Method for detecting average coupling rate of antibody-coupled drug based on small molecule extinction coefficient correction and application of method

Abstract

The invention provides a method for detecting the average coupling rate of an antibody-coupled drug based on small molecule extinction coefficient correction and application of the method. The method comprises the following steps: firstly, measuring the absorbance and extinction coefficient of L-cysteine-linker-toxin molecules at 280nm, and then calculating and correcting to obtain the accurate average coupling rate of the antibody coupling medicine based on the measured absorbance and extinction coefficient and liquid chromatogram conditions. According to the method, in the analysis process, through the extinction coefficient of L-cysteine-connexon-toxin molecules at the wavelength of 280 nm, the area of a liquid chromatogram peak which is collected at the wavelength of 280 nm and is reduced by ADC is corrected, so that the measured average coupling rate result is more accurate and real. Besides, the method is suitable for the antibody-coupled drug obtained in a cysteine coupling mode, and is especially suitable for the antibody-coupled drug which is strong in coupled micromolecule ultraviolet absorption and has obvious influence on a DAR result.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis, and specifically relates to a detection method for average coupling rate of antibody-drug coupling based on small molecule extinction coefficient correction and application thereof. Background Art

[0002] Antibody Drug Conjugate (ADC) is an innovative biotherapeutic drug formed by coupling antibodies or antibody fragments targeting specific antigens with cytotoxic small molecules through linkers. Therefore, ADC has both the high targeting of antibody drugs and the high activity of CLP small molecule drugs to reduce systemic toxicity and more selectively deliver the payload to tumor cells, tumor microenvironment or other target cells. Therefore, antibody drug conjugates have shown excellent efficacy and great potential in the field of tumor treatment, becoming a hot spot in drug research and development.

[0003] The drug-to-antibody ratio (DAR) represents the average value of the effective load coupled to each antibody molecule. It is a key quality attribute of antibody-drug conjugates and directly affects the safety and effectiveness of the product. The appropriate analytical method is selected according to the chemical properties of the linker and the load, the coupling method with the antibody, and the heterogeneity of DAR. Currently, reversed phase liquid chromatography (RP-LC) is a common analytical method for determining the average coupling ratio.

[0004] However, the existing RP-LC method does not take into account the influence of the absorbance contribution of small molecules on the determination of DAR values. As the types of small molecules used for conjugation gradually increase, studies have found that some small molecules have a large absorption at the maximum absorption wavelength of antibodies at 280nm. Ignoring the absorption of small molecules at 280nm will affect the accuracy of the DAR value. Therefore, it is necessary to develop a DAR value analysis method that can detect antibody-drug conjugates. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a detection method for the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction and its application. The present invention provides a more accurate detection method for the average coupling rate of antibody-drug conjugates based on CLP extinction coefficient correction, and calculates and corrects the average coupling rate of antibody-drug conjugates. And the small molecule used for extinction coefficient correction is CLP. By comparing with the mass spectrometry (MS) results, the DAR value obtained by reverse phase chromatography after correction is consistent with the DAR value obtained by mass spectrometry, and the DAR value obtained by reverse phase chromatography without correction is quite different from the DAR value obtained by mass spectrometry. The present invention improves the accuracy of the extinction coefficient of small molecules and the average DAR value of antibody-drug conjugates tested by reverse phase liquid chromatography, which is of great significance for the production and quality control of antibody-drug conjugates.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting the average coupling rate of an antibody-drug conjugate based on small molecule extinction coefficient correction, the method comprising:

[0008] (S1) using reverse phase liquid chromatography to perform chromatographic detection on the antibody-drug conjugate and CLP small molecule to be tested; the structure of the antibody-drug conjugate is antibody-linker-toxin molecule, and the structure of the CLP small molecule is L-cysteine-linker-toxin molecule;

[0009] (S2) Calculating the molar extinction coefficient of the CLP small molecule based on the detection results, and correcting the peak area percentage of the liquid chromatography peak according to the molar extinction coefficients of the antibody light chain, the antibody heavy chain and the CLP small molecule; the correction is performed using the following formula:

[0010] ε LCn =ε LC0 +n×ε CLP

[0011] ε HCn =ε HC0 +n×ε CLP

[0012] Corrected LCn%=LCn%×ε LC0 / ε LCn

[0013] Corrected HCn%=HCn%×ε HC0 / ε HCn

[0014] Among them, ε LCn represents the molar extinction coefficient of the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules; ε HCn represents the molar extinction coefficient of the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules; ε LC0 represents the molar extinction coefficient of antibody light chain; ε HC0 represents the molar extinction coefficient of antibody heavy chain; ε CLPIndicates the molar extinction coefficient of the CLP small molecule; LCn% indicates the percentage of the liquid chromatography peak area of ​​the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules; HCn% indicates the percentage of the liquid chromatography peak area of ​​the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules; CorrectedLCn% indicates the percentage of the corrected liquid chromatography peak area of ​​the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules; CorrectedHCn% indicates the percentage of the corrected liquid chromatography peak area of ​​the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules;

[0015] (S3) Calculate the average antibody-drug conjugate rate based on the corrected liquid chromatography peak area percentage.

[0016] The existing reversed-phase liquid chromatography method separates the substances to be tested based on their hydrophobicity, obtains light chains and heavy chains by reducing antibody-coupled drugs, and calculates the weighted average DAR value by calculating the peak area percentage of each light and heavy chain and the number of small molecules coupled to each peak. However, the existing method does not take into account the contribution of CLP to the peak area at the maximum absorption wavelength of the protein at 280 nm, which will affect the accuracy of the calculated DAR value.

[0017] The present invention develops a DAR value analysis method capable of detecting antibody-drug conjugates, and on the basis of the method, an accurate DAR value is obtained by a small molecule extinction coefficient correction method, thereby eliminating the influence of the ultraviolet absorption of the small molecule on the DAR determination; at the same time, for cysteine-conjugated ADCs, the small molecule used for extinction coefficient correction is L-cysteine-linker-toxin molecule (CLP) rather than linker-toxin molecule (LP), and the ultraviolet absorption of LP conjugated to the antibody is fully simulated by CLP.

[0018] Preferably, in step (S3), the average coupling rate of the antibody-drug conjugate is calculated using the following formula:

[0019] Corrected DAR LC =2×Σn×Corrected LCn% / ΣCorrected LCn%

[0020] Corrected DAR HC =2×Σn×Corrected HCn% / ∑Corrected HCn%

[0021] Corrected DAR=Corrected DAR LC +Corrected DAR HC

[0022] Among them, Corrected DARLC It represents the average coupling rate of antibody-drug conjugated light chain after correction;

[0023] Corrected DAR HC represents the average coupling rate of antibody-drug conjugate heavy chain after correction;

[0024] Corrected DAR represents the corrected average coupling rate.

[0025] Preferably, in step (S1), the chromatographic detection is performed by gradient elution.

[0026] Preferably, the mobile phase used for elution includes mobile phase A and mobile phase B, the mobile phase A is a 0.05%-0.1% trifluoroacetic acid aqueous solution, for example, it can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%, and the mobile phase B is a 0.05%-0.1% trifluoroacetic acid acetonitrile solution, for example, it can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%, etc.

[0027] Preferably, the gradient elution procedure comprises:

[0028] In the first 0-2 minutes, the volume fraction of mobile phase B was 30.0-31.0, and the rest was mobile phase A;

[0029] From 2 to 35 minutes, the volume fraction of mobile phase B changed at a constant rate from 30.0-31.0 to 46.5-47.0, and the rest was mobile phase A;

[0030] At 35-35.1 min, the volume fraction of mobile phase B changed from 46.5-47.0 to 90.0-91.0 at a constant rate, and the rest was mobile phase A;

[0031] From 35.1 to 40 min, the volume fraction of mobile phase B was 90.0-91.0, and the rest was mobile phase A;

[0032] At 40-40.1 min, the volume fraction of mobile phase B changed from 90.0-91.0 to 30.0-31.0 at a constant rate, and the rest was mobile phase A;

[0033] From 40.1 to 42 minutes, the volume fraction of mobile phase B was 30.0-31.0, and the rest was mobile phase A.

[0034] As a preferred embodiment of the present invention, the gradient elution procedure comprises:

[0035]

[0036]

[0037] Preferably, the detection wavelength of the chromatographic detection is λ=280 nm.

[0038] Preferably, the flow rate of the mobile phase is 0.2-0.5 mL / min, for example, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min.

[0039] Preferably, the chromatographic column for chromatographic detection is PLRP-S, 8μm, 2.1×150mm;

[0040] Preferably, the column temperature of the chromatographic column is 65-75°C, for example, 65°C, 70°C or 75°C.

[0041] Preferably, in step (S1), the CLP small molecule is prepared by a method comprising the following steps: oscillating the linker-toxin molecule with L-cysteine ​​to obtain the CLP small molecule.

[0042] In the present invention, the molar ratio of L-cysteine ​​to linker-toxin molecule is (9-10):1.

[0043] Preferably, the rotation speed of the oscillating reaction is 1000-2000 rpm, for example, it can be 1000 rpm, 1500 rpm or 2000 rpm.

[0044] Preferably, the oscillation reaction time is 25-35 min, for example, 25 min, 30 min or 35 min.

[0045] Preferably, the temperature of the oscillating reaction is 20-30°C, for example, 20°C, 25°C or 30°C.

[0046] Preferably, in step (S1), the antibody-drug conjugate to be tested further includes a pretreatment step before chromatographic detection; the pretreatment includes: sequentially mixing a denaturant, a buffer and a reducing agent with the sample and incubating them, and performing chromatographic detection on the incubated sample.

[0047] Preferably, the denaturant is guanidine hydrochloride.

[0048] Preferably, the final concentration of the denaturant in the mixed system is 7-10 mol / L, for example, 7 mol / L, 8 mol / L, 9 mol / L or 10 mol / L.

[0049] Preferably, the buffer is Tris-HCl.

[0050] Preferably, the final concentration of the buffer in the mixed system is 0.5-2 mol / L, for example, 0.5 mol / L, 1 mol / L, 1.5 mol / L or 2 mol / L.

[0051] Preferably, the reducing agent is dithiothreitol.

[0052] Preferably, the final concentration of the reducing agent in the mixed system is 0.5-2 mol / L, for example, 0.5 mol / L, 1 mol / L, 1.5 mol / L or 2 mol / L.

[0053] Preferably, the incubation temperature is 25-30°C, for example, 25°C, 28°C or 30°C.

[0054] Preferably, the incubation time is 25-30 min, for example, 25 min, 28 min or 30 min.

[0055] Preferably, the molar extinction coefficient of the CLP small molecule is calculated using a method comprising the following steps:

[0056] The CLP small molecule test solution was prepared using solvents of different concentrations, and the ultraviolet absorbance of the CLP small molecule at 280 nm was measured. The measured absorbance was fitted with the corresponding solvent concentration value to obtain a fitting equation. The intermediate concentration between the solvent concentration corresponding to the beginning of the first component peak and the solvent concentration corresponding to the end of the last component peak in the chromatogram was selected, and the intermediate concentration was substituted into the above fitting equation to obtain the absorbance of the small molecule, and the mass extinction coefficient of the small molecule was obtained according to the Lambert-Beer law.

[0057] Preferably, the solvent is a mixture of 0.05%-0.1% trifluoroacetic acid acetonitrile solution and 0.05%-0.1% trifluoroacetic acid aqueous solution.

[0058] Preferably, the volume ratio of the trifluoroacetic acid-acetonitrile solution in the mixed solution is 30%-50%, for example, it can be 30%, 35%, 40%, 45% or 50%.

[0059] In a second aspect, the present invention provides an application of the method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction described in the first aspect in detecting the average coupling rate of antibody-drug conjugates.

[0060] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] The average coupling rate of the antibody-drug conjugate of the present invention is corrected by the extinction coefficient of the small molecule for the peak area of ​​the small molecule at the protein absorption wavelength of 280nm, excluding the liquid chromatography peak area contributed by the small molecule; at the same time, the small molecule used to determine the extinction coefficient is CLP, which simulates the ultraviolet absorption of LP coupled to the antibody. This method makes the average coupling rate of the determination have a higher accuracy. The method is suitable for antibody-drug conjugates obtained by cysteine ​​coupling, and is particularly suitable for the determination of the average coupling rate of antibody-drug conjugates when the toxin molecule or linker has a large absorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 It is a schematic diagram of the detection method of the average coupling rate of antibody-drug conjugates.

[0064] Figure 2 It is a liquid chromatogram of the antibody-drug conjugate test sample obtained by the detection method provided by the present invention in Example 1.

[0065] Figure 3 It is a linear curve of MPB%-CLP average absorbance obtained by the detection method provided by the present invention.

[0066] Figure 4 It is a liquid chromatogram of the antibody-drug conjugate test sample obtained by the detection method provided by the present invention in Example 2. DETAILED DESCRIPTION

[0067] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0068] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0069] The present invention provides a method for determining the average coupling ratio in an antibody-drug conjugate, such as Figure 1 As shown, the absorbance and extinction coefficient of the CLP used to form the antibody-drug conjugate at 280nm are determined, and the average coupling rate of the antibody-drug conjugate is calculated and corrected by combining the extinction coefficients of the antibody light chain and heavy chain at 280nm, and the liquid chromatography gradient conditions for detecting the average coupling rate. Among them, the peak area of ​​the light chain and heavy chain of the antibody-drug conjugate coupled with different numbers of small molecules on the liquid chromatography after reduction is obtained by correcting the extinction coefficient of the small molecule, excluding the peak area contributed by the small molecules on the light and heavy chains at 280nm.

[0070] Example 1

[0071] This example uses the antibody-drug conjugate average coupling rate detection method based on small molecule extinction coefficient correction described in the present invention to analyze the average coupling rate of a certain antibody-drug conjugate test sample, and the antibody-drug conjugate coupling method is cysteine ​​coupling. The antibody type used in the present invention is IgG. Specifically comprising the following steps:

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

[0073] Add 40 μg of protein sample to a 1.5 mL centrifuge tube, and add 75 μL of guanidine hydrochloride solution, 5 μL of Tris-HCl buffer, and 2 μL of 1 mol / L dithiothreitol solution in sequence. Make up to a final volume of 100 μL with ultrapure water and mix well. Incubate at 30°C for 30 min. Transfer the obtained sample to a glass insert vial for detection. Table 1 shows the chromatographic conditions for detecting the average coupling rate of antibody-drug conjugates.

[0074] Table 1

[0075]

[0076]

[0077] Figure 2 is the liquid chromatogram of the antibody-drug conjugate test sample obtained through the above pretreatment and chromatographic separation, Figure 2 In the table, LC0 represents the chromatographic peak corresponding to the antibody light chain, HC0 represents the chromatographic peak corresponding to the antibody heavy chain, LC1 represents the chromatographic peak of the light chain coupled to one linker-toxin molecule in the antibody-drug conjugate test sample, LC2 represents the chromatographic peak of the light chain coupled to two linker-toxin molecules in the antibody-drug conjugate test sample, HC1 represents the chromatographic peak of the heavy chain coupled to one linker-toxin molecule in the antibody-drug conjugate test sample, HC2 represents the chromatographic peak of the heavy chain coupled to two linker-toxin molecules in the antibody-drug conjugate test sample, HC3 represents the chromatographic peak of the heavy chain coupled to three linker-toxin molecules in the antibody-drug conjugate test sample, and HC4 represents the chromatographic peak of the heavy chain coupled to four linker-toxin molecules in the antibody-drug conjugate test sample. The identification of each chromatographic peak was confirmed by mass spectrometry.

[0078] (2) Preparation of linker-toxin molecule stock solution:

[0079] Accurately weigh 11.9 mg of LP (linker-toxin molecule) and dilute it to 1.0 mg / mL LP stock solution with an appropriate amount of DMA (dimethylacetamide).

[0080] (3) Preparation of CLP stock solution:

[0081] Prepare diluents with different proportions of mobile phase B: take 300 μL, 350 μL, 400 μL, 450 μL, and 500 μL of mobile phase B respectively, add ultrapure water to make up each diluent to 1 mL; take 20 μL of 1.0 mg / mL LP stock solution, mix it with 10 μL of 100 mM L-cysteine, incubate at 1500 rpm for 30 minutes at 25°C, prepare 7 replicate samples, add 598 μL of the above-mentioned solvents with different proportions of mobile phase B respectively, dilute CLP to 35 μg / mL, and make the absorbance of the diluted sample between 0.2-0.8.

[0082] The absorbance of the above five sample solutions at 280 nm was measured using a Cary 3500 UV-Vis spectrophotometer. As shown in Table 2, each sample was measured three times to calculate the average absorbance. The results are shown in Table 2.

[0083] Table 2

[0084]

[0085] With MPB% in solvent as horizontal axis and average absorbance of CLP as vertical axis, a curve was drawn and fitted. The linear equation is y=0.1052x+0.5173, and the spectrum is shown in Figure 3 .

[0086] (4) Result analysis:

[0087] Figure 2 The retention times of LC0, LC1, and HC3 of the antibody-drug conjugate test product are 15.7 min, 20.0 min, and 24.3 min, respectively. The component with the midpoint retention time is LC1 (HC4 accounts for a small proportion, and HC4 is not considered when selecting the component in the middle of the retention time). According to the elution gradient of the liquid chromatography method, the proportion of mobile phase B corresponding to LC1 elution is 39%.

[0088] Depend on Figure 3 The fitting curve and linear equation show that the absorbance corresponding to the proportion of 39% mobile phase B is 0.558. According to the Lambert-Beer law, the mass extinction coefficient of CLP is 15.95 (mg / mL) -1 cm -1 , and then combined with the molecular weight of CLP to obtain its molar extinction coefficient 21440.70 (mol / L) -1 cm -1 .

[0089] In this example, the antibody type used is IgG, the coupling method of the antibody-drug conjugate is cysteine ​​coupling, and the calculation formula of the average coupling rate and the corrected average coupling rate of the antibody-drug conjugate test sample is:

[0090] DAR=DAR LC +DAR HC

[0091] Corrected DAR=Corrected DAR LC +Corrected DAR HC

[0092] The average coupling rate of the antibody-drug conjugate test product was calculated to be 4.0, and the average coupling rate after correction was 3.3, where:

[0093] ε LCn =ε LC0 +n×ε CLP

[0094] ε HCn =ε HC0 +n×ε CLP

[0095] Corrected LCn%=LCn%×ε LC0 / ε LCn

[0096] Corrected HCn%=HCn%×ε HC0 / ε HCn

[0097] Corrected DAR LC =2×∑n×Corrected LCn% / Corrected LCn%

[0098] Corrected DAR HC =2×∑n×Corrected HCn% / ∑Corrected HCn%

[0099] In the above formula:

[0100] ε LC0 represents the molar extinction coefficient of antibody light chain;

[0101] ε LCn It represents the molar extinction coefficient of the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules, which is calculated by combining the measured molar extinction coefficient of CLP with the molar extinction coefficient of the antibody light chain and the number of coupled small molecules;

[0102] ε HC0 represents the molar extinction coefficient of antibody heavy chain;

[0103] ε HCnrepresents the molar extinction coefficient of the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules, which is calculated by combining the measured molar extinction coefficient of CLP with the molar extinction coefficient of the antibody heavy chain and the number of coupled small molecules;

[0104] Corrected LCn% represents the corrected LC peak area percentage of the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules;

[0105] Corrected HCn% represents the corrected LC peak area percentage of the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules;

[0106] Corrected DAR LC It represents the average coupling rate of antibody-drug conjugated light chain after correction;

[0107] Corrected DAR HC represents the average coupling rate of antibody-drug conjugate heavy chain after correction;

[0108] The peak area of ​​the liquid chromatography peak is collected at a wavelength of 280 nm.

[0109] The peak area of ​​each chromatographic peak at 280 nm obtained by liquid phase, the molar extinction coefficients of the light chain and the heavy chain, and the corrected peak area of ​​each chromatographic peak are shown in Table 3.

[0110] Table 3

[0111]

[0112] The corrected average coupling rates of the antibody-drug conjugate light chain and heavy chain are shown in Table 4.

[0113] Table 4

[0114]

[0115] The corrected mean conjugation ratio of the available ADCs was 3.3, and the uncorrected mean conjugation ratio of the ADCs was 4.0.

[0116] The DAR value of the antibody-drug conjugate measured by mass spectrometry was 3.3. It can be seen that the average conjugation rate of 3.3 obtained by CLP extinction coefficient correction is closer to the actual DAR of the drug.

[0117] Example 2

[0118] This example uses the antibody-drug conjugate average coupling rate detection method based on small molecule extinction coefficient correction described in the present invention to analyze the average coupling rate of a certain antibody-drug conjugate test sample, and the antibody-drug conjugate coupling method is cysteine ​​coupling. The antibody type used in the present invention is IgG. Specifically comprising the following steps:

[0119] (1) Pretreatment method for detecting the average coupling rate of antibody-drug conjugates:

[0120] Add 40 μg of protein sample to a 1.5 mL centrifuge tube, and add 75 μL of guanidine hydrochloride solution, 5 μL of Tris-HCl buffer, and 2 μL of 1 mol / L dithiothreitol solution in sequence. Make up to a final volume of 100 μL with ultrapure water and mix well. Incubate at 30°C for 30 min. Transfer the obtained sample to a glass insert vial for detection. Table 5 shows the chromatographic conditions for detecting the average coupling rate of antibody-drug conjugates.

[0121] Table 5

[0122]

[0123] Figure 4 is the liquid chromatogram of the antibody-drug conjugate test sample obtained through the above pretreatment and chromatographic separation, Figure 4 In the table, LC0 represents the chromatographic peak corresponding to the antibody light chain, HC0 represents the chromatographic peak corresponding to the antibody heavy chain, LC1 represents the chromatographic peak of the light chain coupled to one linker-toxin molecule in the antibody-drug conjugate test sample, HC1 represents the chromatographic peak of the heavy chain coupled to one linker-toxin molecule in the antibody-drug conjugate test sample, HC2 represents the chromatographic peak of the heavy chain coupled to two linker-toxin molecules in the antibody-drug conjugate test sample, HC3 represents the chromatographic peak of the heavy chain coupled to three linker-toxin molecules in the antibody-drug conjugate test sample, and HC4 represents the chromatographic peak of the heavy chain coupled to four linker-toxin molecules in the antibody-drug conjugate test sample. The identification of each chromatographic peak was confirmed by mass spectrometry.

[0124] (2) Preparation of linker-toxin molecule stock solution:

[0125] Accurately weigh 11.9 mg of LP and dilute it with an appropriate amount of DMA (dimethylacetamide) to 1.0 mg / mL LP stock solution.

[0126] (3) Preparation of CLP stock solution:

[0127] Prepare diluents with different proportions of mobile phase B: take 300 μL, 350 μL, 400 μL, 450 μL, and 500 μL of mobile phase B respectively, add ultrapure water to make up each diluent to 1 mL; take 20 μL of 1.0 mg / mL LP stock solution, mix it with 10 μL of 100 mM L-cysteine, incubate at 1500 rpm for 30 minutes at 25°C, prepare 7 replicate samples, add 598 μL of the above-mentioned solvents with different proportions of mobile phase B respectively, dilute CLP to 35 μg / mL, and make the absorbance of the diluted sample between 0.2-0.8.

[0128] The absorbance of the above five sample solutions at 280 nm was measured using a Cary 3500 UV-Vis spectrophotometer. As shown in Table 6, each sample was measured three times to calculate the average absorbance. The results are shown in Table 6.

[0129] Table 6

[0130]

[0131]

[0132] With MPB% in solvent as horizontal axis and average absorbance of CLP as vertical axis, a curve was drawn and fitted. The linear equation is y=0.1052x+0.5173, and the spectrum is shown in Figure 3 .

[0133] (4) Result analysis:

[0134] Figure 4 The retention times of LC0, HC0, and HC3 of the antibody-drug conjugate test product are 15.9 min, 22.0 min, and 28.0 min, respectively. The component with the midpoint retention time is HC0 (HC4 accounts for a small proportion, and HC4 is not considered when selecting the component in the middle of the retention time). According to the elution gradient of the liquid chromatography method, the proportion of mobile phase B corresponding to HC0 elution is 40%.

[0135] Depend on Figure 3 The fitting curve and linear equation show that the absorbance corresponding to the proportion of 40% mobile phase B is 0.559. According to the Lambert-Beer law, the mass extinction coefficient of CLP is 15.98 (mg / mL) -1 cm -1 , and then combined with the molecular weight of CLP to obtain its molar extinction coefficient 21479.68 (mol / L) -1 cm -1 .

[0136] In this example, the antibody type used is IgG, the coupling method of the antibody-drug conjugate is cysteine ​​coupling, and the calculation formula of the average coupling rate and the corrected average coupling rate of the antibody-drug conjugate test sample is:

[0137] DAR=DAR LC +DAR HC

[0138] Corrected DAR=Corrected DAR LC +Corrected DAR HC

[0139] The average coupling rate of the antibody-drug conjugate test product was calculated to be 3.5, and the average coupling rate after correction was 2.9, where:

[0140] ε LCn =ε LC0 +n×ε CLP

[0141] ε HCn =ε HC0 +n×ε CLP

[0142] Corrected LCn%=LCn%×ε LC0 / ε LCn

[0143] Corrected HCn%=HCn%×ε HC0 / ε HCn

[0144] Corrected DAR LC =2×Σn×Corrected LCn% / ΣCorrected LCn%

[0145] Corrected DAR HC =2×Σn×Corrected HCn% / ΣCorrected HCn%

[0146] In the above formula:

[0147] ε LC0 represents the molar extinction coefficient of antibody light chain;

[0148] ε LCn It represents the molar extinction coefficient of the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules, which is calculated by combining the measured molar extinction coefficient of CLP with the molar extinction coefficient of the antibody light chain and the number of coupled small molecules;

[0149] ε HC0represents the molar extinction coefficient of antibody heavy chain;

[0150] ε HCn represents the molar extinction coefficient of the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules, which is calculated by combining the measured molar extinction coefficient of CLP with the molar extinction coefficient of the antibody heavy chain and the number of coupled small molecules;

[0151] Corrected LCn% represents the corrected LC peak area percentage of the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules;

[0152] Corrected HCn% represents the corrected LC peak area percentage of the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules;

[0153] Corrected DAR LC It represents the average coupling rate of antibody-drug conjugated light chain after correction;

[0154] Corrected DAR HC represents the average coupling rate of antibody-drug conjugate heavy chain after correction;

[0155] The peak area of ​​the liquid chromatography peak is collected at a wavelength of 280 nm.

[0156] The peak area of ​​each chromatographic peak at 280 nm obtained by liquid phase, the molar extinction coefficients of the light chain and the heavy chain, and the corrected peak area of ​​each chromatographic peak are shown in Table 7.

[0157] Table 7

[0158]

[0159] The corrected average coupling rates of the antibody-drug conjugate light chain and heavy chain are shown in Table 5.

[0160] Table 5

[0161]

[0162] The average corrected ADC conjugate ratio was 2.9, and the uncorrected average ADC conjugate ratio was 3.5.

[0163] In summary, the present invention provides a method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction, which improves the accuracy of the small molecule extinction coefficient and the average DAR value of antibody-drug conjugates tested by reversed-phase liquid chromatography, and is of great significance for the production and quality control of antibody-drug conjugates.

[0164] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction, characterized in that: The method comprises: (S1) using reverse phase liquid chromatography to perform chromatographic detection on the antibody-drug conjugate and CLP small molecule to be tested; the structure of the antibody-drug conjugate is antibody-linker-toxin molecule, and the structure of the CLP small molecule is L-cysteine-linker-toxin molecule; (S2) Calculating the molar extinction coefficient of the CLP small molecule based on the detection results, and correcting the peak area percentage of the liquid chromatography peak according to the molar extinction coefficients of the antibody light chain, the antibody heavy chain and the CLP small molecule; the correction is performed using the following formula: e LCn =e LC0 +n×e CLP e HCn =e HC0 +n×e CLP Corrected LCn%=LCn%×εL C0 / ε LCn Correctedd HCn%=HCn%×ε HC0 / ε HCn Among them, ε LC n represents the molar extinction coefficient of the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules; ε HC n represents the molar extinction coefficient of the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules; ε LC0 represents the molar extinction coefficient of antibody light chain; ε HC0 represents the molar extinction coefficient of antibody heavy chain; ε CLP Indicates the molar extinction coefficient of the CLP small molecule; LCn% indicates the liquid chromatography peak area percentage of the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules; HCn% indicates the liquid chromatography peak area percentage of the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules; CorrectedLCn% indicates the corrected liquid chromatography peak area percentage of the light chain of the antibody-drug conjugate coupled to n linker-toxin molecules; Corrected HCn% indicates the corrected liquid chromatography peak area percentage of the heavy chain of the antibody-drug conjugate coupled to n linker-toxin molecules; (S3) Calculate the average antibody-drug conjugate rate based on the corrected liquid chromatography peak area percentage.

2. The method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to claim 1, characterized in that: In step (S3), the average coupling rate of the antibody-drug conjugate is calculated using the following formula: Corrected DAR LC =2×Σn×Corrected LCn% / ΣCorrected LCn% Corrected DAR HC =2×Σn×Corrected HCn% / ΣCorrected HCn% Corrected DAR=Corrected DAR LC +Corrected DAR HC Among them, Corrected DAR LC It represents the average coupling rate of antibody-drug conjugated light chain after correction; Corrected DAR HC represents the average coupling rate of antibody-drug conjugate heavy chain after correction; Corrected DAR represents the corrected average coupling rate.

3. The method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to claim 1 or 2, characterized in that: In step (S1), the chromatographic detection is performed by gradient elution; Preferably, the mobile phase used for elution includes mobile phase A and mobile phase B, wherein the mobile phase A is a 0.05%-0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is a 0.05%-0.1% trifluoroacetic acid acetonitrile solution.

4. The method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to claim 3, characterized in that: The gradient elution procedure includes: In the first 0-2 minutes, the volume fraction of mobile phase B was 30.0-31.0, and the rest was mobile phase A; From 2 to 35 minutes, the volume fraction of mobile phase B changed at a constant rate from 30.0-31.0 to 46.5-47.0, and the rest was mobile phase A; At 35-35.1 min, the volume fraction of mobile phase B changed from 46.5-47.0 to 90.0-91.0 at a constant rate, and the rest was mobile phase A; From 35.1 to 40 min, the volume fraction of mobile phase B was 90.0-91.0, and the rest was mobile phase A; At 40-40.1 min, the volume fraction of mobile phase B changed from 90.0-91.0 to 30.0-31.0 at a constant rate, and the rest was mobile phase A; From 40.1 to 42 minutes, the volume fraction of mobile phase B was 30.0-31.0, and the rest was mobile phase A.

5. The method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to any one of claims 1 to 4, characterized in that: The detection wavelength of the chromatographic detection is λ=280nm; Preferably, the flow rate of the mobile phase is 0.2-0.5 mL / min; Preferably, the chromatographic column for chromatographic detection is PLRP-S, 8μm, 2.1×150mm; Preferably, the column temperature of the chromatographic column is 65-75°C.

6. The method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to any one of claims 1 to 5, characterized in that: In step (S1), the CLP small molecule is prepared by a method comprising the following steps: oscillating a linker-toxin molecule with L-cysteine ​​to obtain a coupled CLP small molecule; Preferably, the rotation speed of the oscillating reaction is 1000-2000 rpm; Preferably, the oscillation reaction time is 25-35 min; Preferably, the temperature of the shaking reaction is 20-30°C.

7. The method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to any one of claims 1 to 6, characterized in that: In step (S1), the antibody-drug conjugate to be tested further includes a pretreatment step before chromatographic detection; the pretreatment includes: sequentially mixing a denaturant, a buffer and a reducing agent with the sample and incubating them, and performing chromatographic detection on the incubated sample; Preferably, the denaturant is guanidine hydrochloride; Preferably, the final concentration of the denaturant in the mixed system is 7-10 mol / L; Preferably, the buffer is Tris-HCl; Preferably, the final concentration of the buffer in the mixed system is 0.5-2 mol / L; Preferably, the reducing agent is dithiothreitol; Preferably, the final concentration of the reducing agent in the mixed system is 0.5-2 mol / L; Preferably, the incubation temperature is 25-30°C; Preferably, the incubation time is 25-30 min.

8. The method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to any one of claims 1 to 7, characterized in that: The molar extinction coefficient of the CLP small molecule is calculated using a method comprising the following steps: The CLP small molecule test solution was prepared using solvents of different concentrations, and the ultraviolet absorbance of the CLP small molecule at 280 nm was measured. The measured absorbance was fitted with the corresponding solvent concentration value to obtain a fitting equation. The intermediate concentration between the solvent concentration corresponding to the beginning of the first component peak and the solvent concentration corresponding to the end of the last component peak in the chromatogram was selected, and the intermediate concentration was substituted into the above fitting equation to obtain the absorbance of the small molecule, and the mass extinction coefficient of the small molecule was obtained according to the Lambert-Beer law.

9. The method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to claim 8, characterized in that: The solvent is a mixture of 0.05%-0.1% trifluoroacetic acid acetonitrile solution and 0.05%-0.1% trifluoroacetic acid aqueous solution; Preferably, the volume ratio of the trifluoroacetic acid acetonitrile solution in the mixed solution is 30%-50%.

10. Use of the method for detecting the average coupling rate of antibody-drug conjugates based on small molecule extinction coefficient correction according to any one of claims 1 to 9 in detecting the average coupling rate of antibody-drug conjugates.

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