Correction method for DAR value detection of antibody-coupled drug based on dual-wavelength detection and application of correction method

Abstract

The invention belongs to the technical field of drug detection and analysis, and provides a correction method for DAR value detection of an antibody-coupled drug based on dual-wavelength detection and application of the correction method, and the method comprises the following steps: (S1) carrying out reduction treatment on the antibody-coupled drug, and then carrying out detection by adopting reversed-phase chromatography or affinity chromatography, the detection wavelength is set to be 280 nm and the special absorption wavelength of the selected linker-load is a nm, and obtaining an atlas according with the separation degree; respectively obtaining response peak areas under the two detection wavelengths through integration; and (S2) correcting the corresponding response peak area under 280nm through each response peak area under a nm, calculating the percentage of the corrected peak area through the corrected peak area, and calculating the average DAR value according to the contribution of the similar components to the DAR by using the corrected peak area percentage. The correction method is wide in application range and can be applied to DAR value calculation correction of various types of antibody conjugates.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug detection and analysis, and particularly relates to a correction method for DAR value detection of antibody conjugated drugs based on double-wavelength detection and application thereof. BACKGROUND

[0002] The antibody conjugation rate refers to the proportion of antibodies successfully conjugated with drugs or other functional molecules in total antibodies in biological preparations such as antibody-drug conjugates (ADCs). It is usually expressed in the form of percentage or molar ratio.

[0003] Suitable antibody conjugation rate and accurate determination of antibody conjugation rate are crucial for ADC drugs to exert optimal efficacy. Too low antibody conjugation rate means that each antibody carries too few drug molecules, which may not effectively kill target cells and affect treatment effect. Too high antibody conjugation rate may lead to changes in the structure and function of antibodies, affecting their recognition and binding ability to targets, which is also not conducive to the exertion of efficacy. Only when the antibody conjugation rate is controlled within a certain range, can the ADC drug release a sufficient number of drug molecules after reaching the target cells, thereby efficiently exerting the therapeutic effect. Therefore, the antibody conjugation rate is one of the key indicators for measuring the quality of ADC drugs, which can provide an important basis for the design and optimization of ADC drugs and ensure drug safety.

[0004] At present, the determination methods of antibody conjugation rate mainly include reversed-phase chromatography (or affinity chromatography), hydrophobic interaction chromatography, capillary gel electrophoresis and direct UV-Vis method. Among them, the first three methods are most commonly used, which mainly utilize the good separation degree between components conjugated with different numbers of load-linkers in the chromatogram to calculate the average antibody conjugation rate by calculating the percentage of response peak area of each component. However, if the load-linker has absorption contribution at the target detection wavelength (usually 280 nm for liquid chromatography and 220 nm for capillary gel electrophoresis), the proportion of components conjugated with load-linkers (hereinafter referred to as LP) will be too large, resulting in a measured antibody conjugation rate that is too high. Therefore, it is of great significance to investigate whether the measured antibody conjugation rate needs to be corrected and to select an accurate correction method.

[0005] The most intuitive and basic method for correcting the detection result of the antibody conjugation rate is to obtain the extinction coefficient of HC0 or LC0, and the extinction coefficient (hereinafter referred to as EC) of the heavy chain (HC) or light chain (LC) conjugated with different numbers of load-linkers, correct the response peak area, and then correct the antibody conjugation rate calculation. In this process, since it is difficult to directly obtain the EC value of each component after conjugation, the EC value of each structural unit (such as HC0, LC0, and load-linker) of the antibody conjugated drug needs to be obtained, and then the EC value of each component (such as light chain LC conjugated with one load-linker, and heavy chain HC conjugated with one to four load-linkers) conjugated with different numbers of load-linkers is obtained by summation. Due to the large difference in structural properties, the EC values of the antibody structure and the EC values of the load-linker are obtained in completely different ways, thereby affecting the accuracy and rationality of the direct summation calculation to obtain the EC value of the target conjugated component. Among them, the EC value of the antibody single chain can be obtained by theoretical calculation or experiment (such as amino acid quantification method-hydrolysis derivation method or UV / RI double detector method); and the EC value of the load-linker is usually obtained by detecting the light absorption of the load-linker at the target wavelength according to the Lambert-Beer law, which is highly dependent on the accuracy of the concentration of the detected load-linker sample and the rationality of the selected diluent.

[0006] The existing peak area correction technology for detecting antibody conjugated drugs is less, has limited application range, or is limited by the acquisition channel and method of related parameters such as the extinction coefficient of the antibody single chain and the extinction coefficient of the load-linker, and it is difficult to accurately correct the DAR value (antibody conjugation rate) detection of the antibody conjugated drug. Therefore, how to provide a simple, parameter-easy-to-obtain, and widely-applied correction method for the DAR value detection of the antibody conjugated drug is a problem to be solved at present. SUMMARY

[0007] In view of the deficiencies in the prior art, the purpose of the present application is to provide a correction method for DAR value detection of antibody conjugated drugs based on double wavelength detection and its application.

[0008] To achieve the purpose of the present application, the following technical solutions are adopted:

[0009] In a first aspect, the present application provides a correction method for DAR value detection of antibody conjugated drugs based on double wavelength detection, which comprises:

[0010] (S1) After reducing treatment of the antibody conjugated drug, reverse phase chromatography or affinity chromatography is used for detection, the detection wavelength is set to 280 nm and the special absorption wavelength anm of the selected linker-load, and a spectrum meeting the separation degree is obtained; the peak areas of each response at the two detection wavelengths are obtained by integration;

[0011] (S2) Correct the peak area of each response peak at an nm by the corresponding response peak area at 280 nm, calculate the percentage of the corrected peak area by the corrected peak area, use the percentage of the corrected peak area, calculate the average DAR value according to the contribution of the same component to the DAR;

[0012] The calculation formula of the average DAR value is as follows:

[0013] Corrected DAR LC = 2 x ∑n x Corrected LCn% / ∑c Corrected LCn%

[0014] Corrected DAR HC = 2 x ∑n x Corrected HCn% / ∑ Corrected HCn%

[0015] Corrected DAR = Corrected DAR LC + Corrected DAR HC ;

[0016] Wherein, Corrected DAR represents the average DAR calculated after correction, Corrected DAR LC represents the average DAR related to the light chain LC calculated after correction, Corrected DAR HC represents the average DAR related to the heavy chain HC calculated after correction, Corrected LCn% represents the peak area percentage of LC coupled with n load-connectors after correction, and Corrected HCn% represents the peak area percentage of HC coupled with n load-connectors after correction.

[0017] In the present application, the absorption of LC or HC coupled with different number of load-connectors at 280 nm is obtained by deducting the absorption of load-connector at 280 nm in the target detection wavelength of antibody drug analysis. The specific principle can be represented by the following equation group:

[0018]

[0019] Wherein is the peak area of component x at 280 nm and an nm by analyzing antibody drug by double wavelength reverse phase chromatography or affinity chromatography, wherein, that is, the peak area of component x at 280 nm before correction; and are the peak areas of protein in component x at detection wavelengths 280 nm and an nm, respectively, wherein, The peak area of the corrected component x at 280 nm The peak area of the response of the LP contribution in the x component at the detection wavelength 280 nm, a nm.

[0020]

[0021] Solving the above equation set can obtain a peak area correction calculation formula;

[0022]

[0023] This correction method does not require the extinction coefficients of each component of the antibody, such as light chains and heavy chains, nor the extinction coefficients of small molecules, and can be used for DAR detection correction. The parameters m protein and n LP This correction method can be obtained on the basis of the original detection of antibody conjugation rate (DAR) chromatography method. The correction method has a wide range of applications and can be applied to the DAR value calculation correction of various types of antibody conjugates (various types of antibodies or various types of loads-linkers).

[0024] Preferably, in (S1), the step of reduction treatment comprises: mixing a reducing agent with the antibody conjugated drug and then incubating.

[0025] Preferably, the reducing agent is dithiothreitol.

[0026] Preferably, the use ratio of the reducing agent is 5 μmol DTT corresponding to 1 μg of the antibody conjugated drug.

[0027] In the present application, the step of reverse phase chromatography comprises: using a large-pore reverse phase liquid chromatography column suitable for protein separation, analyzing by high performance liquid chromatography or ultra-high performance liquid chromatography, and setting the detection wavelength to 280 nm and a nm; the mobile phase uses: phase A: aqueous phase; phase B: acetonitrile. The mobile phase gradient starts with a higher proportion of phase A and a lower proportion of phase B, and ends with a lower proportion of phase A and a higher proportion of phase B.

[0028] In one specific embodiment of the present application, the chromatographic conditions of the reverse phase chromatography comprise:

[0029] The chromatographic column is BioResolve RP mAb Polyphenyl, 2.7 μm, 2.1 × 150 mm; the detection wavelength is λ = 280 nm; λ = 370 nm; the column temperature is 70°C; the flow rate is 0.3 mL / min; the mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is 0.1% TFA, 80% ACN and 20% IPA; gradient elution is used, and the elution conditions are as follows:

[0030]

[0031] In the present application, the step of affinity chromatography comprises: using a macroporous affinity liquid chromatography column suitable for protein separation, analyzing by high performance liquid chromatography or ultra-high performance liquid chromatography, and setting the detection wavelength at 280 nm and a nm; the mobile phase uses: A phase: water phase; B phase: acetonitrile. The mobile phase gradient starts with a lower proportion of A phase and a higher proportion of B phase, and ends with a higher proportion of A phase and a lower proportion of B phase.

[0032] In one specific embodiment of the present application, the chromatographic conditions of the affinity chromatography comprise:

[0033] The chromatographic column is Glycoprotein BEH Amide 300A1.7μm 2.1*150mm (Waters / 186007963); the detection wavelength is λ=280nm, λ=374nm; the column temperature is 50℃; the flow rate is 0.3mL / min; the mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution; gradient elution is used, and the elution conditions are as follows:

[0034]

[0035]

[0036] Preferably, in (S1), the special absorption wavelength of the linker-load is selected by scanning the load-linker from 200nm to 500nm by ultraviolet-visible spectrophotometer, and selecting the characteristic absorption wavelength a nm of the load-linker.

[0037] Preferably, the special absorption wavelength of the linker-load is selected to be far away from 280nm, or the maximum absorption wavelength of the load-linker is selected.

[0038] Preferably, in (S1), the separation degree standard is that the separation degree between the chromatographic peak of the to-be-detected substance and the adjacent chromatographic peak is not less than 1.5.

[0039] Preferably, in (S2), the calculation formula for correcting the area of each response peak at 280nm is as follows:

[0040]

[0041] Wherein, the peak area of the response peak of the component x at 280 nm, wherein the component x comprises a light chain LC1 coupled with one loading-linker, or a heavy chain HC1, HC2 or HC3 coupled with one to three loading-linkers, respectively; the peak area of the response peak of the component x at a nm, the peak area of the component x after correction at 280 nm detection, m protein the ratio of the absorption of the uncoupled antibody heavy chain HC0 or the uncoupled antibody light chain LC0 at a nm wavelength to that at 280 nm wavelength, n LP the ratio of the absorption of the loading-linker at a nm wavelength to that at 280 nm wavelength.

[0042] Preferably, the m protein The calculation formula of m is as follows:

[0043]

[0044] The calculation formula of m is as follows: protein the ratio of the absorption of the HC0 or LC0 at a nm wavelength to that at 280 nm wavelength, the detection peak area of the HC0 or LC0 at a nm wavelength, the detection peak area of the HC0 or LC0 at 280 nm.

[0045] Preferably, the detection peak signal-to-noise ratio S / N of the HC0 or LC0 at a nm is less than 10, and the m protein is approximately 0, and the formula can be simplified as:

[0046]

[0047] Preferably, the n LP The calculation formula of n is as follows:

[0048]

[0049] The calculation formula of n is as follows: LP the ratio of the absorption of the loading-linker at a nm wavelength to that at 280 nm wavelength, the detection peak area of the loading-linker at a nm wavelength, the detection peak area of the loading-linker at 280 nm wavelength.

[0050] Preferably, in (S2), the calculation formula of the percentage of the peak area after correction is as follows:

[0051]

[0052] The calculation formula of n is as follows: The percentage of the area of the peak after correction.

[0053] In a second aspect, the application provides the application of the correction method of the DAR value detection of antibody conjugate based on dual-wavelength detection in the detection of antibody conjugate.

[0054] Compared with the prior art, the application has the following beneficial effects:

[0055] (1) DAR detection correction can be performed without extinction coefficients of each component of the antibody, such as light chains and heavy chains, and without the extinction coefficient of the load-linker.

[0056] (2) The absorption ratio parameters of proteins at two wavelengths and the absorption ratio of small molecules at two wavelengths involved in the calculation are easy to obtain.

[0057] (3) Wide application range, which can be applied to DAR value calculation correction of various types of antibody conjugates (various types of antibodies or various types of load-linkers). BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 is a flowchart for correcting the antibody conjugation rate of the antibody conjugate drug.

[0059] Figure 2 is a UV (ultraviolet-visible spectrophotometer) full scan graph of the antibody (Antibody) in the range of 200 nm-500 nm.

[0060] Figure 3 is a UV (ultraviolet-visible spectrophotometer) full scan graph of the load (payload) in the range of 200 nm-500 nm.

[0061] Figure 4 is a chromatogram for detecting the antibody conjugation rate using affinity chromatography.

[0062] Figure 5 is a UV (ultraviolet-visible spectrophotometer) full scan graph of the load (payload) in the range of 200 nm-500 nm.

[0063] Figure 6 is a chromatogram for detecting the antibody conjugation rate using reverse phase chromatography. DETAILED DESCRIPTION

[0064] The technical solutions of the application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the application and should not be regarded as specific limitations on the application.

[0065] Unless otherwise indicated, the techniques or conditions employed in the examples were performed according to the protocols or conditions described in the literature or according to the manufacturer's instructions. Unless otherwise indicated, the reagents or instruments used were conventional products available from regular commercial channels.

[0066] Example 1

[0067] The present example provides a correction method for detecting the DAR value of antibody conjugated drugs based on dual wavelength detection. Figure 1 is a flowchart for correcting the antibody conjugation rate of antibody conjugated drugs.

[0068] 1. Detection of sample and its preparation steps

[0069] Detection sample: bispecific antibody conjugated drug. Payload-linker class: camptothecin derivative with hydrophilic side chain. Its linker has a hydrophilic side chain. The average antibody conjugation rate of three samples A, B and C is detected in this example, and the target conjugation rate of the three samples is different.

[0070] Sample preparation steps: take 30 μL ultrapure water in a 1.5 mL centrifuge tube, add 8 μL 5x fast N-glycosidase F buffer, 2 μL 1 mol / L DTT solution (final concentration 50 mM), mix well, incubate at 75°C for 6 min. Cool the blank sample to room temperature, add 2 μL of fast N-glycosidase F, mix well and incubate at 50°C for 13 min. After incubation, add ultrapure water to make the final volume 102 μL and mix well, and transfer the obtained deglycosylated sample to a glass inner tube injection vial for injection detection.

[0071] The addition of fast N-glycosidase F buffer and fast N-glycosidase F is to achieve the target resolution, that is, to make there is a resolution between the peaks of the components related to the calculation of the antibody conjugation rate.

[0072] 2. Detection method

[0073] 1) Selection of detection wavelength. Use UV-Vis spectrophotometer to scan the payload-linker (LP) in the range of 200 nm-500 nm.

[0074] Figure 2 For antibody conjugated drugs (ADC), the UV (using UV-Vis spectrophotometer) full scan chart of the antibody (Antibody) in the range of 200 nm-500 nm.

[0075] Figure 3 For payload-linker (LP), the UV (using UV-Vis spectrophotometer) full scan chart of the payload in the range of 200 nm-500 nm.

[0076] From Figure 2 andFigure 3 It is known that the specific absorption wavelength of LP is between 370nm and 380nm. In this embodiment, 374nm is selected, that is, a nm = 374nm.

[0077] 2) The parameters for the detection method of antibody conjugation rate are shown in the table below. Table 1 shows the chromatographic parameters for the detection method of antibody conjugation rate.

[0078] Table 1

[0079]

[0080] 3. Test Results

[0081] Figure 4 This is a chromatogram for detecting antibody conjugation rate using affinity chromatography.

[0082] from Figure 4 The integration results show that, based on the detection results at both wavelengths, the absorption response of the uncoupled antibody components LCO and HC0 at 374 nm is negligible compared to that at 280 nm (the signal-to-noise ratio S / N of the detection peak for HC0 or LCO at 374 nm is <10, making it impossible to integrate the effective peak area). Therefore, m LC0 =0,m HC0 =0.

[0083] 4. Correction methods

[0084] (1)n LP The process of obtaining it is as follows.

[0085] The following chromatographic method was used, with detection at wavelengths of 374 nm and 280 nm, and the ratio of peak areas was obtained by integration. Table 2 shows the n... LP To obtain chromatographic parameters.

[0086] Table 2

[0087]

[0088] Table 3 shows n LP Test results.

[0089] Table 3

[0090]

[0091] (2) By Figure 4 The detection results of antibody conjugation rates at two wavelengths indicate that the response absorption of the naked antibody components LCO and HCO at 374 nm is negligible compared to the response absorption at 280 nm. Therefore, m protein It is approximately 0.

[0092] (3) The calculation formula for correcting the area of ​​each response peak at 280nm is shown below.

[0093]

[0094] wherein, Corrected DAR represents the corrected peak area of component x at 280 nm, Corrected DAR represents the corrected peak area of component x at 374 nm, Corrected DAR represents the corrected peak area of component x at 280 nm, n LP Corrected DAR represents the corrected peak area of component x at 280 nm, n

[0095] (4) The formula for calculating the percentage of the corrected peak area is shown below.

[0096]

[0097] wherein, Corrected DAR represents the corrected peak area of component x at 280 nm,

[0098] (5) Using the percentage of the corrected peak area, the average DAR value is calculated according to the contribution of the same component to the DAR.

[0099] The formula for calculating the average DAR value is shown below;

[0100] Corrected DAR LC = 2 x ∑n x Corrected LCn% / ∑Corrected LCn%

[0101] Corrected DAR HC = 2 x ∑n x Corrected HCn% / ∑Corrected HCn%

[0102] Corrected DAR = Corrected DAR LC + Corrected DAR HC ;

[0103] wherein, Corrected DAR represents the corrected peak area of component x at 280 nm, LC Corrected DAR represents the corrected peak area of component x at 280 nm, n HCCorrected LCn% represents the peak area percentage of LC coupled with n loads-linkers after correction, and Corrected HCn% represents the peak area percentage of HC coupled with n loads-linkers after correction.

[0104] Table 4 is test data and peak area correction.

[0105] Table 4

[0106]

[0107] Table 5 is antibody conjugation rate calculation results and comparison before and after correction.

[0108] Table 5

[0109] Sample DAR (mass spec detection) DAR (before correction) DAR (after correction) Whether correction needed Sample A 4.64 5.35 4.84 Yes Sample B 7.24 7.49 7.38 Yes Sample C 3.21 4.21 3.55 Yes

[0110] As can be seen from Table 5, the method has obvious correction effect on antibody conjugation rate, and the correction result is close to the antibody conjugation rate detected by mass spectrometry.

[0111] Example 2

[0112] The embodiment provides a correction method for detecting antibody conjugated drug DAR value based on dual-wavelength detection, and the target antibody conjugation rate is 8.

[0113] 1. Detection of sample and preparation steps thereof.

[0114] Detection sample: IGg1 monoclonal antibody conjugated drug. Load type: camptothecin. In this embodiment, the average antibody conjugation rate of three samples D, E and F is detected, and the target conjugation rates of the three samples are different.

[0115] Sample preparation steps: 40.0 μg of sample, 75 μL of 8 mol / L guanidine hydrochloride (final concentration of 6M), 5 μL of 1 mol / L Tris-HCl (final concentration of 50 mM) and 2 μL of 1 mol / L DTT (final concentration of 20 mM) are added into a 1.5 mL centrifuge tube. The total volume is made up to 100 μL with ultrapure water. After vortex mixing, centrifugation (1 minute, 13000 rpm) is performed. The solution is incubated at 30°C for 30 minutes.

[0116] 2. Detection method

[0117] (1) Selection of detection wavelength and acquisition of n LP . The ultraviolet-visible spectrophotometer is used to perform full scanning of the load-linker (LP) in the range of 200 nm-500 nm, and n LP is acquired.

[0118] Figure 5For the load-connector (LP), the payload 200nm-500nm range of UV (ultraviolet-visible spectrophotometer) full scan.

[0119] From Figure 5 It can be seen that the special absorption wavelength of the LP is between 350nm-400nm, and the embodiment selects 370nm, that is, a nm = 370nm. According to the absorbance at 280nm and 370nm obtained by full scan, n LP = A 370(LP) / A 280(LP) = 2.52.

[0120] 2) The detection method parameters of the antibody coupling rate are shown in the following table, and Table 6 is the chromatographic parameters of the detection method of the antibody coupling rate.

[0121] Table 6

[0122]

[0123] 3, Detection results

[0124] Figure 6 The chromatogram for detecting the antibody coupling rate using reverse phase chromatography.

[0125] From Figure 6 The integral results show that the detection results at two wavelengths can determine that the uncoupled antibody components LC0 and HC0 have negligible response absorption at 370nm compared to the response absorption at 280nm (the detection peak signal-to-noise ratio S / N of HC0 or LC0 at 374nm is less than 10, and the effective peak area cannot be integrated), so m protein = 0 (that is, m LC0 = 0, m HC0 = 0).

[0126] 4, Correction method

[0127] (1) From Figure 6 The detection results of the antibody coupling rate at two wavelengths can determine that the naked antibody components LC0 and HC0 have negligible response absorption at 370nm compared to the response absorption at 280nm, so m protein is approximately 0 (that is, m LC0 = 0, m HC0 = 0).

[0128] (2) The calculation formula for correcting each response peak area at 280nm is as follows.

[0129]

[0130] Wherein, Corrected Peak Area at 280 nm (n) Corrected Peak Area at 370 nm (n) Corrected Peak Area at 280 nm (n) LP Ratio of Absorption at 370 nm to 280 nm

[0131] (4) The formula for calculating the percentage of the corrected peak area is as follows.

[0132]

[0133] wherein, Corrected Peak Area at 280 nm (n)

[0134] (5) Using the percentage of the corrected peak area, the average DAR value is calculated according to the contribution of the same component to the DAR.

[0135] The formula for calculating the average DAR value is as follows;

[0136] Corrected DAR LC = 2 x ∑n x Corrected LCn% / ∑Corrected LCn%

[0137] Corrected DAR HC = 2 x ∑n x Corrected HCn% / ∑Corrected HCn%

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

[0139] wherein, Corrected DAR represents the average DAR calculated after correction, Corrected DAR LC represents the average DAR related to the light chain (LC) calculated after correction, Corrected DAR HC represents the average DAR related to the heavy chain (HC) calculated after correction, Corrected LCn% represents the percentage of the peak area of LC coupled with n load-linkers after correction, and Corrected HCn% represents the percentage of the peak area of HC coupled with n load-linkers after correction.

[0140] Table 7 is the test data and peak area correction.

[0141] Table 7

[0142]

[0143] Table 8 is the calculation result of antibody conjugation rate and comparison before and after correction.

[0144] Table 8

[0145] Sample DAR (mass spec detection) DAR (before correction) DAR (after correction) Whether correction needed Sample D 3.34 3.73 3.53 Yes Sample E 4.73 5.24 5.08 Yes Sample F 7.84 7.83 7.81 No

[0146] From Table 8, it can be seen that the effect of the method for correcting the antibody conjugation rate is obvious, and the result after correction is close to the result of mass spectrometry for detecting the antibody conjugation rate.

[0147] In summary, the application provides a method for correcting the detection result of the antibody conjugation rate of antibody conjugated drugs by using the ratio of the absorption of the load-linker and the antibody at two wavelengths as the main calculation parameter, which is simple and easy to implement, the parameters are easy to obtain, and has a wide application prospect in the detection of the DAR value of antibody conjugated drugs.

[0148] The applicant declares that the above description is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the application can be easily thought of by any person skilled in the art, and all fall within the protection scope and disclosure scope of the application.

Claims

1. A correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection, characterized in that, The method includes: (S1) After reducing the antibody-drug conjugate, it is detected by reversed-phase chromatography or affinity chromatography. The detection wavelength is set to 280 nm and the specific absorption wavelength of the selected linker-loader is an nm to obtain a spectrum that meets the resolution. The response peak area at each detection wavelength is obtained by integration. (S2) Correct the corresponding response peak area at 280 nm by the response peak area at each nm. Calculate the percentage of the corrected peak area using the corrected peak area. Calculate the average DAR value using the percentage of the corrected peak area based on the contribution of similar components to DAR. The formula for calculating the average DAR value is as follows; 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 ; Wherein, Corrected DAR represents the average DAR calculated after correction. LC The corrected DAR represents the average DAR related to light chain LC after calculation. HC The value represents the average DAR related to the heavy chain HC after correction. Corrected LCn% represents the percentage of the peak area of ​​the LC of the n coupled load-connectors after correction, and CorrectedHCn% represents the percentage of the peak area of ​​the HC of the n coupled load-connectors after correction.

2. The correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection according to claim 1, characterized in that, In (S1), the reduction process includes: mixing the reducing agent with the antibody-drug conjugate and then incubating.

3. The correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection according to claim 1 or 2, characterized in that, In (S1), the specific absorption wavelength of the connector-load is selected by performing a full scan of the load-connector from 200 nm to 500 nm using an ultraviolet-visible spectrophotometer, and selecting the characteristic absorption wavelength a nm of the load-connector.

4. The correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection according to claim 3, characterized in that, The specific absorption wavelength of the connector-load is selected from wavelengths that are far from 280nm, or the maximum absorption wavelength of the load-connector is selected.

5. The correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection according to any one of claims 1-4, characterized in that, In (S1), the resolution standard is that the resolution between the chromatographic peak of the analyte and the adjacent chromatographic peak should not be less than 1.

5.

6. The correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection according to any one of claims 1-5, characterized in that, In (S2), the calculation formula for correcting the area of ​​each response peak at 280nm is as follows; in, The peak area of ​​the response at 280 nm represents the peak of component x, wherein component x includes a light chain LC1 coupled with one load-connector, or a heavy chain HC1, HC2 or HC3 coupled with one to three load-connectors respectively. This represents the peak area of ​​component x at a nm. The m represents the peak area of ​​component x after correction at 280 nm detection. protein The ratio of the absorbance of unconjugated antibody heavy chain HC0 or unconjugated antibody light chain LC0 at wavelength a nm to that at wavelength 280 nm, where n is the number of wavelengths used. LP This represents the ratio of the absorption of the load-connector at a wavelength of 0 nm to that at a wavelength of 280 nm.

7. The correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection according to claim 6, characterized in that, The m protein The calculation formula is shown below; Where, m protein This represents the ratio of the absorption of HClO or LCO at a wavelength of 0 nm to that at a wavelength of 280 nm. This represents the detection peak area of ​​HC0 or LCO at a wavelength of a nm. This indicates the detection peak area of ​​HC0 or LCO at 280 nm. Preferably, the signal-to-noise ratio (S / N) of the detection peak of HC0 or LCO at a nm is <10, then m protein Approximately 0, the formula can be simplified to; 8. The correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection according to claim 6, characterized in that, The n LP The calculation formula is shown below; Where, n LP This represents the ratio of the absorption of the load-connector at a wavelength of 'a' nm to that at a wavelength of '280' nm. This represents the detection peak area of ​​the load-connector at a wavelength of a nm. This represents the detection peak area of ​​the load-connector at a wavelength of 280 nm.

9. The correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection according to any one of claims 1-8, characterized in that, In (S2), the formula for calculating the percentage of the corrected peak area is as follows; in, This indicates the percentage of the peak area after correction.

10. The application of the correction method for DAR value detection of antibody-drug conjugates based on dual-wavelength detection as described in any one of claims 1-9 in the detection of antibody-drug conjugates.

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