Method for detecting N-glycoform of antibody

Abstract

The invention relates to the technical field of medicine quality control, in particular to a detection method of an antibody N-glycoform. The detection method comprises the following steps: carrying out qualitative detection on the N-glycoform of an antibody to be detected by adopting mass spectrometry; and carrying out quantitative detection on each N-glycotype which is qualitatively detected by adopting a reverse phase chromatography method. Compared with a traditional detection method, the method can be suitable for detection of drugs with approximate N glycoforms, and an accurate detection result is provided.

Description

Technical Field

[0001] The present application relates to the technical field of drug quality control, and in particular to a method for detecting antibody N-glycoforms. Background Art

[0002] Glycosylation is a structurally diverse and complex post-translational modification of proteins that affects immunoglobulin Fc effector function. Although glycosylation accounts for only 2-3% of the total antibody, a comprehensive assessment of the glycoforms of antibody distribution is key to the development of new mAbs. Different glycoforms such as galactose, fucose, high mannose, N-acetylneuraminic acid (NANA) and N-glycosylneuraminic acid (NGNA) have different effects on the safety / immunogenicity, efficacy / bioactivity and clearance (pharmacodynamic / pharmacokinetic properties (PD / PK)) of antibodies.

[0003] At present, the main analysis methods for N-sugars of biomacromolecules (such as antibodies) are hydrophilic interaction chromatography (HILIC), high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS). HILIC and HPAEC-PAD can well separate various forms of N-sugars and determine the form of N-sugars by mass spectrometry. However, the response of the mass spectrometry signal cannot fully reflect the sample content and cannot provide an accurate content composition. The separated monosaccharides must also pass through other detectors, such as fluorescence detectors, to accurately quantify the composition of each glycoform and achieve simultaneous qualitative and quantitative analysis of glycoforms. Traditional methods are relatively cumbersome. How to provide an integrated method for qualitative and quantitative analysis of N-sugars is crucial to the quality control of biomacromolecules such as antibodies. Summary of the invention

[0004] Based on this, one or more embodiments of the present application provide a method for detecting antibody N-glycoforms, including the following technical solutions:

[0005] One or more embodiments of the present application provide a method for detecting an antibody N-glycoform, the detection method comprising the following steps:

[0006] Qualitative detection of N-glycoforms of the antibody under test by mass spectrometry; and,

[0007] Reverse phase chromatography was used to quantitatively detect the qualitatively detected N-glycoforms.

[0008] In some embodiments of the present application, the conditions of the reverse phase chromatography include:

[0009] The mobile phase includes mobile phase A and mobile phase B.

[0010] The mobile phase A comprises water and trifluoroacetic acid in a volume ratio of 0.1% to 0.2%,

[0011] The mobile phase B comprises water, trifluoroacetic acid accounting for 0.1% to 0.2% by volume, and acetonitrile accounting for 85% to 95% by volume.

[0012] In some embodiments of the present application, the conditions of the reverse phase chromatography include: using a gradient elution method;

[0013] Optionally, the gradient elution procedure includes:

[0014] 0min~10min: the volume proportion of the mobile phase B is maintained at 6%;

[0015] 10min-40min: the volume proportion of the mobile phase B increases from 6% to 18%;

[0016] 40min-42min: the volume proportion of the mobile phase B increases from 18% to 100%;

[0017] 42min-44min: the volume proportion of the mobile phase B decreases from 100% to 6%;

[0018] 44min-60min: the volume proportion of the mobile phase B is maintained at 6%.

[0019] In some embodiments of the present application, the conditions of the reverse phase chromatography further include:

[0020] Column temperature is 22℃~27℃;

[0021] The injection volume is 5μL to 15μL; or / and,

[0022] The detection wavelengths are 225-235nm and 420nm-430nm.

[0023] In some embodiments of the present application, the conditions of the reverse phase chromatography include: the reverse phase chromatography column uses silica gel as the filling matrix, and the specifications are 2.1 mm in diameter, 150 mm in length, and 2.6 μm in particle size.

[0024] In some embodiments of the present application, the conditions of the mass spectrometry method include:

[0025] The positive ion Full MS-SIM scanning mode was used.

[0026] Microscans is set to 10,

[0027] Resolution is set to 17500,

[0028] AGC target is set to 3e6,

[0029] Maximum IT is set to 200ms,

[0030] Scan range is set to 200-2500,

[0031] Sheath gas flow (arb) is set to 35,

[0032] Aux gas flow rate (arb) is set to 10,

[0033] Sweep gas flow rate (arb) is set to 0,

[0034] The spray voltage (kV) is set to 3.8,

[0035] Capillary temperature (℃) is set to 320, and

[0036] S-lens RF level is set to 50.

[0037] In some embodiments of the present application, the antibody to be tested comprises a monoclonal antibody.

[0038] In some embodiments of the present application, the monoclonal antibody comprises an IgG antibody.

[0039] In some embodiments of the present application, the IgG class antibody includes an IgG4 antibody.

[0040] In some embodiments of the present application, the antibody to be tested includes the original antibody and / or a replica antibody of the original antibody.

[0041] In some embodiments of the present application, the antibody to be tested is pretreated using the 2-AA method before detection.

[0042] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0044] Figure 1 Mass spectrometry identification results of mAb A;

[0045] Figure 2 Reverse phase chromatography integration results of mAb A;

[0046] Figure 3 Mass spectrometry identification results of mAb B;

[0047] Figure 4 Reverse phase chromatography integration results of monoclonal antibody B;

[0048] Figure 5 The chromatograms of different elution methods of the original solution;

[0049] Figure 6 Different gradient chromatograms of the original solution;

[0050] Figure 7 The chromatograms of the original solution at different column temperatures;

[0051] Figure 8 Mass spectrometry identification of sugar forms for stock solution and original research;

[0052] Fig. 9 The G1 glycoform was identified for the fucose-deficient sample;

[0053] Fig.10 The chromatogram identification result;

[0054] Fig.11 This is the chromatogram of different reagent amounts of the stock solution;

[0055] Fig.12 Chromatograms of different injection volumes of stock solution;

[0056] Fig.13 It is the linear fitting curve of different injection amounts of stock solution and G0F peak area;

[0057] Fig.14 Detection chromatograms of different chromatographic columns for stock solutions;

[0058] Fig.15 Comparison of glycoform chromatograms between DS buffer and stock solution;

[0059] Fig.16 This is the reproducible chromatogram of the stock solution;

[0060] Fig.17 is the linear chromatogram of the original solution;

[0061] Fig.18 is the linear fitting curve of the original solution;

[0062] Fig.19 This is the durability chromatogram of the stock solution. DETAILED DESCRIPTION

[0063] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0065] the term

[0066] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0067] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0068] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0069] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0070] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0071] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0072] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0073] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0074] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0075] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0076] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0077] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0078] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0079] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0080] At present, the main analytical methods for monoclonal antibody N-glycans are hydrophilic interaction chromatography (HILIC), high-performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) for characterizing and monitoring the glycosylation of monoclonal antibodies. HILIC and HPAEC-PAD can well separate various forms of N-glycans, but in some usage environments, such as the detection of drugs with similar N-glycan forms such as original drugs and generic drugs, the differences between similar drugs may be magnified. If the N-glycan form is determined by mass spectrometry, the response of the mass spectrometry signal cannot fully reflect the sample content and cannot provide accurate content composition.

[0081] One or more embodiments of the present application provide a method for detecting an antibody N-glycoform, the detection method comprising the following steps:

[0082] Qualitative detection of N-glycoforms of the antibody under test by mass spectrometry; and,

[0083] Reverse phase chromatography was used to quantitatively detect the qualitatively detected N-glycoforms.

[0084] Compared with traditional HILIC and HPAEC-PAD detection methods, the detection method of the present application is suitable for the detection of drugs with similar N-glycoforms, such as original drugs and generic drugs, and the detection results are accurate. In some embodiments of the present application, the conditions of the reverse phase chromatography include:

[0085] The mobile phase includes mobile phase A and mobile phase B.

[0086] The mobile phase A comprises water and trifluoroacetic acid in an amount of 0.1% to 0.2% by volume (e.g., 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%),

[0087] The mobile phase B includes water, 0.1% to 0.2% (for example, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%) of trifluoroacetic acid, and 85% to 95% (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%) of acetonitrile by volume.

[0088] In some embodiments of the present application, the conditions of the reverse phase chromatography include: using a gradient elution method;

[0089] Optionally, the gradient elution procedure includes:

[0090] 0min~10min: the volume proportion of the mobile phase B is maintained at 6%;

[0091] 10min-40min: the volume proportion of the mobile phase B increases from 6% to 18%;

[0092] 40min-42min: the volume proportion of the mobile phase B increases from 18% to 100%;

[0093] 42min-44min: the volume proportion of the mobile phase B decreases from 100% to 6%;

[0094] 44min-60min: the volume proportion of the mobile phase B is maintained at 6%.

[0095] In some embodiments of the present application, the conditions of the reverse phase chromatography further include:

[0096] The column temperature is 22°C to 27°C (e.g., 22, 23, 24, 25, 26, 27°C);

[0097] The injection volume is 5 μL to 15 μL (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μL); or / and,

[0098] The detection wavelengths are 225nm to 235nm (for example, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, and 235nm) and 420nm to 430nm (for example, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, and 430nm).

[0099] In some embodiments of the present application, the conditions of the reverse phase chromatography include: the reverse phase chromatography column uses silica gel as the filling matrix, and the specifications are 2.1 mm in diameter, 150 mm in length, and 2.6 μm in particle size.

[0100] In some embodiments of the present application, the conditions of the mass spectrometry method include:

[0101] The positive ion Full MS-SIM scanning mode was used.

[0102] Microscans is set to 10,

[0103] Resolution is set to 17500,

[0104] AGC target is set to 3e6,

[0105] Maximum IT is set to 200ms,

[0106] Scan range is set to 200-2500,

[0107] Sheath gas flow (arb) is set to 35,

[0108] Aux gas flow rate (arb) is set to 10,

[0109] Sweep gas flow rate (arb) is set to 0,

[0110] The spray voltage (kV) is set to 3.8,

[0111] Capillary temperature (℃) is set to 320, and

[0112] S-lens RF level is set to 50.

[0113] In some embodiments of the present application, the antibody to be tested comprises a monoclonal antibody.

[0114] In some embodiments of the present application, the monoclonal antibody comprises an IgG antibody.

[0115] In some embodiments of the present application, the IgG class antibody includes an IgG4 antibody.

[0116] In some embodiments of the present application, the antibody to be tested is pretreated using the 2-AA method before detection.

[0117] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0118] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0119] The present application provides a method for analyzing the N-sugar composition of biomacromolecules by combining high-resolution mass spectrometry and 2-AA reverse phase chromatography. The method identifies the N-sugar forms by high-resolution mass spectrometry and analyzes the separation properties and content ratio of each N-sugar form by 2-AA reverse phase chromatography.

[0120] Example 1

[0121] This embodiment provides a method for detecting antibody N-glycoforms, comprising the following steps:

[0122] 1. Sample pretreatment:

[0123] Sample pretreatment for mass spectrometry identification: dilute the sample to 2.5-7.5 mg / mL with water, label the sample using the Primid 2-AA labeling kit, take the supernatant after high-speed centrifugation and add it to the sample tube for subsequent detection using reverse phase chromatography connected to high-resolution mass spectrometry.

[0124] Sample pretreatment for 2-AA reverse phase chromatography analysis: dilute the sample to 2.5-7.5 mg / mL with water, label the sample using the Primid 2-AA labeling kit, centrifuge at high speed and add the supernatant to the sample tube for subsequent detection using reverse phase chromatography.

[0125] During the sample pretreatment for mass spectrometry identification and the sample pretreatment for 2-AA reverse phase chromatography analysis, the 2-AA labeling sample process is as follows:

[0126] (1) Enzyme digestion: Take 24 μL of sample dilution and place it in an EP tube. Add 4 μL of enzyme digestion buffer and 2 μL of PNGase F enzyme. Mix well and digest at 50°C for 5 min. Then denature at 98°C for 5 min. Centrifuge at 14,000 rpm for 5 min. Take 25 μL of supernatant and place it in a new EP tube.

[0127] (2) Labeling: Add 25 μL of the prepared 2-AA labeling solution to the 25 μL supernatant in the previous step, pipette and mix well, and incubate at 50°C for 15 min.

[0128] (3) Binding: Before labeling is finished, take out the magnetic beads for sugar type purification, vortex and mix, then pipette 200 μL of magnetic bead solution, add it to the EP tube, place it on the magnetic rack, wait for about half a minute for the solution to become clear, then carefully aspirate and discard the liquid, remove the magnetic rack from the magnet, transfer 50 μL of the labeled mixture into the EP tube containing the magnetic beads (when transferring the liquid, it is recommended to blow it along the tube wall where the magnetic beads are located above the magnetic beads, so that the magnetic beads can be smoothly flushed to the bottom of the tube and a magnetic bead suspension can be obtained at the same time) and mix it by blowing. Add 330 μL of acetonitrile and mix it by blowing 3 to 4 times.

[0129] (4) Washing: Place the EP tube on the magnetic rack, wait for about half a minute for the solution to become clear, then carefully aspirate and discard the liquid. Remove the rack from the magnet, add 400 μL of washing solution (add in the same way as adding the labeled mixed solution), and pipette 3 to 4 times to mix. Place the EP tube on the magnetic rack again, wait for about half a minute for the solution to become clear, then carefully aspirate and discard the liquid.

[0130] Repeat the above washing steps at least 2 times.

[0131] (5) Elution: Remove the magnetic stand from the magnet, add 100 μL of 5% acetonitrile, mix well by pipetting, incubate at room temperature for 2 to 3 min, place on the magnetic stand, wait for half a minute for the solution to become clear, carefully pipette 95 μL of the supernatant into a new EP tube, centrifuge at 14,000 rpm for 5 min, take out the supernatant and add it to the sample bottle for detection.

[0132] 2. Detection

[0133] The mass spectrometry detection conditions are as follows: positive ion Full MS-SIM scanning mode is used, Microscans is set to 10, Resolution is set to 17500, AGC target is set to 3e6, Maximum IT is set to 200 ms, Scan range is set to 200-2500, Sheath gas flow (arb) is set to 35, Aux gas flow rate (arb) is set to 10, Sweep gas flow rate (arb) is set to 0, Spray voltage (kV) is set to 3.8, Capillary temperature (℃) is set to 320, and S-lens RF level is set to 50.

[0134] The conditions for reverse phase chromatography were as follows:

[0135] Mobile phase A was an aqueous solution containing 0.15% TFA (trifluoroacetic acid);

[0136] Mobile phase B was an aqueous solution containing 0.15% TFA and 90% acetonitrile (ACN);

[0137] The chromatographic column was a reverse phase column with a diameter of 2.1 mm, a length of 150 mm, and a particle size of 2.6 μm;

[0138] The column temperature was 25°C;

[0139] The injection volume was 10 μL;

[0140] The fluorescence detector wavelength is wavelength Ex230nm / Em425nm;

[0141] The gradient elution conditions were as follows:

[0142] 0-10min: maintain 6% B;

[0143] 10min~40min: from 6% B to 18% B;

[0144] 40min~42min: from 18% B to 100% B;

[0145] 42min~44min: from 100%B to 6%B;

[0146] 44min~60min: maintain 6% B;

[0147] The flow rate was maintained at 0.2 mL / min.

[0148] The antibody sample A detected in this example is a commercially available IgG4 monoclonal antibody drug with two glycosylation sites. The registration standard for the N glycoform is that the total fucose percentage is above 75% (i.e., the sum of the percentages of G0F+G1F+G2F).

[0149] The results are as follows Figure 1 and Figure 2 shown.

[0150] Mass spectrometry identification of the N-glycoform of IgG4 monoclonal antibody in antibody sample A revealed that the chromatographic peak of G0F+G1F+G2F contained not only G0F, G1F and G2F, but also G0F-GN, Man3F and G0FB.

[0151] The total fucose content of the A molecule was calculated using reverse phase chromatography to be G2F+G1F+G0F=1.91%+5.44%+85.46%=92.81%.

[0152] Example 2

[0153] This embodiment is a variation of Embodiment 1, and the only difference compared with Embodiment 1 is that:

[0154] The test sample is sample B, and the IgG4 monoclonal antibody and its N-glycoform in sample B are basically consistent with sample A in Example 1. The operation steps are all referred to the method provided in Example 1. The results are shown in FIG. Figure 3 and Figure 4 shown.

[0155] Mass spectrometry identification of the N-glycoforms of sample B revealed that the chromatographic peak of G0F+G1F+G2F contained not only G0F, G1F and G2F, but also G0F-GN, Man3F and G0FB.

[0156] The total fucose content of sample B was calculated using reverse phase chromatography to be G2F+G1F+G0F=3.40%+4.50%+87.63%=95.53%, which was within the similarity range of the total fucose content of multiple batches of samples A (92.00% to 96.68%).

[0157] According to the test results of Example 1 and Example 2 above, the values ​​obtained by the test are close, which proves that this method can achieve accurate quantitative comparative analysis of sugar forms of similar antibody drugs.

[0158] Example 3

[0159] Unless otherwise specified, the stock solution and sample in this embodiment refer to sample B.

[0160] 1. Elution method optimization

[0161] 1.1 Purpose of the experiment

[0162] In glycoform analysis, the elution method of N-sugars may affect the amount and composition of eluted N-sugars. The elution method in the instructions of the Primid 2-AA labeling kit is for HILIC chromatography, while this application uses reversed-phase chromatography analysis. Therefore, this application needs to optimize the elution method.

[0163] 1.2 Experimental procedures

[0164] The sample was selected as stock solution (batch number 202301), and the sample treatment experimental steps were as described in "1 Sample pretreatment" in Example 1. On this basis, the present example optimized "(5) elution", using the kit elution method (elution method 1) and the following elution method (elution method 2) respectively.

[0165] Elution method 1: Remove the rack from the magnet, add 40μL of elution solution, mix well by blowing, and incubate at room temperature for 2-3 minutes. Place it on the magnetic rack, wait for half a minute for the solution to become clear, carefully pipette 40μL of supernatant into a new EP tube, add 60μL of acetonitrile and mix well. Centrifuge at 14000rpm for 5 minutes and add to the loading bottle.

[0166] Elution method 2: Remove the rack from the magnet, add 100 μL of 5% acetonitrile, mix well by blowing, and incubate at room temperature for 2-3 minutes. Place on the magnetic rack, wait for half a minute for the solution to become clear, and carefully pipette 100 μL of the supernatant into a new EP tube. Centrifuge at 14000 rpm for 5 minutes and add to the loading bottle.

[0167] The two detection methods corresponding to elution method 1 and elution method 2 are the same as Example 1 except for the different elution methods.

[0168] 1.3 Experimental Results

[0169] The results of different elution methods of the original solution are as follows Figure 5 shown. Figure 5 The chromatograms of the original solution with different elution methods are shown.

[0170] 1.4 Results Analysis

[0171] Under the same treatment, elution method 1 has an abnormal peak and a low main peak response, while the spectrum of elution method 2 is normal, so elution method 2 is selected.

[0172] 1.5 Experimental Conclusion

[0173] Select elution method 2 for the experiment and proceed to the next step of optimization.

[0174] 2 Gradient Optimization

[0175] 2.1 Experimental Purpose

[0176] In liquid chromatography experiments, gradient is one of the key factors. The separation results of glycoforms under different gradients may be significantly different. Therefore, this application optimizes the gradient.

[0177] 2.2 Experimental procedures

[0178] The sample selected was stock solution (batch number 202301), and the sample treatment experimental steps refer to "1 Sample pretreatment" in Example 1. The elution method was elution method 2, and the following gradients were used for analysis:

[0179] Table 1, Gradient 1

[0180]

[0181]

[0182] Table 2, Gradient 2

[0183] Time(min) Flow Rate (mL / min) A％ B％ 0.00 0.2 94.0 6.0 10.00 0.2 94.0 6.0 40.00 0.2 85.0 15.0 42.00 0.2 0.0 100.0 44.00 0.2 94.0 6.0 60.00 0.2 94.0 6.0

[0184] Table 3, Gradient 3

[0185] Time(min) Flow Rate (mL / min) A％ B％ 0.00 0.2 94.0 6.0 10.00 0.2 94.0 6.0 40.00 0.2 80.0 20.0 42.00 0.2 0.0 100.0 44.00 0.2 94.0 6.0 60.00 0.2 94.0 6.0

[0186] The three detection methods corresponding to gradient 1, gradient 2 and gradient 3 have the same operations as those in Example 1 except that the gradients are different.

[0187] 2.3 Experimental Results

[0188] The results of different gradient tests of the stock solution are as follows Figure 6 shown. Figure 6 The following are chromatograms of different gradients of the original solution.

[0189] 2.4 Results Analysis

[0190] The peak shapes of the main peaks of gradients 1 to 3 are basically the same. The main peak of gradient 2 is located later. The separation of gradient 3 is worse than that of gradient 1. The separation and retention time of gradient 1 are more similar to the registered spectrum of sample A.

[0191] 2.5 Experimental Conclusion

[0192] Select gradient 1 for the experiment and proceed to the next step of optimization.

[0193] 3 Column temperature optimization

[0194] 3.1 Experimental Purpose

[0195] In liquid chromatography experiments, the chromatographic column is one of the key factors. The temperature of the chromatographic column may affect the separation of peaks, so this application optimizes the temperature of the chromatographic column.

[0196] 3.2 Experimental procedures

[0197] The sample selected was stock solution (batch number 202301), the elution method was elution method 2, the gradient was gradient 1, and the sample treatment experimental steps were as shown in "1 Sample pretreatment" in Example 1. The column temperature was set at 25°C, 35°C, 45°C and 50°C for analysis.

[0198] The four detection methods corresponding to column temperatures of 25°C, 35°C, 45°C and 50°C are the same as those in Example 1 except for the different column temperatures.

[0199] 3.3 Experimental Results

[0200] The results of the original solution at different column temperatures are as follows Figure 7 shown. Figure 7 The chromatograms of the original solution at different column temperatures.

[0201] 2.4 Results Analysis

[0202] Different chromatographic column temperatures were set to detect the sugar types of the original solution. It was found that there were more small peaks near the main peak at 45℃ and 50℃, which was different from the spectrum of sample A. The separation of the main peaks at 25℃ and 35℃ was more similar to the spectrum of sample A, and the separation of the small peaks behind the main peak at 25℃ was better.

[0203] 2.5 Experimental Conclusion

[0204] Select the column temperature of 25°C and proceed to the next step of optimization.

[0205] 4 Glycoform identification

[0206] 4.1 Experimental Purpose

[0207] In glycoform analysis, it is necessary to characterize the glycoform corresponding to the chromatographic peak, so the present application uses liquid chromatography-mass spectrometry to identify the glycoform in the chromatogram.

[0208] 4.2 Experimental procedures

[0209] The samples selected were stock solution (batch number 202301), 3 batches of A sample purchased, and Fut8 fucose excision samples prepared by the process. The sample treatment experimental steps refer to "1 Sample pretreatment" in Example 1. This application selected elution method 2 for elution, gradient 1 was used, the column temperature was 25°C, and the mass spectrometer used was Q Exactive Plus.

[0210] 4.3 Experimental Results

[0211] Mass spectrometry identification of sugar forms in stock solution and original research Figure 8 , Fig. 9 and Fig.10 shown. Figure 8 Mass spectrometry identification of sugar forms for stock solution and original research. Fig. 9 The G1 glycoform was identified for the fucose-depleted sample. Fig.10 The chromatogram identification results.

[0212] 4.4 Results Analysis

[0213] Through QE plus mass spectrometry identification, the present application can determine that the positions of G0F, G1F, G2F, G0 and Man5 correspond to the registered standard. Since the G1 in the original research (molecule A) and the original solution (molecule B) of the present application is extremely low, the position of G1 cannot be determined. Through 2AA-RPLC mass spectrometry analysis of Fut8 fucose excision samples and confirmation by HILIC chromatography, the present application can determine that the position of G1 corresponds to the registered standard and is on the left side of G0.

[0214] Based on the above results, the 2AA-RPLC method established in this application is completely consistent with the glycoforms annotated in the standard spectrum of the registered method, and on the basis of the registered spectrum, a more in-depth study of the low-abundance glycoforms not annotated in the main peak of the registered method was conducted (such as G0FB hidden in G2F, G0F-GN hidden in the G0F peak, Man3 hidden in G0, and G0-GN hidden in G1).

[0215] 4.5 Experimental Conclusion

[0216] The spectral identification was consistent with the registration standard, and the next step of optimization was continued.

[0217] 5. Reagent volume optimization

[0218] 5.1 Experimental Purpose

[0219] In glycoform analysis, the amount of reagents may affect the final spectrum. Taking the cost of the kit into consideration, this application optimizes the amount of reagents.

[0220] 5.2 Experimental procedures

[0221] The sample selected was A molecule stock solution (batch number 202301), and the sample treatment experimental steps refer to Example 1 "1 Sample pretreatment". Elution method 2 was selected for elution, gradient 1 was used as the gradient, and the chromatographic column temperature was 25°C. This application uses halved reagent volume and standard reagent volume for optimization. The halved dosage is based on Example 1, and the dosage of each reagent in the kit used is halved.

[0222] 5.3 Experimental Results

[0223] The test results of different reagent amounts of the stock solution are as follows Fig.11 shown. Fig.11 The chromatograms are of different reagent amounts of the stock solution.

[0224] 5.4 Results Analysis

[0225] The spectra of the two are basically overlapped, and the peaks in the integral range are consistent, and the sum of the percentages of G0F+G1F+G2F is consistent. It can be seen that when testing general process samples, the amount of reagents can be halved to reduce the testing cost (testing cost after halving: 100 yuan / sample).

[0226] 5.5 Experimental Conclusion

[0227] The next step of optimization is to proceed with the standard reagent dosage. During routine testing of process samples, the reagent dosage can be halved to reduce the testing cost, and the results remain consistent with the standard reagent dosage method.

[0228] 6. Injection volume optimization

[0229] 6.1 Experimental Purpose

[0230] In liquid chromatography experiments, the injection volume is one of the key factors. The injection volume may affect the proportion of the main peak area within the integration range, so this application optimizes the injection volume.

[0231] 6.2 Experimental procedures

[0232] The sample selected was stock solution (batch number 202301), the elution method was elution method 2, the gradient was gradient 1, the column temperature was 25°C, the reagent volume was the standard reagent volume, and the sample processing experimental steps refer to the specific operation method in the summary. This application sets the injection volume to 1μL, 5μL, 10μL, 15μL and 20μL for analysis.

[0233] The five detection methods corresponding to 1 μL, 5 μL, 10 μL, 15 μL and 20 μL are the same as Example 1 except for the different injection volumes.

[0234] 6.3 Experimental Results

[0235] The test results of different injection volumes of stock solution are as follows Fig.12 , Fig.13 shown. Fig.12 The chromatograms are of different injection volumes of the original solution. Fig.13 It is the linear fitting curve of different injection amounts of stock solution and G0F peak area.

[0236] Table 4. G0F peak area and G0F+G1F+G2F percentage values ​​corresponding to different injection volumes

[0237] Injection volume (μL) G0F peak area G0F+G1F+G2F(%) 1 289.24 93.66 5 1508.99 93.32 10 3054.17 93.3 15 4576.95 93.16 20 6000.27 95.08

[0238] 6.4 Results Analysis

[0239] Different injection volumes were set for stock solution glycoform analysis. The injection volume was linear with the peak area of ​​the main peak when it was 1-20 μL. However, the retention time was later when 20 μL was injected. The G0F+G1F+G2F percentage value in the integration range was significantly higher than that of other injection volumes. The G0F+G1F+G2F percentage values ​​of other injection volumes were basically the same, and the main peak response was more similar to the registered spectrum when 10 μL was injected, so 10 μL was selected.

[0240] 6.5 Experimental Conclusion

[0241] Select the injection volume as 10 μL and proceed to the next step of optimization.

[0242] 7 Column Optimization

[0243] 7.1 Experimental Purpose

[0244] In liquid chromatography experiments, the chromatographic column is one of the key factors. Inconsistency in chromatographic column batches may affect the retention time and response of the spectrum, and thus affect the final integration results. Therefore, this application optimizes the chromatographic column.

[0245] 7.2 Experimental procedures

[0246] The sample selected was stock solution (batch number 202301), the elution method was elution method 2, the gradient was gradient 1, the column temperature was 25°C, the reagent volume was the standard reagent volume, the injection volume was 10 μL, and the sample processing experimental steps refer to the specific operation method in the summary. This application used LH-RP98 and LH-RP99 for analysis.

[0247] The two detection methods corresponding to LH-RP98 and LH-RP99 are the same as in Example 1 except for the chromatographic columns.

[0248] 7.3 Experimental Results

[0249] The results of different chromatographic columns of the original solution are as follows Fig.14 shown. Fig.14 The chromatograms of the original solution detected by different chromatographic columns.

[0250] 7.4 Results Analysis

[0251] Different chromatographic columns were used to analyze the sugar types of the stock solution, and it was found that the peak shape and the sum of the peak areas of G0F+G1F+G2F were basically the same, so both batches of chromatographic columns could be used.

[0252] 7.5 Experimental Conclusion

[0253] Both batches of chromatographic columns can be used, indicating that this method is applicable to chromatographic columns of the same model but different batches.

[0254] 8 Method Validation

[0255] 8.1 Method Validation

[0256] To ensure that this method is suitable for N-glycan detection, the stock solution used sample batch number 202301 for methodological confirmation.

[0257] 1.1 Specificity research

[0258] Use this method to detect DS buffer and stock solution (DS202301), and compare the chromatograms of buffer and stock solution.

[0259] 1.1.1 Acceptability criteria for experimental protocols

[0260] There is no obvious chromatographic peak in the blank solution within the integration range, and the sum of the peak areas of G0F, G1F, and G2F in the blank solution within the retention time is less than 0.2% of the sum of the sample areas.

[0261] 1.1.2 Experimental Results

[0262] The test results all met the system suitability standards.

[0263] The specificity research results are as follows Fig.15 , Table 5. Fig.15 Comparison of glycoform chromatograms between DS buffer and stock solution.

[0264] Table 5. Calculation table of stock solution specificity

[0265] sample Peak area of ​​G0F+G1F+G2F integration interval DS buffer 0.75 DS202301 1449.44 Area ratio (DSbuffer / DS202301) 0.05％

[0266] 1.1.3 Results Analysis

[0267] There was no obvious interfering peak in the integration range of DS buffer, and the peak area at the retention time of G0F+G1F+G2F was 0.05% of the peak area of ​​the original solution.

[0268] 1.1.4 Experimental Conclusion

[0269] This method meets the requirements for specificity verification.

[0270] 1.2 Precision-Repeatability Study

[0271] This study requires that the same experimenter prepare 6 DS202301 samples in parallel, load them for testing, and calculate the sum of the relative peak area percentages of G0F, G1F, and G2F.

[0272] 1.2.1 Experimental Acceptability Criteria

[0273] The RSD of the sum of the relative peak areas of G0F, G1F and G2F in 6 repeatability test samples was no higher than 3%.

[0274] 1.2.2 Experimental Results

[0275] The test results all met the system suitability standards.

[0276] The results of precision-repeatability study are as follows Fig.16 , Table 6. Fig.16 This is the reproducibility chromatogram of the stock solution.

[0277] Table 6. Calculation table of stock solution repeatability

[0278] sample G2F(%) G1F(%) G0F(%) G0F+G1F+G2F(%) DS202301-rep1 2.07 3.98 86.11 92.16 DS202301-rep2 2.06 3.96 86.35 92.38 DS202301-rep3 2.05 3.96 86.51 92.52 DS202301-rep4 2.03 3.91 86.42 92.36 DS202301-rep5 2.06 3.97 86.47 92.50 DS202301-rep6 2.04 3.93 86.43 92.40 Average 92.39 RSD 0.14％

[0279] 1.2.3 Results Analysis

[0280] The RSD of the sum of relative peak areas of G0F, G1F, and G2F in 6 repeatability test samples was 0.14%.

[0281] 1.2.4 Experimental Conclusion

[0282] This method meets the requirements for precision-repeatability validation.

[0283] 1.3 Linearity and range studies

[0284] The DS202301 sample was diluted to 7.5, 6.25, 5, 3.75, and 2.5 mg / mL to prepare 5 samples. The sum of the absolute peak areas of G0F, G1F, and G2F of each concentration sample was statistically analyzed. A linear fit was performed with the concentration as the horizontal axis and the sum of the absolute peak areas of G0F, G1F, and G2F as the vertical axis. The R of the fitting curve was calculated. 2 The application scope of this method is determined by using the linear experimental results.

[0285] 1.3.1 Experimental Acceptability Criteria

[0286] R 2 The linear experimental results were used to determine the application range of this method.

[0287] 1.3.2 Experimental Results

[0288] The test results all met the system suitability standards.

[0289] The results of the linear research experiment are as follows Fig.17 , Fig.18 . Fig.17 This is the linear chromatogram of the original solution. Fig.18 is the linear fitting curve of the original solution.

[0290] 1.3.3 Results Analysis

[0291] The linear fitting curve R in the range of 2.5~7.5mg / mL 2 =0.9906.

[0292] 1.3.4 Experimental Conclusion

[0293] This method meets the linearity verification requirements. According to the linearity results, the applicable range of this method is 2.5 mg / mL to 7.5 mg / mL.

[0294] 1.4 Accuracy Study

[0295] Using the linear experiment results, accuracy % = (relative peak area of ​​the experimentally measured peak) ÷ (expected relative peak area) × 100%, the expected peak area value is the percentage average (defined) of the sum of the relative peak areas of G0F, G1F, and G2F when the data are repeated 6 times.

[0296] 1.4.1 Experimental Acceptability Criteria

[0297] The sum of the relative peak areas of glycoforms G0F, G1F, and G2F is used as the detection index, and the accuracy should be between 85% and 115%.

[0298] 1.4.2 Experimental Results

[0299] The test results all met the system suitability standards.

[0300] The accuracy study experimental results are shown in Table 7.

[0301] Table 7. Calculation table of stock solution accuracy

[0302] sample G2F(%) G1F(%) G0F(%) G0F+G1F+G2F(%) Accuracy DS202301-2.5 1.91 3.72 84.49 90.11 97.54％ DS202301-3.75 2.03 3.87 85.21 91.10 98.61％ DS202301-5 1.97 3.79 86.01 91.78 99.33％ DS202301-6.25 1.90 3.73 85.60 91.24 98.75％ DS202301-7.5 1.97 3.88 84.81 90.67 98.13％

[0303] 1.4.3 Results Analysis

[0304] For five sample concentrations ranging from 2.5 to 7.5 mg / mL, the accuracy ranged from 97.54% to 99.33%.

[0305] 1.4.4 Experimental Conclusion

[0306] This method meets the accuracy verification requirements.

[0307] 1.5 Durability Study

[0308] The DS202301 sample was placed at 2-8°C for 0 hours, 24 hours and 48 hours for sample analysis. The G0F retention time and the sum of the relative peak areas of G0F, G1F and G2F were recorded. After the sample was placed for 24 hours and 48 hours, the difference in G0F retention time and relative peak area compared with 0 hours was calculated. According to formula (1) and formula (2), the difference ΔT in G0F retention time and the sum of the relative peak areas of G0F, G1F and G2F were calculated.

[0309] |△T|=|AB|(1), where A=0 hour G0F retention time; B=24 hour, 48 hour G0F retention time. Peak area % difference percentage (D%)=(|DC| / C)×100%(2), where C=0 hour G0F peak area %; D=24 hour, 48 hour G0F peak area %.

[0310] 1.5.1 Experimental Acceptability Criteria

[0311] Compared with the 0-hour sample, after the sample was placed at 2-8°C for 24 hours and 48 hours, the RSD of the sum of the relative peak areas of G0F, G1F, and G2F was no higher than 3%, and the retention time of G0F was less than 0.5 min.

[0312] 1.5.2 Experimental Results

[0313] The test results all met the system suitability standards.

[0314] Durability study test results are as follows Fig.19 , Table 8. Fig.19 This is the durability chromatogram of the stock solution.

[0315] Table 8. Calculation table of the durability of the original solution

[0316] sample G2F% G1F% G0F% G0F+G1F+G2F% Peak area % difference G0F ΔRT(min) DS202301-rep1 2.07 3.98 86.11 92.16 29.09 DS202301-rep1_24h 2.05 3.97 86.51 92.53 0.40％ 29.06 0.03 DS202301-rep1_48h 2.06 3.99 86.54 92.59 0.47％ 29.00 0.09

[0317] 1.5.3 Results Analysis

[0318] The differences in peak area after 24 h and 48 h were 0.4% and 0.47%, and the differences in retention time were 0.03 min and 0.09 min, respectively.

[0319] 1.5.4 Experimental Conclusion

[0320] This method meets the durability verification requirements.

[0321] 8.2 Conclusion

[0322] Based on the above research, this application finally determined that the elution method is elution method 2, the gradient is gradient 1, the column temperature is 25°C, the reagent volume is the standard reagent volume, the injection volume is 10 μL, and LH-RP98 and LH-RP99 columns are used for analysis, and condition confirmation experiments are carried out on the above parameters.

[0323] Through methodological pre-validation, the system suitability standards are determined as follows:

[0324] 1) The chromatogram of the system suitability reference is similar to the reference chromatogram.

[0325] 2) The RSD of the fucosylated glycan results of the system suitability reference must be ≤2.0%.

[0326] 3) The reagent blank must have no significant interference at the retention time of any relevant chromatographic peak.

[0327] Analytical Validity Criteria: Replicate results for % fucosylated glycans obtained for the System Suitability Control must have an RSD of ≤2.0% and the overall appearance of the chromatograms must be similar to each other.

[0328] The above conclusions are shown in Table 9:

[0329] Table 9. Summary of the development of glycan analysis detection methods

[0330]

[0331] 8.3 Final Conclusion

[0332] Based on the above research, the method development for sugar form detection was carried out by the laboratory of China Resources Biopharmaceutical Analysis and Quality Control Center, and the recommended detection method parameters are as follows:

[0333] Table 10

[0334] Experimental conditions Method or parameter Elution method Elution method 2 gradient Gradient 1 Column temperature 25℃ Reagent volume Standard reagent volume (method development) halved reagent volume (routine testing) Injection volume 10μL Gain 12 (Beijing); 10 (Shandong)

[0335] After methodological verification, the specificity, precision (repeatability), linearity and range, accuracy and durability of the method all met the verification requirements. Finally, the chromatogram of the original solution after Shandong method verification was compared with the registration standard. Fig.19 The sugar form peaks and separation conditions of the optimized stock solution chromatogram corresponded one to one with the registration standard, proving that the method was successfully developed and can be used for stock solution release testing.

[0336] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0337] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A method for detecting antibody N-glycoforms, characterized in that: The detection method comprises the following steps: Qualitative detection of N-glycoforms of the antibody under test by mass spectrometry; and, Reverse phase chromatography was used to quantitatively detect the qualitatively detected N-glycoforms.

2. The method for detecting antibody N-glycoforms according to claim 1, characterized in that: The conditions of the reverse phase chromatography include: The mobile phase includes mobile phase A and mobile phase B. The mobile phase A comprises water and trifluoroacetic acid in a volume ratio of 0.1% to 0.2%, The mobile phase B comprises water, trifluoroacetic acid accounting for 0.1% to 0.2% by volume, and acetonitrile accounting for 85% to 95% by volume.

3. The method for detecting antibody N-glycoforms according to claim 2, characterized in that: The conditions of the reverse phase chromatography also include: Adopt gradient elution method; Optionally, the gradient elution procedure includes: 0min~10min: the volume proportion of the mobile phase B is maintained at 6%; 10min-40min: the volume proportion of the mobile phase B increases from 6% to 18%; 40min-42min: the volume proportion of the mobile phase B increases from 18% to 100%; 42min-44min: the volume proportion of the mobile phase B decreases from 100% to 6%; 44min-60min: the volume proportion of the mobile phase B is maintained at 6%.

4. The method for detecting antibody N-glycoforms according to claim 1, characterized in that: The conditions of the reverse phase chromatography also include: Column temperature is 22℃~27℃; The injection volume is 5μL to 15μL; or / and, The detection wavelengths are 225-235nm and 420nm-430nm.

5. The method for detecting antibody N-glycoforms according to claim 1, characterized in that: The conditions of the reverse phase chromatography method include: the reverse phase chromatography column uses silica gel as a filling matrix, and the specifications are a diameter of 2.1 mm, a length of 150 mm, and a particle size of 2.6 μm.

6. The method for detecting antibody N-glycoforms according to claim 1, characterized in that: The mass spectrometry conditions include: The positive ion Full MS-SIM scanning mode was used. Microscans is set to 10, Resolution is set to 17500, AGC target is set to 3e6, Maximum IT is set to 200ms, Scan range is set to 200-2500, Sheath gas flow (arb) is set to 35, Aux gas flow rate (arb) is set to 10, Sweep gas flow rate (arb) is set to 0, The spray voltage (kV) is set to 3.8, Capillary temperature (℃) is set to 320, and S-lens RF level is set to 50.

7. The method for detecting antibody N-glycoforms according to any one of claims 1 to 6, characterized in that: The antibody to be tested includes monoclonal antibody.

8. The method for detecting antibody N-glycoforms according to claim 7, characterized in that: The monoclonal antibody includes IgG class antibodies.

9. The method for detecting antibody N-glycoforms according to claim 8, characterized in that: The IgG class antibodies include IgG4 antibodies.

10. The method for detecting antibody N-glycoforms according to any one of claims 1 to 6 and 8 to 9, characterized in that: The antibody to be tested is pretreated by 2-AA method before detection.

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