Method for detecting abbeximab drug concentration in human blood sample based on characteristic peptide fragment

Abstract

The invention provides a method for detecting the drug concentration of Abbeximab in a human blood sample based on a characteristic peptide fragment, the characteristic peptide fragment of Abbeximab is studied, Skyine coarsely screens out the characteristic peptide fragment, and then the concentration of Abbeximab drug in a human blood sample is determined by sequentially investigating the peak appearance condition of liquid chromatography-tandem mass spectrometry, eliminating plasma interference and eliminating interference before and after the peak appearance of the characteristic peptide fragment. Determining ALAAPIEK as a quantitative peptide fragment and QFDVVFGGGGTK as a qualitative peptide fragment; then exploring a treatment method of a human blood sample, determining the dosage of ProteinG magnetic beads, a blood sample form (serum or EDTA-anticoagulant plasma) and incubation conditions, and determining elution, denaturation, enzymolysis and centrifugation conditions of the monoclonal antibody; and the detection conditions of liquid chromatography-mass spectrometry are determined. The method is high in sensitivity, the lowest quantitative concentration can reach 1 microgram / mL, and the abbeximab in human blood can be accurately, rapidly and sensitively measured.

Description

Technical Field

[0001] The present invention belongs to the field of biological quantitative detection technology, relates to the qualitative and quantitative detection of abatacil blood concentration, and specifically relates to a method for detecting the abatacil drug concentration in human blood samples based on characteristic peptide segments. Background Art

[0002] Abelacimab is a novel, highly selective, fully human monoclonal antibody. As an antiplatelet monoclonal antibody, it is primarily used to prevent and treat thrombosis, playing a key role in cardiovascular interventional procedures. It targets coagulation factor XI and locks it in an inactive state, thereby inhibiting both factor XI and its activated form, factor XIa.

[0003] In clinical treatment, especially for patients with critical vascular disease, personalized medication is essential to achieve optimal therapeutic outcomes. The pharmacokinetics (PK) and pharmacodynamics (PD) of monoclonal antibodies are highly complex, characterized by nonlinear dynamics and significant individual exposure variability. Different patients experience differences in drug metabolism, leading to varying drug concentrations even at the same dose. Therefore, by measuring drug concentrations, personalized medication can be implemented to ensure optimal dosing and efficacy.

[0004] The clinical method for measuring monoclonal antibody drugs in human blood samples often uses enzyme-linked immunosorbent assay (ELISA). Based on antigen-antibody specific binding, it uses enzyme-labeled secondary antibody colorimetry to detect intact antibodies, free targets, or anti-drug antibodies (ADA). However, during the detection process, it is easily interfered by endogenous IgG, requiring optimization of the blocking step. In addition, the dynamic range is narrow, usually 1-100 ng / mL, which cannot meet the detection of the peak concentration of abatacizumab.

[0005] In summary, in order to ensure that the drug is within a safe and effective concentration range and also provide data resources for drug metabolism and pharmacokinetic research, the development of a quantitative detection method for abacizumab with a large quantitative detection range and high sensitivity is of great significance for improving its clinical therapeutic effect and ensuring patient safety. Summary of the Invention

[0006] Based on the above problems, the present invention aims to develop and validate an LC-MS / MS analysis method for quantitative analysis of abecizumab concentration using serum or anticoagulated plasma as samples, and apply this method to abecizumab research to provide technical support for clinical individualized treatment and PK / PD research.

[0007] The process of the present method is summarized as follows: First, the characteristic peptides of abatacil were studied and roughly screened using Skyine. Then, by examining the peak elution patterns of liquid chromatography-tandem mass spectrometry, eliminating plasma interference, and eliminating interference before and after the peak elution of the characteristic peptides, ALAAPIEK was identified as the quantitative peptide and QFDVVFGGGTK as the qualitative peptide. Subsequently, methods for processing human blood samples were explored to determine the dosage of Protein G magnetic beads, the blood sample format (serum or EDTA-anticoagulated plasma), incubation conditions, and the elution, denaturation, enzymatic hydrolysis, and centrifugation conditions for the monoclonal antibody. Finally, the liquid chromatography-mass spectrometry detection conditions were determined. This method is highly sensitive, with a minimum quantification concentration of 1 μg / mL, enabling accurate, rapid, and sensitive determination of abatacil in human blood.

[0008] The technical solutions to be protected by the present invention are as follows:

[0009] The method provided by the present invention for detecting the concentration of abecizumab in human blood samples based on characteristic peptide segments comprises the following steps:

[0010] A) Sample preparation

[0011] After the serum or anticoagulated plasma of the blood sample to be tested is incubated with Protein G magnetic beads, the monoclonal antibody is eluted, denatured, and enzymatically hydrolyzed in sequence. A 50% acetonitrile-water solution containing 10% formic acid is added, and the supernatant is collected for analysis after centrifugation.

[0012] Preferably, the solvent of the Protein G magnetic beads is a PBS solution containing 0.05% Tween 20, the volume ratio of the Protein G magnetic beads to the PBS solution is 6:25, and the volume ratio of the Protein G magnetic beads to the blood sample is 3:1;

[0013] The incubation conditions were as follows: washing twice with PBS solution containing 0.1% BSA, washing twice with deionized water, and incubating for 45 min;

[0014] The conditions for mAb elution were as follows: mAb was eluted from the magnetic beads using 0.25% formic acid aqueous solution by high-speed vortexing for 10 min;

[0015] Denaturation conditions were as follows: 1 mol / L ammonium bicarbonate solution was added to the eluate, and denatured at 95°C with shaking for 30 min, wherein the volume ratio of the eluate to the ammonium bicarbonate solution was 90:17;

[0016] The enzymatic hydrolysis conditions were as follows: the denatured solution was allowed to cool to room temperature, 0.5 mg / mL pancreatic enzyme solution was added to the solution, and enzymatic hydrolysis was carried out at 37°C for 4 h. The volume ratio of the solution to the pancreatic enzyme solution was 107:15.

[0017] The volume ratio of the enzymatic hydrolysis solution to a 50% acetonitrile-water solution containing 10% formic acid was 122:23.3. After vortex mixing, the mixture was centrifuged at 15,000 rpm for 5 min to obtain the supernatant.

[0018] B) Liquid chromatography-tandem mass spectrometry detection

[0019] ALAAPIEK was used as the quantitative peptide of abatacil, QFDVVFGGGTK was used as the qualitative peptide, and caniluzumab was used as the internal standard. Liquid chromatography-mass spectrometry was used to detect abatacil in the supernatant in step A), and the content of abatacil was calculated using the standard curve method.

[0020] The concentration of the internal standard cardunolimab was 50 μg / mL, and the volume ratio of blood sample to internal standard was 2:1.

[0021] Liquid chromatography detection conditions are as follows:

[0022] Chromatographic column: Kinetex F5 column, 100 × 3.0 mm, particle size 2.6 μm;

[0023] Mobile phase A: aqueous solution containing 0.1% formic acid;

[0024] Mobile phase B: acetonitrile containing 0.1% formic acid;

[0025] Needle wash solution: 50% methanol;

[0026] Gradient elution was used with a flow rate of 0.3 mL / min, a column temperature of 45°C, and an injection of 20 μL. The gradient elution included: from 0 to 1.0 min, the volume fraction of mobile phase A was maintained at 90%, and the volume fraction of mobile phase B was maintained at 10%; from 1.0 to 7.5 min, the volume fraction of mobile phase A decreased from 90% to 65%, and the volume fraction of mobile phase B increased from 10% to 35%; from 7.5 to 8.0 min, the volume fraction of mobile phase A decreased from 65% to 10%, and the volume fraction of mobile phase B increased from 35% to 90%; from 8.0 to 10.0 min, the volume fraction of mobile phase A was maintained at 10%, and the volume fraction of mobile phase B was maintained at 90%; from 10.0 to 10.1 min, the volume fraction of mobile phase A increased from 10% to 90%, and the volume fraction of mobile phase B decreased from 90% to 10%; and the volume fractions of mobile phase A and mobile phase B were maintained until 12.0 min and stopped.

[0027] The mass spectrometry detection conditions are as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 500° C.; ion source nebulizer gas (GS1): 55 psi; ion source heating auxiliary gas (GS2): 50 psi; curtain gas (CUR): 30 psi; collision gas (CAD): 8 psi; scan mode: MRM,

[0028] The MRM scan parameters for abeticumab and the internal standard were as follows:

[0029]

[0030] Preferably, during the detection process, the abacizumab standard curve contains nine concentration points S1 to S9, which are 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 80 μg / mL, 150 μg / mL, and 200 μg / mL, respectively.

[0031] Furthermore, quality control solutions are required during the detection process, including the lower limit of quantification sample LLOQ with a concentration of 1 μg / mL and low quality control samples LQC with concentrations of 3 μg / mL, 25 μg / mL, and 160 μg / mL, medium quality control samples MQC, and high quality control samples HQC.

[0032] The beneficial protection and effects of the present invention are as follows:

[0033] (1) Large detection range: The present invention uses serum or EDTA-anticoagulant plasma as samples to determine the blood concentration of abecizumab in the range of 1 μg / mL to 200 μg / mL. The large quantitative range can accurately detect the peak concentration of abecizumab and meet the needs of clinical monitoring of peak and trough concentrations.

[0034] (2) High sensitivity and good accuracy. The sensitivity of drug detection using LC-MS / MS has been significantly improved, which can meet the real-world clinical needs. This detection method has been validated and complies with the M10 guidelines of the International Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) on the validation of bioanalytical methods and analysis of research samples and the European Bioanalytical Forum (EBF) bioanalytical method regulations.

[0035] (3) The detection method is stable and efficient, with an LC-MS / MS run time of 12 minutes. The method has been validated and has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 LC-MS / MS chromatograms of the quantitative characteristic peptide ALAAPIEK (top) and the qualitative characteristic peptide QFDVVFGGGTK (bottom) are shown;

[0037] Figure 2 The results of the comparison of the response levels with different magnetic bead dosages are shown;

[0038] Figure 3 The results comparing the response levels of serum and plasma sample matrices are shown;

[0039] Figure 4 It shows the comparison of different incubation results of blood samples and ProteinG magnetic beads;

[0040] Figure 5 The effect of no formic acid or different concentrations of formic acid on the elution of monoclonal antibodies from magnetic beads is shown;

[0041] Figure 6 The effect of different enzymatic digestion times on peptide response is shown;

[0042] Figure 7 It shows that after overnight enzymatic digestion, the response of the quantitative peptide ALAAPIEK decreases and the qualitative peptide QFDVVFGGGTK has more impurity peaks.

[0043] Figure 8 The standard curve of the quantitative signature peptide ALAAPIEK is shown. DETAILED DESCRIPTION

[0044] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, which are well known to those skilled in the art and are described in numerous publications.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention, and the preferred implementation methods and materials described in the specific embodiments are for illustrative purposes only.

[0046] The experimental methods in the following examples, where specific conditions are not specified, are generally carried out under conventional conditions or the conditions recommended by the manufacturers. Unless otherwise specified, the reagents used in the examples of the present invention can be obtained from sales companies.

[0047] 1. Experimental Materials

[0048] 1. Instruments, reagents and consumables

[0049] AB SCIEX Triple Quad 4500MD high performance liquid chromatography tandem triple quadrupole mass spectrometer (ABSCIEX, USA);

[0050] KS-060AL ultrasonic cleaning machine (Shenzhen Kejie Ultrasonic Technology Co., Ltd., China);

[0051] Eppendorf Centrifuge 5430R desktop high-speed refrigerated centrifuge (Eppendorf, Germany);

[0052] EVortex-Genie2 vortex mixer (Scientific Industries, USA);

[0053] Synergy UV ultrapure water preparation system (Merck Millipore, Germany);

[0054] MB100-4A microplate constant temperature oscillator (Shanghai Luodanlun Scientific Instrument Co., Ltd., China)

[0055] XSR204 electronic balance (METTLER TOLEDO, Switzerland).

[0056] The reagents and consumables used in the examples include: abeticumab standard: Shanghai Taoshu Biotechnology Co., Ltd.; methanol: purity ≥99.97%, LCMS grade (catalog number 1.06007.4008, Merk, Germany); acetonitrile: purity ≥99.97%, LCMS grade (catalog number 1.00030.4008, Merk, Germany); 300 μl 96-well plate and sealing mat (Thermo Fisher, USA); 1.5 mL centrifuge tube (Axygen, USA); pipette (Eppendorf, Germany).

[0057] 2. Preparation of working solution

[0058] 2.1 Preparation of internal standard solution

[0059] Dissolve and dilute the cadunolimab stock solution in PBS to a concentration of 1 mg / L. Accurately pipette an appropriate amount of the stock solution and dilute it with PBS to obtain a 50 μg / mL internal standard working solution.

[0060] 2.2 Preparation of standard curve working solution (linear range 1-200 μg / mL)

[0061] The mixed standard stock solution S0 contained 1000 μg / mL of abecizumab; the blank matrix was blank human plasma.

[0062] Prepared from S0 and blank matrix; S0 was thoroughly mixed and diluted with blank matrix to obtain S1, S2, S3, S4, S5, S6, S7, S8 and S9. The preparation method and concentration are shown in Table 1 below. Store sealed in a -80℃ refrigerator.

[0063] Table 1 Preparation of standard curve working solution

[0064]

[0065] 2.3 Preparation of quality control working solution

[0066] Using the same method, prepare a high quality control (HQC) sample at a concentration of 160 μg / mL using S0 working solution, a medium quality control (MQC) sample at a concentration of 25 μg / mL using S8 working solution, and a low quality control (LQC) sample at a concentration of 3 μg / mL and a lower limit of quantification (LLOQ) sample at a concentration of 1 μg / mL using MQC. See Table 2 below for preparation methods and concentrations. Store sealed in a -80°C freezer.

[0067] Table 2 Preparation of quality control working solution

[0068]

[0069] 2. Characteristic peptide screening

[0070] The query sequence for abexin is as follows:

[0071] >SUBUNIT_1 (heavy chain)

[0072] QVQLLESGGGLVQPGGSLRLSCAASGFTFSTAAMSWVRQAPGKGLEWVSGISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELSYLYSGYYFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.1).

[0073] >SUBUNIT_3 (light chain)

[0074] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNDVSWYQQLPGTAPKLLIYKNYNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCSAWDQRQFDVVFGGGTKLTVLG QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQID NO.2).

[0075] Skyline was used to screen the characteristic peptides of abeximab, and a total of 68 peptides were screened out as shown in Table 3 below:

[0076] Table 3 Summary of 68 peptides

[0077]

[0078]

[0079] Plasma containing 100 μg / mL of abecizumab was prepared, and the peaks were examined by liquid chromatography-tandem mass spectrometry. 17 peak peptides were screened out, as shown in Table 4 below:

[0080] Table 4 Summary of 17 peak peptides

[0081]

[0082]

[0083] Compared with the blank plasma of Abasimax, plasma containing 2 μg / mL Abasimax was prepared to eliminate the interference in the plasma, and the six peptides shown in Table 5 below were screened out:

[0084] Table 5 Summary of 6 peak peptides

[0085]

[0086] Plasma containing 1 μg / mL (lower limit of quantification) of abecizumab was prepared, and interference in the plasma and interference before and after the peak of the characteristic peptide were eliminated. The two peptides shown in Table 6 below were screened out. Based on the response, ALAAPIEK (SEQ ID NO. 3) was determined to be the quantitative peptide, and QFDVVFGGGTK (SEQ ID NO. 4) was determined to be the qualitative peptide. The spectra are shown in Table 6. Figure 1 .

[0087] Table 6 Summary of 2 characteristic peptides

[0088]

[0089] 3. Detection of Abasilimab Blood Concentration in Blood Samples

[0090] 3.1 Sample pretreatment

[0091] After the serum or anticoagulated plasma of the blood sample to be tested is incubated with Protein G magnetic beads, the monoclonal antibody is eluted, denatured, and enzymatically hydrolyzed in sequence. A 50% acetonitrile-water solution containing 10% formic acid is added, and the supernatant is collected for analysis after centrifugation.

[0092] Standards were used to screen the treatment conditions as follows:

[0093] (1) Take ProteinG magnetic beads and add 500 μL PBS solution to wash twice; after investigating the amount of magnetic beads, considering the response and cost, it is decided to add 60 μL ProteinG magnetic beads ( Figure 2 ).

[0094] (2) Add 250 μL of PBS solution containing 0.05% Tween 20 to the washed magnetic beads, then add 20 μL of biological sample, and then add 10 μL of internal standard solution (50 μg / mL cardunili PBS solution) and mix at low speed. The sample matrix was investigated. Four serum and plasma samples were selected and abexin was added (final concentration of 20 μg / mL). The results showed that the response in EDTA-anticoagulated plasma samples was slightly higher than that in serum samples, but the response CV% of both samples was less than 15% (Table 7, Figure 3 ), can be considered as no difference, and serum or EDTA-anticoagulant plasma can be selected as test samples in the future.

[0095] Table 7 Comparison of response values ​​of two sample matrices

[0096]

[0097] (3) Incubation: Wash twice with 500 μL PBS solution containing 0.1% BSA and twice with deionized water; The adsorption time of incubation was investigated, and considering the response and time cost, the incubation time of 45 min was selected ( Figure 4 ).

[0098] (4) Use 100 μL of formic acid aqueous solution to vortex at high speed for 10 min to elute the monoclonal antibody from the magnetic beads; the formic acid content of the eluate was investigated, and the results showed that adding acid was beneficial to the elution of the monoclonal antibody. Finally, 0.25% formic acid was added ( Figure 5 ).

[0099] (5) Take 90 μL of eluate, add 17 μL of 1 mol / L ammonium bicarbonate solution, and denature at 95°C with shaking for 30 min;

[0100] (6) The solution was cooled to room temperature, and 15 μL of 0.5 mg / mL pancreatic enzyme solution was added to the solution and enzymatic hydrolysis was carried out at 37°C. The enzymatic hydrolysis time was investigated, and enzymatic hydrolysis was carried out for 1 h, 2 h, 4 h and overnight. The results showed that the response was better when the enzymatic hydrolysis was carried out for 4 h. When the enzymatic hydrolysis was carried out overnight, the response of the quantitative peptide ALAAPIEK decreased and the qualitative peptide QFDVVFGGGTK showed more impurity peaks, which proved that the longer the enzymatic hydrolysis time, the better. The enzymatic hydrolysis time of 4 h was selected for subsequent experiments. Figure 6 、 Figure 7 ).

[0101] (7) Add 23.3 μL of 50% acetonitrile-water solution (containing 10% formic acid) to the enzymatic hydrolyzate, vortex mix, centrifuge at 15,000 rpm for 5 min, and take the supernatant for analysis.

[0102] 3.2 Liquid chromatography-mass spectrometry detection

[0103] LC-MS / MS analysis was performed on an AB SCIEX Triple Quad 4500MD high-performance liquid chromatograph coupled with a triple quadrupole mass spectrometer. The concentration of abecizumab in pretreated serum samples was calculated using a standard curve method with canidolimab as the internal standard.

[0104] 3.2.1 Chromatographic conditions

[0105] Chromatographic column: Kinetex F5 column, 100 × 3.0 mm, particle size 2.6 μm;

[0106] Mobile phase A: water containing 0.1% formic acid;

[0107] Mobile phase B: acetonitrile containing 0.1% formic acid;

[0108] Needle wash solution: 50% methanol;

[0109] Gradient elution was used with a flow rate of 0.3 mL / min, a column temperature of 45°C, and an injection of 20 μL;

[0110] The gradient elution comprises: from 0 to 1.0 min, the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%; from 1.0 to 7.5 min, the volume fraction of mobile phase A decreases from 90% to 65%, and the volume fraction of mobile phase B increases from 10% to 35%; from 7.5 to 8.0 min, the volume fraction of mobile phase A decreases from 65% to 10%, and the volume fraction of mobile phase B increases from 35% to 90%; from 8.0 to 10.0 min, the volume fraction of mobile phase A is maintained at 10%, and the volume fraction of mobile phase B is maintained at 90%; from 10.0 to 10.1 min, the volume fraction of mobile phase A increases from 10% to 90%, and the volume fraction of mobile phase B decreases from 90% to 10%; and the volume fractions of mobile phase A and mobile phase B are maintained until 12.0 min and then stopped.

[0111] 3.2.2 Mass spectrometry conditions

[0112] The mass spectrometry conditions were as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 500°C; ion source nebulizer gas (GS1): 55 psi; ion source heating auxiliary gas (GS2): 50 psi; curtain gas (CUR): 30 psi; collision gas (CAD): 8 psi; scan mode: MRM.

[0113] The MRM scanning parameters of the mass spectrometer compounds are shown in Table 8 below:

[0114] Table 8 Summary of compound MRM scanning parameters for mass spectrometry

[0115]

[0116] * indicates the quantitative ion pair; cadunolimab was used as the internal standard.

[0117] 4. Validation of Methodology

[0118] Method validation was performed according to the guidelines for bioanalytical method validation and sample analysis in the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), including linearity, limit of quantification, accuracy and precision.

[0119] 4.1 Linearity

[0120] Prepare blank samples (without the analyte and internal standard), zero-point samples (without the analyte but with the internal standard), and nine calibration points. Perform method validation for three consecutive analytical runs (at least two days). Blank and zero-concentration samples are used to determine if there is any endogenous interference or sample contamination and are not included in the standard curve calculation. Use the weighted least squares method to perform linear regression, with the analyte concentration as the horizontal axis and the peak area ratio of the analyte to the internal standard as the vertical axis. The resulting linear regression equation is the standard curve. Each non-zero sample in the standard curve is calculated using this standard curve, and the deviation between the obtained concentration and the set concentration must fall within an acceptable range.

[0121] Require:

[0122] 1) The concentration deviation at LLOQ (lowest limit of quantification STD1) is within ±20%, and the other concentration deviations are within ±15%

[0123] within;

[0124] 2) At least 75% of the non-zero samples should meet the above conditions;

[0125] 3) The correlation coefficient R should be greater than or equal to 0.990.

[0126] Test results:

[0127] Table 9 Summary of standard curve parameters

[0128]

[0129] See Table 10 for back-calculated concentrations of the standard curve:

[0130] Table 10 Back-calculated concentrations from the standard curve

[0131]

[0132] The validation results show that the validation results of the three batches of standard curves fully meet the acceptance criteria. Figure 8 .

[0133] 4.2 Precision and Accuracy

[0134] Precision refers to the closeness of the target analyte concentration values ​​repeatedly measured by the analytical method, and is expressed as the relative standard deviation (CV%). Accuracy refers to the ratio of the concentration value actually measured by the method to the known theoretical concentration value, and is expressed as the relative error (RE%). Serum quality control samples of four concentrations, LLOQ, LQC, MQC, and HQC, were taken and pretreated for LC-MS / MS analysis. Each of the three analytical batches (completed over at least two days) was verified to analyze six samples at each concentration of the four QC samples, namely the lower limit of quantification (LLOQ), low (LQC), medium (MQC), and high (HQC). The intra-batch precision, inter-batch precision, intra-batch accuracy, and inter-batch accuracy were calculated. See Table 11 below for details:

[0135] Table 11. Minimum limit of quantification, accuracy, and precision of abexin in human serum by LC-MS / MS

[0136]

[0137] Any undescribed parts of the present invention are the same as or implemented using existing technologies. The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the detailed methods of the present invention, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A method for detecting the concentration of abexin in human blood samples based on characteristic peptide segments, characterized in that: The following steps are involved: A) Sample preparation After incubating the serum or anticoagulated plasma of the blood sample to be tested with Protein G magnetic beads, the monoclonal antibody is eluted, denatured, and enzymatically hydrolyzed in sequence. Then, a 50% acetonitrile-water solution containing 10% formic acid is added, and the supernatant is collected for analysis after centrifugation. B) Liquid chromatography-tandem mass spectrometry detection ALAAPIEK was used as the quantitative peptide of abecizumab, QFDVVFGGGTK was used as the qualitative peptide, and cadunolimab was used as the internal standard. Liquid chromatography-mass spectrometry was used to detect abecizumab in the supernatant in step A), and the content of abecizumab was calculated using the standard curve method. Among them, the liquid chromatography detection conditions are as follows: Chromatographic column: Kinetex F5 column, 100 × 3.0 mm, particle size 2.6 μm; Mobile phase A: aqueous solution containing 0.1% formic acid; Mobile phase B: acetonitrile containing 0.1% formic acid; Needle wash solution: 50% methanol; Gradient elution was used with a flow rate of 0.3 mL / min, a column temperature of 45°C, and an injection of 20 μL. The gradient elution included: from 0 to 1.0 min, the volume fraction of mobile phase A was maintained at 90%, and the volume fraction of mobile phase B was maintained at 10%; from 1.0 to 7.5 min, the volume fraction of mobile phase A decreased from 90% to 65%, and the volume fraction of mobile phase B increased from 10% to 35%; from 7.5 to 8.0 min, the volume fraction of mobile phase A decreased from 65% to 10%, and the volume fraction of mobile phase B increased from 35% to 90%; from 8.0 to 10.0 min, the volume fraction of mobile phase A was maintained at 10%, and the volume fraction of mobile phase B was maintained at 90%; from 10.0 to 10.1 min, the volume fraction of mobile phase A increased from 10% to 90%, and the volume fraction of mobile phase B decreased from 90% to 10%; the volume fractions of mobile phase A and mobile phase B were maintained until 12.0 min, and then stopped. The mass spectrometry detection conditions are as follows: ion source: electrospray ion source, positive ion mode; capillary voltage: 5500 V; ion source temperature (TEM): 500°C; ion source nebulizer gas (GS1): 55 psi; ion source heating auxiliary gas (GS2): 50 psi; curtain gas (CUR): 30 psi; collision gas (CAD): 8 psi; scan mode: MRM, The MRM scan parameters for abeticumab and the internal standard were as follows: 。 2. The method for detecting the concentration of abecizumab in human blood samples based on characteristic peptide segments according to claim 1, characterized in that: in, In step A), the incubation matrix of Protein G magnetic beads is 0.05% Tween 20 in PBS solution, the volume ratio of Protein G magnetic beads to PBS solution is 6:25, and the volume ratio of Protein G magnetic beads to blood sample is 3:

1. The incubation conditions were as follows: washing twice with PBS solution containing 0.1% BSA, washing twice with deionized water, and incubating for 45 min.

3. The method for detecting the concentration of abecizumab in human blood samples based on characteristic peptide segments according to claim 1, characterized in that: in, In step A), the conditions for eluting the monoclonal antibody are as follows: 0.25% formic acid aqueous solution is vortexed at high speed for 10 min to elute the monoclonal antibody from the magnetic beads; Denaturation conditions were as follows: 1 mol / L ammonium bicarbonate solution was added to the eluate, and denatured at 95°C with shaking for 30 min, wherein the volume ratio of the eluate to the ammonium bicarbonate solution was 90:17; The enzymatic hydrolysis conditions were as follows: the denatured solution was allowed to cool to room temperature, 0.5 mg / mL pancreatic enzyme solution was added to the solution, and enzymatic hydrolysis was carried out at 37°C for 4 h. The volume ratio of the solution to the pancreatic enzyme solution was 107:

15. The volume ratio of the enzymatic hydrolysis solution to a 50% acetonitrile-water solution containing 10% formic acid was 122:23.

3. After vortex mixing, the mixture was centrifuged at 15,000 rpm for 5 min to obtain the supernatant.

4. The method for detecting the concentration of abecizumab in human blood samples based on characteristic peptide segments according to claim 1, characterized in that: in, In step B), the concentration of the internal standard cadunolimab was 50 μg / mL, and the volume ratio of the blood sample to the internal standard was 2:

1.

5. The method for detecting the concentration of abecizumab in human blood samples based on characteristic peptide segments according to claim 1, characterized in that: in, In step B), the standard curve contains nine concentration points S1 to S9, namely 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 80 μg / mL, 150 μg / mL, and 200 μg / mL.

6. The method for detecting the concentration of abecizumab in human blood samples based on characteristic peptide segments according to claim 1, characterized in that: in, Quality control fluid is also required during the detection process, including the lower limit of quantification sample LLOQ with a concentration of 1μg / mL and low quality control samples LQC with concentrations of 3μg / mL, 25μg / mL, and 160μg / mL, medium quality control samples MQC, and high quality control samples HQC.

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