Multi-attribute mass spectrometry method of antibody

Abstract

According to the multi-attribute mass spectrometry analysis method of the antibody, deep pre-characterization analysis is carried out on the antibody through MAM based on high-resolution mass spectrometry, and the main mass attribute of the antibody is successfully obtained. The pre-characterization information is then used for MAM development based on a single quadrupole. According to the method, various product quality attributes are effectively monitored in single analysis, and the accuracy of the method is confirmed through a method based on high-resolution mass spectrometry. And then strict methodological verification is carried out on the method. A verification result shows that the method has good specificity, linearity, accuracy, precision and the like. The MAM based on the single quadrupole not only effectively solves the problem of multi-attribute detection of the glycosylation site monoclonal antibody drug, but also shows certain method universality, and provides a new and simple technical scheme for quality control of different glycosylation site antibody drugs. The research result is expected to promote the improvement of related technical platforms and the updating of concepts, so that the overall capability of product development and quality control is enhanced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a multi-attribute mass spectrometry analysis method for antibodies. Background Technology

[0002] Due to their highly complex molecular structures, therapeutic antibody drugs are susceptible to various physicochemical and biochemical factors during production and storage. Posttranslational modifications (PTMs) such as glycosylation, oxidation, deamidation, and terminal modification often occur at specific sites, forming various variants. These variants are an important component of product quality attributes (PQA), and even minor variations can significantly affect the product's physicochemical properties, biological activity, and stability, and may even lead to immunogenicity issues. Therefore, identifying and quantifying certain quality attributes of antibodies is objectively necessary to effectively ensure the safety and efficacy of the drug.

[0003] Antibody drugs with polysaccharide-sylated sites typically exhibit higher modification complexity, which is often further amplified by different expression systems. This poses a greater challenge to the quality control of polysaccharide-sylated antibody drugs. Although traditional analytical methods, such as capillary electrophoresis (CE), ion exchange chromatography (IEX), hydrophilic interaction chromatography (HILIC), reversed-phase high-performance liquid chromatography (RP), size exclusion chromatography (SEC), and hydrophobic interaction chromatography (HIC), are widely used in product quality control, they are usually only targeted at specific quality attributes and have limitations in detecting polysaccharide-sylated sites. Especially when detecting a single quality attribute of complex proteins, complex peaks containing multiple components are often generated, which may mask the true characteristics of a single critical quality attribute (CQA). Given these challenges, developing new technologies that can provide a wider range of detection dimensions, higher sensitivity, and higher specificity becomes particularly important. Adopting a "broad-spectrum, rapid, and accurate" quality control concept and platform technology can more accurately identify and quantify multiple quality attributes, thereby improving overall quality control efficiency and strengthening drug safety assurance.

[0004] In recent years, with the advancement of new technologies, especially the significant improvement in resolution and stability of mass spectrometry, multi-attribute analysis methods (MAM) based on high-resolution mass spectrometry have begun to be used directly or indirectly for the control of multiple quality attributes of monoclonal antibody products. This method can obtain data on multiple quality attributes from a single analysis, significantly reducing the number of analyses required for product quality control, thus contributing to lower analytical costs and improved efficiency. MAM data can also provide the same or deeper understanding of product quality as traditional methods. MAM methods based on high-resolution mass spectrometry can perform quality control of antibody drugs at the intact and peptide map levels. Intact protein multi-attribute methods (IMAM) can perform identification analysis through deconvolution results and chromatographic retention times, and achieve attribute quantification analysis through response intensity. Moreover, it exhibits consistency with traditional detection methods on multiple quality attributes; for example, the quantification results of site-specific glycosylation are essentially consistent with the traditional HILIC method. Classical peptide map-based mass spectrometry (MAM) analysis provides the richest product quality information. Although this "bottom-up" analytical strategy sacrifices some overall protein information and increases operational complexity, these issues can be effectively addressed by establishing automated sample processing platforms and optimizing sample processing methods. Furthermore, it can simultaneously monitor antibody drug oxidation, deamidation, isomerization, and glycosylation modifications, offering broader possibilities for the application of peptide map-based MAM. However, high-resolution mass spectrometry, with its superior performance, often comes with higher equipment maintenance costs and more complex data analysis, limiting its widespread adoption in current pharmaceutical industry quality control (QC) laboratories. In contrast, single quadrupole detectors / mass spectrometers, with their low cost, small footprint, and ease of operation, have gained some application in macromolecular drug QC. This device tracks the mass-to-charge ratio (m / z) of target peptides through flexible MS full scan or selected ion scan (SIR) modes, increasing the detection dimension and improving detection sensitivity and selectivity compared to liquid chromatography. The limit of quantitation (LOQ) in Scan mode is close to 1%, and SIR mode may be even better. Single quadrupole MAM analysis shows broad applicability. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a multi-attribute mass spectrometry analysis method for antibodies.

[0006] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows: a multi-attribute mass spectrometry analysis method for antibodies, characterized in that a single quadrupole mass spectrometer is used for multi-attribute analysis.

[0007] The sample processing procedure for the multi-attribute analysis method is as follows: a. Replace the antibody sample using an ultrafiltration centrifuge tube to obtain the replaced sample; b. Denaturation, reduction, alkylation, and termination of the reaction of the substituted sample; c. Further desalting, enzyme digestion, and termination of enzyme digestion reaction of the sample; d. After centrifugation, the processed samples were subjected to multi-attribute analysis using a single quadrupole centrifuge.

[0008] The antibody mentioned in step a is a cetuximab monoclonal antibody.

[0009] In step a, the sample replacement involves replacing the sample three or more times in a dilution buffer solution using a 30KD ultrafiltration centrifuge tube, with a final sample volume of 95-115 µL.

[0010] In step b, the denaturation of the replaced sample is performed by adding 350-380 µL of denaturing buffer to the replaced sample; the reduction is performed by adding 7.0-7.5 µL of 0.95-1.15 mol / L DTT to the denatured sample and incubating at 35-39°C for 60±5 min; the alkylation is performed by adding 8.0-8.7 µL of 2.9 mol / L sodium iodoacetate to the reduced sample and incubating at room temperature in the dark for 43-47 min, and then terminating the reaction by adding 16.2-18.4 µL of 0.95-1.15 mol / L DTT.

[0011] In step c, the further desalting of the sample is carried out by using a Sephadex G25 column to desalt the sample into 48-53 mmol / L pH 7.8-8.1 ammonium bicarbonate buffer. The enzyme digestion and termination of the enzyme digestion reaction are carried out by adding trypsin at a ratio of 1:10 to 1:14 (m / m), incubating at 35-39℃ for 4h±10min, and then adding 1.8-2.5 µL FA to terminate the reaction.

[0012] In step d, the processed sample is centrifuged and then subjected to multi-attribute analysis on a single quadrupole analyzer. This involves centrifuging the sample at 17,000 g for 10 min and analyzing it on a 1290 Infinity II LC-Infinity Lab LC / MSDiO single quadrupole analyzer.

[0013] The analytical methods and parameters were set as follows: the chromatographic column was a UPLC BEH 13 nm C18 1.7 μm 2.1×150 mm column (Waters); the mobile phases were A: 0.1% FA aqueous solution and B: 0.1% FA acetonitrile solution; the liquid phase gradient program was that the mobile phase B increased from 2% to 40% within 90 min; the flow rate was 0.2 mL / min; and the column temperature was 60 ℃. The MSD iQ detector parameters were set as follows: capillary voltage, 3.5 kV; desiccator temperature, 325 ℃; nebulizer gas pressure, 40 psi; desiccator gas flow rate, 10 L / min; scan range, 50–1450 u; and data were acquired using Scan and SIM modes. Agilent OpenLab Data Analysis software was used to control the LC-UV-MSD iQ system and perform data analysis. Attached Figure Description

[0014] Figure 1 : MAM based on a single quadrupole for monitoring key peptides of cetuximab monoclonal antibody Figure 2 The impact of FA on key quality attributes of anti-PD-1 antibodies detected by a single quadrupole mass spectrometer. Figure 3 The impact of TFA on the detection of key quality attributes of anti-PD-1 antibodies using a single quadrupole mass spectrometer. Figure 4 Methodological validation of linear graphs.

[0015] Example 1: Identifying Key Quality Attributes and Screening for the Optimal Mass-to-charge Ratio Data on glycosylated, glycosylated, oxidized, deamination-treated, and terminally modified peptides, as well as characteristic peptides (CDR peptides), in cetuximab monoclonal antibody (CMAB009) were obtained using a UPLC Xevo G2-S Q-TOF (Waters) and a 1290 Infinity II / 6545XT AdvanceBio Q-TOF (Agilent) LC-MS / MS system (Shanghai Maitaijunao Biotechnology Co., Ltd.). Mobile phases: A1 (99.9% water + 0.1% formic acid) (FA, LC-MS grade, Thermo Fisher Scientific (China) Co., Ltd.), B1 (99.9% ACN + 0.1% FA). Analytical column: UPLC BEH 13 nm C18 1.7 μm 2.1 × 100 mm column (Waters), linear gradient from 1% to 37% in mobile phase B over 80 min. Flow rate: 0.2 mL / min, loading volume: 5 μL. Data acquisition was performed in positive ion mode, with a range of 150–2000 Da. Data analysis was conducted using appropriate mass spectrometry software.

[0016] Since the single quadrupole detector (QDA) is a single quadrupole mass spectrometer controlled by the chromatographic data software Empower 3, it has a relatively low acquisition range (50–1250 Da), and Empower 3 software lacks deconvolution functionality. Therefore, high-resolution mass spectrometry is used to directly determine the retention time (RT) and mass-to-charge ratio (m / z) of the target peptide. In this study, a Q-TOF high-resolution mass spectrometer was used to pre-characterize CMAB009 after trypsin digestion, identifying and obtaining information such as the sequences, molecular weights, retention times, and modification levels of peptides including CDR peptides, terminal modified peptides, oxidized peptides, deamidated peptides, and glycosylated peptides, as shown in Tables 1, 2, 3, and 4. Because the scanning mass range of Q-TOF differs from that of the single quadrupole detector, high-response m / z values ​​obtained on Q-TOF may not necessarily fall within the QDA scanning range, requiring m / z screening. The m / z values ​​of the multi-charged ions of the target peptide were calculated using the molecular weight calculation formula. The total ion chromatogram was obtained in QDA Fullscan mode. The m / z values ​​of multiple charges of the target peptide were extracted to screen for high-response m / z values ​​within the single quadrupole range.

[0017] Table 1 CDR Peptide Information

[0018] Table 2 Information on post-translational modified peptides

[0019] Table 3 Fc Glycopeptide Information

[0020] Table 4 Fab Glycopeptide Information

[0021] Example 2: Monitoring of key peptides in cetuximab monoclonal antibody using a single quadrupole-based MAM. 500 μg CMAB009 (≥5 mg / mL) in a 30KD ultrafiltration centrifuge tube was replaced with dilution buffer, and the buffer was added to a final volume of 100 µL. After dilution, 375 µL of denaturing buffer was added, followed by 7.2 µL of 1 mol / L dithiothreitol (DTT) (Shanghai Aladdin Biochemical Technology Co., Ltd.). The mixture was incubated at 37°C for 60 min. After cooling to room temperature, 8.5 µL of 2.9 mol / L iodoacetamide (MIA) (Shanghai Aladdin Biochemical Technology Co., Ltd.) was added, and the mixture was incubated at room temperature in the dark for 45 min. The reaction was terminated by adding 17.8 µL of 1 mol / L DTT. Sephadex G25 (Cytiva) was used to desalt the solution to 50 mmol / L ammonium bicarbonate buffer at pH 8.0. Trypsin (Shanghai Zhangjiang Biotechnology Co., Ltd.) was added at a 1:10 (m / m) ratio, and the mixture was incubated at 37°C for 4 hours. The reaction was terminated by adding 2 µL of FA. The mixture was centrifuged at 17000 g for 10 min before analysis. UPLC-single quadrupole detector method and parameter settings: The chromatographic column was a UPLC BEH 13 nm C18 1.7 μm 2.1×100 mm column (Waters). The mobile phases were A: 0.1% FA aqueous solution and B: 0.1% FA acetonitrile solution (LC-MS grade, Sigma-Aldrich (Shanghai) Trading Co., Ltd.). The liquid phase gradient program was 1% to 37% of mobile phase B over 80 min. The flow rate was 0.2 mL / min, and the column temperature was 45 ℃. The QDA detector parameters were set as follows: capillary voltage, 0.8 kV; cone voltage, 20 V; source temperature, 600 ℃; desolvation gas flow rate, 800 L / h; scan range, 300-~1250 u. Full scan and SIR modes were used for data acquisition. Empower 3 software was used to control the LC-UV-QDA system (Waters) and for data analysis.

[0022] like Figure 1As shown, for CDR peptides, oxidized peptides, deamidated peptides, and terminal modified peptides, high-response target peptides m / z were extracted and screened by creating a derivation channel after scanning with a single quadrupole mass spectrometer. All six CDR peptides were identifiable, but the H:T9 and H:T2 peptides, due to their similar polarities, could not be separated at the baseline. Monitoring of modified peptides allowed input of the acquisition switching time in the derivation channel, switching at specific times to extract different m / z values, thus avoiding peak overlap due to small molecular weight differences. Tables 5 and 6 show that the detection results of glycosylation, deamination, oxidation, C-terminal lysine removal, and N-terminal pyroglutamic acid cyclization based on high-resolution mass spectrometry (MAM) and single quadrupole MAM are comparable. The single quadrupole MAM method primarily extracts the ion with the highest response m / z within the target peptide range, using the high-response m / z value to represent the overall modification level. Compared to MAM results based on high-resolution mass spectrometry (HMS), the H:T18 oxidation modification level detected by single quadrupole MAM was higher and had a higher CV (21.13%). Oxidation sites may introduce additional modifications during sample processing, leading to increased oxidation levels and higher variability. Compared to MAM results based on HMS, L:T11 deamidation showed a higher CV (15.80%). The L:T11 deamidated peptide had a lower response and peak area, indicating that instrument noise had a slightly greater impact on integrated quantification.

[0023] Table 5. Detection results of PTMs for CMAB009 monoclonal antibody using different MAM methods.

[0024] Table 6 Comparison of CMAB009 monoclonal antibody glycosylation modification detection results

[0025] Example 3: The effect of FA on key quality properties of anti-PD-1 antibodies detected by a single quadrupole mass spectrometer To confirm the universality of this method, we also performed key quality attribute monitoring on an IgG4 subtype anti-PD-1 antibody (Shanghai Maitaijunao Biotechnology Co., Ltd.). Sample processing was as described in Example 1. The single quadrupole detector method and parameter settings were as follows: 1290 Infinity II LC-Infinity Lab LC / MSD iO single quadrupole detector (Agilent) method and parameter settings: HPLC column was UPLC BEH 13 nm C18 1.7 μm 2.1×150 mm Column (Waters), mobile phase was A: 0.1% FA aqueous solution and B: 0.1% FA acetonitrile solution; the liquid phase gradient program was that mobile phase B increased from 2% to 40% within 90 min. The flow rate was 0.2 mL / min, and the column temperature was 60 ℃. The MSD iQ detector parameters were set as follows: capillary voltage, 3.5 kV; dryer temperature, 325 °C; nebulizer gas pressure, 40 psi; dryer gas flow rate, 10 L / min; scan range, 50~1450 u; data were acquired using Scan and SIM modes. Agilent OpenLab Data Analysis software was used to control the LC-UV-MSD iQ system and perform data analysis.

[0026] like Figure 2 As shown, this method can monitor and quantify different key peptides. This indicates that the method has good versatility for different antibodies or different instrument brands.

[0027] Example 4: Effect of TFA on Key Quality Attributes of Anti-PD-1 Antibody Detection Using a Single Quadrupole Mass Spectrometer Trifluoroacetic acid (TFA) (LC-MS grade, Sigma-Aldrich (Shanghai) Trading Co., Ltd.) is an important ion-pairing reagent in liquid chromatography, especially suitable for the analysis of peptides and small proteins. However, in LC-MS detection systems, TFA can reduce detection sensitivity by inhibiting the ionization of analytes in the electrospray ionization (ESI) source. To examine whether TFA is also suitable for this method, we replaced FA with TFA and tested the impact of TFA usage.

[0028] Sample preparation was as described in Example 1. The single quadrupole detector method and parameter settings were as follows: 1290 Infinity IILC-Infinity Lab LC / MSD iQ single quadrupole detector method and parameter settings: UPLC BEH 13 nm C18 1.7 μm 2.1 × 150 mm Column (Waters); mobile phase: A: 0.1% TFA aqueous solution and B: 0.1% TFA acetonitrile solution; liquid phase gradient program: mobile phase B increased from 2% to 40% within 90 min; flow rate: 0.2 mL / min; column temperature: 60℃. MSDiQ detector parameters were set as follows: capillary voltage, 3.5 kV; dryer temperature, 325℃; nebulizer gas pressure, 40 psi; dryer gas flow rate, 10 L∙ / min⁻¹; scan range, 50~1450 u; data acquisition using Scan and SIM modes. Agilent OpenLab Data Analysis software was used to control the LC-UV-MSD iQ system and perform data analysis.

[0029] like Figure 3 The results showed that after TFA replacement, all key modified peptides could be detected well, with only a slight decrease in signal intensity. Figure 2 As can be seen, the detection rate of some oxidized peptides may be higher under TFA conditions, so the consistency of each modification needs to be discussed on a case-by-case basis.

[0030] Example 5 Methodological Validation Using the polysaccharide-sylation site antibody drug CMAB009 as a model monoclonal antibody, and a single quadrupole detector as a representative, the methodology was validated.

[0031] A blank control was prepared using 50 mmol / L ammonium bicarbonate solution with an appropriate amount of trypsin. The blank control should be free of interference. The system suitability control solution was injected six times repeatedly. The levels of oxidized, deamidated, terminally modified, Fc-glycosylated fucoidan, core-free fucose, high-mannose (Man5), and total sialic acid glycotypes in the Fab glycosylation were examined in each control solution, and the RSD% values ​​were calculated (n=6). The system suitability results are shown in Table 7. The RSDs of the low-response L:T11 oxidized peptide and Fc glycopeptide Man5 detected in the six repeated injections of the system suitability control solution were 14.85% and 12.51%, respectively. Other target peptides all had small RSD values ​​(RSD ≤ 20%). The blank control did not interfere with the multi-attribute monitoring of CMAB009. The results indicate good system suitability and specificity.

[0032] Table 7 System Applicability Results

[0033] Repeatability: Six parallel sample solutions were prepared to examine the total sialic acid glycotype levels in oxidized, deamidated, Fc-terminated fucoidan oligosaccharides, core-free fucoidan, high-mannose (Man5), and Fab glycosylated solutions, and the RSD% value was calculated (n=6). Intermediate precision: Six parallel sample solutions were prepared on different days to examine the total sialic acid levels in oxidized, deamidated, Fc-terminated fucoidan oligosaccharides, core-free fucoidan, high-mannose (Man5), and Fab glycosylated solutions, totaling 12 sample solutions, and the RSD% value was calculated (n=12). The repeatability results are shown in Table 8. The total sialic acid levels in oxidized, deamidated, Fc-terminated fucoidan oligosaccharides, core-free fucoidan, high-mannose (Man5), and Fab glycosylated solutions in the six parallel sample solutions all showed good RSD% (RSD ≤ 20%). The intermediate precision results are shown in Table 9. Six sample solutions prepared in parallel by the same personnel on different days were examined, and the total sialic acid levels in oxidized, deamidated, terminally modified Fc glycosylated fucoidan, core-free fucose, high-mannose (Man5), and Fab glycosylated samples were investigated in a total of 12 sample solutions. All samples had good RSD n=12% (RSD ≤ 20%). Low levels of PTMs may show higher RSD values.

[0034] Table 8 Repeatability Test Results

[0035] Table 9 Intermediate Precision Test Results

[0036] The results of total sialic acid in oxidation, deamidation, Fc-terminated fucose biantennary oligosaccharides, core-free fucose, high-mannose (Man5), and Fab glycosylation detected by single quadrupole MAM were compared with those of Q-TOF MAM. The glycosylation level in single quadrupole MAM was also compared with the HILIC results of traditional free oligosaccharide levels. To ensure that QDA-based MAM has comparable or higher quality attribute monitoring capabilities than traditional methods or high-resolution MAM, the results of total sialic acid in oxidation, deamidation, Fc-terminated fucose biantennary oligosaccharides, core-free fucose, high-mannose (Man5), and Fab glycosylation detected by QDA MAM were compared with the results obtained by Q-TOF MAM as true values. Simultaneously, the glycoform detection results were compared with those of traditional free oligosaccharide levels. The accuracy results are shown in Table 10. The detection results of the QDA-based MAM method for various quality attributes are basically consistent with the detection results of high-resolution MAM and traditional HILIC.

[0037] Table 10 Accuracy Test Results

[0038] After desalting, the sample was concentrated to 400 μg / mL. Following enzymatic digestion, the sample was diluted to concentrations of 300, 200, 100, 75, 50, 40, 20, and 10 μg / mL. Linear regression was performed using the peak area of ​​the L:T11 deamidinated peptide, which showed significant separation from the unmodified peptide and exhibited the lowest response, against the sample concentration. A range was determined by considering linearity, accuracy, and precision. Within this range, the L:T11 deamidinated peptide should demonstrate good linearity, accuracy, and precision. The L:T11 deamidinated peptide, showing significant separation from the unmodified peptide and exhibiting the lowest response, was used as a representative for linearity assessment. Linear regression was performed using peak area against sample concentration. The linear fit graph is shown below. Figure 4 As shown, linear analysis was performed with the enzyme digestion sample concentration as the x-axis and the peak area of ​​the L:T11 deamidated peptide as the y-axis. The linear regression equation was y = 315.3x + 237.56; the correlation coefficient R² = 0.9921 (≥ 0.990). The results indicate that the method exhibits good linearity within the range of 50–400 μg / mL. At lower sample concentrations, the peak area of ​​the L:T11 deamidated peptide is significantly affected by noise, impacting the accuracy of quantification.

[0039] The limits of detection (LOD) and quantitation (LOQ) were determined based on the signal-to-noise ratio (SNR) of the extracted ion spectrum of L:T11 deamidinated peptides. An acceptable SNR of 3:1 was found for the LOD, and 10:1 for the LOQ. L:T11 deamidinated peptides with lower signal responses were used as representative peptides, and their LOD and LOQ were determined using the extracted ion spectrum. An acceptable SNR of 3:1 was found for the LOD, and 10:1 for the LOQ. The results indicate that the LOD of this method is 20 μg / mL, and the LOD is 100 μg / mL.

[0040] Samples were placed in an 8°C autosampler for 4 and 24 h, and the column temperature was varied (40, 45, 50°C). Under constant conditions, three injections were performed for each treatment to examine the total sialic acid levels in oxidized, deamidated, Fc-terminated fucose bianthraquinone oligosaccharides, core-free fucose, high-mannose (Man5), and Fab-glycosylated samples, and the RSD% values ​​were calculated (n=9). Robustness results are shown in Table 11. The total sialic acid levels in 12 sample solutions under the three treatments—oxidized, deamidated, Fc-terminated fucose bianthraquinone oligosaccharides, core-free fucose, high-mannose (Man5), and Fab-glycosylated—all exhibited good RSD% (RSD ≤20%). Oxidized and deamidated peptides showed a slight increase during storage, resulting in significant variability. Table 12 shows the robustness results of varying column temperatures (40, 45, and 50 °C). Under the three column temperature conditions, the total sialic acid levels in the 12 sample solutions for oxidation, deamidation, Fc-terminated fucose bianthraquinone, core-free fucose, high-mannose (Man5), and Fab glycosylation all exhibited good RSD% (RSD ≤ 20%). Studies have shown that increasing temperature leads to increased protein oxidation and deamidation levels, with deamidation being more susceptible to temperature changes. In conclusion, this method demonstrates good robustness.

[0041] Table 11 Durability_Storage Time

[0042] Table 12 Durability Results_Column Temperature

Claims

1. A multi-attribute mass spectrometry analysis method for antibodies, characterized in that, Multi-attribute analysis was performed using a single quadrupole mass spectrometer.

2. The multi-attribute analysis method as described in claim 1, characterized in that: a. Replace the antibody sample using an ultrafiltration centrifuge tube to obtain the replaced sample; b. Denaturation, reduction, alkylation, and termination of the reaction of the substituted sample; c. Further desalting, enzyme digestion, and termination of enzyme digestion reaction of the sample; d. After centrifugation, the processed samples were subjected to multi-attribute analysis using a single quadrupole centrifuge.

3. The multi-attribute analysis method as described in claim 2, characterized in that, The antibody mentioned in step a is cetuximab monoclonal antibody.

4. The multi-attribute analysis method as described in claim 2, characterized in that, The sample replacement in step a involves replacing the sample three or more times in a dilution buffer solution using a 30KD ultrafiltration centrifuge tube, with a final sample volume of 95-115 µL.

5. The multi-attribute analysis method as described in claim 2, characterized in that, In step b, the denaturation of the replaced sample is performed by adding 350-380 µL of denaturing buffer to the replaced sample; the reduction is performed by adding 7.0-7.5 µL of 0.95-1.15 mol / L DTT to the denatured sample and incubating at 35-39°C for 60±5 min; the alkylation is performed by adding 8.0-8.7 µL of 2.9 mol / L sodium iodoacetate to the reduced sample and incubating at room temperature in the dark for 43-47 min, and then terminating the reaction by adding 16.2-18.4 µL of 0.95-1.15 mol / L DTT.

6. The multi-attribute analysis method as described in claim 2, characterized in that, The further desalting of the sample in step c is performed by using a Sephadex G25 column to desalt the sample into 48-53 mmol / L pH 7.8-8.1 ammonium bicarbonate buffer; the enzyme digestion and termination of the enzyme digestion reaction are performed by adding trypsin at a ratio of 1:10-1:14 (m / m), incubating at 35-39℃ for 4h±10min, and then adding 1.8-2.5 µL FA to terminate the reaction.

7. The multi-attribute analysis method as described in claim 2, characterized in that, After the processed sample in step d is centrifuged, multi-attribute analysis is performed on a single quadrupole analyzer. The sample is centrifuged at 17000 g for 10 min and analyzed on a 1290 Infinity II LC-Infinity Lab LC / MSD iO single quadrupole analyzer.

8. The multi-attribute analysis method as described in claim 6, characterized in that, The analytical methods and parameters were set as follows: The chromatographic column was a UPLC BEH 13 nm C18 1.7 μm 2.1 × 150 mm column (Waters); the mobile phases were A: 0.1% FA aqueous solution and B: 0.1% FA acetonitrile solution; the liquid phase gradient program was 2% to 40% of mobile phase B over 90 min; the flow rate was 0.2 mL / min; and the column temperature was 60 ℃. The MSD iQ detector parameters were set as follows: capillary voltage, 3.5 kV; desiccator temperature, 325 ℃. Nebulizer gas pressure: 40 psi; dryer gas flow rate: 10 L / min; scan range: 50–1450 u; data were acquired using Scan and SIM modes. Agilent OpenLab Data Analysis software was used to control the LC-UV-MSD iQ. system and for data analysis.

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