Universal Mass Spectrometry-Based Quantitative Analysis of Antibodies

Abstract

The present invention provides a general quantitative analysis method for antibodies based on mass spectrometry. Wherein, the above-mentioned quantitative analysis method includes: a) performing mass spectrometry detection on a general peptide segment to obtain a standard curve of the concentration of the general peptide segment and the mass spectrometry response intensity; b) performing a first enzymatic hydrolysis on the antibody to be tested to obtain a first enzymatic hydrolysis system containing a polypeptide fragment to be tested; c) performing mass spectrometry detection on the first enzymatic hydrolysis system to screen the response information of the polypeptide fragment to be tested that has the same parent-daughter ion pair as the general peptide segment; according to the response information and the standard curve of the general peptide segment, the content of the antibody to be tested is calculated; wherein, the general peptide segment includes a heavy chain general peptide segment and / or a light chain general peptide segment, having a sequence shown in SEQ ID NOs: 1-7. It can solve the problem of lack of a general method for quantitative detection of antibodies to be tested in the prior art, and is applicable to the field of antibody analysis.

Description

Technical Field

[0001] The present invention relates to the field of antibody analysis, and in particular to a universal quantitative analysis method for antibodies based on mass spectrometry. Background Art

[0002] In the field of biomedicine, especially in the clinical research, development, and stability studies of antibody drugs, accurate quantification of the antibody to be tested is a key step to ensure the safety and effectiveness of the drug. Existing technologies mainly rely on ultraviolet visible spectrophotometry (UV / Vis) to achieve this goal. However, the sensitivity of the UV / Vis method can usually only reach mg / mL, and it is impossible to quantify low concentrations or trace amounts of protein. For trace or low concentration protein quantification, ELISA is an alternative to UV / Vis, which can achieve a quantification limit of μg / mL or even ng / ml, but the ELISA method has many challenges and limitations. In recent years, the use of liquid chromatography-mass spectrometry has become more and more widespread, and its use has also become an alternative option for quantification.

[0003] Traditional LC-MS quantitative analysis is based on the characteristic peptides of each protein. It can quantify specific proteins in a complex matrix background, and requires the development of a specific analysis method for each protein molecule. This means that for each new antibody molecule to be tested, researchers need to start from scratch and determine suitable analysis conditions through a complex experimental design and optimization process. This process is not only time-consuming and labor-intensive, but also costly. For example, in the quantitative analysis of the antibody to be tested, it is usually necessary to perform theoretical analysis on the target protein and confirm the optimal enzymatic hydrolysis to obtain peptides that can be used for quantification. This step involves screening a large number of peptides to determine which peptides are representative and can accurately reflect the characteristics of the drug molecule. In actual analysis work, re-screening and testing of peptides based on each antibody to be tested greatly increases the workload and affects the efficiency of analysis. Summary of the invention

[0004] The main purpose of the present invention is to provide a universal quantitative analysis method for antibodies based on mass spectrometry to solve the problem of lack of a universal method for quantitative detection of antibodies to be tested in the prior art.

[0005] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a universal quantitative analysis method for antibodies based on mass spectrometry is provided, and the above-mentioned quantitative analysis method comprises: a) performing mass spectrometry detection on universal peptide segments to obtain a standard curve of the concentration of universal peptide segments and the mass spectrometry response intensity; b) performing a first enzymatic hydrolysis on the antibody to be tested to obtain a first enzymatic hydrolysis system containing the polypeptide fragment to be tested; c) performing mass spectrometry detection on the first enzymatic hydrolysis system to screen the response information of the polypeptide fragment to be tested having the same parent-daughter ion pair as the universal peptide segments; and calculating and obtaining the content of the antibody to be tested according to the response information and the standard curve of the universal peptide segments; wherein the universal peptide segments include heavy chain universal peptide segments and / or light chain universal peptide segments according to their sources, and the heavy chain universal peptide segments include any one or more of the first universal peptide segment, the second universal peptide segment, the third universal peptide segment or the fourth universal peptide segment; the light chain universal peptide segment includes any one or more of the fifth universal peptide segment, the sixth universal peptide segment or the seventh universal peptide segment; the first universal peptide segment has the sequence shown in SEQ ID NO: 1; the second universal peptide segment has the sequence shown in SEQ ID NO: 2; the third universal peptide segment has the sequence shown in SEQ ID NO: NO:3; the fourth universal peptide segment has the sequence shown in SEQ ID NO:4; the fifth universal peptide segment has the sequence shown in SEQ ID NO:5; the sixth universal peptide segment has the sequence shown in SEQ ID NO:6; and the seventh universal peptide segment has the sequence shown in SEQ ID NO:7.

[0006] Furthermore, the universal peptide segment is derived from the enzymatic hydrolysis system of the universal peptide segment standard solution or the standard antibody, and correspondingly, a) includes: a1) performing mass spectrometry detection on multiple universal peptide segment standard solutions with known concentrations respectively to obtain a standard curve of the concentration of the universal peptide segment and the mass spectrometry response intensity; or a2) performing a second enzymatic hydrolysis on multiple solutions with known concentrations and containing the standard antibody respectively to obtain a second enzymatic hydrolysis system containing the universal peptide segment, performing mass spectrometry detection on the second enzymatic hydrolysis system to obtain a standard curve of the concentration of the universal peptide segment and the mass spectrometry response intensity, wherein the standard antibody is an antibody containing the universal peptide segment and capable of obtaining the universal peptide segment after the second enzymatic hydrolysis; preferably, c) includes: performing mass spectrometry detection on the first enzymatic hydrolysis system to screen the polypeptide fragments to be tested that have the same parent-daughter ion pairs as the universal peptide segment, and calculating the content of the antibody to be tested based on the response information of the polypeptide fragments to be tested and the standard curve of the universal peptide segment; preferably, the content of the antibody to be tested is the average value after the contents are calculated separately using multiple universal peptide segments.

[0007] Further, the parent-daughter ion pair includes a parent ion and a daughter ion; the parent-daughter ion pair of the first universal peptide segment includes a first parent ion and a first daughter ion, wherein the m / z value of the first parent ion is 593.8280±0.5, and the m / z value of the first daughter ion is any one or more of 418.23±0.5, 489.27±0.5, 699.40±0.5 or 846.47±0.5; the parent-daughter ion pair of the second universal peptide segment includes a second parent ion and a second daughter ion, wherein the m / z value of the second parent ion is 661.3430±0.5, and the m / z value of the second daughter ion is 576.32±0.5 , 689.40±0.5, 760.44±0.5 or 831.48±0.5, any one or more thereof; the parent-daughter ion pair of the third universal peptide segment includes a third parent ion and a third daughter ion, wherein the m / z value of the third parent ion is 419.7550±0.5, and the m / z value of the third daughter ion is any one or more of 327.69±0.5, 486.29±0.5, 557.33±0.5 or 654.38±0.5; the parent-daughter ion pair of the fourth universal peptide segment includes a fourth parent ion and a fourth daughter ion, wherein the m / z value of the fourth parent ion is 937.9660±0.5 , the m / z value of the fourth daughter ion is any one or more of 397.21±0.5, 836.42±0.5, 1015.47±0.5 or 1378.65±0.5; the parent-daughter ion pair of the fifth universal peptide segment includes a fifth parent ion and a fifth daughter ion, wherein the m / z value of the fifth parent ion is 709.6950±0.5, and the m / z value of the fifth daughter ion is any one or more of 600.35±0.5, 763.40±0.5, 1138.56±0.5 or 1251.65±0.5; the parent-daughter ion pair of the sixth universal peptide segment includes a sixth parent ion and a sixth daughter ion, Among them, the m / z value of the sixth parent ion is 1068.9860±0.5, and the m / z value of the sixth daughter ion is any one or more of 400.18±0.5, 707.32±0.5, 893.42±0.5 or 1408.62±0.5; the parent-daughter ion pair of the seventh universal peptide segment includes the seventh parent ion and the seventh daughter ion, wherein the m / z value of the seventh parent ion is 751.8840±0.5, and the m / z value of the seventh daughter ion is any one or more of 836.47±0.5, 949.56±0.5, 1199.66±0.5 or 1036.58±0.5.

[0008] Further, a) includes: using lysine protease to enzymatically hydrolyze the antibody to be tested to obtain a polypeptide fragment to be tested.

[0009] Further, a) comprises: after mixing the antibody to be tested with a buffer to obtain an antibody solution to be tested, performing pretreatment to obtain a polypeptide fragment to be tested; the pretreatment comprises: sequentially adding a surfactant, a reducing agent, an alkylating agent and a lysine protease to the antibody solution to be tested, and incubating after each addition; preferably, the reducing agent comprises dithiothreitol; preferably, the alkylating agent comprises iodoacetamide and / or bromoacetamide.

[0010] Further, the buffer comprises Tris-HCl buffer, preferably 0.1-1.0 M Tris-HCl buffer; preferably, the pH of the buffer is 7.0-8.0.

[0011] Furthermore, the surfactant includes one or more of RapiGest SF, SDS or Triton-100.

[0012] Furthermore, the method for preparing universal peptides for mass spectrometry detection in b) includes: configuring universal peptide standards into standard curve solutions with known concentrations and different concentration gradients, and then incubating using the same pretreatment method as in a) to obtain universal peptides.

[0013] Furthermore, the antibody to be tested is a protein containing a universal peptide segment; preferably, the antibody to be tested is an IgG recombinant antibody, an IgM recombinant antibody, an IgA recombinant antibody or an IgE recombinant antibody; more preferably, the IgG recombinant antibody includes an IgG1 recombinant antibody, an IgG2 recombinant antibody, an IgG3 recombinant antibody or an IgG4 recombinant antibody.

[0014] Further, the mass spectrometry conditions in b) and c) are:

[0015]

[0016] By applying the technical scheme of the present invention and utilizing the quantitative analysis method of the recombinant protein, standard curves of the universal peptide chain of the heavy chain and / or the universal peptide chain of the light chain are established based on the constant region sequence of the antibody respectively; the antibody to be tested for quantitative analysis is enzymatically hydrolyzed to obtain an enzymatic hydrolysis system containing at least one polypeptide fragment to be tested; the enzymatic hydrolysis system is then subjected to mass spectrometry detection, and the polypeptide fragment to be tested having the same parent-daughter ion pair as the universal peptide chain is screened from the mass spectrometry results, such polypeptide fragment to be tested has the same amino acid sequence as the corresponding universal peptide chain, and the mass spectrometry response information of the polypeptide fragment to be tested is substituted into the standard curve of the corresponding universal peptide chain to achieve quantification of the antibody to be tested. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 The result graph of the standard curve solution of the first universal peptide segment according to Example 1 of the present invention is shown.

[0019] Figure 2 The result graph of the standard curve solution of the second universal peptide segment according to Example 1 of the present invention is shown.

[0020] Figure 3 The result graph of the standard curve solution of the third universal peptide segment according to Example 1 of the present invention is shown.

[0021] Figure 4 The result graph of the standard curve solution of the fourth universal peptide segment according to Example 1 of the present invention is shown.

[0022] Figure 5 The result graph of the standard curve solution of the fifth universal peptide segment according to Example 1 of the present invention is shown.

[0023] Figure 6 The result graph of the standard curve solution of the sixth universal peptide segment according to Example 1 of the present invention is shown.

[0024] Figure 7 The result graph of the standard curve solution of the seventh universal peptide segment according to Example 1 of the present invention is shown.

[0025] Figure 8 The figure shows the detection result of the first universal peptide segment in the antibody to be tested according to Example 1 of the present invention.

[0026] Fig. 9 The figure shows the detection result of the second universal peptide segment in the antibody to be tested according to Example 1 of the present invention.

[0027] Fig.10 The figure shows the detection result of the third universal peptide segment in the antibody to be tested according to Example 1 of the present invention.

[0028] Fig.11 The figure shows the detection result of the fourth universal peptide segment in the antibody to be tested according to Example 1 of the present invention.

[0029] Fig.12 The figure shows the detection result of the fifth universal peptide segment in the antibody to be tested according to Example 1 of the present invention.

[0030] Fig.13 The figure shows the detection result of the sixth universal peptide segment in the antibody to be tested according to Example 1 of the present invention.

[0031] Fig.14 The figure shows the detection result of the seventh universal peptide segment in the antibody to be tested according to Example 1 of the present invention.

[0032] Fig.15The figure shows the detection result of the first universal peptide segment in the antibody to be tested according to Example 2 of the present invention.

[0033] Fig.16 The figure shows the detection result of the second universal peptide segment in the antibody to be tested according to Example 2 of the present invention.

[0034] Fig.17 The figure shows the detection result of the fourth universal peptide segment in the antibody to be tested according to Example 2 of the present invention.

[0035] Fig.18 The figure shows the detection result of the fifth universal peptide segment in the antibody to be tested according to Example 2 of the present invention.

[0036] Fig.19 The figure shows the detection result of the sixth universal peptide segment in the antibody to be tested according to Example 2 of the present invention.

[0037] Fig. 20 The figure shows the detection result of the seventh universal peptide segment in the antibody to be tested according to Example 2 of the present invention.

[0038] Fig.21 The result graph of the standard curve solution of the polypeptide represented by SEQ ID NO: 14 according to the comparative example of the present invention is shown.

[0039] Fig. 22 The result graph of the standard curve solution of the polypeptide represented by SEQ ID NO: 15 according to the comparative example of the present invention is shown. DETAILED DESCRIPTION

[0040] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0041] Terminology explanation:

[0042] Parent-daughter ion pair: refers to the process in which a molecular ion (parent ion) undergoes a dissociation process to generate daughter ions during mass spectrometry analysis. The parent ion and daughter ions are collectively called a parent-daughter ion pair. The parent ion is the charged form of the original molecule, and the daughter ion is the product of the parent ion after energy fragmentation. The mass of the daughter ion is less than the mass of the parent ion. By analyzing the m / z values ​​of the parent ion and the daughter ion in the parent-daughter ion pair, the structural information of the molecule can be obtained, including but not limited to the composition of the molecule, the connection method, and the possible chemical bond breakage mode. Two mass spectrometry samples with the same parent-daughter ion pair both contain the same compound corresponding to the parent-daughter ion pair.

[0043] The terms "first", "second", etc. in this application are not intended to imply a specific orientation or order, but are used to distinguish one component from another. For example, the terms "first universal peptide segment" and "first universal peptide segment" in this application are only used to distinguish universal peptide segments with different amino acid sequences.

[0044] As mentioned in the background technology, for each antibody molecule to be tested, researchers need to start from scratch and determine suitable analysis conditions through complex experimental design and optimization processes. In order to overcome these challenges and improve the efficiency of quantitative analysis of antibodies to be tested, it is urgent to develop a more general and automated analysis method. Therefore, in the present application, the inventors based on LC-MS, after amino acid sequence analysis of a large number of antibody-related drugs, screened out several universal peptides (containing this part of amino acid sequence in most antibody-related drugs) located in the conserved region of the molecule for quantitative analysis, and established a general, lower cost, more efficient antibody to be tested based on mass spectrometry universal quantitative analysis method for antibodies, thus proposing a series of protection schemes of the present application.

[0045] In a first typical embodiment of the present application, a universal quantitative analysis method for antibodies to be tested based on mass spectrometry is provided, and the above-mentioned quantitative analysis method comprises: a) performing mass spectrometry detection on universal peptide segments to obtain a standard curve of the concentration and mass spectrometry response intensity of universal peptide segments; b) performing a first enzymatic hydrolysis on the antibody to be tested to obtain a first enzymatic hydrolysis system containing the polypeptide fragment to be tested; c) performing mass spectrometry detection on the first enzymatic hydrolysis system to screen the response information of the polypeptide fragment to be tested having the same parent-daughter ion pair as the universal peptide segment; according to the response information and the standard curve of the universal peptide segment, the content of the antibody to be tested is calculated; wherein the universal peptide segment includes a heavy chain (HC) universal peptide segment and / or a light chain (LC) universal peptide segment according to its source, and the heavy chain universal peptide segment includes any one or more of the first universal peptide segment, the second universal peptide segment, the third universal peptide segment or the fourth universal peptide segment; the light chain universal peptide segment includes any one or more of the fifth universal peptide segment, the sixth universal peptide segment or the seventh universal peptide segment; the first universal peptide segment has the sequence shown in SEQ ID NO: 1; the second universal peptide segment has the sequence shown in SEQ ID NO: 2; the third universal peptide segment has the sequence shown in SEQ ID NO:3; the fourth universal peptide segment has the sequence shown in SEQ ID NO:4; the fifth universal peptide segment has the sequence shown in SEQ ID NO:5; the sixth universal peptide segment has the sequence shown in SEQ ID NO:6; and the seventh universal peptide segment has the sequence shown in SEQ ID NO:7.

[0046] SEQ ID NO: 1: GSVFPLAPSSK;

[0047] SEQ ID NO: 2: STSGGTAALGCLVK;

[0048] SEQ ID NO: 3: ALPAPIEK;

[0049] SEQ ID NO: 4: TTPPVLDSDGSFFLYSK;

[0050] SEQ ID NO: 5: SGTASVVCLLNNFYPREAK;

[0051] SEQ ID NO: 6: VDNALQSGNSQESVTEQDSK;

[0052] SEQ ID NO: 7: DSTYSLSSTLTLSK.

[0053] In the above quantitative analysis method, step a) and step b) are performed independently.

[0054] In step a), the universal peptides are analyzed and detected by mass spectrometry to establish a standard curve for the universal peptides, including but not limited to: using a universal peptide standard, preparing a plurality of universal peptide standard curve solutions with known concentrations and a concentration gradient (including but not limited to 1.0 μg / mL, 5.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL and 100.0 μg / mL), and then performing mass spectrometry analysis on the above standard curve solutions to establish a standard curve for the universal peptides.

[0055] In step b), the antibody to be tested is first enzymatically hydrolyzed by an enzyme to obtain a plurality of polypeptide fragments to be tested, and the concentration of one or more polypeptide fragments to be tested is subsequently measured, so that the antibody to be tested can be quantitatively analyzed.

[0056] The above-mentioned universal peptides are several peptides located in the conserved region of the molecule that were screened out by the inventors after amino acid sequence analysis of a large number of antibody-related drugs. The mutation probability is low, and this part of the amino acid sequence is contained in most antibody-related drugs. Therefore, the use of the above-mentioned universal peptides can realize universal analysis of the antibodies to be tested, without the need to redevelop detection methods based on the specific antibodies to be tested. And it can realize quantitative analysis of antibodies to be tested with unknown sequences.

[0057] Moreover, for many peptides, it is difficult to perform qualitative and quantitative analysis by mass spectrometry, as not all peptides can produce stable and measurable parent-daughter ion pairs. Therefore, in the present application, the inventors also considered the above issues in the screening of universal peptides, and obtained the first to seventh universal peptides after a large number of experimental studies.

[0058] The aforementioned universal peptides of heavy chains or light chains are only used to help identify the positions of peptides in common antibodies. If changes in antibody types or engineering modifications are involved, changes in positions in the antibodies to be tested do not affect the quantification of the recombinant protein.

[0059] In step c), the polypeptide fragments to be tested obtained by the first enzymatic hydrolysis are detected by mass spectrometry, and the polypeptide fragments to be tested having the same parent-daughter ion pair as the universal peptide segment are screened. The sequence of the polypeptide fragments to be tested obtained by screening is consistent with the sequence of the corresponding universal peptide segment, so the standard curve of the universal peptide segment is also the standard curve of the polypeptide fragments to be tested. According to the mass spectrometry quantitative information of the peak (including but not limited to the peak area or peak height, etc.) and the above standard curve, the concentration of the polypeptide fragment to be tested can be calculated, and the molar amount of the polypeptide fragment to be tested is further calculated according to the known or measurable volume of the first enzymatic hydrolysis system, molar amount = concentration × volume.

[0060] In the actual quantitative analysis method, any one or more of the above-mentioned universal peptides can be used to calculate the quantitative analysis of the antibody to be tested. When multiple universal peptides are used for detection and multiple quantitative results are obtained by calculation respectively, the average value of the multiple quantitative results is selected as the quantitative result of the antibody to be tested; preferably, the average value is calculated after excluding abnormal values.

[0061] Preferably, one peptide is selected from each of the heavy chain and light chain universal peptides for use. Furthermore, when the mass spectrum data of the first enzymatic hydrolysis system can be screened to obtain data that are identical to the parent-daughter ion pairs of the heavy chain universal peptide and the light chain universal peptide, respectively, it means that the match is successful and quantitative calculation can be achieved. More preferably, more peptides are selected from each of the heavy chain and light chain universal peptides for use, and when the mass spectrum data of the first enzymatic hydrolysis system can match at least one heavy chain universal peptide and at least one heavy chain universal peptide, quantitative calculation can be achieved. The more successful matches, the higher the credibility of this analysis method.

[0062] Preferably, in the above-mentioned quantitative analysis method, a person skilled in the art may optionally connect a liquid chromatography device in front of a mass spectrometer to facilitate the separation of the polypeptide fragments to be tested and then the mass spectrometer detection. Alternatively, a variety of mass spectrometers capable of achieving quantification in the prior art may be used for quantitative analysis, and multiple mass spectrometers may be optionally connected in series. In the above-mentioned first enzymatic hydrolysis and mass spectrometer analysis process, a person skilled in the art may flexibly set and adjust according to the prior art.

[0063] The VH1 domain of the antibody light chain (Kappa configuration, >sp|P01834|IGKC_HUMAN Immunoglobulin cappa constant OS=Homo sapiens OX=9606GN=IGKC PE=1SV=2) has the amino acid sequence shown in SEQ ID NO: 8. The CH1 domain of the antibody heavy chain (>sp|P01857|IGHG1_HUMAN Immunoglobulin heavy constant gamma 1 OS=Homo sapiens OX=9606GN=IGHG1 PE=1SV=2) has the amino acid sequence shown in SEQ ID NO: 9.

[0064] SEQ ID NO: 8: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0065] SEQ ID NO: 9: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPELQLEESCAEAQDGELDGLWTTITIFITLFLLSVCYSATVTFFKVKWIFSSVVDLKQTIIPDYRNMIGQGA.

[0066] The sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 are generally common in antibody subtypes (especially IgG). In the quantitative analysis method of the present application, the heavy chain universal peptide segment and / or light chain universal peptide segment can be used to achieve quantitative detection of most antibodies to be tested.

[0067] It should be noted that the quantitative analysis method disclosed in the present application is not a method that can detect all antibodies to be tested. The above quantitative analysis method realizes the quantitative detection of most antibodies to be tested, especially antibody drugs. The detection method is simple, and the time and cost required for detection are less. For antibodies to be tested with unknown sequences, those skilled in the art can first try to use the above quantitative analysis method for detection. If the detection can be achieved, the experimental step of developing a separate detection method for such antibodies to be tested can be omitted. For antibodies that do not contain the above-mentioned heavy chain universal peptides and light chain universal peptides, those skilled in the art can flexibly select other methods in the prior art for detection.

[0068] In a preferred embodiment, the universal peptide segment is derived from the enzymatic hydrolysis system of the universal peptide segment standard solution or the standard antibody, and correspondingly, a) includes: a1) performing mass spectrometry detection on multiple universal peptide segment standard solutions with known concentrations to obtain a standard curve of the concentration of the universal peptide segment and the mass spectrometry response intensity; or a2) performing a second enzymatic hydrolysis on multiple solutions with known concentrations and containing the standard antibody to obtain a second enzymatic hydrolysis system containing the universal peptide segment, performing mass spectrometry detection on the second enzymatic hydrolysis system to obtain a standard curve of the concentration of the universal peptide segment and the mass spectrometry response intensity, wherein the standard antibody is an antibody containing the universal peptide segment and can obtain the universal peptide segment after the second enzymatic hydrolysis; preferably, c) includes: performing mass spectrometry detection on the first enzymatic hydrolysis system to screen the polypeptide fragments to be tested that have the same parent-daughter ion pairs as the universal peptide segment, and calculating the content of the antibody to be tested based on the response information of the polypeptide fragments to be tested and the standard curve of the universal peptide segment.

[0069] In the quantitative analysis method of the present application, the universal peptide can be derived from a universal peptide standard solution, that is, a standard solution with a known concentration obtained by dissolving the prepared universal peptide standard in a solvent; or it can be obtained by enzymatic hydrolysis from a standard antibody, which is an antibody containing the universal peptide, including but not limited to Trastuzumab, Loncastuximab or other antibodies, and the antibody to be tested can also be used as the standard antibody. The antibody concentration in the solution of the standard antibody is known.

[0070] In the prior art, for antibody solutions with higher concentrations (such as the order of mg / mL), it is simple and accurate to determine the antibody concentration therein, and those skilled in the art can flexibly select methods in the prior art for detection. However, for antibody solutions with lower concentrations (such as the order of μg / mL, or even 1 μg / mL), the above methods in the prior art are difficult to achieve accurate detection. However, using the above-mentioned quantitative analysis method of the present application, using the standard curve of the mass spectrometer and the response information of the sample to be tested, accurate detection of low-concentration antibody solutions can be achieved.

[0071] In a preferred embodiment, the content of the antibody to be tested is the average value of the contents calculated using multiple universal peptide segments.

[0072] In the above quantitative analysis method, in order to improve the reliability of the detection, the mass spectrometry information of the enzymatic hydrolysis system is screened for polypeptide fragments that can match the heavy chain universal peptide segment and the light chain universal peptide segment (i.e., have the same parent-daughter ion pair). If both can match, it proves that the above quantitative analysis method has higher reliability. When calculating the antibody content to be tested, any of the polypeptide fragments obtained by screening can be used for calculation.

[0073] In a preferred embodiment, the parent-daughter ion pair comprises a parent ion and a daughter ion;

[0074] The parent-daughter ion pair of the first universal peptide segment includes a first parent ion and a first daughter ion, wherein the m / z value of the first parent ion is 593.8280±0.5, and the m / z value of the first daughter ion is any one or more of 418.23±0.5, 489.27±0.5, 699.40±0.5 or 846.47±0.5;

[0075] The parent-daughter ion pair of the second universal peptide segment includes a second parent ion and a second daughter ion, wherein the m / z value of the second parent ion is 661.3430±0.5, and the m / z value of the second daughter ion is any one or more of 576.32±0.5, 689.40±0.5, 760.44±0.5 or 831.48±0.5;

[0076] The parent-daughter ion pair of the third universal peptide segment includes a third parent ion and a third daughter ion, wherein the m / z value of the third parent ion is 419.7550±0.5, and the m / z value of the third daughter ion is any one or more of 327.69±0.5, 486.29±0.5, 557.33±0.5 or 654.38±0.5;

[0077] The parent-daughter ion pair of the fourth universal peptide comprises a fourth parent ion and a fourth daughter ion, wherein the m / z value of the fourth parent ion is 937.9660±0.5, and the m / z value of the fourth daughter ion is any one or more of 397.21±0.5, 836.42±0.5, 1015.47±0.5 or 1378.65±0.5;

[0078] The parent-daughter ion pair of the fifth universal peptide segment includes a fifth parent ion and a fifth daughter ion, wherein the m / z value of the fifth parent ion is 709.6950±0.5, and the m / z value of the fifth daughter ion is any one or more of 600.35±0.5, 763.40±0.5, 1138.56±0.5 or 1251.65±0.5;

[0079] The parent-daughter ion pair of the sixth universal peptide segment includes a sixth parent ion and a sixth daughter ion, wherein the m / z value of the sixth parent ion is 1068.9860±0.5, and the m / z value of the sixth daughter ion is any one or more of 400.18±0.5, 707.32±0.5, 893.42±0.5 or 1408.62±0.5;

[0080] The parent-daughter ion pair of the seventh universal peptide segment includes a seventh parent ion and a seventh daughter ion, wherein the m / z value of the seventh parent ion is 751.8840±0.5, and the m / z value of the seventh daughter ion is any one or more of 836.47±0.5, 949.56±0.5, 1199.66±0.5 or 1036.58±0.5.

[0081] In the process of detecting parent-daughter ion pairs using mass spectrometry, the error generated is generally ±0.5, so if the detection result is within the ±0.5 range of the above m / z value, the signal is also considered to be the corresponding parent ion or daughter ion.

[0082] The parent and daughter ion pair information of the above-mentioned common peptides is shown in Table 1.

[0083] Table 1

[0084]

[0085] Note: The above carbamidomethylation is Carbamidomethylation. The universal peptides shown in SEQ ID NO: 2 and SEQ ID NO: 5 contain carbamidomethylation modification, that is, the cysteine ​​on the polypeptide is modified.

[0086] After pre-treatment of the test antibody to be tested (including but not limited to alkylation treatment with an alkylating agent and enzymatic hydrolysis with a lysine protease), a polypeptide fragment to be tested is obtained. If the polypeptide fragment to be tested contains a polypeptide fragment identical to SEQ ID NO: 2 or SEQ ID NO: 5, the polypeptide fragment derived from the test antibody to be tested also contains the same carbamidomethyl modification. The charge state in the above Table 1 is also called "valence state", and the charge states in Table 1 are all positive.

[0087] In a preferred embodiment, a) includes: enzymatically digesting the antibody to be tested with lysine protease or other methods that can obtain the same sequence as the universal peptide fragment to treat the antibody to be tested, so as to obtain the polypeptide fragment to be tested.

[0088] In the above quantitative analysis method, the antibody to be tested is enzymatically hydrolyzed by lysine protease to form multiple polypeptide fragments to be tested, shorten the sequence length, reduce the molecular weight, and facilitate subsequent mass spectrometry detection.

[0089] In a preferred embodiment, a) comprises: mixing the antibody to be tested with a buffer to obtain an antibody solution to be tested, and then performing a pretreatment to obtain a polypeptide fragment to be tested; the pretreatment comprises: sequentially adding a surfactant (destroying the high-order structure of the protein to facilitate the reaction of the reagent), a reducing agent (capable of reducing disulfide bonds within and between protein chains), an alkylating agent (blocking the sulfhydryl groups generated by the reduction of disulfide bonds to prevent the reformation of disulfide bonds) and a lysine protease (a highly specific enzyme that cleaves lysine in the protein sequence to produce peptides that can be used for quantification) to the antibody solution to be tested, and incubating after each addition; preferably, the buffer comprises a Tris-HCl buffer, more preferably a 0.1-1.0 M Tris-HCl buffer; preferably, the pH of the buffer is 7.0-8.0; preferably, the surfactant includes but is not limited to one or more of RapiGest SF, SDS or Triton-100; preferably, the reducing agent includes but is not limited to dithiothreitol; preferably, the alkylating agent includes but is not limited to iodoacetamide and / or bromoacetamide.

[0090] In a preferred embodiment, the method for preparing universal peptides for mass spectrometry detection in b) includes: preparing universal peptide standards into standard curve solutions with known concentrations and different concentration gradients, and then incubating using the same pretreatment method as in a) to obtain universal peptides.

[0091] For samples to be detected by mass spectrometry, including universal peptides and enzymatic hydrolysis systems, the same pretreatment method is used to prevent different pretreatment methods from affecting the accuracy of quantification. The preparation method of universal peptides for mass spectrometry detection in b) includes: after obtaining the standard curve solution, adding surfactant, dithiothreitol, iodoacetamide and lysine protease to the standard curve solution in sequence, incubating after each addition, and the parameters such as the proportion of added reagents, incubation time, incubation temperature, etc. are consistent with the pretreatment method used in a).

[0092] In a preferred embodiment, preferably, the antibody to be tested is an antibody drug developed with IgG, IgM, IgA or IgE as the framework, that is, the antibody to be tested is an IgG recombinant antibody, an IgM recombinant antibody, an IgA recombinant antibody or an IgE recombinant antibody; more preferably, the IgG recombinant antibody includes an IgG1 recombinant antibody, an IgG2 recombinant antibody, an IgG3 recombinant antibody or an IgG4 recombinant antibody.

[0093] In the development of antibody drugs, "as a framework" usually refers to the use of existing antibody structures as a basis or skeleton to construct new antibody drugs. The "framework" here refers to the basic structure of antibody molecules, especially their constant regions (such as Fc regions), which are relatively conservative parts of antibody molecules and usually do not make large-scale changes to the sequence. For an antibody drug obtained by artificial design, technicians in this field can judge which immunoglobulin is used as the framework based on the homology of its constant region with human IgG, IgM, IgA or IgE (generally speaking, the homology of the Fc region is greater than 80%), and judge the universal peptides that can be used based on the parent-daughter ion pairs of the peptides in the enzyme cleavage system.

[0094] In a preferred embodiment, the mass spectrometry conditions in b) and c) are as shown in Table 2.

[0095] Table 2

[0096]

[0097] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0098] Example 1

[0099] Sample preparation method:

[0100] Preparation of standard curve solution: Take the first universal peptide, the second universal peptide, the third universal peptide, the fourth universal peptide, the fifth universal peptide, the sixth universal peptide, and the seventh universal peptide, and use the same solution as the system of the antibody to be tested (Trastuzumab) to dilute the above universal peptides respectively to obtain standard curve solutions with concentrations of 1.0 μg / mL, 5.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL, and 100.0 μg / mL, respectively, for pretreatment.

[0101] The heavy chain sequence of the test antibody Trastuzumab is shown in SEQ ID NO: 10, and the light chain sequence is shown in SEQ ID NO: 11.

[0102] SEQ ID NO: 10:

[0103] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0104] SEQ ID NO: 11:

[0105] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0106] Pretreatment process of standard curve solution and test antibody: Take 100 μL of standard curve solution or test antibody in a centrifuge tube, add 5 μL of 1M Tris-HCl solution (pH 7.5) and mix well, add 1 μL of 5% RapiGest SF and mix well, then incubate at 95°C for 7 minutes, add 1 μL of 1M DTT and mix well, then incubate at 37°C for 30 minutes, add 4 μL of 0.5M IAM and mix well, then incubate at room temperature in the dark for 30 minutes. Add 4 μL of 1.0 μg / μL Lys-C protease solution, incubate at 37°C for 4 hours, and then test the sample.

[0107] Analytical methods:

[0108] Mobile Phase:

[0109] MPA: 0.1% (v / v) FA (formic acid), 0.02% (v / v) TFA (trifluoroacetic acid) in H 2 O;

[0110] MPB: 0.08% (v / v) FA, 0.02% (v / v) TFA in ACN (acetonitrile).

[0111] The UPLC analysis method and elution program are shown in Tables 3 and 4, and the MS analysis method is shown in Table 5.

[0112] Table 3

[0113] Flow / flow rate (mL / min) 0.25 Injection Volume / Injection Volume (μL) 10 Column Temperature / column temperature (℃) 50 Sample Room Temperature / Sample Room Temperature (℃) 10 Detection Wavelength / Detection Wavelength (nm) 214 Run Time / Run Time (min) 10

[0114] Table 4

[0115]

[0116] Table 5

[0117]

[0118]

[0119] Results for the standard curve solution: The standard curve solution of SEQ ID NO: 1 is as follows: Figure 1 As shown, the standard curve solution of SEQ ID NO: 2 is as follows Figure 2 As shown, the standard curve solution of SEQ ID NO: 3 is as follows Figure 3 As shown, the standard curve solution of SEQ ID NO: 4 is as follows Figure 4 As shown, the standard curve solution of SEQ ID NO: 5 is as Figure 5 As shown, the standard curve solution of SEQ ID NO: 6 is as follows Figure 6 As shown, the standard curve solution of SEQ ID NO: 7 is as Figure 7 shown.

[0120] For the test results of the antibody to be tested: the test results of SEQ ID NO: 1 are as follows Figure 8 As shown, the detection result of SEQ ID NO: 2 is as follows Fig. 9 As shown, the detection result of SEQ ID NO: 3 is as follows Fig.10 As shown, the detection result of SEQ ID NO: 4 is as follows Fig.11 As shown, the detection result of SEQ ID NO: 5 is as follows Fig.12 As shown, the detection result of SEQ ID NO: 6 is as follows Fig.13As shown, the detection result of SEQ ID NO: 7 is as follows Fig.14 shown.

[0121] The quantitative results are shown in Table 6.

[0122] Table 6

[0123] SEQ ID NO: Peptide information Quantitative results (μg / mL) 1 GPSVFPLAPSSK 39.6 2 STSGGTAALGCLVK 39.9 3 ALPAPIEK 38.3 4 TTPPVLDSDGSFFLYSK 40.9 5 SGTASVVCLLNNFYPREAK 40.4 6 VDNALQSGNSQESVTEQDSK 40.4 7 DSTYSLSSTLTLSK 43.6 average value / 40.4 Theoretical value / 40.0

[0124] Example 2

[0125] Sample preparation method:

[0126] Preparation of standard curve solution: Take the first universal peptide, the second universal peptide, the fourth universal peptide, the fifth universal peptide, the sixth universal peptide, and the seventh universal peptide, and use the same solution as the system of the antibody to be tested (Loncastuximab) to dilute the above universal peptides respectively to obtain standard curve solutions with concentrations of 1.0 μg / mL, 5.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL, and 100.0 μg / mL, respectively, for pretreatment.

[0127] The heavy chain sequence of the test antibody Loncastuximab is shown in SEQ ID NO: 12, and the light chain sequence is shown in SEQ ID NO: 13.

[0128] SEQ ID NO: 12:

[0129] QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQAPGQGLEWIGEIDPSDSYTNYNQNFQGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQ GTSVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0130] SEQ ID NO: 13:

[0131] EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0132] Pretreatment process of standard curve solution and test antibody: Take 100 μL of standard curve solution or test antibody in a centrifuge tube, add 5 μL of 1M Tris-HCl solution (pH 7.5) and mix well, add 1 μL of 5% RapiGest SF and mix well, then incubate at 95°C for 7 minutes, add 1 μL of 1M DTT and mix well, then incubate at 37°C for 30 minutes, add 4 μL of 0.5M IAM and mix well, then incubate at room temperature in the dark for 30 minutes. Add 4 μL of 1.0 μg / μL Lys-C protease solution, incubate at 37°C for 4 hours, and then test the sample.

[0133] Analytical methods:

[0134] Mobile Phase:

[0135] MPA: 0.1% (v / v) FA (formic acid), 0.02% (v / v) TFA (trifluoroacetic acid) in H 2 O;

[0136] MPB: 0.08% (v / v) FA, 0.02% (v / v) TFA in ACN (acetonitrile).

[0137] The UPLC analysis method and elution program are shown in Tables 3 and 4 above, and the MS analysis method is shown in Table 5 above.

[0138] Standard curves of the universal peptides shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 were prepared respectively. For the test results of the antibody to be tested: the test results of SEQ ID NO: 1 are as follows Fig.15 As shown, the detection result of SEQ ID NO: 2 is as follows Fig.16 As shown, the detection result of SEQ ID NO: 4 is as follows Fig.17 As shown, the detection result of SEQ ID NO: 5 is as follows Fig.18 As shown, the detection result of SEQ ID NO: 6 is as follows Fig.19 As shown, the detection result of SEQ ID NO: 7 is as follows Fig. 20 shown.

[0139] The quantitative results are shown in Table 7.

[0140] Table 7

[0141] SEQ ID NO: Peptide information Quantitative results (μg / mL) 1 GPSVFPLAPSSK 47.9 2 STSGGTAALGCLVK 51.1 4 TTPPVLDSDGSFFLYSK 53.4 5 SGTASVVCLLNNFYPREAK 50.6 6 VDNALQSGNSQESVTEQDSK 52.5 7 DSTYSLSSTLTLSK 53.0 average value / 51.4 Theoretical value / 50.0

[0142] Comparative Example

[0143] Preparation of standard curve solution: Take the fragment VYACEVTHQGLSSPVTK (SEQ ID NO: 14) in the light chain and the fragment FNWYVDGVEVHNAK (SEQ ID NO: 15) in the heavy chain, dilute the above universal peptides respectively, and obtain standard curve solutions with concentrations of 1.0 μg / mL, 5.0 μg / mL, 25.0 μg / mL, 50.0 μg / mL, and 100.0 μg / mL, respectively, for pretreatment.

[0144] Pretreatment process of standard curve solution and test antibody (same as Example 2, test antibody Loncastuximab): take 100 μL of standard curve solution and test antibody in a centrifuge tube, add 5 μL of 1M Tris-HCl solution (pH 7.5) and mix well, add 1 μL of 5% RapiGest SF and mix well, then incubate at 95°C for 7 min, add 1 μL of 1M DTT and mix well, then incubate at 37°C for 30 min, add 4 μL of 0.5M IAM and mix well, then incubate at room temperature in the dark for 30 min. Add 4 μL of 1.0 μg / μL Lys-C protease solution, incubate at 37°C for 4 hours, and then test the sample.

[0145] Analytical methods:

[0146] Mobile Phase:

[0147] MPA: 0.1% (v / v) FA (formic acid), 0.02% (v / v) TFA (trifluoroacetic acid) in H 2 O;

[0148] MPB: 0.08% (v / v) FA, 0.02% (v / v) TFA in ACN (acetonitrile).

[0149] The UPLC analysis method and elution program are shown in Tables 3 and 4 in Example 1 above, and the MS analysis method is shown in Table 5 in Example 1 above.

[0150] Results for the standard curve solution: The standard curve solution of the light chain fragment VYACEVTHQGLSSPVTK (SEQ ID NO: 14) is as follows: Fig.21 As shown in Figure 2, the chromatographic peak integration of the quantitative peptide was disturbed, and the linear correlation coefficient of the standard curve was R 2 ≤0.990. Results for the standard curve solution: The standard curve solution of the heavy chain fragment FNWYVDGVEVHNAK (SEQ ID NO: 15) is as follows Fig. 22 As shown, the linear correlation coefficient of the standard curve is R 2 ≤0.990, the results of the standard curve solution showed that the peptides in the comparative example had a poor linear relationship with the peptide response at different concentrations, and the quantitative process lacked accuracy.

[0151] The peptides shown in SEQ ID NO: 14 and SEQ ID NO: 15 are also derived from the conserved regions of antibodies. As shown in the above test data, it is difficult to achieve accurate quantification of the antibodies to be tested. The inventors speculate that the following reasons may lead to only the universal peptides in this application being able to achieve accurate quantitative analysis of the antibodies to be tested:

[0152] 1. The peptide contains KP site. Because the theoretical cleavage site of Lys-C enzyme is the carbon end of lysine (K), when the carbon end of lysine (K) is linked to proline (P), the cleavage efficiency will be different (there will be missed cleavage), so theoretically, in samples of different concentrations and under different treatment times, the cleavage efficiency of the KP position will be different, thus affecting the quantitative accuracy;

[0153] 2. Shorter amino acid peptides have weak liquid phase retention, insufficient product ions, and some product ions have poor quality;

[0154] 3. When there are easily modified sites in the peptide segment, when there is a difference in the modification ratio between the standard sample and the sample to be tested, the quantitative results will also be affected.

[0155] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: in the above-mentioned universal quantitative analysis method of antibodies based on mass spectrometry for the antibodies to be tested, the present application has developed 4 universal peptides for heavy chains and 3 universal peptides for light chains. At least one of the above two universal peptides can be taken to achieve quantitative analysis of the antibodies to be tested. This kind of quantitative analysis is a general analysis method. There is no need to develop a specific analysis method for a specific sequence as in the prior art. This quantitative analysis method can be used to quantify recombinant proteins containing universal peptides, greatly reducing the cost and workload required for analysis. And the above-mentioned quantitative analysis method uses mass spectrometry for quantification. Compared with the UV method commonly used in the prior art, the sensitivity can be increased by 2-3 orders of magnitude, greatly improving the analysis capability for low-concentration samples.

[0156] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A universal quantitative analysis method for antibodies based on mass spectrometry, characterized in that: The quantitative analysis method comprises: a) performing mass spectrometry detection on the universal peptide segment to obtain a standard curve of the concentration of the universal peptide segment and the mass spectrometry response intensity; b) performing a first enzymatic hydrolysis on the antibody to be tested to obtain a first enzymatic hydrolysis system containing the polypeptide fragment to be tested; c) performing mass spectrometry detection on the first enzymatic hydrolysis system to screen the response information of the polypeptide fragment to be tested that has the same parent-daughter ion pair as the universal peptide segment; and calculating the content of the antibody to be tested based on the response information and the standard curve of the universal peptide segment; Wherein, the universal peptide segment includes a heavy chain universal peptide segment and / or a light chain universal peptide segment, The heavy chain universal peptide segment includes any one or more of the first universal peptide segment, the second universal peptide segment, the third universal peptide segment or the fourth universal peptide segment; The light chain universal peptide segment includes any one or more of the fifth universal peptide segment, the sixth universal peptide segment, or the seventh universal peptide segment; The first universal peptide segment has a sequence shown in SEQ ID NO: 1; The second universal peptide segment has a sequence shown in SEQ ID NO: 2; The third universal peptide segment has a sequence shown in SEQ ID NO: 3; The fourth universal peptide segment has a sequence shown in SEQ ID NO: 4; The fifth universal peptide segment has a sequence shown in SEQ ID NO: 5; The sixth universal peptide segment has a sequence shown in SEQ ID NO: 6; The seventh universal peptide segment has a sequence shown in SEQ ID NO:

7.

2. The quantitative analysis method according to claim 1, characterized in that The universal peptide segment is derived from the enzymatic hydrolysis system of the universal peptide segment standard solution or the standard antibody. Correspondingly, the a) includes: a1) performing mass spectrometry detection on a plurality of solutions of the universal peptide standard with known concentrations to obtain a standard curve of the concentration of the universal peptide and the mass spectrometry response intensity; or a2) performing a second enzymatic hydrolysis on a plurality of solutions with known concentrations and containing the standard curve antibody to obtain a second enzymatic hydrolysis system containing the universal peptide segment, performing mass spectrometry detection on the second enzymatic hydrolysis system to obtain a standard curve of the concentration and mass spectrometry response intensity of the universal peptide segment, wherein the standard curve antibody is an antibody containing the universal peptide segment and capable of obtaining the universal peptide segment after the second enzymatic hydrolysis; Preferably, said c) comprises: performing mass spectrometry detection on the first enzymatic hydrolysis system, Screening the polypeptide fragment to be tested that has the same parent-daughter ion pair as the universal peptide segment, Calculating the content of the antibody to be tested based on the response information of the polypeptide fragment to be tested and the standard curve of the universal peptide segment; Preferably, the content of the antibody to be tested is the average value of the contents calculated using a plurality of the universal peptide segments.

3. The quantitative analysis method according to claim 1 or 2, characterized in that: The parent-daughter ion pair comprises a parent ion and a daughter ion; The parent-daughter ion pair of the first universal peptide segment includes a first parent ion and a first daughter ion, wherein: The m / z value of the first parent ion is 593.8280±0.5, The m / z value of the first daughter ion is any one or more of 418.23±0.5, 489.27±0.5, 699.40±0.5 or 846.47±0.5; The parent-daughter ion pair of the second universal peptide segment includes a second parent ion and a second daughter ion, wherein: The m / z value of the second parent ion is 661.3430±0.5, The m / z value of the second product ion is any one or more of 576.32±0.5, 689.40±0.5, 760.44±0.5 or 831.48±0.5; The parent-daughter ion pair of the third universal peptide segment includes a third parent ion and a third daughter ion, wherein: The m / z value of the third parent ion is 419.7550±0.5, The m / z value of the third product ion is any one or more of 327.69±0.5, 486.29±0.5, 557.33±0.5 or 654.38±0.5; The parent-daughter ion pair of the fourth universal peptide segment includes a fourth parent ion and a fourth daughter ion, wherein: The m / z value of the fourth parent ion is 937.9660±0.5, The m / z value of the fourth daughter ion is any one or more of 397.21±0.5, 836.42±0.5, 1015.47±0.5 or 1378.65±0.5; The parent-daughter ion pair of the fifth universal peptide segment includes a fifth parent ion and a fifth daughter ion, wherein: The m / z value of the fifth parent ion is 709.6950±0.5, The m / z value of the fifth daughter ion is any one or more of 600.35±0.5, 763.40±0.5, 1138.56±0.5 or 1251.65±0.5; The parent-daughter ion pair of the sixth universal peptide segment includes a sixth parent ion and a sixth daughter ion, wherein: The m / z value of the sixth parent ion is 1068.9860±0.5, The m / z value of the sixth daughter ion is any one or more of 400.18±0.5, 707.32±0.5, 893.42±0.5 or 1408.62±0.5; The parent-daughter ion pair of the seventh universal peptide segment includes a seventh parent ion and a seventh daughter ion, wherein: The m / z value of the seventh parent ion is 751.8840±0.5, The m / z value of the seventh daughter ion is any one or more of 836.47±0.5, 949.56±0.5, 1199.66±0.5 or 1036.58±0.

5.

4. The quantitative analysis method according to claim 1, characterized in that: The step a) comprises: performing enzymatic hydrolysis on the antibody to be tested by using lysine protease to obtain the polypeptide fragment to be tested.

5. The quantitative analysis method according to claim 4, characterized in that: The a) comprises: mixing the antibody to be tested with a buffer to obtain an antibody solution to be tested, and then performing a pre-treatment to obtain the polypeptide fragment to be tested; The pretreatment comprises: sequentially adding a surfactant, a reducing agent, an alkylating agent and the lysine protease to the antibody solution to be tested, and incubating after each addition; Preferably, the reducing agent comprises dithiothreitol; Preferably, the alkylating agent comprises iodoacetamide and / or bromoacetamide.

6. The quantitative analysis method according to claim 5, characterized in that: The buffer comprises Tris-HCl buffer, preferably 0.1-1.0M Tris-HCl buffer; Preferably, the pH of the buffer solution is 7.0-8.

0.

7. The quantitative analysis method according to claim 5, characterized in that: The surfactant includes one or more of RapiGestSF, SDS or Triton-100.

8. The quantitative analysis method according to any one of claims 5 to 7, characterized in that: The method for preparing the universal peptide segment for mass spectrometry detection in b) comprises: configuring the universal peptide segment standard into a standard curve solution with known concentration and different concentration gradients, and then incubating using the same pretreatment method as in a) to obtain the universal peptide segment.

9. The quantitative analysis method according to claim 1, characterized in that: The antibody to be tested is a protein containing the universal peptide segment; Preferably, the antibody to be tested is an IgG recombinant antibody, an IgM recombinant antibody, an IgA recombinant antibody or an IgE recombinant antibody; More preferably, the IgG recombinant antibody includes IgG1 recombinant antibody, IgG2 recombinant antibody, IgG3 recombinant antibody or IgG4 recombinant antibody.

10. The quantitative analysis method according to claim 1, characterized in that: The mass spectrometry conditions in b) and c) are:

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