113] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that the enzyme used in step (C) enzymatic digestion is Glu-C enzyme; the remaining steps are the same as in Example 1.

[0114] Comparative Example 7

[0115] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that the enzyme used in step (C) enzymatic digestion is Lys-C enzyme; the remaining steps are the same as in Example 1.

[0116] Comparative Example 8

[0117] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that the enzyme used in step (C) enzymatic digestion is Asp-N enzyme; the remaining steps are the same as in Example 1.

[0118] Comparative Example 9

[0119] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C) enzymatic digestion, the enzymes used are trypsin, chymotrypsin, and Glu-C enzyme, which are used for enzymatic digestion respectively; the remaining steps are the same as in Example 1.

[0120] Comparative Example 10

[0121] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C) enzymatic digestion, the enzymes used are trypsin, chymotrypsin, and pepsin, and the digestion is performed using trypsin, chymotrypsin, and pepsin, respectively; the remaining steps are the same as in Example 1.

[0122] Comparative Example 11

[0123] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C) enzymatic digestion, the enzymes used are trypsin, chymotrypsin, and Lys-C enzyme, and the digestion is performed using trypsin, chymotrypsin, and Lys-C enzyme, respectively; the remaining steps are the same as in Example 1.

[0124] Comparative Example 12

[0125] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C) enzymatic digestion, the enzymes used are trypsin, chymotrypsin, pepsin, and Glu-C enzyme, respectively. The remaining steps are the same as in Example 1.

[0126] Comparative Example 13

[0127] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C) enzymatic digestion, the enzymes used are trypsin, chymotrypsin, pepsin, and elastase, respectively. The remaining steps are the same as in Example 1.

[0128] Comparative Example 14

[0129] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C) enzymatic digestion, the enzymes used are trypsin, chymotrypsin, pepsin, elastase, and Lys-C enzyme. The enzymatic digestion is performed using trypsin, chymotrypsin, pepsin, elastase, and Lys-C enzyme, respectively. The remaining steps are the same as in Example 1.

[0130] Comparative Example 15

[0131] This comparative example provides a method for de novo sequencing of monoclonal antibodies. The only difference between this method and Example 1 is that in step (C) enzymatic digestion, the enzymes used are trypsin, chymotrypsin, pepsin, elastase, Glu-C enzyme, and Lys-C enzyme. The enzymatic digestion is performed using trypsin, chymotrypsin, pepsin, elastase, Glu-C enzyme, and Lys-C enzyme, respectively. The remaining steps are the same as in Example 1.

[0132] Comparative Example 16

[0133] This comparative example provides a method for sequencing monoclonal antibodies. The difference between this method and Example 1 is that the specific steps of the sequencing method are as follows:

[0134] Step S1: Take 100 μg of monoclonal antibody sample, add 6M guanidine hydrochloride buffer and 1M dithiothreitol to a final concentration of 10 mM, and react at 56°C for 30 minutes for denaturation and reduction. The amount of dithiothreitol (DTT) added is to a final concentration of 10 mM.

[0135] Step S2: After the denatured and reduced monoclonal antibody sample is cooled to room temperature, 1M iodoacetamide is added until the final concentration of iodoacetamide is 20mM. The sample is then reacted at room temperature in the dark for 30 minutes to carry out alkylation.

[0136] Step S3: The alkylated monoclonal antibody sample is desalted using a Zeba desalting column.

[0137] Step S4: After desalting, add trypsin to the monoclonal antibody sample and hydrolyze it at room temperature for 10-18 hours. The mass ratio of monoclonal antibody to trypsin is 50:1. Adjust the pH of the solution to acidic to terminate the hydrolysis and obtain the polypeptide sample.

[0138] Step S5: Take the peptide sample obtained in step S4, separate the peptides using reversed-phase chromatography, and then perform mass spectrometry detection.

[0139] The chromatographic conditions were as follows: C18 column, column temperature 55℃, sample chamber temperature 5℃, sample loading volume 2μL, mobile phase A was 0.1% FA aqueous solution, mobile phase B was 0.1% FA acetonitrile solution, flow rate was 0.3mL / min, and gradient elution was used.

[0140]

[0141] The mass spectrometry conditions were as follows: spray voltage of 3.8 kV, acquisition in positive ion mode, capillary temperature of 320 °C, S-lens RF level of 50; precursor ion scan range: 300-2000 m / z; secondary mass spectrometry selected the top ten precursor ions in intensity under data-dependent mode (DDA) for dissociation, with normalized collision energy (NCE) of 27%.

[0142] The raw data were analyzed using the commercial de novo sequencing software Peaks AB. Data refinement was performed using the Correct precursor-Mass only option; the enzymatic digestion method was specified by each sample; the fixed modification was Carbamidomethylation (+57.0215 Da); the maximum number of variable modifications allowed for each peptide was 3; the peptides were de novo sequenced using the precise mass number of the primary precursor ion and the abundant fragment ions in the secondary spectrum, and then the peptide sequences were reassembled into monoclonal antibody sequences.

[0143] Test Example 1

[0144] This test case used Examples 1-7 and Comparative Examples 1-16 to detect three monoclonal antibodies. Each monoclonal antibody underwent three parallel enzymatic digestion steps, and each sample was injected once for testing. The test results are shown in Table 1. The sequences of the three monoclonal antibodies to be tested are shown below:

[0145] Antibody 1: Heavy chain (SEQ ID No. 1):

[0146] EVQLEESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVALIRSKSNNYETNYADSVKDRFSISRDDSENMLYLQMNNLKSEDSAMYYCVRHGAVVEGAWFPYWGQGTLVTVSAASTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK。

[0147] Antibody 1: Light chain (SEQ ID No.2):

[0148] DIVMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGNSPQLLVYAATNLADGVPSRFSGSVSGTQYSLKINSLQSEDFGIYYCQHFWGSPPTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0149] Antibody 2: Heavy chain (SEQ ID No.3):

[0150] EVQLEQSGPELVKPGASVKLSCKASGYSFTAYYIHWVKQSHGNILDWIGYIYPYNGLSNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREKTTVEGTWFAYWGQGTLVTVSAASTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK。

[0151] Antibody 2: Light chain (SEQ ID No.4):

[0152] DIVMTQTTSSLSASLGDRVTISCSASQGIHNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKPPYTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0153] Antibody 3: Heavy chain (SEQ ID No.5):

[0154] QVQLQQSGAELVRPGTSVRVSCKASGYAFTDYFIEWVKQRPGQGLEWIGVINPGSGGSNSYEKFKGQATLTADKASSTAYMQITSLTSEDSAVYFCARSLRLHRYFDYWGQGT AITVSSASTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPC PPLKECPPCAAPDLLGGPSVFIFPPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEK TISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK.

[0155] Antibody 3: Light chain (SEQ ID No. 6):

[0156] DIVLTQSPASLAVSLGQRATISCRASQSVSSSGYSYIHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYSCQHSWELPWTFGGGTKLD IKRTDAAPVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0157] Table 1

[0158]

[0159] A comparison of Examples 1 with Examples 4 and 5 shows that the concentration of enzyme in the reaction solution of the enzymatic hydrolysis reaction affects the final fragments of the antibody. A suitable enzyme concentration can fully degrade the antibody, which is beneficial for subsequent mass spectrometry detection.

[0160] The comparison between Example 1 and Examples 6-7 shows that the enzymatic hydrolysis time should not be too short, otherwise the enzymatic hydrolysis will be incomplete and the measured results will be inaccurate. If it exceeds 18 hours, it will also affect the enzyme itself, introduce impurities, and interfere with subsequent detection.

[0161] The comparison between Example 1 and Comparative Example 1 shows that using different enzymes for separate digestion allows monoclonal antibodies to be digested into suitable peptides, which is more beneficial for subsequent mass spectrometry detection. Simultaneous detection with mixed enzymes will result in mutual interference.

[0162] A comparison of Example 1 and Comparative Examples 2-16 shows that different enzyme combinations have different detection effects. For some enzyme combinations, the detection effect is good for all three antibodies; however, for other enzyme combinations, the detection effect is only good for some antibodies. The enzyme combinations are not universal. The enzyme combination in Example 1 is more universal and has a higher detection coverage than other combinations. Comparative Example 15 used a combination of 6 enzymes to prepare the test sample. The results show that further increasing the number of enzymes does not help to improve the detection effect, but instead increases the detection cost to a certain extent.

[0163] Test Example 2

[0164] The sequencing methods used in Comparative Example 16 and Example 1 were used to compare the experimental results of antibody sample (antibody 1) under the same equipment conditions.

[0165] After software sequencing and assembly, a monoclonal antibody sequence coverage map was obtained. Figure 1 and Figure 2 In this sequence, every amino acid was reliably sequenced, with a de novo score greater than 85%. The experimental results for Comparative Example 16 are as follows: Figure 1 The results showed that the monoclonal antibody had a heavy chain coverage of 98% and a light chain coverage of 100%, while the experimental results of Example 1 were as follows: Figure 2 The results showed that the monoclonal antibody had 100% heavy chain coverage and 100% light chain coverage.

[0166] In summary, this invention utilizes an established antibody sequencing quality assessment platform to rapidly evaluate the quality of corresponding antibodies, accurately detect antibody mutation sites, significantly simplify the operation of antibody reagent quality assessment, and shorten the antibody quality assessment time. Furthermore, starting with and breaking through the antibody sequencing quality assessment platform, the focus on developing antibody-related quality assessment technologies and antigen-related quality assessment technologies lays the foundation for establishing a scientific and comprehensive research and regulatory system for antigen and antibody product quality assessment, and will play a crucial role in the development of the IVD raw material industry.

[0167] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for de novo sequencing of monoclonal antibodies, characterized in that, The method includes: (1) Monoclonal antibody samples were sequentially denatured, reduced, alkylated, and digested to prepare polypeptide samples; (A) Denaturation and reduction: The monoclonal antibody sample is mixed with urea and dithiothreitol and reacted to obtain the denatured and reduced monoclonal antibody; (B) Alkylation: The denatured and reduced monoclonal antibody is subjected to an alkylation reaction with iodoacetamide solution to obtain an alkylation reaction solution; (C) Enzymatic hydrolysis: The alkylation reaction solution was divided into 5 equal portions, and trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme were added to each portion for enzymatic hydrolysis at 34-37℃ for 9-12 hours. The final concentrations of trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme in the reaction solution were 0.005-0.05 mg / mL, 0.005-0.05 mg / mL, 0.005-0.05 mg / mL, and 0.005-0.05 mg / mL, respectively. The reaction was terminated by adjusting the pH to acidic using formic acid, yielding the polypeptide sample to be tested. (2) The peptide sample to be tested was separated by high performance liquid chromatography and mass spectrometry to obtain mass spectrometry data of the peptide sample; the peptide sequence was reassembled into a monoclonal antibody sequence using de novo sequencing software.

2. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (A), the concentration of the monoclonal antibody sample in the reaction solution of the mixed reaction is 0.1-0.5 mg / mL.

3. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (A), the concentration of urea in the reaction solution of the mixed reaction is 2-4 M.

4. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (A), the concentration of dithiothreitol in the reaction solution of the mixed reaction is 4-10 mM.

5. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (A), the temperature of the mixing reaction is 35-65°C and the time is 20-60 minutes.

6. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (B), the concentration of iodoacetamide in the reaction solution of the alkylation reaction is 10-20 mM.

7. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (B), the alkylation reaction is carried out under light-protected conditions for 20-60 minutes.

8. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (2), the chromatographic column used for high performance liquid chromatography separation is C18.

9. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (2), the mobile phase used in the high performance liquid chromatography separation consists of mobile phase A and mobile phase B; mobile phase A is a 0.08-0.12% formic acid aqueous solution, and mobile phase B is a formic acid aqueous solution-acetonitrile solution.

10. The method for de novo sequencing of monoclonal antibodies according to claim 9, characterized in that, The concentration of the formic acid aqueous solution in the formic acid-acetonitrile solution is 0.08-0.12%.

11. The method for de novo sequencing of monoclonal antibodies according to claim 9, characterized in that, The volume ratio of formic acid aqueous solution to acetonitrile solution in the formic acid aqueous solution-acetonitrile solution is (18-22):(78-82).

12. The method for de novo sequencing of monoclonal antibodies according to claim 9, characterized in that, The flow rate of the mobile phase is 280-320 nL / min.

13. The method for de novo sequencing of monoclonal antibodies according to claim 9, characterized in that, In step (2), the high-performance liquid chromatography separation employs gradient elution, and the gradient elution procedure is as follows: Time / minutes Mobile phase A Mobile phase B 098%2% 0-292-98%2-8% 2-3265-92%8-35% 32-375-65%35-95% 37-405%95%。 14. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (2), the mass spectrometry detection is performed using an ultra-high resolution mass spectrometer.

15. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, In step (2), the spray voltage in the mass spectrometry detection is 3.8-4.0 kV, the acquisition is performed in positive ion mode, the capillary temperature is 300-320℃, the Slens RF level is 20-40; the precursor ion scanning range is 350-1500 m / z; the secondary mass spectrometry selects the top ten precursor ions in intensity in data-dependent mode for dissociation, and the normalized collision energy is 20-40%.

16. The method for de novo sequencing of monoclonal antibodies according to claim 1, characterized in that, The method includes: (1) Monoclonal antibody samples were sequentially denatured, reduced, alkylated, and digested to prepare polypeptide samples; (A) Denaturation and reduction: The monoclonal antibody sample is mixed with urea and dithiothreitol and reacted at 35-65℃ for 20-60 minutes. The concentration of the monoclonal antibody sample in the reaction solution is 0.1-0.5 mg / mL, the concentration of urea is 2-4 M, and the concentration of dithiothreitol is 4-10 mM, to obtain the denatured and reduced monoclonal antibody. (B) Alkylation: The denatured and reduced monoclonal antibody is reacted with iodoacetamide solution in the dark for 20-60 minutes. The concentration of iodoacetamide in the reaction solution of the alkylation reaction is 10-20 mM to obtain the alkylation reaction solution. (C) Enzymatic hydrolysis: The alkylation reaction solution was divided into 5 equal portions, and trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme were added to each portion for enzymatic hydrolysis at 34-37℃ for 9-18 hours. The final concentrations of trypsin, chymotrypsin, pepsin, elastase, and Glu-C enzyme in the reaction solution were 0.005-0.05 mg / mL, 0.005-0.05 mg / mL, 0.005-0.05 mg / mL, and 0.005-0.05 mg / mL, respectively. The reaction was terminated by adjusting the pH to acidic using formic acid to obtain the polypeptide sample to be tested. (2) The peptide sample to be tested was separated by high performance liquid chromatography and mass spectrometry to obtain mass spectrometry data of the peptide sample; the peptide sequence was reassembled into a monoclonal antibody sequence using de novo sequencing software. The high-performance liquid chromatography (HPLC) separation uses a C18 column; the mobile phase consists of mobile phase A and mobile phase B; mobile phase A is a 0.08-0.12% formic acid aqueous solution, and mobile phase B is a formic acid aqueous solution-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 280-320 nL / min; gradient elution is used, and the gradient elution program is as follows: Time / minutes Mobile phase A Mobile phase B 098%2% 0-292-98%2-8% 2-3265-92%8-35% 32-375-65%35-95% 37-405%95%； Mass spectrometry detection was performed using an ultra-high resolution mass spectrometer; the spray voltage was 3.8-4.0 kV, acquisition was carried out in positive ion mode, the capillary temperature was 300-320℃, and the S lens RF level was 20-40; the precursor ion scanning range was 350-1500 m / z; for secondary mass spectrometry, the top ten precursor ions in intensity were selected for dissociation in data-dependent mode, and the normalized collision energy was 20-40%.

17. The method of de novo sequencing of monoclonal antibodies according to any one of claims 1-16 is used in monoclonal antibody sequencing.

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