Capillary zone electrophoresis general method for analyzing charge heterogeneity of antibody-coupled drug and application of capillary zone electrophoresis general method

Abstract

The invention provides a general capillary zone electrophoresis method for analyzing the charge heterogeneity of an antibody-coupled drug and application of the general capillary zone electrophoresis method, and the method comprises the following steps: diluting a test sample to 0.5-2.0 mg / mL with ultrapure water for CZE analysis; sequentially adopting a hydrochloric acid solution, ultrapure water and a separation buffer solution to pre-treat the capillary tube; the separation buffer solution is prepared from 0.04 to 0.06 percent of HPMC (Hydroxy Propyl Methyl Cellulose), 360 to 400 mM of EACA (Ethylene-Aminoacetic Acid) and 1.8 to 2.0 mM of TETA (Tetraacetic Acid) according to concentration, and the pH (Potential of Hydrogen Washing the capillary tube by adopting a hydrochloric acid solution, ultrapure water and a separation buffer solution in sequence before sample injection, and injecting the sample for 10 seconds at the pressure of 0.5 psi; and the voltage of 20-30 kV is separated for 10 minutes. The method disclosed by the invention has good specificity, repeatability and universality, and also has the advantages of high resolution, small sample size, high analysis speed, high column efficiency, good separation degree and low cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis, and particularly relates to a general capillary zone electrophoresis method for analyzing charge heterogeneity of antibody-drug conjugates and its application. Background Art

[0002] Antibody Drug Conjugate (ADC) is a biotherapeutic drug formed by coupling an antibody and a cytotoxic small molecule via a bioactive linker. Therefore, ADC has both the high targeting of antibodies and the high activity of small molecule drugs, achieving precise attacks on tumor cells while reducing damage to normal cells. It has shown great potential and broad application prospects in the field of tumor treatment.

[0003] ADCs exhibit complex charge heterogeneity. During production or storage, variations in antibody surface charge often occur due to transcriptional modification or autodegradation, resulting in charge heterogeneous variants. For example, modifications such as C-terminal lysine modification, amidation, oxidation, or succinimide can produce basic variants, while modifications such as phosphorylation, sialylation, or N-terminal glutamine cyclization can produce acidic variants. These modifications are closely related to the efficacy, pharmacokinetics, and safety of ADCs. Therefore, charge heterogeneity analysis of ADCs is a critical quality attribute (CQA) of the entire production process, and the use of advanced analytical techniques for in-depth characterization and assessment of ADC charge heterogeneity is essential.

[0004] Currently, the main methods used to analyze ADC charge heterogeneity include cation exchange chromatography (CEX), capillary isoelectrophoretic focusing (cIEF), and imaged capillary isoelectrophoretic focusing (icIEF). However, these methods typically require extensive method development and experimental optimization, resulting in relatively long analysis times. Furthermore, specific methods must be developed for different molecules, resulting in limited versatility. Capillary zone electrophoresis (CZE), due to its versatility, has become a commonly used detection tool for biopharmaceutical analysis.

[0005] Capillary zone electrophoresis (CZE) is a free-solution method that separates analytes based on differences in charge and mass (charge-to-mass ratio). In charge heterogeneity analysis of ADCs, since variants are mostly derived from amino acid modifications and changes, the mass differences between variants are minimal, with differences primarily reflected in surface charge. Therefore, the results of CZE separation are similar to those of isoelectric focusing (IEF), which is based on isoelectric point separation, but it is simpler, more convenient, and more versatile than CIEF. Another advantage of CZE is its ease of connection to a mass spectrometer. Combining CZE with MS allows for structural analysis of individual charge variants separated by CE, providing crucial information for variant identification. Furthermore, since 2010, CZE has garnered significant attention in the biopharmaceutical industry, with numerous pharmaceutical companies, including Pfizer and Roche, employing CZE for rapid charge heterogeneity analysis of monoclonal antibody drugs. However, as ADCs are a new hot topic in oncology therapy, there are currently few reports on the application of CZE to ADC analysis.

[0006] Considering the above limitations of existing analytical technologies, it is necessary to develop a universal CZE method for the charge heterogeneity analysis of antibody-drug conjugates, so as to obtain test results quickly and accurately, save time and cost, and achieve quality control and identification of ADCs. Summary of the Invention

[0007] To address the shortcomings of the prior art, the present invention aims to provide a universal capillary zone electrophoresis method for analyzing charge heterogeneity in antibody-drug conjugates and its application. This invention provides, for the first time, a universal method for detecting charge heterogeneity in antibody-drug conjugates. The method comprises: using capillary zone electrophoresis to separate antibody-drug conjugates of various conjugate types in a separation buffer to obtain capillary zone electrophoresis spectra. Based on the capillary zone electrophoresis spectra, the charge heterogeneity of the various antibody-drug conjugate types is determined.

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

[0009] In a first aspect, the present invention provides a general method for capillary zone electrophoresis for analyzing charge heterogeneity of antibody-drug conjugates, the method comprising:

[0010] (1) Dilute the sample to 0.5-2.0 mg / mL with ultrapure water, for example, 0.5 g / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL for CZE analysis;

[0011] (2) pre-treating the capillary with hydrochloric acid solution, ultrapure water, and separation buffer in sequence; the separation buffer comprises, by concentration, 0.04-0.06% HPMC, 360-400 mM EACA (ε-aminocaproic acid), and 1.8-2.0 mM TETA (triethylenetetramine), pH = 5.65-5.75;

[0012] (3) Before injection, the capillary was flushed with hydrochloric acid solution, ultrapure water, and separation buffer in sequence, and the injection pressure was 0.5 psi for 10 s; the separation voltage was 20-30 kV (for example, 20 kV, 25 kV, or 30 kV, etc.) for 10 min.

[0013] The concentration of HPMC in the separation buffer may be, for example, 0.04%, 0.05% or 0.06%; the concentration of EACA may be, for example, 360 mM, 370 mM, 380 mM, 390 mM or 400 mM; the concentration of TETA may be, for example, 1.8 mM, 1.9 mM or 2.0 mM; and the pH may be, for example, 5.65, 5.70 or 5.75.

[0014] The present invention improves the separation degree of zone electrophoresis by optimizing the concentration of the separation buffer. According to the method for determining the charge heterogeneity of antibody-drug conjugates according to the embodiment of the present invention, various coupling types of ADCs can be analyzed simultaneously, including cysteine-coupled ADCs, engineered site-coupled ADCs (such as THIOLMAB technology), and disulfide-bridged ADCs. The method of the present invention can be applied to ADCs with different drug-antibody coupling rates and different small molecule types, making this analysis method very versatile and universal for different antibody-drug conjugates. At the same time, the method of the present invention has high resolution, small sample volume, fast analysis speed, high column efficiency, good separation, low sample consumption (only nanoliter level), and greatly reduces experimental costs.

[0015] Preferably, the separation buffer comprises, by concentration, 0.04-0.06% HPMC, 370-390 mM EACA and 1.8-2.0 mM TETA, with a pH of 5.65-5.75.

[0016] The concentration of EACA may be, for example, 370 mM, 373 mM, 375 mM, 377 mM, 380 mM, 383 mM, 385 mM, 387 mM or 390 mM.

[0017] In the present invention, the separation buffer has a significant effect on the separation effect of capillary zone electrophoresis. The concentration of the separation buffer affects the size of the buffer capacity. A high-concentration separation buffer can increase the buffer capacity, but as the concentration increases, the migration speed of the solute in the capillary decreases, resulting in a prolonged migration time. When the concentration of the separation buffer is too low, it will lead to poor buffer capacity. The electrolysis generated during the electrophoresis process may cause pH changes, affecting the surface charge of the inner wall of the capillary, thereby affecting the size of the electroosmotic flow and the adsorption of the sample by the inner wall of the capillary. Therefore, it is very important to optimize the concentration of the separation buffer. The addition of EACA can reduce the interaction between the analyte and the capillary wall and generate less Joule heat. The present invention chose to investigate the EACA concentration in the running buffer and found that with the increase of the buffer concentration, the migration time tends to increase. This is because high concentration can compress the double layer, thereby reducing the Zeta potential and reducing the electroosmotic flow. For six test samples with different coupling methods, different toxin molecule types, and different antibody-drug coupling ratios, CZE resolution was optimal at 380 mM EACA, with a sharp main peak and good separation between acidic and basic regions. Therefore, the present invention ultimately selected an EACA concentration of 380 mM. By comparing and optimizing the running buffer concentrations, the present invention improved capillary zone electrophoresis resolution.

[0018] Preferably, the concentration of the hydrochloric acid solution is 0.1 mol / L.

[0019] Preferably, in step (2), the method for pretreating the capillary comprises: flushing with 0.1 mol / L hydrochloric acid solution at 50 psi pressure for 10 min; flushing with ultrapure water at 30 psi pressure for 2 min; flushing with separation buffer at 50 psi pressure for 5 minutes; and applying a voltage of 20-30 kV (for example, 20 kV, 25 kV, or 30 kV, etc.) for balancing for 30 min.

[0020] Preferably, in step (3), the step of flushing the capillary comprises: flushing with 0.1 mol / L hydrochloric acid solution at a pressure of 50 psi for 10 minutes; and flushing with separation buffer at a pressure of 50 psi for 5 minutes.

[0021] Preferably, the connection mode between the antibody and the toxin molecule in the antibody-drug conjugate is cysteine coupling, engineered cysteine site coupling or sulfide bridge coupling; the toxin molecule type is camptothecin or Dxd.

[0022] Preferably, the capillary used in the capillary zone electrophoresis analysis is a fused silica capillary with an inner diameter of 50 μm, a total length of 30.0 cm, and an effective length of 20.0 cm.

[0023] The capillary selected in the present invention has good separation effect and is particularly suitable for charge heterogeneity analysis of antibody-drug conjugates. Different inner diameters or coatings of the capillary will affect the separation effect.

[0024] Preferably, the detection wavelength of the capillary zone electrophoresis analysis is set to 214 nm.

[0025] Preferably, the sampling frequency of the capillary zone electrophoresis analysis is set to 2 Hz.

[0026] Preferably, the injection mode of the capillary zone electrophoresis analysis is set to pressure injection.

[0027] Preferably, the electrophoresis analysis further includes a data processing step; the data processing includes: grouping and integrating the spectra of different components, the grouping of peaks in the capillary zone electrophoresis spectrum includes: alkaline peak area, main peak area and acidic peak area; calculating the charge isoform content of each component in the sample based on the time-corrected peak area percentage.

[0028] In the present invention, the principle of CZE is that in capillary zone electrophoresis, charge variants refer to different variants of ADC molecules formed due to differences in mass-to-charge ratio. Due to different surface charges, these components have different migration rates in the electric field, which in turn appear as separated peaks in CZE.

[0029] The main peak region refers to the component with the largest percentage of time-corrected peak area in the capillary zone electrophoresis spectrum, the basic peak region refers to the component with a migration time before the main peak in the capillary zone electrophoresis spectrum, and the acidic peak region refers to the component with a migration time after the main peak region in the capillary zone electrophoresis spectrum.

[0030] In a second aspect, the present invention provides the use of the general capillary zone electrophoresis method for analyzing charge heterogeneity of antibody-drug conjugates described in the first aspect in detecting charge heterogeneity of antibody-drug conjugates.

[0031] The numerical range described in the present invention includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values included in the range.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention provides, for the first time, a universal capillary zone electrophoresis method for charge heterogeneity analysis of antibody-drug conjugates. The universal charge heterogeneity analysis method for ADC drugs provided by the present invention has good specificity, repeatability, and durability. The method is fast, achieving high-resolution analysis within 10 minutes, and has low injection volume, high column efficiency, good analytical reproducibility, and low sample consumption (only at the nanoliter level), greatly reducing experimental costs.

[0034] (2) The present invention improves the separation degree of zone electrophoresis by optimizing the concentration of the running buffer. The method for determining the charge heterogeneity of antibody-drug conjugates according to the embodiment of the present invention can simultaneously analyze various types of ADCs, including cysteine-conjugated ADCs, engineered site-conjugated ADCs (such as THIOLMAB technology), and disulfide-bridged ADCs. The method can also be applied to ADCs with different drug-antibody conjugation rates and different small molecule types, making this analysis method highly versatile and universal for different antibody-drug conjugates. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the CZE electrophoresis spectrum of the test sample 1 in Example 1 showing the effect of different EACA concentrations on separation.

[0036] Figure 2 This is the CZE electrophoresis spectrum of the separation effect of different EACA concentrations in Example 1 for the second test sample.

[0037] Figure 3 This is the CZE electrophoresis spectrum of the effect of different EACA concentrations on the separation of test sample 3 in Example 1.

[0038] Figure 4 This is the CZE electrophoresis spectrum of the effect of different EACA concentrations on the separation of test sample 4 in Example 2.

[0039] Figure 5 This is the CZE electrophoresis spectrum of the separation effect of different EACA concentrations in Example 2 on the test sample 5.

[0040] Figure 6 This is the CZE electrophoresis spectrum of the effect of different EACA concentrations on the separation of test sample 6 in Example 2.

[0041] Figure 7 This is an overlay of CZE electrophoresis spectra analyzing the charge heterogeneity of six different cysteine-conjugated ADCs, engineered site-conjugated ADCs, and sulfide-bridge-conjugated ADCs in Example 2.

[0042] Figure 8 This is an overlay of the CZE electrophoresis spectra from the charge heterogeneity specificity experiment of analyzing cysteine-conjugated ADC (ADC-6) in Example 2.

[0043] Figure 9 This is an overlay of the CZE electrophoresis spectra from the charge heterogeneity specificity experiment of analyzing the cysteine-conjugated ADC (ADC-1) in Example 2.

[0044] Figure 10 This is an overlay of the CZE electrophoresis spectra from the charge heterogeneity specificity experiment of analyzing the sulfide-bridge coupled ADC (ADC-1) in Example 2.

[0045] Figure 11 This is an overlay of CZE electrophoresis spectra from the repeatability experiment for analyzing charge heterogeneity of cysteine-conjugated ADC (ADC-6) in Example 2. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] Example 1

[0049] This example uses a universal capillary zone electrophoresis method to analyze the charge heterogeneity of six antibody-drug conjugates. The antibody-drug conjugates in this example are linked to the toxin molecule via cysteine conjugation, engineered cysteine conjugation, or sulfide bridge conjugation. The antibody used in this example is IgG, and the toxin molecule is camptothecin, Dxd, etc. The specific steps include:

[0050] Pretreatment methods for analyzing charge heterogeneity of antibody-drug conjugates:

[0051] 1. Solution preparation

[0052] (1) Preparation of 600 mM EACA + 2 mM TETA (pH = 5.7) buffer.

[0053] (2) Preparation of 1% HPMC solution.

[0054] (3) Preparation of 0.05% HPMC, 380 mM EACA, and 1.9 mM TETA separation buffer.

[0055] (4) Preparation of 0.05% HPMC, 570 mM EACA, and 1.9 mM TETA separation buffer.

[0056] (5) 0.1 mol / L hydrochloric acid solution.

[0057] 2. Sample pretreatment:

[0058] The test samples were diluted to 1 mg / mL with ultrapure water for CZE analysis.

[0059] 3. Capillary zone electrophoresis analysis method:

[0060] (1) Capillary: 50 μm inner diameter fused silica capillary, total length 30.0 cm, effective length 20.0 cm.

[0061] (2) Detection wavelength: 214 nm.

[0062] (3) Sampling frequency: 2Hz.

[0063] (4) Injection mode: pressure injection.

[0064] (5) Cartridge temperature: 25°C.

[0065] (6) Sample chamber temperature: 15°C.

[0066] (7) Capillary pretreatment method: flush with 0.1 mol / L hydrochloric acid solution at 50 psi pressure for 10 min; flush with ultrapure water at 30 psi pressure for 2 min; flush with separation buffer at 50 psi pressure for 5 minutes; apply 30 kV voltage for 30 min.

[0067] (8) Capillary zone electrophoresis analysis method: flushing with 0.1 mol / L hydrochloric acid solution at 50 psi pressure for 10 min; flushing with separation buffer at 50 psi pressure for 5 min; injection at 0.5 psi pressure for 10 s; separation at 30 kV voltage for 20 min.

[0068] The effect of separation buffer concentration on CZE separations is primarily manifested in the following aspects: The concentration of the separation buffer affects the buffer capacity. While a high concentration of separation buffer can increase the buffer capacity, increasing the concentration decreases the migration velocity of solutes within the capillary, resulting in a prolonged migration time. A low concentration of the separation buffer results in poor buffer capacity, and the electrolysis generated during electrophoresis may cause pH shifts, affecting the surface charge of the capillary wall, thereby affecting the electroosmotic flow and the adsorption of the sample onto the capillary wall. Therefore, optimizing the concentration of the separation buffer is crucial.

[0069] Test product 1 is ADC-1, whose antibody type is IgG, whose toxin molecule type is camptothecin, whose conjugation type is cysteine, and whose target DAR value is 8. Test product 2 is ADC-2, whose antibody type is THIOMAB IgG1, whose conjugation type is engineered cysteine site, and whose target DAR value is 2. Test product 3 is ADC-3, whose antibody type is IgG, whose toxin molecule type is Dxd, whose conjugation type is cysteine, and whose target DAR value is 8. Test product 4 is ADC-4, whose antibody type is IgG, whose toxin molecule type is Dxd, whose conjugation type is cysteine, and whose target DAR value is 4. Test product 5 is ADC-5, whose antibody type is IgG, whose toxin molecule type is Dxd, whose conjugation type is sulfide bridge, and whose target DAR value is 8. Test product 6 is ADC-6, whose antibody type is IgG, whose toxin molecule type is camptothecin, whose conjugation type is cysteine, and whose target DAR value is 8.

[0070] Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 、 Figure 6 The CZE electrophoresis spectra of the six test samples obtained by the two separation buffer preparation methods provided in this example are as follows: Separation buffer 1 comprises 0.05% HPMC, 570mM EACA, and 1.9mM TETA, and separation buffer 2 comprises 0.05% HPMC, 380mM EACA, and 1.9mM TETA.

[0071] This example examined EACA concentrations of 380 mM and 570 mM in the running buffer and found that migration time increased with increasing buffer concentration. This is because high concentrations can compress the electrical double layer, thereby reducing the zeta potential and electroosmotic flow. For six test samples with different coupling methods, different toxin molecule types, and different antibody-drug coupling ratios, CZE separation was optimal at 380 mM EACA, with a sharp main peak shape and good separation between acidic and basic regions. Therefore, this example ultimately selected an EACA concentration of 380 mM. This example improved the separation of capillary zone electrophoresis by comparing and optimizing the concentration of the running buffer.

[0072] After optimizing the experimental results, the experimental conditions were finally determined as follows: operating voltage 30 kV; injection pressure 0.5 psi for 5 s; separation temperature 25°C; running buffer 0.05% HPMC, 380 mM EACA, 1.9 mM TETA, pH 5.7.

[0073] Example 2

[0074] This example analyzes different types of ADC samples

[0075] 1. Solution preparation

[0076] (1) Preparation of 600 mM EACA + 2 mM TETA (pH = 5.7) buffer.

[0077] (2) Preparation of 1% HPMC solution.

[0078] (3) Preparation of 0.05% HPMC, 380 mM EACA, and 1.9 mM TETA separation buffer.

[0079] (4) 0.1 mol / L hydrochloric acid solution.

[0080] 2. Sample pretreatment:

[0081] The test samples were diluted to 1 mg / mL with ultrapure water for CZE analysis.

[0082] 3. Capillary zone electrophoresis analysis method:

[0083] (1) Capillary: 50 μm inner diameter fused silica capillary, total length 30.0 cm, effective length 20.0 cm.

[0084] (2) Detection wavelength: 214 nm.

[0085] (3) Sampling frequency: 2Hz.

[0086] (4) Injection mode: pressure injection.

[0087] (5) Cassette temperature: 25°C.

[0088] (6) Sample chamber temperature: 15°C.

[0089] (7) Capillary pretreatment method: flush with 0.1 mol / L hydrochloric acid solution at 50 psi pressure for 10 min; flush with ultrapure water at 30 psi pressure for 2 min; flush with separation buffer at 50 psi pressure for 5 minutes; apply 30 kV voltage for 30 min.

[0090] (8) Capillary zone electrophoresis analysis method: flushing with 0.1 mol / L hydrochloric acid solution at 50 psi pressure for 10 min; flushing with separation buffer at 50 psi pressure for 5 min; injection at 0.5 psi pressure for 10 s; separation at 30 kV voltage for 20 min.

[0091] 3. Experimental results

[0092] Different types of ADC samples were analyzed under the conditions of zone electrophoresis. Figure 7This is an overlay of CZE electrophoresis spectra analyzed for charge heterogeneity across six different cysteine-conjugated ADCs, engineered site-conjugated ADCs, and sulfide-bridge-conjugated ADCs in this example. The results demonstrate good separation between the main peak and its acidic and basic variants in the CZE spectra. Each ADC exhibits a characteristic electrophoretic pattern and migration time. This method is highly versatile and can be used to evaluate the charge heterogeneity of ADCs with different conjugation methods, toxin molecule types, and antibody-drug conjugation ratios.

[0093] Data processing: The spectra of different components were grouped and integrated. The groups included: basic peak area, main peak area and acidic peak area; the charge isomer content of each component in the sample was calculated based on the time-corrected peak area percentage.

[0094] Figure 7 The experimental results of time-corrected peak area percentage of six different ADCs are shown in Table 1.

[0095] Table 1

[0096] Sample name Main peak area percentage Acidic peak area percentage Basic peak area percentage ADC-6 74.2 18.3 7.5 ADC-5 21.9 47.4 30.7 ADC-4 55.5 35.9 8.6 ADC-3 67.4 26.0 6.6 ADC-2 54.8 34.4 10.8 ADC-1 72.5 20.2 7.3

[0097] Figure 8 This is an overlay of CZE electrophoresis spectra from the charge heterogeneity-specificity experiment analyzing a cysteine-conjugated ADC (ADC-6) in this example. ADC-6 was incubated at 56°C for 26 hours. The optimized CZE method was used to separate and detect ADC-6, a temperature-degraded ADC-6 sample, and the corresponding antibody. Figure 8 Table 2 shows the time-corrected peak area percentage results of the ADC-6 charge heterogeneity specificity experiment. Significant differences were observed in the CZE spectra of the ADC and the corresponding antibody, demonstrating the method's high specificity. Furthermore, the proportion of acidic peaks significantly increased, while the proportion of the main peak decreased, in the temperature-degraded sample. This demonstrates the method's ability to sensitively detect changes in charge heterogeneity within ADC samples.

[0098] Table 2

[0099] Sample name Main peak area percentage Acidic peak area percentage Basic peak area percentage mAb-6 68.8 19.4 11.8 ADC-6 74.2 18.3 7.5 ADC-6_56℃_6h 64.7 26.4 8.9

[0100] Figure 9 This is an overlay of CZE electrophoresis spectra from the charge heterogeneity-specificity experiment analyzing a cysteine-conjugated ADC (ADC-1) in this example. ADC-6 was incubated at 56°C for 26 hours, and ADC-1, a temperature-degraded sample of ADC-1, and the corresponding antibody were separated and detected using the optimized CZE method. Figure 9Table 3 shows the time-corrected peak area percentage results of the ADC-1 charge heterogeneity specificity experiment. Significant differences were observed in the CZE spectra of the ADC and the corresponding antibody, demonstrating the method's high specificity. Furthermore, the proportion of acidic peaks increased and the proportion of the main peak decreased in the temperature-degraded sample, demonstrating the method's ability to sensitively monitor charge heterogeneity in ADC samples.

[0101] Figure 10 This is an overlay of CZE electrophoresis spectra from the charge heterogeneity specificity experiment analyzing the sulfide-bridged ADC (ADC-5) in this example. ADC-5 and its corresponding antibody were separated and detected using the optimized CZE method. The CZE spectra for the ADC and its corresponding antibody show significant differences, demonstrating the method's excellent specificity.

[0102] Table 3

[0103] Sample name Main peak area percentage Acidic peak area percentage Basic peak area percentage mAb-1 68.8 19.4 11.8 ADC-1 72.5 20.2 7.3 ADC-1_56℃_6h 64.7 27.5 7.8

[0104] Figure 11 This is an overlay of CZE electrophoresis spectra from the repeatability experiment for analyzing charge heterogeneity of cysteine-conjugated ADC (ADC-6) in this example; Figure 11 The results of the time-corrected peak area percentage experiment for the repeatability of ADC-6 charge heterogeneity are shown in Table 4. Six replicate injections of the same sample demonstrated that the method demonstrated good precision in the analysis of acid-base variants. The RSD values for the time-corrected peak area percentage of the main peak were less than 5%, the RSD values for the time-corrected peak area percentage of the acidic peak were less than 15%, and the RSD values for the time-corrected peak area percentage of the basic peak were less than 20%, meeting the requirements for method validation.

[0105] Table 4

[0106] Sample name Main peak area percentage Acidic peak area percentage Basic peak area percentage ADC-6 Repeatability-1 73.7 19.3 7.0 ADC-6 Repeatability-2 72.8 19.4 7.8 ADC-6 Repeatability-3 74.3 18.6 7.1 ADC-6 Repeatability-4 72.8 19.6 7.6 ADC-6 Repeatability-5 72.8 20.0 7.2 ADC-6 Repeatability-6 74.2 18.3 7.5 Average(n=6) 73.4 19.2 7.4 RSD (%, n=6) 1.0 3.3 4.3

[0107] In summary, the present invention provides for the first time a universal method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates. The method has good specificity, repeatability and universality, and can simultaneously analyze various types of ADCs, including cysteine-coupled ADCs, engineered site-coupled ADCs (such as THIOLMAB technology), and disulfide-bridged ADCs. The method of the present invention can be applied to ADCs with different drug-antibody coupling rates and different small molecule types, and has different characteristic electrophoretic patterns and migration times for each ADC, making this analysis method very versatile for different antibody-drug conjugates. In addition, the method has a fast analysis speed, and high-resolution analysis can be achieved within 10 minutes, with a small injection volume, high column efficiency, and low sample consumption (only nanoliter level), which greatly reduces the experimental cost. The present invention has important application prospects in charge heterogeneity analysis and is of great significance for the production and quality control of antibody-drug conjugates.

[0108] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A general method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates, characterized in that: The method comprises: (1) Dilute the sample to 0.5-2.0 mg / mL with ultrapure water for CZE analysis; (2) pre-treating the capillary with hydrochloric acid solution, ultrapure water, and separation buffer in sequence; the separation buffer comprises, by concentration, 0.04-0.06% HPMC, 360-400 mM EACA, and 1.8-2.0 mM TETA, pH = 5.65-5.75; (3) Before injection, the capillary was flushed with hydrochloric acid solution, ultrapure water, and separation buffer in sequence. The injection pressure was 0.5 psi for 10 s and the separation voltage was 20-30 kV for 10 min.

2. The universal method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates according to claim 1, characterized in that: The separation buffer comprises, by concentration, 0.04-0.06% HPMC, 370-390 mM EACA, and 1.8-2.0 mM TETA, pH = 5.65-5.75; Preferably, the concentration of the hydrochloric acid solution is 0.1 mol / L.

3. The general method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates according to claim 1 or 2, characterized in that: In step (2), the method for pretreating the capillary includes: flushing with 0.1 mol / L hydrochloric acid solution at 50 psi pressure for 10 minutes; flushing with ultrapure water at 30 psi pressure for 2 minutes; flushing with separation buffer at 50 psi pressure for 5 minutes; and applying 20-30 kV voltage for balancing for 30 minutes.

4. The general method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates according to any one of claims 1 to 3, characterized in that: In step (3), the step of flushing the capillary comprises: flushing with 0.1 mol / L hydrochloric acid solution at a pressure of 50 psi for 10 minutes; and flushing with separation buffer at a pressure of 50 psi for 5 minutes.

5. The general method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates according to any one of claims 1 to 4, characterized in that: The connection mode between the antibody and the toxin molecule in the antibody-drug conjugate is cysteine coupling, engineered cysteine site coupling or sulfide bridge coupling; the toxin molecule type is camptothecin or Dxd.

6. The general method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates according to any one of claims 1 to 5, characterized in that: The capillary used in the capillary zone electrophoresis analysis is a fused silica capillary with an inner diameter of 50 μm, a total length of 30.0 cm, and an effective length of 20.0 cm.

7. The general method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates according to any one of claims 1 to 6, characterized in that: The detection wavelength of the capillary zone electrophoresis analysis was set to 214 nm.

8. The general method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates according to any one of claims 1 to 7, characterized in that: The sampling frequency of the capillary zone electrophoresis analysis was set to 2 Hz; Preferably, the injection mode of the capillary zone electrophoresis analysis is set to pressure injection.

9. The general method for capillary zone electrophoresis for charge heterogeneity analysis of antibody-drug conjugates according to any one of claims 1 to 8, characterized in that: The electrophoresis analysis also includes a data processing step; the data processing includes: grouping and integrating the spectra of different components, the peak grouping in the capillary zone electrophoresis spectrum includes the basic peak area, the main peak area and the acidic peak area; and calculating the charge isoform content of each component in the sample based on the time-corrected peak area percentage.

10. Use of the general capillary zone electrophoresis method for analyzing charge heterogeneity of antibody-drug conjugates according to any one of claims 1 to 9 in detecting charge heterogeneity of antibody-drug conjugates.

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