Rapid multi-attribute analysis method for antibody drug

Abstract

The invention discloses a rapid multi-attribute analysis method for an antibody drug, belongs to the field of biological medicines, and is used for quality characterization of the antibody drug, especially characterization of post-translational modifications such as succinimide. The method comprises the following steps: carrying out enzyme digestion on an antibody by using IdeS protease under the condition that the pH is 6.6, then reducing, and carrying out MAM analysis by using a method based on LC-MS (Liquid Chromatography-Mass Spectrometer). Compared with a peptide map method, the method has the advantages that the level of succinimide can be represented more accurately, time is saved, and multi-attribute detection can be provided; compared with hydrophilic chromatography, the method can provide positioning information of post-translational modification. And the method is suitable for various forms of antibody drugs such as IgG1, IgG2, IgG4, Fc fusion protein and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for multi-attribute analysis of antibody drugs. Background Technology

[0002] Therapeutic antibody drugs have undergone multiple generations of development, with continuously improving efficacy, leading to sustained growth in global demand for these biopharmaceuticals. During production and storage, these antibodies undergo extensive post-translational modifications (PTMs), such as glycosylation, oxidation, deamidation, isomerization, pyroglutamate cyclization, and terminal lysine cleavage, resulting in significant molecular heterogeneity. These modifications collectively contribute to over a hundred forms of heterogeneity in antibody drugs, posing a substantial challenge to quality control.

[0003] Among the many key peptide markers (PTMs), succinimide (Asu) is a crucial intermediate in the deamidation process of asparagine (Asn). Under neutral to alkaline conditions, Assu rapidly hydrolyzes to produce aspartic acid (Asp) and isoaspartic acid (isoAsp) residues. This instability complicates accurate detection because the most commonly used Assu analysis method—traditional peptide mapping (PM)—is prone to introducing artifacts. PM typically requires trypsin digestion at 37°C and a slightly alkaline pH for several hours to overnight. This process can lead to Assu hydrolysis, resulting in false negatives or false positives. Therefore, reliable Assu quantification is difficult to achieve using traditional methods.

[0004] Other PTMs, such as the oxidation of methionine (Met) and tryptophan (Trp), can also affect antibody quality. These variability highlights the necessity of performing comprehensive PTM profiling analysis.

[0005] Multi-Attribute Method (MAM) is a highly efficient biopharmaceutical quality control technology that has emerged in recent years. By simultaneously detecting multiple key quality indicators of antibody drugs (such as glycosylation and oxidation), it can rapidly assess product stability and process consistency, helping to ensure drug quality. Generally, MAM relies on liquid chromatography to separate sample components, mass spectrometry for precise detection, enzymatic digestion to improve sensitivity, and integrates multimodal analysis and automated data processing technologies.

[0006] Traditional LC-MS (liquid chromatography-tandem mass spectrometry) typically only addresses differences in a single aspect (such as molecular weight, glycosylation, or peptide map). MAM, on the other hand, monitors multiple key quality attributes (such as sequence, glycoform, oxidation, deamidation, C-terminal lysine deletion, etc.) on a single platform. By using peptide maps and database comparison, it can directly replace traditional IEF, CE-SDS, HILIC, and other multi-item detection methods, achieving multiple detections with a single method. Summary of the Invention

[0007] This invention provides a rapid subunit-level MAM analysis method for antibody drugs, which can accurately characterize succinimide (Asu) while performing comprehensive PTM analysis. This method employs a moderate degradation (Middow Down) strategy.

[0008] The technical solution of the present invention is as follows: The antibody samples to be tested were sequentially digested and reduced with IdeS (Immunoglubulin G-degrading enzyme of Streptococcus pyogenes), and then detected using reversed-phase liquid chromatography-tandem mass spectrometry.

[0009] Specifically, the method is as follows: The antibody sample to be tested (including monoclonal antibodies, Fc fusion proteins, antibody-drug conjugates, and other macromolecules containing the antibody Fc fragment) is dissolved or replaced in 50 mmol / L ammonium formate buffer with a pH of 6.0-7.0, preferably 6.6, and the final antibody concentration is 0.5 mg / mL.

[0010] Then perform the following operations in sequence:

[0011] (1) Enzyme digestion: Add enzyme at a ratio of 4 μg IdeS enzyme per 200 μg antibody (i.e., antibody to enzyme mass ratio of 50:1), and incubate at 37°C for 20-40 minutes, preferably 30 minutes.

[0012] (2) Reduction: Add 5.3 μL of dithiothreitol (1 mmol / L) to each 100 μL sample and reduce at 37℃ for 10 minutes.

[0013] (3) Centrifuge the sample at 17000 g for 10 minutes at 4℃. After centrifugation, perform LC-MS analysis under the following conditions:

[0014] 3.1 Reversed-phase chromatographic conditions A Waters Acquity UPLC BEH C4 column was used (the stationary phase matrix is ​​an ethylene-bridged hybrid siloxane framework material with C4 alkyl chain ligands bonded to the surface, i.e., butyl ligands; column inner diameter 2.1 mm, length 50 mm, particle size 1.7 μm). The column temperature was set to 60 °C. The mobile phase A for reversed-phase liquid chromatography was an aqueous solution containing 0.1% formic acid, and the mobile phase B was an acetonitrile solution containing 0.1% formic acid. Preferably, the linear gradient increased from 25% B to 35% B within 6 minutes, which could be optimized according to different antibody subunits. The total run time was 16 minutes. The flow rate was 0.4 mL / min, and the injection volume was 1 μL.

[0015] 3.2 Mass Spectrometry Conditions Data was acquired using positive ion mode (electrospray ionization, ESI), with a detection range of m / z 500–2500. Ion source parameters were set as follows: capillary voltage: 3.0 kV; desolvation temperature: 300℃; source temperature: 80℃; desolvation gas flow rate: 800 L / h. The instrument was calibrated using NaI in a 50% isopropanol aqueous solution within the m / z 500–2000 range.

[0016] The system was controlled by MassLynx 4.2 and UNIFI was used for data analysis to obtain subunit experimental data.

[0017] Preferably, the above chromatographic mass spectrometry analysis was performed using a Waters ACQUITY UPLC RDa system.

[0018] The present invention has the following beneficial technical effects: Employing a "middle-down" strategy, this approach integrates IdeS enzyme-mediated subunit-level antibody degradation with optimized LC-MS conditions, achieving an optimal balance between analytical depth and speed. It provides higher PTM site resolution compared to whole subunit analysis, while significantly reducing sample processing time and the risk of artificial modification compared to bottom-up PM analysis. Operation at pH 6.6 maximizes the preservation of easily degradable Asu intermediates.

[0019] The method has been validated to show excellent repeatability and intermediate precision (CV <5% and <15%, respectively), and quantitative results that are highly consistent with orthogonal techniques (HILIC, peptide mapping). It also shows good linearity (R² > 0.98) for Asu and oxidative modification under various forced degradation conditions. It is well tolerant to fluctuations in key parameters such as column temperature, loading volume, and scan time, making it very suitable for routine QC deployment.

[0020] Furthermore, this method demonstrates broad applicability in biopharmaceuticals with extremely high structural diversity: it has been successfully applied to IgG1, IgG2, and IgG4 subclasses; it is adapted to antibody-drug conjugates (ADCs) and Fc fusion proteins; and it can simultaneously quantify multiple key quality attributes (CQAs) such as glycoform, Asu, oxidation, and N- / C-terminal modifications. Attached Figure Description

[0021] Figure 1 The structure of monoclonal antibodies (mAbs) and their key post-translational modifications (PTMs). Figure 2 Quantitative results of Asu content based on peptide mapping under different pH conditions Figure 3 Representative total ion current (TIC) chromatograms obtained from peptide mapping analysis at pH 5.0 and 8.0, 5-55 minutes. Figure 4 Representative total ion current (TIC) chromatograms obtained from peptide mapping analysis at pH 5.0 and 8.0 conditions (55-100 minutes) Figure 5 The effect of incubation time at pH 6.6 on the analysis of succinimide (Asu) content using the subunit-based MAM method. Figure 6 The effect of incubation time at pH 8.0 on the analysis of succinimide (Asu) content using the subunit-based MAM method. Figure 7 Linear relationship of Asu content determination by dithiothreitol (DTT) reducing subunit method under pH 5.0 conditions. Figure 8 Linear relationship of Asu content determination by dithiothreitol (DTT) reducing subunit method under pH 6.0 conditions. Figure 9 Linear relationship of Asu content detection by dithiothreitol (DTT) reducing subunit method under pH 6.6 conditions. Figure 10 Linear relationship of Asu content determination by dithiothreitol (DTT) reducing subunit method under pH 7.0 conditions. Figure 11 Linear relationship of Asu content determination by dithiothreitol (DTT) reducing subunit method under pH 8.0 conditions. Figure 12 Linear relationship of Asu content determination by dithiothreitol (DTT) reducing subunit method under pH 9.0 conditions. Figure 13 The linear relationship of Asu content was detected using the IdeS-based subunit MAM method at pH 5.0. Figure 14 The linear relationship of Asu content was detected using the IdeS-based subunit MAM method at pH 6.0. Figure 15 The linear relationship of Asu content was detected using the IdeS-based subunit MAM method at pH 6.6. Figure 16 The linear relationship of Asu content was detected using the IdeS-based subunit MAM method at pH 7.0. Figure 17 The linear relationship of Asu content was detected using the IdeS-based subunit MAM method at pH 8.0. Figure 18 The linear relationship of Asu content was detected using the IdeS-based subunit MAM method at pH 9.0. Figure 19 Cetuximab digestion results at different IdeS ratios Figure 20 Enzyme digestion results of cetuximab at different IdeS incubation times Figure 21 Enzyme digestion results of golimumab at different protein to IdeS ratios Figure 22 Enzyme digestion results of golimumab at different incubation times Figure 23 LC-MS quantitative analysis of the relative abundance of Fc fragments of cetuximab and golimumab under different IdeS restriction enzyme digestion conditions Figure 24 LC-MS quantitative analysis of the relative abundance of Fc fragments of cetuximab and golimumab under different IdeS incubation times Figure 25 Flowchart of the Subunit-Based Fast Multi-Attribute Method (MAM) Figure 26 LC-MS deconvolution validation plot for cetuximab specificity detected by MAM method Figure 27 LC-MS deconvolution validation map for detecting golimumab specificity using the MAM method Figure 28 The HILIC / PM method was used to validate the linear regression of subunit-based MAM. Figure 29 Linear regression analysis of Asu quantification of golimumab using the MAM method and the dilution ratio of the stock solution. Figure 30 Linear regression analysis of Asu quantification and peptide mapping values ​​of golimumab using the MAM method Figure 31 Linear regression analysis of MAM and PM methods for detecting 0.1% AAPH-induced cetuximab scFc subunit oxidation. Figure 32 Linear regression analysis of MAM and PM methods for detecting 0.1% AAPH-induced oxidation of cetuximab Fd subunits. Figure 33 Linear regression analysis of MAM and PM methods for detecting 0.1% AAPH-induced cetuximab LC subunit oxidation. Figure 34 Linear regression analysis of MAM and PM methods for detecting 0.1% t-BHP-induced cetuximab scFc subunit oxidation. Figure 35 Linear regression analysis of MAM and PM methods for detecting 0.01% H2O2-induced cetuximab scFc subunit oxidation. Figure 36 Representative spectra of mAb1 (cetuximab) characterized using the rapid subunit-level MAM method. Figure 37 Representative spectra of mAb2 (golimumab) characterized using the rapid subunit level MAM method. Figure 38 Representative spectra of mAb3 characterized using the rapid subunit-level MAM method Figure 39 Representative spectra of mAb4 characterized using the rapid subunit-level MAM method Figure 40 Representative spectra of mAb5 characterized using the rapid subunit-level MAM method Figure 41 Representative spectra of mAb6 characterized using the rapid subunit-level MAM method Figure 42 Representative spectra of mAb7 characterized using the rapid subunit-level MAM method Figure 43 Representative spectra of mAb8 characterized using the rapid subunit-level MAM method Detailed Implementation

[0022] Unless otherwise specified, the reagents, materials, and instruments used in the following procedures are as follows:

[0023] Reagents and materials: Hydrogen peroxide, tert-butyl hydroperoxide (t-BHP), 2,2'-azobis(2-amidinylpropane) dihydrochloride (AAPH), dithiothreitol (DTT), guanidine hydrochloride (GuHCl), iodoacetamide (IAM), ammonium formate (NH4FA), ammonium bicarbonate (NH4HCO3), 30% sodium N-lauroyl glycinate, and formic acid (FA, mass spectrometry grade) were purchased from Sigma-Aldrich (USA).

[0024] Acetonitrile (ACN, LC / MS grade) was supplied by Thermo Scientific, Inc. (USA).

[0025] Cetuximab (mAb1) and golimumab (mAb2) were prepared and expressed by Shanghai Zhangjiang Biotechnology Co., Ltd. using recombinant CHO cells (Chinese hamster ovary cells). The structures of these two monoclonal antibodies (mAbs) and their key post-translational modifications (PTMs) are described below. Figure 1 .

[0026] Endo F2 (endonuclease F2), glycosidase PNGase F, trypsin (sequencing grade), and IdeS enzyme were purchased from Shanghai Zhangjiang Biotechnology Co., Ltd.

[0027] The reversed-phase column used was a Waters Acquity UPLC BEH C4 column (2.1 x 50 mm, 1.7 μm), catalog number 186004495.

[0028] instrument: Chromatographic separation and mass spectrometric analysis of subunit-level MAMs and moderately degraded MAMs were performed on a Waters ACQUITY UPLC RDa system (Waters, Massachusetts, USA). This system is equipped with a quaternary solvent manager, sample manager, and UV detector. Mass spectrometric detections were performed using a compact mass spectrometer detector with an electrospray ionization (ESI) source.

[0029] Data acquisition and deconvolution were performed using Waters UNIFI software (version 3.12.0.548), which uses the integrated MaxEnt1 algorithm to process the mass spectra.

[0030] Confirmatory MS / MS measurements were performed on a Waters Premier UPLC XEVO G3 Q-TOF system (Waters, Massachusetts, USA).

[0031] Glycan analysis was performed using an Agilent 1260 HPLC system equipped with a fluorescence detector (Agilent, California, USA).

[0032] Example 1: Rapid subunit level MAM (using Middow Down moderate degradation strategy)

[0033] The specific implementation method is as follows.

[0034] 1.1 Buffer exchange:

[0035] The antibody sample to be tested (such as cetuximab or golimumab) was replaced with 50 mmol / L ammonium formate buffer (pH 6.6) to a final concentration of 0.5 mg / mL.

[0036] 1.2 Enzyme digestion: Add the enzyme at a ratio of 4 μg IdeS enzyme per 200 μg of therapeutic antibody, and incubate at 37°C for 30 minutes.

[0037] 1.3 Restoration: Add 5.3 μL of DTT (1 M) to every 100 μL of sample and reduce at 37°C for 10 minutes. Centrifuge the sample at 17000 g for 10 minutes at 4°C and collect the supernatant for loading.

[0038] 1.4 MAM Analysis: Perform under the following conditions: Reversed-phase liquid chromatography (RP-LC) conditions: A Waters UPLC ACQUITY RDa system and a Waters Acquity UPLCBEH C4 column (2.1 x 50 mm, 1.7 μm) were used, with the column temperature set at 60 °C. The mobile phases for the UPLC system were: A1 (99.9% H2O + 0.1% FA) and B1 (99.9% ACN + 0.1% FA). A linear gradient of 25%–35% B was applied over 0–6 minutes, with a total run time of 16 minutes. The flow rate was 0.4 mL / min, and the injection volume was 1 μL.

[0039] Mass spectrometry conditions: Data were acquired via positive ion mode (electrospray ionization, ESI), with a detection range of 500–2500 Da. Ion source parameters were set as follows: capillary voltage: 3.0 kV; desolvation temperature: 300 °C; source temperature: 80 °C; desolvation gas flow rate: 800 L / h. The instrument was calibrated using NaI in a 50% isopropanol aqueous solution within the m / z range of 500–2000. The system was controlled by a MassLynx 4.2, and data analysis was performed using UNIFI to obtain subunit experimental data.

[0040] Example 2: Assessment of the effect of pH on Asu and development of novel mass spectrometry characterization methods

[0041] We first investigated the effect of pH on Asu in traditional peptide mapping methods for characterizing antibody quality. Then, we developed and compared two mass spectrometry-based methods: subunit-level MAM and middle-down MAM. We named the MAM using the middle-down strategy "rapid subunit-level MAM". Both strategies are designed to reduce sample complexity compared to whole protein mass analysis, but they differ in the resolution of structural details, which directly affects the localization ability of post-translational modifications (PTMs) and analytical throughput.

[0042] 2.1 Evaluation of the effect of pH on Asu intermediate levels in peptide mapping (PM)

[0043] We first used the gold standard method for Asu analysis—the traditional peptide mapping (PM) method—to assess the effect of pH on Asu intermediate levels.

[0044] A traditional bottom-up peptide mapping method was employed. The sample was concentrated to 5 mg / mL and dissolved in 50 mM ammonium formate solution. Denaturation, DTT reduction, and IAM alkylation were performed sequentially. Subsequently, the sample was replaced with 50 mM NH4HCO3 buffer using a G25 desalting column (Cytiva, USA), and the pH was adjusted to different values ​​using hydrochloric acid or ammonia. Trypsin (enzyme:substrate = 1:20 w / w) was added, and digestion was carried out at 37°C for 4 hours. After the reaction was complete, 0.1% formic acid was added to terminate the digestion.

[0045] The obtained peptides were separated on a Waters Acquity UPLC BEH C18 column (2.1 x 100 mm, 1.7 μm, 130 Å) at a column temperature of 45 °C. A relatively long 80-minute linear gradient elution was used, with the proportion of mobile phase B (ACN containing 0.1% FA) in mobile phase A (water containing 0.1% FA) increasing from 1% to 37%, at a flow rate of 0.20 mL / min. Mass spectrometry was performed in positive ion ESI mode, with a scan range of 100 to 2000 Da. Due to the low flow rate, the ion source parameters were adjusted: desolvation temperature was 450 °C, and source temperature was 120 °C.

[0046] Peptide profiling results at different pH levels are as follows Figure 2 As shown, the Asu content gradually decreased as the pH increased from 5.0 to 9.0, exhibiting relative stability only in the pH range of 5.0 to 6.0. However, under these weakly acidic conditions, the digestive efficiency of trypsin decreased significantly.

[0047] This is clearly evident in the total ion current chromatogram (TIC): Figure 3 and 4Representative total ion current (TIC) chromatograms obtained from peptide mapping analysis at pH 5.0 and pH 8.0 are presented, illustrating the trade-off between enzymatic cleavage efficiency and Asu stability. At pH 8.0 ( Figure 3 The enzyme digestion was more complete, and more peptide peaks appeared in the first 50 minutes; conversely, under pH 5.0 conditions ( Figure 4 The higher signal response in the later stages of the chromatographic gradient indicates the presence of incompletely digested peptides, confirming low enzymatic cleavage efficiency. This leads to a fundamental trade-off: while alkaline conditions favor trypsin activity, they accelerate Asu hydrolysis; conversely, while acidic conditions protect Asu, they inhibit the enzymatic cleavage reaction. Both of these conditions introduce measurement biases, such as false negatives or nonspecific peptide modifications. These limitations highlight the inadequacy of traditional peptide mapping methods in monitoring therapeutic antibody Asu.

[0048] 2.2 Development of a Novel Subunit-Level MAM Method

[0049] To overcome the limitations of the peptide mapping (PM) method, we first developed a subunit-level MAM method. This method uses DTT reduction to generate subunits, followed by MAM analysis. This reduced subunit method shortens sample processing time and operates under optimized pH conditions to maintain the integrity of Asu. The method is as follows: The monoclonal antibody sample was replaced with buffer in 50 mM NH4FA at different pH levels (adjusted with ammonia) until a final concentration of 0.5 mg / mL was achieved. The sample was then reconstituted by adding DTT to a final concentration of 50 mM and incubating at 37°C for 10 minutes. The reconstituted sample was centrifuged at 17,000 × g for 10 minutes at 4°C, and the supernatant was subsequently used for LC-MS analysis, as described in Section 1.4 of Example 1.

[0050] We evaluated the stability of Asu over time at pH 6.6 and pH 8.0, and the results are shown in [Figure number missing]. Figure 5 , Figure 6 At pH 8.0, Asu levels began to decrease significantly after more than 1 hour of incubation; while at pH 6.6, Asu remained stable for 24 hours and showed very little degradation in the first 6 hours.

[0051] Based on this stable window period, we further evaluated the linear response of Asu detection by subunit-level MAM assay within the pH range of 5.0–9.0. The results are shown in [Figure number missing]. Figures 7 to 12 Linear regression analysis confirmed that it exhibited excellent linearity across the entire test pH range, with a significant deviation only at pH 9.0 due to accelerated Asu hydrolysis under strongly alkaline conditions.

[0052] 2.3 Development of a more optimized method for moderately degrading MAM (i.e., rapid subunit-level MAM)

[0053] To achieve a balance between high-resolution modification localization and protection of unstable modifications such as Asu, we further developed a middle-down strategy based on the IdeS protease, which we named "Rapid Subunit Level MAM". The IdeS protease can cleave the heavy chain (HC) into well-defined fragments (Fab-derived Fd and Fc fragments), thereby reducing molecular complexity and significantly improving site-specific localization compared to intact subunit analysis.

[0054] The method is as follows: The monoclonal antibody sample buffer was replaced with 50 mM NH4FA at different pH values ​​(adjusted with ammonia) to a final concentration of 0.5 mg / mL. IdeS protease was added at a ratio of 1:50 (w / w, enzyme to antibody) and incubated at 37°C for 30 min. Then, the reaction was reduced by adding DTT to a final concentration of 50 mM and incubating at 37°C for 10 min. Finally, the sample was centrifuged at 17,000 g for 10 min at 4°C, and the supernatant was subsequently used for LC-MS analysis, as described in Section 1.4 of Example 1.

[0055] The linear calibration curve constructed using this IdeS-cutting subunit method is shown in [reference needed]. Figures 13 to 18 The results showed that Asu detection exhibited excellent linearity within the pH range of 5.0–7.0. Although a rapid decrease in Asu content was observed under alkaline conditions (more than 30% loss at pH 9.0 compared to pH 7.0) – confirming the instability of Asu even in the absence of trypsin – this method successfully maintained Asu stability within the optimized pH window. More importantly, this moderate degradation strategy not only preserved the integrity of Asu under appropriate conditions but also simultaneously monitored multiple quality properties, achieving an optimal balance between analytical depth, modification stability, and multi-attribute coverage.

[0056] 2.4 Optimization of protease ratio and digestion time in the moderate degradation method of MAM

[0057] For the critical step of IdeS enzyme digestion efficiency, we used two model monoclonal antibodies—cetuximab (mAb1) and golimumab (mAb2)—to evaluate six different protein-to-enzyme ratios (1:10 to 1:100) and five incubation times (20 to 40 minutes). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed. Figure 5 See results Figures 19 to 22 , Figure 19Figures 2 and 20 show the enzyme digestion results of cetuximab under different protein to IdeS ratios and different incubation times, while Figures 21 and 22 show the corresponding enzyme digestion results of golimumab. The results show that under all test conditions, both antibodies achieved complete and consistent cleavage, and no significant differences were observed.

[0058] To supplement this qualitative assessment, the relative abundance of the Fc fragment was quantified using subunit-level MAM. Figure 23 , Figure 24 The results show the relative abundance of Fc fragments of cetuximab and golimumab under different IdeS digestion conditions and incubation times. The results confirm that the variability of digestion efficiency is extremely small, verifying the robustness of this digestion step.

[0059] Based on the aforementioned research, in order to strictly control the total sample processing time within 1 hour and retain Asu to the maximum extent, the pH of the buffer solution was evaluated within the range of 5.0–7.0, and pH 6.6 was finally selected because it achieves the best balance between DTT reduction efficiency, IdeS activity and Asu stability. Based on the combined SDS-PAGE and MAM data, the optimal IdeS conditions were determined to be: enzyme to antibody mass ratio of 1:50, incubation at 37°C for 30 min.

[0060] The optimized process flow for moderately degrading MAM is shown below. Figure 25 The process includes steps such as IdeS enzyme digestion, DTT reduction, and LC-MS detection, as detailed in Example 1.

[0061] Example 3 Validation of the method for moderately degrading MAM

[0062] 3.1 Specificity

[0063] The method described in Example 1 was used to simultaneously analyze blank matrix (50 mmol / L ammonium formate, pH 6.6, with IdeS enzyme added) and cetuximab (mAb1) and golimumab (mAb2) samples to verify the specificity of the method. The deconvolution LC-MS mass spectra are shown below. Figure 26 (Cetuximab) Figure 27 (Golimucilab) showed no interfering peaks at the retention times of the target subunits (LC, Fd, and Fc) of mAb1 and mAb2. This confirms the high specificity of the method, which can clearly distinguish analyte signals from potential matrix impurities, a prerequisite for reliable quality control (QC).

[0064] 3.2 Precision

[0065] The precision of the rapid subunit method described in Example 1 was evaluated from two perspectives: repeatability (intra-day) and intermediate precision (inter-day, different operators, different dates). The analysis focused on five representative post-translational modifications (PTMs) of the antibody: Asu, oxidation, glycosylation, N-terminal pyroglutamate (Pyr), and C-terminal lysine variation (-K).

[0066] The analytical results for cetuximab (mAb1) and golimumab (mAb2) are shown in Table 1. This method demonstrated excellent precision on both antibodies. All repeatability coefficients of variation (CV) were ≤4.74%, and the intermediate precision CV values ​​ranged from 0.15% to 13.55%, significantly lower than the industry-recognized PTM quantification standards (typically CV < 15%, repeatability < 5%). This low variability ensures reliable quantification of critical quality attributes (CQA) even under routine operational fluctuations.

[0067] Table 1. Precision parameters of key PTMs in mAb1 and mAb2 analyzed by MAM method

[0068]

[0069] 3.3 Accuracy

[0070] The method of Example 1 was compared with an orthogonal reference method to analyze cetuximab (mAb1) and golimumab (mAb2) to evaluate the accuracy of the method. The orthogonal reference method included: determining terminal modifications using peptide mapping (PM) and determining glycosylation using HILIC (hydrophilic chromatography). The peptide mapping procedure was the same as described in 2.1 of Example 2, and the HILIC procedure was as follows: First, mAb1 was digested with the IdeS enzyme, and the Fc and F(ab')2 fragments were separated using a Protein A column (Shanghai Zhangjiang Biotechnology Co., Ltd.). The N-glycans released from each fragment were fluorescently labeled with 2-aminobenzamide (2-AB). After purification, the labeled glycans were analyzed on a Waters UPLC BEH Amide column (2.1 × 150 mm, 1.7 μm) at 60 °C. Mobile phase A was 50 mM ammonium formate solution (pH 4.5), and mobile phase B was acetonitrile (ACN). Gradient elution was used, with mobile phase B decreasing from 78% to 55.9% within 38.5 min at a flow rate of 0.5 mL / min, and detection was performed using a fluorescence detector.

[0071] Table 2 summarizes the data comparing the MAM method with the PM and HILIC methods. The results show that the two methods yield consistent results for most attributes. Linear regression analysis of the paired results is shown in [reference needed]. Figure 28The results showed a strong correlation, confirming the good accuracy of the method. The recovery rates of some low-abundance PTMs were slightly lower, which is common when comparing methods with different detection principles and dynamic ranges.

[0072] Table 2. Accuracy comparison of the rapid subunit level MAM method with traditional PM and HILIC methods for analyzing key PTMs of mAb1 and mAb2.

[0073]

[0074] 3.4 Linear

[0075] The linear relationship between Asu and oxidative modification was verified.

[0076] For the quantification of Asu in mAb1, a calibration series was created by adding an Asu-rich stock solution to a low-Asu matrix, and then analyzed using the rapid subunit level MAM method (Example 1) and the PM method (2.1 in Example 2). The analytical results are shown in […]. Figure 29 , Figure 30 The results showed that the subunit level MAM (capturing total Asu on the Fd subunit) exhibited excellent linearity with dilution ratio (R² = 0.99). Figure 29 Correlation with PM data (R² = 0.75), Figure 30 The results were good, with one PM outlier likely due to Asu degradation during its long treatment period, highlighting the advantages of MAM in terms of speed and protection.

[0077] For oxidative modification, three different oxidants (AAPH, t-BHP, and H2O2) were used to force the degradation of mAb2. The results were then analyzed using the rapid subunit-level MAM method (Example 1) and the PM method (section 2.1 of Example 2). The analytical results are shown in [Figure 1]. Figures 31 to 35 The results showed that the rapid subunit level MAM exhibited a strong linear correlation with the PM results across different subunits (scFc, Fd, and LC subunits). This confirms that the method has a broad reportable range and can reliably quantify oxidative modifications induced by different stressors.

[0078] 3.5 Robustness

[0079] The robustness of the rapid subunit-level MAM method (Example 1) was tested by varying key parameters (mass spectrometry scan time, column temperature, protein loading amount, and sample placement time). The results are shown in Table 3. Under all test conditions, the quantification results for multiple attributes remained consistent, with a maximum CV of 12.13%. This low variability demonstrates the method's strong tolerance to slight but expected fluctuations in analytical conditions, making it well-suited for routine QC environments.

[0080] Table 3. Robustness verification data of fast subunit level MAM

[0081]

[0082] 3.6 Wide applicability across multiple mAb subtypes

[0083] To clearly demonstrate the broad applicability of our subunit-based rapid MAM, we applied the method described in Example 1 to characterize eight different therapeutic monoclonal antibodies, covering IgG1, IgG2, and IgG4 subclasses, including an antibody-drug conjugate (ADC) and an Fc fusion protein, as shown in Table 4. These antibodies were all prepared and expressed by Shanghai Zhangjiang Biotechnology Co., Ltd. using recombinant CHO cells (Chinese hamster ovary cells). The characterization results are shown in Table 5, and representative maps are shown in [Table 5]. Figures 36 to 43 The results showed that this method successfully quantified a wide range of PTMs across all these different antibody subtypes, including glycosylated variants, Asu, oxidation, and terminal modifications. Notably, it seamlessly adapts to different glycosylation patterns, such as the dual-antenna glycan on the mAb1 Fab region. This method demonstrated consistent and reliable performance on this structurally diverse family of biopharmaceuticals, highlighting its potential as a versatile and broad-spectrum tool for therapeutic antibody QC.

[0084] Table 4. Methodological applications of different subtypes of monoclonal antibody drugs

[0085]

[0086] Table 5. Summary of PTM results for different antibody isotypes characterized by the rapid subunit level MAM method.

[0087]

Claims

1. A rapid multi-attribute analysis method for antibody drugs, characterized in that, Includes the following steps: Dissolve or displace the antibody in a weakly acidic buffer solution, and then perform the following steps in sequence: Enzyme digestion: Add IdeS enzyme for digestion; Reduction: Dithiothreitol is added to reduce the disulfide bond; Analysis: Liquid chromatography-tandem mass spectrometry analysis.

2. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The weakly acidic buffer solution is an ammonium formate buffer solution with a pH of 6.0 to 7.

0.

3. The rapid multi-attribute analysis method for antibody drugs according to claim 2, characterized in that, The weakly acidic buffer solution is a 50 mmol / L ammonium formate buffer solution with a pH of 6.

6.

4. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The final concentration of the antibody in the weakly acidic buffer was 0.5 mg / mL.

5. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The ratio of IdeS enzyme added is antibody:enzyme = 50:1 (mass ratio).

6. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The enzyme digestion conditions for the IdeS enzyme are incubation at 37°C for 20-40 minutes.

7. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The enzyme digestion conditions for the IdeS enzyme are incubation at 37°C for 30 minutes.

8. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The reduction conditions were as follows: 5.3 μL of 1 mmol / L dithiothreitol was added to every 100 μL of sample, and the sample was reduced at 37°C for 10 minutes.

9. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, Before liquid chromatography-tandem mass spectrometry analysis, the sample was centrifuged at 17000 g for 10 minutes at 4°C, and the supernatant was used for analysis.

10. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The liquid chromatography-tandem mass spectrometry column used is packed with ethylene-bridged hybrid siloxane packing material with C4 alkyl chain ligands bonded to the surface. The column has an inner diameter of 2.1 mm, a length of 50 mm, and a particle size of 1.7 μm.

11. The rapid multi-attribute analysis method for antibody drugs according to claim 10, characterized in that, The chromatographic column used in the liquid chromatography-tandem mass spectrometry is a Waters Acquity UPLC BEH C4 column.

12. The rapid multi-attribute analysis method for antibody drugs according to claim 10, characterized in that, In liquid chromatography, mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid.

13. The rapid multi-attribute analysis method for antibody drugs according to claim 10, characterized in that, The linear gradient for liquid chromatography was from 25% B to 35% B within 6 minutes.

14. The rapid multi-attribute analysis method for antibody drugs according to claim 10, characterized in that, The column temperature for liquid chromatography was set to 60℃.

15. The rapid multi-attribute analysis method for antibody drugs according to claim 10, characterized in that, The flow rate of the liquid chromatography was 0.4 mL / min, the injection volume was 1 μL, and the total running time was 16 minutes.

16. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The mass spectrometry conditions are as follows: positive ion mode (electrospray ionization, ESI), detection range of m / z 500–2500, and ion source parameters are set as follows: capillary voltage: 3.0 kV; desolvation temperature: 300℃; source temperature: 80℃; desolvation gas flow rate: 800 L / h.

17. The rapid multi-attribute analysis method for antibody drugs according to claim 1, characterized in that, The liquid chromatography-tandem mass spectrometry equipment used was the Waters ACQUITY UPLC RDa system.

18. The rapid multi-attribute analysis method for antibody drugs according to claim 17, characterized in that, The system was controlled by MassLynx 4.2 and UNIFI was used for data analysis to obtain subunit experimental data.

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