A method for confirming the molecular weight structure of monoclonal antibodies by deglycosylation

Abstract

The present invention provides a method for confirming the desugared molecular weight structure of a monoclonal antibody, using high performance liquid chromatography-high resolution mass spectrometry to detect the desugared intact molecular weight and desugared reduced molecular weight of the monoclonal antibody, and using BIO Finder software to deconvolute and confirm the results, and confirm the structural integrity of the monoclonal antibody through the results of the confirmation and analysis of the desugared intact molecular weight and the desugared reduced molecular weight, and finally determine the type of the monoclonal antibody. The present invention shortens the time of monoclonal antibody pretreatment, is suitable for the determination of bevacizumab monoclonal antibody, sindili monoclonal antibody, carrelizumab monoclonal antibody, toripalil monoclonal antibody, rituximab monoclonal antibody, this detection method has the characteristics of accuracy, strong anti-interference ability, small error, simple operation and generality, and is suitable for the detection of multiple monoclonal antibodies.

Description

Technical Field

[0001] The invention belongs to the field of monoclonal antibody drug analysis, and particularly relates to a method for determining the desugared intact molecular weight and the desugared reduced molecular weight of a monoclonal antibody. Background Art

[0002] Monoclonal antibody (abbreviated as MAb) is a complex high-molecular glycoprotein with a complex three-dimensional structure, in which glycosylation mainly occurs in the endoplasmic reticulum and Golgi apparatus, and is a complex post-translational modification. According to the mechanism of action of monoclonal antibodies, as well as the complexity of glycoprotein structure and glycan structure, antibody drugs are actually a mixture of proteins with different glycoforms, but when confirming the structure of monoclonal antibodies, the overall structural level of glycoproteins is usually tested. At this time, different glycoforms will directly affect the accuracy of structural confirmation. Since the accuracy of the molecular weight of monoclonal antibodies is directly related to the protein level and glycoform of monoclonal antibodies, it is very necessary to study their structure after sugar cleavage. In the similarity study between biosimilars and original drugs, the similarity study between biosimilars and original drugs should at least include quality attribute similarity study and stability similarity study. Among them, the quality attribute similarity study usually includes protein structure confirmation and physicochemical properties, purity and impurities, biological activity, immunological properties, etc. Protein structure confirmation is the focus of quality attribute similarity research, and the structure of glycoproteins, in addition to the similarity of the overall glycoprotein structure, needs to examine the similarity of protein structures produced by different cell lines. Therefore, the confirmation of the post-deglycosylation structure is also one of the important contents of the similarity assessment between antibody biosimilars and original drugs. Most of the currently approved monoclonal antibodies are based on IgG (Immunoglobulin G). IgG antibodies have a conserved N-glycosylation site in the CH2 domain of the Fc part, usually located at Asn297. Another part of the IgG glycosylation site is located in the Fab region. Both sites are located on the heavy chain. Since the production process of glycoproteins is biosynthetic in nature, different cells produce different glycoforms, and the glycosylation sites and glycosylation of the two heavy chains are asymmetric, it is necessary to increase the structural study of the protein after removing the glycans on the basis of studying the complete protein. The combination of high performance liquid chromatography and high resolution mass spectrometry to determine the molecular weight of monoclonal antibodies has the characteristics of high resolution, strong sensitivity and good specificity. Among them, the ionization effect of ESI is softer and less likely to produce fragments. It is the most commonly used mass spectrometry mode in the characterization of macromolecules. The basic principle of mass spectrometry is to ionize the components in the sample in the ion source to generate charged ions with different charge-to-mass ratios. After the action of the accelerating electric field, an ion beam is formed and enters the mass analyzer. In the mass analyzer, the electric field and magnetic field are used to cause opposite velocity dispersion, and they are focused separately to obtain a mass spectrum to determine their mass. Due to the particularity of the mass spectrometry principle, the use of ion pair reagents and non-volatile reagents needs to be reduced in the pretreatment process, and the use of mass-to-charge ratio reagents that affect protein binding in a high temperature environment needs to be avoided. Therefore, it is necessary to develop a system and method with good mass spectrometry compatibility. PNGase F is an amidase, which is the most effective enzymatic method for removing N-sugars from glycoproteins. It can cut between the innermost GlcNAc and asparagine residues. There are many options on the market, including fast enzymes and ordinary enzymes that require overnight digestion, and they are all equipped with digestion buffers, whose components are basically SDS, triton ion pair reagents, protease inhibitors, and pH buffers. However, in actual operation, the supporting buffer system is complex and uncontrolled, and the ion pair reagents in the buffer system will affect the subsequent analysis of other protein products and are incompatible with protein spectrum analysis. Therefore, the desugaring system needs to be optimized during the research process. Due to the high folding of proteins and the morphology of spatial stereostructure, denaturation treatment is required during the structural confirmation process, but improper denaturation can easily change the solubility of proteins, and protein precipitation occurs during the pretreatment process. If the desugaring reduction molecular weight of monoclonal antibodies needs to be measured, the system becomes more complicated due to the addition of reducing agents and alkylating agents during the reduction and alkylation process. If precipitation occurs in the experiment, the protein concentration will change during subsequent operations, which will have a great impact on subsequent research. When denaturation and sugar cleavage are processed simultaneously, the concentration of the denaturant can easily affect the efficiency of sugar cleavage, resulting in incomplete sugar cleavage, mis-cleavage or missed cleavage, thus making the morphology of glycoproteins in the sample more complex. Therefore, it is urgent to establish an efficient and rapid pre-treatment method for the deglycosylated molecular weight and reduced molecular weight of the sample. Summary of the invention

[0003] Technical problem to be solved: The purpose of the present invention is to provide a method for confirming the deglycosylated molecular weight structure of a monoclonal antibody, shorten the time for pretreatment of the test sample, reduce the possibility of protein precipitation during the treatment process, and use high performance liquid chromatography-high resolution mass spectrometry to detect the deglycosylated intact molecular weight and deglycosylated reduced molecular weight of the monoclonal antibody.

[0004] Technical solution: A method for confirming the desugared molecular weight structure of a monoclonal antibody, wherein the desugared molecular weight includes the desugared intact molecular weight and the desugared reduced molecular weight; The specific steps include: S1. Preparation of desugared intact molecular weight sample: Take 100-300μg monoclonal antibody, add to 80-150mg guanidine hydrochloride, add 100μL 50mM NH4HCO3, dissolve by ultrasonication, shake for 20-40min, transfer to 20-100kD ultrafiltration tube, centrifuge and change the solution, add 500μL 50mM NH4HCO3 to dilute 10 times, centrifuge and change the solution again, transfer the solution in the tube to a centrifuge tube, add PNgase F enzyme, and digest for 10-20min to obtain the desugared intact molecular weight sample; S2. Preparation of desugared reduced molecular weight sample: Take 100-300 μg monoclonal antibody, add to 80-150 mg guanidine hydrochloride and 10-20 mg thiourea, add 3-5 μL 100 mg / mL dithiothreitol solution, add 50 mM NH4HCO3100 μL, dissolve by ultrasonication, shake for 10-20 min, add 3-5 μL 1M alkylation reagent, place in dark for 10-20 min, transfer to 5-20 kD ultrafiltration tube, centrifuge and change the liquid, add 500 μL 50 mM NH4HCO3 to dilute 10 times, centrifuge and change the liquid, transfer the solution in the tube to a centrifuge tube, add PNgase F enzyme, and digest for 20 min to obtain the desugared reduced molecular weight sample; S3. The deglycosylated intact molecular weight of the monoclonal antibody was detected by high performance liquid chromatography-high resolution mass spectrometry, and the results were deconvoluted and confirmed by BIO Finder software; S4. The deglycosylated reduced molecular weight of the monoclonal antibody was detected by high performance liquid chromatography-high resolution mass spectrometry, and the results were deconvoluted and confirmed by BIO Finder software; S5. Confirm the analysis results by using the deglycosylated intact molecular weight and deglycosylated reduced molecular weight to confirm the structural integrity of the monoclonal antibody and ultimately determine the type of the monoclonal antibody. Furthermore, the monoclonal antibodies include bevacizumab monoclonal antibody, sintilide monoclonal antibody, carrelizumab monoclonal antibody, toripalil monoclonal antibody and rituximab monoclonal antibody. Furthermore, in step S1, the ultrasonic temperature is 32-43° C., the ultrasonic time is 5-10 min, and the shaking temperature is 37-45° C. Furthermore, in step S2, the alkylating agent includes iodoacetamide and iodoacetic acid; the ultrasonic temperature is 32-43° C., the ultrasonic time is 5-10 min; and the shaking temperature is 37-45° C. Furthermore, in the steps S1 and S2, the amount of PNgase F enzyme used is 2-5 μL, and the enzyme cleavage temperature is 50-60°C. Furthermore, the conditions for the centrifugal liquid exchange in steps S1 and S2 are: the centrifugal speed is 13000 rpm, the centrifugal time is 3-10 min, and the outer tube solution is discarded after centrifugation. Furthermore, in step S3, (1) the high performance liquid chromatography conditions are: Column: MAbPacTM RP, 2.1x 50mm, 4μm; Mobile phase: A-0.1% formic acid in water, B-0.1% formic acid in acetonitrile; Flow rate: 0.2-0.4 mL / min; Column temperature: 60-80℃; Injection volume: 2-5 μL Gradient elution program: 0-1 min, 30% B; 1-6 min, 30%→90% B; 6-7 min, 90% B; 7-7.01 min, 90%→30% B; 7.01-10 min, 30% B; (2) Mass spectrometry conditions are: Mass spectrometry resolution: 17500; Spray voltage: 3-3.8kV; Capillary temperature: 250-400℃; Sheath gas flow rate: 20-35arb; Auxiliary gas flow rate: 10arb; Probe temperature: 200°C; AGC: 3e6; Precursor ion scanning range: m / z 1800-4000; S Lens:50. Furthermore, in step S4, (1) the high performance liquid chromatography conditions are: Column: MAbPacTM RP, 2.1x 50mm, 4μm; Mobile phase: A-0.1% formic acid in water, B-0.1% formic acid in acetonitrile; Flow rate: 0.2-0.4 mL / min; Column temperature: 60-80℃; Injection volume: 2-5 μL; Gradient elution program: 0-3 min, 25% B; 3-13 min, 25%→35% B; 13-14 min, 35%→80% B; 14-17 min, 80% B; 17-18 min, 80%→25% B; 18-22 min, 25% B; (2) Mass spectrometry conditions are: Mass spectrometry resolution: 17500; Spray voltage: 3-3.8kV; Capillary temperature: 250-400℃; Sheath gas flow rate: 20-35arb; Auxiliary gas flow rate: 10arb; Probe temperature: 200°C; AGC: 3e6; Precursor ion scan range: m / z 600-3000. Beneficial effects: 1. The present invention treats the test sample by temperature-controlled ultrasound plus oscillation, which greatly shortens the test sample treatment time. No protein precipitation occurs during the experiment, thus avoiding the impact of protein precipitation on subsequent experimental research. 2. High performance liquid chromatography-high resolution mass spectrometry is used to detect the deglycosylated intact molecular weight and deglycosylated reduced molecular weight of monoclonal antibodies including bevacizumab monoclonal antibody, sintili monoclonal antibody, carrelizumab monoclonal antibody, toripalil monoclonal antibody, and rituximab monoclonal antibody. This detection method is accurate, has strong anti-interference ability, small error, simple operation and is universal, and is suitable for the detection of multiple monoclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The experimental flow chart of the detection method. Figure 2 The figures are the intact molecular weight multi-charged mass spectrum of bevacizumab monoclonal antibody, the intact molecular weight mass spectrum total ion current spectrum of bevacizumab monoclonal antibody and the deglycosylated intact molecular weight identification result of bevacizumab monoclonal antibody in Example 1, wherein A is the intact molecular weight multi-charged mass spectrum of bevacizumab monoclonal antibody, B is the intact molecular weight mass spectrum total ion current spectrum of bevacizumab monoclonal antibody, and C is the deglycosylated intact molecular weight identification result of bevacizumab monoclonal antibody. Figure 3 The deglycosylated reduced molecular weight mass spectrum total ion current spectrum of bevacizumab monoclonal antibody, the deglycosylated reduced molecular weight heavy chain multiply charged mass spectrum of bevacizumab monoclonal antibody and the deglycosylated molecular weight heavy chain identification result of bevacizumab monoclonal antibody in Example 1, wherein A is the deglycosylated reduced molecular weight mass spectrum total ion current spectrum of bevacizumab monoclonal antibody, B is the deglycosylated reduced molecular weight heavy chain multiply charged mass spectrum of bevacizumab monoclonal antibody, and C is the deglycosylated molecular weight heavy chain identification result of bevacizumab monoclonal antibody. Figure 4 These are the multi-charged mass spectrum of the intact molecular weight of Sindili monoclonal antibody, the total ion current spectrum of the intact molecular weight mass spectrum of Sindili monoclonal antibody and the identification result of the deglycosylated intact molecular weight of Sindili monoclonal antibody in Example 2, wherein A is the multi-charged mass spectrum of the intact molecular weight of Sindili monoclonal antibody, B is the total ion current spectrum of the intact molecular weight mass spectrum of Sindili monoclonal antibody, and C is the identification result of the deglycosylated intact molecular weight of Sindili monoclonal antibody. Figure 5These are the multi-charged mass spectrum of the deglycosylated reduced molecular weight heavy chain of the Sindili monoclonal antibody, the total ion current spectrum of the deglycosylated reduced molecular weight mass spectrum of the Sindili monoclonal antibody and the identification result of the deglycosylated molecular weight heavy chain of the Sindili monoclonal antibody in Example 2, wherein A is the multi-charged mass spectrum of the deglycosylated reduced molecular weight heavy chain of the Sindili monoclonal antibody, B is the total ion current spectrum of the deglycosylated reduced molecular weight mass spectrum of the Sindili monoclonal antibody, and C is the identification result of the deglycosylated molecular weight heavy chain of the Sindili monoclonal antibody. Figure 6 These are the intact molecular weight multi-charged mass spectrum of the carrelli beads monoclonal antibody, the intact molecular weight mass spectrum total ion current spectrum of the carrelli beads monoclonal antibody and the deglycosylated intact molecular weight identification result of the carrelli beads monoclonal antibody of Example 3, wherein A is the intact molecular weight multi-charged mass spectrum of the carrelli beads monoclonal antibody, B is the intact molecular weight mass spectrum total ion current spectrum of the carrelli beads monoclonal antibody, and C is the deglycosylated intact molecular weight identification result of the carrelli beads monoclonal antibody. Figure 7 These are the multi-charged mass spectrum of the deglycosylated reduced molecular weight heavy chain of carrelizumab, the total ion current spectrum of the deglycosylated reduced molecular weight mass spectrum of carrelizumab and the identification result of the deglycosylated molecular weight heavy chain of carrelizumab in Example 3, wherein A is the multi-charged mass spectrum of the deglycosylated reduced molecular weight heavy chain of carrelizumab, B is the total ion current spectrum of the deglycosylated reduced molecular weight mass spectrum of carrelizumab, and C is the identification result of the deglycosylated molecular weight heavy chain of carrelizumab. DETAILED DESCRIPTION The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Drugs and reagents: Acetonitrile and formic acid (Merck) were of mass spectrometry grade; urea (Sigma), dithiothreitol, iodoacetamide, and thiourea were of analytical grade; PNgase F enzyme (NEB) was a fast enzyme; The experimental sample was bevacizumab monoclonal antibody injection (100 mg / 4 mL / bottle). instrument: Bio-liquid chromatography-mass spectrometry (Thermo Fisher), analytical balance (Mettler), refrigerated centrifuge (Epend), thermostat (Ausun), ultrasound machine (Kedao) Example 1 The detection of the desugared molecular weight of bevacizumab monoclonal antibody includes the following steps: S1. Detection of the intact molecular weight of bevacizumab monoclonal antibody after deglycosylation: S11. Preparation of bevacizumab monoclonal antibody desugared samples: Take 100 μg of bevacizumab monoclonal antibody, add it to a centrifuge tube containing 100 mg of guanidine hydrochloride, add 100 μL of 50 mM NH4HCO3, dissolve it by ultrasonication at 40°C for 5 min, shake it at 40°C for 20 min, transfer the sample to a 100 kD ultrafiltration tube, centrifuge it at 13000 rpm for 5 min, add 500 μL of 50 mM NH4HCO3 to dilute it 10 times, centrifuge it at 13000 rpm for 5 min, transfer the solution in the tube to a centrifuge tube, add 5 μL of PNgase F enzyme, and digest it at 60°C for 20 min to obtain the desugared complete molecular weight sample of bevacizumab monoclonal antibody; S12. The desugared intact molecular weight of bevacizumab monoclonal antibody was detected by high performance liquid chromatography-high resolution mass spectrometry, wherein the conditions of high performance liquid chromatography were: Column: MAbPacTM RP, 2.1x 50mm, 4μm; Mobile phase: A-0.1% formic acid in water, B-0.1% formic acid in acetonitrile; Flow rate: 0.2 mL / min; Column temperature: 60°C; Injection volume: 5 μL; UV detector wavelength: 280nm; Gradient elution program: 0-1 min, 30% B; 1-6 min, 30%→90% B; 6-7 min, 90% B; 7-7.01 min, 90%→30% B; 7.01-10 min, 30% B; The mass spectrometry conditions were: Mass spectrometry resolution: 17500; Spray voltage: 3.8 kV; Capillary temperature: 320℃; Sheath gas flow rate: 35arb; Auxiliary gas flow rate: 10arb; Probe temperature: 200°C; AGC: 3e6; Precursor ion scanning range: m / z 1800-4000; S Lens:50. S13: Sample Quantification The sample was processed according to the above step S11, and the chromatographic conditions of step S12 were used for determination, the mass spectrum acquisition time was 2-7 min, the main peak was at 5.76 min, and the main peak was deconvoluted by Bio Finder software, Measure 95%, Mass Tolerance 10 ppm, Max. Number of Modifications 4; S2. Detection of the desugared reduced molecular weight of bevacizumab monoclonal antibody, specifically comprising the following steps: S21. Preparation of bevacizumab monoclonal antibody desugaring reduction sample: Take 200μg of bevacizumab monoclonal antibody, add to 80mg guanidine hydrochloride, 10mg thiourea, add 3μL 100mg / mL DTT solution, add 50mM NH4HCO3100μL, dissolve by ultrasonic at 40℃ for 5min, shake at 40℃ for 20min, add 1M iodoacetic acid 3μL and place in dark for 10min. Transfer the sample to a 20kD ultrafiltration tube, centrifuge at 13000rpm for 5min, add 500μL 50mM NH4HCO3 to dilute 10 times, centrifuge at 13000rpm for 5min, transfer the solution in the tube to a centrifuge tube, add 5μL PNgase F enzyme, and digest at 50℃ for 20min to obtain the bevacizumab monoclonal antibody desugaring reduction sample; S22. The desugared reduced molecular weight of bevacizumab monoclonal antibody was detected by high performance liquid chromatography-high resolution mass spectrometry, wherein the conditions of high performance liquid chromatography were: Column: MAbPacTM RP, 2.1x 50mm, 4μm; Mobile phase: A-0.1% formic acid in water, B-0.1% formic acid in acetonitrile; Flow rate: 0.25 mL / min; Column temperature: 80°C; Injection volume: 2 μL; UV detector wavelength: 280nm; Gradient elution program: 0-3 min, 25% B; 3-13 min, 25%→35% B; 13-14 min, 35%→80% B; 14-17 min, 80% B; 17-18 min, 80%→25% B; 18-22 min, 25% B; The mass spectrometry conditions were: Mass spectrometry resolution: 17500; Spray voltage: 3.8 kV; Capillary temperature: 320℃; Sheath gas flow rate: 30arb; Auxiliary gas flow rate: 10arb; Probe temperature: 200°C; AGC: 3e6; Precursor ion scan range: m / z 600-2600. S23. Sample Quantification The sample was processed according to the above step S21 and measured under the chromatographic conditions of step S22. The mass spectrometry acquisition time was 2-15 min, the heavy chain peak time was 11.40 min, and the heavy chain peak was deconvoluted by Bio Finder software. The setting mode was ReSpect, and the analysis parameters were Peak Detection Quality Measure 95%, Mass Tolerance 20 ppm. See the full molecular weight multi-charged mass spectrum of bevacizumab monoclonal antibody, the full molecular weight mass spectrum total ion current spectrum of bevacizumab monoclonal antibody and the identification results of the desugared full molecular weight of bevacizumab. Figure 2 The ion flow diagram shows that there is only one main peak after bevacizumab monoclonal antibody is deglycosylated, and the identification result shows that the accurate molecular weight of the protein after bevacizumab monoclonal antibody is 146306.25Da. Compared with the theoretical sequence, the identification result error is 4.4ppm, which is consistent with the theoretical sequence. The total ion flow spectrum of the mass spectrum of the deglycosylated reduced molecular weight of bevacizumab monoclonal antibody, the multi-charged mass spectrum of the deglycosylated reduced molecular weight heavy chain of bevacizumab monoclonal antibody and the identification results of the deglycosylated molecular weight heavy chain of bevacizumab are shown in Figure 3 The identification results were all the desugared forms of bevacizumab monoclonal antibody. The results showed that the molecular weight of the heavy chain of the reduced molecular weight of bevacizumab monoclonal antibody was 50297.84Da, with an error of 4.3ppm, which was consistent with the theoretical sequence. Based on the above confirmation analysis results of the desugared intact molecular weight and the desugared reduced molecular weight, the structural integrity of the bevacizumab monoclonal antibody was confirmed, and the monoclonal antibody was finally determined to be bevacizumab. Example 2 The detection of the desugared molecular weight of Sindily monoclonal antibody includes the following steps: S1. Detection of the intact molecular weight of the desugared Sindily monoclonal antibody, specifically comprising the following steps: S11. Preparation of deglycosylated samples of Sindily monoclonal antibody Take 300 μg of Sindili monoclonal antibody, add it to a centrifuge tube containing 100 mg of guanidine hydrochloride, add 100 μL of 50 mM NH4HCO3, dissolve it by ultrasonication at 37°C for 10 min, shake it at 37°C for 20 min, transfer the sample to a 100 kD ultrafiltration tube, centrifuge it at 13000 rpm for 3 min, add 500 μL of 50 mM NH4HCO3 to dilute it 10 times, centrifuge it at 13000 rpm for 10 min, transfer the solution in the tube to a centrifuge tube, add 5 μL of PNgase F enzyme, and digest it at 60°C for 20 min to obtain the desugared sample of Sindili monoclonal antibody; S12. The intact molecular weight of the desugared Sindily monoclonal antibody was detected by HPLC-HRMS, and the HPLC and mass spectrometry conditions were consistent with those in step S12 of Example 1; S13. Sample Quantification The sample was processed according to the above step S11, and the chromatographic conditions of step S12 were used for determination, the mass spectrum acquisition time was 2-7 min, the main peak was at 5.11 min, and the main peak was deconvoluted by Bio Finder software, Measure 95%, Mass Tolerance 10 ppm, Max. Number of Modifications 4; S2. Detection of the desugared reduced molecular weight of the Sindily monoclonal antibody, specifically comprising the following steps: S21. Preparation of Deglycosylated and Reduced Samples of Sindily Monoclonal Antibody Take 200μg of Sindily monoclonal antibody, add it to 80mg of guanidine hydrochloride and 10mg of thiourea, add 3μL of 100mg / mL DTT solution, add 100μL of 50mM NH4HCO3, dissolve it by ultrasonic treatment at 45℃ for 5min, shake it at 45℃ for 20min, add 3μL of 1M iodoacetamide and place it in the dark for 15min. Transfer the sample to a 10kD ultrafiltration tube, centrifuge it at 13000rpm for 10min, change the liquid, add 500μL of 50mM NH4HCO3 to dilute it 10 times, centrifuge it at 13000rpm for 10min, transfer the solution in the tube to a centrifuge tube, add 2μL of PNgase F enzyme, and digest it at 60℃ for 20min to obtain the desugared and reduced sample of Sindily monoclonal antibody; S22. The deglycosylated reduced molecular weight of the Sindili monoclonal antibody was detected using high performance liquid chromatography-high resolution mass spectrometry, and the high performance liquid chromatography and mass spectrometry conditions were consistent with step S22 in Example 1. S23. Sample Quantification The sample was processed according to the above step S21 and measured under the chromatographic conditions of step S22. The mass spectrometry acquisition time was 2-9 min, the heavy chain peak time was 6.26 min, and the heavy chain peak was deconvoluted by Bio Finder software. The setting mode was ReSpect, and the analysis parameters were Peak Detection Quality Measure 95%, Mass Tolerance 20 ppm. See the full molecular weight multi-charged mass spectrum of Sindili monoclonal antibody, the full molecular weight mass spectrum total ion current spectrum of Sindili monoclonal antibody and the identification results of the desugared full molecular weight of Sindili monoclonal antibody for details. Figure 4 The identification results show that the accurate molecular weight of Sindili monoclonal antibody is 143498.75Da. Compared with the theoretical sequence, the identification result error is 6.8ppm, which is consistent with the theoretical sequence. The multi-charged mass spectrum of the deglycosylated molecular weight heavy chain of Sindili monoclonal antibody, the total ion current spectrum of the deglycosylated molecular weight mass spectrum of Sindili monoclonal antibody and the identification results of the deglycosylated molecular weight heavy chain of Sindili monoclonal antibody are shown in Figure 5The results showed that the molecular weight of the reduced molecular weight heavy chain of the Sindili monoclonal antibody was 49295.10Da, with an error of 2.9ppm, which was consistent with the theoretical sequence. Based on the above-mentioned confirmation analysis results of the desugared intact molecular weight and the desugared reduced molecular weight, the structural integrity of the Sindili monoclonal antibody was confirmed, and the monoclonal antibody was finally determined to be Sindili. Example 3 The detection of the desugared molecular weight of the carrelizumab monoclonal antibody includes the following steps: S1. Detection of the intact molecular weight of the desugared monoclonal antibody of carrelizumab, specifically comprising the following steps: S11. Preparation of Carrelizumab Monoclonal Antibody Deglycosylation Samples Take 300 μg of carrelizumab monoclonal antibody, add it to a centrifuge tube containing 100 mg of guanidine hydrochloride, add 100 μL of 50 mM NH4HCO3, dissolve it by ultrasonication at 37°C for 10 min, shake it at 37°C for 20 min, transfer the sample to a 100 kD ultrafiltration tube, centrifuge it at 13000 rpm for 3 min, add 500 μL of 50 mM NH4HCO3 to dilute it 10 times, and centrifuge it at 13000 rpm for 10 min. Transfer the solution in the tube to a centrifuge tube, add 5 μL of PNgase F enzyme, and digest it at 60°C for 20 min to obtain the desugared sample of carrelizumab monoclonal antibody. S12. The intact molecular weight of the desugared Sindily monoclonal antibody was detected by HPLC-HRMS, and the HPLC and mass spectrometry conditions were consistent with those of S12 in Example 1; S13. Sample Quantification The sample was processed according to the above step S11, and the chromatographic conditions of step S12 were used for determination, the mass spectrum acquisition time was 2-7 min, the main peak was at 5.32 min, and the main peak was deconvoluted by Bio Finder software, Measure 95%, Mass Tolerance 10 ppm, Max. Number of Modifications 4; S2. Detection of the desugared reduced molecular weight of the carrelizumab monoclonal antibody, specifically comprising the following steps: S21. Preparation of Carrelizumab Monoclonal Antibody Deglycosylation Sample Take 200μg of carrelizumab monoclonal antibody, add it to 80mg guanidine hydrochloride, 10mg thiourea, add 3μL 100mg / mL DTT solution, add 50mM NH4HCO3100μL, dissolve it by ultrasonic at 45℃ for 5min, shake it at 45℃ for 20min, add 1M iodoacetamide 3μL and place it in the dark for 15min. Transfer the sample to a 10kD ultrafiltration tube, centrifuge it at 13000rpm for 10min, change the liquid, add 500μL 50mM NH4HCO3 to dilute it 10 times, centrifuge it at 13000rpm for 10min, transfer the solution in the tube to a centrifuge tube, add 2μL PNgase F enzyme, and digest it at 60℃ for 20min to obtain the desugared and reduced sample of carrelizumab monoclonal antibody; S22. The deglycosylated reduced molecular weight of the Sindili monoclonal antibody was detected using high performance liquid chromatography-high resolution mass spectrometry, and the high performance liquid chromatography and mass spectrometry conditions were consistent with step S22 in Example 1. S23. Sample Quantification The sample was processed according to the above step S21 and measured under the chromatographic conditions of step S22. The mass spectrometry acquisition time was 3-9 min, the heavy chain peak time was 6.05 min, and the heavy chain peak was deconvoluted by Bio Finder software. The setting mode was ReSpect, and the analysis parameters were Peak Detection Quality Measure 95%, Mass Tolerance 20 ppm. See the full molecular weight multi-charged mass spectrum of the carrelizumab monoclonal antibody, the full molecular weight mass spectrum total ion current spectrum of the carrelizumab monoclonal antibody, and the identification results of the desugared full molecular weight of the carrelizumab monoclonal antibody for details. Figure 6 The identification results showed that the desugared intact molecular weight was 143453.47Da, with an error of 2.5ppm, which was consistent with the theoretical sequence. The multi-charged mass spectrum of the desugared reduced molecular weight heavy chain of carrelizumab, the total ion current spectrum of the desugared reduced molecular weight mass spectrum of carrelizumab, and the identification results of the desugared molecular weight heavy chain of carrelizumab are shown in Figure 7 The results showed that the molecular weight of the reduced molecular weight heavy chain of the carrelix beads monoclonal antibody was 49084.62Da, with an error of 3.1ppm, which was consistent with the theoretical sequence. Based on the above confirmation analysis results of the desugared intact molecular weight and desugared reduced molecular weight, the structural integrity of the carrelix beads monoclonal antibody was confirmed, and the monoclonal antibody was finally determined to be a carrelix beads. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for confirming the desugared molecular weight structure of a monoclonal antibody, characterized in that: The desugared molecular weight includes the desugared complete molecular weight and the desugared reduced molecular weight; The specific steps include: S1. Preparation of desugared intact molecular weight sample: Take 100-300μg monoclonal antibody, add to 80-150mg guanidine hydrochloride, add 100μL 50mM NH4HCO3, dissolve by ultrasonication, shake for 20-40min, transfer to 20-100kD ultrafiltration tube, centrifuge and change the solution, add 500μL 50mM NH4HCO3 to dilute 10 times, centrifuge and change the solution again, transfer the solution in the tube to a centrifuge tube, add PNgase F enzyme, and digest for 10-20min to obtain the desugared intact molecular weight sample; S2. Preparation of desugared reduced molecular weight sample: Take 100-300 μg monoclonal antibody, add to 80-150 mg guanidine hydrochloride and 10-20 mg thiourea, add 3-5 μL 100 mg / mL dithiothreitol solution, add 50 mM NH4HCO3100 μL, dissolve by ultrasonication, shake for 10-20 min, add 3-5 μL 1M alkylation reagent, place in dark for 10-20 min, transfer to 5-20 kD ultrafiltration tube, centrifuge and change the liquid, add 500 μL 50 mM NH4HCO3 to dilute 10 times, centrifuge and change the liquid, transfer the solution in the tube to a centrifuge tube, add PNgase F enzyme, and digest for 20 min to obtain the desugared reduced molecular weight sample; S3. The deglycosylated intact molecular weight of the monoclonal antibody was detected by high performance liquid chromatography-high resolution mass spectrometry, and the results were deconvoluted and confirmed by BIO Finder software; S4. The deglycosylated reduced molecular weight of the monoclonal antibody was detected by high performance liquid chromatography-high resolution mass spectrometry, and the results were deconvoluted and confirmed by BIO Finder software; S5. Confirm the analysis results by using the deglycosylated intact molecular weight and deglycosylated reduced molecular weight to confirm the structural integrity of the monoclonal antibody and ultimately determine the type of the monoclonal antibody.

2. The method for confirming the desugared molecular weight structure of a monoclonal antibody according to claim 1, characterized in that: The monoclonal antibodies include bevacizumab monoclonal antibody, sintilide monoclonal antibody, carrelizumab monoclonal antibody, toripalil monoclonal antibody and rituximab monoclonal antibody.

3. The method for confirming the desugared molecular weight structure of a monoclonal antibody according to claim 1, characterized in that: In step S1, the ultrasonic temperature is 32-43°C, the ultrasonic time is 5-10 min, and the shaking temperature is 37-45°C.

4. The method for confirming the desugared molecular weight structure of a monoclonal antibody according to claim 1, characterized in that: In step S2, the alkylating agent includes iodoacetamide and iodoacetic acid; the ultrasonic temperature is 32-43° C., the ultrasonic time is 5-10 min; and the shaking temperature is 37-45° C.

5. The method for confirming the desugared molecular weight structure of a monoclonal antibody according to claim 1, characterized in that: In the steps S1 and S2, the amount of PNgase F enzyme used is 2-5 μL, and the enzyme cleavage temperature is 50-60° C.

6. The method for confirming the desugared molecular weight structure of a monoclonal antibody according to claim 1, characterized in that: The conditions for the centrifugal liquid exchange in steps S1 and S2 are: the centrifugal speed is 13000 rpm, the centrifugal time is 3-10 min, and the outer tube solution is discarded after centrifugation.

7. The method for confirming the desugared molecular weight structure of a monoclonal antibody according to claim 1, characterized in that: In step S3, (1) the HPLC conditions are: Column: MAbPacTM RP, 2.1x 50mm, 4μm; Mobile phase: A-0.1% formic acid in water, B-0.1% formic acid in acetonitrile; Flow rate: 0.2-0.4 mL / min; Column temperature: 60-80℃; Injection volume: 2-5 μL Gradient elution program: 0-1 min, 30% B; 1-6 min, 30%→90% B; 6-7 min, 90% B; 7-7.01 min, 90%→30% B; 7.01-10 min, 30% B; (2) Mass spectrometry conditions are: Mass spectrometry resolution: 17500; Spray voltage: 3-3.8kV; Capillary temperature: 250-400℃; Sheath gas flow rate: 20-35arb; Auxiliary gas flow rate: 10arb; Probe temperature: 200°C; AGC: 3e6; Precursor ion scanning range: m / z 1800-4000; S Lens:

50.

8. The method for confirming the desugared molecular weight structure of a monoclonal antibody according to claim 1, characterized in that: In step S4, (1) the HPLC conditions are: Column: MAbPacTM RP, 2.1x 50mm, 4μm; Mobile phase: A-0.1% formic acid in water, B-0.1% formic acid in acetonitrile; Flow rate: 0.2-0.4 mL / min; Column temperature: 60-80℃; Injection volume: 2-5 μL; Gradient elution program: 0-3 min, 25% B; 3-13 min, 25%→35% B; 13-14 min, 35%→80% B; 14-17 min, 80% B; 17-18 min, 80%→25% B; 18-22 min, 25% B; (2) Mass spectrometry conditions are: Mass spectrometry resolution: 17500; Spray voltage: 3-3.8kV; Capillary temperature: 250-400℃; Sheath gas flow rate: 20-35arb; Auxiliary gas flow rate: 10arb; Probe temperature: 200°C; AGC: 3e6; Precursor ion scan range: m / z 600-3000.

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