Method for quantifying antigen-binding molecule bound to cell

Abstract

To provide a method for accurately quantifying an antigen-binding molecule bound to a cell expressing a target antigen.SOLUTION: A method for quantifying an antigen-binding molecule bound to a cell comprises: a preparation step of preparing a biological sample derived from the cell, where the biological sample is homogenized, the cell expresses a target antigen to which the antigen-binding molecule specifically binds, and the cell has been exposed to the antigen-binding molecule; an extraction step of adding an extraction solution to the biological sample to obtain an analysis sample, where the extraction solution contains an organic acid and a nonionic surfactant, and a pH of the extraction solution is 1 or more and 3 or less; and a quantification step of analyzing the analysis sample to quantify the antigen-binding molecule.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for quantifying antigen-binding molecules bound to cells.

Background Art

[0002] In recent years, the development of pharmaceuticals using antibodies (hereinafter sometimes referred to as "antibody pharmaceuticals") has been actively carried out. Antibodies have the property of specifically binding to specific antigens (antigen specificity). Utilizing this property, for example, an antibody that specifically binds only to a receptor (antigen) specifically expressed on cancer cells is administered to a cancer tissue containing the cancer cells to induce an immune response and treat cancer. For example, cetuximab is known as an anticancer agent that specifically binds to the epidermal growth factor receptor (EGFR) and suppresses the growth of cancers such as colorectal cancer.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In general, an antibody drug first binds to the target antigen in order to exert its pharmacological effect. Therefore, in order to evaluate an antibody drug under development, it is desired to develop a technique for precisely measuring the accumulation level of the antibody that has reached the target tissue or target cells. For example, Non-Patent Document 1 discloses a technique for measuring the amount of ADC (antibody drug conjugate) distributed in a tissue using a mass spectrometer by sharing the XY-axis direction with the mass spectrometry side based on imaging data obtained by microscopy, and obtaining a molecular distribution image thereof. Non-Patent Document 2 discloses a technique for analyzing the accumulation level of an ADC in a formalin-fixed paraffin-embedded tissue section (FFPE tissue section) using an anti-human antibody and an anti-payload antibody from a tissue staining image.

[0006] However, in the prior art, since the antibody bound to the target tissue is indirectly detected, there remains room for improvement in terms of quantitative accuracy. The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for accurately and sensitively quantifying an antigen-binding molecule bound to a cell expressing a target antigen.

Means for Solving the Problems

[0007] As a result of intensive research, the present inventors have found that an antigen-binding molecule bound to the above cells can be accurately quantified by adding a predetermined extraction solution to a biological sample derived from the cells expressing the target antigen to obtain an analysis sample, and have completed the present invention.

[0008] A first aspect of the present invention is a method for quantifying an antigen-binding molecule bound to a cell, comprising: a preparation step of preparing a biological sample derived from the above cells, wherein the above biological sample is homogenized, the above cells express a target antigen to which the above antigen-binding molecule specifically binds, the above cells have been exposed to the above antigen-binding molecule, and the preparation step; an extraction step of adding an extraction solution to the above biological sample to obtain an analysis sample, wherein The extraction solution contains an organic acid and a nonionic surfactant, in an extraction step where the pH of the extraction solution is 1 or more and 3 or less, in a quantification step where the sample for analysis is analyzed to quantify the antigen-binding molecule, and includes

Advantages of the Invention

[0009] According to the present invention, it becomes possible to provide a method for accurately quantifying an antigen-binding molecule bound to cells expressing a target antigen.

Brief Description of the Drawings

[0010]

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[0011] Hereinafter, an embodiment of the present invention (hereinafter referred to as "this embodiment") will be described. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of the range (that is, A or more and Z or less). When there is no unit description for A and there is a unit description only for Z, the units of A and Z are the same.

[0012] Method for Quantifying Antigen-Binding Molecules Bound to Cells The first aspect of this embodiment is a method for quantifying antigen-binding molecules bound to cells, comprising: A preparation step of preparing a biological sample derived from the above cells, wherein: the biological sample is homogenized, the cells express a target antigen to which the antigen-binding molecule specifically binds, the cells have been exposed to the antigen-binding molecule, and the preparation step; An extraction step of adding an extraction solution to the biological sample to obtain an analysis sample, wherein: the extraction solution contains an organic acid and a nonionic surfactant, the pH of the extraction solution is 1 or more and 3 or less, and the extraction step; A quantification step of analyzing the analysis sample to quantify the antigen-binding molecule; and comprising:

[0013] <Preparation Step> In this step, a biological sample derived from cells is prepared. In the present embodiment, the "biological sample derived from cells" means the cells themselves or a biological sample containing substances (cell membrane, cell nucleus, cytoplasm, etc.) constituting the cells. The biological sample is homogenized. "Homogenized" means a state in which the components in the biological sample are uniform and there is no partial bias in the components. The biological sample may be in a solid state or a liquid state, but is preferably in a liquid state.

[0014] The above cells express a target antigen to which the above antigen-binding molecule specifically binds. In the present embodiment, the "target antigen" mainly refers to a protein expressed on the surface of the above cells. In one aspect of the present embodiment, the above target antigen can also be understood as a "target biomolecule" or a "target protein". Examples of such proteins include receptors expressed on the surface of the above cells, enzymes expressed inside the above cells, tumor necrosis factor receptors, insulin receptors, vascular endothelial growth factor receptors, and the like. Examples of receptors expressed on the surface of the above cells include epidermal growth factor receptor (EGFR), HER2, CD30, PD-L1, RANKL, tumor necrosis factor receptor, insulin receptor, vascular endothelial growth factor receptor, and the like. In one aspect of the present embodiment, the above target antigen preferably includes at least one selected from the group consisting of EGFR, HER2, CD30, PD-L1, RANKL, tumor necrosis factor receptor, insulin receptor, and vascular endothelial growth factor receptor.

[0015] The above cells have been exposed to the above antigen-binding molecule. In the present embodiment, the "antigen-binding molecule" refers to a protein or peptide that specifically binds to the above target antigen. Examples of the above antigen-binding molecule include antibodies, peptide aptamers, and the like. In one aspect of the present embodiment, the above antigen-binding molecule preferably includes an antibody or a peptide aptamer. In another aspect of the present embodiment, the above antigen-binding molecule preferably includes at least one selected from the group consisting of cetuximab, trastuzumab, brentuximab, denosumab, infliximab, adalimumab, etanercept, aflibercept, ramucirumab, atezolizumab, avelumab, and insulin analogs.

[0016] In this embodiment, "having been exposed to the antigen-binding molecule" means that the cell or the tissue containing the cell was present in the environment where the antigen-binding molecule was present, or was artificially exposed to the antigen-binding molecule, before collecting the cell or the tissue containing the cell. For example, culturing the cell in a medium containing the antigen-binding molecule or administering the antigen-binding molecule to the tissue containing the cell corresponds to "having been exposed to the antigen-binding molecule".

[0017] In this embodiment, the cell is preferably a cell derived from a cancer tissue or a cell derived from a diseased tissue.

[0018] In this embodiment, the method for homogenizing the biological sample is not particularly limited and may be homogenized by a known method. Examples of the homogenization method include a method of using beads for cell disruption (e.g., Figure 1) by placing the cell or the tissue containing the cell (preferably, a thin slice of the tissue) in a predetermined buffer (e.g., in D-PBS), a method by ultrasonic treatment, a method by freeze-thawing, and a method using a French press.

[0019] In one aspect of this embodiment, the biological sample may further contain an enzyme inhibitor. By including the enzyme inhibitor in the biological sample, it is possible to suppress the antigen-binding molecule from being decomposed by an enzyme. Examples of the enzyme inhibitor include a phosphatase inhibitor, a protease inhibitor, and the like.

[0020] <Extraction step> In this process, an extraction solution is added to the biological sample to obtain an analysis sample. In the present embodiment, the "extraction solution" means a solution used to dissociate the antigen-binding molecule bound to the target antigen from the target antigen. The extraction solution contains an organic acid and a nonionic surfactant. In the present embodiment, the "analysis sample" means a sample derived from the biological sample and used for analysis in the quantification process described later. In this specification, the pretreatment method performed in the extraction process may be referred to as the "ACES method" (Acidic Conditions Extraction supported with Surfactant method).

[0021] In the present embodiment, the "organic acid" means an organic compound showing acidity. The organic acid is preferably an organic compound having a carboxyl group. Examples of the organic acid include arginine, citrulline, and glycine. In one aspect of the present embodiment, the organic acid preferably contains at least one selected from the group consisting of arginine, citrulline, and glycine. When the antigen-binding molecule is an antibody, from the viewpoint of maintaining the stability of the antibody, the organic acid is preferably arginine.

[0022] The concentration of the organic acid is preferably 20 mM or more and 1000 mM or less, more preferably 100 mM or more and 500 mM or less, with respect to the extraction solution.

[0023] In this embodiment, the "nonionic surfactant" means a surfactant that does not ionize even when dissolved in water. Examples of the nonionic surfactant include alkyl glycosides in which sugar and a higher alcohol are glycosidically bonded. Examples of the alkyl glycoside include n-octyl-β-D-thioglucopyranoside, n-octyl-β-D-glucoside, n-octyl-β-D-maltoside, n-decyl-β-D-glucoside, n-decyl-β-D-maltoside, n-dodecyl-β-D-glucoside, n-heptyl-β-D-thioglucoside, and n-nonyl-β-D-thiomaltoside, trehalose C12, and the like. In one aspect of this embodiment, it is preferable that the nonionic surfactant contains an alkyl glycoside. The alkyl glycoside preferably contains at least one selected from the group consisting of n-octyl-β-D-thioglucopyranoside, n-octyl-β-D-glucoside, n-octyl-β-D-maltoside, n-decyl-β-D-glucoside, n-decyl-β-D-maltoside, n-dodecyl-β-D-glucoside, n-heptyl-β-D-thioglucoside, and n-nonyl-β-D-thiomaltoside. More preferably, the alkyl glycoside contains n-octyl-β-D-thioglucopyranoside.

[0024] The concentration of the nonionic surfactant is preferably 0.1% by mass or more and 10% by mass or less, more preferably 0.5% by mass or more and 5% by mass or less, based on the extraction solution. In one aspect of this embodiment, the concentration of the nonionic surfactant is preferably higher than the critical micelle concentration of the nonionic surfactant.

[0025] When the nonionic surfactant is n-octyl-β-D-thioglucopyranoside, the concentration of n-octyl-β-D-thioglucopyranoside is preferably 0.2% by mass or more and 10% by mass or less, more preferably 0.5% by mass or more and 5% by mass or less, based on the extraction solution.

[0026] The type and concentration of the above-mentioned organic acid, and the type and concentration of the above-mentioned nonionic surfactant are determined by analyzing the above-mentioned extraction solution with a liquid chromatograph and a mass spectrometer.

[0027] The pH of the above-mentioned extract is 1 or more and 3 or less, preferably 1.5 or more and 2.5 or less. The pH of the above-mentioned extract can be measured with a commercially available pH meter.

[0028] In this embodiment, the addition amount of the above-mentioned extraction solution is not particularly limited as long as the effects of the present invention are achieved. For example, it is preferably 10 times or more and 50 times or less, more preferably 20 times or more and 40 times or less, based on the mass of the above-mentioned biological sample. At this time, the density of the above-mentioned extraction solution is regarded as 1 mg / 1 μL, and the addition amount of the above-mentioned extract is determined accordingly.

[0029] In this embodiment, the above-mentioned extraction solution may be directly added to the above-mentioned biological sample to obtain an analysis sample (for example, FIG. 1). Or after separating the above-mentioned biological sample into a liquid component and a solid component and recovering them respectively, the above-mentioned extraction solution may be added to the above-mentioned solid component to obtain an analysis sample. The method for separating the above-mentioned biological sample into a liquid component and a solid component is not particularly limited. For example, a method of separating into a supernatant (liquid component) and a precipitate (solid component) by centrifugation can be mentioned.

[0030] In one aspect of this embodiment, the above-mentioned extraction step may include separating the above-mentioned biological sample into a liquid component and a solid component, adding the above-mentioned extraction solution to the above-mentioned solid component to obtain a first analysis sample, and obtaining the above-mentioned liquid component as a second analysis sample.

[0031] In another aspect of this embodiment, the above-mentioned preparation step and the above-mentioned extraction step may be performed simultaneously. For example, a method of performing homogenization after adding the above-mentioned extract to the above-mentioned biological sample can be mentioned.

[0032] In one aspect of the present embodiment, the extraction solution may further contain an enzyme inhibitor, an internal standard substance, or an adsorption inhibitor. Here, the "adsorption inhibitor" means a reagent that suppresses the physical adsorption of the antigen-binding molecule to the wall surface of the microtube or the like. Examples of the enzyme inhibitor include a phosphatase inhibitor, a protease inhibitor, and the like. Examples of the internal standard substance include an antigen-binding molecule labeled with an isotope. Examples of the adsorption inhibitor include BSA, mouse IgG, and the like.

[0033] <Quantification step> In this step, the analysis sample is analyzed to quantify the antigen-binding molecule. In one aspect of the present embodiment, "analyzing the analysis sample" includes directly subjecting the analysis sample to analysis and subjecting the analysis sample to analysis after removing solid components (for example, the remaining precipitate) in the analysis sample.

[0034] In the present embodiment, the method for quantifying the antigen-binding molecule may directly detect and quantify the antigen-binding molecule, or may detect and quantify a peptide derived from the antigen-binding molecule.

[0035] In the present embodiment, the method for quantifying the antigen-binding molecule is not particularly limited, and examples thereof include a quantification method by ELISA, a quantification method by a liquid chromatograph tandem mass spectrometer (LC-MS analyzer or LC-MS / MS analyzer), and the like.

[0036] The column of LC in the LC-MS analyzer is not particularly limited, and a hydrophobic column such as C30, C18, C8, C4, etc., which are generally used for the analysis of proteins or peptides, or a column containing a carrier for hydrophilic affinity chromatography can be appropriately selected and used. If necessary, after performing treatments such as desalting, solubilization, extraction, concentration, drying, etc., the sample may be used for mass spectrometry.

[0037] The ionization method in mass spectrometry is not particularly limited, and methods such as electron ionization (EI) method, chemical ionization (CI) method, field desorption (FD) method, fast atom bombardment (FAB) method, matrix-assisted laser desorption ionization (MALDI) method, electrospray ionization (ESI) method, etc. can be adopted. The analysis method of the ionized sample is also not particularly limited, and magnetic field deflection type, quadrupole (Q) type, ion trap (IT) type, time-of-flight (TOF) type, Fourier transform ion cyclotron resonance (FT-ICR) type, etc. can be appropriately determined according to the ionization method. It is also possible to perform MS / MS analysis, multi-stage mass spectrometry of MS3 or higher, or multiple reaction monitoring (MRM) using a triple quadrupole type mass spectrometer or the like.

[0038] The device particularly suitable for the quantification method of this embodiment is not particularly limited, and examples thereof include LCMS-8030, LCMS-8040, LCMS-8050, LCMS-8060, LCMS-9030, LCMS-IT-TOF (all manufactured by Shimadzu Corporation).

[0039] In one aspect of this embodiment, when the antigen-binding molecule is an antibody, the quantification step preferably includes limiting the peptide derived from the variable region of the antibody by the nSMOL method and analyzing the peptide by LC-MS / MS analysis method. The nSMOL method can be carried out, for example, using the nSMOL Antibody BA Kit (product name, manufactured by Shimadzu Corporation).

[0040] In the prior art, since an antigen-binding molecule (e.g., an antibody) bound to a cell expressing a target antigen is indirectly detected, there has been room for improvement in terms of quantification accuracy. In the present invention, by recovering the antigen-binding molecule bound to the cell from the cell using an extraction solution containing an organic acid and a non-surfactant, the antigen-binding molecule can be directly detected, and the quantification accuracy can be improved. According to the quantification method of the present invention, not only can the accumulation state of an antigen-binding molecule (e.g., an antibody drug) in a target tissue (e.g., a cancer tissue) be analyzed, but also whether the antigen-binding molecule accumulates in an unintended tissue (e.g., a normal tissue) can be analyzed. Therefore, the quantification method according to the present invention is also useful for elucidating the mechanism of side effects caused in normal tissues.

Examples

[0041] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[0042] (Experiment 1: Quantification of antibodies present in cancer tissues) The antibodies present in cancer tissues were quantified according to the following procedure. Here, the cancer tissue corresponds to a tissue containing cells expressing a target antigen, and the antibody corresponds to an antigen-binding molecule.

[0043] (Example: Extraction by the ACES method) (Preparation step, extraction step) First, the following cancer tissues and preparation solutions were prepared. Cancer tissue: Cancer tissues (esophageal cancer) collected from patients administered with cetuximab and cancer tissues of experimental model mice administered with cetuximab (1) Tissue washing solution: D-PBS Phosphatase inhibitor (1:50 (volume ratio), PhosSTOP) Protease inhibitor mix (1:50 (volume ratio), cOmplete ULTRA) (2) Inhibitor solution: 200 mM Arginine-HCl aqueous solution (pH 2.1) Phosphatase inhibitor (1:50 (volume ratio), PhosSTOP) Protease inhibitor mix (1:50 (volume ratio), cOmplete ULTRA) (3) Internal standard IS solution: Inhibitory solution SILuMAb cetuximab (final concentration 0.25 μg / ml) BSA (final concentration 0.1%) mouse IgG1 (clone 2545) (final concentration 50 μg / ml) (4) Extraction solution: 200 mM Arginine-HCl aqueous solution (pH 2.1) n-Octyl-β-D-thioglucopyranoside (final concentration 2%)

[0044] First, the above cancer tissue was sliced with a slicer to prepare thin sections (thickness 5 μm). The above thin sections (10 mg) were transferred to a low-adsorption microtube, and the cooled tissue washing solution (200 μl) was added thereto. Beads for tissue disruption (zirconia beads, 2 mm in diameter, 8 pieces) were further added to the above microtube.

[0045] Under cooling (less than 2°C), the cancer tissue in the above microtube was disrupted (homogenized) at 5,000 rpm × 1 minute with a bead homogenizer (MicroSmash MS-100R). Then, it was allowed to stand for 1 minute in the apparatus to cool the above microtube. This operation was performed a total of 4 times. By the above procedure, a sample containing the disrupted cancer tissue (homogenized biological sample) was prepared (preparation step). The above biological sample is a sample derived from cancer cells.

[0046] The sample containing the disrupted cancer tissue (homogenized biological sample) was transferred in its entirety to another low-adsorption microtube. Tissue washing solution (100 μl) was further added to the microtube to wash and collect the disrupted cancer tissue (twice). The collected sample was sonicated for 5 minutes using an ultrasonic cleaner under ice-cooling (0 °C) to remove air bubbles. The microtube containing the sample was centrifuged (16,000 g × 5 minutes, 4 °C) to separate it into a supernatant and a precipitate. The entire supernatant was recovered into another low-adsorption microtube and designated as the "free antibody sample" (sample for the second analysis). The "free antibody sample" was stored at 4 °C.

[0047] On the other hand, for the precipitate, an inhibition solution (160 μl), an internal standard IS solution (40 μl), and an extraction solution (200 μl) were added to obtain a suspension of the precipitate. The obtained suspension was allowed to stand for 1 hour under ice-cooling. Thereafter, the suspension was resuspended by pipetting and then allowed to stand overnight at 4 °C (extraction step, ACES method). The tube containing the suspension was centrifuged (16,000 g × 30 minutes, 4 °C) to separate it into a supernatant and a precipitate. The supernatant was recovered and centrifugally filtered (10,000 g × 2 minutes, 4 °C) using Ultrafree MC (0.22 μm) to obtain a filtrate. The obtained filtrate was recovered into another low-adsorption microtube and designated as the "bound antibody sample" (sample for analysis, or sample for the first analysis). The bound antibody sample was stored at 4 °C.

[0048] (Quantification step) The following preparation solutions were prepared. (1) Standard dilution solution: Inhibition solution BSA (final concentration 0.1%) mouse IgG1 (clone 2454) (final concentration 50 μg / ml) (2) Washing solution: D-PBS n-Octyl-β-D-thioglucopyranoside (final concentration 0.1%) (3) Neutralization solution: 250 mM Na 2 HPO 4 aqueous solution n-Octyl-β-D-thioglucopyranoside (final concentration 1%) (4) Reaction solution: 25 mM Tris-HCl aqueous solution (pH 8.0) (5) Initial mobile phase: MilliQ water (0.1% formic acid) + acetonitrile (volume ratio 99:1)

[0049] Calibration curve samples and QC samples with the following compositions were prepared. Calibration curve samples: All were prepared with standard product dilution solutions. Blank sample 0 ng / ml cetuximab Calibration curve 1 313 ng / ml cetuximab Calibration curve 2 625 ng / ml cetuximab Calibration curve 3 1.25 μg / ml cetuximab Calibration curve 4 2.50 μg / ml cetuximab Calibration curve 5 5.00 μg / ml cetuximab Calibration curve 6 10.0 μg / ml cetuximab Calibration curve 7 20.0 μg / ml cetuximab Calibration curve 8 40.0 μg / ml cetuximab

[0050] QC samples: All were prepared with standard product dilution solutions. LLOQ 313 ng / ml cetuximab LQC 938 ng / ml cetuximab MQC 3.75 μg / ml cetuximab HQC 12.5 μg / ml cetuximab HLOQ 40.0 μg / ml cetuximab

[0051] 20 μL each of the calibration curve sample and the QC sample were added to 160 μL of a matrix solution (a solution obtained by subjecting interfering components derived from the measurement tissue to extraction treatment in the same manner as the measurement sample using an inhibition solution and an extraction solution), and further 20 μL of an internal standard IS solution was added. These correspond to the "calibration curve sample (with IS)" and "QC sample (with IS)" described below. Also, in the above preparation, samples were prepared by adding 20 μL of the matrix solution instead of 20 μL of the internal standard IS solution. These correspond to the "calibration curve sample (without IS)" and "QC sample (without IS)" described below.

[0052] Quantitative analysis of cetuximab (antigen-binding molecule) in the analytical sample was performed by generating a peptide specific to cetuximab (a peptide derived from the variable region) by the nSMOL method and detecting the peptide with a mass spectrometer. The specific procedure is as follows.

[0053] (Generation of peptide by nSMOL method) 200 μl each of the bound antibody sample, the calibration curve sample (with or without IS), and the QC sample (with or without IS) were dispensed into low-adsorption microtubes. Next, 600 μl of a washing solution and 200 μl of a neutralization solution were added to each microtube. Also, 200 μl of the free antibody sample was transferred to a low-adsorption microtube, and 20 μl of the internal standard IS solution was further added. Next, 760 μl of the washing solution and 20 μl of the neutralization solution were added to the above microtube.

[0054] After thoroughly suspending the Immunoglobulin collection resin in the nSMOL Antibody BA Kit (manufactured by Shimadzu Corporation), 12.5 μl was collected and added to each tube. Each tube was stirred for 30 minutes using a self-standing tube mixer. Thereafter, each tube was centrifuged (15,000 g × 5 minutes, 4 °C). 700 μl of the supernatant was gently removed from each tube, taking care not to aspirate the resin. The suspension (resin and remaining liquid) remaining in each tube was transferred to Ultrafree MC (0.22 μm) respectively. After transferring the above suspension, a washing solution (300 μl) was added to each tube, and the resin remaining in each tube was recovered and transferred to the above Ultrafree MC (0.22 μm) respectively.

[0055] Centrifugal filtration (10,000 g × 1 minute, 25 °C) was performed on each Ultrafree MC to remove the filtrate. A washing solution (300 μl) was added to each Ultrafree MC, and centrifugal filtration (10,000 g × 1 minute, 25 °C) was performed to remove the filtrate (a total of 2 times). D-PBS (300 μl) was added to each Ultrafree MC, and centrifugal filtration (10,000 g × 1 minute, 25 °C) was performed to remove the filtrate (a total of 3 times).

[0056] A reaction solution (85 μl) was added to each Ultrafree MC. After thoroughly suspending the FG beads Trypsin DART in the nSMOL Antibody BA Kit, 5 μl was collected and added to each Ultrafree MC. Thereafter, incubation was performed at 52 °C for 5 hours under saturated vapor pressure to carry out the nSMOL reaction.

[0057] The Stop solution (10 μl) in the nSMOL Antibody BA Kit was added to each Ultrafree MC to stop the nSMOL reaction. Subsequently, centrifugal filtration (10,000 g × 1 minute, 25°C) was performed on each Ultrafree MC, and the total volume of the filtrate was collected in separate microtubes. Each microtube was placed upright on a magnetic stand and left standing for 2 minutes to remove excess magnetic beads. Then, the supernatant (90 μl) in each microtube was collected into low-adsorption microtubes. An additional 90 μl of the initial mobile phase was added to the low-adsorption microtubes. They were placed into HPLC polypropylene vials and degassed. A peptide specific to cetuximab (a peptide derived from the variable region) was generated by the above procedure.

[0058] (Detection of Peptides by Mass Spectrometer) The peptide specific to cetuximab (SEQ ID NO: 1: ASQSIGTNIHWYQQR (one-letter notation)) was quantified by LCMS analysis under the conditions shown below.

[0059] List of Preparation Solutions: Mobile Phase A: MilliQ (LCMS grade) Mobile Phase B: 0.1% Formic Acid + Acetonitrile (LCMS grade)

[0060] LCMS Analysis Conditions: Column Used: Shimpack GISS C18, 2.1 mm ID × 50 mm Column Temperature: 50°C Sample Injection Volume: 10 μl Nebulizer Gas Flow Rate: 3 l / min Drying Gas Flow Rate: 10 l / min Heating Gas Flow Rate: 10 l / min ESI Interface Temperature: 300°C Heat Block Temperature: 400°C DL Temperature: 250°C CID Gas Pressure: 270 kPa

[0061] HPLC Conditions: HPLC: Shimadzu Nexera X2 Flow rate: 0.4 ml / min 0 - 1.5 min: 1%B 1.5 - 4.9 min: 1 - 38%B gradient 4.9 - 6.5 min: 38%B 6.5 - 7.0 min: 38 - 95%B gradient 7.0 - 9.0 min: 95%B 9.0 - 9.5 min: 95 - 1%B 9.5 - 13.5 min: 1%B LCMS analysis time: 3.0 - 6.0 min

[0062] LCMS MRM analysis transition conditions: LCMS: Shimadzu LCMS - 8060 Cetuximab, ASQSIGTNIHWYQQR 596.95(+++) > 651.80 (y10++) for quantification 596.95(+++) > 751.85 (y12++) confirmation ion 596.95(+++) > 708.35 (y11++) confirmation ion 13C6,15N4 - Cetuximab, 13C6,15N4 - ASQSIGTNIHWYQQR 600.45 (+++) > 657.10 (y10++) for quantification (Ab_IS) 600.45 (+++) > 757.30 (y12++) confirmation ion 600.45 (+++) > 713.15 (y11++) confirmation ion

[0063] (Comparative example) Using ProteinWorks Auto-eXpress Digest Kits (manufactured by Waters), thin sections (about 10 mg) of the above cancer tissue were enzymatically treated. The obtained enzymatically treated product was centrifuged (800 g × 15 minutes, 10 °C), and the supernatant (about 160 μl) was collected. The collected supernatant was analyzed by LCMS analysis under the same conditions as above.

[0064] (Results) The results of LCMS analysis of the binding antibody sample in the example and the sample in the comparative example are shown in Figure 2. In Figure 2, the horizontal axis represents the retention time in LC, and the vertical axis represents the detection intensity when the peptide corresponding to that retention time was mass analyzed. From the results of Figure 2, it was found that only the peptide specific to cetuximab was detected by the methods using the ACES method and the sMOL method (the left graph in Figure 2). That is, the method of the example was able to detect cetuximab present in cancer cells. On the other hand, a plurality of peaks were detected in the sample obtained by enzymatically treating the entire cancer tissue (the right graph in Figure 2).

[0065] Figure 3 shows the results of LCMS analysis of the calibration curve sample (blank) in the example (with IS (the second graph from the top), or without IS (the first graph from the top)) and the QC sample (LLOQ) (with IS (the fourth graph from the top), or without IS (the third graph from the top)). From the results of Figure 3, it was found that cetuximab was detectable even at a concentration of 313 ng / ml under the analysis conditions of the example. In addition, it was confirmed that the same results were obtained for the confirmation ion (596.95(+++) > 751.85 (y12++)) (Figure 5).

[0066] The calibration curve graph obtained from the analysis results of the calibration curve samples (blank, calibration curves 1 to 8) (with IS) in the examples is shown in Fig. 4. In Fig. 4, the horizontal axis represents the concentration ratio of cetuximab in each calibration curve sample (based on the concentration of IS), and the vertical axis represents the ratio of the peak area of the analyte to the peak area of IS in mass spectrometry. The results in Fig. 4 suggest that cetuximab can be quantified under the analysis conditions of the examples.

[0067] (Experiment 2: Quantification of antibodies bound to the surface of cell lines) Using four types of cell lines (A-431, FaDu, TE-4, and SW620), the antibodies bound to the surface of each cell line were quantified by the following procedure. First, each cell was seeded at 4×10 5 cells / well in a 12-well plate and allowed to adhere in an incubator (37°C, 5% CO 2 2). 1 ml of 5 mg / ml (excess amount) of Cetuximab was added to each well and left standing for 30 minutes under ice-cooling. After the reaction, Cetuximab was aspirated and removed, and each well was washed twice with D-PBS and aspirated and removed.

[0068] To each well, an inhibition solution (160 μl), an internal standard IS solution (40 μl), and an extraction solution (200 μl) were added and pipetted well, and then left standing for 1 hour under ice-cooling (extraction step, ACES method). The above suspension was collected in a tube, centrifuged (16,000 g × 30 minutes, 4°C), and separated into a supernatant and a precipitate. The supernatant was collected and centrifugally filtered (10,000 g × 2 minutes, 4°C) using Ultrafree MC (0.22 μm) to obtain a filtrate. Thereafter, it was processed and quantified in the same manner as the quantification step in "Experiment 1: Quantification of antibodies present in tissue samples". The results are shown in Fig. 6 (lower left graph and lower right graph). The results in Fig. 6 show that the antibodies (Cetuximab) bound to each cell surface can be quantified in correlation with the expression level of EGFR in the quantification by the QIF kit (manufactured by DAKO) (upper left and upper right in Fig. 6).

[0069] (Experiment 3: Quantification of antibodies bound to tissue in a model tumor) SW620 strain or A431 strain was transplanted subcutaneously into nude mice (BALB / c, female, 5 - 8 weeks old). The number of cells transplanted at this time was 1×10 7 cells per mouse. Normal breeding was carried out for 14 - 21 days until the transplanted cells became established. Thereafter, cetuximab (0 - 1.0 mg / body) was intravenously injected into the above - mentioned mice. 24 hours after the intravenous injection, the transplanted cells (model tumors) were collected from the mice (Figure 7, left side).

[0070] The collected model tumors were subjected to tissue staining with HE and anti - EGFR antibody (Figure 7, right side). In the model tumors collected from the mice transplanted with A431 strain, it was confirmed that EGFR was expressed.

[0071] Also, the collected model tumors were subjected to tissue staining with DAPI and anti - human IgG antibody (Figure 8, right side). In the model tumors collected from the mice transplanted with A431 strain, it was confirmed that the amount stained with anti - human IgG antibody increased depending on the amount of cetuximab administered.

[0072] The collected model tumors were processed by the same method as in Experiment 1 to quantify cetuximab bound to the model tumors. The samples used for quantification were the samples corresponding to the "bound antibody samples" in Experiment 1. The results are shown in Figure 8 (left side). In the model tumors collected from the mice transplanted with A431 strain, it was confirmed that the quantification value of cetuximab using the ACES method increased depending on the amount of cetuximab administered. This result was also correlated with the result of tissue staining, which is a conventional method (Figure 8). On the other hand, in the model tumors collected from the mice transplanted with SW620 strain, almost no cetuximab was detected.

[0073] Using the sample corresponding to the "free antibody sample" in Experiment 1, cetuximab present in the model tumor but not bound to EGFR was quantified (Figure 9, left). More cetuximab was detected in the model tumors collected from mice transplanted with the SW620 strain than in the model tumors collected from mice transplanted with the A431 strain.

[0074] (Experiment 4: Extraction by ACES method from cancer tissues administered with Trastuzumab-Deruxtecan (T-DXd)) (Preparation step, extraction step) First, the following cancer tissues and preparation solutions were prepared. Cancer tissues: Cancer tissues of experimental model mice administered with T-DXd (1) Tissue washing solution, (2) inhibition solution, and (4) extraction solution used solutions with the same composition as in "Experiment 1: Quantification of antibodies present in tissue samples". (3) Internal standard IS solution: Inhibition solution SILuMAb Trastuzumab (trastuzumab) (final concentration 0.25 μg / ml) BSA (final concentration 0.1%) mouse IgG1 (clone 2545) (final concentration 50 μg / ml)

[0075] First, the above cancer tissues were sliced with a slicer to prepare thin sections (thickness 5 μm). The above thin sections (10 mg) were transferred to a low-adsorption microtube and treated in the same manner as in "Experiment 1: Quantification of antibodies present in tissue samples". By the above procedure, a sample containing disrupted cancer tissues (homogenized biological sample) was prepared (preparation step). The above biological sample is a sample derived from cancer cells. Thereafter, it was treated in the same manner as in "Experiment 1: Quantification of antibodies present in tissue samples" to obtain a "bound antibody sample" (analysis sample, or first analysis sample) and a "free antibody sample" (second analysis sample) from the sample containing the disrupted cancer tissues.

[0076] (Quantification step) The following preparation solutions were prepared. (1) The standard dilution solution, (2) the washing solution, (3) the neutralization solution, and (4) the reaction solution used were the same as those in "Experiment 1: Quantification of Antibodies Present in Tissue Samples". (5) Initial mobile phase: MilliQ water (0.1% acetic acid) + acetonitrile (volume ratio 99:1)

[0077] Calibration curve samples and QC samples with the following compositions were prepared. Calibration curve samples: All were prepared with the standard dilution solution. Blank sample 0 ng / ml Trastuzumab Calibration curve 1 250 ng / ml Trastuzumab Calibration curve 2 500 ng / ml Trastuzumab Calibration curve 3 1.00 μg / ml Trastuzumab Calibration curve 4 2.00 μg / ml Trastuzumab Calibration curve 5 4.00 μg / ml Trastuzumab Calibration curve 6 8.0 μg / ml Trastuzumab Calibration curve 7 16.0 μg / ml Trastuzumab Calibration curve 8 32.0 μg / ml Trastuzumab

[0078] QC samples: All were prepared with the standard dilution solution. LLOQ 250 ng / ml Trastuzumab LQC 750 ng / ml Trastuzumab MQC 2.50 μg / ml Trastuzumab HQC 12.0 μg / ml Trastuzumab HLOQ 32.0 μg / ml Trastuzumab

[0079] 20 μL each of the calibration curve sample and the QC sample were added to 160 μL of a matrix solution (a solution obtained by subjecting interfering components derived from the measurement tissue to extraction treatment in the same manner as the measurement sample using an inhibition solution and an extraction solution), and further 20 μL of an internal standard IS solution was added. These correspond to the "calibration curve sample (with IS)" and "QC sample (with IS)" described below. In addition, in the preparation of the above calibration curve sample, a sample was also prepared by adding 20 μL of the matrix solution instead of 20 μL of the internal standard IS solution. This corresponds to the "calibration curve sample (without IS)" described below.

[0080] Quantitative analysis of Trastuzumab (antigen-binding molecule) in the analytical sample was performed by generating a peptide specific to Trastuzumab (a peptide derived from the variable region) by the nSMOL method and detecting the peptide with a mass spectrometer. The specific procedure is as follows.

[0081] (Generation of peptide by nSMOL method) 200 μl each of the bound antibody sample, calibration curve sample (with or without IS), and QC sample (with IS) were dispensed into low-adsorption microtubes, and thereafter treated in the same manner as in "Experiment 1: Quantification of antibodies present in tissue samples". A peptide specific to Trastuzumab (a peptide derived from the variable region) was generated by the above procedure.

[0082] (Detection of peptide by mass spectrometer) The peptide specific to Trastuzumab (SEQ ID NO: 2: IYPTNGYTR (one-letter notation)) was quantified by LCMS analysis under the conditions shown below.

[0083] List of conditioning solutions: Mobile phase A: MilliQ water (0.1% acetic acid) (LCMS grade) Mobile phase B: acetonitrile (LCMS grade)

[0084] LCMS analysis conditions: Performed in the same manner as in "Experiment 1: Quantification of antibodies present in tissue samples".

[0085] HPLC conditions: HPLC: Shimadzu Nexera X2 Flow rate: 0.4 ml / min 0 - 1.5 min: 1%B 1.5 - 5.5 min: 1 - 42%B gradient 5.5 - 6.5 min: 95%B 6.5 - 8.5 min: 1%B LCMS analysis time: 2.5 - 6.5 min

[0086] LCMS MRM analysis transition conditions: LCMS: Shimadzu LCMS - 8060 Trastuzumab, IYPTNGYTR 542.90 (++) > 404.80 (y7++) for quantification 542.90 (+++) > 808.50 (y7+) confirmation ion 13C6,15N4 - Trastuzumab, 13C6,15N4 - IYPTNGYTR 547.90 (++) > 409.70 (y7++) for quantification (Ab_IS) 547.90 (++) > 818.50 (y7+) confirmation ion

[0087] (Results) Figure 10 shows the results of LCMS analysis of the calibration curve samples (blank) (with IS (the second graph from the top) or without IS (the first graph from the top)) and the QC sample (LLOQ) (with IS (the third graph from the top)) in the examples. From the results in Figure 10, it was found that under the analysis conditions of the examples, Trastuzumab could be detected even at a concentration of 250 ng / ml, similar to the examples of Cetuximab.

[0088] The calibration curve graph obtained from the analysis results of the calibration curve samples (blank, calibration curves 1-8) (with IS) in the examples is shown in Fig. 11. In Fig. 11, the horizontal axis represents the concentration ratio of Trastuzumab in each calibration curve sample (based on the concentration of IS), and the vertical axis represents the ratio of the peak area of the analyte to the peak area of IS in mass spectrometry. From the results in Fig. 11, it was suggested that Trastuzumab could be quantified under the analysis conditions of the examples.

[0089] (Experiment 5: Quantification of the antibody portion (Trastuzumab) of ADC (T-DXd) bound to tissues in model tumors) SW620 strain or OE-19 strain was transplanted subcutaneously into nude mice (BALB / c, female, 5-8 weeks old). Here, the SW620 strain is a cell line that does not express HER2 (the antigen of Trastuzumab), and the OE-19 strain is a cell line that expresses HER2. The number of cells transplanted at this time was 1×10 7 cells per mouse. Normal breeding was carried out for 14-21 days until the transplanted cells became established. Thereafter, T-Dxd (0 or 10 mg / Kg) was intravenously injected into the above mice. 24 hours after the intravenous injection, the transplanted cells (model tumors) were collected from the mice in the same manner as in "Experiment 3: Quantification of antibodies bound to tissues in model tumors". In addition, the collected model tumors were stained with DAPI and anti-human IgG antibody (right side of Fig. 12). In the model tumors collected from the mice transplanted with the OE-19 strain, the antibody portion of T-DXd was stained in the tissues administered with T-DXd.

[0090] The collected model tumors were processed in the same manner as in Experiment 1 to quantify the antibody portion of T-DXd bound to the model tumors. The samples used for quantification were the samples corresponding to the "bound antibody samples" in Experiment 1. The results are shown in Fig. 12 (left side). In the model tumors collected from the mice transplanted with the OE-19 strain, Trastuzumab was detected only from the tissues administered with T-DXd. On the other hand, in the model tumors collected from the mice transplanted with the SW620 strain, almost no Trastuzumab was detected.

[0091] Using the sample corresponding to the "free antibody sample" in Experiment 4, the antibody portion of T-DXd that is present in the model tumor but not bound to HER2 was quantified (Figure 13, left). An antibody portion of T-DXd equivalent to that in the model tumor transplanted with the OE-19 strain was also detected in the model tumor collected from the mouse transplanted with the SW620 strain (however, for the lower limit of quantification value, the calculation was performed by substituting a value half of the LLOQ (12.5 ng / ml)).

[0092] (Experiment 6: Extraction by ACES method from cancer tissues administered with Trastuzumab) (Preparation step, extraction step) First, the following cancer tissues and preparation solutions were prepared. Cancer tissues: Cancer tissues of experimental model mice administered with Trastuzumab (1) Tissue washing solution, (2) inhibition solution, and (4) extraction solution used solutions with the same composition as in "Experiment 1: Quantification of antibodies present in tissue samples". (3) Internal standard IS solution: Inhibition solution SILuMAb Trastuzumab (Trastuzumab) (final concentration 0.25 μg / ml) BSA (final concentration 0.1%) mouse IgG1 (clone 2545) (final concentration 50 μg / ml)

[0093] First, the above cancer tissues were sliced with a slicer to prepare thin sections (thickness 5 μm). The above thin sections (10 mg) were transferred to a low-adsorption microtube and treated in the same manner as in "Experiment 1: Quantification of antibodies present in tissue samples". By the above procedure, a sample containing disrupted cancer tissues (homogenized biological sample) was prepared (preparation step). The above biological sample is a sample derived from cancer cells. Thereafter, it was treated in the same manner as in "Experiment 1: Quantification of antibodies present in tissue samples" to obtain a "bound antibody sample" (analysis sample, or first analysis sample) and a "free antibody sample" (second analysis sample) from the sample containing the disrupted cancer tissues.

[0094] (Quantitative Engineering) The following preparation solutions were prepared. (1) Standard dilution solution, (2) washing solution, (3) neutralization solution, and (4) reaction solution used the same solutions as in "Experiment 1: Quantification of Antibodies Present in Tissue Samples". (5) Initial mobile phase: MilliQ water (0.1% acetic acid) + acetonitrile (volume ratio 99:1)

[0095] Calibration curve samples and QC samples with the following compositions were prepared. Calibration curve samples: All were prepared with the standard dilution solution. Blank sample 0 ng / ml Trastuzumab Calibration curve 1 250 ng / ml Trastuzumab Calibration curve 2 500 ng / ml Trastuzumab Calibration curve 3 1.00 μg / ml Trastuzumab Calibration curve 4 2.00 μg / ml Trastuzumab Calibration curve 5 4.00 μg / ml Trastuzumab Calibration curve 6 8.0 μg / ml Trastuzumab Calibration curve 7 16.0 μg / ml Trastuzumab Calibration curve 8 32.0 μg / ml Trastuzumab

[0096] QC samples: All were prepared with the standard dilution solution. LLOQ 250 ng / ml Trastuzumab LQC 750 ng / ml Trastuzumab MQC 2.50 μg / ml Trastuzumab HQC 12.0 μg / ml Trastuzumab HLOQ 32.0 μg / ml Trastuzumab

[0097] 20 μL each of the calibration curve sample and the QC sample were added to 160 μL of a matrix solution (a solution obtained by subjecting interfering components derived from the measurement tissue to extraction treatment in the same manner as the measurement sample using an inhibition solution and an extraction solution), and further 20 μL of an internal standard IS solution was added. These correspond to the "calibration curve sample (with IS)" and "QC sample (with IS)" described later. Also, in the preparation of the above calibration curve sample, a sample was prepared by adding 20 μL of the matrix solution instead of 20 μL of the internal standard IS solution. This corresponds to the "calibration curve sample (without IS)" and "QC sample (without IS)" described later.

[0098] Quantitative analysis of Trastuzumab (antigen-binding molecule) in the analytical sample was performed by generating a peptide specific to Trastuzumab (a peptide derived from the variable region) by the nSMOL method and detecting the peptide with a mass spectrometer. The specific procedure is as follows.

[0099] (Generation of peptide by nSMOL method) 200 μl each of the binding antibody sample, calibration curve sample (with or without IS), and QC sample (with or without IS) were dispensed into low-adsorption microtubes, and thereafter treated in the same manner as in "Experiment 1: Quantification of antibodies present in tissue samples". A peptide specific to Trastuzumab (a peptide derived from the variable region) was generated by the above procedure.

[0100] (Detection of peptide by mass spectrometer) The peptide specific to Trastuzumab (SEQ ID NO: 2: IYPTNGYTR (one-letter notation)) was quantified by LCMS analysis under the conditions shown below.

[0101] List of conditioning solutions: Mobile phase A: MilliQ water (0.1% acetic acid) (LCMS grade) Mobile phase B: acetonitrile (LCMS grade)

[0102] LCMS analysis conditions: It was carried out in the same manner as in "Experiment 1: Quantification of Antibodies Present in Tissue Samples".

[0103] HPLC conditions: HPLC: Shimadzu Nexera X2 Flow rate: 0.4 ml / min 0 - 1.5 min: 1%B 1.5 - 5.5 min: 1 - 42%B gradient 5.5 - 6.5 min: 95%B 6.5 - 8.5 min: 1%B LCMS analysis time: 2.5 - 6.5 min

[0104] LCMS MRM analysis transition conditions: LCMS: Shimadzu LCMS - 8060 Trastuzumab, IYPTNGYTR 542.90 (++) > 404.80 (y7++) for quantification 542.90 (+++) > 808.50 (y7+) confirmation ion 13C6,15N4 - Trastuzumab, 13C6,15N4 - IYPTNGYTR 547.90 (++) > 409.70 (y7++) for quantification (Ab_IS) 547.90 (++) > 818.50 (y7+) confirmation ion

[0105] (Results) Figure 14 shows the results of LCMS analysis of the calibration curve samples (blank) (with IS (the second graph from the top) or without IS (the first graph from the top)) and QC samples (LLOQ) (with IS (the fourth graph from the top) or without IS (the third graph from the top)) in the examples. From the results in Figure 14, it was found that under the analysis conditions of the examples, Trastuzumab was detectable even at a concentration of 250 ng / ml, similar to the examples of Cetuximab.

[0106] A calibration curve graph was created (not shown) from the analysis results of the calibration curve samples (blank, calibration curves 1 to 8) (with IS) in the examples. From the results of the graph, it was suggested that Trastuzumab could be quantified under the analysis conditions of the examples.

[0107] (Experiment 7: Quantification of Trastuzumab Bound to Tissues in Model Tumors SW620 cells or OE-19 cells were transplanted subcutaneously into nude mice (BALB / c, female, 5 - 8 weeks old). Here, the SW620 cell line is a cell line that does not express HER2 (the antigen of Trastuzumab), and the OE-19 cell line is a cell line that expresses HER2. The number of cells transplanted at this time was 1×10 7 cells per mouse. Normal breeding was carried out for 14 - 21 days until the transplanted cells became established. Thereafter, Trastuzumab (0, 10, or 20 mg / Kg) was intravenously injected into the above mice. 24 hours after the intravenous injection, the transplanted cells (model tumors) were collected from the mice in the same manner as in "Experiment 3: Quantification of Antibodies Bound to Tissues in Model Tumors" (Figure 15, left side).

[0108] In addition, the collected model tumors were tissue-stained with DAPI and anti-human IgG antibody (Figure 16, right side). In the model tumors collected from the mice transplanted with the OE-19 cell line, Trastuzumab was stained in the tissues administered with Trastuzumab.

[0109] The collected model tumors were processed in the same manner as in Experiment 1 to quantify the antibody portion of Trastuzumab bound to the model tumors. The samples used for quantification were the samples corresponding to the "bound antibody samples" in Experiment 1. The results are shown in Figure 16 (left side). In the model tumors collected from the mice transplanted with the OE-19 cell line, Trastuzumab was detected only from the tissues administered with Trastuzumab. On the other hand, in the model tumors collected from the mice transplanted with the SW620 cell line, almost no Trastuzumab was detected.

[0110] Using the sample corresponding to the "free antibody sample" in Experiment 6, Trastuzumab present in the model tumor but not bound to HER2 was quantified (Figure 17, left). Trastuzumab equivalent to that in the model tumor transplanted with the OE-19 strain was also detected in the model tumor collected from the mouse transplanted with the SW620 strain (however, for the lower limit of quantification value, the calculation was performed by substituting a value half of the LLOQ (12.5 ng / ml)).

[0111] [Aspect] It will be understood by those skilled in the art that the above-described plurality of exemplary embodiments and examples are specific examples of the following aspects.

[0112] (Item 1) A method for quantifying an antigen-binding molecule according to one aspect is a method for quantifying an antigen-binding molecule bound to a cell, comprising: a preparation step of preparing a biological sample derived from the cell, wherein the biological sample is homogenized, the cell expresses a target antigen to which the antigen-binding molecule specifically binds, and the cell has been exposed to the antigen-binding molecule; an extraction step of adding an extraction solution to the biological sample to obtain an analysis sample, wherein the extraction solution contains an organic acid and a nonionic surfactant, and the pH of the extraction solution is 1 or more and 3 or less; and a quantification step of analyzing the analysis sample to quantify the antigen-binding molecule. According to the quantification method described in Item 1, it is possible to provide a method for accurately quantifying an antigen-binding molecule bound to a cell expressing a target antigen.

[0113] (Item 2) In the quantification method according to Item 1, the organic acid contains at least one selected from the group consisting of arginine, citrulline, and glycine. According to the quantification method described in Item 2, the antigen-binding molecule can be extracted in a stable state.

[0114] (Item 3) In the quantification method according to claim 1 or 2, the nonionic surfactant contains alkyl glycoside. According to the quantification method described in claim 3, antigen-binding molecules can be extracted in a stable state.

[0115] (Claim 4) In the quantification method according to any one of claims 1 to 3, the target antigen contains at least one selected from the group consisting of EGFR, HER2, CD30, PD-L1, RANKL, tumor necrosis factor receptor, insulin receptor, and vascular endothelial growth factor receptor. According to the quantification method described in claim 4, antigen-binding molecules that bind to a predetermined target antigen can be accurately quantified.

[0116] (Claim 5) In the quantification method according to any one of claims 1 to 4, the antigen-binding molecule contains an antibody or a peptide aptamer. According to the quantification method described in claim 5, an antibody or a peptide aptamer can be accurately quantified.

[0117] (Claim 6) In the quantification method according to claim 5, the antigen-binding molecule contains at least one selected from the group consisting of cetuximab, trastuzumab, brentuximab, denosumab, infliximab, adalimumab, etanercept, aflibercept, ramucirumab, atezolizumab, avelumab, and insulin analog. According to the quantification method described in claim 6, a predetermined antibody can be accurately quantified.

[0118] (Claim 7) In the quantification method according to any one of claims 1 to 6, the cell is a cell derived from a cancer tissue or a cell derived from a diseased tissue. According to the quantification method described in claim 7, the accumulation amount of antigen-binding molecules in a cancer tissue or a diseased tissue can be accurately quantified.

[0119] (Claim 8) In the quantification method according to any one of Items 1 to 7, the antigen-binding molecule is an antibody, and the quantification step includes generating a peptide derived from the variable region of the antibody by the nSMOL method and analyzing the peptide by an LC-MS analyzer. According to the quantification method described in Item 8, the antibody can be quantified with higher accuracy.

[0120] As described above, the embodiments and examples of the present invention have been explained. However, it has also been planned from the beginning to appropriately combine the configurations of the above-described embodiments and examples.

[0121] The embodiments and examples disclosed this time should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is shown not by the above-described embodiments and examples but by the claims, and it is intended that all modifications within the meaning and scope equivalent to the claims are included.

Claims

1. A method for quantifying an antigen-binding molecule bound to a cell, comprising the steps of: A preparation step of preparing a biological sample derived from the cells, The biological sample is homogenized; The cell expresses a target antigen to which the antigen-binding molecule specifically binds, A preparation step in which the cells have been exposed to the antigen-binding molecule; An extraction step of adding an extraction solution to the biological sample to obtain an analytical sample, The extraction solution comprises an organic acid and a nonionic surfactant; an extraction step, in which the pH of the extract is 1 or more and 3 or less; a quantification step of quantifying the antigen-binding molecule by analyzing the analytical sample; A quantitative method comprising:

2. The quantitative method according to claim 1 , wherein the organic acid comprises at least one selected from the group consisting of arginine, citrulline, and glycine.

3. The quantitative method according to claim 1 or 2, wherein the nonionic surfactant comprises an alkyl glycoside.

4. The method according to claim 1 or 2, wherein the target antigen comprises at least one selected from the group consisting of EGFR, HER2, CD30, PD-L1, RANKL, a tumor necrosis factor receptor, an insulin receptor, and a vascular endothelial growth factor receptor.

5. The method of claim 1 or 2, wherein the antigen-binding molecule comprises an antibody or a peptide aptamer.

6. The method of claim 5, wherein the antigen-binding molecule comprises at least one selected from the group consisting of cetuximab, trastuzumab, brentuximab, denosumab, infliximab, adalimumab, etanercept, aflibercept, ramucirumab, atezolizumab, avelumab, and insulin analogues.

7. The method for quantification according to claim 1 or 2, wherein the cells are cells derived from cancer tissue or diseased tissue.

8. the antigen-binding molecule is an antibody, 3. The method according to claim 1, wherein the quantification step comprises generating peptides derived from the variable region of the antibody by an nSMOL method, and analyzing the peptides by an LC-MS analyzer.

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