Method for detecting average coupling rate of high-hydrophilicity micromolecule high-glycosylation antibody coupling medicine and application of method for detecting average coupling rate of high-hydrophilicity micromolecule high-glycosylation antibody coupling medicine

Abstract

The invention belongs to the technical field of detection and analysis, and provides a method for detecting the average coupling rate of a high-hydrophilicity small-molecule high-glycosylation antibody coupling drug and application of the method for detecting the average coupling rate of the high-hydrophilicity small-molecule high-glycosylation antibody coupling drug. The method comprises the following steps: firstly, carrying out desugaring treatment on a high-glycosylation coupling medicine, then coupling desugarized high-hydrophilicity toxin molecules with an antibody medicine, analyzing by using hydrophilic interaction chromatography conditions, and calculating to obtain the accurate average coupling rate of the antibody coupling medicine. According to the method, through desugaring treatment and hydrophilic interaction chromatography detection in the analysis process, the defect that the separation degree of high-hydrophilicity micromolecules with different coupling ratios is low due to hydrophobicity difference in a traditional method is overcome, so that the measured average coupling rate result is more accurate and real. The method is suitable for coupling a high-hydrophilicity payload drug with a high-glycosylation antibody drug, and is especially suitable for an antibody coupling drug which is high in micromolecule hydrophilicity and has obvious influence on the separation degree of a traditional DAR liquid phase analysis method.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis technology, specifically relating to a method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs) are innovative biotherapeutic drugs formed by conjugating antibodies or antibody fragments targeting specific antigens to small molecule payloads with cytotoxicity via linkers. Therefore, ADCs combine the high targeting ability of antibody drugs with the high activity of linker-payload (LP) small molecule drugs, using a single antibody as a carrier to deliver the small molecule cytotoxic drug more selectively and efficiently to target tumor cells, the tumor microenvironment, or other target cells to achieve therapeutic goals. Antibody-drug conjugates have demonstrated excellent efficacy and great potential in the field of cancer treatment, becoming a hot topic in drug development.

[0003] Drug-to-antibody ratio (DAR) represents the average effective payload conjugated to each antibody molecule and is a key quality attribute of antibody-drug conjugates, directly affecting the safety and efficacy of the product. Appropriate analytical methods are selected based on the chemical properties of the linker and payload, the conjugation mode with the antibody, and the degree of heterogeneity in DAR.

[0004] Currently, hydrophobic interaction chromatography (DAR) is a common analytical method for determining the average conjugation ratio (DAR). ADC payloads must cross lipid membranes to inhibit DNA replication, protein synthesis, enzymatic activity, or cell division. Therefore, payloads are typically highly hydrophobic, such as MMAE and camptothecin. However, with the diversification of therapeutic applications, the types of conjugated small molecules are increasing. Studies have found that some highly hydrophilic small molecules exhibit low separation rates in ADCs conjugated in different proportions using common assays. Low separation rates of heterogeneous components with different DAR values ​​can affect the accuracy of DAR values. Furthermore, with the increasing variety of antibodies, the high hydrophilicity of antibodies due to high glycosylation can also affect the performance of conventional liquid chromatography methods. Therefore, there is a need to develop an analytical method for detecting the DAR values ​​of highly hydrophilic small molecule, highly glycosylated antibody-drug conjugates. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting the average conjugation rate of highly hydrophilic small-molecule, highly glycosylated antibody-drug conjugates (ADCs) and its application. This invention provides a more accurate method for detecting the average conjugation rate of highly hydrophilic, highly glycosylated ADCs, calculating and deriving the average conjugation rate. This invention improves the accuracy of liquid chromatography testing for the average DAR value of highly hydrophilic small-molecule, highly glycosylated ADCs, which is of great significance for ADC production and quality control.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates, the method comprising:

[0008] (S1) The antibody-drug conjugate to be tested is deglycosylated using N-glycosidase F to obtain a deglycosylated antibody-drug conjugate. The structure of the antibody-drug conjugate is antibody-linker-toxin molecule. The antibody has high glycosylation properties, and the toxin molecule has high hydrophilic properties. The deglycosylation treatment includes non-reductive deglycosylation treatment or reductive deglycosylation treatment.

[0009] (S2) The antibody-drug conjugate to be tested is detected by hydrophilic interaction chromatography; the chromatographic column used for the chromatographic detection is a hydrophilic interaction chromatographic column, and the packing material of the chromatographic column is an amide-bonded phase;

[0010] (S3) Calculate the average conjugation rate of antibody-drug conjugates based on the percentage of peak area obtained from the liquid chromatography analysis; for antibody-drug conjugates that have undergone non-reducing desaccharification treatment, the average conjugation rate is calculated using the following formula:

[0011] ;

[0012] Where Dn% represents the percentage of peak area of ​​the protein in liquid chromatography conjugated with n linker-toxin molecules by the antibody-drug conjugate; (This formula is applicable to the calculation of antibody conjugation rate in samples where the disulfide bond content of glycoproteins in the sample is low or the spatial structure has little effect on deglycosylation, and the reducing agent is not added (i.e. the final concentration is 0 mol / L).

[0013] The average conjugation rate of antibody-drug conjugates that have undergone reduction and desaccharification treatment is calculated using the following formula:

[0014] ;

[0015] Wherein, Dn% represents the percentage of the peak area of ​​the protein conjugated with n linker-toxin molecules in liquid chromatography; LCn% represents the percentage of the peak area of ​​the light chain conjugated with n linker-toxin molecules in liquid chromatography; HCn% represents the percentage of the peak area of ​​the heavy chain conjugated with n linker-toxin molecules in liquid chromatography; (This formula is applicable to the calculation scenario of antibody conjugation rate when the disulfide bond content of the glycoprotein in the sample is high or the spatial structure has a significant impact on deglycosylation, and a reducing agent is added (i.e., the final concentration is >0 mol / L).

[0016] Most existing small molecules have highly hydrophobic structures. Therefore, hydrophobic exchange chromatography can separate antibody-drug conjugates (ADCs) according to the degree of hydrophobicity based on the number of conjugated small molecules. By using non-reduced or reduced antibody-drug conjugates, targeting the intact antibody or the reduced light and heavy chains, the weighted average DAR value is calculated by determining the percentage of peak areas corresponding to different numbers of conjugated small molecules (either intact or light / heavy chains). However, existing methods do not consider the high hydrophilicity of small molecules or antibodies caused by high glycosylation, making effective separation impossible in commonly used hydrophobic chromatography, thus affecting the accuracy of DAR value calculation.

[0017] In this invention, desaccharification treatment can eliminate the high hydrophilicity caused by high glycosylation, thereby avoiding differences in the average antibody conjugation rate due to high glycosylation. Because of the hydrophilicity caused by high glycosylation, low-DAR components may have similar retention times to high-DAR components, thus being misjudged as high-DAR components. By comparing with commonly used phenyl-bonded phase hydrophobic chromatographic columns and combining the results with mass spectrometry (MS) analysis, the hydrophilic interaction chromatographic method developed based on amide-bonded phase columns yields chromatograms with higher resolution, and the obtained DAR values ​​are consistent with those obtained by mass spectrometry.

[0018] Preferably, in (S1), the desugaring process includes: sequentially mixing the denaturant, buffer, reducing agent and desugaring enzyme with the sample and incubating them, and then performing reduction desugaring or desugaring treatment on the incubated sample.

[0019] Preferably, the denaturant is 5× rapid N-glycosidase F buffer.

[0020] In this invention, the denaturant serves to create optimal biochemical conditions for the deglycosylation reaction of the sample, ensuring reaction efficiency, enzyme stability, and the direct applicability of the product. Specifically, the denaturant can disrupt the spatial structure of glycoproteins in the sample, fully exposing the N-glycosylation sites originally encapsulated within the protein, thereby improving the contact efficiency between the deglycosease and the substrate and avoiding the problem of enzyme cleavage site masking caused by protein folding. Simultaneously, a moderate denaturing environment can reduce the aggregation between glycoprotein molecules, maintaining a uniform dispersion of the substrate in the reaction system and providing a homogeneous environment for the enzymatic reaction. Furthermore, the denaturant can enhance the conformational stability of the enzyme during the reaction process without destroying the structure of the enzyme's active site, reducing enzyme activity loss due to fluctuations in system conditions (such as changes in temperature and ionic strength), thus ensuring the controllability and reproducibility of the deglycosylation reaction.

[0021] Preferably, the final concentration of the denaturant in the mixed system is 1×.

[0022] Preferably, the reducing agent is dithiothreitol.

[0023] Preferably, the final concentration of the reducing agent in the mixed system is 0-0.5 mol / L, for example, it can be 0 mol / L, 0.25 mol / L or 0.5 mol / L, etc.

[0024] In this invention, the reducing agent functions to reduce the disulfide bonds between the sample chains, reducing the intact protein into heavy and light chain components. This process further releases the covalent cross-linking constraints within the protein molecules, allowing the higher-order structure of the glycoprotein to unfold more fully, thereby assisting the denaturing agent in more thoroughly exposing N-glycosylation sites and further improving the efficiency of deglycosylation enzymes. Simultaneously, the reduced light and heavy chain components exhibit more stable chromatographic behavior in subsequent separation and detection, reducing peak shape anomalies or retention time fluctuations caused by protein conformational differences and improving the accuracy of detection results. Furthermore, for complex glycoproteins containing multiple disulfide bonds, the appropriate use of the reducing agent can avoid the formation of insoluble complexes due to disulfide bond-mediated intermolecular polymerization, ensuring a uniform liquid phase environment in the reaction system and guaranteeing the efficient enzymatic reaction. When the disulfide bond content of the glycoprotein in the sample is low or the spatial structure has little impact on deglycosylation, the reducing agent can be omitted (i.e., the final concentration is 0 mol / L).

[0025] Preferably, the buffer is Tris-HCl with a pH of 7.8-8.0.

[0026] Preferably, the final concentration of the buffer in the mixed system is 0-0.015 mol / L.

[0027] Preferably, the final concentration of the reducing agent is 0 mol / L, and the incubation conditions are: temperature 50°C, time 10-15 min, for example, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.; the final concentration of the reducing agent is greater than 0 mol / L, and the incubation conditions are: temperature 75°C, time 5-8 min, for example, 5 min, 6 min, 7 min or 8 min, etc.; temperature 50°C, time 10-15 min, for example, 10 min, 11 min, 12 min, 13 min, 14 min or 15 min, etc.

[0028] Preferably, the desaccharifying enzyme is rapid N-glycosidase F.

[0029] In this invention, the rapid N-glycosidase F deglycosylates the sample, eliminating the high hydrophilicity of the sample caused by high glycosylation.

[0030] Preferably, the final concentration of the desaccharidase in the mixed system is 0.08-0.20 U, for example, it can be 0.08 U, 0.10 U or 0.20 U.

[0031] In this invention, the upper limit of the applicable desaccharifying enzyme can be appropriately adjusted according to the scale of the reaction system, the concentration of the substrate (such as glycoprotein / glycopeptide), and the reaction efficiency requirements, and usually does not exceed 0.20 U. If the concentration of the desaccharifying enzyme is too high, it may not only increase the experimental cost, but may also interfere with subsequent detection (such as chromatographic analysis and protein quantification) due to the enzyme protein itself. At the same time, the interaction between excessive enzyme molecules may also slightly affect the specificity of the enzyme-catalyzed reaction. Therefore, considering both economy and reaction effect, it is better to control the upper limit within 0.20 U.

[0032] Preferably, in (S2), the mobile phase used for chromatographic detection includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05%-0.1% aqueous solution of trifluoroacetic acid and mobile phase B is a 0.05%-0.1% acetonitrile solution of trifluoroacetic acid.

[0033] In this invention, the concentration of trifluoroacetic acid in the mobile phase must be strictly controlled between 0.05% and 0.1%. Too low a concentration will lead to broadened peak shapes and unstable retention times, while too high a concentration may damage the stationary phase of the chromatographic column and increase the risk of ion suppression effects in subsequent mass spectrometry detection (LC-MS).

[0034] The concentration of the trifluoroacetic acid aqueous solution can be, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%; the concentration of the trifluoroacetic acid acetonitrile solution can be, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%.

[0035] Preferably, the chromatographic column used for the chromatographic detection is an Acquity Premier Glycoprotein BEHAmide column, 1.7 μm, 300 Å, 2.1 × 150 mm.

[0036] Preferably, in (S2), the chromatographic detection is performed using gradient elution; the gradient elution procedure includes:

[0037] Over 0-2 minutes, the volume fraction of mobile phase A changed uniformly from 20.0-21.0% to 30.0-31.0%, with the remainder being mobile phase B.

[0038] Over 2-27 minutes, the volume fraction of mobile phase A changed uniformly from 30.0-31.0% to 40.0-41.0%, with the remainder being mobile phase B.

[0039] At 27-28 min, the volume fraction of mobile phase A changed uniformly from 40.0-41.0% to 100.0%, with the remainder being mobile phase B;

[0040] For 28-30 min, the volume fraction of mobile phase A is maintained at 100.0%, with the remainder being mobile phase B;

[0041] Over 30-31 minutes, the volume fraction of mobile phase A changes uniformly from 100.0% to 20.0-21.0%, with the remainder being mobile phase B.

[0042] For 31-45 min, the volume fraction of mobile phase A is maintained at 20.0-21.0%, with the remainder being mobile phase B.

[0043] In this invention, the elution program follows the liquid chromatography gradient design framework of "equilibrium-elution-strong elution-reset". The core objective is to ensure the separation of the target components while taking into account the elution efficiency and system stability.

[0044] Preferably, the chromatographic column used for the chromatographic detection is a Glycoprotein BEH Amide 300 Å 1.7µm 2.1*150mm.

[0045] Preferably, in (S2), the chromatographic detection is performed using gradient elution; the gradient elution procedure is as follows:

[0046] For 0-3 min, the volume fraction of mobile phase A is maintained at 20.0-20.1%, with the remainder being mobile phase B;

[0047] Over 3-33 minutes, the volume fraction of mobile phase A changed uniformly from 20.0-20.1% to 50.0-51.0%, with the remainder being mobile phase B.

[0048] At 33-34 min, the volume fraction of mobile phase A changed uniformly from 50.0-51.0% to 100%, with the remainder being mobile phase B;

[0049] For 34-40 minutes, the volume fraction of mobile phase A is maintained at 100%, with the remainder being mobile phase B;

[0050] At 40-41 min, the volume fraction of mobile phase A changes uniformly from 100% to 20.0-20.1%, with the remainder being mobile phase B;

[0051] For 41-46 min, the volume fraction of mobile phase A is maintained at 20.0-20.1%, with the remainder being mobile phase B.

[0052] Preferably, the detection wavelength λ of the chromatographic detection is 210 / 280 nm.

[0053] Preferably, the flow rate of the mobile phase is 0.2-0.5 mL / min, for example, it can be 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min, etc.

[0054] Preferably, the column temperature of the chromatographic column is 30-50°C, for example, it can be 30°C, 40°C or 50°C.

[0055] Secondly, the present invention provides the application of the method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates described in the first aspect in the detection of the average conjugation rate of antibody-drug conjugates.

[0056] This invention develops a method for detecting the average conjugation rate of highly hydrophilic small molecule antibody-drug conjugates (ADCs). Based on the hydrophobic interaction chromatography method, it optimizes the use of a hydrophilic exchange column with an amide-bonded phase to eliminate the influence of highly hydrophilic small molecules on DAR determination. At the same time, for ADCs with highly glycosylated characteristics, a deglycosylation treatment is used to eliminate the influence of high glycosylation on the method's resolution.

[0057] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The average conjugation rate of the antibody-drug conjugates described in this invention is achieved through a deglycosylation pretreatment process, eliminating the high hydrophilicity caused by high glycosylation and thus avoiding differences in the average conjugation rate due to high glycosylation. Simultaneously, the use of a hydrophilic exchange column with an amide-bonded phase allows for gradient elution separation of highly hydrophilic small molecules as the proportion of the aqueous mobile phase increases uniformly, effectively improving the resolution of conventional reversed-phase chromatography and enhancing the accuracy of DAR determination. This method results in high accuracy in the determination of the average conjugation rate. This method is suitable for determining the average antibody conjugation rate of antibody-drug conjugates containing highly glycosylated sites or conjugated highly hydrophilic small molecules. Attached Figure Description

[0060] Figure 1 This is a flowchart of a method for detecting the average conjugation rate of highly hydrophilic small molecule, highly glycosylated antibody-drug conjugates.

[0061] Figure 2 The image shows the liquid chromatogram obtained by hydrophobic column detection of the antibody-drug conjugate test sample in Example 1.

[0062] Figure 3 This is a hydrophilic exchange column liquid chromatogram of the antibody-drug conjugate test sample from Example 1.

[0063] Figure 4 This is a hydrophilic exchange column liquid chromatogram of the antibody-drug conjugate test sample from Example 2.

[0064] Figure 5 The liquid chromatograms of the antibody-drug conjugate test sample in Example 3 were obtained using hydrophilic exchange columns of different specifications.

[0065] Figure 6 The image shows the liquid chromatograms of the antibody-drug conjugate test sample in Example 4 using hydrophilic exchange column chromatography under different liquid phase separation conditions. Detailed Implementation

[0066] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0067] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0068] This invention provides a method for determining the average conjugation rate in highly hydrophilic small molecule, highly glycosylated antibody-drug conjugates, such as... Figure 1As shown, a pretreatment method for highly hydrophilic small-molecule, highly glycosylated antibody-drug conjugates (ADCs) was determined. Combining hydrophilic exchange interaction chromatography and liquid chromatography gradient conditions for detecting the average conjugation rate, the corresponding peak areas of different numbers of small molecules conjugated to the antibody light chain, heavy chain, or intact antibody were obtained, and the accurate average conjugation rate of the ADC was calculated. Specifically, the peak areas of the ADC after deglycosylation or reductive deglycosylation, conjugated to different numbers of small molecules on liquid chromatography, were obtained through separation using an amide-bonded phase column, thus eliminating the influence of low resolution of conventional hydrophobic interaction columns on the calculation of the average conjugation rate.

[0069] This invention improves upon traditional methods by employing desaccharification treatment and hydrophilic interaction chromatographic conditions during the analytical process. This addresses the low separation of highly hydrophilic small molecules with varying coupling ratios due to differences in hydrophobicity, resulting in more accurate and reliable average coupling rate measurements. This method is suitable for conjugating highly hydrophilic payload drugs with highly glycosylated antibody drugs, and is particularly suitable for conjugating highly hydrophilic small molecules that significantly affect the separation of traditional DAR liquid chromatography methods.

[0070] Example 1

[0071] This embodiment uses the method described above for detecting the average conjugation rate of a specific antibody-drug conjugate sample based on highly hydrophilic small molecule glycosylated antibody-drug conjugates to analyze the average conjugation rate (wherein, the antibody is a human serum fusion protein, and the drug is GalNAc (a tricarbon sugar), randomly conjugated through lysine residues). The specific steps include the following:

[0072] (1) Pretreatment method for detecting the average conjugation rate of antibody-drug conjugates.

[0073] Add 20 µg of protein sample to a 1.5 mL centrifuge tube, followed by 4 µL of 5X Rapid N-glycosidase F buffer. Make up the volume to 20 µL with 20 mM Tris HCl and mix well. Incubate at 75 °C for 6 min. Cool the denatured sample to room temperature, add 5 µL of Rapid N-glycosidase F, and mix well. Incubate at 50 °C for 15 min. Cool the deglycosylated sample to room temperature and make up the volume to 51 µL with 20 mM Tris HCl. Transfer the resulting sample to a glass vial for analysis.

[0074] (2) Liquid chromatography separation.

[0075] Table 1 shows the chromatographic conditions for detecting the average conjugation rate of antibody-drug conjugates using a hydrophobic column.

[0076] Table 1

[0077]

[0078] Figure 2 This is a liquid chromatogram of the antibody-drug conjugate sample obtained by desaccharification and hydrophobic interaction chromatography. Figure 2 The chromatograms show that conventional hydrophobic interaction columns have low resolution for highly hydrophilic molecules, making it impossible to calculate the average coupling rate.

[0079] Table 2 shows the chromatographic conditions for detecting the average conjugation rate of antibody-drug conjugates using a hydrophilic exchange column.

[0080] Table 2

[0081]

[0082] Figure 3 This is a liquid chromatogram of the antibody-drug conjugate test sample obtained by desaccharification treatment and hydrophilic exchange chromatography. Figure 3 In the diagram, D0 represents the chromatographic peak of the corresponding antibody; D1 represents the chromatographic peak of the antibody-drug conjugate (ADC) sample conjugated with one linker-toxin molecule; D2 represents the chromatographic peak of the ADC sample conjugated with two linker-toxin molecules; D3 represents the chromatographic peak of the ADC sample conjugated with three linker-toxin molecules; D4 represents the chromatographic peak of the ADC sample conjugated with four linker-toxin molecules; D5 represents the chromatographic peak of the ADC sample conjugated with five linker-toxin molecules; D6 represents the chromatographic peak of the ADC sample conjugated with six linker-toxin molecules; D7 represents the chromatographic peak of the ADC sample conjugated with seven linker-toxin molecules; D8 represents the chromatographic peak of the ADC sample conjugated with eight linker-toxin molecules; and D9 represents the chromatographic peak of the ADC sample conjugated with nine linker-toxin molecules. The identification of each chromatographic peak was confirmed by mass spectrometry.

[0083] (3) Results analysis.

[0084] In this embodiment, the pretreatment method used is non-reducing desugaring treatment, and the average conjugation rate of the antibody-drug conjugate test sample is calculated using the following formula:

[0085] .

[0086] The average conjugation rate of the antibody-drug conjugate test sample was calculated to be 6.2%; in the above formula: Dn% represents the percentage of peak area of ​​the liquid chromatography peak of the protein conjugated with n linker-toxin molecules by the antibody-drug conjugate.

[0087] The peak areas of the liquid chromatography peaks were collected at a wavelength of 210 nm. The percentage of peak area of ​​each chromatographic peak obtained by liquid chromatography at 210 nm is shown in Table 3.

[0088] Table 3

[0089]

[0090] The DAR value of the antibody-drug conjugate, measured by mass spectrometry, was 6.2. This indicates that the average conjugation rate obtained using desaccharification treatment combined with hydrophilic exchange interaction chromatography is approximately equal to the actual DAR of the drug.

[0091] Example 2

[0092] This embodiment uses the method described above for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates to analyze the average conjugation rate of a certain antibody-drug conjugate sample (where the antibody is IgG, the drug is Glycan, and the conjugation method is cysteine ​​chain disulfide bond conjugation). Specifically, it includes the following steps:

[0093] (1) Pretreatment method for detecting the average conjugation rate of antibody-drug conjugates.

[0094] Add 40 µg of protein sample to a 1.5 mL centrifuge tube, followed by 8 µL of 5× Rapid N-glycosidase F buffer and 2 µL of 1M DTT. Make up the volume to 40 µL with ultrapure water and mix well. Incubate at 75 °C for 6 min. Cool the denatured and reduced sample to room temperature, add 2 µL of Rapid N-glycosidase F, and mix well. Incubate at 50 °C for 13 min. Cool the deglycosylated sample to room temperature and make up the volume to 102 µL with ultrapure water. Transfer the resulting sample to a glass vial for analysis.

[0095] (2) Liquid chromatography separation.

[0096] Table 4 shows the chromatographic conditions for detecting the average conjugation rate of antibody-drug conjugates using a hydrophilic exchange column.

[0097] Table 4

[0098]

[0099] Figure 4 This is a liquid chromatogram of the antibody-drug conjugate test sample obtained by reductive desugaring treatment and hydrophilic exchange chromatography. Figure 4 In the table, LC0 represents the chromatographic peak corresponding to the light chain of the antibody, HC0 represents the chromatographic peak corresponding to the heavy chain of the antibody-drug conjugate sample, HC1 represents the chromatographic peak of the heavy chain of the antibody-drug conjugate sample conjugated with one linker-toxin molecule, and HC2 represents the chromatographic peak of the heavy chain of the antibody-drug conjugate sample conjugated with two linkers-toxin molecules. The identification of each chromatographic peak is confirmed by mass spectrometry.

[0100] (3) Results analysis.

[0101] In this embodiment, the pretreatment method used is reductive desugaring treatment, and the average conjugation rate of the antibody-drug conjugate test sample is calculated using the following formula:

[0102] ;

[0103] The calculated average conjugation rate of the antibody-drug conjugate (ADC) test sample was 2.0. In the above formula, LCn% represents the percentage of the peak area of ​​the light chain of the ADC conjugated with n linker-toxin molecules in liquid chromatography; HCn% represents the percentage of the peak area of ​​the heavy chain of the ADC conjugated with n linker-toxin molecules in liquid chromatography.

[0104] The peak areas of the liquid chromatography peaks were collected at a wavelength of 280 nm. The percentage of peak area of ​​each chromatographic peak obtained by liquid chromatography at 280 nm is shown in Table 5.

[0105] Table 5

[0106]

[0107] The DAR value of the antibody-drug conjugate, measured by mass spectrometry, was 1.9. This indicates that the average conjugation rate obtained using desaccharification reduction treatment combined with hydrophilic interaction chromatography is approximately equal to the actual DAR of the drug.

[0108] Example 3

[0109] This embodiment investigates the hydrophilic exchange chromatography column.

[0110] This embodiment uses the method described above for the average conjugation rate of antibody-drug conjugates based on highly hydrophilic small molecule glycosylated antibodies, but analyzes the average conjugation rate of the antibody-drug conjugate test sample in Example 1 using different types of hydrophilic exchange chromatographic columns. Specifically, it includes the following steps:

[0111] (1) Pretreatment method for detecting the average conjugation rate of antibody-drug conjugates.

[0112] Add 20 µg of protein sample to a 1.5 mL centrifuge tube, followed by 4 µL of 5X Rapid N-glycosidase F buffer. Make up the volume to 20 µL with 20 mM Tris HCl and mix well. Incubate at 75 °C for 6 min. Cool the denatured sample to room temperature, add 5 µL of Rapid N-glycosidase F, and mix well. Incubate at 50 °C for 15 min. Cool the deglycosylated sample to room temperature and make up the volume to 51 µL with 20 mM Tris HCl. Transfer the resulting sample to a glass vial for analysis.

[0113] (2) Liquid chromatography separation, chromatographic conditions are shown in Table 6.

[0114] Table 6

[0115]

[0116] Figure 5 This is a liquid chromatogram of the antibody-drug conjugate sample obtained by desaccharification treatment and alternative hydrophilic exchange chromatography 1. Figure 5 The chromatograms show that other specifications of hydrophilic exchange interaction columns have low resolution for highly hydrophilic molecules, making it impossible to calculate the average coupling rate.

[0117] Example 4

[0118] This embodiment examines gradient elution conditions.

[0119] This embodiment uses the method described above for the average conjugation rate of antibody-drug conjugates based on highly hydrophilic small molecule glycosylated antibodies, but analyzes the average conjugation rate of the antibody-drug conjugate test samples in Example 1 using different elution gradients. Specifically, it includes the following steps:

[0120] (1) Pretreatment method for detecting the average conjugation rate of antibody-drug conjugates.

[0121] Add 20 µg of protein sample to a 1.5 mL centrifuge tube, followed by 4 µL of 5X Rapid N-glycosidase F buffer. Make up the volume to 20 µL with 20 mM Tris HCl and mix well. Incubate at 75 °C for 6 min. Cool the denatured sample to room temperature, add 5 µL of Rapid N-glycosidase F, and mix well. Incubate at 50 °C for 15 min. Cool the deglycosylated sample to room temperature and make up the volume to 51 µL with 20 mM Tris HCl. Transfer the resulting sample to a glass vial for analysis.

[0122] (2) Liquid chromatography separation, the chromatographic conditions are shown in Table 7.

[0123] Table 7

[0124]

[0125] Figure 6 The liquid chromatogram of the antibody-drug conjugate test sample obtained by desaccharification and hydrophilic exchange chromatography. Figure 6 The spectra showed low separation of highly hydrophilic molecules by different liquid phase elution gradients, and co-elution of the rapid desaccharidase F and the antibody-drug conjugate component, making it impossible to calculate the average conjugation rate.

[0126] In summary, this invention provides a method for detecting the average conjugation rate in highly hydrophilic small molecule highly glycosylated antibody-drug conjugates, which improves the accuracy of liquid chromatography testing of the average DAR value of antibody-drug conjugates and is of great significance for the production and quality control of antibody-drug conjugates.

[0127] The applicant declares that the above description is only a specific embodiment 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 conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates, characterized in that, The method includes: (S1) The antibody-drug conjugate to be tested is deglycosylated using N-glycosidase F to obtain a deglycosylated antibody-drug conjugate. The structure of the antibody-drug conjugate is antibody-linker-toxin molecule. The antibody has high glycosylation properties, and the toxin molecule has high hydrophilic properties. The deglycosylation treatment includes non-reductive deglycosylation treatment or reductive deglycosylation treatment. (S2) The antibody-drug conjugate to be tested is detected by hydrophilic interaction chromatography; the chromatographic column used for the chromatographic detection is a hydrophilic interaction chromatographic column, and the packing material of the chromatographic column is an amide-bonded phase; (S3) Calculate the average conjugation rate of antibody-drug conjugates based on the percentage of peak area obtained from the liquid chromatography analysis; for antibody-drug conjugates that have undergone non-reducing desaccharification treatment, the average conjugation rate is calculated using the following formula: ； Wherein, Dn% represents the percentage of peak area of ​​the protein in liquid chromatography conjugated with n linker-toxin molecules by the antibody-drug conjugate; The average conjugation rate of antibody-drug conjugates that have undergone reduction and desaccharification treatment is calculated using the following formula: ； Wherein, Dn% represents the percentage of peak area of ​​the protein in liquid chromatography of the antibody-drug conjugate conjugate with n linker-toxin molecules; LCn% represents the percentage of peak area of ​​the light chain in liquid chromatography of the antibody-drug conjugate conjugate with n linker-toxin molecules; and HCn% represents the percentage of peak area of ​​the heavy chain in liquid chromatography of the antibody-drug conjugate conjugate with n linker-toxin molecules.

2. The method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates according to claim 1, characterized in that, In (S1), the desugaring process includes: mixing denaturant, buffer, reducing agent and desugaring enzyme with the sample in sequence and incubating, and then performing reduction desugaring or desugaring treatment on the incubated sample.

3. The method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates according to claim 2, characterized in that, The denaturing agent is 5× rapid N-glycosidase F buffer; Preferably, the final concentration of the denaturant in the mixed system is 1×; Preferably, the reducing agent is dithiothreitol; Preferably, the final concentration of the reducing agent in the mixed system is 0-0.5 mol / L.

4. The method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates according to any one of claims 1-3, characterized in that, The buffer is Tris-HCl, pH 7.8-8.0; Preferably, the final concentration of the buffer in the mixed system is 0-0.015 mol / L; Preferably, the final concentration of the reducing agent is 0 mol / L, and the incubation conditions are: temperature 50°C, time 10-15 min; or the final concentration of the reducing agent is greater than 0 mol / L, and the incubation conditions are: temperature 75°C, time 5-8 min, or temperature 50°C, time 10-15 min.

5. The method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates according to any one of claims 1-4, characterized in that, The desaccharifying enzyme is rapid N-glycosidase F; Preferably, the final concentration of the desaccharifying enzyme in the mixed system is 0.08-0.20 U.

6. The method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates according to any one of claims 1-5, characterized in that, In (S2), the mobile phase used for chromatographic detection includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05%-0.1% trifluoroacetic acid aqueous solution and mobile phase B is a 0.05%-0.1% trifluoroacetic acid acetonitrile solution.

7. The method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates according to any one of claims 1-6, characterized in that, The chromatographic column used for the chromatographic detection was an Acquity Premier Glycoprotein BEH Amide, 1.7 μm, 300 Å, 2.1 × 150 mm. Preferably, in (S2), the chromatographic detection is performed using gradient elution; the gradient elution procedure is as follows: Over 0-2 minutes, the volume fraction of mobile phase A changed uniformly from 20.0-21.0% to 30.0-31.0%, with the remainder being mobile phase B. Over 2-27 minutes, the volume fraction of mobile phase A changed uniformly from 30.0-31.0% to 40.0-41.0%, with the remainder being mobile phase B. At 27-28 min, the volume fraction of mobile phase A changed uniformly from 40.0-41.0% to 100.0%, with the remainder being mobile phase B; For 28-30 min, the volume fraction of mobile phase A is maintained at 100.0%, with the remainder being mobile phase B; Over 30-31 minutes, the volume fraction of mobile phase A changes uniformly from 100.0% to 20.0-21.0%, with the remainder being mobile phase B. For 31-45 min, the volume fraction of mobile phase A is maintained at 20.0-21.0%, with the remainder being mobile phase B.

8. The method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates according to any one of claims 1-6, characterized in that, The chromatographic column used for the chromatographic detection was a Glycoprotein BEH Amide 300 Å 1.7µm 2.1*150mm; Preferably, in (S2), the chromatographic detection is performed using gradient elution; the gradient elution procedure is as follows: For 0-3 min, the volume fraction of mobile phase A is maintained at 20.0-20.1%, with the remainder being mobile phase B; Over 3-33 minutes, the volume fraction of mobile phase A changed uniformly from 20.0-20.1% to 50.0-51.0%, with the remainder being mobile phase B. At 33-34 min, the volume fraction of mobile phase A changed uniformly from 50.0-51.0% to 100%, with the remainder being mobile phase B; For 34-40 minutes, the volume fraction of mobile phase A is maintained at 100%, with the remainder being mobile phase B; At 40-41 min, the volume fraction of mobile phase A changes uniformly from 100% to 20.0-20.1%, with the remainder being mobile phase B; For 41-46 min, the volume fraction of mobile phase A is maintained at 20.0-20.1%, with the remainder being mobile phase B.

9. The method for detecting the average conjugation rate of highly hydrophilic small molecule highly glycosylated antibody-drug conjugates according to any one of claims 1-8, characterized in that, The detection wavelength for the chromatographic detection is λ = 210 / 280 nm; Preferably, the flow rate of the mobile phase is 0.2-0.5 mL / min; Preferably, the column temperature of the chromatographic column is 30-50°C.

10. The method for detecting the average conjugation rate of a highly hydrophilic small molecule highly glycosylated antibody-drug conjugate according to any one of claims 1-9 is used in the detection of the average conjugation rate of antibody-drug conjugates.

Images