Method for determining the extinction coefficient of toxin linker in antibody-drug conjugates and method for detecting drug-antibody conjugation ratio in antibody-drug conjugates

Abstract

The present invention provides a method for determining the extinction coefficient of a toxin linker in an antibody-drug conjugate and a method for detecting the drug-antibody conjugation ratio in an antibody-drug conjugate. The method for determining the extinction coefficient comprises: a) dissolving the toxin linker in a solvent to obtain a toxin linker solution; b) mixing the concentrated toxin linker solution and the solvent with an excess antibody solution to obtain a toxin linker reaction solution and an antibody negative control solution; c) allowing the toxin linker to react completely with the antibody; d) using the antibody negative control solution as a reference solution for baseline calibration and scanning the absorption spectrum of the toxin linker reaction solution; and e) plotting a linear curve of the toxin linker absorbance and toxin linker concentration, fitting the curve to obtain a linear equation, and the slope of the linear equation is the extinction coefficient of the toxin linker after conjugation. This method can solve the problem of the difficulty in accurately determining the extinction coefficient of the toxin linker in an antibody-drug conjugate in the prior art and is suitable for the field of antibody-drug conjugate analysis.

Description

Technical Field

[0001] The present invention relates to the field of antibody-drug conjugate analysis, and in particular to a method for determining the extinction coefficient of a toxin linker in an antibody-drug conjugate and a method for detecting the drug-antibody conjugation ratio in an antibody-drug conjugate. Background Art

[0002] The drug-to-antibody ratio (DAR) is a core quality metric for antibody-drug conjugates (ADCs). This ratio is directly related to the therapeutic efficacy and safety of ADCs. Traditional DAR measurement techniques, such as reversed-phase chromatography (RP-HPLC), hydrophobic chromatography (HIC-HPLC), and mass spectrometry (MS), while capable of providing relatively accurate DAR values, are limited by their long development cycles, complex procedures, and high costs, making them unsuitable for the rapid sample analysis required in the early stages of ADC conjugation process development. In particular, RP-HPLC and HIC require chromatographic separation of the ADC components, a technically demanding and time-consuming process that is unsuitable for the frequent sample analysis required in early process development. While mass spectrometry offers high versatility, its reliance on high-resolution biomass spectrometry leads to extremely high requirements for equipment and specialized operators, increasing testing costs and limiting its widespread application in R&D.

[0003] In contrast, UV spectroscopy, due to its minimal equipment requirements and ease of operation, has become a preferred method for determining the DAR value of ADC samples. UV spectrometry measures the absorbance of the ADC at specific wavelengths and, combined with the known extinction coefficients of the antibody and toxin linker at these wavelengths, rapidly calculates the DAR value. This method not only reduces sample testing costs but, thanks to its high-precision absorbance measurement, can sensitively reflect the impact of changes in conjugation process parameters on the product DAR value. However, the extinction coefficient of the toxin linker used to calculate the drug-antibody conjugation ratio in antibody-drug conjugates in the prior art is based on the measurement of the unconjugated starting material before the conjugation reaction. However, after conjugation to the antibody (e.g., by reacting with lysine, cysteine, or glycosylation sites on the antibody), the toxin linker's molecular structure or microenvironment (e.g., pH, solvent effects, etc.) may change. This can cause the absorption spectrum of the antibody-bound toxin linker after the conjugation reaction to differ significantly from that of the free toxin linker dissolved in an organic solvent before the conjugation reaction, thereby affecting its extinction coefficient. Continuing to use the extinction coefficient of the free toxin linker measured before the conjugation reaction will inevitably introduce significant errors in the DAR calculation, especially for complex toxin linkers. Existing methods for determining the extinction coefficient are difficult to accurately determine, significantly impacting the accuracy of subsequent DAR measurements. Summary of the Invention

[0004] The main purpose of the present invention is to provide a method for determining the extinction coefficient of the toxin linker in an antibody-drug conjugate and a method for detecting the drug-antibody conjugation ratio in an antibody-drug conjugate, so as to solve the problem in the prior art that it is difficult to accurately determine the extinction coefficient of the toxin linker in the antibody-drug conjugate.

[0005] To achieve the above objectives, according to a first aspect of the present invention, a method for determining the extinction coefficient of a toxin linker in an antibody-drug conjugate is provided, the method comprising: a) dissolving the toxin linker in a solvent to obtain toxin linker solutions of known concentrations; b) taking a certain volume of solution from the toxin linker solutions of different concentrations and the solvent used to dissolve the toxin linker, respectively, mixing them with excess antibody solution at the same volume ratio, fixing the volumes, and correspondingly obtaining toxin linker reaction solutions of known concentrations and antibody negative control solutions; c) oscillating the toxin linker reaction solution and the antibody negative control solution in the dark to allow the toxin linker to react completely with the antibody; d) using the antibody negative control solution as a reference solution for ultraviolet spectral scanning to perform baseline calibration, and scanning the absorption spectra of toxin linker reaction solutions of different concentrations; e) drawing linear curves of toxin linker absorbance and toxin linker concentration at wavelengths 1 and 2, and obtaining a linear equation by linear fitting, the slope of which is the extinction coefficient of the toxin linker after conjugation.

[0006] Furthermore, the antibody solution includes an activated antibody solution.

[0007] Furthermore, the activation includes: according to the type of coupling reaction between the antibody and the toxin linker, activating the antibody into an antibody capable of performing the coupling reaction.

[0008] Furthermore, the coupling reaction includes lysine coupling, glycosylation site coupling or cysteine ​​coupling.

[0009] To achieve the above objectives, according to a second aspect of the present invention, a method for detecting the drug-antibody conjugation ratio in an antibody-drug conjugate is provided, comprising: i) measuring the extinction coefficient of the conjugated toxin linker at wavelength 1 and wavelength 2 using the above-mentioned method for measuring the extinction coefficient of the toxin linker in the antibody-drug conjugate; ii) measuring the extinction coefficient of the antibody at wavelength 1 and wavelength 2; iii) measuring the ratio of the response signals of the antibody-drug conjugate at wavelength 1 and wavelength 2 using a liquid chromatography or electrophoresis separation technique that can separate the antibody-drug conjugate, free toxin linker, and impurities; and iv) calculating the drug-antibody conjugation ratio using the ratio of the response signals, the extinction coefficient in i), and the extinction coefficient in ii).

[0010] By applying the technical solution of the present invention, the extinction coefficient of the toxin linker after coupling can be accurately measured in the above method. Furthermore, based on this accurate extinction coefficient, the DAR value of the ADC product can be detected with high precision, providing strong analytical support for the early process development of ADC. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0012] Figure 1 The characteristic absorption spectra of the toxin linker according to Example 1 of the present invention in two different solvents are shown.

[0013] Figure 2 The characteristic absorption spectrum of the toxin linker on the ADC molecule according to Example 1 of the present invention is shown.

[0014] Figure 3 The UV spectra of toxin linker solutions with different concentrations after quantitative reaction with excess antibodies according to Example 1 of the present invention are shown.

[0015] Figure 4 The figure shows a linear fitting graph (least square method) of absorbance at two different wavelengths and toxin linker solution concentration after quantitative reaction of toxin linker solutions with different concentrations and excess antibody according to Example 1 of the present invention. DETAILED DESCRIPTION

[0016] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0017] As mentioned in the background art, the traditional cuvette UV method faces two major challenges when determining the drug-antibody conjugation ratio (DAR): first, it cannot accurately reflect the change in the extinction coefficient after toxin linker conjugation, especially when its molecular structure or microenvironmental conditions change; second, due to the lack of ability to distinguish between ADC molecules and free toxin linkers, the absorbance of free toxin linkers or certain process impurities in the sample interferes with the accurate determination of the DAR value. Therefore, in this application, the inventors attempted to develop a new analytical method that can overcome the problems of changes in the extinction coefficient after toxin linker conjugation and spectral interference from free toxin linkers or process impurities, providing a more accurate and rapid means of detecting DAR values ​​for early-stage process development of ADCs. Based on this, a series of protection schemes for this application are proposed.

[0018] In a first typical embodiment of the present application, a method for determining the extinction coefficient of a toxin linker in an antibody-drug conjugate is provided, the method comprising: a) dissolving the toxin linker in a solvent to obtain toxin linker solutions of known concentrations; b) taking a certain volume of solution from the toxin linker solutions of different concentrations and the solvent used to dissolve the toxin linker, respectively, mixing them with excess antibody solution in the same volume ratio, fixing the volumes, and correspondingly obtaining toxin linker reaction solutions and antibody negative control solutions of known concentrations; c) oscillating the toxin linker reaction solution and the antibody negative control solution in the dark to allow the toxin linker to react completely with the antibody; d) using the antibody negative control solution as a reference solution for ultraviolet spectral scanning to perform baseline calibration, and scanning the absorption spectra of toxin linker reaction solutions of different concentrations; e) drawing linear curves of toxin linker absorbance and toxin linker concentration at wavelengths 1 and 2, and obtaining a linear equation by linear fitting, the slope of which is the extinction coefficient of the toxin linker after conjugation.

[0019] The method for determining the extinction coefficient of the conjugated toxin linker achieves technical improvement through the following steps:

[0020] 1. Accurate extinction coefficient determination: This method involves dissolving the toxin conjugate in a specific solvent to prepare a series of solutions of known concentrations. These solutions are then mixed with an excess of antibody to ensure complete conjugation of the toxin conjugate to the antibody. This allows for accurate determination of the extinction coefficient of the conjugated toxin conjugate at a specific wavelength and in a specific solution medium, avoiding the potential error associated with using the extinction coefficient of the toxin conjugate prior to conjugation.

[0021] 2. Eliminate background interference: Introducing an antibody negative control solution for baseline calibration effectively eliminates the background absorption of the antibody itself, ensuring that the measured absorbance is attributed only to the toxin linker, thereby improving the accuracy of the measurement results.

[0022] 3. Establishing a linear relationship: By plotting the absorbance versus concentration curves of the toxin linker at wavelengths 1 and 2 and performing a linear fit, the molar extinction coefficient of the toxin linker at the two detection wavelengths can be obtained. This linear relationship provides a solid foundation for the subsequent calculation of the DAR value.

[0023] In summary, by measuring the extinction coefficient of the toxin linker after coupling, this method can provide more accurate and reliable DAR value determination results, effectively supporting the early process development of ADCs, especially in the application of structurally complex toxin linkers, overcoming the limitations of traditional UV spectroscopy and achieving significant technological progress.

[0024] Preferably, the above method is applicable to stable toxin linkers, i.e., toxin linkers that do not undergo significant side reactions (e.g., hydrolysis) during the conjugation process, and toxin linkers whose side reaction products do not interfere with the UV absorption spectrum of the conjugated toxin linker. Generally, the toxin linker structure is stable, and significant side reactions are generally not observed during the conjugation reaction. This means that the above method is applicable to the vast majority of antibody-drug conjugates.

[0025] In a preferred embodiment, the linear fitting method includes but is not limited to the least squares method.

[0026] In a preferred embodiment, the antibody solution includes an activated antibody solution.

[0027] In a preferred embodiment, the activation comprises: activating the antibody into an antibody capable of performing a coupling reaction according to the type of coupling reaction between the antibody and the toxin linker.

[0028] In a preferred embodiment, the coupling reaction includes lysine coupling, glycosylation site coupling or cysteine ​​coupling. Correspondingly, the activation corresponding to cysteine ​​coupling includes reducing disulfide bonds in the antibody to free sulfhydryl groups.

[0029] In step b) of the above-mentioned method for determining the extinction coefficient, activation of the antibody solution may be necessary depending on the type of coupling reaction. For example, for cysteine ​​coupling, disulfide bonds in the antibody must first be reduced to free sulfhydryl groups available for coupling. Those skilled in the art can flexibly select an appropriate activation reaction based on the selected coupling reaction and antibody.

[0030] In a preferred embodiment, wavelength 1 is the characteristic absorption wavelength of the antibody or toxin linker between 240 nm and 400 nm; correspondingly, wavelength 2 is the characteristic absorption wavelength of the toxin linker or antibody between 240 nm and 400 nm.

[0031] In the methods of the present application, those skilled in the art can flexibly select wavelengths 1 and 2 to correspond to the corresponding compounds. This includes: wavelength 1 being the wavelength of the antibody, and wavelength 2 being the wavelength of the toxin linker; or wavelength 1 being the wavelength of the toxin linker, and wavelength 2 being the wavelength of the antibody. The correspondence between wavelengths and different types of compounds does not affect the calculation results and detection accuracy of the detection method.

[0032] In a preferred embodiment, the characteristic absorption wavelength includes ±20 nm of the maximum absorption wavelength between 240 nm and 400 nm, more preferably ±15 nm, ±10 nm; preferably, the absolute value of the difference between wavelength 1 and wavelength 2 is ≥30 nm, more preferably ≥40 nm.

[0033] Preferably, wavelength 1 and wavelength 2 are selected from wavelengths at which the antibody and toxin linker can produce characteristic absorption within the wavelength range of 240 nm to 400 nm (i.e., characteristic absorption wavelengths), and the characteristic absorption wavelengths are located within ±20 nm of the maximum absorption wavelength of the compound within the wavelength range of 240 nm to 400 nm. For example, if the maximum absorption wavelength of an antibody within the wavelength range of 240 nm to 400 nm is 280 nm, then the characteristic absorption wavelength of the antibody is 260-300 nm.

[0034] Preferably, the absolute value of the difference between wavelength 1 and wavelength 2 is ≥30 nm, which can ensure that the parameters such as the extinction coefficient measured in actual detection have sufficient discrimination, thereby ensuring the accuracy of the method for detecting the drug-antibody conjugate ratio in the antibody-drug conjugate.

[0035] In a second typical embodiment of the present application, a method for detecting the drug-antibody coupling ratio in an antibody-drug conjugate is provided, the method comprising: i) using the above method for determining the extinction coefficient of the toxin linker in the antibody-drug conjugate, measuring the extinction coefficient of the toxin linker after coupling at wavelength 1 and wavelength 2, respectively. and ii) Determine the extinction coefficient of the antibody at wavelength 1 and wavelength 2, respectively. and iii) utilizing a liquid chromatography or electrophoresis separation technique capable of separating the antibody-drug conjugate, free toxin linker, and impurities, to determine the ratio of the response signals of the liquid chromatography or electrophoresis detector of the antibody-drug conjugate at wavelength 1 to that at wavelength 2, where the ratio is represented by k; iv) utilizing the measured values, including the ratio of the response signals, the extinction coefficient in i), and the extinction coefficient in ii), to calculate the drug-antibody conjugation ratio.

[0036] In a preferred embodiment, the above calculation includes: drug-antibody coupling ratio = .

[0037] It should be noted that in order to simplify the description of relevant parameters in this application, some parameters are expressed in the form of abbreviations and letters, such as 、 、 、 Through the above explanation, this application hopes to emphasize that the use of abbreviations and letter symbols should not be regarded as limiting the scope of implementation of the technical solution, but is a way of expression intended to make the technical disclosure more concise and easy to understand.

[0038] The calculation formula is: drug-antibody coupling ratio = .

[0039] Using the Lambert-Beer law, the calculation formula for the above DAR value is derived as follows:

[0040] ,

[0041] ,

[0042] ,

[0043] ,

[0044] ,

[0045] Transform the above formula to obtain the calculation formula of DAR.

[0046] .

[0047] in:

[0048] The subscripts w1 and w2 represent two wavelengths,

[0049] A w1*ADC , A w2*ADC Indicates: absorbance of ADC at wavelength 1 and wavelength 2,

[0050] , Indicates: the molar extinction coefficient of the antibody in ADC at wavelength 1 and wavelength 2,

[0051] , Indicates: the molar extinction coefficient of the toxin linker in ADC at wavelength 1 and wavelength 2,

[0052] C mAb Indicates: the molar concentration of the antibody in the ADC,

[0053] C PL represents: the molar concentration of the toxin linker in the ADC,

[0054] L represents: optical path,

[0055] k represents the response signal ratio at dual wavelengths.

[0056] In the above method, the ratio used to evaluate the difference in absorbance levels is calculated using the parameter associated with wavelength 1 as the numerator and the parameter associated with wavelength 2 as the denominator.

[0057] In a preferred embodiment, the ratio = the absorbance level at wavelength 1 ÷ the absorbance level at wavelength 2.

[0058] In the above-mentioned method for determining the ratio, there is no need to introduce strict absolute quantitative analysis in the liquid phase method or the capillary electrophoresis method, and there is no need to prepare a standard curve. The ratio k can be determined by a simple detector signal intensity ratio.

[0059] In a preferred embodiment, the method for determining the extinction coefficient of the antibody in the above ii) includes but is not limited to: UV spectrophotometry, amino acid sequence analysis, SEC-UV-RI-MALS method or denaturation treatment method.

[0060] In a preferred embodiment, the ultraviolet spectrophotometry method includes preparing an antibody solution of known concentration, scanning the absorption spectrum, and calculating the extinction coefficient of the antibody based on the absorbance at a specific wavelength, the antibody concentration, and the optical path length; preferably, the amino acid sequence analysis method includes: calculating the extinction coefficient of the antibody at a wavelength of 280 nm based on the protein sequence of the antibody.

[0061] The basic principle of the SEC-UV-RI-MALS method is to separate antibodies by size exclusion chromatography (SEC). The eluted antibodies are then detected simultaneously using an ultraviolet (UV) detector, a refractive index detector (RI), and a multi-angle laser light scattering detector (MALS). The extinction coefficient of the antibody is calculated based on the absorbance measured by the UV detector and the molecular weight measured by the MALS detector.

[0062] The basic principle of the denaturation method is to denature the antibody using reagents such as guanidine hydrochloride, unfold the higher-order folded structure of the antibody, and treat it as a ternary system of tryptophan, tyrosine, and "SS" bonds. The extinction coefficient of the antibody is calculated by detecting the absorbance values ​​of folded and unfolded antibodies at the same concentration at an ultraviolet wavelength of 280 nm.

[0063] In a preferred embodiment, an analytical technique that can effectively separate antibody-drug conjugates from spectrally interfering impurities is applicable, for example, to antibody affinity chromatography, hydrophobic chromatography, hydrophilic interaction chromatography, etc. Preferably, the capillary electrophoresis includes capillary zone electrophoresis (CZE), sodium dodecyl sulfate polyacrylamide gel capillary electrophoresis (CE-SDS), or capillary isoelectric focusing electrophoresis.

[0064] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0065] Example 1

[0066] The ADC platform molecule (antibody-conjugated camptothecin ADC molecule) of Shanghai Ailiying Biotechnology Co., Ltd. was used to conduct experiments according to the method described in the present invention.

[0067] 1) The characteristic absorption spectra of the camptothecin toxin linker in different solvents are significantly different (e.g. Figure 1), which exhibits an obvious fine structure in the wavelength range of 300nm ~ 400nm. After the toxin linker is coupled to the antibody, its fine structure in the wavelength range of 300nm ~ 400nm becomes less obvious (as shown in Figure 2 As shown), the absorption spectrum and Figure 1 Compared with the absorption spectrum before coupling, the absorption spectrum of Figure 1 The extinction coefficients shown for the toxin linker prior to conjugation cannot be used to accurately determine the DAR values ​​for the ADC molecules.

[0068] 2) According to the quantitative coupling reaction method of the present invention, the absorption spectra of the toxin linker with different concentrations after quantitative reaction with excess antibody are obtained, and the characteristic absorption in the wavelength range of 300nm to 400nm is completely consistent with that of the ADC molecule (such as Figure 3 As shown in Figure 2), the absorbance showed a good linear relationship with the concentration of the toxin linker (as shown in Figure 2). Figure 4 The extinction coefficient was calculated based on the concentration of the toxin conjugate after conjugation and the absorbance at two wavelengths.

[0069] 3) Liquid chromatography is used to separate the ADC and the free toxin linker small molecule, and the UV detector signal ratio of the ADC at two wavelengths is obtained.

[0070] 4) Substituting the extinction coefficient of the conjugated toxin linker and the detector signal ratio at dual wavelengths of the ADC into the formula described in the present invention, the DAR value of the ADC sample was measured to be 7.99, which is consistent with the target value of 8 for this ADC product.

[0071] Comparative Example 1

[0072] According to the traditional method, the extinction coefficient of the unconjugated toxin linker (i.e., the toxin linker before conjugation) is measured and the DAR value is calculated using the extinction coefficient as follows:

[0073] 1) Weigh a certain amount of toxin linker (raw material for ADC conjugation), fully dissolve it in DMSO solvent, and then further dilute it to approximately 0.01 mg / mL with DMSO solvent;

[0074] 2) Use DMSO solvent as the reference solution for UV spectral scanning for baseline calibration;

[0075] 3) Measure the absorbance of the toxin linker DMSO solution at two wavelengths and calculate the extinction coefficient of the toxin linker based on the concentration;

[0076] 4) Dilute the ADC sample to approximately 0.3 mg / mL with purified water and measure its absorbance at two wavelengths using a UV spectrometer.

[0077] 5) Substituting the extinction coefficients of the toxin linker and antibody, as well as the absorbance of the ADC, into the calculation formula, the calculated DAR value was approximately 6.23, significantly lower than the actual value of 8, indicating a large method error.

[0078] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: in the above method, by measuring the ratio of the extinction coefficient of the toxin linker after conjugation, the extinction coefficient of the antibody, and the absorbance level of the antibody-drug conjugate, a unique quantitative conjugation reaction and chromatography method for eliminating impurity interference can effectively eliminate the influence of the change in the extinction coefficient of the toxin linker after conjugation and the interference of free toxin linkers. This method overcomes the problem of poor accuracy of the traditional UV method in determining the DAR value when the toxin linker structure changes. In particular, for toxin linkers whose structure or microenvironmental conditions change significantly after conjugation, it can provide more realistic and accurate extinction coefficient measurements, thereby improving the reliability of DAR value determination. Moreover, by directly measuring the extinction coefficient of the toxin linker after conjugation, there is no need to rely on pre-conjugation data, which simplifies the calculation process of the DAR value and reduces the calculation error introduced by data mismatch. This method has a short development cycle and is relatively simple to operate. It can quickly adapt to the early stages of ADC conjugation process development, providing R&D personnel with a real-time and effective means of monitoring DAR values ​​and accelerating the development process of ADC products.

[0079] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for determining the extinction coefficient of a toxin linker in an antibody-drug conjugate, characterized in that: The method comprises: a) dissolving the toxin linker in a solvent to obtain toxin linker solutions with known concentrations; b) taking a certain volume of solution from each of the toxin linker solutions of different concentrations and the solvent used to dissolve the toxin linker, respectively, and mixing them with excess antibody solution at the same volume ratio, and fixing the volumes to obtain toxin linker reaction solutions of known concentrations and antibody negative control solutions, respectively; c) shaking the toxin linker reaction solution and the antibody negative control solution in the dark to allow the toxin linker to react completely with the antibody; d) using the antibody negative control solution as a reference solution for ultraviolet spectrum scanning to perform baseline calibration, and scanning the absorption spectra of the toxin linker reaction solution at different concentrations; e) plotting linear curves of toxin linker absorbance and toxin linker concentration at wavelength 1 and wavelength 2, and obtaining a linear equation by linear fitting, wherein the slope of the linear equation is the extinction coefficient of the conjugated toxin linker; the wavelength 1 and the wavelength 2 are the characteristic absorption wavelengths of the antibody and the toxin linker between 240 nm and 400 nm, respectively.

2. The method according to claim 1, characterized in that The antibody solution includes an activated antibody solution.

3. The method according to claim 2, characterized in that The activation includes: according to the type of the coupling reaction between the antibody and the toxin linker, activating the antibody into an antibody capable of performing the coupling reaction.

4. The method according to claim 3, characterized in that The coupling reaction includes lysine coupling, glycosylation site coupling or cysteine ​​coupling.

5. A method for detecting the drug-antibody coupling ratio in an antibody-drug conjugate, characterized in that: The method comprises: i) using the method for determining the extinction coefficient of the toxin linker in the antibody-drug conjugate according to any one of claims 1 to 4, measuring the extinction coefficient of the conjugated toxin linker at wavelength 1 and wavelength 2; ii) determining the extinction coefficient of the antibody at wavelength 1 and wavelength 2; iii) using a liquid chromatography or electrophoresis separation technique capable of separating the antibody-drug conjugate, free toxin linker, and impurities, to determine the ratio of the liquid chromatography or electrophoresis detector response signals of the antibody-drug conjugate at wavelength 1 to that at wavelength 2; iv) calculating the drug-antibody coupling ratio using the ratio of the response signals, the extinction coefficient in i) and the extinction coefficient in ii); Wherein, the wavelength 1 and the wavelength 2 are the characteristic absorption wavelengths of the antibody and the toxin linker between 240nm and 400nm, respectively.

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