Method for simultaneously detecting antibody conjugate purity and drug antibody coupling ratio based on size exclusion chromatography and application thereof

Abstract

The invention provides a method for simultaneously detecting the purity of an antibody conjugate and the coupling ratio of a drug antibody based on a size exclusion chromatography method and application of the method. The method comprises the following steps: detecting and analyzing the purity of an ADC (Analog to Digital Converter) sample to be detected and the coupling ratio of the drug antibody by adopting an SEC-UPLC (Single Emission Computation-Ultra Performance Liquid Chromatography) serially connected with an ultraviolet detector; collecting a signal of the sample under the antibody characteristic ultraviolet absorption wavelength and the small molecule characteristic ultraviolet absorption wavelength at the same time; calculating the DAR value and the monomer purity of the ADC molecule according to the chromatographic absorption peak area; a mobile phase adopted by the SEC-UPLC is a sodium chloride-phosphate buffer solution containing 0-10vol% of an organic solvent; the ADC sample to be detected is an ADC molecule coupled with Dxd type small molecules. The invention provides an analysis method which is widely applicable to determination of the DAR value and the monomer purity of the ADC molecule coupled with the Dxd small molecules. The method can simultaneously solve the problems that the DAR value detection and analysis flux of the Dxd small molecules is low and the applicability is limited.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for simultaneously detecting the purity of an antibody conjugate and the drug-antibody conjugate ratio based on size exclusion chromatography and its application. Background Art

[0002] An antibody-drug conjugate (ADC) is a combinatorial drug formed by conjugating a monoclonal antibody and a cytotoxic small molecule through a bioactive linker. The antibody-drug conjugate first specifically targets cancer cells through the monoclonal antibody, and then the conjugated small molecule chemical drug kills the cancer cells. It combines the high specificity and targeting of monoclonal antibody drugs and the high efficiency of small molecule chemical drugs in eliminating cancer cells, can synergistically exert the respective advantages of antibody drugs and chemical drugs, reduce the harm to the human biological system, and has great application value in the field of tumor treatment and other fields.

[0003] The drug to antibody ratio (DAR) represents the average number of small molecule drugs conjugated to each antibody. Generally, ADC molecules with low DAR values have weak drug efficacy. As the DAR value increases, the drug efficacy increases while the clearance rate also increases. In short, the drug to antibody conjugate ratio is directly related to the drug efficacy and safety. The molecular design of ADC drugs is complex, and the production process is relatively complex. The inherent properties of proteins cause them to easily generate fragments or aggregates during the production process, resulting in a decrease in the purity of the final product. The purity of ADC drugs can reflect the level of the production process. Therefore, the purity of samples needs to be monitored at each step during the process development to provide a basis for subsequent development. More importantly, ADC products with higher purity have better pharmacokinetic properties and safety. In summary, the DAR value and monomer purity of ADC molecules are key quality parameters of ADC drugs.

[0004] Currently, liquid chromatography or ultraviolet spectroscopy (UV) is usually used to detect the DAR value of ADC molecules. Liquid chromatography mainly includes hydrophobic interaction chromatography (HIC) and reverse phase liquid chromatography (RPLC). Sodium dodecyl sulfate-capillary electrophoresis (SDS-CE) and size exclusion chromatography (SEC) are used for the purity detection of ADC drugs.

[0005] HIC separates based on the number and hydrophobicity of the small molecules conjugated to the ADC drug. Since the conjugated small molecule drugs usually have high hydrophobicity, the more small molecules are conjugated, the stronger the hydrophobicity of the ADC molecule and the longer the retention time. During the HIC separation process, ADC molecules with different DAR values are separated in sequence, so that the DAR distribution information of the ADC molecules can be obtained. At the same time, the average DAR value of the ADC molecules can be obtained by calculating the peak area. For samples with high DAR values, such as DAR 8 molecules, the HIC method has poor applicability. At this time, the RP method can be selected for DAR value analysis. When using the RP method for DAR value analysis, the ADC sample needs to be pretreated with a reducing agent before reduction. The reduced sample is separated by RP into light chains, heavy chains, and light and heavy chains with different numbers of small molecule drugs. The average DAR value can be obtained by calculating the area percentage of each peak. Although the measurement accuracy of the HIC and RP analysis methods is relatively high, due to the need to meet the resolution of chromatographic peaks, there are many method development parameters, a long development cycle, and often a low analysis throughput.

[0006] The UV method is based on the Beer-Lambert law, and uses the extinction coefficients of antibodies and small molecules to measure the molar concentrations of antibodies and small molecule drugs respectively, so as to calculate the DAR value of the ADC drug. Although the UV method is relatively simple to operate, has a short detection time, and high throughput compared with liquid phase methods, due to the lack of separation, the test is easily interfered by other components in the ADC solution, such as free small molecules, resulting in relatively large detection errors.

[0007] CE is a separation technique with a high-voltage DC electric field as the driving force and a capillary as the separation channel. After sodium dodecyl sulfate binds to proteins, it can make the proteins carry a large amount of negative charges, neutralize the charges of the proteins themselves, eliminate the influence of the sample charge effect, and make different components in the sample migrate at different speeds in the capillary according to their molecular sizes and be separated, and finally reach the UV detector for detection. Its disadvantages are: (1) long analysis time and low throughput. (2) Pretreatment of samples that need to be denatured may produce fragment molecules of unstable ADC molecules due to denaturing conditions, affecting the accuracy of analysis.

[0008] SEC mainly separates according to the size of the substances to be separated. Macromolecular substances are eluted first, followed by small molecules with longer retention times. When using SEC for separation, not only can the monomer, high molecular weight components (HMWS) and low molecular weight components (LMWS) in the ADC sample be separated, but also the ADC can be separated from the impurity components in the sample (such as free small molecules). And compared with CE and other purity analysis methods, the analysis and test time of SEC is often shorter, the sample does not need pretreatment, and the analysis throughput is relatively high.

[0009] In summary, based on SEC chromatographic separation and the UV absorption differences between the antibody and small molecule components of the ADC molecule, a high-throughput and versatile dual-wavelength SEC analysis method for simultaneously analyzing the DAR value and monomer purity of ADC molecules has important application value in the detection of ADC drugs. Summary of the Invention

[0010] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method for simultaneously detecting the purity of antibody conjugates and the drug-to-antibody conjugate ratio based on size exclusion chromatography and its application. The present invention provides an analytical method that is widely applicable to the determination of the DAR value and monomer purity of ADC molecules conjugated with Dxd-class small molecules, and the method can simultaneously solve the problems of low detection and analysis throughput and limited applicability of the DAR value of Dxd-class ADC molecules.

[0011] To achieve the purpose of this invention, the following technical solutions are adopted:

[0012] First invention, the present invention provides a method for simultaneously detecting the purity of antibody conjugates and the drug-to-antibody conjugate ratio based on size exclusion chromatography, and the method includes:

[0013] Using SEC-UPLC tandem with a UV detector to detect and analyze the purity and drug-to-antibody conjugate ratio of the ADC sample to be tested; simultaneously collecting the signals of the sample at the antibody characteristic UV absorption and small molecule characteristic UV absorption wavelengths; calculating the DAR value and monomer purity of the ADC molecule according to the chromatographic absorption peak area;

[0014] The mobile phase used in the SEC-UPLC is a sodium chloride-phosphate buffer solution containing 0-10 vol% organic solvent (for example, it can be 0 vol%, 1 vol%, 2 vol%, 3 vol%, 4 vol%, 5 vol%, 6 vol%, 7 vol%, 8 vol%, 9 vol% or 10 vol%, etc.);

[0015] The ADC sample to be tested is an ADC molecule conjugated with a Dxd-class small molecule.

[0016] In the present invention, the principle of the detection method is to separate according to the size of the components to be tested, and sequentially separate the high molecular components, monomers, low molecular components and residual small molecule drugs in the antibody conjugate sample, and combine the dual-wavelength scanning method to obtain the corresponding values of the sample at the antibody characteristic absorption wavelength and the small molecule drug characteristic absorption wavelength, so as to simultaneously obtain the sample purity and DAR value information. The advantages are simple sample pretreatment, high analysis throughput and the ability to obtain the analysis results of two key quality attributes, purity and DAR.

[0017] Preferably, the sodium chloride-phosphate buffer comprises 45-55 mM phosphate buffer and 290-310 mM sodium chloride by concentration, with a pH of 6.8 ± 0.1.

[0018] Among them, the 45-55 mM phosphate buffer can be, for example, 45 mM, 47 mM, 49 mM, 50 mM, 51 mM, 53 mM or 55 mM, etc.; the 290-310 mM sodium chloride can be, for example, 290 mM, 300 mM or 310 mM, etc.

[0019] In the present invention, the molar concentration of phosphate ions in the phosphate buffer is 45-55 mM.

[0020] Preferably, the organic solvent is selected from isopropanol or acetonitrile.

[0021] Preferably, the flow rate of the mobile phase is 0.4-0.5 mL / min, which can be, for example, 0.4 mL / min or 0.5 mL / min, etc.

[0022] Preferably, the SEC-UPLC adopts an isocratic elution method, and the running time is 8.0-9.0 min, which can be, for example, 8.0 min, 8.5 min or 9.0 min, etc.

[0023] Preferably, the characteristic ultraviolet absorption wavelength of the antibody is 270-290 nm, which can be, for example, 270 nm, 280 nm or 290 nm, etc.; the characteristic ultraviolet absorption wavelength of the small molecule is 360-380 nm, which can be, for example, 360 nm, 370 nm or 380 nm, etc.

[0024] Preferably, the pore size of the chromatographic column used in the SEC-UPLC is a super high performance liquid chromatography column with a particle size of 1.7-2.5 μm and an inner diameter of 4-5 mm.

[0025] In specific detection, a suitable chromatographic column is selected according to the molecular weight of the antibody conjugate to be analyzed.

[0026] Preferably, the chromatographic column is Waters ACQUITY UPLC Protein BEH SEC.

[0027] Preferably, the column temperature of the chromatographic column is 19-31 °C, which can be, for example, 19 °C, 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, 30 °C or 31 °C, etc.

[0028] Preferably, the injection volume of the SEC-UPLC is 5-15 μg, which can be, for example, 5 μg, 7 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg or 15 μg, etc.

[0029] Preferably, the Dxd-based small molecule is a Dxd-based payload-linker, and the Dxd-based small molecule includes MC-GGFG-Dxd or MC-Gly-PAB-Exatecan-D-glucuronic acid.

[0030] Preferably, the concentration of the ADC sample to be measured is less than or equal to 5.0 mg / mL.

[0031] In the present invention, when the concentration of the ADC sample to be measured is higher than 5.0 mg / mL, it needs to be diluted, and the sample is diluted to 5.0 mg / mL with ultrapure water and then injected for analysis.

[0032] Preferably, the calculation formula for the DAR value is:

[0033]

[0034] where A 280 is the ultraviolet absorption peak area at the characteristic absorption wavelength of 280 nm of the antibody, and A λ(drug) is the ultraviolet absorption peak area at the characteristic absorption wavelength of the small molecule, is the extinction coefficient of the antibody at the characteristic absorption wavelength of 280 nm, is the extinction coefficient of the antibody at the characteristic absorption wavelength of the small molecule, is the extinction coefficient of the small molecule at the characteristic absorption wavelength of 280 nm of the antibody, is the extinction coefficient of the small molecule at the characteristic absorption wavelength, MW mAb is the molecular weight of the antibody, MW LP is the molecular weight of the small molecule.

[0035] In the present invention, the is obtained by theoretical calculation according to the theoretical amino acid sequence.

[0036] In the present invention, the is obtained by theoretical calculation according to the theoretical amino acid sequence.

[0037] In the present invention, the is actually measured using SoloVPE.

[0038] In the present invention, the is actually measured using SoloVPE.

[0039] Preferably, the calculation formula for the purity is:

[0040]

[0041] where: Area monomerIt is the peak area of the antibody conjugate monomer at the ultraviolet absorption wavelength of 280 nm for the ADC sample, Area total It is the total peak area of the ADC sample at the ultraviolet absorption wavelength of 280 nm.

[0042] In the present invention, three peaks of high molecular weight components (HMWs), monomer (Monomer), and low molecular weight components (LMWs) are detected, and the monomer is the main peak.

[0043] In a second aspect, the present invention provides the application of the method for simultaneously detecting the purity of an antibody conjugate and the drug-antibody conjugate ratio based on size exclusion chromatography described in the first aspect in the detection of antibody-drug conjugates.

[0044] The numerical ranges described in the present invention not only include the above-listed point values, but also any point values between the above numerical ranges not listed. Due to space limitations and for the sake of brevity, the specific point values included in the described ranges are not exhaustively listed in the present invention.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention provides a dual-wavelength size exclusion chromatography method for simultaneously detecting the monomer purity of an antibody conjugate and the DAR value, and uses SEC-UPLC for detection and analysis at the ultraviolet absorption wavelength characteristic of the antibody and the ultraviolet absorption wavelength characteristic of the small molecule. Through the detection of dual wavelengths, the SEC purity information of the Dxd class ADC sample and the DAR value of the Dxd class ADC with the native structure maintained can be obtained simultaneously. The entire analysis process does not require sample pretreatment, and SEC can effectively separate the ADC monomer and interfering substances in the components, which is more conducive to more high-throughput and accurate determination of the DAR value of the ADC sample while realizing the detection of monomer purity. Description of the Drawings

[0047] Figure 1 It is the ultraviolet absorption spectra of the antibody (Herceptin) and the small molecule (MC-GGFG-Dxd).

[0048] Figure 2 It is the SEC chromatogram of the ADC (Herceptin-MC-GGFG-Dxd).

[0049] Figure 3 It is the ultraviolet absorption spectra of the antibody (Herceptin) and the small molecule (MC-Gly-PAB-Exatecan-D-glucuronic acid).

[0050] Figure 4It is the SEC chromatogram of ADC (Herceptin-MC-Gly-PAB-Exatecan-D-glucuronic acid).

[0051] Figure 5 It is the chromatogram detected under two column temperature conditions of 22 °C and 28 °C.

[0052] Figure 6 It is the chromatogram detected under the liquid phase condition with a flow rate of 0.5 mL / min.

[0053] Figure 7 It is the chromatogram detected under the MP-1 mobile phase condition.

[0054] Figure 8 It is the chromatogram detected under the MP-2 mobile phase condition.

[0055] Figure 9 It is the result of the linear chromatogram of 50%-150%.

[0056] Figure 10 It is the chromatogram result of the repeatability detection of low, medium and high (50%, 100%, 150%).

[0057] Figure 11 It is the result of the specificity chromatogram.

[0058] Figure 12 It is the result of the solution stability chromatogram. Specific implementation mode

[0059] The technical solution of the present invention will be further described below through specific implementation modes. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0060] For those not specifying specific technologies or conditions in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular channels of commercial purchase.

[0061] The sources of the experimental materials used in the following embodiments are as follows:

[0062] Herceptin-MC-GGFG-Dxd sample (trastuzumab-MC-GGFG-Dxd sample): Synthesized by intermolecular disulfide bond coupling in the laboratory.

[0063] Herceptin-MC-Gly-PAB-Exatecan-D-glucuronic acid sample (trastuzumab-MC-Gly-PAB-exatecan-D-glucuronic acid sample): Synthesized by intermolecular disulfide bond coupling in the laboratory.

[0064] Example 1

[0065] 1. Sample preparation

[0066] Use a pipette to transfer an appropriate amount of trastuzumab-MC-GGFG-Dxd sample into a liquid phase injection vial for analysis.

[0067] 2. Chromatographic conditions

[0068] The mobile phase is 90% 50 mM phosphate buffer, 300 mM sodium chloride, pH 6.8 ± 0.1, and 10% isopropanol. The specific liquid phase conditions are shown in Table 1.

[0069] Table 1

[0070]

[0071] 3. Result analysis

[0072] The antibody of the trastuzumab-MC-GGFG-Dxd sample has a characteristic ultraviolet absorption at 280 nm, and the small molecule (LP) MC-GGFG-Dxd has a characteristic ultraviolet absorption at 370 nm. The spectrum is shown in Figure 1 . The theoretical EC value (extinction coefficient) of the antibody, the measured EC value of LP (10 mg / mL), and the molecular weight information are shown in Table 2.

[0073] Table 2

[0074]

[0075] The sample can be separated by SEC, and the absorption of the ADC molecule at 280 nm and 370 nm can be detected. The DAR value can be calculated by measuring the absorption peak area. The representative spectra of the SEC-UPLC detection of two repeated experiments are shown in Figure 2 , and the detection results are shown in Table 3.

[0076] Table 3

[0077]

[0078] As Figure 2 shown, the separation degree of the high molecular weight component and the monomer of trastuzumab-MC-GGFG-Dxd is good, and no low molecular weight component is detected. Under the existing gradient conditions, the ADC sample is eluted before 4 minutes, and the overall peak shape is good, and the separation of the high molecular weight component and the monomer is good. The DAR value of the sample can be calculated to be 7.2 through the DAR value calculation formula. The SEC monomer purity of the sample can be calculated to be 98.7% through the purity calculation formula.

[0079] Example 2

[0080] 1. Sample Preparation

[0081] Use a pipette to transfer an appropriate amount of trastuzumab-MC-Gly-PAB-exatecan-D-glucuronic acid sample into a liquid chromatography injection vial for analysis.

[0082] 2. Chromatographic Conditions

[0083] The mobile phase is 90% 50 mM phosphate buffer, 300 mM sodium chloride, pH 6.8 ± 0.1, and 10% isopropanol. The specific liquid chromatography conditions are shown in Table 1.

[0084] 3. Result Analysis

[0085] The antibody in the trastuzumab-MC-Gly-PAB-exatecan-D-glucuronic acid sample has a characteristic ultraviolet absorption at 280 nm, and the small molecule MC-Gly-PAB-Exatecan-D-glucuronic acid (MC-Gly-PAB-exatecan-D-glucuronic acid) has a characteristic ultraviolet absorption at 370 nm. The chromatogram is shown in Figure 3 . The theoretical EC value of the antibody, the measured EC value of LP (10 mg / mL), and the molecular weight information are shown in Table 4.

[0086] Table 4

[0087]

[0088] The sample can be separated by SEC and the absorption of the ADC molecule at 280 nm and 370 nm can be detected. The DAR value can be calculated by measuring the absorption peak area. The representative chromatograms of two repeated SEC-UPLC detections are shown in Figure 4 , and the detection results are shown in Table 5.

[0089] Table 5

[0090]

[0091] As Figure 4 and shown in Table 5, the separation degree of the high molecular weight component and the monomer in the trastuzumab-MC-Gly-PAB-exatecan-D-glucuronic acid sample is good, and no low molecular weight component is detected. Under the existing gradient conditions, the ADC sample is eluted before 4 minutes, and the overall peak shape is good, and the separation of the high molecular weight component and the monomer is good. The DAR value of the sample can be calculated to be 6.1 by the DAR value calculation formula. The SEC monomer purity of the sample can be calculated to be 98.5% by the purity calculation formula.

[0092] Example 3

[0093] In this example, the column temperature was investigated.

[0094] The experimental steps are as follows:

[0095] 1. Sample preparation

[0096] Use a pipette to transfer an appropriate amount of trastuzumab-MC-GGFG-Dxd sample and place it in a liquid phase injection vial for analysis.

[0097] 2. Chromatographic conditions

[0098] The mobile phase is 90% 50 mM phosphate buffer, 300 mM sodium chloride, pH 6.8 ± 0.1, and 10% isopropanol. The specific liquid phase conditions are shown in Table 6.

[0099] Table 6

[0100]

[0101] 3. Result analysis

[0102] The chromatograms detected under two column temperature conditions of 22 °C and 28 °C are as Figure 5 shown. The calculated results of purity and DAR value are shown in Table 7, indicating that the method has good durability when the column temperature is 19 - 31 °C.

[0103] Table 7

[0104]

[0105] Example 4

[0106] This example examines the flow rate

[0107] The experimental steps are as follows:

[0108] 1. Sample preparation

[0109] Use a pipette to transfer an appropriate amount of trastuzumab-MC-GGFG-Dxd sample and place it in a liquid phase injection vial for analysis.

[0110] 2. Chromatographic conditions

[0111] The mobile phase is 90% 50 mM phosphate buffer, 300 mM sodium chloride, pH 6.8, and 10% isopropanol. The specific liquid phase conditions are shown in Table 8.

[0112]

[0113] 3. Result analysis

[0114] The chromatogram detected under the liquid phase condition of a flow rate of 0.5 mL / min is as Figure 6 shown. The calculated results of purity and DAR value are shown in Table 9, indicating that the method has good durability when the liquid phase flow rate is 0.5 mL / min.

[0115] Table 9

[0116]

[0117] Example 5

[0118] This example examines the composition of the mobile phase

[0119] The experimental procedure is as follows:

[0120] 1. Sample preparation

[0121] Use a pipette to transfer an appropriate amount of trastuzumab-MC-GGFG-Dxd sample and place it in a liquid-phase injection vial for analysis.

[0122] 2. Chromatographic conditions

[0123] In the SEC analysis of ADC samples in which camptothecin-based small molecules are coupled through interchain disulfide bonds, it was found that when more than 10% isopropanol (organic solvent) is added to the mobile phase, the high-molecular-weight components (HMWS) of the sample will deaggregate. In addition, the addition of a high proportion of organic solvents will irreversibly damage the SEC chromatographic column. Therefore, the conditions for the mobile phase investigation in this study are as follows:

[0124] The mobile phase is MP-1: 50 mM phosphate buffer, 300 mM sodium chloride, pH 6.8 ± 0.1; or MP-2: 90% 50 mM phosphate buffer, 300 mM sodium chloride, pH 6.8 ± 0.1, 10% ACN (acetonitrile).

[0125] The specific liquid-phase conditions are shown in Table 10.

[0126] Table 10

[0127]

[0128] The chromatograms detected under two different mobile phase conditions of MP-1 and MP-2 are as shown in Figure 7 and Figure 8 shown. The calculation results of purity and DAR value are shown in Table 11, indicating that this method is also suitable for both pure water phase systems and mobile phase systems with 10% ACN organic solvent added, and the method has a certain universality.

[0129] Table 11

[0130]

[0131] Example 6

[0132] This example conducted a methodological verification of the dual-wavelength size-exclusion chromatography method for detecting the purity of antibody conjugates and the drug-antibody conjugate ratio, including linearity, repeatability, specificity, and stability experiments. The analysis chromatograms are as shown inFigure 9 and 10 As shown in Table 12 for 11 and 12 and the calculation results of purity and DAR value, the results indicate that this method has good linearity in the linear range of 50%-150% and good repeatability. The specificity results show that this method has good specificity. In addition, this method has a solution stability of 24 hours and is suitable for long-sequence analysis.

[0133] Table 12

[0134]

[0135]

[0136] In summary, based on the chromatographic separation of SEC and the ultraviolet absorption differences between the antibody and small molecule that make up the ADC molecule, the present invention develops a dual-wavelength SEC analysis method for high-throughput and general simultaneous analysis of the DAR value and monomer purity of ADC molecules. The entire analysis process does not require sample pretreatment and can quickly and accurately complete the detection of monomer purity while more highly and accurately determining the DAR value of ADC samples.

[0137] The applicant declares that the above description is only the specific implementation manner 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 fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for simultaneously detecting the purity of an antibody conjugate and the drug-antibody conjugation ratio based on size exclusion chromatography, characterized in that: The method comprises: The purity and drug-antibody coupling ratio of the ADC samples to be tested were detected and analyzed using a SEC-UPLC tandem UV detector; the signal of the sample was collected at the characteristic UV absorption wavelengths of the antibody and the small molecule; the DAR value and monomer purity of the ADC molecule were calculated based on the chromatographic absorption peak area; The mobile phase used in the SEC-UPLC is a sodium chloride-phosphate buffer containing 0-10 vol% organic solvent; The ADC sample to be tested is an ADC molecule coupled with a Dxd-type small molecule.

2. The method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to claim 1, characterized in that: The sodium chloride-phosphate buffer comprises, by concentration, 45-55 mM phosphate buffer, 290-310 mM sodium chloride, and a pH of 6.8±0.1; Preferably, the organic solvent is selected from isopropanol or acetonitrile.

3. The method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to claim 2, characterized in that: The flow rate of the mobile phase is 0.4-0.5 mL / min; Preferably, the SEC-UPLC adopts an isocratic elution method with a running time of 8.0-9.0 min; Preferably, the characteristic ultraviolet absorption wavelength of the antibody is 270-290 nm; the characteristic ultraviolet absorption wavelength of the small molecule is 360-380 nm.

4. The method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to any one of claims 1 to 3, characterized in that: The pore size of the chromatographic column used in the SEC-UPLC is Ultra-high performance liquid chromatography columns with a particle size of 1.7-2.5 μm and an inner diameter of 4-5 mm; Preferably, the chromatographic column is Waters ACQUITY UPLC Protein BEH SEC.

5. The method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to any one of claims 1 to 4, characterized in that: The column temperature of the chromatographic column is 19-31°C; Preferably, the injection volume of the SEC-UPLC is 5-15 μg.

6. The method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to any one of claims 1 to 5, characterized in that: The Dxd-type small molecules are payload-linkers based on Dxd, and the Dxd-type small molecules include MC-GGFG-Dxd or MC-Gly-PAB-Exatecan-D-glucuronic acid.

7. The method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to any one of claims 1 to 6, characterized in that: The concentration of the ADC sample to be tested is less than or equal to 5.0 mg / mL.

8. The method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to any one of claims 1 to 7, characterized in that: The calculation formula of the DAR value is: Among them, A 280 A is the UV absorption peak area of ​​the antibody at the characteristic absorption wavelength of 280 nm. λ(drug) is the ultraviolet absorption peak area at the characteristic absorption wavelength of small molecules, is the extinction coefficient of the antibody at the characteristic absorption wavelength of 280 nm, is the extinction coefficient of the antibody at the characteristic absorption wavelength of small molecules, is the extinction coefficient of the small molecule at the antibody characteristic absorption wavelength of 280nm, is the extinction coefficient of small molecules at the characteristic absorption wavelength, MW mAb is the molecular weight of the antibody, MW LP is the molecular weight of a small molecule.

9. The method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to any one of claims 1 to 8, characterized in that: The calculation formula of the purity is: Where: Area monomer Area is the peak area of ​​the antibody conjugate monomer of the ADC sample at 280nm UV absorption wavelength. total It is the total peak area of ​​ADC sample at 280nm ultraviolet absorption wavelength.

10. Use of the method for simultaneously detecting the purity of antibody conjugates and the drug-antibody conjugation ratio based on size exclusion chromatography according to any one of claims 1 to 9 in the detection of antibody-drug conjugates.

Images