Method for determining extinction coefficient of toxin connexon in antibody-coupled drug and method for detecting drug-antibody coupling ratio in antibody-coupled drug

Abstract

The invention provides a method for determining an extinction coefficient of a toxin connexon in an antibody-coupled drug and a method for detecting a drug-antibody coupling ratio in the antibody-coupled drug. The method for measuring the extinction coefficient comprises the following steps: a) dissolving a toxin connexon in a solvent to obtain a toxin connexon solution; b) respectively mixing the concentration toxin connexon solution and the solvent with the excessive antibody solution to obtain a toxin connexon reaction solution and an antibody negative control solution; c) completely reacting the toxin connexon with the antibody; d) carrying out baseline calibration by using the antibody negative control solution as a reference solution and scanning a toxin linker reaction solution absorption spectrum; and e) drawing a linear curve of the absorbance of the toxin connexon and the concentration of the toxin connexon, fitting to obtain a linear equation, and the slope of the linear equation being the extinction coefficient of the coupled toxin connexon. The method can solve the problem that the extinction coefficient of the toxin connexon in the antibody-coupled drug is difficult to accurately determine in the prior art, and is suitable for the field of antibody-coupled drug analysis.

Description

Technical Field

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

[0002] The drug-to-antibody ratio (DAR) is a core indicator for measuring the quality of antibody-drug conjugates (ADCs). This ratio is directly related to the therapeutic effect and safety of ADCs. Traditional DAR value detection techniques, such as reverse-phase chromatography (RP-HPLC), hydrophobic interaction chromatography (HIC-HPLC), and mass spectrometry (MS), although able to provide relatively accurate DAR values, have limitations in that the method development cycle is long, the operation is complex, and the cost is high, making it difficult to meet the rapid sample detection needs in the initial stage of ADC conjugation process development. In particular, reverse-phase chromatography and hydrophobic interaction chromatography require chromatographic separation of the components of ADCs. This process not only has high technical requirements but also takes a long time, and is not suitable for the frequent sample analysis requirements in the early process development stage. Although mass spectrometry has high versatility, its dependence on high-resolution bio-mass spectrometry results in extremely high requirements for equipment and professional operators, thus increasing the detection cost and limiting its wide application in the R & D stage.

[0003] In contrast, ultraviolet spectroscopy has become a preferred method for detecting the DAR value of ADC samples due to its low requirements for equipment and simplicity of operation. An ultraviolet spectrometer can quickly calculate the DAR value by detecting the absorbance of an ADC at specific wavelengths and combining the known extinction coefficients of the antibody and toxin linker at these wavelengths. This method not only reduces the sample detection cost but also, due to its high-precision detection of absorbance, can sensitively reflect the impact of changes in conjugation process parameters on the DAR value of the product. However, in the prior art, the extinction coefficient of the toxin linker used to calculate the drug-to-antibody ratio of an antibody-drug conjugate is based on the detection results of the unconjugated raw materials before the conjugation reaction. However, when the toxin linker is conjugated to an antibody (e.g., reacts with lysine, cysteine, or glycosylation sites on the antibody), its molecular structure or the microenvironment it is in (such as pH value, different solvent effects, etc.) may change, which can cause a significant change in the absorption spectrum of the toxin linker conjugated to the antibody after the conjugation reaction compared to the absorption spectrum of the free toxin linker dissolved in an organic solvent before the conjugation reaction, thereby affecting its extinction coefficient. If the extinction coefficient of the free toxin linker measured before the conjugation reaction is still used, it will inevitably introduce a large error in the calculation of the DAR value, especially for toxin linkers with complex structures. It is difficult to accurately determine the extinction coefficient using the extinction coefficient determination methods in the prior art, which will also have a greater impact on the accuracy of subsequent DAR value determination. Summary of the Invention

[0004] The main object 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 an antibody-drug conjugate.

[0005] To achieve the above object, according to the first aspect of the present invention, a method for determining the extinction coefficient of the toxin linker in an antibody-drug conjugate is provided. The method includes: a) dissolving the toxin linker in a solvent to prepare toxin linker solutions with different known concentrations; b) taking a certain volume of solution from the toxin linker solutions with different concentrations and the solvent used to dissolve the toxin linker respectively, mixing them with an excessive antibody solution according to the same volume ratio, and making up the volume to obtain toxin linker reaction solutions with different known concentrations and antibody negative control solutions correspondingly; c) subjecting the toxin linker reaction solutions and the antibody negative control solutions to light-shielded oscillation to make the toxin linker react completely with the antibody; d) using the antibody negative control solution as a reference solution for ultraviolet spectrum scanning to calibrate the baseline, and scanning the absorption spectra of the toxin linker reaction solutions with different concentrations; e) plotting a linear curve of the absorbance of the toxin linker and the concentration of the toxin linker at wavelength 1 and wavelength 2, performing linear fitting to obtain a linear equation, and the slope of the linear equation is the extinction coefficient of the conjugated toxin linker.

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

[0007] Further, the activation includes: activating the antibody into an antibody capable of undergoing a conjugation reaction according to the type of the conjugation reaction between the antibody and the toxin linker.

[0008] Further, the conjugation reaction includes lysine conjugation, glycosylation site conjugation or cysteine conjugation.

[0009] To achieve the above object, according to the second aspect of the present invention, a method for detecting the drug-antibody conjugation ratio in an antibody-drug conjugate is provided. The method includes: i) using the method for determining the extinction coefficient of the toxin linker in an antibody-drug conjugate as described above to determine the extinction coefficients of the conjugated toxin linker at wavelength 1 and wavelength 2; ii) determining the extinction coefficients 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 and wavelength 2; 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] Applying the technical solution of the present invention, the extinction coefficient of the conjugated toxin linker can be accurately determined in the above method. Further, based on this accurate extinction coefficient, high-precision detection of the DAR value of the ADC product can be achieved, providing strong analytical support for the early process development of ADC. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0012] Figure 1 Shows the characteristic absorption spectra of the toxin linker according to Embodiment 1 of the present invention in two different solvents.

[0013] Figure 2 Shows the characteristic absorption spectra of the toxin linker on the ADC molecule according to Embodiment 1 of the present invention.

[0014] Figure 3 Shows the ultraviolet spectra after the quantitative reaction of different concentrations of toxin linker solutions with an excess of antibody according to Embodiment 1 of the present invention.

[0015] Figure 4 Shows the linear fitting graphs (least squares method) of the absorbance at two different wavelengths and the concentration of the toxin linker solution after the quantitative reaction of different concentrations of toxin linker solutions with an excess of antibody according to Embodiment 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments 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 in determining the drug-antibody conjugation ratio (DAR): one is that it cannot accurately reflect the change in the extinction coefficient after the conjugation of the toxin linker, especially when its molecular structure or microenvironmental conditions change; the other is that due to the lack of the 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 attempt to develop a new analytical method that can overcome the change in the extinction coefficient after the conjugation of the toxin linker and the spectral interference of free toxin linkers or process impurities, provide a more accurate and faster means for detecting the DAR value for the early process development of ADC, and based on this, a series of protection schemes of this application are proposed.

[0018] In the 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 above method includes: a) dissolving the toxin linker in a solvent to prepare toxin linker solutions with different known concentrations; b) taking a certain volume of solution from the toxin linker solutions with different concentrations and the solvent used to dissolve the toxin linker respectively, mixing them with an excess antibody solution according to the same volume ratio, and making up the volume to obtain toxin linker reaction solutions with different known concentrations and antibody negative control solutions correspondingly; c) subjecting the toxin linker reaction solutions and the antibody negative control solutions to light-shielded oscillation to ensure complete reaction between the toxin linker and the antibody; d) using the antibody negative control solution as a reference solution for ultraviolet spectrum scanning to calibrate the baseline, and scanning the absorption spectra of the toxin linker reaction solutions with different concentrations; e) plotting a linear curve of the absorbance of the toxin linker against the concentration of the toxin linker at wavelength 1 and wavelength 2, performing linear fitting to obtain a linear equation, and the slope of the linear equation is the extinction coefficient of the conjugated toxin linker.

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

[0020] 1. Precise extinction coefficient determination: By dissolving the toxin linker in a specific solvent and preparing a series of solutions with known concentrations, and mixing with an excess of antibody, it ensures that the toxin linker is completely conjugated to the antibody. In this way, the extinction coefficient of the conjugated toxin linker in a specific solution medium at a specific wavelength can be accurately determined, avoiding the errors that may be brought by using the extinction coefficient of the toxin linker before conjugation.

[0021] 2. Elimination of 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 only attributed to the toxin linker, and improving the accuracy of the measurement results.

[0022] 3. Establishment of linear relationship: By plotting a linear curve of the absorbance of the toxin linker against the concentration at wavelength 1 and wavelength 2 and performing linear fitting, the molar extinction coefficient of the toxin linker at the two detection wavelengths can be obtained. The establishment of this linear relationship provides a solid foundation for the subsequent calculation of the DAR value.

[0023] In summary, by determining the extinction coefficient of the conjugated toxin linker, this method can provide more accurate and reliable DAR value measurement results, effectively supporting the early process development of ADCs, especially in the application of structurally complex toxin linkers, overcoming the limitations of traditional ultraviolet spectroscopy methods 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 (such as hydrolysis) during the conjugation process, and toxin linkers in which the ultraviolet absorption spectrum of the side reaction products does not interfere with the ultraviolet absorption spectrum of the conjugated toxin linker. Under normal circumstances, the structure of the toxin linker is stable, and there are generally no obvious side reactions during the conjugation reaction. That is, the above method can be applied 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 the activated antibody solution.

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

[0028] In a preferred embodiment, the conjugation reaction includes lysine conjugation, glycosylation site conjugation, or cysteine conjugation. Correspondingly, the activation corresponding to cysteine conjugation includes reducing the disulfide bond in the antibody to a free thiol group.

[0029] In step b) of the method for measuring the extinction coefficient described above, according to the type of conjugation reaction, it may be necessary to activate the antibody solution. For example, for cysteine conjugation, the disulfide bond in the antibody needs to be reduced to a free thiol group that can undergo a conjugation reaction first. Those skilled in the art can flexibly select an appropriate activation reaction according to the selected conjugation reaction and the antibody.

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

[0031] In the method of the present application, for the selection of wavelength 1 and wavelength 2, those skilled in the art can flexibly select the compounds corresponding to wavelength 1 and wavelength 2. It includes: wavelength 1 is the wavelength of the antibody, and wavelength 2 is the wavelength of the toxin linker; or wavelength 1 is the wavelength of the toxin linker, and wavelength 2 is the wavelength of the antibody. The corresponding relationship between the wavelength and different types of compounds does not affect the calculation results and detection accuracy in 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 ≥ 30 nm, more preferably ≥ 40 nm.

[0033] Preferably, wavelength 1 and wavelength 2 are selected from the wavelengths at which the antibody-toxin linker can produce characteristic absorption in the wavelength range of 240 nm to 400 nm (i.e., characteristic absorption wavelengths), and the characteristic absorption wavelengths are within ±20 nm of the maximum absorption wavelength of the compound within 240 nm to 400 nm. For example, if the maximum absorption wavelength of a certain antibody in 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 ≥ 30 nm, which can ensure that parameters such as the extinction coefficient measured in actual detection have sufficient discrimination, thereby ensuring the accuracy of the detection of the drug-to-antibody conjugate ratio in the antibody-drug conjugate by this method.

[0035] In the second typical embodiment of the present application, a method for detecting the drug-to-antibody conjugate ratio in an antibody-drug conjugate is provided. The above method includes: i) Using the above method for measuring the extinction coefficient of the toxin linker in the antibody-drug conjugate, measure the extinction coefficients of the conjugated toxin linker at wavelength 1 and wavelength 2, respectively represented by and ; ii) Measure the extinction coefficients of the antibody at wavelength 1 and wavelength 2, respectively represented by and ; iii) Using a liquid chromatography or electrophoresis separation technique capable of separating the antibody-drug conjugate, free toxin linker, and impurities, measure the ratio of the liquid chromatography or electrophoresis detector response signals of the antibody-drug conjugate at wavelength 1 and wavelength 2, and the ratio is represented by k; iv) Using each measured value, including the ratio of the response signals, the extinction coefficient in i), and the extinction coefficient in ii), calculate to obtain the drug-to-antibody conjugate ratio.

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

[0037] It should be noted that in the present application, for the sake of simplicity in the expression of relevant parameters, some parameters are represented in the form of abbreviations or letters, such as , , , , k, etc. Such a simple expression is not a limitation of the actual range. Through the above description, what the present application hopes to emphasize is that the use of abbreviations and letter symbols should not be regarded as a limitation of the implementation scope of the technical solution, but a way of expression aiming to make the technical disclosure more refined and easy to understand.

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

[0039] Calculated through the Lambert-Beer law, the formula for calculating the above DAR value is derived as follows:

[0040] ,

[0041] ,

[0042] ,

[0043] ,

[0044] ,

[0045] Convert the above formula to obtain the calculation formula for DAR.

[0046] .

[0047] Where:

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

[0049] A w1*ADC , A w2*ADC represent the absorbance of the ADC at wavelengths 1 and 2,

[0050] , represent the molar extinction coefficients of the antibody in the ADC at wavelengths 1 and 2,

[0051] , represent the molar extinction coefficients of the toxin linker in the ADC at wavelengths 1 and 2,

[0052] C mAb represents 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 the optical path,

[0055] k represents the ratio of the response signals at two wavelengths.

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

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

[0058] In the above method for measuring the ratio, there is no need to introduce strict absolute quantitative analysis in the liquid phase method or capillary electrophoresis method, and there is no need to prepare a standard curve. The measurement of the ratio k can be achieved through the simple ratio of detector signal intensities.

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

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

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

[0062] The basic principle of the denaturation treatment method is to use reagents such as guanidine hydrochloride to denature the antibody, open the folded higher-order structure of the antibody, regard it as a ternary system of tryptophan, tyrosine, and "S-S" bonds, and calculate the extinction coefficient of the antibody by detecting the absorbance values of the folded and unfolded antibodies at a wavelength of 280 nm in the ultraviolet at the same concentration.

[0063] In a preferred embodiment, an analytical technique that can effectively separate the antibody-drug conjugate from spectral interference impurities, such as suitable for 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 in conjunction with specific embodiments.

[0065] Example 1

[0066] The ADC platform molecule (antibody-conjugated camptothecin ADC molecule) of Shanghai ChemPartner Biotechnology Co., Ltd. was used for 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 (such as Figure 1As shown, it exhibits obvious fine structure in the wavelength range of 300 nm to 400 nm. After the toxin linker is conjugated to the antibody, its fine structure in the wavelength range of 300 nm to 400 nm becomes unclear (as Figure 2 shown), and the absorption spectrum has a relatively significant change compared with the absorption spectrum before conjugation as Figure 1 shown, resulting in that the extinction coefficient of the toxin linker before conjugation as Figure 1 shown cannot be used to accurately determine the DAR value of the ADC molecule.

[0068] 2) According to the quantitative conjugation reaction method of the present invention, the absorption spectra after the quantitative reaction of the toxin linker with different concentrations and the excessive antibody are obtained. Its characteristic absorption in the wavelength range of 300 nm to 400 nm is completely consistent with that of the ADC molecule (as Figure 3 shown), and the absorbance has a good linear relationship with the concentration of the toxin linker (as Figure 4 shown), indicating that the quantitative conjugation reaction is complete. Calculate the extinction coefficient through the concentration of the toxin linker after conjugation and the absorbance at two wavelengths.

[0069] 3) Use liquid chromatography to separate the ADC and the free toxin linker small molecule, and obtain the ratio of the UV detector signals of the ADC at two wavelengths.

[0070] 4) Substitute the extinction coefficient of the toxin linker after conjugation obtained and the ratio of the detector signals of the ADC at two wavelengths into the formula for calculation according to the method described in the present invention. The measured DAR value of the ADC sample is 7.99, which is consistent with the target value of 8 of this ADC product.

[0071] Comparative Example 1

[0072] According to the traditional method, use the unconjugated toxin linker (i.e., the toxin linker before conjugation) to measure the extinction coefficient, and calculate the DAR value with this extinction coefficient. The steps are as follows:

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

[0074] 2) Use DMSO solvent as the reference solution for UV spectrum scanning to calibrate the baseline;

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

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

[0077] 5) Substitute the extinction coefficients of the toxin linker and the antibody, as well as the absorbance of the ADC, into the calculation formula. The calculated DAR value is about 6.23, which is significantly lower than the actual value of 8, showing 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, through a unique quantitative conjugation reaction and a method of eliminating impurity interference by chromatography, it is possible to effectively eliminate the influence of the change in the extinction coefficient after conjugation of the toxin linker and the interference of the free toxin linker. This method overcomes the problem of poor accuracy in measuring the DAR value by the traditional UV method when the structure of the toxin linker changes. Especially for those toxin linkers whose structure or microenvironment conditions change significantly after conjugation, it can provide a more real and accurate extinction coefficient measurement value, thereby improving the reliability of DAR value measurement. And by directly measuring the extinction coefficient of the toxin linker after conjugation, there is no need to rely on the data before conjugation, 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 relatively simple operation, can quickly adapt to the early stage of ADC conjugation process development, provides a real-time and effective means for monitoring the DAR value for R & D personnel, and accelerates the development process of ADC products.

[0079] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope 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 includes: a) Dissolve the toxin linker in a solvent to prepare toxin linker solutions with different known concentrations; b) Take a certain volume of solution from the toxin linker solutions with different concentrations and the solvent used to dissolve the toxin linker respectively, mix them with an excessive antibody solution according to the same volume ratio, and make up the volume to correspondingly obtain toxin linker reaction solutions with different known concentrations and an antibody negative control solution; c) Shake the toxin linker reaction solutions and the antibody negative control solution in the dark to ensure complete reaction between the toxin linker and the antibody; d) Use the antibody negative control solution as a reference solution for ultraviolet spectrum scanning to calibrate the baseline, and scan the absorption spectra of the toxin linker reaction solutions with different concentrations; e) Draw a linear curve of the absorbance of the toxin linker and the concentration of the toxin linker at wavelength 1 and wavelength 2, perform linear fitting to obtain a linear equation, and the slope of the linear equation is the extinction coefficient of the conjugated toxin linker; wavelength 1 and wavelength 2 are respectively the characteristic absorption wavelengths of the antibody and the toxin linker between 240 nm and 400 nm.

2. The method according to claim 1, wherein The antibody solution includes an activated antibody solution.

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

4. The method according to claim 3, wherein The conjugation reaction includes lysine conjugation, glycosylation site conjugation or cysteine conjugation.

5. A method for detecting the drug-antibody conjugate ratio in an antibody-drug conjugate, characterized in that, The method includes: i) Use the method for determining the extinction coefficient of the toxin linker in the antibody-drug conjugate according to any one of claims 1-4 to determine the extinction coefficient of the conjugated toxin linker at wavelength 1 and wavelength 2; ii) Determine the extinction coefficient of the antibody at wavelength 1 and wavelength 2; iii) Use 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 and wavelength 2; iv) Calculate the drug-antibody conjugation ratio using the ratio of the response signals, the extinction coefficient in i) and the extinction coefficient in ii); wherein, wavelength 1 and wavelength 2 are respectively the characteristic absorption wavelengths of the antibody and the toxin linker between 240 nm and 400 nm.

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