LC-ms / ms quantitative determination method for Anti-novel coronavirus bispecific antibody drug

Abstract

The present application relates to the technical field of antibody drug determination, and provides a liquid chromatography-tandem mass spectrometry (LC-MS / MS) quantitative determination method for an anti-novel coronavirus bispecific antibody drug. According to the method, characteristic peptide segments EAYGMDVWGQGTTVTVSSASTK and SLSLSPGGGSQVQLVQSGAEVK are used, and on the basis that the characteristic peptide segments comprise a stable isotope internal standard, the content of the anti-novel coronavirus bispecific antibody drug is accurately quantified on an LC-MS / MS platform by means of the process of protein precipitation, enzymolysis and solid-phase extraction. According to the present application, the method and the characteristic peptide segments are highly selective and specific for anti-novel coronavirus bispecific antibody YBSW015, without being interfered by a pre-existing anti-novel coronavirus antibody in a patient, have the characteristics of high efficiency and simplicity, and can be used for clinical monitoring of the anti-novel coronavirus bispecific antibody.

Description

LC-MS / MS quantitative detection method for anti-novel coronavirus bispecific antibody drugs

[0001] This application requests the priority of the Chinese patent application filed with the China Patent Office on June 9, 2023, with application number 202310683669.4 and invention name “LC-MS / MS quantitative detection method for anti-new coronavirus bispecific antibody drugs”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of antibody drug detection technology, and specifically relates to a technology for quantitatively determining the concentration of anti-novel coronavirus bispecific antibodies in biological samples using liquid chromatography-tandem mass spectrometry. Background Art

[0003] The anti-novel coronavirus bispecific antibody (YBSW015) is a monoclonal antibody developed by Yabao Pharmaceutical Group Co., Ltd. for the treatment of coronavirus disease 2019 (COVID-19), caused by infection with the novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). YBSW015 is derived by modifying two parental monoclonal antibodies isolated from recovered COVID-19 patients. It binds to two distinct epitopes on the novel coronavirus's Spike protein (S protein), blocking the binding of the S protein to human ACE2. This antibody exhibits significantly higher selectivity and neutralizing activity against SARS-CoV-2 than the parental monoclonal antibodies, demonstrating excellent safety and efficacy.

[0004] Monitoring antibody drug concentrations in biological samples is an important indicator for studying drug pharmacokinetic properties and formulating dosing regimens. Patients receiving YBSW015 therapy may be infected with or have previously been infected with the novel coronavirus, as well as those who have received a novel coronavirus vaccine. These patients may have a large number of pre-existing anti-SARS-CoV-2 antibodies that are highly homologous to the YBSW015 antibody, and may also have pre-existing antibodies with sequences identical to the complementarity-determining regions (CDRs) of the YBSW015 antibody. Therefore, drug monitoring methods that can highly selectively and specifically monitor YBSW015 drug concentrations are needed. Currently, two clinical drug monitoring methods for antibody therapeutics include ligand-binding assays (LBAs) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). The LBA method is currently a commonly used drug monitoring method in clinical practice, but it has problems such as a narrow linear measurement range, the inability to jointly test multiple indicators, and the long development time and difficulty in obtaining the required high-specificity antibodies. The conventional LC-MS / MS method has factors such as multiple operational processes and long operation time, and it is difficult to distinguish between pre-existing anti-new coronavirus antibodies and YBSW015 antibodies in patients, which seriously limits its application in clinical practice (Zhong, X., et al., Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Assay for Quantitative Measurement of Therapeutic Antibody Cocktail REGEN-COV Concentrations in COVID-19 Patient Serum. Anal Chem, 2021. 93(38): p. 12889-12898.). Therefore, it is necessary to provide a new technology that can highly selectively and specifically distinguish between pre-existing anti-new coronavirus antibodies and YBSW015 antibodies in patients, and efficiently, quickly and accurately monitor the concentration of YBSW015 antibody drugs.

[0005] Summary of the Invention

[0006] Based on the above-mentioned problems existing in the prior art, the present application establishes an LC-MS / MS quantitative detection method for anti-novel coronavirus bispecific antibody drugs to achieve high selectivity and specificity for the YBSW015 antibody, without interference from pre-existing anti-novel coronavirus antibodies, and with the characteristics of high efficiency and simplicity, which can be used for clinical monitoring of anti-novel coronavirus bispecific antibodies.

[0007] For detailed information about the YBSW015 antibody described in this application, please refer to CN 113563475A. The YBSW015 antibody is BS-mAb-2 in CN 113563475A. The entire content of this patent document is incorporated into this application by reference.

[0008] In the present application, blood samples can be pre-treated using methods known in the art to obtain a supernatant of the sample to be tested, such as protein precipitation, enzymatic hydrolysis, solid phase extraction (SPE), etc.; LC-MS / MS is then used to quantitatively detect the concentration of the bispecific antibody by detecting characteristic peptides.

[0009] The first aspect of the present application provides an LC-MS / MS quantitative detection method for an anti-novel coronavirus bispecific antibody drug, comprising the following steps:

[0010] (1) Performing protein precipitation (PPT) on the blood sample to obtain precipitated protein;

[0011] (2) enzymatically hydrolyzing the precipitated protein to obtain an enzymatic hydrolysis product;

[0012] (3) extracting the enzymatic hydrolysis product using a solid phase extraction plate (SPE plate) to obtain a supernatant of the sample to be tested;

[0013] (4) using a liquid chromatography tandem mass spectrometry platform to quantify the characteristic peptides in the supernatant of the sample to be tested by an internal standard method, thereby quantifying the concentration of the anti-novel coronavirus bispecific antibody; wherein,

[0014] The characteristic peptide segment in step (4) is EAYG or SLSL, and its amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2, respectively;

[0015] In a preferred embodiment, in step (4), the concentration of the anti-novel coronavirus bispecific antibody is quantified based on EAYG and / or SLSL, using EAYG-IS and SLSL-IS as internal standards;

[0016] EAYG-IS and SLSL-IS are isotope-labeled peptides based on the characteristic peptides;

[0017] EAYG-IS: EAYGMDVWGQGTTVTV(+6)SSASTK(+8); or

[0018] SLSL-IS:SLSLSPGGGSQVQL(+7)VQSGAEVK(+8);

[0019] The above +6, +7, and +8 refer to the mass numbers increased after isotope labeling.

[0020] The present application does not impose any particular limitation on the isotope labeling method of the characteristic peptide segment, as long as it can achieve the purpose of the present application, for example, a solid phase synthesis method is used.

[0021] In a preferred embodiment, the blood sample is selected from at least one of whole blood, serum and plasma.

[0022] In a preferred embodiment, step (1) can be performed using any method known in the art for protein precipitation. Preferably, a 1% (w / v) trichloroacetic acid (TCA)-isopropanol solution is used for protein precipitation. In this application, "% (w / v)" represents the mass-to-volume ratio, in units of g / 100 mL.

[0023] In a preferred embodiment, the protein precipitation treatment method is: taking a blood sample, adding 100 μL of 1% (w / v) trichloroacetic acid-isopropanol at a volume ratio of 1:1, mixing, centrifuging and discarding the supernatant; adding NH4HCO3 solution to the precipitate, washing twice, and discarding the supernatant.

[0024] In a preferred embodiment, step (2) can use any method known in the art to enzymatically hydrolyze the precipitated protein; preferably, the precipitated protein is enzymatically hydrolyzed using trypsin.

[0025] In a preferred embodiment, after adding NH4HCO3 solution to the precipitated protein in step (1) and mixing, an internal standard of a characteristic peptide (i.e., an isotope-labeled peptide) and trypsin are added, mixed, and incubated at 500-600 rpm and 60°C for 2 hours; the reaction is terminated by adding 20% ​​(v / v) formic acid aqueous solution, and centrifuged at 4°C and 10,000g for 5 minutes. In this application, "v / v" means a volume ratio, for example, "20% (v / v) formic acid aqueous solution" means that formic acid accounts for 20% of the volume of the aqueous solution.

[0026] In a preferred embodiment, the isotope-labeled peptide can be configured as an isotope-labeled peptide working solution during use, which is not particularly limited in this application, as long as the purpose of this application can be achieved. The trypsin can be configured as a trypsin solution during use, which is not particularly limited in this application, as long as the purpose of this application can be achieved.

[0027] In a preferred embodiment, step (3) can be performed using any extraction method known in the art. Preferably, the enzymatic hydrolysis product is extracted using an SPE plate.

[0028] In a preferred embodiment, the process of step (3) is as follows: loading the enzymatic hydrolysis product, allowing the enzymatic hydrolysis product to slowly pass through the SPE plate, and discarding the filtrate; washing the SPE plate with 2% (v / v) formic acid aqueous solution and 90% (v / v) methanol aqueous solution in sequence, and discarding the washing solution; then eluting twice with acetonitrile: water: NH3H2O: isopropanol solution, and collecting the filtrate.

[0029] In a preferred embodiment, the enzymatic hydrolysis product is selected from the supernatant after enzymatic hydrolysis.

[0030] In the present application, the ratio of "acetonitrile: water: NH3H2O: isopropanol solution" is not particularly limited, as long as the purpose of the present application can be achieved. For example, the volume ratio of acetonitrile: water: NH3H2O: isopropanol is 35:55:5:5.

[0031] In a preferred embodiment, before use, the SPE plate is activated with methanol and then equilibrated with purified water.

[0032] In a preferred embodiment, in step (4), any suitable liquid chromatography column can be used. Preferably, the liquid chromatography column is ACE C18- (2.1×50mm, 3μm).

[0033] In the LC-MS / MS method of the present application, any suitable mobile phase and flow rate can be used; preferably, the flow rate of the mobile phase is 0.1 to 1.5 mL / min, for example, the flow rate of the mobile phase is 0.5 to 0.8 mL / min. In a preferred embodiment, the mobile phase is formic acid water-formic acid acetonitrile, for example, mobile phase A is 0.1% (v / v) formic acid in water, and phase B is 0.1% (v / v) formic acid acetonitrile.

[0034] In the LC-MS / MS method of the present application, any suitable temperature may be used; preferably, the column temperature is 50°C; and the autosampler temperature is 4°C.

[0035] In a preferred embodiment, the linear range of the characteristic peptide SLSL standard used in the liquid chromatography is 2.00 μg / mL, 5.00 μg / mL, 10.0 μg / mL, 50.0 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 800 μg / mL, and 1000 μg / mL, and the linear range concentration of the characteristic peptide EAYG standard is 5.00 μg / mL, 10.0 μg / mL, 50.0 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 800 μg / mL, and 1000 μg / mL.

[0036] In a preferred embodiment, the mass spectrometry conditions are:

[0037] The second aspect of the present application provides a characteristic peptide segment selected from EAYG or SLSL, whose amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2, respectively. The characteristic peptide segment is used to quantitatively detect the concentration of anti-novel coronavirus bispecific antibodies.

[0038] The third aspect of the present application provides an isotope-labeled peptide segment, which is:

[0039] EAYG-IS: EAYGMDVWGQGTTVTV(+6)SSASTK(+8); or

[0040] SLSL-IS:SLSLSPGGGSQVQL(+7)VQSGAEVK(+8).

[0041] The fourth aspect of the present application provides a composition comprising the characteristic peptide segment described in the second aspect of the present application and / or the isotope-labeled peptide segment described in the third aspect of the present application.

[0042] The fifth aspect of the present application provides a kit comprising the characteristic peptide segment described in the second aspect of the present application and / or the isotope-labeled peptide segment described in the third aspect of the present application.

[0043] The sixth aspect of the present application provides the use of the characteristic peptide described in the second aspect of the present application, the isotope-labeled peptide described in the third aspect of the present application, the composition described in the fourth aspect of the present application, or the kit described in the fifth aspect of the present application for quantitatively detecting the concentration of YBSW015 antibodies, or for preparing a product for quantitatively detecting the concentration of YBSW015 antibodies.

[0044] The methods and peptides of the present application are highly selective and specific for the YBSW015 antibody, are not interfered with by pre-existing anti-new coronavirus antibodies, and are efficient and simple, and can be used for clinical monitoring of anti-new coronavirus bispecific antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figures 1A to 1F show the selectivity of candidate peptides (Figures 1A, 1C, and 1E are characteristic spectra of double-blank (DB) samples under predicted MRM conditions; Figures 1B, 1D, and 1F are characteristic spectra of candidate peptide peaks in matrix samples containing bispecific antibodies under predicted MRM conditions; Figures 1A and 1B: SLSL; Figures 1C and 1D: EAYG; Figures 1E and 1F: IEFP);

[0046] Figures 2A to 2D are characteristic spectra of the specificity investigation of the characteristic peptide EAYG (Figures 2A and 2B: DB sample; Figures 2C and 2D: LLOQ sample; Figures 2A and 2C: EAYG MRM characteristic spectra; Figures 2B and 2D: EAYG-IS MRM characteristic spectra);

[0047] Figures 3A to 3D are characteristic spectra of the SLSL specificity investigation of the characteristic peptide segment (Figures 3A and 3B: DB sample; Figures 3C and 3D: LLOQ sample; Figures 3A and 3C: SLSL MRM characteristic spectra; Figures 3B and 3D: SLSL-IS MRM characteristic spectra);

[0048] Figures 4A and 4B are standard curves of characteristic peptides of bispecific antibodies (Figure 4A: EAYG; Figure 4B: SLSL);

[0049] FIG5 is a YBSW015 antibody concentration-time curve in healthy human serum (logarithmic graph);

[0050] Note: The signal strength unit "CPS" in Figures 1A to 1F, 2A to 2D, and 3A to 3D stands for "counts per second." DETAILED DESCRIPTION

[0051] To make the purpose, technical solutions, and advantages of this application more clearly understood, the present application is further described in detail below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of them. All other embodiments obtained by those skilled in the art based on this application fall within the scope of protection of this application. The present application will be explained below through specific embodiments, but the content of this application is not limited thereto.

[0052] The following examples are provided to further illustrate the embodiments of the present application. Various tests and evaluations were performed according to the following methods. Unless otherwise specified, the reagents and instruments used in the following examples were commercially available and used according to conventional techniques.

[0053] 1. Overview

[0054] Through in-depth research, this application has established an LC-MS / MS quantitative detection method for clinical monitoring of the anti-new coronavirus bispecific antibody YBSW015 antibody. In order to establish this method, the inventors first studied the YBSW015 antibody. Pre-existing anti-SARS-CoV-2 antibodies in patients infected or infected with the new coronavirus and patients who have been vaccinated with the new coronavirus vaccine, and the complex components of biological samples may seriously interfere with the accurate quantification of the YBSW015 antibody in the sample to be tested. It is particularly important to obtain specific characteristic peptides that can represent the YBSW015 antibody. The characteristic peptide needs to have characteristics such as stability (such as avoiding peptides that are easily modified) and the ability to distinguish interference from pre-existing anti-SARS-CoV-2 antibodies.

[0055] The inventors predicted the peptides after enzymatic hydrolysis of the YBSW015 antibody using Skyline software 22.2 and screened them through LC-MS / MS. In particular, they used the serum of recovered COVID-19 patients for specificity investigation, and obtained characteristic peptides with high selectivity and specificity, EAYGMDVWGQGTTVTVSSASTK (EAYG, whose amino acid sequence is shown in SEQ ID NO: 1) and SLSLSPGGGSQVQLVQSGAEVK (SLSL, whose amino acid sequence is shown in SEQ ID NO: 2). Then, based on the characteristic peptides, their isotope internal standards were synthesized. The isotope internal standard of EAYG is EAYGMDVWGQGTTVTV(+6)SSASTK(+8)(EAYG-IS), and the isotope internal standard of SLSL is SLSLSPGGGSQVQL(+7)VQSGAEVK(+8)(SLSL-IS). The samples were subjected to protein precipitation and enzymatic hydrolysis, and the enzymatic hydrolysis products were extracted using SPE plates. Finally, a liquid chromatography-tandem mass spectrometry platform was used to quantify highly specific characteristic peptides by the internal standard method to accurately quantify the YBSW015 antibody concentration. The lower limit of quantification of this method can be as low as 2μg / mL, which can meet the needs of clinical monitoring. Subsequently, the established method was systematically validated in accordance with the requirements of the National Medical Products Administration (NMPA) and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, and the drug concentration of YBSW015 antibody in the serum of the subjects was detected to preliminarily verify the clinical applicability of the method. The validation results showed that the method was precise, accurate, reproducible, and had good specificity, achieving efficient, simple, highly selective, and specific monitoring of YBSW015 antibody drug concentration in human serum.

[0056] 2. Materials and Methods

[0057] 2.1 Standards and Reagents

[0058] YBSW015 antibody injection standards were provided by Yabao Pharmaceutical Group Co., Ltd., and isotope internal standards were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. Oasis MCX 96-well plates (10 mg) were purchased from Waters, trypsin from Sigma-Aldrich, chromatography-grade acetonitrile, methanol, and isopropanol from Thermo Fisher Scientific, and ultrapure water was prepared using a MicroPure UV / UF ultrapurifier (Thermo Fisher Scientific). Blank serum was obtained from healthy volunteers or recovered COVID-19 patients. All other reagents were of commercial analytical grade.

[0059] 2.2 Instruments and Equipment

[0060] Liquid chromatograph: LC-30AD (Shimadzu, Japan);

[0061] Mass spectrometer: QTRAP 6500 (AB Sciex);

[0062] Processing software: Analyst 1.6.2 (AB Sciex);

[0063] 2.3 Liquid quality conditions

[0064] 2.3.1 Chromatographic conditions

[0065] Column: ACE C18- (2.1×50mm, 3μm);

[0066] Column temperature: 50°C;

[0067] Autosampler temperature: 4°C;

[0068] Flow rate: 0.8 mL / min;

[0069] Injection volume: 3.0 μL;

[0070] Mobile phase: Phase A: aqueous solution containing 0.1% (v / v) formic acid; Phase B: acetonitrile solution containing 0.1% (v / v) formic acid;

[0071] The elution gradient is as follows:

[0072] 2.3.2 Mass spectrometry conditions

[0073] The ion source parameters of the mass spectrometer are as follows:

[0074] The compound monitoring mode adopts the multiple reaction monitoring (MRM) mode, and the parameters are shown in the following table, where "Q1" is the mass-to-charge ratio of the first quadrupole in the mass spectrometer, "Q3" is the mass-to-charge ratio of the second quadrupole in the mass spectrometer, "Dwell Time (ms)" is the dwell time (milliseconds), "DP" is the declustering potential, "CXP" is the collision cell exit voltage, "CE" is the collision energy, and "EP" is the entrance voltage.

[0075] 2.4 Preparation of internal standard stock solution and working solution

[0076] Prepare a 1000 μg / mL stock solution of the isotope-labeled internal standard peptide in 50% (v / v) acetonitrile-water solution. Prepare a mixed isotope internal standard working solution in 100 mM NH₄HCO₃ (pH 8.0) containing 4 μg / mL SLSL-IS and 10 μg / mL EAYG-IS. Store at -10 to -30°C until use.

[0077] 2.5 Preparation of standard curve and quality control samples

[0078] Anti-novel coronavirus bispecific antibodies stored at -10 to -30°C were diluted with human blank serum to a series of standard curve concentrations and quality control sample concentrations. The linear range for EAYG was 5.00 μg / mL, 10.0 μg / mL, 50.0 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 800 μg / mL, and 1000 μg / mL, and the linear range for SLSL was 2.00 μg / mL, 5.00 μg / mL, 10.0 μg / mL, 50.0 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, 800 μg / mL, and 1000 μg / mL. The concentrations of EAYG quality control (QC) samples were 5.00 μg / mL (lower limit of quantification, LLQC QC), 15.0 μg / mL (low concentration quality control, LQC), 450 μg / mL (medium concentration quality control, MQC), and 750 μg / mL (high concentration quality control, HQC); the concentrations of SLSL quality control samples were 2.00 μg / mL (LLQC QC), 6.00 μg / mL (LQC), 60.0 μg / mL (MQC-1), 450 μg / mL (MQC-2), and 750 μg / mL (HQC).

[0079] 2.6 Serum sample pretreatment

[0080] 2.6.1 Protein precipitation

[0081] Accurately measure 100 μL of serum sample into a 2.0 mL low-absorption Eppendorf tube. Add 100 μL of 1% (w / v) trichloroacetic acid-isopropanol. Mix for 5 minutes. Centrifuge at 500 g for 5 minutes at 4°C. Discard an equal volume of the supernatant. Add 300 μL of 100 mM NH4HCO3 (pH 8.0) to the pellet. Vortex mix for at least 3 minutes. Centrifuge at 1000 g for 5 minutes at 4°C. Discard the supernatant. Add another 300 μL of 100 mM NH4HCO3 (pH 8.0) to the pellet. Repeat the vortex centrifugation procedure. Discard the supernatant.

[0082] 2.6.2 Enzymatic hydrolysis

[0083] Add 200 μL of 100 mM NH4HCO3 (pH 8.0) to the protein precipitate and vortex for at least 5 minutes. Add 20 μL of internal standard working solution (i.e., working solution containing isotope-labeled peptides, with 4 μg / mL SLSL-IS and 10 μg / mL EAYG-IS) and 20 μL of trypsin solution (15 mg / mL), vortex for 5 minutes, and incubate at 500-600 rpm and 60°C for 2 hours. Terminate the reaction by adding 15 μL of 20% (v / v) formic acid-water solution and centrifuge at 10,000 g for 5 minutes at 4°C.

[0084] 2.6.3 Solid Phase Extraction

[0085] An Oasis MCX 96-well plate (10 mg) was activated with 400 μL of methanol and then equilibrated with 400 μL of purified water. The supernatant from the enzymatic digestion was loaded onto the plate and slowly passed through the plate, discarding the filtrate. The plate was rinsed sequentially with 400 μL of 2% (v / v) formic acid in water and 400 μL of 90% (v / v) methanol in water, discarding the washes. The plate was then eluted twice with 100 μL of acetonitrile:water:NH3H2O:isopropanol (v / v / v / v, 35:55:5:5). The filtrate was collected for LC-MS / MS analysis.

[0086] 2.7 Methodological Validation

[0087] The established method was systematically validated according to the "9012 Guidelines for Validation of Quantitative Analysis Methods for Biological Samples" in the Chinese Pharmacopoeia (2020 Edition) and ICH M10 "Bioanalytical Method Validation and Study Sample Analysis." Validation included selectivity, standard curves, intra- and inter-batch precision and accuracy, matrix effects, recovery, carryover, dilution reliability, and stability.

[0088] Example

[0089] Example 1. Characteristic peptide screening

[0090] The amino acid sequence information of the bispecific antibody was imported into Skyline software 22.2 to predict the information of peptide fragments that may be produced under the action of proteases. The peptide information was compared and the peptides located in the complementary determining regions (CDRs) were selected. The information of the screened candidate peptides is shown in Table 1. The screened peptide ion pair information was then imported into Analyst 1.6.2. The enzymatically hydrolyzed double blank sample (i.e., healthy volunteer serum) and the matrix sample containing the YBSW015 antibody (i.e., the anti-new coronavirus bispecific antibody was added to the serum of healthy volunteers) were subjected to LC-MS / MS analysis to screen out peptides with good selectivity and sensitivity. The results are shown in Figure 1. The 100 μg / mL bispecific antibody candidate peptides EAYG, SLSL and IEFP have good sensitivity and are expected to meet the method sensitivity requirements. The three specific peptides were further investigated. The specificity of candidate characteristic peptides was further investigated using the serum of recovered COVID-19 patients, and characteristic peptides that could resist the interference of endogenous anti-SARS-CoV-2 virus antibodies were selected for further investigation. The results are shown in Table 2. The endogenous antibodies present in the serum of recovered COVID-19 patients (coded A to J) interfere with the determination of IEFP, but do not interfere with the determination of EAYG and SLSL. Through pre-validation investigation of specificity and sensitivity, EAYG and SLSL were selected for methodological validation. The characteristic peptides EAYG and SLSL and their isotope internal standards were synthesized, and the isotope internal standard sequences are shown in Table 1.

[0091] Table 1 Information of screened candidate peptides

[0092] In Table 1, “Q1” is the mass-to-charge ratio of the first quadrupole of the mass spectrometer, “Q3” is the mass-to-charge ratio of the second quadrupole of the mass spectrometer, “DP” is the declustering voltage, and “CE” is the collision energy.

[0093] Table 2 Interference of serum from recovered COVID-19 patients in the determination of anti-novel coronavirus bispecific antibodies

[0094] In Table 2, “LLOQ” is the lower limit of quantification.

[0095] Example 2, selectivity

[0096] Six different healthy volunteer sera (coded A to F), one hemolytic serum, and one hyperlipidemia serum (100 μL each) were collected and processed according to "2.6 Serum Sample Pretreatment" except that no internal standard was added. LC-MS / MS analysis was then performed. The results are shown in Table 3, Figures 2 and 3. The responses of interfering components in healthy volunteer serum, hemolytic serum, and hyperlipidemia serum were less than 20% of the lower limit of quantification (LLOQ) responses of the characteristic peptides EAYG and SLSL, and less than 5% of the responses of the isotopic internal standards EAYG-IS and SLSL-IS, demonstrating the good specificity of the method.

[0097] Table 3 Results of the selectivity study of anti-novel coronavirus bispecific antibodies

[0098] In Table 3, “LLOQ” is the lower limit of quantification.

[0099] Example 3, standard curve

[0100] Prepare a series of standard curve samples using healthy volunteer serum according to the method in "2.5 Preparation of Standard Curve and Quality Control Samples". Then proceed according to "2.6 Serum Sample Pretreatment". Use the bispecific antibody concentration as the horizontal axis (X) and the peak area ratio of the characteristic peptide and its isotope-labeled internal standard as the vertical axis (Y) to perform linear regression using the weighted least squares method, with a weight of 1 / X. 2 The results are shown in Table 4 and Figure 4 (Figure 4 shows the first linear equation of each characteristic peptide in Table 4). The results show that EAYG has a good linear relationship in the concentration range of 5.00 to 1000 μg / mL; SLSL has a good linear relationship in the concentration range of 2.00 to 1000 μg / mL.

[0101] Table 4 Linearity results

[0102] Example 4. Precision and Accuracy

[0103] Using healthy volunteer serum, quality control samples and LLOQ samples were prepared according to the method of "2.5 Preparation of standard curve and quality control samples". Five copies were prepared for each concentration as a batch of precision and accuracy samples, and the samples were processed according to "2.6 Serum sample pretreatment". Three batches of precision and accuracy samples were determined within at least two days and named Run1, Run2, and Run3 respectively. The inter-run results are the combined analysis of the results of runs 1 to 3, which are called inter-run precision and accuracy. Precision and accuracy are expressed as RSD% and RE%, respectively. The results are shown in Tables 5 and 6. The absolute values ​​of RE% and RSD% of the quality control concentrations of the characteristic peptides EAYG and SLSL were less than 15%, and the absolute values ​​of RE% and RSD% of the LLOQ concentration were less than 20%, indicating that the precision and accuracy of this method are good.

[0104] Table 5 Results of EAYG precision and accuracy inspection

[0105] Table 6 SLSL precision and accuracy inspection results

[0106] Example 5: Matrix Effect

[0107] The matrix effect was investigated using serum from six different healthy volunteers, as well as hemolytic serum and high-lipid serum. LQC and HQC (n=3) were prepared according to "2.5 Preparation of Standard Curve and Quality Control Samples", and sample processing was performed according to "2.6 Serum Sample Pretreatment". The results are shown in Table 7. The accuracy RE% of the characteristic peptide SLSL was within the range of ±20%, and the precision was within 20%; the accuracy RE% of the characteristic peptide EAYG was within the range of ±20%, and the precision was within 20%. This shows that the matrix effect of the method of the present application is good.

[0108] Table 7 Matrix effect investigation results

[0109] In Table 7, “LQC” refers to low-concentration quality control, and “HQC” refers to high-concentration quality control.

[0110] Example 6, recovery rate

[0111] The recovery evaluation included protein precipitation recovery and solid phase extraction recovery.

[0112] 6.1 Protein precipitation recovery

[0113] YBSW015 antibody was added to the serum of healthy volunteers, and the sample was processed according to "2.6 Serum Sample Pretreatment". LC-MS / MS analysis was performed to obtain the peak area A1 of the protein precipitation sample mass spectrometry analysis.

[0114] The serum of healthy volunteers was processed according to "2.6.1 Protein Precipitation" to obtain precipitated protein. YBSW015 antibody was added to the precipitated protein. The precipitated protein was treated according to the processes of "2.6.2 Enzymatic Hydrolysis" and "2.6.3 Solid Phase Extraction". Then, LC-MS / MS analysis was performed to obtain the mass spectrometry peak area A2 of the sample without protein precipitation.

[0115] The protein precipitation recovery was evaluated using the A1 / A2 ratio. As shown in Table 8, the EAYG protein precipitation recovery ranged from 109.4% to 124.1%, with an overall RSD of 9.5%. The SLSL protein precipitation recovery ranged from 99.1% to 135.0%, with an overall RSD of 14.9%.

[0116] 6.2 SPE recovery

[0117] Serum from healthy volunteers was processed according to "2.6.1 Protein Precipitation" and "2.6.2 Enzymatic Hydrolysis" to obtain enzymatic hydrolysis samples. EAYG and SLSL synthetic specific peptides were added to the enzymatic hydrolysis samples. The enzymatic hydrolysis samples were processed according to "2.6.3 Solid Phase Extraction" and subsequently analyzed by LC-MS / MS to obtain the mass spectrometry peak area A1 of the SPE-treated samples.

[0118] The serum of healthy volunteers was processed according to "2.6 Serum Sample Pretreatment" to obtain SPE elution filtrate. EAYG and SLSL synthetic specific peptides were added to the obtained filtrate, and LC-MS / MS analysis was performed to obtain the mass spectrometry peak area A2 of the sample without SPE treatment.

[0119] The SPE recovery rate was evaluated by the A1 / A2 ratio. As shown in Table 8, the SPE recovery rate of EAYG ranged from 71.9% to 81.0%, with an overall RSD% of 6.3%. The SPE recovery rate of SLSL ranged from 57.8% to 70.3%, with an overall RSD% of 11.7%.

[0120] Table 8 Recovery rate investigation

[0121] In Table 8, “LQC” refers to low-concentration quality control, “MQC” refers to medium-concentration quality control, and “HQC” refers to high-concentration quality control. The MQC inspected by SLSL is MQC-2.

[0122] Example 7, Residue Investigation

[0123] The effect of sample carryover was investigated by injecting healthy volunteer serum samples processed according to "2.6 Serum Sample Pretreatment" after injection at the upper limit of quantification (ULOQ). The carryover of EAYG and SLSL in the blank sample at the upper limit of quantification should not exceed 20% of the EAYG or SLSL response at the LLOQ, or 5% of the internal standard, respectively. The results are shown in Table 9. The EAYG-IS carryover was less than 5% of the internal standard, meeting the validation criteria. The carryover of SLSL and its internal standard also met the validation criteria.

[0124] Table 9 Residue investigation

[0125] In Table 9, “LLOQ” is the lower limit of quantification.

[0126] Example 8: Dilution reliability

[0127] Quality control samples exceeding the upper limit of quantitation (3000 μg / mL) were prepared using healthy volunteer serum. These samples were diluted with healthy volunteer serum at dilution factors of 20 and 5, respectively. At least five replicates were prepared for each dilution factor. Samples were then processed according to "2.6 Serum Sample Pretreatment" and quantified using a freshly prepared standard curve. The results are shown in Table 10. At dilution factors of 20 and 5, respectively, the quantification using the characteristic peptides EAYG and SLSL demonstrated precision (RSD%) less than 15% and accuracy (RE%) within ±15%.

[0128] Table 10 Dilution reliability investigation results

[0129] Example 9, Stability

[0130] 9.1 Whole Blood Stability

[0131] Prior to stability sample preparation, fresh whole blood from healthy volunteers will be incubated at 37°C for 10-15 minutes. YBSW015 antibody will be added to the whole blood to prepare LQC and HQC concentration whole blood samples, and gently mixed to avoid hemolysis. The whole blood samples will then be incubated at 37°C for 15 minutes to allow the analyte to reach equilibrium between blood cells and plasma. The zero-time sample (the freshly prepared stability test sample) will be immediately centrifuged at 4°C, 1500g for 10 minutes to separate the plasma, and the plasma sample will be stored at -60 to -90°C until use. Other stability test whole blood samples will be kept at room temperature for 2 hours, then centrifuged at 4°C, 1500g for 10 minutes to separate the plasma, and the plasma sample will be stored at -60 to -90°C until use as the whole blood stability sample. Stability samples will be processed according to "2.6 Serum Sample Pretreatment". The results are shown in Table 11. After the whole blood samples were placed at room temperature for 2 hours, the RE% and RSD% determined by SLSL were both less than 15%, and the RE% and RSD% determined by EAYG were both less than 25%.

[0132] 9.2 Room temperature stability

[0133] Using serum from healthy volunteers, LQC and HQC concentrations were prepared according to "2.5 Preparation of Standard Curves and Quality Control Samples." The quality control samples were stored at room temperature for 24 hours. Samples were then processed according to "2.6 Serum Sample Pretreatment." The stability of the plasma samples after 24 hours at room temperature was investigated. The results are shown in Table 11. The RE% for the SLSL assay was within ±15% and the RSD% was less than 15%. The RE% for the EAYG assay was within ±20%, and the RSD% was less than 15%.

[0134] 9.3 Freeze-thaw stability

[0135] The stability of samples stored at -20°C and -80°C after five freeze-thaw cycles was investigated. Samples at low quality control (LQC) and high quality control (HQC) concentrations were prepared using serum from healthy volunteers according to "2.5 Preparation of Standard Curves and Quality Control Samples" and stored at -20°C or -80°C for at least 24 hours. The samples were then removed from the refrigerator, allowed to equilibrate at room temperature, and returned to the refrigerator to complete one freeze-thaw cycle. After each freeze-thaw cycle, the samples were returned to the refrigerator and stored for at least 12 hours before being thawed at room temperature. After five freeze-thaw cycles, the samples were processed according to "2.6 Serum Sample Preparation" to assess freeze-thaw stability. The results are shown in Table 11. The RE% determined by SLSL was within ±15% and the RSD% was less than 15%. The RE% determined by EAYG was within ±20%, with the RSD% both less than 20%.

[0136] Table 11 Stability test results

[0137] In Table 11, “LQC” refers to low-concentration quality control, and “HQC” refers to high-concentration quality control.

[0138] Example 10. Clinical Applicability

[0139] Participants in the Phase I clinical trial (NCT05369754) of a bispecific antibody against the novel coronavirus were administered YBSW015 via intravenous infusion at doses of 180 mg, 450 mg, 900 mg, and 1800 mg. Whole blood samples were collected from the clinic, allowed to stand at room temperature, and centrifuged 30 to 60 minutes after collection to separate the upper serum layer. The centrifugation conditions were 15-25°C and 2000-4000g for 5-10 minutes. The separated upper serum layer was frozen and stored at -80°C. The applicability of the assay was preliminarily verified by measuring the blood concentration of the YBSW015 antibody in participants who received the drug. The specific method is to collect 100μL of serum samples from each enrolled subject, process the serum samples according to the steps in "2.6 Serum Sample Pretreatment", and perform LC-MS / MS analysis according to "2.3 Liquid Mass Spectrometry Conditions" to obtain the YBSW015 antibody drug concentration in each serum sample. The YBSW015 antibody drug-time curve for each dose group was calculated (Figure 5). No interference with blood drug concentration was observed due to pre-existing antibodies in the enrolled subjects, and the blood drug concentration at the final sampling point of each dose group, including the lowest dose group, was less than 1 / 20 of the corresponding peak concentration. This shows that this method is suitable for clinical drug concentration monitoring of the YBSW015 antibody.

[0140] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A LC-MS / MS quantitative detection method for anti-novel coronavirus bispecific antibody drugs, wherein The following steps are involved: (1) performing protein precipitation treatment on a blood sample to obtain precipitated protein; (2) enzymatically hydrolyzing the precipitated protein to obtain an enzymatic hydrolysis product; (3) extracting the enzymatic hydrolysis product using a solid phase extraction plate to obtain a supernatant of the sample to be tested; (4) using a liquid chromatography-tandem mass spectrometry platform to quantify the characteristic peptides in the supernatant of the sample to be tested by an internal standard method, thereby quantifying the concentration of the anti-novel coronavirus bispecific antibody; wherein, The characteristic peptide segment in step (4) is EAYG or SLSL, and its amino acid sequence is shown in SEQ ID NO: 1 or SEQ ID NO: 2, respectively; Preferably, in step (4), the concentration of the anti-novel coronavirus bispecific antibody is quantified based on EAYG and / or SLSL, using EAYG-IS and SLSL-IS as internal standards; EAYG-IS and SLSL-I S are isotope-labeled peptides based on the characteristic peptides; EAYG-IS: EAYGMDVWGQGTTVTV(+6)SSASTK(+8); or SLSL-IS:SLSLSPGGGSQVQL(+7)VQSGAEVK(+8); +6, +7, and +8 refer to the mass numbers that increase after isotope labeling.

2. The method according to claim 1, in, The blood sample is selected from at least one of whole blood, serum and plasma.

3. The method according to claim 1 or 2, in, In step (1), a 1% (w / v) trichloroacetic acid-isopropanol solution is used for protein precipitation. Preferably, the protein precipitation method comprises: taking a blood sample, adding 100 μL of 1% (w / v) trichloroacetic acid-isopropanol in a volume ratio of 1:1, mixing, centrifuging and discarding the supernatant; adding NH 4 HCO 3 The solution was washed twice and the supernatant was discarded.

4. The method according to claim 1 or 2, in, In step (2), trypsin is used to enzymatically hydrolyze the precipitated protein; preferably, NH 4 HCO 3 After the solution is mixed, isotope-labeled peptides and trypsin are added, mixed, and incubated at 500-600 rpm and 60° C. for 2 h; 20% (v / v) formic acid aqueous solution is added to terminate the reaction, and centrifuged at 4° C. and 10,000 g for 5 min.

5. The method according to claim 1 or 2, in, The step (3) uses a solid phase extraction plate to extract the enzymatic hydrolysis product, which includes: loading the enzymatic hydrolysis product, allowing the enzymatic hydrolysis product to slowly pass through the solid phase extraction plate, and discarding the filtrate; washing the solid phase extraction plate with a 2% (v / v) formic acid aqueous solution and a 90% (v / v) methanol aqueous solution in sequence, and discarding the washing solution; and then using acetonitrile: water: NH 3 H 2 O: eluted twice with isopropanol solution and the filtrate was collected; preferably, the solid phase extraction plate was activated with methanol before use and then balanced with purified water.

6. The method according to claim 1 or 2, in, In step (4), the liquid chromatography column used in the liquid chromatography is ACE C18- (2.1×50mm, 3μm); preferably, the flow rate of the mobile phase is 0.1-1.5mL / min, more preferably, the flow rate of the mobile phase is 0.5-0.8mL / min; preferably, the mobile phase is formic acid water-formic acid acetonitrile, more preferably, mobile phase A is 0.1% (v / v) formic acid water, and phase B is 0.1% (v / v) formic acid acetonitrile; preferably, the column temperature is 50°C; the automatic sampler temperature is 4°C.

7. The method according to claim 6, in, The linear range of the characteristic peptide SLSL standard used in the liquid chromatography is 2.00μg / mL, 5.00μg / mL, 10.0μg / mL, 50.0μg / mL, 100μg / mL, 250μg / mL, 500μg / mL, 800μg / mL, and 1000μg / mL. The linear range concentration of the characteristic peptide EAYG standard is 5.00μg / mL, 10.0μg / mL, 50.0μg / mL, 100μg / mL, 250μg / mL, 500μg / mL, 800μg / mL, and 1000μg / mL.

8. A characteristic peptide segment, whose amino acid sequence is shown as SEQ ID NO: 1 or SEQ ID NO:

2.

9. An isotope-labeled peptide segment: EAYG-IS: EAYGMDVWGQGTTVTV(+6)SSASTK(+8); or SLSL-IS: SLSLSPGGGSQVQL(+7)VQSGAEVK(+8).

10. A composition comprising the characteristic peptide segment according to claim 8 and / or the isotope-labeled peptide segment according to claim 9.

11. A kit comprising the characteristic peptide segment according to claim 8 and / or the isotope-labeled peptide segment according to claim 9.

12. Use of the characteristic peptide segment according to claim 8, the isotope-labeled peptide segment according to claim 9, the composition according to claim 10, or the kit according to claim 11 for quantitatively detecting the concentration of YBSW015 antibody, or for preparing a product for quantitatively detecting the concentration of YBSW015 antibody.

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