Detection method for neutralizing antibody of therapeutic monoclonal antibody and application

Abstract

The present application provides a detection method and use for neutralizing antibodies for therapeutic monoclonal antibodies, which is a CLB NAb assay developed through target-based drug depletion and drug-based NAb extraction for CMAB009 with high drug and target tolerance. Acid is typically used in the pretreatment of a sample to dissociate the drug / NAb complex. In order to improve the treatment effect, a two-step method involving an amphiphilic method and a bead method is adopted. Initially, EGFR-coated magnetic beads (target beads) are used to eliminate the majority of free drugs in a sample. Subsequently, after acid dissociation of the sample, NAb is captured with beads. NAb is neutralized and eluted by acid, and a biotinylated drug is added into an EGFR-coated flat plate for detecting NAb. A comprehensive experiment strategy is implemented, meanwhile, drug interference is relieved, and target tolerance is enhanced. Through troubleshooting and optimization, NAb determination is verified to be used for clinical sample analysis, so that the NAb is successfully applied to III-stage clinical immunogenicity NAb detection of CMAB009. The result shows that the integrated detection optimization strategy can greatly improve the key performance characteristics of CLB NAb detection, so that the potential immunogenicity and the influence of the potential immunogenicity on better biological development can be evaluated more reliably.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for detecting therapeutic monoclonal antibody neutralizing antibody. Background Art

[0002] Therapeutic proteins are considered the most important biologics due to their clinical utility. However, therapeutic proteins have the potential to induce unwanted anti-drug antibody (ADA) immune responses. ADA responses to therapeutic proteins can be divided into neutralizing antibodies (NAbs) and non-neutralizing antibodies (non-NAbs). NAbs are a subset of ADAs that bind to key epitopes and hinder the biological activity of therapeutic proteins by preventing the drug from binding to its intended target. In some cases, NAbs can also cause adverse clinical consequences, reduce drug efficacy and affect patient safety. Therefore, regulatory agencies require the detection of NAb activity to evaluate the immunogenicity of therapeutic protein products, especially bioimproved products, during development.

[0003] NAb detection formats are generally divided into two types: cell-based assays and non-cell-based assays, among which non-cell-based includes competitive ligand binding (CLB) assays. Cell-free assays for NAb detection are reproducible, easy to perform, and cost-effective. Generally, the choice of cell-based or CLB assay format depends on the mechanism of action (MOA) of the drug. If the drug directly binds to and neutralizes the target, the CLB format is preferred. However, since drugs or targets in the sample can interfere with the accuracy of NAb detection, NAb detection formats with sufficient drug and target tolerance are required. Several methods have been successfully used to mitigate drug and target interference, including affinity capture elution (ACE), solid phase acid dissociation (SPEAD), and biotin-drug extraction and acid dissociation (BEAD).

[0004] Cetuximab (Erbitux) is a recombinant human-mouse chimeric monoclonal IgG1 antibody targeting epidermal growth factor receptor (EGFR). It is the first antibody drug approved for the treatment of irinotecan-refractory and / or oxaliplatin-refractory metastatic colorectal cancer (mCRC). Cetuximab is also the only EGFR-targeted therapy approved by the FDA for the treatment of head and neck squamous cell carcinoma (HNSCC). The drug is designed to inhibit tumor proliferation by blocking the binding of EGF to EGFR expressed by tumor cells. This original mAb drug (Ebitux), produced by SP2 / 0 murine myeloma cells, is N-glycosylated in both the Fc and Fab fragments, which has been shown to affect safety pharmacokinetics / pharmacodynamics. In addition, the terminal sialic acid form of Ebitux is mainly N-glycolylneuraminic acid (NGNA), which is also potentially immunogenic. It is relevant to the development of bioimprovers and next-generation antibodies.

[0005] CMAB009 is a potential biological candidate for cetuximab with the same amino acid sequence as Ebitux. In vitro analysis showed that CMAB009 has similar abilities to Ebitux in mediating antibody-dependent cellular cytotoxicity and inhibiting cell proliferation. CMAB009 is expressed by Chinese hamster ovary (CHO) cells, which are characterized by the absence of α-Gal and the presence of predominantly N-acetylneuraminic acid (NANA) as sialic acid. This ideal human-type sialylation suggests that CMAB009 may have better clinical tolerability and a lower likelihood of active hypersensitivity reactions compared to Ebitux. Reducing potential immunogenicity is critical for developing a biologically improved version of cetuximab, as the development of an immune response may reduce therapeutic efficacy and potential active hypersensitivity reactions. One of the requirements for clinical studies of CMAB009 is the development of a NAb assay, and the assay requires adequate drug and target tolerance. Currently, a highly drug / target-tolerant NAb assay for CMAB009 has not been reported. Summary of the invention

[0006] The present invention aims to provide a method for detecting therapeutic monoclonal antibody neutralizing antibodies, in particular a method for detecting EGFR therapeutic monoclonal antibody neutralizing antibodies, and a method for determining NAbs with high drug / target tolerance. The EGFR therapeutic monoclonal antibody is preferably cetuximab, and the further preferred cetuximab is CMAB009.

[0007] The operation includes pretreatment, dissociation, binding, secondary dissociation, competitive binding and detection. The specific process is as follows: 1. Pretreatment: add target magnetic beads to the sample to be tested, shake at 800 rpm for 1 hour at room temperature, and after the free drug in the sample is fully combined with the target magnetic beads, remove the target magnetic bead-drug complex to obtain the pretreated supernatant, and then transfer the supernatant to a new plate.

[0008] 2. Dissociation: Dissociate the pretreated supernatant with 300 mmol / L HAc at room temperature for 5 minutes and shake at 800 rpm to obtain a dissociation solution.

[0009] 3. Binding, add excess drug magnetic beads to the dissociation solution, add 1 M Tris to neutralize the solution at room temperature for 1 hour to obtain drug magnetic beads-NAb complex.

[0010] 4. Secondary dissociation: The drug magnetic beads-NAb complex was eluted with 150 mmol / L (pH 2.5, 80 μL) HAc at room temperature for 5 minutes and shaken at 800 rpm to obtain a secondary dissociation solution.

[0011] 5. Competitive binding: add 0.65 M Tris (pH 9.5, 30 μL) solution to the secondary dissociation solution for neutralization, add an equal volume of biotin-drug and incubate at 37°C for 1 hour to obtain the sample to be tested.

[0012] 6. Detection, a) Drug targets were coated into 96-well plates at 4°C overnight. After washing three times with PBST, the plates were blocked with 5% BSA for 2 hours at room temperature with shaking at 300 rpm. b) Add the sample to be tested to the microplate prepared in step a, incubate at 37°C for 1 hour, wash three times with PBST, add 100 μL / well of avidin HRP (1:1000) and incubate at 37°C for 1 hour, discard the supernatant, wash three times with PBST, add TMB substrate and develop color at room temperature in the dark for 10 minutes; c) Use H2SO4 to stop the color development and use a microplate reader set to dual wavelengths (main wavelength 450nm, reference wavelength 570nm) to detect the OD value. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : NAb detection with sample pretreatment.

[0014] Figure 2 : NAb detection without sample pretreatment.

[0015] Figure 3 : To evaluate the cut-off value and sensitivity of CLB NAb assay.

[0016] Figure 4 : Assessing drug and target tolerance.

[0017] Figure 5 : Evaluation of the selectivity and specificity of the CLB NAb assay.

[0018] Figure 6 : Assayed hook effect assessment. Implementation

[0019] This application study developed a new method combining target-based drug depletion and drug-based NAb extraction to detect the immunogenicity of CMAB009, a better biological candidate for cetuximab, using a competitive ligand binding assay. However, the presence of free drug still interferes with sample detection. In order to improve the therapeutic effect, a two-step method involving dual affinity beads was used. Initially, EGFR-coated magnetic beads (target beads) were used to eliminate most of the free drug in the sample. Subsequently, drug beads were used to capture NAb after sample acid dissociation. NAbs were then neutralized and acid-eluted, and biotinylated drugs were added to EGFR-coated plates for NAb detection. A comprehensive experimental strategy was implemented to simultaneously mitigate drug interference and enhance target tolerance. After troubleshooting and optimization, the NAb assay was validated for clinical sample analysis and successfully applied to the Phase III clinical immunogenicity NAb detection of CMAB009. The results show that the integrated assay optimization strategy can greatly improve the key performance characteristics of CLB NAb detection, enabling more reliable assessment of potential immunogenicity and its impact on better biological development.

[0020] Example 1 CLB NAb Detection Procedure (1) CLB NAb assay procedure, no sample pretreatment

[0021] The CLB assay on the ELISA platform was used to evaluate the NAb response to CMAB009. Soluble EGFR was seeded (0.1 μg / ml in CBS) in a 96-well plate at 4°C overnight. After washing three times with PBST, the plate was blocked with 300 μL 5% BSA for 2 h at room temperature with shaking at 300 rpm. At the same time, 110 μL of sample was co-incubated with 110 μL biotin drug (0.0025 μg / mL) at 37°C for 1 h. Then, 100 μL of sample was transferred to the microplate wells in duplicate and incubated at 37°C for 1 h. The free biotin drug was allowed to fully bind to the EGFR at the bottom of the plate. Then, 100 μL / well of avidin HRP (1:1000) was added to the plate and incubated at 37°C for 1 h. TMB substrate was added and incubated at room temperature for 10 min. After incubation, the reaction was stopped by H2SO4 and the OD was measured using a microplate reader (Bio Tek) at 450 nm with wavelength correction at 570 nm.

[0022] (2) CLB NAb assay procedure and target magnetic bead pretreatment samples

[0023] To improve drug and target tolerance, sample pretreatment with target (EGFR) beads was performed prior to running samples in the acid hydrolysis-based CLB NAb assay.

[0024] Free drug was removed from 10 μL sample using 15 μL target beads at room temperature with shaking at 800 rpm for 1 hour. The supernatant was then transferred to a new plate and dissociated with 300 mmol / L HAc (pH 2.5, 75 μL) at room temperature for 5 minutes with shaking at 800 rpm. 1 M Tris (pH 9.5, 15 μL) was then added to neutralize the solution at room temperature for 1 hour with shaking at 300 rpm. Finally, 110 μL pre-treated sample was obtained.

[0025] (3) CLBNAb assay procedure and drug magnetic bead pretreatment samples

[0026] To improve drug and target tolerance, samples were pre-treated with drug (CMAB009) beads before running them in the acid hydrolysis-based CLB NAb assay.

[0027] 10 μL of sample was dissociated using 300 mmol / L HAc (pH 2.5, 100 μL) at room temperature for 5 minutes and shaken at 800 rpm. Then 15 μL of drug beads were added to capture NAb under acidic conditions, and then 1 M Tris (pH 9.5, 30 μL) was added to neutralize for 1 hour at room temperature. The drug beads were eluted with 150 mmol / L (pH 2.5, 80 μL) HAc at room temperature for 5 minutes, shaken at 800 rpm, and a solution of 0.65 M Tris (pH 9.5, 30 μL) was added to neutralize the eluted sample for 1 hour at room temperature and shaken at 300 rpm. Finally, 110 μL of pre-treated sample was obtained.

[0028] (4) CLB NAb Assay Procedure with Target and Bead Pretreatment Samples

[0029] To further improve drug and target tolerance, sample pretreatment with target (EGFR) and drug (CMAB009) beads was performed before running samples in the acid hydrolysis-based CLB NAb assay ( Figure 2 ).

[0030] First, free drug was removed from 10 μL sample using 15 μL target beads for 1 h at RT with shaking at 800 rpm. The supernatant was transferred to a new plate and dissociated with 300 mmol / L HAc (pH 2.5, 100 μL) for 5 min at RT with shaking at 800 rpm. Then 15 μL drug beads were added to capture NAbs under acidic conditions, and then 1 M Tris (pH 9.5, 30 μL) was added to neutralize for 1 h at RT. The drug beads were eluted with 150 mmol / L (pH 2.5, 80 μL) HAc for 5 min at RT with shaking at 800 rpm, and a solution of 0.65 M Tris (pH 9.5, 30 μL) was added to neutralize the eluted sample for 1 h at RT with shaking at 300 rpm. Finally, 110 μL pre-treatment sample was obtained.

[0031] (5) Dot film

[0032] Samples were prepared by spiking healthy human serum containing 20 μg / mL NAb at concentrations of 70, 17.5, and 0 μg / ml of drug.

[0033] During the pretreatment process, samples were collected at different stages: (1) untreated samples; (2) supernatant of samples after treatment with target beads; (3) samples acidified and eluted from target beads and subsequently neutralized; (4) supernatant of samples treated with drug beads; (5) samples acidified and eluted from drug beads and subsequently neutralized. These samples were detected by dot blotting. Briefly, 1.2 μL of sample was spotted on a nitrocellulose membrane (NC membrane), which was then blocked with blocking buffer for 1 h at room temperature. After washing, sheep anti-rabbit antibody (1:1000) prepared in PBS was soaked and incubated at room temperature for 1 h. The intensity of the resulting color varied, with darker colors indicating higher NAb concentrations and lighter colors indicating lower concentrations.

[0034] (6) Effect of sample pretreatment To evaluate drug tolerance under different pretreatment conditions, NC and PC samples (2 or 8 μg / mL) were prepared by spiking with 0 or 70 μg / mL of drug. When there was no drug interference, NAb PC tested positive and NC tested negative ( Figure 1 ). The NC sample containing 70 μg / mL of drug was identified as a false positive ( Figure 2 ).

[0035] After pre-treatment of the samples with target beads, the NC sample was negative, while the PC sample containing 70 μg / mL of drug was identified as a false negative ( Figure 2However, after pre-treatment of samples with the ACE method using drug beads, PC samples containing 70 μg / mL of drug showed positive results for NAb, while NC samples containing 70 μg / mL of drug showed negative results for NAb ( Figure 2 ).

[0036] To improve the therapeutic efficacy, a two-step approach involving dual affinity beads was used. Initially, target beads were used to eliminate most of the free drug in the sample. Subsequently, drug beads were used to capture NAbs after sample acid dissociation. After applying sample pretreatment with target-based drug depletion and drug-based NAb extraction, PC samples containing 70 μg / mL of drug showed positive results with a low signal-to-noise ratio, while NC samples tested negative. This indicates a significant increase in sensitivity and drug tolerance ( Figure 2 ).

[0037] The combined application of target and drug beads was evaluated using dot blots, and the results showed that when 20 μg / mL NAb PC samples were treated with target beads, no significant differences were observed in NAb concentrations, regardless of whether they contained 70 μg / mL or 17.5 μg / mL of drug. This suggests that NAbs were retained in the supernatant. After samples were treated with 40 μL of target beads, the supernatant was treated with drug beads, 20 μg / mL of NAb PC, 17.5 μg / mL of drug, and the results were similar to the 20 μg / mL NAb PC control group. This indicates that NAbs were successfully extracted using drug beads after target-based drug depletion.

[0038] Example 2 Establishment of NAb assay cutoff point The assay cutoff determines whether a sample test result is positive or negative, and it is critical to establish it correctly to reduce the chance of obtaining a false negative result. To generate the assay cutoff point, serum samples from 30 pre-dose patients were analyzed in triplicate on two days by two different analysts. The average signal for each sample in the six runs was calculated and used for cut point evaluation. Analytical outliers were removed using Grubbs, while biological outliers were removed using Boxplot. The normality of the distribution was assessed using the Shapiro-Wilk test. The data set was analyzed so that 1% of the measured samples scored positive in the CLB NAb assay. The cut point was calculated as follows: NAb detection cut-off point (SCP) = mean (S / N) - 2.33* overall standard deviation (S / N) The signal-to-noise ratio of each individual spiked sample was calculated as the OD of the spiked sample / OD of the normal serum (NC) in the pool. Sample signal-to-noise ratio values ​​below the cut point indicate a positive result for NAb, while values ​​above the cut point indicate a negative result for NAb.

[0039] The cut point of the assay was based on the signal-to-noise ratio. If the signal-to-noise ratio of the sample was equal to or lower than the cut point, it was classified as positive. On the other hand, if the signal-to-noise ratio was higher than the cut point, the sample was classified as negative. A total of 30 naive serum samples were processed using the target-based drug depletion and drug-based NAb extraction NAb assays and analyzed by two analysts on three days. Individual sample response ODs were converted to signal-to-noise ratios, resulting in 180 data points. After excluding outliers, the normality of the dataset was assessed using the Shapiro-Wilk test or the cut point factor. For healthy individuals, the dataset was determined to be normally distributed (p = 0.745, >0.05) and the validated screening cutoff point was calculated to be 0.861 ( Figure 3 A).

[0040] Example 3 Sensitivity PC samples were prepared with a final concentration range of 31.25 to 2000 ng / ml by diluting rabbit anti-CMAB009 in healthy human serum. Unspiked serum pool was used as negative control (NC). Sensitivity was defined as the lowest concentration of antibody that consistently produced a positive result or equaled the assay cutoff point.

[0041] To determine assay sensitivity, NAbs were spiked into healthy human serum at a final concentration of 2000 ng / mL and then serially diluted. Assay sensitivity was determined by interpolating the lowest concentration of PC at the dilution that yielded an S / N value equal to or below the assay cut point. Assay sensitivity was estimated to be 92 ng / mL ( Figure 3 B), which is the lowest detectable concentration of NAb in this assay (using 0.861 as the cut-off factor).

[0042] Example 4 Drug tolerance and target tolerance Drug tolerance samples were prepared by spiking different levels of drug (70, 35, 17.5, 8.75, 4.375, 2.1875, 1.09375 and 0 μg / ml) into serum mixed with 0, 1 or 4 μg / ml NAb and mixing for 1 h at room temperature. Then stored at -80°C for 24 h. Based on the calculated cutoff point for each plate, the concentration of added drug that increases the signal above a certain threshold can be determined. This concentration turns a positive result into a negative result, called assay drug tolerance, is determined by identifying the highest concentration of added drug, in the absence of PC, that produces a signal above the cutoff point value.

[0043] To assess target tolerance, different concentrations of EGFR (1000, 500, 250, 125, 62.5, and 0 ng / ml) were spiked into NC. Target interference may cause the assay signal to be below the cutoff point and generate target-mediated false positive signals. Therefore, the signal-to-noise value of the assay results decreases with increasing EGFR concentration. Target tolerance is defined as the highest concentration of the target that consistently produces a negative result or equals the cutoff point.

[0044] NAb assays were performed on samples to assess drug and target tolerance in this study. Drug concentrations that inhibited PC assays were measured to assess drug tolerance. Figure 4 A shows that the highest concentration of drug that tested positive for NAb (below the plate cut point) at a PC of 1000 ng / mL was 70 μg / mL. The results indicate that a PC of 1000 ng / mL can tolerate at least 70 μg / mL of drug.

[0045] To assess target tolerance, NCs were pre-incubated with soluble target (EGFR) diluted to 0, 62.5, 125, 250, 500, and 1000 ng / mL. At the highest concentration of the target assessed, NCs gave false positive NAbs. The assay was calculated to have target tolerance up to 798 ng / mL ( Figure 4 B), which was significantly higher than the serum soluble EGFR level of 25.7 ng / mL in patients after administration.

[0046] Example 5 Selectivity Rabbit anti-CMAB009 and polyclonal antibody (PC) were spiked into 10 healthy individual human serum samples, 5 hemolytic serum samples, and 5 hyperlipidemic serum samples at a concentration of 110 ng / mL. Twenty individual serum samples, both spiked and unspiked, were tested and analyzed simultaneously in duplicate (two wells).

[0047] To evaluate selectivity, 10 healthy individual human sera, 5 hemolytic sera, and 5 hyperlipidemic sera were spiked or not with LPC. When not spiked with LPC, all 20 individuals tested negative for NAb with a signal-to-noise ratio above the plate cut point. Furthermore, when spiked, all 20 individuals tested positive for NAb with a signal-to-noise ratio below the plate cut point ( Figure 5 AC).

[0048] Example 6 Specificity Isotype control IgG of CMAB009 was spiked into NC, LPC or HPC samples at a concentration of 2 μg / ml, NC refers to pooled normal healthy human serum, LPC refers to 110 ng / ml NAb PC, and HPC refers to 2000 ng / ml NAb PC. The assay was performed at -80°C after 24 h. The results are expressed as signal-to-noise ratio values.

[0049] The specificity of the NAb assay was evaluated by spiking the drug isotype control IgG at a final concentration of 2 μg / mL into NC, LPC, or HPC. All NC samples showed negative NAb results with a signal-to-noise ratio higher than the plate cut-off point. In cases where the NAb detection in LPC or HPC samples was positive and the S / N was lower than the plate cut-off point, it was further confirmed that the sample matrix had no matrix interference ( Figure 5 D).

[0050] Example 7 Hook effect Rabbit anti-CMAB009 neutralizing polyclonal antibody was spiked into pooled healthy human serum at various concentrations (1, 10, 20, 40, 60, 80, 100, and 120 μg / ml) respectively. Analyzed in one replicate (two wells), the results were expressed as signal-to-noise ratios.

[0051] To obtain the hook effect of the assay, NAb PC was spiked into healthy human serum at a final concentration of 120 μg / mL and then diluted at 8 different concentrations. The signal-to-noise ratio started to decline from the concentration of 1 μg / mL and continued up to the highest tested concentration. The results showed that there was no hook effect as the increase in NAb concentration did not lead to an increase in the signal-to-noise ratio ( Figure 6 )

[0052] Example 8 Stability To evaluate the stability of the samples, LPC and HPC were subjected to specific conditions: stored at 4°C for 7 days, freeze-thawed 8 times, and kept on the bench for 24 h. Then the analysis results were compared with fresh positive control samples. If the signal-to-noise ratio results of the tested samples met the criteria of HPC < LPC < SCP, it was considered stable.

[0053] The stability of the samples was evaluated under different conditions, including bench storage for 24 h, refrigeration for 7 days, or 8 freeze-thaw cycles. No significant change in the signal-to-noise ratio was detected compared with the fresh positive control samples. The stability test results are shown in Table 1 and all samples met the stability acceptance criteria.

[0054] Table 1. Bench, refrigeration, and freeze-thaw stability

[0055] Example 9 Precision Intra-assay and inter-assay precision of the assay were tested on HPC, LPC, and NC samples, and each sample was tested in 6 independent experiments. The coefficient of variation (CV) between replicates was calculated using the formula "%CV = Std. Dev / Avg. × 100" and compared with the preset acceptance criterion of not more than 20%.

[0056] The intra-assay and inter-assay precision of the NAb assay for each sample was evaluated in six independent experiments by calculating the standard deviation and the corresponding coefficient of variation. The results shown in Tables 2 and 3 show that the %CV of the intra- and inter-plate measurements ranged from 7.6–12%. The calculated CV values ​​did not exceed 20%, which is within the acceptable standard range.

[0057] Table 2. Intra-assay and inter-assay precision

[0058] Example 10 System Applicability The mean signal-to-noise ratio of LPC or HPC ± 3*SD and the mean OD of NC + 3*SD were used as the acceptance criteria for negative and positive controls, respectively, to evaluate the suitability of the system.

[0059] HPC, LPC, and NC data were collected from 10 analyses to establish the system suitability control acceptance criteria (details are shown in Table 4). 3*SD±mean S / N values ​​were used for quality control of HPC (0.119 ≤ S / N ≤ 0.209), LPC (S / N 0.560 ≤ ≤ 0.861), and NC (≥ 0.722), respectively.

[0060] Table 3. System suitability System suitability Mean (SNR) SD(SNR) Mean-3*SD Mean+3*SD NC* 1.149 0.127 0.768 / LPC 0.727 0.055 0.560 0.861 HPC 0.164 0.015 0.119 0.209 *The mean and SD values ​​of NC were calculated based on the measured OD.

[0061] Example 11 Application to clinical samples ADA-positive patient sera were analyzed using the cutoff points established during method validation, allowing the identification of NAb-positive samples based on the signal-to-noise ratio. Among individuals who tested positive for NAb, excluding those with pre-existing anti-CMAB009 NAbs at the T0 (drug-naive) time point, the incidence of NAbs after CMAB009 treatment could be calculated.

Claims

1. A method for detecting therapeutic monoclonal antibody neutralizing antibody, comprising pretreatment, dissociation, binding, secondary dissociation, competitive binding and detection, wherein the pretreatment is characterized in that: Add target magnetic beads to the sample to be tested, shake at 800 rpm for 1 hour at room temperature, and after the free drugs in the sample are fully combined with the target magnetic beads, remove the target magnetic beads-drug complex to obtain the pretreated supernatant, which is then transferred to a new plate.

2. The detection method according to claim 1, wherein the dissociation is characterized in that: The pretreated supernatant was acidified with an acidifying solution at room temperature to obtain a dissociation solution.

3. The detection method according to claim 1, wherein the combination is characterized in that: An excess of drug magnetic beads was added to the dissociation solution, and 1 M Tris was added to neutralize the solution at room temperature for 1 hour to obtain a drug magnetic bead-NAb complex.

4. The detection method according to claim 1, wherein the secondary dissociation is characterized in that: The drug magnetic beads-NAb complex was eluted with 150 mmol / L (pH 2.5, 80 μl) HAc at room temperature for 5 minutes and shaken at 800 rpm to obtain a secondary dissociation solution.

5. The detection method according to claim 1, wherein the competitive binding is characterized in that: 0.65 M Tris (pH 9.5, 30 μl) solution was added to the secondary dissociation solution for neutralization, and an equal volume of biotin-drug was added and incubated at 37°C for 1 hour to obtain the sample to be tested.

6. The detection method according to claim 1, wherein the detection is characterized in that: a) Drug targets were coated into 96-well plates at 4°C overnight. After washing three times with PBST, the plates were blocked with 5% BSA for 2 hours at room temperature with shaking at 300 rpm. b) Add the sample to be tested to the microplate prepared in step a, incubate at 37°C for 1 hour, wash three times with PBST, add 100 μl / well of avidin HRP (1:1000) and incubate at 37°C for 1 hour, discard the supernatant, wash three times with PBST, add TMB substrate and develop color at room temperature in the dark for 10 minutes; c) Use H2SO4 to stop the color development and use a microplate reader set to dual wavelengths (main wavelength 450nm, reference wavelength 570nm) to detect the OD value.

7. The detection method according to claims 1-6, wherein the anti-therapeutic monoclonal antibody comprises an anti-EGFR therapeutic monoclonal antibody.

8. The EGFR therapeutic monoclonal antibody according to claim 7, which is cetuximab.

9. The acidified liquid as claimed in claim 2 is HAc, the acidified conditions are a concentration of 300 mmol / L and oscillation at 800 rpm.

10. The detection method according to claims 1 to 7, characterized in that: Used for CMAB009 for highly drug / target tolerant NAb assays.

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