Method for detecting glycoform content of antibody based on liquid chromatography-mass spectrometry

Abstract

The invention provides a method for detecting the glycoform content of an antibody based on liquid chromatography-mass spectrometry, which comprises the following steps: (1) carrying out reduction reaction on an antibody sample to be detected and a reducing agent to obtain an antibody reduction subunit solution; and (2) detecting the antibody reduction subunit solution by adopting ultra-high performance liquid chromatography-mass spectrometry, and calculating to obtain the glycoform content of the antibody. According to the detection method provided by the invention, the sample pretreatment process is simple and rapid, the ultra-high performance liquid chromatography-mass spectrometer is also very efficient in determination, the detection time of a single sample is greatly shortened, the detection flux is improved, and strong and powerful support is provided for product development of biological drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical analysis and detection, and specifically relates to a method for detecting the glycoform content of an antibody based on liquid chromatography-mass spectrometry, and in particular to a method for rapidly quantifying the glycosylation modification of an antibody using an ultra-high performance liquid chromatography-mass spectrometry instrument, and in particular to a method for rapidly quantifying the glycosylation modification of a monospecific antibody, a bispecific antibody, and a heavy chain antibody. Background Art

[0002] Glycosylation is a very important post-translational modification of proteins, which can be divided into N-glycosylation and O-glycosylation according to the modification site. Currently, most of the antibody drugs on the market have undergone N-glycosylation modification, located at Asn-297. Since glycosylation modification affects the properties of antibodies, such as conformation, stability, pharmacokinetics, activity and immunogenicity, glycosylation modification is one of the key quality attributes for evaluating antibody drug products.

[0003] Glycosylation modification is completed in the Golgi apparatus within the cell, so antibodies expressed by mammalian cells are greatly affected by the cell growth environment. In the process of antibody drug development, the antibody production is improved by optimizing the cell culture conditions. Therefore, under different culture conditions, the glycosylation modification of antibodies will also vary greatly, requiring a stable and reliable method for timely monitoring and analysis.

[0004] Currently, the hydrophilic interaction chromatography included in the 2020 edition of the Chinese Pharmacopoeia is the most widely used quantitative analysis method for glycosylation modification, which is to first use N-glycosidase F (PNGase F) to enzymatically cut N-sugar from the antibody (≥20h), then use ice ethanol for protein precipitation, and then use 2-aminobenzamide (2-AB) for labeling (≥4h), and then use solid phase extraction for purification, and finally use a hydrophilic interaction chromatography column for separation and detection on a liquid chromatography-coupled fluorescence detector (1h). The operation steps of this method are cumbersome and complicated. Not only are the reagents and consumables used expensive, but the detection cycle is also long.

[0005] CN109358131B discloses a rapid analysis method for monoclonal antibody N-sugar glycoforms, that is, the antibody is first cut into smaller fragments using IdeS enzyme, then a reducing agent is added for reduction reaction, and finally a chip capillary electrophoresis coupled with a mass spectrometer is used for detection and analysis. This method introduces enzyme cleavage, which is not only expensive in cost, but also requires strict and careful operation. The activity of the enzyme will affect the effect of enzyme cleavage. In addition, it also needs to rely on a mass spectrometry-compatible chip capillary electrophoresis instrument that is new in technology and has not yet been widely used.

[0006] Therefore, how to provide a method for rapid quantification of glycosylation modification of antibodies has become an urgent problem to be solved. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for detecting the glycoform content of antibodies based on liquid chromatography-mass spectrometry. The method provided by the present invention has the advantages of simple pretreatment, short detection cycle, low cost, etc., which greatly helps the product development of antibody drugs, and is particularly suitable for the cell line development process and cell culture optimization process.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for detecting antibody glycoform content based on liquid chromatography-mass spectrometry, the method comprising the following steps:

[0010] (1) subjecting the antibody sample to be tested to a reduction reaction with a reducing agent, terminating the reaction, and obtaining an antibody reduced subunit sample;

[0011] (2) The antibody reduced subunit sample is detected by ultra-high performance liquid chromatography-mass spectrometry, and the antibody glycoform content is obtained through calculation.

[0012] In the present invention, in step (1), before the antibody sample to be tested and the reducing agent undergo reduction reaction, the antibody sample to be tested is also centrifuged and diluted, specifically comprising: centrifuging the antibody sample to be tested, taking the supernatant, and diluting it to obtain a diluted antibody sample to be tested.

[0013] Preferably, the solvent used for the dilution includes ultrapure water.

[0014] Preferably, the concentration of the antibody in the diluted antibody sample to be tested is 0.5-2.0 mg / mL (for example, it can be 0.5 mg / mL, 1 mg / mL, 1.3 mg / mL, 1.6 mg / mL, 1.9 mg / mL, 2 mg / mL, etc.), preferably 0.8-1.0 mg / mL (for example, it can be 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, etc.).

[0015] In the present invention, in step (1), the reducing agent includes any one of dithiothreitol (DTT), β-mercaptoethanol (β-Me), tris(2-carboxyethyl)phosphine (TCEP) or mercaptoethylamine, or a combination of at least two thereof, preferably dithiothreitol.

[0016] Preferably, in step (1), the concentration of the reducing agent in the reaction system for the reduction reaction is 10-50 mM (for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, etc.).

[0017] Preferably, in step (1), the reduction reaction temperature is 25-60°C (for example, 25°C, 35°C, 37°C, 45°C, 55°C, 60°C, etc.), and the time is 20-120min (for example, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min, etc.), preferably 30-60min (for example, 30min, 35min, 40min, 45min, 50min, 55min, 60min, etc.).

[0018] In the present invention, in step (1), the termination reaction is carried out using an aqueous formic acid solution.

[0019] Preferably, the volume fraction of formic acid in the formic acid aqueous solution is 5%-20% (for example, it can be 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 20%, etc.).

[0020] In the present invention, in step (2), the injection volume in the ultra-high performance liquid chromatography detection is 1-5 μL (for example, it can be 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, etc.), preferably 1-2 μL (for example, it can be 1 μL, 1.2 μL, 1.4 μL, 1.6 μL, 1.8 μL, 2 μL, etc.).

[0021] Preferably, in step (2), the chromatographic column used in the ultra-high performance liquid chromatography detection includes Waters BioResolve TM RP mAb Polyphenyl column or other equivalent alternative columns.

[0022] Preferably, in step (2), the column temperature of the chromatographic column in the ultra-high performance liquid chromatography detection is 60-80°C (for example, it can be 60°C, 63°C, 66°C, 69°C, 73°C, 76°C, 79°C, 80°C, etc.).

[0023] In the present invention, in step (2), the detector used in the ultra-high performance liquid chromatography detection is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 280±2nm.

[0024] In the present invention, in step (2), in the ultra-high performance liquid chromatography detection, formic acid aqueous solution is used as mobile phase A, and formic acid acetonitrile solution is used as mobile phase B.

[0025] Preferably, the volume fraction of formic acid in the formic acid aqueous solution is 0.05%-0.1% (for example, it may be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc.).

[0026] Preferably, the volume fraction of formic acid in the formic acid-acetonitrile solution is 0.05%-0.1% (for example, it may be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc.).

[0027] Preferably, in step (2), gradient elution is used in the ultra-high performance liquid chromatography detection, and the gradient of the elution is:

[0028] 0-8min, 25%-45% (for example, 25%, 27%, 29%, 33%, 36%, 39%, 42%, 45%, etc.) mobile phase B, flow rate of 0.2-0.3mL / min (for example, 0.2mL / min, 0.23mL / min, 0.26mL / min, 0.29mL / min, 0.3mL / min, etc.);

[0029] 8-10 min, 45%-80% (for example, 45%, 55%, 65%, 75%, 80%, etc.) mobile phase B, flow rate of 0.2-0.45 mL / min (for example, 0.2 mL / min, 0.23 mL / min, 0.26 mL / min, 0.29 mL / min, 0.3 mL / min, 0.32 mL / min, 0.35 mL / min, 0.39 mL / min, 0.41 mL / min, 0.43 mL / min, 0.45 mL / min, etc.);

[0030] 10-11min, 25%-80% (for example, it can be 25%, 35%, 45%, 55%, 65%, 75%, 80%, etc.) mobile phase B, and the flow rate is 0.35-0.45mL / min (for example, it can be 0.35mL / min, 0.39mL / min, 0.41mL / min, 0.43mL / min, 0.45mL / min, etc.).

[0031] Alternatively, gradient elution is used in the detection of the ultra-high performance liquid chromatography, and the gradient of the elution is:

[0032] 0-3min, 25%-60% (for example, 25%, 35%, 45%, 55%, 60%, etc.) mobile phase B, flow rate of 0.45-0.55mL / min (for example, 0.45mL / min, 0.47mL / min, 0.49mL / min, 0.51mL / min, 0.53mL / min, 0.55mL / min, etc.);

[0033] 3-4 min, 60%-80% mobile phase B (for example, 60%, 63%, 66%, 69%, 73%, 76%, 79%, 80%, etc.), flow rate 0.45-0.55 mL / min (for example, 0.45 mL / min, 0.47 mL / min, 0.49 mL / min, 0.51 mL / min, 0.53 mL / min, 0.55 mL / min, etc.);

[0034] 4-5min, 25%-80% (for example, it can be 25%, 35%, 45%, 55%, 65%, 75%, 80%, etc.) mobile phase B, and the flow rate is 0.45-0.55mL / min (for example, it can be 0.45mL / min, 0.47mL / min, 0.49mL / min, 0.51mL / min, 0.53mL / min, 0.55mL / min, etc.).

[0035] Preferably, in step (2), gradient elution is used in the ultra-high performance liquid chromatography detection, and the gradient of the elution is:

[0036] time Flow rate mL / min A％ B％ 0.0 0.2-0.3 70-80 30-20 2.0 0.2-0.3 70-80 30-20 8.0 0.2-0.3 50-60 50-40 8.1 0.35-0.45 15-25 85-75 10.0 0.35-0.45 15-25 85-75 10.1 0.35-0.45 70-80 30-20 11.0 0.35-0.45 70-80 30-20

[0037] In the present invention, in step (2), in the mass spectrometry detection, a positive ion mode is adopted, the temperature of the ion transfer tube is 310-330°C (for example, it can be 310°C, 315°C, 320°C, 325°C, 330°C, etc.), and the m / z range is 600-3000.

[0038] In the present invention, the calculation specifically includes the following steps:

[0039] The antibody reduced subunit solution was detected by ultra-high performance liquid chromatography-mass spectrometry to obtain a total ion current chromatogram; the mass chromatogram of the antibody heavy chain was extracted, and then each mass number and the corresponding ion signal intensity were obtained after deconvolution processing. Finally, the ion signal intensity was normalized and the proportion of the ion signal intensity of each mass number was calculated. The proportion of the ion signal intensity of each mass number is the content of different glycoforms.

[0040] In the present invention, the antibody includes any one of a monospecific antibody, a bispecific antibody or a heavy chain antibody, or a combination of at least two of them.

[0041] The antibodies described in the present invention are all of IgG1 type, and have only one conserved N-glycosylation site in their Fc segment.

[0042] The method for detecting antibody glycoform content based on liquid chromatography-mass spectrometry provided by the present invention comprises the steps of antibody sample pretreatment and ultra-high performance liquid chromatography-mass spectrometry measurement.

[0043] As a preferred technical solution of the present invention, the method for detecting the content of monospecific antibody glycoforms based on liquid chromatography-mass spectrometry comprises the following specific steps:

[0044] (1) centrifuging the antibody sample to be tested, taking the supernatant, and diluting it to obtain a diluted antibody sample to be tested;

[0045] Wherein, the solvent used for the dilution includes ultrapure water, and the concentration of the antibody in the antibody sample to be tested after the dilution is 0.5-2.0 mg / mL;

[0046] (2) subjecting the diluted antibody sample to be tested to a reduction reaction with a reducing agent to obtain an antibody reduced subunit solution;

[0047] Wherein, the reducing agent includes any one of dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine or mercaptoethylamine or a combination of at least two thereof, the concentration of the reducing agent in the reaction system for the reduction reaction is 10-50 mM, the temperature of the reduction reaction is 25-60° C., and the time is 20-120 min;

[0048] (3) The antibody reduced subunit solution is detected by ultra-high performance liquid chromatography-mass spectrometry to obtain a total ion current chromatogram; then the mass chromatogram of the antibody heavy chain is extracted, and each mass number and the corresponding ion signal intensity are obtained by deconvolution processing, and finally the ion signal intensity is normalized and the ion signal intensity is calculated to obtain the ion signal intensity ratio of each mass number. Among them, the antibody heavy chain has a corresponding theoretical mass number after different glycosylation modifications, which can be used to attribute each mass number measured by mass spectrometry, and the ion signal intensity ratio of the mass number is the ratio of glycosylation modification;

[0049] The parameters of the ultra-high performance liquid chromatography detection are as follows: the injection volume is 1-5 μL; the chromatographic column includes Waters BioResolve TMRP mAb Polyphenyl chromatographic column or equivalent alternative chromatographic column, column temperature is 60-80°C; UV detector is used, and the detection wavelength of the UV detector is 280nm; 0.05%-0.1% formic acid aqueous solution is used as mobile phase A, and 0.05%-0.1% formic acid acetonitrile solution is used as mobile phase B; gradient elution is used, and the elution gradient is: 0-8min, 25%-45% mobile phase B, flow rate is 0.2-0.3mL / min; 8-10min, 45%-80% mobile phase B, flow rate is 0 .35-0.45mL / min; 10-11min, 25%-80% mobile phase B, flow rate is 0.2-0.3mL / min; or, the elution gradient is: 0-3min, 25%-60% mobile phase B, flow rate is 0.45-0.55mL / min; 3-4min, 60%-80% mobile phase B, flow rate is 0.45-0.55mL / min; 4-5min, 80%-25% mobile phase B, flow rate is 0.45-0.55mL / min.

[0050] The parameters of the mass spectrometer detection are as follows: positive ion mode is adopted, the temperature of the ion transfer tube is 310-330° C., and the m / z range is 600-3000.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The sample pretreatment steps are simplified. The sample only needs to be simply diluted and reduced. The entire sample preparation process can be completed within 1 hour, which greatly improves the analysis throughput and the degree of automation of the analysis, avoids complicated derivatization or enzyme cutting operations, and avoids potential human errors or mistakes.

[0053] (2) In the ultra-high performance liquid chromatography-mass spectrometry method, the sample loading amount is only at the microgram level, so only a small amount of sample is needed to achieve the detection purpose.

[0054] (3) The time for UPLC-MS to detect samples is 10 minutes, and it only takes 2 hours from sample pretreatment to reporting results, which greatly improves the detection efficiency. The high sensitivity of UPLC-MS technology provides strong support for cell line development and optimization of cell culture conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The results of the reduced HC mass number, glycoforms and ratio of the monospecific antibody after deconvolution measured by the method provided in Example 1.

[0056] Figure 2 The results of the reduced HC mass, glycoforms and ratios of the heavy chain antibody after deconvolution measured by the method provided in Example 2.

[0057] Figure 3 The results of the reduced HC mass, glycoforms and ratio of the bispecific antibody after deconvolution measured by the method provided in Example 3. DETAILED DESCRIPTION

[0058] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0059] In the following examples, unless otherwise specified, the reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0060] The Kabat (light chain variable region) amino acid sequence of the monospecific antibody sample in Example 1 is shown in SEQ ID NO:1, and the Kabat (heavy chain variable region) amino acid sequence is shown in SEQ ID NO:2.

[0061] SEQ ID NO: 1:

[0062] DIQMTQSPSSSLSASVGDRVTITCKASQDINTYLTWFQQKPGKAPKSLIYRANILVDGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCLQYDEFPLTFGGGTKVEIK.

[0063] SEQ ID NO:2:

[0064] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQGLEWMGRINPYNG ATTYNQNFKDRVTMTVDKSTSTAYMELRSLRSDDTAVYYCARFHYGAPDYWGQGTLVTV SS.

[0065] The light chain variable region and heavy chain variable region were constructed into the eukaryotic expression vector of IgG1, respectively, and transfected into CHO-K1 cells. After culturing for 3-7 days, the supernatant was collected and purified with a protein A column to obtain the antibody protein.

[0066] The amino acid sequence of the heavy chain variable region of the heavy chain antibody (Nanobody) in Example 2 is shown in SEQ ID NO:3.

[0067] SEQ ID NO:3:

[0068] QVQLVESGGGLVQPGGSLRLSCAASGSIFNIPVMGWYRQAPGKQRELVAGISTGGTTN YGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVLVVSGIGSTLEVWGQGTLVTV SS.

[0069] The heavy chain variable region is constructed into the eukaryotic expression vector of IgG1, transfected into CHO-K1 host cells, and the supernatant is collected after culturing for 3-7 days, and purified with a protein A column to obtain the antibody protein.

[0070] The amino acid sequence of the light chain variable region of the antigen binding site one of the bispecific antibody in Example 3 is shown in SEQ ID NO: 4, the amino acid sequence of the heavy chain variable region of the antigen binding site one is shown in SEQ ID NO: 5, and the amino acid sequence of the heavy chain variable region of the antigen binding site two is shown in SEQ ID NO: 6.

[0071] SEQ ID NO:4:

[0072] DIQMTQSPSSSLSASVGDRVTITCRASQDISNHLNWYQQKPGKAPKLLIYYTSRIHSGVP SSFRGSGSGTDYTLTISSLQPEDIATYFCQQGYTLPFTFGSGTKLEIK.

[0073] SEQ ID NO:5:

[0074] QVQLVQSGAEVKKPGASVKVSCKASGFNIKDIYIYWVRQAPGQGLEWIGKIDPANGN TKYDQKFQGRATITADTSTNTAYLELSSLRSEDTAVYYCARGYGSGFAYWGQGTLVTVSS.

[0075] SEQ ID NO:6:

[0076] QVQLVESGGGLVQPGGSLRLSCAASGSIFNIPVMGWYRQAPGKQRELVAGISTGGTTNYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNVLVVSGIGSTLEVWGQGTLVTVSS.

[0077] The variable regions of the above light and heavy chains were constructed into eukaryotic expression vectors of IgG1, respectively, and transfected into CHO-K1 cells. After culturing for 3-7 days, the supernatant was collected and purified with a protein A column to obtain the antibody protein.

[0078] The monospecific antibodies, heavy chain antibodies and bispecific antibodies used in the following examples are all products independently prepared by our company.

[0079] Example 1

[0080] This embodiment provides a method for detecting the content of monospecific antibody glycoforms based on liquid chromatography-mass spectrometry, and the specific steps are as follows:

[0081] (1) Pretreatment: Centrifuge the monospecific antibody sample at 10,000 rpm for 2 min, take the supernatant, use NanoDrop micro-volume UV-visible spectrophotometer to determine the antibody concentration, and then use ultrapure water to dilute the antibody sample to 1.0 mg / mL. Add 1.0 μL of 1M DTT solution to 50 μL of the diluted antibody solution to a final concentration of 20 mM / L, vortex to mix evenly, incubate in a 37°C water bath for 1 h, and add 2 μL of 10% formic acid solution to terminate the reaction. Prepare the reduced LC and HC subunit solutions.

[0082] (2) Ultra-high performance liquid chromatography-mass spectrometry detection:

[0083] The ultra-high performance liquid chromatograph was Thermo Ultimate 3000, and the high-resolution mass spectrometer was Thermo Q-Exactive.

[0084] 1.0 μL of the LC and HC subunit solutions treated with monospecific antibodies were injected respectively, and the samples were separated by ultra-high performance liquid chromatography and then detected by high-resolution mass spectrometer. The mass spectrometer was collected in primary scanning mode. The ultra-high performance liquid chromatography parameters were set as follows:

[0085] The parameters of the ultra-high performance liquid chromatography are shown in Table 1 below:

[0086] Table 1

[0087]

[0088]

[0089] The formic acid and acetonitrile were of mass spectrometry grade.

[0090] The high-resolution mass spectrometer parameter settings are shown in Table 2 below:

[0091] Table 2

[0092] Mass spectrometry parameters Parameter Value Scan Type Full MS Scan range 600 to 3000 m / z Resolution 17500 Scan Mode Positive In-source cracking energy 0eV Mass spectrum overlay quantity 5 Automatically obtain control values 3e6 Maximum ion implantation time 200ms

[0093] (3) Data processing: The Intact Protein Analysis module of the BioPharma Finder software was used to process the data. The main N-sugar glycoform modification type was determined based on the mass numbers of antibody molecules with various glycoform modifications given by the BioPharma Finder software. The ion signal intensity was normalized and calculated to obtain the ion signal intensity ratio of each mass number. Among them, the antibody HC has a corresponding theoretical mass number after different glycosylation modifications, which can be used to attribute the mass numbers determined by mass spectrometry. The ion signal intensity ratio of the mass number is the ratio of glycosylation modification.

[0094] The same monospecific antibody was analyzed using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled to a fluorescence detector detection method, and the detection results were compared. The results of the main glycoforms and their ratios of the monospecific antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography method are shown in Table 3 below:

[0095] Table 3

[0096]

[0097] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 3 are the results of three replicates of the monospecific antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0098] The 2-AB labeling results in Table 3 are the results of a single experiment.

[0099] The deviations of the two methods in Table 3 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0100] Figure 1 The results of the reduced HC mass number, glycoforms and ratio of the monospecific antibody after deconvolution measured by the method provided in Example 1.

[0101] Example 2

[0102] This example provides a method for detecting the glycoform content of heavy chain antibodies based on liquid chromatography-mass spectrometry. The only difference from Example 1 is that the monospecific antibody is replaced by a heavy chain antibody, and the other steps are the same as Example 1.

[0103] The same heavy chain antibody was analyzed and the detection results were compared using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography is shown in Table 4 below:

[0104] Table 4

[0105]

[0106] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 4 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0107] The 2-AB labeling results in Table 4 are the results of a single experiment.

[0108] The deviations of the two methods in Table 4 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0109] Figure 2 The results of the reduced HC mass number, glycoform and ratio of the heavy chain antibody after deconvolution measured by the method provided in Example 2.

[0110] Example 3

[0111] This example provides a method for detecting the glycoform content of a bispecific antibody based on liquid chromatography-mass spectrometry. The only difference from Example 1 is that the monospecific antibody is replaced by a bispecific antibody, and the other steps are the same as Example 1.

[0112] The same bispecific antibody was analyzed using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled to a fluorescence detector detection method, and the detection results were compared. The results of the main glycoforms and their ratios of the bispecific antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography method are shown in Table 5 below:

[0113] Table 5

[0114]

[0115] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 5 are the results of three replicates of the bispecific antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation. The 2-AB labeling method results in Table 5 are the results of a single experiment.

[0116] The deviations of the two methods in Table 5 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0117] Figure 3 The results of the reduced HC mass number, glycoforms and ratio of the bispecific antibody after deconvolution measured by the method provided in Example 3.

[0118] As can be seen from the data in Tables 3 to 5, the present invention can quickly measure the glycosylation modification and content results of different types of antibody samples, and the detection results are close to the results of 2-AB labeling high performance liquid chromatography. The method provided by the present invention has simple pretreatment, short detection cycle, low cost, and accurate results. It has high application value for detecting glycosylation modification of antibodies at different research and development stages, and is particularly suitable for cell line development and cell culture optimization processes.

[0119] Example 4

[0120] This example provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Example 1 only in that the Waters BioResolve TM The RP mAb Polyphenyl column was replaced with a Waters ACQUITYUPLC Protein BEH C4 Column, and the other steps were the same as in Example 1.

[0121] The same heavy chain antibody was analyzed and the detection results were compared using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography is shown in Table 6 below:

[0122] Table 6

[0123]

[0124]

[0125] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 6 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0126] The 2-AB labeling results in Table 6 are the results of a single experiment.

[0127] The deviations of the two methods in Table 6 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0128] Example 5

[0129] This embodiment provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Example 1 only in that the flow rate of 0-8 min in the ultra-high performance liquid chromatography elution process is changed to 0.40 mL / min, and the other steps are the same as Example 1.

[0130] The same heavy chain antibody was analyzed and the detection results were compared using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography is shown in Table 7 below:

[0131] Table 7

[0132]

[0133] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 7 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0134] The 2-AB labeling results in Table 7 are the results of a single experiment.

[0135] The deviations of the two methods in Table 7 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0136] Example 6

[0137] This embodiment provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Example 1 only in that the flow rate of 0-8 min in the ultra-high performance liquid chromatography elution process is changed to 0.25 mL / min, and the other steps are the same as Example 1.

[0138] The same heavy chain antibody was analyzed and the detection results were compared using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector. The comparison of the results of the main glycoforms and their ratios of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography is shown in Table 8 below:

[0139] Table 8

[0140]

[0141] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 8 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0142] The 2-AB labeling results in Table 8 are the results of a single experiment.

[0143] The deviations of the two methods in Table 8 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0144] Example 7

[0145] This embodiment provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Embodiment 1 only in that the column temperature of the liquid chromatography column is changed to 55° C., and the other steps are the same as Embodiment 1.

[0146] The ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with fluorescence detector detection method were used to analyze the same heavy chain antibody and compare the detection results. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected by ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography method is shown in Table 9 below:

[0147] Table 9

[0148]

[0149]

[0150] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 9 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0151] The 2-AB labeling results in Table 9 are the results of a single experiment.

[0152] The deviations of the two methods in Table 9 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0153] Example 8

[0154] This embodiment provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Embodiment 1 only in that the column temperature of the liquid chromatography column is changed to 85° C., and the other steps are the same as Embodiment 1.

[0155] The same heavy chain antibody was analyzed and the detection results were compared using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography is shown in Table 10 below:

[0156] Table 10

[0157]

[0158] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 10 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0159] The 2-AB labeling results in Table 10 are the results of a single experiment.

[0160] The deviations of the two methods in Table 10 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0161] Example 9

[0162] This embodiment provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Embodiment 1 only in that the injection volume in ultra-high performance liquid chromatography detection is changed to 0.5 μL, and the other steps are the same as Embodiment 1.

[0163] The ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with fluorescence detector detection method were used to analyze the same heavy chain antibody and compare the detection results. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected by ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography method is shown in Table 11 below:

[0164] Table 11

[0165]

[0166] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 11 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0167] The 2-AB labeling results in Table 11 are the results of a single experiment.

[0168] The deviations of the two methods in Table 11 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0169] Example 10

[0170] This embodiment provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Embodiment 1 only in that the injection volume in ultra-high performance liquid chromatography detection is changed to 2.5 μL, and the other steps are the same as Embodiment 1.

[0171] The same heavy chain antibody was analyzed using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector detection method, and the detection results were compared. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography method is shown in Table 12 below:

[0172] Table 12

[0173]

[0174] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 12 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0175] The 2-AB labeling results in Table 12 are the results of a single experiment.

[0176] The deviations of the two methods in Table 12 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0177] Embodiment 11

[0178] This example provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Example 1 only in that the elution gradient is as follows:

[0179]

[0180] The other steps are the same as in Example 1.

[0181] The same heavy chain antibody was analyzed using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector detection method, and the detection results were compared. The comparison of the results of the main glycoforms and their ratios of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography method is shown in Table 13 below:

[0182] Table 13

[0183]

[0184] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 13 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0185] The 2-AB labeling results in Table 13 are the results of a single experiment.

[0186] The deviations of the two methods in Table 13 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0187] Example 12

[0188] This example provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Example 1 only in that the elution gradient is as follows:

[0189]

[0190]

[0191] The other steps are the same as in Example 1.

[0192] The same heavy chain antibody was analyzed and the detection results were compared using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography is shown in Table 14 below:

[0193] Table 14

[0194]

[0195] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 14 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0196] The 2-AB labeling results in Table 14 are the results of a single experiment.

[0197] The deviations of the two methods in Table 14 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0198] Embodiment 13

[0199] This example provides a method for detecting the glycoform content of a monospecific antibody based on liquid chromatography-mass spectrometry, which is different from Example 1 only in that the elution gradient is as follows:

[0200]

[0201]

[0202] The other steps are the same as in Example 1.

[0203] The same heavy chain antibody was analyzed using the ultra-high performance liquid chromatography-mass spectrometry technology provided by the present invention and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector detection method, and the detection results were compared. The comparison of the results of the main glycoforms and their proportions of the heavy chain antibody detected using ultra-high performance liquid chromatography-mass spectrometry technology and the traditional 2-AB labeling high performance liquid chromatography method is shown in Table 15 below:

[0204] Table 15

[0205]

[0206] The ultra-high performance liquid chromatography-mass spectrometry detection results in Table 15 are the results of three replicates of heavy chain antibody LC and HC subunit solutions using ultra-high performance liquid chromatography-mass spectrometry, and the final results are reported as mean ± standard deviation.

[0207] The 2-AB labeling results in Table 15 are the results of a single experiment.

[0208] The deviations of the two methods in Table 15 are the deviations of the detection results of ultra-high performance liquid chromatography-mass spectrometry technology compared with the detection results of 2-AB labeling method high performance liquid chromatography.

[0209] Test Case

[0210] The methods provided in Examples 1 and 4-13 and the traditional 2-AB labeling high performance liquid chromatography coupled with a fluorescence detector detection method were used to analyze the same monospecific antibody and compare the detection results.

[0211] The methods provided in Example 1 and Examples 4-13 were repeated 3 times and the average value was taken; the results of the 2-AB labeling method were the results of a single experiment. For the contents of different glycoforms of G0, G0F, G1F and G2F, the deviations between the average values ​​determined by the methods provided in Example 1 and Examples 4-13 and the results of the 2-AB labeling method were calculated respectively.

[0212] The statistical results of the deviation calculation of the methods provided in Example 1 and Examples 4-13 and the traditional 2-AB labeling high performance liquid chromatography are shown in Table 16 below:

[0213] Table 16

[0214]

[0215]

[0216] It can be seen from the data in Table 16 that the type of chromatographic column, elution flow rate, column temperature, injection volume and elution gradient in ultra-high performance liquid chromatography-mass spectrometry detection will affect the accuracy of the detection results.

[0217] The present invention can quickly measure the glycosylation modification and content results of different types of antibody samples, and the glycoform results are close to the results of 2-AB labeling high performance liquid chromatography. The method provided by the present invention has simple pretreatment, short detection cycle, low cost, and accurate results. It has high application value for detecting the glycosylation modification of antibodies at different research and development stages, and is particularly suitable for cell line development and cell culture optimization processes.

[0218] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting antibody glycoform content based on liquid chromatography-mass spectrometry, characterized in that: The method comprises the following steps: (1) subjecting the antibody sample to be tested to a reduction reaction with a reducing agent, terminating the reaction, and obtaining an antibody reduced subunit sample; (2) The antibody reduced subunit sample is detected by ultra-high performance liquid chromatography-mass spectrometry, and the antibody glycoform content is obtained through calculation.

2. The method according to claim 1, characterized in that In step (1), before the antibody sample to be tested and the reducing agent undergo a reduction reaction, the antibody sample to be tested is also centrifuged and diluted, specifically comprising: centrifuging the antibody sample to be tested, taking the supernatant, and diluting it to obtain a diluted antibody sample to be tested; Preferably, the solvent used for the dilution includes ultrapure water; Preferably, the concentration of the antibody in the diluted antibody sample to be tested is 0.5-2.0 mg / mL, preferably 0.8-1.0 mg / mL.

3. The method according to claim 1 or 2, characterized in that: In step (1), the reducing agent includes any one of dithiothreitol, β-mercaptoethanol, tris(2-carboxyethyl)phosphine or mercaptoethylamine, or a combination of at least two thereof; Preferably, in step (1), the concentration of the reducing agent in the reaction system for the reduction reaction is 10-50 mM; Preferably, in step (1), the reduction reaction temperature is 25-60°C, and the time is 20-120 min, preferably 30-60 min.

4. The method according to any one of claims 1 to 3, characterized in that In step (1), the termination reaction is carried out using an aqueous formic acid solution; Preferably, the volume fraction of formic acid in the formic acid aqueous solution is 5%-20%.

5. The method according to any one of claims 1 to 4, characterized in that In step (2), the injection volume in the ultra-high performance liquid chromatography detection is 1-5 μL, preferably 1-2 μL; Preferably, in step (2), the chromatographic column used in the ultra-high performance liquid chromatography detection includes Waters BioResolve TM RP mAb Polyphenyl column or other equivalent alternative columns; Preferably, in step (2), the column temperature of the chromatographic column in the ultra-high performance liquid chromatography detection is 60-80°C.

6. The method according to any one of claims 1 to 5, characterized in that In step (2), the detector used in the ultra-high performance liquid chromatography detection is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 280±2nm.

7. The method according to any one of claims 1 to 6, characterized in that In step (2), in the ultra-high performance liquid chromatography detection, aqueous formic acid solution is used as mobile phase A, and formic acid acetonitrile solution is used as mobile phase B; Preferably, the volume fraction of formic acid in the formic acid aqueous solution is 0.05%-0.1%; Preferably, the volume fraction of formic acid in the formic acid acetonitrile solution is 0.05%-0.1%; Preferably, in step (2), gradient elution is used in the ultra-high performance liquid chromatography detection, and the gradient of the elution is: 0-8min, 25%-45% mobile phase B, flow rate 0.2-0.3mL / min; 8-10 min, 45%-80% mobile phase B, flow rate 0.2-0.45 mL / min; 10-11 min, 25%-80% mobile phase B, flow rate 0.35-0.45 mL / min; Alternatively, in step (2), gradient elution is used in the ultra-high performance liquid chromatography detection, and the gradient of the elution is: 0-3min, 25%-60% mobile phase B, flow rate 0.45-0.55mL / min; 3-4 min, 60%-80% mobile phase B, flow rate 0.45-0.55 mL / min; 4-5min, 25%-80% mobile phase B, flow rate 0.45-0.55mL / min.

8. The method according to any one of claims 1 to 7, characterized in that In step (2), in the mass spectrometry detection, a positive ion mode is used, the temperature of the ion transfer tube is 310-330° C., and the m / z range is 600-3000.

9. The method according to any one of claims 1 to 8, characterized in that The calculation specifically comprises the following steps: The antibody reduced subunit solution was detected by ultra-high performance liquid chromatography-mass spectrometry to obtain a total ion current chromatogram; the mass chromatogram of the antibody heavy chain was extracted, and then each mass number and the corresponding ion signal intensity were obtained after deconvolution processing. Finally, the ion signal intensity was normalized and the proportion of the ion signal intensity of each mass number was calculated. The proportion of the ion signal intensity of each mass number is the content of different glycoforms.

10. The method according to any one of claims 1 to 9, characterized in that The antibody includes any one of a monospecific antibody, a bispecific antibody or a heavy chain antibody, or a combination of at least two of them.

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