A method for detecting the coupling effect of antibody-drug conjugate

Abstract

The present invention discloses a method for detecting the coupling effect of an antibody drug conjugate (ADC). The method comprises the steps of: detecting a sample to be tested by LC-SEC, and detecting the sample to be tested by LC-Q-TOF after treating the sample with a reducing agent, which can analyze the overall coupling effect of the ADC drug and can characterize the number of couplings on the light and heavy chains in detail, monitor the coupling process of the ADC drug in a timely and accurate manner, and the method is fast, simple, highly sensitive, and highly specific.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting the coupling effect of an antibody-drug conjugate and an application thereof. Background Art

[0002] Antibody-drug conjugate (ADC) is a chemical link that connects a small molecule drug with biological activity to a monoclonal antibody. The monoclonal antibody acts as a carrier to transport the small molecule drug to the target cells. The antibody molecule mainly plays a role in targeted delivery, the small molecule drug plays a role in efficacy, and the linker plays a role in connecting the antibody and the small molecule cytotoxic drug.

[0003] An important indicator for evaluating ADC is DAR (drug-antibody ratio): the number of drug molecules that can be carried on a unit number of antibodies. A DAR value that is too high or too low will affect the effectiveness, safety and stability of ADC drugs. Non-site-coupled ADC drugs have multiple coupling sites, which will form a mixture with different DAR values. A method with high sensitivity and strong separation ability is needed to monitor the coupling effect of ADC drugs. Summary of the invention

[0004] In view of this, the present invention provides a method for detecting the coupling effect of an antibody-drug conjugate and its application.

[0005] In a first aspect of the present invention, a method for detecting the coupling effect of an antibody-drug conjugate is provided, comprising the steps of detecting a sample to be tested by LC-SEC, and detecting the sample to be tested by LC-Q-TOF after treating the sample to be tested with a reducing agent.

[0006] Optionally, the method further comprises the step of complementing the obtained LC-SEC detection results and LC-Q-TOF detection results.

[0007] Specifically, the stationary phase of the LC-SEC can be TSKgel SW XL Series columns, such as TSKgelG2000SW XL 、G3000SW XL and G4000SW XL , especially TSKgel G3000SW XL .

[0008] Specifically, the particle size of the stationary phase of the LC-SEC can be 1-10 μm (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μm), especially 5, 8 μm.

[0009] In some embodiments of the present invention, the stationary phase of LC-SEC is TSKgel G3000SW XL , 7.8mmI.D.*30cm, 5μm.

[0010] In some embodiments of the present invention, the instrument used for LC-SEC detection is Nexera XR LC-20AD.

[0011] Specifically, the column temperature of the LC-SEC can be 20-30°C (eg, 20, 22, 24, 25, 26, 28, 30°C), in particular room temperature.

[0012] Specifically, the mobile phase of the LC-SEC is an aqueous solution of sodium phosphate and sodium chloride, wherein the concentration of sodium phosphate can be 20-100mmol / L (e.g., 20, 40, 50, 60, 80, 100mmol / L), particularly 40-60mmol / L, for example, 50mmol / L; the concentration of sodium chloride can be 100-500mmol / L (e.g., 100, 150, 200, 250, 280, 300, 320, 350, 400, 450, 500mmol / L), particularly 250-350mmol / L, for example, 300mmol / L.

[0013] Specifically, the flow rate of the mobile phase is 0.1-5 mL / min (e.g., 0.1, 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 mL / min), in particular 0.5-2 mL / min, for example 1 mL / min.

[0014] Specifically, the LC-SEC detection adopts an isocratic elution procedure; more specifically, the elution time can be 10-60 min (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min), in particular 15-30 min, for example 20 min.

[0015] Specifically, the detector for LC-SEC detection is an ultraviolet detector; more specifically, the detection wavelength is 250-300 nm (eg, 250, 260, 270, 280, 290, 300 nm), especially 280 nm.

[0016] Specifically, in the LC-SEC detection, the injection volume is 1-20 μL (such as 1, 5, 6, 8, 10, 12, 14, 15, 16, 18, 20 μL), especially 5-15 μL, for example 10 μL.

[0017] Specifically, the step of treating the sample to be tested with a reducing agent and then detecting it by LC-Q-TOF includes:

[0018] (1) mixing the sample to be tested with a reducing agent to perform a reduction reaction;

[0019] (2) The product obtained in step (1) is detected by LC-Q-TOF.

[0020] Specifically, if necessary, the sample to be tested can be diluted and mixed with a reducing agent, and the diluent can be a buffer solution, such as NH 4 HCO 3 Solution.

[0021] Specifically, the reducing agent can be selected from: β-mercaptoethanol, tris(2-carboxyethyl)phosphine, dithiothreitol (DTT) or a combination thereof, especially DTT.

[0022] In one embodiment of the present invention, the reducing agent is DTT, and its concentration is 100-1000mmol / L (e.g., 100, 200, 300, 400, 500, 600, 800, 1000mmol / L), for example, 200mmol / L.

[0023] Specifically, the temperature of the reduction reaction in step (1) is 35-40°C (e.g., 35, 36, 37, 38, 39, 40°C), especially 37°C.

[0024] Specifically, the reduction reaction time in step (1) is 10-30 minutes (eg, 10, 15, 20, 25, 30 minutes), especially 20 minutes.

[0025] Specifically, the reduction reaction in step (1) is carried out under shaking conditions.

[0026] In one embodiment of the present invention, the stationary phase of the LC-Q-TOF is a MAbPac RP chromatographic column.

[0027] Specifically, the particle size of the stationary phase of the LC-Q-TOF can be 1-10 μm (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μm), especially 4 μm.

[0028] In some embodiments of the present invention, the stationary phase of the LC-Q-TOF is MAbPac RP 4um, 2.1*150mm.

[0029] In some embodiments of the present invention, the liquid chromatograph used for the LC-Q-TOF detection is a SCIEX Exion LC liquid phase system.

[0030] In some embodiments of the present invention, the mass spectrometer used for the LC-Q-TOF detection is a SCIEX Triple TOF6600 system.

[0031] Specifically, the column temperature of the LC-Q-TOF is 60-100°C (e.g., 60, 65, 70, 75, 76, 78, 80, 82, 84, 85, 90, 95, 100°C), in particular 75-85°C, e.g., 80°C.

[0032] Specifically, the mobile phase of the LC-Q-TOF consists of a mobile phase aqueous phase (A) and a mobile phase organic phase (B).

[0033] Specifically, mobile phase A is an aqueous formic acid solution with a concentration of 0.01-1% (volume percentage) (e.g. 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.4%, 0.5%, 1%), particularly 0.05-0.2%, e.g. 0.1%.

[0034] Specifically, mobile phase B is an acetonitrile solution of formic acid with a concentration of 0.01-1% (volume percentage) (e.g. 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.4%, 0.5%, 1%), particularly 0.05-0.2%, e.g. 0.1%.

[0035] Specifically, the flow rate of the mobile phase is 0.05-1 mL / min (e.g., 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mL / min), in particular 0.1-0.5 mL / min, for example 0.2 mL / min.

[0036] Specifically, the liquid chromatography detection of the LC-Q-TOF adopts gradient elution; more specifically, the elution procedure includes:

[0037] 0 min, mobile phase B, 5-15%, mobile phase A, balance;

[0038] 0-10min, mobile phase B, 5-15%→90%, mobile phase A, balance;

[0039] 10-20min, mobile phase B, 90%;

[0040] 20-20.1min, mobile phase B, 90% → 5-15%, mobile phase A, balance;

[0041] 20.1-35min, mobile phase B, 5-15%→stop, mobile phase A, balance.

[0042] In some embodiments of the invention, the elution procedure comprises:

[0043] 0 min, mobile phase B, 10%, mobile phase A, balance;

[0044] 0-10min, mobile phase B, 10%→90%, mobile phase A, balance;

[0045] 10-20min, mobile phase B, 90%;

[0046] 20-20.1min, mobile phase B, 90%→10%, mobile phase A, balance;

[0047] 20.1-35min, mobile phase B, 10%→stop, mobile phase A, remainder.

[0048] Specifically, the injection volume of the LC-Q-TOF detection is 1-10 μL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μL), especially 2 μL.

[0049] Specifically, for the LC-Q-TOF detection, the ion source of the mass spectrometer is ESI, and the ionization mode is positive ion.

[0050] Specifically, for the LC-Q-TOF detection, the scanning mass range of the mass spectrometer is 100-5000 m / z.

[0051] Specifically, for the LC-Q-TOF detection, the accumulation time of the mass spectrum was 0.15 seconds.

[0052] Specifically, for the LC-Q-TOF detection, the ion source temperature of the mass spectrometer is 450°C.

[0053] Specifically, for the LC-Q-TOF detection, the ion spray voltage of the mass spectrometer was 5500V.

[0054] Specifically, for the LC-Q-TOF detection, the curtain gas voltage of the mass spectrometer was 35 psi.

[0055] Specifically, for the LC-Q-TOF detection, the declustering voltage (DP) of the mass spectrometer was 300V.

[0056] Specifically, for the LC-Q-TOF detection, the fragmentation voltage of the mass spectrometer is 10V.

[0057] Specifically, for the LC-Q-TOF detection, the Time bins to sum of the mass spectrum is 80.

[0058] Specifically, the samples to be tested include ADC samples and antibody samples.

[0059] Specifically, the ADC consists of three parts: an antibody, a drug, and a linker.

[0060] In some embodiments of the present invention, the linker of the ADC is a polymer structure part, especially a water-soluble polymer, such as polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyoxazoline, polyacrylamide morpholine, polysialic acid, dextran, etc., especially polyethylene glycol.

[0061] Specifically, the molecular weight of the polymer is 400-100000 Daltons (e.g., 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, 50000, 100000 Daltons), especially 400-4000 Daltons.

[0062] Specifically, the polyethylene glycol is a linear or branched polyethylene glycol, especially a linear polyethylene glycol.

[0063] Specifically, the polyethylene glycol includes an activating group that is activated at one end, two ends or multiple ends, especially an activating group that is activated at one end; more specifically, the activating group includes, but is not limited to, an aldehyde group, a carboxyl group, an ester group, an amide group, a thiol group, an epoxy group, a maleimide group, succinimidyl carbonate, succinimidyl acetate, succinimidyl propionate, carbonyl aldehyde, hydrazide, amino group, etc.

[0064] Specifically, the antibody is a monoclonal antibody; in one embodiment of the present invention, the antibody is an EGFR monoclonal antibody, such as Necitumumab.

[0065] In one embodiment of the present invention, the drug is a small molecule anti-tumor drug, such as ixabepilone, mitomycin, plicamycin, bleomycin, pyrantelone, amrubicin, valrubicin, pirarubicin, mitoxantrone, idarubicin, daunorubicin, aclarubicin, epirubicin, daunorubicin, doxorubicin, dactinomycin, trabectedin, cabazitaxel, paclitaxel polyaniline, docetaxel, paclitaxel, demecillin, teniposide, etoposide, vinforin , vinflunine, vinorelbine, vindesine, vincristine, vinblastine, camptothecin derivatives (e.g., 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin (SN-38), 9-nitrocamptothecin, 9-aminocamptothecin, irinotecan, topotecan and belotecan, exatecan, lurtotecan, diflomotecan, gimatecan, karenitecin), floxuridine, trifluridine, tegafur, fluorouracil, decitabine, azacitidine, capecitabine, gemcitabine, carmofur, cytarabine, nelarabine, clofarabine, fludarabine, cladribine, thioguanine, mercaptopurine, pralatrexate, pemetrexed, raltitrexed, methotrexate Amine, dacarbazine, temozolomide, pipobromide, mitropol, etoside, uracil mustard, ranimustine, nimustine, flutemustine, streptozotocin, semustine, lomustine, carmustine, carboquinone, triazine quinone, thiotepa, mannosulfuron, trothiosulfuron, busulfan, bendamustine, prednimustine, trofosfamide, ifosfamide, methylchlorethylamine, melphalan, chlorambucil, cyclophosphamide, cisplatin, carboplatin, oxaliplatin, satraplatin and polyplatin, etc.

[0066] In some embodiments of the present invention, the drug is a camptothecin derivative, such as 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin (SN-38), 9-nitrocamptothecin, 9-aminocamptothecin, irinotecan, topotecan, belotecan, exatecan, lurtotecan, diflomotecan, gimatecan, karenitecin, in particular 7-ethyl-10-hydroxycamptothecin or irinotecan.

[0067] In one embodiment of the present invention, the ADC is

[0068]

[0069] Among them, Ab is the monoclonal antibody part, and D is the small molecule drug part.

[0070] Specifically, n is an integer of 1-100 (e.g., 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100), especially an integer of 1-20.

[0071] Specifically, m is an integer of 1-100 (e.g., 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100), especially an integer of 1-20.

[0072] Specifically, l is an integer of 1-50 (e.g., 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50), especially an integer of 1-10.

[0073] In some embodiments of the present invention, the ADC is

[0074]

[0075] In some embodiments of the present invention, Ab is Necitumumab.

[0076] In some embodiments of the present invention, the D portion of the ADC is selected from the following structures:

[0077]

[0078] In a second aspect of the present invention, there is provided application of the method described in the first aspect in quality control and quality evaluation of ADC.

[0079] Specifically, the ADC has the definition described in the first aspect of the present invention.

[0080] The detection method of the present invention is based on high-sensitivity and high-resolution LC-Q-TOF, combined with the LC-SEC method for efficient separation based on molecular weight differences, which can analyze the overall coupling effect of ADC drugs and can characterize the number of couplings on the light and heavy chains in detail, and timely and accurately monitor the coupling process of ADC drugs. The method is fast, simple, highly sensitive and highly specific. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Shown is the LC-SEC spectrum of the ADC obtained by detection in Example 1.

[0082] Figure 2 Shown is the Q-TOF spectrum of the ADC (1:20) light chain detected in Example 2.

[0083] Figure 3 Shown is the Q-TOF spectrum of the heavy chain of ADC (1:20) detected in Example 2.

[0084] Figure 4 Shown is the Q-TOF spectrum of the ADC (1:10) light chain detected in Example 2.

[0085] Figure 5 Shown is the Q-TOF spectrum of the heavy chain of ADC (1:10) detected in Example 2. DETAILED DESCRIPTION

[0086] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0087]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0088] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0089] Example 1: LC-SEC method to analyze the overall coupling effect of ADC

[0090] The ADC prepared by the inventor is used as an example to illustrate the detection method and its detection effect of the present invention. The antibody in the ADC is EGFR monoclonal antibody Necitumumab, purchased from Eli Lilly. The ADC is a monoclonal antibody to EGFR (hereinafter referred to as mAb) and SPA-(CH 2 CH 2 O) 4 -DBCO-(CH 2 CH 2 O) 8 -VC-PAB-CO-D (provided by Beijing Jiankai Technology Co., Ltd., with the structural formula as follows) was obtained by reacting with a feed ratio of 1:10 and 1:20 respectively.

[0091]

[0092] 1. Experimental methods

[0093] The ADC samples were directly injected into the LC-SEC system. The pure EGFR monoclonal antibody was tested and analyzed under the same conditions.

[0094] Table 1 Instruments and parameters

[0095] HPLC-UV SHIMADZU Nexera XR LC-20AD, PDA detector SPD-M40 Chromatographic columns <![CDATA[TOSOH TSKgel G3000SW XL ,7.8mmI.D.*30cm,5μm,P / N:0008541]]> Flow rate 1mL / min Column temperature Room temperature Isocratic 100% A for 20 min Mobile phase MPA: 50mmol / L sodium phosphate + 300mmol / L sodium chloride aqueous solution UV wavelength 280nm Injection volume 10μL Sample concentration Stock solution injection

[0096] 2. Experimental results

[0097] The LC-SEC spectrum of ADC samples is as follows: Figure 1 As shown, compared with the pure monoclonal antibody, it can be seen that the ADC drug has multiple new peaks in the larger molecular weight region, which are the monoclonal antibody conjugate peaks with different numbers of Linker+drugs coupled.

[0098] In addition, by Figure 1 It can also be seen that the new peak intensity generated by ADC (1:20) is stronger than that of ADC (1:10), indicating that ADC (1:20) is coupled with more Linker+drugs than ADC (1:10).

[0099] Example 2: Q-TOF method to analyze the coupling effect of ADC light and heavy chains

[0100] 1. Experimental methods

[0101] 50 μL of ADC sample was added with 2.5 μL of 200 mM DTT solution, shaken at 37°C for 30 minutes, and directly injected into the Q-TOF system.

[0102] Table 2 Instruments and parameters

[0103]

[0104] 2. Experimental results

[0105] The Q-TOF spectra of ADC (1:20) light chain and heavy chain were analyzed as follows: Figure 2 and 3 The mass spectrometry data of ADC was deconvoluted and compared with the pure monoclonal antibody, it can be seen that ADC is conjugated with 1 to 6 drugs on the light chain and 1 to 2 drugs on the heavy chain.

[0106] The Q-TOF spectra of ADC (1:10) light chain and heavy chain are analyzed as follows: Figure 4 and 5 The mass spectrometry data of ADC was deconvoluted and compared with the pure monoclonal antibody, it can be seen that ADC is conjugated with 4 drugs on the light chain and 1 to 2 drugs on the heavy chain.

[0107] The number of couplings and coupling peak intensity of ADC (1:10) coupled with Linker+drug on the light chain are less than those of ADC (1:20), which is consistent with the LC-SEC results.

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0109] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0110] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A method for detecting the coupling effect of an antibody-drug conjugate, comprising: The samples to be tested are tested by LC-SEC, and the overall coupling effect of ADC is obtained based on the molecular weight difference; The sample to be tested is treated with a reducing agent and then detected by LC-Q-TOF. The mass spectrum data of the ADC is deconvoluted and compared with the pure monoclonal antibody to obtain the coupling effect of the light chain and heavy chain of the ADC; the obtained LC-SEC detection results and LC-Q-TOF detection results are complemented with each other; The samples to be tested include ADC samples and antibody samples; the ADC consists of three parts: antibody, drug and linker; the linker is polyethylene glycol; the antibody is EGFR monoclonal antibody; the drug is a camptothecin derivative; The reducing agent is selected from: a combination of one or more of β-mercaptoethanol, tris(2-carboxyethyl)phosphine, and dithiothreitol; The stationary phase of the LC-SEC was TSKgel G2000SW XL 、G3000SW XL or G4000SW XL ; The mobile phase of the LC-SEC is an aqueous solution of sodium phosphate and sodium chloride, wherein the concentration of sodium phosphate is 20-100 mmol / L, and the concentration of sodium chloride is 100-500 mmol / L; The stationary phase of the LC-Q-TOF is a MAbPac RP chromatographic column.

2. The method according to claim 1, characterized in that The step of treating the sample to be tested with a reducing agent and then detecting it by LC-Q-TOF comprises: (1) Mix the sample to be tested with the reducing agent to carry out a reduction reaction; (2) The product obtained in step (1) is detected by LC-Q-TOF.

3. The method according to claim 1, characterized in that The particle size of the stationary phase of the LC-SEC is 1-10 μm.

4. The method according to claim 3, characterized in that The particle size of the stationary phase of the LC-SEC is 5 or 8 μm.

5. The method according to claim 1, characterized in that The column temperature of the LC-SEC was 20-30°C.

6. The method according to claim 1, characterized in that The concentration of the sodium phosphate is 40-60 mmol / L.

7. The method according to claim 1, characterized in that The concentration of the sodium chloride is 250-350 mmol / L.

8. The method according to claim 1, characterized in that The flow rate of the mobile phase of the LC-SEC is 0.1-5 mL / min.

9. The method according to claim 8, characterized in that The flow rate of the mobile phase of the LC-SEC is 0.5-2 mL / min.

10. The method according to claim 1, characterized in that The LC-SEC detection adopted an isocratic elution program.

11. The method according to claim 2, characterized in that The temperature of the reduction reaction in step (1) is 35-40°C.

12. The method according to claim 2, characterized in that The reduction reaction time in step (1) is 10-30 minutes.

13. The method according to claim 1, characterized in that The particle size of the stationary phase of the LC-Q-TOF is 1-10 μm.

14. The method according to claim 1, wherein: The column temperature of the LC-Q-TOF is 60-100°C.

15. The method according to claim 14, characterized in that The column temperature of the LC-Q-TOF was 75-85°C.

16. The method according to claim 1, wherein: The mobile phase of the LC-Q-TOF consists of mobile phase A and mobile phase B, wherein the mobile phase A is a formic acid aqueous solution with a volume percentage concentration of 0.01-1%; the mobile phase B is a formic acid acetonitrile solution with a volume percentage concentration of 0.01-1%.

17. The method according to claim 16, characterized in that The mobile phase A is a formic acid aqueous solution with a volume percentage concentration of 0.05-0.2%.

18. The method according to claim 16, characterized in that The mobile phase B is an acetonitrile solution of formic acid, and its volume percentage concentration is 0.05-0.2%.

19. The method according to claim 16, characterized in that The LC-Q-TOF adopts gradient elution, and the elution procedure includes: 0 min, mobile phase B, 5-15%, mobile phase A, balance; 0-10min, mobile phase B, 5-15%→90%, mobile phase A, balance; 10-20 min, mobile phase B, 90%; 20-20.1min, mobile phase B, 90%→5-15%, mobile phase A, balance; 20.1-35min, mobile phase B, 5-15%→stop, mobile phase A, balance.

20. The method according to any one of claims 1 to 19, characterized in that The camptothecin derivative is selected from: 10-hydroxycamptothecin, 7-ethyl-10-hydroxycamptothecin (SN-38), 9-nitrocamptothecin, 9-aminocamptothecin, irinotecan, topotecan, belotecan, exatecan, rutotecan, diflutecan, gimatidecan or carnitine.

21. The method according to any one of claims 1 to 19, characterized in that: The ADC is a monoclonal antibody -COCH2CH2(CH2CH2O) n -DBCO-(CH2CH2O) m -VC-PAB-CO-D', n is an integer of 1-100, m is an integer of 1-100, and D' is a small molecule drug moiety.

22. The method according to claim 21, characterized in that The D' portion is selected from: 。 23. Use of the method according to any one of claims 1 to 22 in quality control and quality assessment of ADC.

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