Method for determining DAR value of antibody-conjugated drug

Abstract

The invention discloses a method for determining the DAR value of an antibody-conjugated drug. The method comprises the following steps: carrying out antibody capture, enzymolysis and detection on a to-be-detected sample; the enzymolysis is to carry out enzymolysis on the antibody coupling medicine by using at least one protease to obtain a characteristic peptide fragment of a CDR region and free micromolecule toxin; the antibody capture is to capture an antibody coupling drug in a sample by using a capture antibody. The problem of high background value during ADC detection in the prior art is solved, and the problem of poor sensitivity caused by high-resolution detection of a small molecule toxin antibody ratio (DAR) value can also be solved. Bioanalytical strategies associated with a range of pharmacokinetic (PK) and anti-drug antibody (ADA) assessments can be provided to producers.

Description

Technical Field

[0001] The present invention relates to the technical field of preclinical and clinical bioanalysis of drugs, and in particular to a method for determining a DAR value of an antibody-drug conjugate. Background Art

[0002] Antibody-drug conjugates (ADCs) have become a hot topic in the development of new cancer drugs because of their stronger targeting and cell-killing ability. The ADC structure consists of antibodies or antibody fragments, linkers, and conjugated payloads (small molecule compounds). They can selectively target antigens on the surface of cancer cells through antibody-mediated high-affinity delivery, which are then engulfed by tumor cells into lysosomes and release effector small molecules. Since the FDA approved the first ADC in 2000, more than 100 candidate drugs are currently being studied in the clinical stage. At the same time, with the continuous advancement of ADC components such as antibody engineering, site-specific conjugation, and new cytotoxic drugs, the development of various ADC platforms has been promoted. The drug-to-antibody ratio (DAR) is a unique and important attribute of ADC drugs, which represents the average number of antibody-conjugated small molecule toxic drugs. When ADC enters the body, it is accompanied by the release of toxin molecules. The average DAR value will decrease over time. The rate of change of the DAR value can reflect the efficiency of toxin molecules dissociating from ADC. Therefore, the DAR value reflects the content of effective drugs in the body and is of great significance for pharmacology and efficacy evaluation.

[0003] The determination of DAR value of ADC in preclinical and clinical studies mainly relies on high-resolution mass spectrometry, but its disadvantage is that it is not sensitive enough and cannot test the DAR value of low-concentration samples. There are also methods to determine the total antibody in ADC by ELISA and the conjugated payload in ADC by LC-MS, and then calculate the DAR value. The operation is cumbersome and involves many platforms. Compared with the ELISA method, the method of using LC-MS / MS method to quantify the total antibody and the conjugated payload and then calculate the DAR not only improves the sensitivity of the method, but also the method development and transfer are relatively simple, and multiple candidate drugs can be detected at the same time. Summary of the invention

[0004] In order to solve the problems of high background value and poor sensitivity in the prior art when detecting ADC, the present invention provides a method for determining the DAR value of an antibody-drug conjugate.

[0005] Specifically, the present invention provides a method for determining the DAR value of an antibody-drug conjugate, which comprises antibody capture, enzymatic hydrolysis and detection of a sample to be tested.

[0006] In some embodiments, the enzymatic hydrolysis is to use at least one protease to enzymatically hydrolyze the antibody-drug conjugate to obtain characteristic peptide segments in the CDR region and free small molecule toxins.

[0007] In some embodiments, the antibody capture is to capture the antibody-drug conjugate in the sample using a capture antibody.

[0008] In some embodiments, in the antibody capture, the molar ratio of the capture antibody to the antibody-drug conjugate is >2:1.

[0009] In some embodiments, the molar ratio of the capture antibody to the antibody-drug conjugate is ≥4:1.

[0010] In some specific embodiments, the molar ratio of the capture antibody to the antibody-drug conjugate is (4-8):1.

[0011] In some embodiments, the capture antibody is a capture antibody carrying a label, and the label is selected from a fluorescent label, a biotin label, an enzyme-linked label, a radioisotope label, and an affinity tag.

[0012] In some embodiments, the antibody capture conditions are shaking incubation at room temperature for <4 h or shaking incubation at 2-8° C. overnight.

[0013] In some specific embodiments, the antibody capture condition is incubation with shaking at room temperature for 1 to 2 hours.

[0014] In some embodiments, the protease is selected from the group consisting of a serine protease, a cysteine ​​protease, an aspartic acid protease.

[0015] In some embodiments, the protease is selected from the group consisting of trypsin, pepsin, cathepsin, and papain;

[0016] In some specific embodiments, the proteases are trypsin and papain.

[0017] In some embodiments, in the antibody-drug conjugate, the connection mode between the antibody and the small molecule toxin is selected from polypeptide connection, disulfide bond connection and cysteine ​​connection.

[0018] In some specific embodiments, the linkage is cysteine ​​linkage.

[0019] In some specific embodiments, the enzymatic hydrolysis is: using trypsin to enzymatically hydrolyze the antibody-drug conjugate to generate characteristic peptide segments in the CDR region.

[0020] In some embodiments, the final concentration of trypsin is 10-70 μg / mL.

[0021] In some embodiments, the final concentration of trypsin is 30-70 μg / mL.

[0022] In some specific embodiments, the final concentration of trypsin is 35 μg / mL.

[0023] In some specific embodiments, papain is used to enzymatically hydrolyze the antibody-drug conjugate to produce a small molecule toxin.

[0024] In some embodiments, the final concentration of papain is 100-1000 μg / mL.

[0025] In some embodiments, the final concentration of papain is 500-1000 μg / mL.

[0026] In some specific embodiments, the final concentration of papain is 666 μg / mL.

[0027] In some embodiments, the detection is to detect the characteristic peptide segment and the small molecule toxin using LC-MS / MS to obtain the detection peak area.

[0028] In some embodiments, a linear least squares regression calculation is performed on the theoretical concentration of total antibody using the ratio of the characteristic peptide detection peak area of ​​a series of known concentrations of antibody-drug conjugates to the isotope internal standard peak area to obtain a regression equation, and the obtained regression equation is used to calculate the actual concentration of the total antibody content of the analyte.

[0029] In some embodiments, a linear least squares regression calculation is performed on the theoretical concentration of the small molecule toxin using the ratio of the detection peak area of ​​the small molecule toxin of a series of antibody-drug conjugates of known concentrations to the peak area of ​​the isotope internal standard to obtain a regression equation, and the actual concentration of the small molecule toxin content of the test object is calculated using the obtained regression equation.

[0030] In some embodiments, the DAR value is calculated based on the content of the small molecule toxin and the total antibody; the DAR value calculation formula is as follows:

[0031] DAR = theoretical DAR value * small molecule toxin content / total antibody content.

[0032] In some embodiments, the detection further comprises the steps of enriching and eluting the captured antibodies prior to enzymatic digestion.

[0033] In some embodiments, the enrichment is magnetic bead enrichment.

[0034] In some embodiments, the elution is acid elution.

[0035] In some embodiments, the acid is a 0.1%-0.5% (v / v) formic acid aqueous solution.

[0036] In some specific embodiments, the acid is a 0.1% (v / v) formic acid aqueous solution.

[0037] In some specific embodiments, the antibody-drug conjugate is trastuzumab.

[0038] In some embodiments, the capture antibody is an Anti-human Fab antibody or an Anti-human IgG antibody.

[0039] In some embodiments, the capture antibody is an Anti-human Fab antibody.

[0040] In some specific embodiments, the capture antibody is Anti-Trastuzumab Antibody 11C4.

[0041] In some embodiments, the sample to be tested is plasma.

[0042] In some specific embodiments, the plasma is human plasma or plasma from preclinical animals.

[0043] In some specific embodiments, the preclinical animal is selected from a mouse, a rat, and a cynomolgus monkey.

[0044] In some embodiments, the concentration of the antibody-drug conjugate in the test sample ranges from 0.1 to 500 pmol / mL.

[0045] In some embodiments, the concentration of the antibody-drug conjugate in the sample ranges from 0.327 to 130.8 pmol / mL.

[0046] In some specific embodiments, the concentration of the antibody-drug conjugate in the sample ranges from 26.16 to 130.8 pmol / mL.

[0047] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0048] The reagents and raw materials used in the present invention are commercially available.

[0049] The positive and progressive effects of the present invention are:

[0050] A method for detecting the DAR value of antibody-drug conjugates with high accuracy and sensitivity is provided, which solves the problem of high background value in the detection of ADC in the existing technology, and also solves the problem of poor sensitivity caused by the use of high-resolution detection of small molecule toxin antibody ratio (DAR) value. It can provide manufacturers with a series of bioanalytical strategies related to pharmacokinetic (PK) and anti-drug antibody (ADA) evaluation. DETAILED DESCRIPTION

[0051] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0052] Example 1

[0053] Main reagents, consumables and instruments

[0054] Human plasma (from Kangya Hospital, Yiyang, Hunan, EDTA-K2 anticoagulant, half plasma from 10 men and half from women)

[0055] Trastuzumab deruxtecan (Manufacturer: MCE, Product No.: HY-138298)

[0056] Anti-Trastuzumab Antibody (11C4), Mouse (Manufacturer: Genscript, Catalog No.: A02032)

[0057] EZ-Link Sulfo-NHS-LC-Biotin, No-Weigh Format (Manufacturer: Thermo Scientific, Catalog Number: A39257)

[0058] Zeba™ Dye and Biotin Removal Spin Columns, 0.5 mL (Manufacturer: Thermo Scientific, Catalog No.: A44296S)

[0059] Pierce Streptavidin-coated magnetic beads (Manufacturer: Thermo Scientific, Catalog No.: 88817)

[0060] Rapigest SF (Manufacturer: Waters, Catalog Number: 186001861)

[0061] DL-1,4-dithiothreitol (Manufacturer: Sigma-Aldrich, Catalog No.: D9779)

[0062] Iodoacetamide (Manufacturer: Sigma-Aldrich, Catalog Number: I6125)

[0063] Trypsin is from bovine pancreas (Manufacturer: Sigma-Aldrich, Catalog No.: T1005)

[0064] Papain (Manufacturer: Sigma-Aldrich, Catalog No.: 76216)

[0065] Dxd (Manufacturer: MCE, Part Number: HY-13631D)

[0066] Dxd-d5 (Manufacturer: MCE, Part Number: HY-13631DS)

[0067] FTISADTS-K*,K * Depend on 13 C6, 15 N2 marker. (Manufacturer: Shanghai Bioengineering Co., Ltd.)

[0068] EDTA (Manufacturer: Sigma, Product No.: E9884)

[0069] Tween 20 (Manufacturer: Aladdin, Product No.: T104863)

[0070] PBS buffer (Manufacturer: Gibco, Catalog No.: 10010023)

[0071] Amicon® Ultra filter, 10 kDa MWCO (Manufacturer: Millipore, Catalog No.: UFC5010)

[0072] Instrument (AB SCIEX TRIPLE QUAD TM 6500+ LC-MS

[0073] Chromatographic column (ACQUITY UPLC BEH 300 C18 1.7 μm 2.1x100 mm, Part No.: 186003686)

[0074] Step 1. Label specific antibodies with biotin

[0075] S1: Pipette 0.5 mL of 2 mg / mL Anti-Trastuzumab Antibody into a 0.5 mL 10 kDa MWCO ultrafiltration tube and centrifuge at 10,000 g for 15 min; add 0.3 mL of phosphate buffered saline (PBS) solution to the ultrafiltration tube and centrifuge under the same conditions, repeat three times;

[0076] S2: Pipette 0.5 mL of PBS solution into the ultrafiltration tube in S1, pipette repeatedly, and transfer to a new centrifuge tube;

[0077] S3: Add 15 μL of 10 mmol / L Sulfo-NHS-LCBiotin solution to 0.5 mL of the labeled solution, mix immediately, and incubate at 24-26 °C for 40 min.

[0078] S4: Take the Zeba™ dye and biotin removal centrifuge column, centrifuge at 1500 g for 1 min, and discard the filtrate; add 0.3 mL PBS, centrifuge at 1500 g for 1 min, and discard the filtrate; repeat 2 to 3 times;

[0079] S5: Take a new 1.5 mL EP tube, slowly add the reaction solution in S3 drop by drop to the Zeba™ dye and biotin removal centrifuge column, centrifuge at 1500 g for 2 min, and collect the filtrate in the EP tube.

[0080] Step 2. Bind biotinylated specific antibodies to streptavidin magnetic beads

[0081] S1: Shake the vial to completely resuspend the magnetic beads; pipette 50 μL of 10 mg / mL magnetic beads into a new 1.5 mL test tube, place the test tube on a magnetic separator for 2 min, and discard the supernatant. Add 0.5 mL of PBS solution containing 0.05% (m / v) Tween 20 to the centrifuge tube, invert it to resuspend the magnetic beads, place the test tube on a magnetic separator for 2 min, and discard the supernatant. Repeat three times;

[0082] S2: Add 150 μL of PBS solution to the magnetic beads in S1, shake up and down to resuspend the magnetic beads, pipette 50 μL of 0.523nmol / mL labeled Anti-Trastuzumab Antibody into the centrifuge tube, gently invert the tube to mix. Incubate the centrifuge tube at room temperature for 60 min with mixing and shaking;

[0083] S3: After the incubation, place the centrifuge tube on a magnetic separator for 2 min, discard the supernatant, add 0.5 mL of PBS solution containing 0.05% (m / v) Tween 20 to the centrifuge tube, turn it upside down to resuspend the magnetic beads, place the tube on a magnetic separator for 2 min, discard the supernatant, and repeat three times;

[0084] Step 3. Use the specific antibody bound to the streptavidin magnetic beads to immunocapture the Trastuzumab deruxtecan drug in the plasma, and purify and enrich it;

[0085] S1: Trastuzumab deruxtecan linear solutions with concentrations of 0.327, 0.654, 1.64, 3.27, 6.54, 26.16, 65.4, and 130.8 pmol / mL were prepared by gradient dilution using human plasma.

[0086] S2: Prepare trastuzumab deruxtecan solution with a concentration of 3.27 pmol / mL using human plasma and place it at 2-8 °C for 24 h for testing.

[0087] S3: Add 150 μL of PBS solution to the magnetic beads in step 2, invert the tube to resuspend the beads, then pipette 50 μL of the sample to be tested and the linear solution into the centrifuge tube, invert the tube to mix, and incubate at room temperature for 60 min.

[0088] S4: Add 0.5 mL of PBS solution containing 0.05% (m / v) Tween 20 to the centrifuge tube, invert it to resuspend the magnetic beads, place the tube on a magnetic separator for 2 min, discard the supernatant, and repeat three times;

[0089] Step 4. eluting the purified and enriched Trastuzumab deruxtecan with an elution buffer, and adjusting the pH with a neutralization buffer;

[0090] S1: Add 100 µL of 0.1% (v / v) formic acid in water to the centrifuge tube, vortex to mix, and incubate at room temperature with shaking for 10 min. Repeat twice.

[0091] S2: Use a magnetic separator to collect the magnetic beads and save the supernatant containing Trastuzumab deruxtecan, aspirate 100 μL of the eluted Trastuzumab deruxtecan, add 10 μL of 1 M ammonium bicarbonate solution, and mix well.

[0092] Step 5. Denature and reduce the eluted Trastuzumab deruxtecan and divide it into two portions;

[0093] S1: Pipette 110 μL of the neutralized Trastuzumab deruxtecan solution, add 10 μL of 0.4% Rapigest SF solution and 4 μL of 200 mM DTT solution, vortex mix, and incubate in a 60 ℃ constant temperature water bath for 30 min.

[0094] Step 6. One of the samples was further subjected to alkylation, neutralization, trypsinization and termination reaction, and then the internal standard was added and centrifuged for testing;

[0095] S1: Take 60 µL of the reduced solution in step 5 and restore it to room temperature, add 5 µL of 200 mM IAM solution, vortex to mix, and incubate at room temperature in the dark for 30 min;

[0096] S2: Add 2 µL of 100 mM DTT solution to the alkylated solution in S1 and incubate at room temperature for 30 min.

[0097] S3: Add 5 µL of 0.5 mg / mL trypsin solution to the neutralized solution, vortex to mix, and incubate in a 37 °C constant temperature water bath overnight;

[0098] S4: Return the digested solution to room temperature, add 2 µL of 50% formic acid, vortex to mix, add 5 µL of 0.5 µg / mL FTISADTS-K* solution, vortex to mix, centrifuge at 22,000 rpm for 10 min, and take the supernatant for testing.

[0099] Step 7. Add papain to the other portion for enzymatic digestion and termination of the reaction, add the internal standard and centrifuge before testing;

[0100] S1: Pipette 60 µL of the Trastuzumab deruxtecan solution in step 5 above, add 10 µL of 15 mM EDTA solution, add 5 µL of 10 mg / mL papain, vortex to mix, and incubate in a 37 ℃ constant temperature water bath overnight.

[0101] S2: After the reaction, add 150 µL of 0.2% (v / v) formic acid in acetonitrile solution, vortex mix, then add 10 µL of 2 µg / ml Dxd-d5 solution, vortex mix, centrifuge at 22,000 rpm for 10 min, and take the supernatant for testing.

[0102] Step 8. Detect the characteristic peptides and Dxd after enzymatic hydrolysis using LC-MS / MS and calculate the DAR value;

[0103] S1: When the characteristic peptides are detected by LC-MS / MS as described in step 8, the steps are as follows: the UPLC system is used for separation, and the analysis sample is placed in a 4 ℃ automatic sampler, the column temperature is 40 ℃, the flow rate is 300 μL / min, and the injection volume is 3 μL; the relevant liquid phase gradient is as follows: 0-0.5 min, the B phase is maintained at 2%; 0.5-4.0 min, the B phase changes linearly from 2% to 15%; 4.0-6.0 min, the B phase changes linearly from 15% to 90%; 6.0-7.0 min, the B phase is maintained at 90%; 7.0-7.01 min, the B phase changes linearly from 90% to 2%; 7.01-8.0 min, the B phase is maintained at 2%. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. The chromatographic column model was ACQUITY UPLC BEH300 C18 1.7 μm 2.1x100 mm.

[0104] S2: The mass spectrometry conditions for detecting characteristic peptides using LC-MS / MS as described in step 8 are set as follows:

[0105] Using AB SCIEX TRIPLE QUAD TM The 6500+ LC / MS instrument was used for mass spectrometry analysis in the positive ion mode. The ESI source positive ion conditions were as follows: Collision Gas: 9; source temperature: 500°C; ion Source Gas1 (Gas1): 50; Ion Source Gas2 (Gas2): 50; Curtain gas (CUR): 40; Ion Sapary Voltage Floating (ISVF): 5500 V. The MRM mode was used to detect the ion pairs to be measured. The MRM ion pair information is shown in Table 1.

[0106] Table 1 MRM ion pair information of characteristic peptides

[0107]

[0108] S3: When using LC-MS / MS to detect Dxd as described in step 8, the steps are as follows: Use UPLC system for separation, place the analytical sample in a 4 ℃ autosampler, column temperature 40 ℃, flow rate 500 μL / min, injection volume 6 μL; the relevant liquid phase gradient is as follows: 0-0.3 min, B phase is maintained at 20%; 0.3-1.5 min, B phase changes linearly from 20% to 95%; 1.5-1.95min, B phase is maintained at 95%; 1.95-1.96 min, B phase changes linearly from 95% to 20%; 1.96-2.5 min, B phase is maintained at 20%. Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid; the chromatographic column model is ACQUITY UPLC ® Peptide BEH C18 300Å (2.1mm×100mm, 1.7 μm).

[0109] S4: The mass spectrometry conditions for Dxd detection using LC-MS / MS described in step 8 are set as follows:

[0110] Using AB SCIEX TRIPLE QUAD TM The 6500+ LC / MS instrument was used for mass spectrometry analysis in the positive ion mode. The ESI source positive ion conditions were as follows: Collision Gas: 9; source temperature: 500°C; ion Source Gas1 (Gas1): 60; Ion Source Gas2 (Gas2): 50; Curtain gas (CUR): 40; Ion Sapary Voltage Floating (ISVF): 5500 V. The MRM mode was used to detect the ion pairs to be measured. The MRM ion pair information is shown in Table 2.

[0111] Table 2 MRM ion pair information of small molecule toxin Dxd

[0112]

[0113] Step 8 is to use Analyst software to analyze the data, use the peak area of ​​the analyte to the peak area of ​​the isotope internal standard to perform a linear least squares regression calculation on the theoretical concentration of the analyte in the standard curve, and use the obtained regression equation to calculate the measured concentration of the analyte, with a weight factor of 1 / x 2 ; The DAR value calculation formula is as follows

[0114] DAR value = 8*the content of coupled small molecule toxin / the content of total antibody

[0115] The results are shown in Table 3, which shows that the average DAR value is close to the theoretical DAR value of 8.

[0116] Table 3 DAR value results of Trastuzumab deruxtecan in human plasma in Example 1

[0117]

[0118] Comparative Example 1

[0119] The specific implementation of Comparative Example 1 is the same as that of Example 1, except that the concentration of Trastuzumabderuxtecan used is 130.8 pmol / mL, and the ratios of the specific antibody to Trastuzumab deruxtecan are 2:1, 4:1 and 8:1. The results are shown in Table 4. When the ratio of the specific antibody to Trastuzumab deruxtecan is 2:1, the average ratio of the peptide peak area of ​​T-Dxd to the internal standard peptide is less than the ratios of the specific antibody to Trastuzumab deruxtecan of 4:1 and 8:1, indicating that when the ratio of the specific antibody to Trastuzumab deruxtecan is lower than 4:1, T-Dxd may not be fully captured.

[0120] Table 4 Capture of biotin-labeled specific antibodies in Comparative Example 1 at different ratios to Trastuzumab deruxtecan concentrations in human plasma

[0121]

[0122] Comparative Example 2

[0123] The specific implementation of Comparative Example 2 is the same as that of Example 1, except that the concentration of Trastuzumabderuxtecan used is 26.16 pmol / mL, and the specific antibodies are Anti-Trastuzumab Antibody (AY5b) and Anti-Trastuzumab Antibody (11C4). The results are shown in Table 5. When capturing Trastuzumabderuxtecan in plasma, Anti-Trastuzumab Antibody (11C4) is superior to Anti-Trastuzumab Antibody (AY5b).

[0124] Table 5 Capture of Trastuzumab deruxtecan in human plasma by different types of specific antibodies in Comparative Example 2

[0125]

[0126] Comparative Example 3

[0127] The specific implementation of Comparative Example 3 is the same as that of Example 1, except that the concentration of Trastuzumab deruxtecan used is 65.4 pmol / mL, and the capture time is 1 hour, 2 hours, 4 hours and overnight. The results are shown in Table 6. When the capture time is 1 hour, all the cells are captured.

[0128] Table 6 Capture of Trastuzumab deruxtecan in human plasma at different capture times in Comparative Example 3

[0129]

[0130] Comparative Example 4

[0131] The specific implementation of Comparative Example 4 is the same as that of Example 1, except that the concentration of Trastuzumabderuxtecan used is 523.2 pmol / m, and the enzymes for enzymatically hydrolyzing the linker are cathepsin B, papain, Ides protease, and IdeZ protease. The results are shown in Table 7, and cathepsin B, Ides protease, and IdeZ protease did not completely cleave the linker.

[0132] Table 7 Enzymatic cleavage of the linker in Trastuzumab deruxtecan in human plasma by different proteases in Comparative Example 4

[0133]

[0134] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. A method for determining the DAR value of an antibody-drug conjugate, characterized in that: The method comprises antibody capture, enzymatic hydrolysis and detection of the sample to be tested; The enzymatic hydrolysis is to use at least one protease to enzymatically hydrolyze the antibody-drug conjugate to obtain characteristic peptide segments in the CDR region and free small molecule toxins; The antibody capture is to use a capture antibody to capture the antibody-coupled drug in the sample.

2. The method according to claim 1, characterized in that In the antibody capture, the molar ratio of the capture antibody to the antibody-drug conjugate is >2:1, preferably ≥4:1, and more preferably (4-8):1; And / or, the capture antibody is a capture antibody carrying a label, and the label is selected from the group consisting of a fluorescein label, a biotin label, an enzyme-linked label, a radioisotope label and an affinity tag; And / or, the antibody capture condition is shaking incubation at room temperature for <4 h or shaking incubation at 2-8° C. overnight, preferably shaking incubation at room temperature for 1-2 h.

3. The method according to claim 1 or 2, characterized in that The protease is selected from the group consisting of serine protease, cysteine ​​protease, and aspartic acid protease; Preferably, the protease is selected from trypsin, pepsin, cathepsin and papain; More preferably, the proteases are trypsin and papain.

4. The method according to any one of claims 1 to 3, characterized in that In the antibody-drug conjugate, the connection mode between the antibody and the small molecule toxin is selected from polypeptide connection, disulfide bond connection and cysteine ​​connection; preferably, the connection mode is cysteine ​​connection; and / or, The enzymatic hydrolysis is: using trypsin to enzymatically hydrolyze the antibody-drug conjugate to generate characteristic peptide segments in the CDR region; preferably, the final concentration of the trypsin is 10-70 μg / mL or 30-70 μg / mL, for example, 35 μg / mL; And / or, using papain to enzymatically hydrolyze the antibody-drug conjugate to produce free small molecule toxins; preferably, the final concentration of the papain is 100-1000 μg / mL or 500-1000 μg / mL, for example, 666 μg / mL.

5. The method according to any one of claims 1 to 4, characterized in that The detection is to detect the characteristic peptide segment and the small molecule toxin using LC-MS / MS to obtain the detection peak area; The theoretical concentration of the total antibody is calculated by linear least squares regression using the ratio of the characteristic peptide detection peak area of ​​a series of antibody-drug conjugates with known concentrations to the isotope internal standard peak area to obtain a regression equation, and the actual concentration of the total antibody content of the analyte is calculated using the obtained regression equation; The theoretical concentration of the small molecule toxin is calculated by linear least squares regression using the ratio of the detection peak area of ​​the small molecule toxin of a series of antibody-drug conjugates with known concentrations to the peak area of ​​the isotope internal standard to obtain a regression equation, and the actual concentration of the small molecule toxin content of the test object is calculated using the obtained regression equation; The DAR value is calculated according to the content of the small molecule toxin and the total antibody; the DAR value calculation formula is as follows: DAR = theoretical DAR value * small molecule toxin content / total antibody content.

6. The method according to any one of claims 1 to 5, characterized in that The detection also includes the steps of enriching and eluting the captured antibodies before enzymatic hydrolysis; Preferably, the enrichment is magnetic bead enrichment; and / or the elution is acid elution, and the acid is preferably 0.1%-0.5% (v / v) formic acid aqueous solution.

7. The method according to any one of claims 1 to 6, characterized in that The antibody-drug conjugate is trastuzumab.

8. The method according to any one of claims 1 to 7, characterized in that The capture antibody is an Anti-human Fab antibody or an Anti-human IgG antibody; Preferably, the capture antibody is an Anti-human Fab antibody; More preferably, the capture antibody is Anti-Trastuzumab Antibody 11C4.

9. The method according to any one of claims 1 to 8, characterized in that The sample to be tested is plasma; Preferably, the plasma is human plasma or plasma from preclinical animals; More preferably, the preclinical animal is selected from the group consisting of mice, rats and cynomolgus monkeys.

10. The method according to any one of claims 2 to 9, characterized in that The concentration range of the antibody-drug conjugate in the test sample is 0.1-500 pmol / mL; Preferably, the concentration of the antibody-drug conjugate in the sample is in the range of 0.327 to 130.8 pmol / mL; More preferably, the concentration range of the antibody-drug conjugate in the sample is 26.16-130.8 pmol / mL.

Images