Method for determining average coupling rate of antibody coupling medicine based on capillary gel electrophoresis and deglycosylation pretreatment and application of method

Abstract

The invention relates to a method for determining the average coupling rate of an antibody-coupled drug based on capillary gel electrophoresis and deglycosylation pretreatment and application thereof, and the method comprises the following steps: carrying out deglycosylation and reduction pretreatment on an antibody-coupled drug test sample, carrying out capillary gel electrophoresis, carrying out peak attribution on an electrophoretogram, and determining the average coupling rate of the antibody-coupled drug according to the peak attribution. And calculating the average coupling rate of the antibody coupling drug. According to the method, enzymatic deglycosylation pretreatment is combined with the CGE, and meanwhile, deglycosylation reaction conditions are optimized, so that the separation effect is remarkably improved, the complexity of a CE-SDS electrophoresis spectrogram is reduced, and the accuracy of ADC molecule average coupling rate analysis is greatly improved. The method is suitable for high-throughput analysis requirements in antibody coupling drug research and development, production quality control and stability research.

Description

Technical Field

[0001] The present invention relates to the technical field of detection and analysis, and in particular to a method for determining the average conjugation ratio of antibody-drug conjugates based on capillary gel electrophoresis and deglycosylation pretreatment and its application. Background Art

[0002] An antibody-drug conjugate (ADC) is an innovative biotherapeutic drug formed by conjugating an antibody targeting a specific antigen and a cytotoxic small molecule through a linker. Therefore, ADC combines the high specificity of antibody drugs and the high activity of cytotoxic drugs, achieving precise targeting of tumor cells while reducing damage to normal cells, showing great potential and broad application prospects in the field of tumor treatment.

[0003] The characteristics of conjugation chemistry determine that ADC is essentially a mixture of antibodies with different drug loads. The drug-to-antibody ratio (DAR) represents the average value of the payload conjugated to each antibody molecule and is a key quality attribute for measuring antibody-drug conjugates, directly affecting the safety and effectiveness of the product. A low drug load reduces efficacy, while a high drug load has a negative impact on pharmacokinetics. Therefore, it is crucial to develop effective analytical methods to monitor the DAR value based on factors such as the physicochemical properties and conjugation process of ADC.

[0004] Currently, common analytical methods for the average conjugation ratio and drug distribution include ultraviolet-visible spectrophotometry (UV-Vis), reverse-phase high-performance liquid chromatography (RP-HPLC), hydrophobic interaction chromatography (HIC-HPLC), and liquid chromatography-mass spectrometry (LC-MS).

[0005] Ultraviolet-visible spectrophotometry is a relatively simple and stable analytical method for analyzing DAR based on Lambert-Beer's law. Three prerequisite conditions are required for this method: 1) The cytotoxic small molecule must have an ultraviolet / visible chromophore; 2) The maximum absorption wavelengths of the cytotoxic small molecule and the antibody are different; 3) The formation of the ADC molecule by the binding of the cytotoxic small molecule and the antibody does not affect the absorption spectra of these two components. Although this method is relatively simple, it has important limitations. For example, the presence of free drug in the ADC sample may lead to an overestimation of the DAR value, and this method can only obtain the average conjugation ratio and cannot provide any information such as drug load distribution.

[0006] Hydrophobic interaction chromatography (HIC-HPLC) separates based on the different hydrophobicities between antibodies conjugated with different numbers of cytotoxic small molecules, maintaining the structural integrity of the ADC molecule. The average conjugation rate is calculated by the percentage of chromatographic peak area and the corresponding drug load, and information on drug loading distribution can also be obtained. However, this method cannot analyze antibody-drug conjugates where there is no difference in hydrophobicity between the cytotoxic small molecule and the antibody.

[0007] Reverse-phase high-performance liquid chromatography is a common analytical method for determining the average conjugation rate. Compared with hydrophobicity-based characterization, RP-HPLC is compatible with mass spectrometry due to the volatility of the mobile phase. This method usually requires reducing the ADC to obtain the light chain and heavy chain, and then separating the light and heavy chains with different numbers of small molecules based on polarity differences. The average conjugation rate of the antibody-drug conjugate, as well as the drug loading distribution on the light and heavy chains, is calculated through the percentage of chromatographic peak area and the corresponding drug load. However, this method has limitations in analyzing ADC components with small polarity differences; in addition, when measuring the DAR value by RP-HPLC, the required elevated temperature and reduction pretreatment of the sample may cause degradation of the ADC, resulting in a DAR value calculated by RP-HPLC being lower than that of hydrophobic interaction chromatography or other analytical methods.

[0008] Mass spectrometry separates molecules with different DAR values by generating ions with different mass-to-charge ratios in the ion source and separates them in the mass analyzer, and determines the average conjugation rate of the ADC molecule based on the intensity of the corresponding mass spectrometry signal peaks. However, the assumption of mass spectrometry analysis is that the ionization efficiency of all substances is the same as that of the unconjugated antibody regardless of the drug load. Therefore, the premise of analyzing the DAR value by mass spectrometry is to assume that all substances have the same recovery rate and ionization situation, but this is not always the case in reality because the conjugation of the drug with a positively charged amine will change its charge and hydrophobicity. In addition, mass spectrometry is costly and difficult to meet the requirements of high-throughput detection in production quality control.

[0009] Considering the above limitations of existing DAR value analysis techniques, it is necessary to develop an analytical method that can determine the average conjugation of antibody-conjugated drugs and drug distribution. Summary of the Invention

[0010] To solve the above technical problems, the present invention provides a method for determining the average conjugation rate of antibody-drug conjugates based on capillary gel electrophoresis and deglycosylation pretreatment and its application. For the first time, the ADC is pretreated by deglycosylation, and then separated according to the molecular size of the antibody-conjugated drug after reduction by using the separation principle of capillary gel electrophoresis. The average conjugation rate of the antibody-conjugated drug is calculated, and information such as drug distribution can be obtained.

[0011] To achieve this purpose, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a method for determining the average conjugation ratio of an antibody-drug conjugate based on capillary gel electrophoresis and deglycosylation pretreatment. The determination method includes: performing deglycosylation and reduction pretreatment on the antibody-drug conjugate test sample, performing capillary gel electrophoresis, attributing the peaks in the electrophoresis pattern, and calculating the average conjugation ratio of the antibody-drug conjugate.

[0013] Capillary gel electrophoresis (CGE), as a high-resolution, low-sample-demand, and rapid separation technique, has been applied in the purity analysis of monoclonal antibodies and ADCs. However, there are few reports on the use of capillary gel electrophoresis for DAR determination. In addition, the glycosylation modification of ADCs may interfere with the separation effect of CGE. For example, glycan heterogeneity masks the true DAR signal (such as the charge difference at N-glycosylation sites); the migration rates of heavy and light chains shift, interfering with the identification of drug-loading peaks, all of which will lead to inaccurate DAR value determination. Therefore, developing a combined method of capillary gel electrophoresis and removal of glycosylation interference is of great significance for the accurate determination of the DAR value and drug distribution of antibody-drug conjugates.

[0014] The present invention first performs deglycosylation treatment on the ADC sample, uses glycosidase to specifically remove the N-linked glycans in the Fc region of the antibody. The deglycosylation pretreatment of ADC eliminates the complexity and heterogeneity brought by glycosylation modification, eliminates the interference of glycans on the electrophoresis migration rate, improves the separation effect, and improves the accuracy and resolution of the analysis of the DAR values of antibody-drug conjugates with different conjugation degrees. At the same time, the deglycosylation pretreatment can, to a certain extent, eliminate the influence brought by glycosylation differences, making this analysis method have better generality and stability for different antibody-drug conjugates. Subsequently, capillary gel electrophoresis is used to separate the components with different drug-antibody conjugation ratios, and the peak areas of the components after ADC reduction are quantified by combining with an ultraviolet detector, and the average conjugation ratio and drug distribution are calculated simultaneously.

[0015] By combining enzymatic deglycosylation pretreatment with CGE and optimizing the deglycosylation reaction conditions, the present invention significantly improves the separation effect, reduces the complexity of the CE-SDS electrophoresis pattern, and greatly improves the accuracy of the analysis of the average conjugation ratio of ADC molecules. In addition, compared with other commonly used methods for analyzing the average conjugation ratio, this method can simultaneously provide information on DAR values and drug-loading distributions; as a low-sample-demand method, capillary electrophoresis can achieve separation and detection with only nanoliter samples. This method is applicable to the high-throughput analysis requirements in the research and development, production quality control, and stability research of antibody-drug conjugates.

[0016] Preferably, the connection mode between the antibody and the drug in the antibody-drug conjugate includes any one of lysine conjugation, cysteine conjugation, or site-specific conjugation.

[0017] Preferably, the molecular weight of the drug in the antibody-drug conjugate is not less than 1800 Da.

[0018] Preferably, AdvanceBio Gly-X is used for the deglycosylation treatment.

[0019] Preferably, the deglycosylation and reduction pretreatment includes the following steps:

[0020] (1) Mix the antibody-drug conjugate test sample with the Gly-X denaturing agent, perform the first incubation, and take the supernatant after cooling and centrifugation;

[0021] (2) Add the N-glycosidase working solution, perform the second incubation, and take the supernatant after cooling and centrifugation;

[0022] (3) Add the SDS sample buffer and the reducing agent, perform the third incubation, and take the supernatant after cooling and centrifugation.

[0023] Preferably, the mass of the antibody-drug conjugate test sample is 75-85 μg (such as 75 μg, 80 μg or 85 μg, etc.), and the volume of the Gly-X denaturing agent is 3-5 μL (such as 3 μL, 4 μL or 5 μL).

[0024] Preferably, the temperature of the first incubation is 85-95 °C (such as 85 °C, 90 °C or 95 °C, etc.), and the time is 5-10 min (such as 5 min, 6 min, 7 min, 8 min, 9 min or 10 min, etc.).

[0025] Preferably, the N-glycosidase working solution contains Gly-X N-glycosidase and Gly-X enzymatic digestion buffer.

[0026] Preferably, the volume ratio of the Gly-X N-glycosidase to the Gly-X enzymatic digestion buffer is 1:(0.5-2).

[0027] The specific point values of the above (0.5-2) can be 0.5, 1, 1.5 or 2, etc.

[0028] Preferably, the volume of the N-glycosidase working solution is 3-5 μL (such as 3 μL, 4 μL or 5 μL, etc.).

[0029] Preferably, the temperature of the second incubation is 45-55 °C (such as 45 °C, 50 °C or 55 °C, etc.), and the time is 10-15 min (such as 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.).

[0030] Preferably, the SDS sample buffer is a disodium hydrogen phosphate - citric acid solution containing 0.5% - 2% (such as 0.5%, 1%, 1.5% or 2% etc.) (w / v) SDS and 15 - 25 mM (such as 15 mM, 20 mM or 25 mM etc.).

[0031] Preferably, the reducing agent includes thiol - type reducing agents.

[0032] Preferably, the thiol - type reducing agents include β - mercaptoethanol and / or dithiothreitol, and further preferably β - mercaptoethanol.

[0033] Preferably, the volume of the SDS sample buffer is 70 - 80 μL (such as 70 μL, 75 μL or 80 μL etc.), and the volume of the reducing agent is 4 - 6 μL (such as 4 μL, 5 μL or 6 μL etc.).

[0034] Preferably, the temperature of the third incubation is 65 - 75 °C (such as 65 °C, 70 °C or 75 °C etc.), and the time is 5 - 15 min (such as 5 min, 10 min or 15 min etc.).

[0035] Preferably, the conditions of the capillary gel electrophoresis include: using a 50 - μm inner diameter fused - silica capillary with a total length of 30 cm and an effective length of 20 cm, a detection wavelength of 220 nm, a sampling frequency of 2 Hz, an injection mode of electrokinetic injection, a cassette temperature of 25 °C, and a sample chamber temperature of 15 ± 3 °C.

[0036] Preferably, the capillary pretreatment method of the capillary gel electrophoresis includes: flushing with 0.1 mol / L sodium hydroxide solution at a pressure of 20 psi for 10 min; flushing with 0.1 mol / L hydrochloric acid solution at a pressure of 20 psi for 5 min; flushing with ultrapure water at a pressure of 20 psi for 2 min; flushing with SDS gel separation buffer at a pressure of 70 psi for 10 min, and the SDS gel separation buffer is SDS - MW gel buffer; balancing at - 15 kV for 10 min with a voltage rise time of 5 min.

[0037] Preferably, the sample detection method of the capillary gel electrophoresis includes: flushing with 0.1 mol / L sodium hydroxide solution at a pressure of 70 psi for 3 min; flushing with 0.1 mol / L hydrochloric acid solution at a pressure of 70 psi for 1 min; flushing with ultrapure water at a pressure of 70 psi for 1 min; flushing with SDS gel separation buffer at a pressure of 70 psi for 10 min; after dipping the inlet / outlet ends in ultrapure water, electrokinetic injection at - 5 kV for 20 s; after dipping the inlet end in ultrapure water, separating at - 15 kV for 30 min while applying a pressure of 20 psi at both ends of the capillary.

[0038] Preferably, the peak attribution of the electrophoresis map includes: comparing the migration times of the CE-SDS electrophoresis maps of the corresponding antibodies after the same deglycosylation and reduction pretreatment, performing peak attribution based on the migration times of the light chain and heavy chain of the antibody without conjugated drug, and reading the time-corrected peak area percentages of the antibody conjugated drug test samples with different conjugation rates.

[0039] Preferably, the calculation formula for calculating the average conjugation rate of the antibody conjugated drug is as follows:

[0040]

[0041] DAR = DAR LC + DAR HC

[0042] wherein, DAR LC represents the average conjugation rate of the light chain of the antibody conjugated drug; DAR HC represents the average conjugation rate of the heavy chain of the antibody conjugated drug; LC n represents the time-corrected peak area percentage of the light chain of the antibody conjugated drug conjugated with n drugs in CE-SDS; HC n represents the time-corrected peak area percentage of the heavy chain of the antibody conjugated drug conjugated with n drugs in CE-SDS; DAR represents the average conjugation rate of the antibody conjugated drug.

[0043] In a second aspect, the present invention provides the application of the detection method as described in the first aspect in detecting the drug distribution of the antibody-drug conjugate.

[0044] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention for the first time provides a method for determining the average conjugation ratio of antibody-drug conjugates based on capillary gel electrophoresis and deglycosylation pretreatment. Through the pretreatment of enzymatic deglycosylation and the DAR value analysis of CE-SDS, the complexity and heterogeneity brought by antibody glycosylation modification are removed, the separation effect is significantly improved, the complexity of the CE-SDS electrophoresis pattern is reduced, and the accuracy of the analysis of the average conjugation ratio of ADC molecules is greatly improved. In addition, compared with other commonly used methods for analyzing the average conjugation ratio (such as ultraviolet-visible spectrophotometry (UV-Vis), reverse-phase high-performance liquid chromatography, and liquid chromatography-mass spectrometry), this method can provide information on the drug loading distribution. As a method with low sample requirements, capillary electrophoresis can achieve separation and detection only with nanoliter samples. At the same time, the deglycosylation pretreatment can, to a certain extent, eliminate the influence brought by glycosylation differences, making this analysis method have better generality and stability for different antibody-drug conjugates. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is a schematic diagram of a method for detecting the average conjugation ratio of antibody-drug conjugates and the drug loading distribution.

[0048] Figure 2 It is the CE-SDS electrophoresis pattern of a test sample obtained by the glycosylation pretreatment method recommended by the detection method and kit provided by the present invention.

[0049] Figure 3 It is the CE-SDS electrophoresis pattern of a low-DAR test sample 1 obtained by the detection method provided by the present invention and a test sample without deglycosylation pretreatment.

[0050] Figure 4 It is the CE-SDS electrophoresis pattern of a low-DAR test sample 2 obtained by the detection method provided by the present invention and a test sample without deglycosylation pretreatment.

[0051] Figure 5 It is the CE-SDS electrophoresis pattern of an ADC molecule, a low-DAR test sample 3, a low-DAR test sample 4, and the antibody corresponding to the antibody-drug conjugate obtained by the detection method provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0052] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention.

[0053] For those without specific techniques or conditions indicated in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained by purchasing through regular channels.

[0054] The reagents used in the following examples are shown in Table 1:

[0055] Table 1

[0056] Number Reagent Name Manufacturer / Catalog Number 1 SDS-MW Gel Buffer SCIEX / A30341 2 Gly-X Deblocking Kit Agilent / GX96-100 3 Sodium Hydroxide Shanghai Experiment Reagent Co., Ltd. / 01016787 4 Hydrochloric Acid Huasen Professional Products Co., Ltd. / 53100278

[0057] Example 1

[0058] In this example, the average conjugation rate and drug distribution of five antibody-drug conjugate test samples were analyzed using the determination method provided by the present invention. The schematic diagram of the determination method is as shown in Figure 1 shown. The conjugation mode of the antibody-drug conjugate is cysteine conjugation, and the antibody type used in the present invention is IgG. The specific steps are as follows:

[0059] S1. Solution preparation:

[0060] (1) N-glycosidase working solution: Mix Gly-X N-glycosidase with Gly-X enzymatic digestion buffer at a ratio of 1:1 (v / v);

[0061] (2) SDS sample buffer: 20 mM disodium hydrogen phosphate citrate solution containing 1% (w / v) SDS;

[0062] (3) SDS gel separation buffer: SDS-MW gel buffer;

[0063] (4) 0.1 mol / L hydrochloric acid solution;

[0064] (5) 0.1 mol / L sodium hydroxide solution.

[0065] S2. Deglycosylation and pre-reduction treatment of the sample:

[0066] Take 80 μg of the test sample, add ultrapure water to 17 μL, add 4 μL of Gly-X denaturant, incubate at 90 °C for 5 min; after cooling and centrifuging, take the supernatant, add 4 μL of N-glycosidase working solution, incubate at 50 °C for 10 min; after cooling and centrifuging, take the supernatant, add 75 μL of SDS sample buffer, add 5 μL of β-mercaptoethanol, incubate at 70 °C for 10 min, cool to room temperature and then centrifuge, and take the supernatant for analysis.

[0067] S3. Capillary gel electrophoresis analysis method:

[0068] (1) Capillary: 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;

[0069] (2) Detection wavelength: 220 nm;

[0070] (3) Sampling frequency: 2 Hz;

[0071] (4) Injection mode: Electrokinetic injection;

[0072] (5) Cartridge temperature: 25 °C;

[0073] (6) Sample chamber temperature: 15 ± 3 °C;

[0074] (7) Capillary pretreatment method: Rinse with 0.1 mol / L sodium hydroxide solution at 20 psi pressure for 10 min; rinse with 0.1 mol / L hydrochloric acid solution at 20 psi pressure for 5 min; rinse with ultrapure water at 20 psi pressure for 2 min; rinse with SDS gel separation buffer at 70 psi pressure for 10 min; balance with -15 kV voltage for 10 min, and the voltage rise time is 5 min;

[0075] (8) Sample detection method: Rinse with 0.1 mol / L sodium hydroxide solution at 70 psi pressure for 3 min; rinse with 0.1 mol / L hydrochloric acid solution at 70 psi pressure for 1 min; rinse with ultrapure water at 70 psi pressure for 1 min; rinse with SDS gel separation buffer at 70 psi pressure for 10 min; after dipping the inlet / outlet ends in ultrapure water, perform -5 kV electrokinetic injection for 20 s; after dipping the inlet end in ultrapure water, separate with -15 kV voltage for 30 min, and apply 20 psi pressure at both ends of the capillary simultaneously.

[0076] S4. CE - SDS peak attribution: The peak attribution of the CE - SDS electrophoresis map of the antibody - drug conjugate test sample is completed by comparing the migration time of the CE - SDS electrophoresis map of the corresponding antibody after the same deglycosylation and reduction pretreatment. The peak attribution is carried out through the migration time of the light chain and heavy chain of the uncoupled drug molecule, and the time - corrected peak area percentage of the ADC test sample with different DAR values is read.

[0077] S5. Data analysis:

[0078] The average conjugation rate of the antibody - drug conjugate based on capillary gel electrophoresis and deglycosylation pretreatment is calculated using the following formula:

[0079]

[0080] DAR = DAR LC + DAR HC

[0081] where, DAR LC represents the average conjugation rate of the light chain of the antibody - drug conjugate; DARHC represents the average conjugation ratio of the antibody-drug conjugate heavy chain; LC n represents the percentage of the time-corrected peak area of the light chain of the antibody-drug conjugate conjugated with n drug molecules in CE-SDS; HC n represents the percentage of the time-corrected peak area of the heavy chain of the antibody-drug conjugate conjugated with n drug molecules in CE-SDS; DAR represents the average conjugation ratio of the antibody-drug conjugate.

[0082] Figure 2 is the CE-SDS electrophoresis pattern of a test sample obtained by the glycosylation pretreatment method provided by the present invention and recommended by the kit. Condition 1 is the detection method provided by the present invention, that is, the CE-SDS electrophoresis pattern of the test sample obtained by the optimized enzymatic deglycosylation method of the present invention. Condition 2 is the CE-SDS electrophoresis pattern of the test sample obtained by the enzymatic deglycosylation method recommended by the kit. The sample deglycosylation method used is as follows: take 80 μg of the test sample, add ultrapure water to 17 μL, add 4 μL of Gly-X denaturant, incubate at 90 °C for 3 min; after cooling and centrifugation, add 4 μL of N-glycosidase working solution, incubate at 50 °C for 5 min; after cooling and centrifugation, add 75 μL of SDS sample buffer, add 5 μL of β-mercaptoethanol, incubate at 70 °C for 10 min, cool to room temperature and then centrifuge, and take the supernatant for analysis.

[0083] Figure 2 In it, LC0 represents the CE-SDS peak of the corresponding antibody light chain, LC1 represents the CE-SDS peak of the light chain conjugated with one drug molecule in the antibody-drug conjugate test sample, LC2 represents the CE-SDS peak of the light chain conjugated with two drug molecules in the antibody-drug conjugate test sample, HC0 represents the CE-SDS peak of the corresponding antibody heavy chain, HC1 represents the CE-SDS peak of the heavy chain conjugated with one drug molecule in the antibody-drug conjugate test sample, HC2 represents the CE-SDS peak of the heavy chain conjugated with two drug molecules in the antibody-drug conjugate test sample, HC3 represents the CE-SDS peak of the heavy chain conjugated with three drug molecules in the antibody-drug conjugate test sample, and HC4 represents the CE-SDS peak of the heavy chain conjugated with four drug molecules in the antibody-drug conjugate test sample. Peak 1 is the CE-SDS peak of the incompletely deglycosylated heavy chain obtained after pretreatment by two enzymatic deglycosylation methods.

[0084] In Condition 1, the percentage of the time-corrected peak area occupied by Peak 1 is 8.9%, and the glycosylation efficiency under this enzymatic hydrolysis condition is 91.1%; in Condition 2, the percentage of the time-corrected peak area occupied by Peak 1 is 0.9%, and the glycosylation efficiency under this enzymatic hydrolysis condition is 99.1%. It can be seen from this that the glycosylation efficiency obtained by the optimized enzymatic deglycosylation method of the present invention is higher.

[0085] Figure 3CE - SDS electrophoretogram of the low - DAR test sample 1 obtained by the detection method provided by the present invention and the test sample without deglycosylation pretreatment. Figure 4 CE - SDS electrophoretogram of the low - DAR test sample 2 obtained by the detection method provided by the present invention and the test sample without deglycosylation pretreatment. The attribution of each peak was completed by comparing the migration time of the CE - SDS electrophoretogram of the corresponding antibody after the same deglycosylation and reduction pretreatment. As shown in the figure, the enzymatic deglycosylation pretreatment significantly improved the separation effect of the low - DAR test sample 1. The resolution of each component peak of the heavy chain (HC0, HC1, HC2, HC3) in the CE - SDS electrophoretogram was significantly improved, reducing the complexity of the CE - SDS electrophoretogram, and greatly improving the accuracy of the analysis of the average conjugation rate of the ADC molecule and the drug - loading distribution.

[0086] Figure 5 CE - SDS electrophoretograms of an ADC molecule, the low - DAR test sample 3, the low - DAR test sample 4, and the antibody corresponding to the antibody - drug conjugate obtained by the detection method provided by the present invention. As shown in the figure, the migration times of the light chains and heavy chains of the low - DAR test sample 3 and the low - DAR test sample 4 are the same as those of the light chain and heavy chain of the corresponding antibody. The corresponding peaks of the drug - conjugated molecules in the ADC molecule, the low - DAR test sample 3, and the low - DAR test sample 4 can be attributed, and the time - corrected peak area percentages of ADC test samples with different DAR values can be read. Figure 5 The time - corrected peak area percentages of each component peak of the ADC molecule, the low - DAR test sample 3, and the low - DAR test sample 4 in

[0087] Figure 5 The results of the DAR values corresponding to the light chain, the DAR values corresponding to the heavy chain, and the DAR value of the antibody - drug conjugate of the ADC molecule, the low - DAR test sample 3, and the low - DAR test sample 4 are shown in Table 2.

[0088] Table 2

[0089]

[0090] In summary, the present invention provides for the first time a method for determining the average conjugation rate of antibody - drug conjugates and its application based on the combination of capillary gel electrophoresis and deglycosylation technology. Through specific deglycosylation, the interference of glycosylation heterogeneity on the DAR analysis results and the complexity of the CE - SDS electrophoretogram are reduced, the separation effect of CGE is significantly improved, the accuracy of the analysis of the average conjugation rate of ADC molecules is greatly improved, and information on DAR values and drug - loading distribution can be provided simultaneously, realizing the precise and rapid analysis of the key quality attributes of ADCs, which is of great significance for the production and quality control of antibody - drug conjugates.

[0091] The applicant declares that the above description is only a specific implementation of the present invention, but the protection scope 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 protection scope and the disclosure scope of the present invention.

Claims

1. A method for determining the average conjugation rate of antibody-drug conjugates based on capillary gel electrophoresis and deglycosylation pretreatment, characterized in that, The determination method includes: subjecting the antibody-drug conjugate test sample to deglycosylation and reduction pretreatment, performing capillary gel electrophoresis, performing peak attribution on the electrophoresis pattern, and calculating the average conjugation rate of the antibody-drug conjugate.

2. The measurement method according to claim 1, wherein The connection mode between the antibody and the drug in the antibody-drug conjugate includes any one of lysine conjugation, cysteine conjugation, or site-directed conjugation; Preferably, the molecular weight of the drug in the antibody-drug conjugate is not less than 1800 Da.

3. The determination method according to claim 1 or 2, characterized in that The deglycosylation and reduction pretreatment includes the following steps: (1) Mix the antibody-drug conjugate test sample with a Gly-X denaturing agent, perform the first incubation, and take the supernatant after cooling and centrifugation; (2) Add an N-glycosidase working solution, perform the second incubation, and take the supernatant after cooling and centrifugation; (3) Add an SDS sample buffer and a reducing agent, perform the third incubation, and take the supernatant after cooling and centrifugation.

4. The measurement method according to claim 3, characterized in that The mass of the antibody-drug conjugate test sample is 75-85 μg, and the volume of the Gly-X denaturing agent is 3-5 μL; Preferably, the temperature of the first incubation is 85-95 °C, and the time is 5-10 min.

5. The measurement method according to claim 3, characterized in that, The N-glycosidase working solution contains Gly-X N-glycosidase and Gly-X enzymatic digestion buffer; Preferably, the volume ratio of Gly-X N-glycosidase to Gly-X enzymatic digestion buffer is 1:(0.5-2); Preferably, the volume of the N-glycosidase working solution is 3-5 μL; Preferably, the temperature of the second incubation is 45-55 °C, and the time is 10-15 min.

6. The measurement method according to claim 3, wherein The SDS sample buffer is a 15-25 mM disodium hydrogen phosphate citrate solution containing 0.5%-2% (w / v) SDS; Preferably, the reducing agent includes a thiol reducing agent; Preferably, the thiol reducing agent includes β-mercaptoethanol and / or dithiothreitol, and further preferably β-mercaptoethanol; Preferably, the volume of the SDS sample buffer is 70-80 μL, and the volume of the reducing agent is 4-6 μL; Preferably, the temperature of the third incubation is 65-75 °C, and the time is 5-15 min.

7. The measurement method according to any one of claims 1 to 6, characterized in that, The conditions for the capillary gel electrophoresis include: using a 50 μm inner diameter fused silica capillary, with a total length of 30 cm, an effective length of 20 cm, a detection wavelength of 220 nm, a sampling frequency of 2 Hz, an injection mode of electrokinetic injection, a cartridge temperature of 25 °C, and a sample chamber temperature of 15±3 °C; Preferably, the capillary pretreatment method for the capillary gel electrophoresis includes: flushing with 0.1 mol / L sodium hydroxide solution at a pressure of 20 psi for 10 min; flushing with 0.1 mol / L hydrochloric acid solution at a pressure of 20 psi for 5 min; flushing with ultrapure water at a pressure of 20 psi for 2 min; flushing with an SDS gel separation buffer at a pressure of 70 psi for 10 min, and the SDS gel separation buffer is an SDS-MW gel buffer; balancing at -15 kV voltage for 10 min, and the voltage rise time is 5 min; Preferably, the sample detection method for capillary gel electrophoresis includes: rinsing with 0.1 mol / L sodium hydroxide solution under a pressure of 70 psi for 3 min; rinsing with 0.1 mol / L hydrochloric acid solution under a pressure of 70 psi for 1 min; rinsing with ultrapure water under a pressure of 70 psi for 1 min; rinsing with SDS gel separation buffer under a pressure of 70 psi for 10 min; after dipping the inlet / outlet ends in ultrapure water, electrokinetic injection at -5 kV for 20 s; after dipping the inlet end in ultrapure water, separation at -15 kV for 30 min, and simultaneously applying a pressure of 20 psi at both ends of the capillary.

8. The measurement method according to any one of claims 1 to 7, characterized in that, The peak attribution for the electrophoresis pattern includes: comparing the migration times of the CE-SDS electrophoresis patterns of the corresponding antibodies after the same deglycosylation and reduction pretreatment, performing peak attribution based on the migration times of the light and heavy chains of the antibody without conjugated drug, and reading the time-corrected peak area percentages of the antibody conjugated drug test samples with different conjugation rates.

9. The measurement method according to any one of claims 1 to 8, characterized in that, The calculation formula for calculating the average conjugation rate of the antibody conjugated drug is as follows: DAR = DAR LC + DAR HC Among them, DAR LC represents the average conjugation ratio of the light chain of the antibody-drug conjugate; DAR HC represents the average conjugation ratio of the heavy chain of the antibody-drug conjugate; LC n represents the CE-SDS time-corrected peak area percentage of the light chain of the antibody-drug conjugate conjugated with n drugs; HC n represents the CE-SDS time-corrected peak area percentage of the heavy chain of the antibody-drug conjugate conjugated with n drugs; DAR represents the average conjugation ratio of the antibody-drug conjugate.

10. Use of the detection method according to any one of claims 1 to 9 in detecting the drug distribution of antibody-drug conjugates.

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