How to analyze antibodies

Abstract

The present invention provides a method that enables quantification of an antibody by mass spectrometry without constructing a calibration curve using the target antibody. Provided is a method for analyzing an antibody, said method comprising digesting a biological protein and the target antibody with a protease, mass spectrometrically analyzing peptide fragments of the thus digested biological protein and target antibody, and quantifying the target antibody on the basis of the ratio of the detection intensity of the biological protein and the detection intensity of the target antibody obtained by the mass spectrometry, wherein: the digestion with the protease comprises immobilizing the biological protein and the target antibody in pores of a carrier and placing the carrier closer to fine particles which have a diameter greater than the diameter of the pores and on the surface of which the protease is immobilized; and the biological protein has a domain which can be immobilized in the pores.

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing an antibody, and more particularly to a method for quantifying an antibody using mass spectrometry. [Background technology]

[0002] ELISA (Enzyme-Linked ImmunoSorbent Assay) is a commonly used method for quantifying proteins. However, ELISA has some drawbacks, such as the need to prepare specific antibodies for each protein to be detected and the inability to detect multiple proteins simultaneously. Therefore, methods using mass spectrometry are being developed as an alternative to ELISA for quantifying proteins.

[0003] The present inventors and their group have discovered a method for regioselectively digesting proteins such as antibodies with proteases to obtain peptide fragments optimal for specific protein detection using mass spectrometry. The resulting peptide fragments can be effectively detected and quantified by liquid chromatography-mass spectrometry (Patent Document 1: International Publication No. 2015 / 033479; Patent Document 2: International Publication No. 2016 / 194114; Non-Patent Document 1: Iwamoto N et al., Analyst. 2014 Feb 7; 139(3): 576-80. doi: 10.1039 / c3an02104a). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2015 / 033479 [Patent Document 2] International Publication No. 2016 / 194114 [Non-patent literature]

[0005] [Non-Patent Document 1] Iwamoto N et.al., Selective detection of complementarity-determining regions of monoclonal antibody by limiting protease access to the substrate: nano-surface and molecular-orientation limited proteolysis, Analyst. 2014 Feb 7; 139(3): 576-80. DOI: 10.1039 / c3an02104a Summary of the Invention [Problem to be solved by the invention]

[0006] To quantify antibodies by mass spectrometry, it is common to create a calibration curve using the antibody to be measured. However, when measuring multiple antibodies, creating a calibration curve for each antibody to be measured is inefficient. Furthermore, for antibodies such as neutralizing antibodies and novel clones, which are important in functional analysis, it is often difficult to obtain antibodies for which a stable calibration curve can be created. The present invention provides a method that enables antibody quantification by mass spectrometry without creating a calibration curve using the antibody to be measured. [Means for solving the problem]

[0007] The present invention provides digesting the biological protein and the target antibody with a protease; Mass spectrometry is performed on the digested peptide fragments of the biological protein and the target antibody; quantifying the target antibody based on a ratio between the detection intensity of the biological protein obtained by the mass spectrometry and the detection intensity of the target antibody; The digestion with the protease comprises: Immobilizing the biological protein and the target antibody within the pores of a carrier; and bringing the carrier into close proximity to microparticles having a diameter larger than the diameter of the pores and having the protease immobilized on the surface thereof, The biological protein has a domain that can be immobilized in the pore. It relates to a method for analyzing antibodies.

[0008] The present invention provides digesting a biological protein and a sample containing at least two target antibodies with a protease; Mass spectrometry is performed on the digested peptide fragments of the biological protein and the target antibody; quantifying the target antibody based on the ratio of the detection intensity of the biological protein to the detection intensity of the target antibody; The biological protein is a monoclonal antibody. It relates to a method for analyzing antibodies.

[0009] The present invention provides A program used in the method for analyzing an antibody, The program receiving a mass spectrum output from a mass spectrometer; reading out from a storage unit a ratio between the detection intensity of the biological protein and the detection intensity of the target antibody; correcting the detected amount of the biological protein calculated from the mass spectrum; The present invention relates to a program that causes a processing device to execute a process for calculating the amount of the target antibody. [Effects of the Invention]

[0010] According to the present invention, an antibody can be quantified by mass spectrometry without preparing a calibration curve using the antibody to be measured. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows an example of a method for site-selectively cleaving an antibody. [Figure 2]1 is a table showing the signature peptide sequences and CDR3 sequences of the antibodies used in the Examples. [Figure 3] 1 is a table showing the signature peptide sequences of the antibodies used in the Examples. [Figure 4] FIG. 10 is a diagram illustrating the procedure for digesting an antibody with a protease in Experiment 3. [Figure 5] This graph compares the ion counts (cps) obtained by mass spectrometry of a single sample with the cps of each antibody obtained by mass spectrometry of a mixed sample in Experiment 3. The horizontal axis shows the abbreviation for each antibody, and the vertical axis shows cps. [Figure 6] This is a graph comparing the values ​​obtained by correcting the detected amount of a single sample using an internal standard with the values ​​obtained by correcting the detected amount of each antibody in a mixed sample using an internal standard in Experiment 3. The horizontal axis shows the abbreviation for each antibody, and the vertical axis shows the values ​​corrected using the internal standard (internal standard corrected values). [Figure 7] 10 is a graph showing the cps ratio of each antibody to the cps of trastuzumab in Experiment 4. The horizontal axis shows the concentration of each antibody, and the vertical axis shows the cps ratio of each antibody to the cps of trastuzumab. [Figure 8] 10 is a graph showing the cps ratio of each antibody to the cps of trastuzumab in Experiment 4. The horizontal axis shows the concentration of each antibody, and the vertical axis shows the cps ratio of each antibody to the cps of trastuzumab. [Figure 9] 10 is a graph showing the cps ratio of each antibody to the cps of trastuzumab in Experiment 4. The horizontal axis shows the concentration of each antibody, and the vertical axis shows the cps ratio of each antibody to the cps of trastuzumab. [Figure 10] 10 is a graph comparing the concentration of ipilimumab (horizontal axis) quantified based on a calibration curve using trastuzumab with the concentration of ipilimumab (vertical axis) quantified based on a calibration curve using ipilimumab in Experiment 5. [Figure 11]10 is a graph comparing the concentration of pembrolizumab (horizontal axis) quantified based on a calibration curve using trastuzumab with the concentration of pembrolizumab (vertical axis) quantified based on a calibration curve using pembrolizumab in Experiment 5. [Figure 12] 10 is a graph comparing the concentration of ipilimumab (horizontal axis) quantified based on a calibration curve using ipilimumab with the concentration of ipilimumab (vertical axis) quantified based on a calibration curve using trastuzumab in Experiment 6. [Figure 13] 1 is a graph comparing the concentration of pembrolizumab (horizontal axis) quantified based on a calibration curve using pembrolizumab with the concentration of pembrolizumab (vertical axis) quantified based on a calibration curve using trastuzumab in Experiment 6. [Figure 14A] 10 is a graph comparing the concentration of ipilimumab (horizontal axis) quantified based on a calibration curve using ipilimumab with the concentration of ipilimumab (vertical axis) quantified based on calibration curves using each reference protein in Experiment 6. [Figure 14B] 10 is a graph comparing the concentration of ipilimumab (horizontal axis) quantified based on a calibration curve using ipilimumab with the concentration of ipilimumab (vertical axis) quantified based on calibration curves using each reference protein in Experiment 6. [Figure 15A] 10 is a graph comparing the concentration of pembrolizumab (horizontal axis) quantified based on a calibration curve using pembrolizumab with the concentration of pembrolizumab (vertical axis) quantified based on a calibration curve using each reference protein in Experiment 6. [Figure 15B] 10 is a graph comparing the concentration of pembrolizumab (horizontal axis) quantified based on a calibration curve using pembrolizumab with the concentration of pembrolizumab (vertical axis) quantified based on a calibration curve using each reference protein in Experiment 6. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Methods for analyzing antibodies] The method for analyzing an antibody according to this embodiment includes the steps of: digesting the biological protein and the target antibody with a protease; Mass spectrometry is performed on the digested peptide fragments of the biological protein and the target antibody; and quantifying the target antibody based on the ratio of the detection intensity of the biological protein obtained by the mass spectrometry to the detection intensity of the target antibody.

[0013] In this embodiment, the term "biological protein" refers to a protein derived from a living organism, such as a cell or a biological tissue, or a protein artificially synthesized from such a protein. In one aspect of this embodiment, the biological protein can also be understood as a "reference protein" because it is used as a reference substance in the method for analyzing an antibody according to this embodiment. The biological protein is preferably a biologic. In this embodiment, "biologics" refers to pharmaceuticals manufactured, extracted, synthesized, etc., using living organisms. The biologics can also be understood as "biopharmaceuticals" or "biological preparations." In another aspect of this embodiment, examples of the biological protein include antibodies (e.g., reference antibodies, etc.), Fc fusion proteins, and monoclonal antibodies derived from mice, rats, rabbits, etc., as described below. In this embodiment, the biological protein preferably includes a reference antibody or an Fc fusion protein.

[0014] In one aspect of this embodiment, the biological protein preferably includes a reference antibody.

[0015] That is, the method for analyzing an antibody according to this embodiment includes the steps of: digesting the reference antibody and the target antibody with a protease; mass spectrometry of the digested peptide fragments of the reference antibody and the target antibody; It is preferable to include a step of quantifying the target antibody based on the ratio between the detection intensity of the reference antibody and the detection intensity of the target antibody.

[0016] The method according to this embodiment eliminates the need to create a calibration curve for each measurement target, which is expected to reduce the number of antibodies required to create the calibration curve and simplify mass spectrometry. In the field of antibody drugs, the importance of easily quantifying the amount of antibody in vivo, for example, in blood or tissue, is increasing. According to this embodiment, even if a calibration curve cannot be created for the antibody being measured, the concentration of the antibody drug in the sample can be calculated using another antibody as a reference. Furthermore, the method according to this embodiment allows the use of a single, general-purpose antibody as a reference, which is also useful when two or more types of antibodies contained in a sample need to be measured simultaneously. The method according to this embodiment is expected to simplify pharmacokinetic testing and reduce costs.

[0017] In addition to antibodies administered as pharmaceuticals (e.g., monoclonal antibodies), many antibodies are produced in vivo. It is not easy to create a calibration curve for antibodies produced in vivo, and it has traditionally been difficult to quantify such antibodies by mass spectrometry. According to the method of this embodiment, it is possible to quantify neutralizing antibodies, autoantibodies, and their clonotypes produced in vivo by mass spectrometry.

[0018] <Step of digesting antibodies with protease> To easily and accurately detect and quantify proteins by mass spectrometry, it is effective to regioselectively cleave the target protein to efficiently produce peptide fragments specific to that protein and reduce the amount of other peptide fragments produced. For example, when the target protein is an antibody, the antibody can be detected with high sensitivity by regioselectively digesting the Fab domain or the variable region of the Fab domain while suppressing digestion of the Fc domain.

[0019] In the method of this embodiment, the target antibody is the antibody to be quantified, and the reference antibody is the antibody used for comparison to quantify the target antibody. The reference antibody corresponds to the biological protein. Herein, the target antibody and reference antibody are sometimes collectively referred to as "antibody." One example of an antibody is an immunoglobulin IgG in which an Fc domain and a Fab domain are connected via a hinge region. The two heavy chains and two light chains that make up an antibody molecule each have a constant region and a variable region. The constant region has an amino acid sequence that is common to most antibodies derived from the same species. Each variable region has three sites with specific sequences called complementarity-determining regions (CDRs). The three-dimensional structure defined by these CDRs (CDR1, CDR2, and CDR3) determines the specificity with the antigen.

[0020] The protease digestion of the reference antibody and the protease digestion of the target antibody may be performed one after the other, but are preferably performed simultaneously. The target antibody and the reference antibody may be contained in the same sample or in different samples.

[0021] The target antibody may include a manufactured antibody or an antibody produced in vivo. The target antibody may include, for example, a monoclonal antibody or a polyclonal antibody. The target antibody may include an antibody produced in vivo by an immune response, and may include a neutralizing antibody, an autoantibody, and / or a clonotype thereof. The target antibody may include two or more types of antibodies. The number of types of antibodies that can be simultaneously quantified may be three, four, five, six, seven, eight, nine, ten, fifteen, twenty, forty, sixty, eighty, or more. Multiple target antibodies may be contained in the same sample or in different samples.

[0022] The target antibody may be an antibody contained in cultured cells, cultured tissue, or its culture supernatant, but is preferably an antibody contained in a biological sample. The biological sample may be blood obtained from a subject, or may be biological tissue, urine, feces, sweat, saliva, lymph, amniotic fluid, breast milk, pleural effusion, ascites, cerebrospinal fluid, tears, etc. The blood-derived biological sample may be whole blood, but is preferably plasma or serum. The sample may be diluted with physiological saline, buffer, etc. The subject may be an animal, for example, a mammal, including a human. The subject may be a patient administered a monoclonal antibody as an antibody drug. The biological sample may be collected appropriately depending on the type of biological sample and may be stored at room temperature or low temperature after collection. When the target antibody is an antibody contained in a biological sample, the concentration of the target antibody in the living body can be determined by the method of this embodiment.

[0023] The reference antibody can be an antibody exemplified as the target antibody. The reference antibody is an antibody different from the target antibody. In one aspect of this embodiment, the reference antibody can also be understood as an antibody that differs from the target antibody in the amino acid sequence of at least the variable region. An antibody used as a reference antibody in one analysis may be the target antibody in another analysis. It is preferable that the concentration of the reference antibody is specified. A calibration curve can be created using reference antibodies with multiple concentrations. The reference antibody may or may not be labeled with a stable isotope amino acid.

[0024] The reference antibody is preferably an antibody that is easily available, and is preferably a monoclonal antibody from the viewpoint of ease of identifying the concentration and amino acid sequence. Monoclonal antibodies include, but are not limited to, fully human antibodies, humanized antibodies, or chimeric antibodies, such as trastuzumab, trastuzumab-DM1, bevacizumab, cetuximab, rituximab, nivolumab, brentuximab, pembrolizumab, ipilimumab, mogamulizumab, ramucirumab, atezolizumab, durvalumab, avelumab, infliximab, tocilizumab, adalimumab, golimumab, mepolizumab, ustekinumab, eculizumab, panitumumab, ofatumumab, omalizumab, gemtuzumab, palivizumab, ranibizumab, certolizumab, ocrelizumab, and basiliximab. The molecular diameter of monoclonal antibodies is approximately 14.5 nm. The monoclonal antibodies of the method of this embodiment also include conjugates that maintain the specificity of a monoclonal antibody while adding additional functions, such as antibody-drug conjugates (e.g., brentuximab-vedotin, gemtuzumab-ozogamicin, trastuzumab-emtansine, etc.). Prior to analysis, the conjugate may be dissociated and only the antibody portion may be subjected to analysis, or the conjugate may be subjected to analysis in its original form.

[0025] In this embodiment, the biological protein may be an Fc fusion protein. Here, "Fc fusion protein" refers to a recombinant protein in which a functional protein or a domain thereof is fused with the Fc domain of immunoglobulin. Examples of the functional protein or a domain thereof include the extracellular domain of human cytotoxic T-lymphocyte antigen-4 (CTLA-4), the extracellular domain of human vascular endothelial growth factor (VEGF) receptor 1 and 2 proteins, and tumor necrosis factor (TNF) receptor. Examples of the Fc fusion protein include abatacept (a fusion protein of the extracellular domain of CTLA-4 and the Fc domain of immunoglobulin), aflibercept (a fusion protein of the extracellular domain of VEGF receptor 1 and 2 proteins and the Fc domain of immunoglobulin), and etanercept (a fusion protein of a TNF receptor and the Fc domain of immunoglobulin). In one aspect of this embodiment, the Fc fusion protein is preferably selected from the group consisting of abatacept, aflibercept, and etanercept.

[0026] In one aspect of this embodiment, the step of digesting with a protease preferably comprises: (a) immobilizing a biological protein and a target antibody within the pores of a carrier; (b) bringing microparticles having a diameter larger than the diameter of the pores and having a protease immobilized on the surface thereof into close proximity with the carrier.

[0027] In another aspect of this embodiment, the step of digesting the antibody with a protease to regioselectively digest the Fab domain (or functional protein domain in an Fc fusion protein) preferably comprises: (a) immobilizing a reference antibody (or an Fc fusion protein) and a target antibody within the pores of a support; (b) bringing microparticles having a diameter larger than the diameter of the pores and having a protease immobilized on the surface thereof into close proximity with the carrier.

[0028] (a) immobilizing a reference antibody and a target antibody within the pores of a support; The carrier for immobilizing the antibody may be a porous body having many pores, and the antibody can be bound to the pores. The material of the carrier is not particularly limited, and examples that can be used include activated carbon, porous membranes, porous resin beads, and metal particles.

[0029] The shape of the pores is not particularly limited, but may be hemispherical or may penetrate the carrier. The size of the pores is preferably determined taking into account the molecular diameter of the antibody, so that when the antibody is immobilized, the tip of the antibody, i.e., the site to be selectively digested, is located near the opening of the pore. The average pore diameter of the carrier is appropriately set, for example, in the range of about 10 nm to 500 nm, and smaller than the average particle size of the microparticles. The average pore diameter of the carrier may be, for example, 20 nm or more, 30 nm or more, 40 nm or more, or 50 nm or more, and may be 200 nm or less, 150 nm or less, 120 nm or less, or 100 nm or less. The average pore diameter of the carrier can be determined by electron microscopy.

[0030] To immobilize an antibody within the pores of a carrier, a carrier is preferably used in which a linker that interacts site-specifically with the antibody is immobilized within the pores. Examples of interactions between the antibody and the linker include chemical bonds, hydrogen bonds, ionic bonds, complex formation, hydrophobic interactions, van der Waals interactions, electrostatic interactions, and stereoselective interactions. From the viewpoint of ease of immobilization within the pores, the reference antibody, and more preferably the reference antibody and target antibody, have a domain that can be immobilized within the pores. The domain that can be immobilized within the pores is, for example, the Fc domain of an antibody. In one aspect of this embodiment, the biological protein has a domain that can be immobilized within the pores.

[0031] As the linker, preferably used is Protein A, Protein G, or the like, which binds site-specifically to the Fc domain of an antibody. By using a support in which these linkers are immobilized within pores, the Fc domain of the antibody is immobilized within the pores, and the Fab domain is positioned near the opening of the pore. By controlling the orientation of the antibody within the pores, site-selective digestion of the Fab domain by protease becomes possible.

[0032] Carriers that can be suitably used in this embodiment include, but are not limited to, Protein G Ultralink resin (manufactured by Pierce), Toyopearl TSKgel (manufactured by Tosoh Corporation), Toyopearl AF-rProtein A HC-650F resin (manufactured by Tosoh Corporation), Protein A Sepharose (GE Healthcare), and KanCapA (KANEKA).

[0033] The method for immobilizing an antibody within the pores of a carrier is not particularly limited. For example, when immobilizing an antibody on a carrier with Protein A or Protein G immobilized within the pores, the antibody can be easily immobilized within the pores by mixing a suspension of the carrier with a solution containing the antibody. The quantitative ratio of the carrier to the antibody can be appropriately set depending on the purpose. The carrier is then washed with an appropriate buffer to remove nonspecifically bound antibodies and molecules contained in the sample. Examples of buffers that can be used include phosphate buffered saline (PBS) and PBS containing n-octylthioglycoside (OTG). Washing can be performed automatically, for example, by suction or centrifugation using a filter-integrated plate, and multiple samples can be simultaneously washed.

[0034] (a') carrying out a reduction reaction under acidic conditions After the above step (a) of immobilizing the reference antibody and target antibody within the pores of the support, a step (a') of performing a reduction reaction on the antibodies under acidic conditions may be carried out. Antibodies may have highly rigid regions due to some mechanism, such as the presence of a cysteine ​​knot structure formed by an S-S bond (disulfide bond), which may make them resistant to proteases. For example, approximately 20-30% of monoclonal antibodies are known to have localized protease resistance. For such rigid antibodies, acidic reduction treatment can sometimes improve the efficiency of protease digestion. Because the bond between protein A or protein G and the Fc domain does not dissociate under acidic reduction conditions, it is thought that the cysteine ​​knot structure can be unraveled while the antibody molecule is retained on the support.

[0035] The reduction reaction under acidic conditions can be carried out by incubation in the presence of an organophosphorus reducing agent. Examples of organophosphorus reducing agents include tris(2-carboxyethyl)phosphine (TCEP) or its hydrochloride, tributylphosphine, etc. These reducing agents are available from Sigma-Aldrich, Nakarai Tesque, Funakoshi, etc. The concentration of TCEP in the reaction solution can be, for example, 100 mM to 500 mM, e.g., 250 mM.

[0036] The reduction reaction is preferably carried out under strongly acidic conditions of pH 2.5 or less, such as pH 1.5, pH 2.0, or pH 2.5. The reduction reaction time is not limited, but a period of 10 to 60 minutes is sufficient, such as 20, 30, 40, 45, or 50 minutes. It is also possible to carry out the reduction reaction for longer than 60 minutes, but this will increase the working time required for measurement. The reaction temperature is not limited, but may be, for example, 15 to 30°C, and can be room temperature. Preferably, the reaction temperature is about 25°C.

[0037] A sample that has not been subjected to acidic reduction treatment and a sample that has been subjected to acidic reduction treatment may be subjected to mass spectrometry simultaneously. Specifically, a solution containing a carrier and an antibody solution is divided into two, and one portion is washed as is. The other portion is subjected to acidic reduction treatment and then washed. After washing, the two carriers are combined and subjected to the next step of protease digestion. This allows both peptide fragments generated from the antibody that has been subjected to TCEP acidic reduction treatment and peptide fragments generated from the antibody that has not been subjected to TCEP acidic reduction treatment to be subjected to mass spectrometry as a single sample.

[0038] (b) bringing microparticles having a diameter larger than the diameter of the pores and having a protease immobilized on the surface thereof into close proximity with the carrier; The type of protease immobilized on the microparticles can be appropriately selected depending on the type of antibody to be quantified by mass spectrometry. For example, trypsin, chymotrypsin, lysyl endopeptidase, V8 protease, AspN protease (Asp-N), ArgC protease (Arg-C), papain, pepsin, and dipeptidyl peptidase can be used alone or in combination. Trypsin is preferred as the protease. Examples of proteases that can be suitably used in the method of this embodiment include Trypsin Gold (Promega) and Trypsin TPCK-treated (Sigma).

[0039] Microparticles are used to immobilize proteases on their surfaces to control the access of proteases to antibodies immobilized within the pores of the support. Therefore, microparticles with an average particle size larger than the average pore size of the support are used so that they do not penetrate deep into the pores of the support. The average particle size of the microparticles may be 1.2 times or more, 1.5 times or more, 1.8 times or more, or even about twice the average pore size of the support. The average particle size of the microparticles may be 50 nm or more, 100 nm or more, 120 nm or more, 150 nm or more, or 170 nm or more, and may be 500 nm or less, or 300 nm or less. The average particle size of the microparticles can be determined by electron microscopy.

[0040] The shape of the microparticles is not particularly limited, but from the viewpoint of uniform access of the protease to the carrier, a spherical shape is preferable. The microparticles preferably have high dispersibility and a uniform average particle size.

[0041] The material of the microparticles is not particularly limited as long as it can immobilize a protease on the surface, and metals, resins, etc. can be used appropriately. Microparticles with a metal surface coated with resin, or microparticles with a resin surface coated with metal, etc. can also be used.

[0042] The fine particles are preferably magnetic nanoparticles that can be dispersed or suspended in an aqueous medium and can be easily recovered from the dispersion or suspension by magnetic separation or magnetic precipitation separation. From the viewpoint of reducing aggregation, it is preferable that the surface of the particles is coated with an organic polymer. Specific examples of magnetic nanobeads coated with an organic polymer include FG beads, SG beads, Adembeads, and nanomag. A commercially available product that can be used is, for example, FG beads (polymer magnetic nanoparticles with a particle size of approximately 200 nm, made by coating ferrite particles with polyglycidyl methacrylate (polyGMA)) manufactured by Tamagawa Seiki Co., Ltd.

[0043] The microparticles are preferably modified with a spacer capable of binding to a protease to suppress nonspecific protein adsorption and selectively immobilize the protease. Immobilizing the protease via the spacer suppresses detachment of the protease from the microparticle surface, thereby enhancing the site selectivity of digestion by the protease. Adjusting the molecular size of the spacer also allows the protease to selectively access a desired site on the antibody, enhancing site selectivity.

[0044] The spacer is preferably one that can bind to the protease and does not inactivate the protease. From the viewpoint of controlling the access range of the protease, the spacer preferably has a small molecular diameter. The molecular diameter of the spacer is preferably 5 nm or less, more preferably 3 nm or less, and even more preferably 2 nm or less. The molecular weight of the spacer is preferably 2000 or less, more preferably 1500 or less, and even more preferably 1000 or less.

[0045] The spacer is preferably non-protein, and is preferably a molecule having a functional group at its terminus, such as an amino group, a carboxyl group, an ester group, an epoxy group, a tosyl group, a hydroxyl group, a thiol group, an aldehyde group, a maleimide group, a succinimide group, an azide group, biotin, avidin, or a chelate. For example, for immobilizing trypsin, it is preferable to use a spacer having an activated ester group. The spacer arm portion other than the above functional group is preferably a hydrophilic molecule such as polyethylene glycol, polypropylene glycol, polyacrylamide, polyethyleneimine, poly(ethylene oxide), polyethylene terephthalate, or a derivative thereof.

[0046] Examples of microparticles surface-modified with spacers include microparticles modified with spacers having ester groups (active ester groups) activated with N-hydroxysuccinimide, which are commercially available under the trade name "FG beads NHS" (manufactured by Tamagawa Seiki Co., Ltd.).

[0047] The method for immobilizing proteases on the surface of microparticles is not particularly limited, and an appropriate method can be adopted depending on the properties of the protease and the microparticles (or the spacer that modifies the microparticle surface). For example, microparticles with proteases immobilized on their surfaces can be obtained by mixing a suspension of spacer-modified microparticles with a solution containing protease. As a commercially available product of microparticles with trypsin immobilized as the protease, "FG beads Trypsin DART (registered trademark)," included in the LC / MS / MS pretreatment kit "nSMOL Antibody BA Kit" (manufactured by Shimadzu Corporation), can also be suitably used.

[0048] By bringing the carrier on which the antibody is immobilized into close proximity to or into contact with the microparticle on which the protease is immobilized, the variable region of the antibody is selectively digested by the protease.

[0049] Digestion with a protease can be carried out, for example, in a buffer solution adjusted to near the optimal pH of the protease. Digestion with a protease is preferably carried out in the pH range of 7 to 9, more preferably about pH 8.0. Digestion with a protease may be carried out in an environment of about 37°C, but is preferably carried out under saturated vapor pressure at about 50°C. The reaction time may be, for example, 30 minutes to 20 hours, for example, 1 hour or more or 3 hours or more, or 5 hours or less or 8 hours or less. To prevent evaporation of the reaction solution, the reaction is preferably carried out under saturated vapor pressure.

[0050] In the step of digesting with a protease, the reaction solution may be stirred to promote contact between the carrier and the fine particles. The reaction solution may be stirred throughout the entire reaction time, or only for a part of the reaction time, for example, only at the beginning of the reaction.

[0051] Peptide fragments generated by protease digestion are released into the reaction mixture. To subject the peptide fragments to mass spectrometry, it is necessary to remove the carrier and microparticles. This can be achieved by subjecting the reaction mixture after protease digestion to filtration, centrifugation, magnetic separation, dialysis, or other procedures.

[0052] Separation of the carrier / particles from the peptide fragments can be easily carried out by filtration using, for example, a polyvinylidene fluoride (PVDF) filtration membrane (low-binding hydrophilic PVDF, pore size 0.2 μm, manufactured by Millipore) or a polytetrafluoroethylene (PTFE) filtration membrane (low-binding hydrophilic PTFE, pore size 0.2 μm, manufactured by Millipore). Further centrifugation allows for rapid and easy filtration.

[0053] The above-mentioned process of digesting antibodies with proteases can be performed using the nano-surface and molecular-orientation limited proteolysis (nSMOL) method (registered trademark) previously developed by the present inventors' group, and the LC / MS / MS pretreatment kit "nSMOL Antibody BA Kit" (Shimadzu Corporation) can be used. Details of the nSMOL method are described, for example, in International Publication No. 2015 / 033479 and Iwamoto N et al., Analyst. 2014 Feb 7;139(3):576-80, doi:10.1039 / c3an02104a. Improved techniques for the nSMOL method are disclosed, for example, in International Publication Nos. 2016 / 143223, 2016 / 143224, 2016 / 143226, 2016 / 143227, 2019 / 130549, 2019 / 155576, Iwamoto N et al., Bioanalysis, doi: 10.4155 / bio-2016-0018, and Iwamoto N et al., Biological & Pharmaceutical Bulletin, 2016, doi: 10.1248 / bpb.b16-00230. The disclosures of these documents are incorporated herein by reference.

[0054] An example of a protocol for the nSMOL method is as follows: <Step (a)> 1. Dilute 5-10 μL of antibody-containing biological sample in approximately 10-20 volumes of PBS + 0.1% n-octylthioglycoside (OTG). 2. Add 25 μL of Immunoglobulin Collection Resin (TOYOPEARL AF-rProtein A HC-650F, 50% slurry) to the diluted biological sample to obtain a mixed solution. 3. Gently vortex the resulting mixture for approximately 5 minutes. 4. Collect the entire mixture onto an UltraFree low-protein binding Durapore PFDF (0.22 μm). 5. Centrifuge (10,000 g, 1 minute) to remove the supernatant. 6. Add 300 μL of PBS + 0.1% OTG, and centrifuge (10,000 g, 1 minute) to remove the supernatant. 7. Repeat step 6. 8. To remove the surfactant (OTG), add 300 μL of PBS and centrifuge (10,000 g, 1 minute) to remove the supernatant. 9. Repeat step 8. 10. Add 75-100 μL of Reaction solution or Enhanced Reaction Solution. 10 fmol / μL of P14R synthetic peptide may also be added to this solution.

[0055] <Step (b)> 11. Add 5-10 μL of FG beads (0.5 mg / ml FG bead suspension) onto which chemically modified trypsin has been immobilized. 12. React at 50°C under saturated vapor pressure with gentle stirring for 4-6 hours. 13. Stop the reaction by adding 10 μL of 10% formic acid to the reaction mixture. 14. Perform centrifugal filtration (10,000 g, 1 minute) and recover the solution. 15. Place the tube containing the solution on a magnetic stand and let it stand for about 1-2 minutes to remove excess resin from the solution.

[0056] Figure 1 shows an example of the regioselective cleavage of an antibody using the nSMOL method. A protease 15 is immobilized on the surface of a microparticle 10 with an average particle diameter D1. The protease 15 is immobilized on the microparticle via a spacer 11. A support 20 has numerous pores 29 with an average pore diameter D2, to which linkers 21 capable of specifically binding to antibodies are immobilized. The antibodies 25 are immobilized within the pores via the linkers 21. The dotted lines near the pores 29 represent the boundaries of the area accessible to the protease 15. By restricting the protease's access to the Fc domain of the antibody bound to the surface of the pore, the probability of the protease accessing the Fab domain located near the opening, away from the pore binding site, is relatively increased. This allows the protease to regioselectively cleave the antibody's Fab domain to obtain peptide fragments.

[0057] <Step of mass spectrometry of peptide fragments> The peptide fragments obtained in the above steps are subjected to mass spectrometry. The peptide fragments of the reference antibody and the peptide fragments of the target antibody may be contained in the same sample or in different samples. The peptide fragments obtained from multiple target antibodies may be contained in the same sample or in different samples.

[0058] To ensure the separation of peptide fragments and improve analytical accuracy, it is preferable to separate and concentrate samples prior to mass spectrometry using a liquid chromatograph (LC). When separating samples using LC, the LC eluate may be directly ionized and subjected to mass spectrometry. Analysis can also be performed using LC / MS / MS or LC / MSn, which combine LC and tandem mass spectrometry. The LC eluate may be first fractionated and then subjected to mass spectrometry. The LC column is not particularly limited; hydrophobic columns such as C30, C18, C8, and C4 columns commonly used in peptide analysis, as well as supports for hydrophilic affinity chromatography, can be appropriately selected and used. If necessary, the sample may be subjected to desalting, solubilization, extraction, concentration, drying, or other processes before being used for mass spectrometry.

[0059] The ionization method used in mass spectrometry is not particularly limited, and examples include electron ionization (EI), chemical ionization (CI), field desorption (FD), fast atom bombardment (FAB), matrix-assisted laser desorption / ionization (MALDI), and electrospray ionization (ESI). The analytical method for the ionized sample is also not particularly limited, and can be selected appropriately depending on the ionization method, including magnetic deflection, quadrupole (Q), ion trap (IT), time-of-flight (TOF), and Fourier transform ion cyclotron resonance (FT-ICR). MS / MS analysis, multistage mass spectrometry (MS3 or higher), and multiple reaction monitoring (MRM) can also be performed using a triple quadrupole mass spectrometer or the like.

[0060] Apparatuses particularly suitable for the method of this embodiment include, but are not limited to, LCMS-8030, LCMS-8040, LCMS-8050, LCMS-8060, LCMS-9030, and LCMS-IT-TOF (all manufactured by Shimadzu Corporation).

[0061] Mass spectrometry can identify target antibodies by detecting peptide fragments containing the amino acid sequences of the Fab region specific to the target antibody, such as the CDR1, CDR2, and CDR3 amino acid sequences of the heavy and / or light chains. Existing databases can be used to identify target antibodies. The nSMOL method uses peptide fragments obtained by site-specific protease digestion of antibodies, thereby increasing the hit rate and data accuracy of database searches. Among multiple peptide fragments derived from the Fab region, peptide fragments that are particularly suitable for antibody identification can be selected as analysis targets. Peptide fragments selected in this way are called "signature peptides."

[0062] When identifying an antibody based on the detection results of a specific peptide fragment containing a CDR sequence, the peptide fragment to be detected preferably has approximately 5 to 30 amino acid residues, and more preferably approximately 7 to 25. If the number of amino acid residues is too small, it may be difficult to distinguish it from contaminants or peptide fragments derived from other parts of the same protein, which may result in erroneous detection. Furthermore, if the number of amino acid residues is too large, detection may become difficult or quantitative accuracy may decrease due to factors such as difficulty in ionization.

[0063] <Step of quantifying target antibodies> The target antibody is quantified based on the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody obtained by mass spectrometry. In this embodiment, "detection intensity" refers to the peak area or peak intensity derived from the peptide fragment of interest in the mass spectrum obtained by mass spectrometry. That is, "detection intensity of the reference antibody" refers to the peak area or peak intensity derived from the peptide fragment of the reference antibody in the mass spectrum obtained by mass spectrometry. "Detection intensity of the target antibody" refers to the peak area or peak intensity derived from the peptide fragment of the target antibody in the mass spectrum obtained by mass spectrometry. The target antibody can be quantified based on the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody, and the ratio of the detected amount of the reference antibody to the detected amount of the target antibody. The detected amount of the target antibody or the detected amount of the reference antibody can be corrected based on the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody. The detected amounts of the reference antibody and the target antibody are calculated based on the peak area or peak intensity of the mass spectrum obtained by mass spectrometry of the peptide fragments of the above-mentioned reference antibody and target antibody.

[0064] For example, the detected amount of the reference antibody can be converted to a standard amount of the target antibody based on the ratio between the detection intensity of the reference antibody and the detection intensity of the target antibody, and the converted amount can be compared with the actual detected amount of the target antibody to quantify the target antibody. For example, the detected amount of the target antibody can be converted to a standard amount of the reference antibody based on the ratio between the detection intensity of the reference antibody and the detection intensity of the target antibody, and the converted amount can be compared with the actual detected amount (standard amount) of the reference antibody to quantify the target antibody. The reference antibody may be subjected to mass spectrometry at a single concentration point, or at multiple concentration points. When the reference antibody is subjected to mass spectrometry at multiple concentrations, a calibration curve for the reference antibody can be created based on the results of mass spectrometry. For example, the calibration curve for the reference antibody can be converted to a calibration curve for the target antibody based on the ratio between the detection intensity of the reference antibody and the detection intensity of the target antibody, and the target antibody can be quantified.

[0065] The ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody can be determined by mass spectrometry of a reference antibody having a predetermined concentration and a target antibody having a predetermined concentration. For example, equal amounts of the reference antibody and the target antibody are digested with a protease under the same conditions, and then subjected to mass spectrometry. The ratio of the peak area or peak intensity of the mass spectrum obtained can be used as the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody. The ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody can be the median or average of the ratios obtained by performing five independent analyses. Typically, the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody is constant regardless of the concentration. The ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody can be determined in advance. By determining the ratio in advance, the target antibody can be quantified easily. The ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody can be universally used as a constant for quantifying the target antibody.

[0066] That is, in one aspect of this embodiment, the method for analyzing an antibody includes: mass spectrometry of the biological protein having a predetermined concentration and the target antibody having a predetermined concentration; It is preferable that the method further comprises determining in advance a ratio between the detection intensity of the biological protein and the detection intensity of the target antibody based on the results of the mass spectrometry.

[0067] In another aspect of this embodiment, the method for analyzing an antibody comprises: mass spectrometry of the reference antibody having a predetermined concentration and the target antibody having a predetermined concentration; Preferably, the method further comprises predetermining a ratio between the detection intensity of the reference antibody and the detection intensity of the target antibody.

[0068] The mass analysis of a reference antibody having a predetermined concentration and a target antibody having a predetermined concentration is not particularly limited and can be performed by the method described in the step of performing mass analysis of the reference antibody and the target antibody. The mass analysis of a reference antibody having a predetermined concentration and a target antibody having a predetermined concentration may be performed simultaneously with the step of performing mass analysis of the reference antibody and the target antibody, or may be performed before the step of performing mass analysis of the reference antibody and the target antibody. The step of performing mass analysis of the reference antibody and the target antibody is preferably performed under the same conditions as those for the mass analysis of the reference antibody having a predetermined concentration and the target antibody having a predetermined concentration.

[0069] [kit] The kit according to this embodiment includes: The biological protein; microparticles having the protease immobilized on their surfaces; and a carrier having the pores formed therein.

[0070] The kit is used in the above-mentioned method for analyzing antibodies. The kit according to this embodiment includes: a reference antibody; microparticles having proteases immobilized on their surfaces; and a carrier having pores formed therein.

[0071] The kit may be a kit used in the above-mentioned method for analyzing antibodies. The biological protein, reference antibody, microparticles, and carrier may be those described in the above-mentioned methods. The kit may further include reaction vessels such as microplates and tubes that can be used in the above-mentioned methods, filter-integrated plates, buffers, washing solutions, protease reaction solutions, acidic reducing agents, filtration membranes, chips, and analytical columns. The kit may include instructions describing how to use the kit and / or mass spectrometry conditions for antibody detection. The kit may also include a correspondence table showing the ratio of the detection intensity of the biological protein to the detection intensity of the target antibody, or a correspondence table showing the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody.

[0072] By using the kit according to this embodiment, the operation of preparing peptide fragments for antibody detection can be performed more easily and can be automated using an apparatus. By using the kit according to this embodiment, mass spectrometry and quantification of target antibodies can also be performed easily.

[0073] [program] The program according to this embodiment is A program used in the method for analyzing an antibody, The above program is receiving a mass spectrum output from a mass spectrometer; reading out from a memory unit a ratio between the detection intensity of the biological protein and the detection intensity of the target antibody; correcting the detected amount of the biological protein calculated from the mass spectrum; The program may be a program that causes a processing device to execute a process for calculating the amount of the target antibody.

[0074] The program according to this embodiment is receiving a mass spectrum output from a mass spectrometer; reading out the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody from the memory unit; correcting the detected amount of the reference antibody calculated from the mass spectrum; The processing device executes a process for calculating the amount of the target antibody.

[0075] The program according to this embodiment may be a program that realizes the step of quantifying the target antibody in the above-described method for analyzing an antibody.

[0076] One example of the program identifies the reference antibody and the target antibody from the mass spectrum output from the mass spectrometer and calculates the detected amount of each. The type and concentration of the reference antibody can be received from the user. Depending on the received reference antibody, the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody is read from the memory unit. The ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody may be a value determined by mass spectrometry of a reference antibody having a predetermined concentration and a target antibody having a predetermined concentration, as described above. This value is preferably stored in the memory unit in advance. Based on the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody, the detected amount of the reference antibody calculated from the mass spectrum can be multiplied by a constant and converted to a standard amount for the target antibody. This value can be compared with the detected amount of the target antibody obtained from the mass spectrum to determine the concentration of the target antibody.

[0077] The program may be recorded on a computer-readable recording medium. The recording medium on which the program according to this embodiment is recorded and the recording medium on which data for performing the mass spectrometry described below are recorded may be the same or different. The program recorded on the recording medium may be loaded into a computer system and executed. In this specification, the term "computer system" includes an operating system (OS) and peripheral hardware. The term "computer-readable recording medium" includes portable recording media such as flexible disks, magneto-optical disks, optical disks, and memory cards, as well as storage devices such as hard disks built into a computer system. The term "computer-readable recording medium" may include a medium that dynamically stores a program for a short period of time, such as a communication line when transmitting a program via a network such as the Internet or a communication line such as a telephone line, or a medium that stores a program for a certain period of time, such as volatile memory within a computer system that serves as a server or client in such a case. The program may execute processing in combination with a program already recorded in the computer system.

[0078] [Recording medium on which data for performing mass spectrometry is recorded] This embodiment may also provide a computer-readable recording medium having recorded thereon data for performing mass spectrometry, for use in the method according to this embodiment. The data may include, but is not limited to, data on parent ions, fragment ions, predicted retention times, and voltages in each of the triple quadrupoles (first quadrupole, second quadrupole, third quadrupole) for one or more peptide fragments having amino acid sequences unique to each antibody. The data may include information related to a specific antibody. The above-mentioned predicted retention time, voltage data, etc. are values ​​that vary depending on the equipment used, measurement conditions, etc., and are preferably provided according to the equipment. For values ​​that vary depending on conditions, it is preferable to also provide the range of variation, as will be understood by those skilled in the art.

[0079] [Method Package] This embodiment may also provide a method package for detecting and quantifying a target antibody by liquid chromatography-mass spectrometry (LC-MS), which includes a recording medium on which the above-mentioned program and / or data is recorded.

[0080] As used herein, the term "method package" refers to a standalone distribution item that contains, in a readable format, analytical conditions for liquid chromatography-mass spectrometry for a specific measurement target and / or a program for quantifying a target antibody. Importing the data contained in the method package into an LC-MS enables analysis under optimal measurement conditions determined after careful consideration. Executing the program contained in the method package enables quantification of the target antibody. The method package may also include instructions for use of the recording medium.

[0081] While mass spectrometry allows for extremely high-precision analysis, the analytical conditions vary greatly depending on the target ion, making it extremely important to set appropriate analytical conditions, which is extremely difficult and takes a huge amount of time. Preparing these conditions in advance can improve convenience for users who actually perform mass spectrometry.

[0082] A method package may describe data common to a plurality of mass spectrometers, or may describe various data suited to analysis by a specific mass spectrometer.

[0083] The recording medium or method package can be provided together with the kit of this embodiment described above, or separately from the kit. [Example]

[0084] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0085] <Experiment 1> We investigated whether multiple antibodies digested with proteases using the nSMOL method could be simultaneously identified by mass spectrometry. Antibodies fractionated from serum were digested using the nSMOL method. The resulting peptide fragments were subjected to mass spectrometry to identify their sequences. A human standard serum reagent (a mixed sample from 10 subjects, manufactured by Innovative Research, product name "Pooled Human AB Serum Plasma Derived") was used. An LCMS-9030 mass spectrometer (manufactured by Shimadzu Corporation) was used, and the acquired MS spectra were output to the mzML file format and analyzed using Peaks Studio version 10.5. Since antibody sequence information (amino acid sequence information) from individual humans is not available, spectral matching and searches were performed against a public database (IMGT database).

[0086] Digestion of antibodies by the nSMOL method was carried out as follows using the "nSMOL Antibody BA Kit" (Shimadzu Corporation). (1) 10 μL of serum was diluted with approximately 10 times the volume of PBS + 0.1% OTG. (2) 25 μL of immunoglobulin collection resin (a carrier in which Protein A is immobilized within the pores, pore size 100 nm) was added to the diluted serum to obtain a mixed solution. (3) The resulting mixture was gently vortexed for 5 minutes. (4) The entire amount of the mixture was collected in an UltraFree low-protein binding Durapore PVDF (0.22 μm) (a centrifugal ultrafiltration filter). (5) The supernatant was removed by centrifugation (10,000 g, 1 minute). (6) 300 μL of PBS+0.1% OTG was added, and the supernatant was removed by centrifugation (10,000 g, 1 minute). (7) Step (6) was repeated. (8) To remove the surfactant (OTG), 300 μL of PBS was added, and the supernatant was removed by centrifugation (10,000 g, 1 minute). (9) Step (8) was repeated. (10) 100 μL of Enhanced reaction solution was added. (11) 5 μL of FG beads DART (diameter 200 nm) (microparticles with protease immobilized on the surface) with chemically modified trypsin immobilized on the surface was added. (12) The reaction was carried out for 5 hours at 50°C under saturated vapor pressure with gentle stirring. (13) The reaction was stopped by adding 10 μL of 10% formic acid to the reaction solution. (14) The solution was collected by centrifugal filtration (10,000 g, 1 minute). (15) The tube containing the above solution was placed on a magnetic stand and left to stand for about 1 minute, and excess magnetic beads were removed from the above solution. (16) The above solution was subjected to LCMS analysis.

[0087] As a comparative example, peptide fragments obtained by digesting an antibody with pepsin (manufactured by Promega, product name "Pepsin") or IdeS enzyme (manufactured by Promega, product name "IdeS Protease") were subjected to mass spectrometry. The results are shown in Table 1.

[0088] [Table 1]

[0089] The nSMOL method was confirmed to be capable of simultaneously identifying multiple antibodies, and was able to identify more sequences with a smaller amount of serum than the pepsin and IdeS prosease methods.

[0090] <Experiment 2> We further verified whether multiple antibodies digested by the nSMOL method could be simultaneously identified by mass spectrometry using monoclonal antibodies with known sequences. Twenty monoclonal antibodies, including trastuzumab, bevacizumab, cetuximab, rituximab, nivolumab, brentuximab, pembrolizumab, ipilimumab, mogamulizumab, ramucirumab, atezolizumab, durvalumab, avelumab, infliximab, tocilizumab, adalimumab, golimumab, mepolizumab, ustekinumab, and eculizumab, were mixed with human serum (Innovative Research, product name "Pooled Human AB Serum Plasma Derived"). This sample was digested using the same nSMOL method as in Experiment 1, and the resulting peptide fragments were subjected to mass spectrometry. An LCMS-9030 (Shimadzu Corporation) mass spectrometer was used. The acquired MS spectra were exported to mzML file format and then spectrally matched and searched against an in-house monoclonal antibody FASTA database using Peaks Studio version 10.5. As a result, signature peptides and CDR3 sequences were identified for all antibodies used. This suggests that mass spectrometry using the nSMOL method can be universally applied to antibody quantification. The signature peptide sequences and CDR3 sequences (SEQ ID NOs: 1 to 43) for each antibody are shown in Figure 2.

[0091] <Experiment 3> The error between quantitative values ​​obtained when antibodies were analyzed individually and when multiple antibodies were analyzed simultaneously was examined. The quantitative values ​​obtained from a single sample containing one antibody were compared with the quantitative values ​​obtained from a mixed sample containing multiple antibodies. The single sample was a mixture of one of 21 monoclonal antibodies or Fc fusion proteins (brentuximab, cetuximab, rituximab, infliximab, atezolizumab, bevacizumab, pembrolizumab, trastuzumab, eculizumab, mepolizumab, tocilizumab, avelumab, durvalumab, ipilimumab, nivolumab, ramucirumab, adalimumab, golimumab, abatacept, aflibercept, and etanercept) in human serum. The mixed sample contained all of these antibodies and Fc fusion proteins. The concentration of each antibody and Fc fusion protein was 50 μg / mL. The signature peptide sequences of each antibody are shown in Figure 2 and Figure 3. In the following description, monoclonal antibodies and Fc fusion proteins may be collectively referred to as "antibodies."

[0092] This sample was digested using the improved nSMOL method (Figure 4) described below. As a modification from Experiment 2, in the improved nSMOL method, the reaction was performed on a microplate to process a large number of samples. A microplate with a built-in filter was used to simplify the centrifugation procedure. To analyze antibodies that exhibited protease resistance, the antibodies were pretreated under strong acid reduction conditions. The efficiency of protease treatment was improved by analyzing antibodies that had undergone strong acid reduction treatment together with those that had not.

[0093] The specific operations are as follows: (1) 10 μL of sample was diluted with 90 μL of PBS + 0.1% OTG. (2) The diluted sample was transferred to a low-binding microplate (Eppendorf, product name "Protein LoBind Plate"), and 25 μL of IgG collection resin (a carrier with Protein A immobilized within the pores, pore diameter 100 nm) was further added. (3) The IgG collection resin was divided into equal portions. One portion was washed directly with PBS + 0.1% OTG, while the other portion was washed after acidic reduction treatment (250 mM TCEP-HCl). Washing was performed on a microplate with a built-in filter (Whatman, "UNIFILTER filter plate," pore size 20 μm), and the waste liquid was treated using a vacuum manifold. (4) The IgG collection resin on the filter-embedded microplate was suspended in Enhanced Reaction Solution, then placed in the same well of the low-adsorption microplate, and the resins under the two conditions were mixed. (5) 10 μL of FG beads Trypsin DART (diameter 200 nm) (microparticles with protease immobilized on the surface) was added. (6) The reaction was carried out for 5 hours under saturated vapor pressure at 50°C with gentle stirring. (7) After the reaction, 10 μL of 10% formic acid was added to stop the reaction, and the solution was collected on a microplate with a built-in filter (manufactured by Nippon Pall Corporation, "AcroPrep advance 96-well Filter plate," pore size 200 nm). (8) The mixture was centrifuged (1500 g, 10 minutes) to recover a solution containing peptide fragments.

[0094] LCMS was carried out under the following conditions. [LC] NexeraX2 system (Shimadzu Corporation) Column: Shim-pack GISS C18 (50 mm x 2.1 mm) Column temperature: 50℃ Solvent A: 0.1% formic acid / water Solvent B: 0.1% formic acid / acetonitrile Gradient: 1% B (1 min) / 1-50% B (5 min) / 95% B (1.5 min) / 1% B (10.5 min) Flow rate: 0.4mL / min Injection volume: 10μL [MS] LCMS-9030 (Shimadzu Corporation) Ionization: ESI Positive DL temperature: 250℃ Heat block temperature: 400℃ Interface temperature: 300℃ Nebulizer gas: 3L / min Drying gas: 10L / min Heating gas: 10L / min The mass spectrometer and analytical method were the same as in Experiment 2.

[0095] First, we confirmed that the calibration curves for single samples (N = 3) and mixed samples (N = 3) were sufficiently reproducible and accurate. Comparisons between single and mixed samples were performed using absolute ion counts and values ​​corrected using an internal standard. Figure 5 shows the results of comparing the ion counts (cps) of a single sample with the cps of each antibody obtained by mass spectrometry of a mixed sample. Figure 6 shows the results of comparing the values ​​corrected for the amount of detection in a single sample using an internal standard with the values ​​corrected for the amount of detection in each antibody in a mixed sample using an internal standard. The P14R synthetic peptide (PPPPPPPPPPPPPPPR: SEQ ID NO: 47) was used as the internal standard. Figures 5 and 6 show the relative values ​​of each antibody in a mixed sample, with the value for a single sample set at 100. The quantitative accuracy of the quantitative analysis of antibodies individually and simultaneously was 6.7% (0.62%-15.9%, median 6.5%). The margin of error, according to the U.S. Food and Drug Administration (FDA) validation guidance standards, is within ±15%. The quantitative values ​​obtained by simultaneously subjecting multiple antibodies to mass spectrometry were confirmed to be highly accurate. This demonstrates that multiple antibodies can be simultaneously quantified by mass spectrometry, even in clinical samples obtained from living organisms.

[0096] <Experiment 4> The ratio of the detection intensity of one antibody to the detection intensity of other antibodies was calculated. Using a mixed sample in which all 21 monoclonal antibodies and Fc fusion proteins described in Experiment 3 were added to serum, the cps ratio of other antibodies to trastuzumab cps was calculated. The ratio of the detection intensity of one antibody to the detection intensity of other antibodies can also be calculated using antibodies other than trastuzumab as the standard. The antibody protease digestion method and mass spectrometry conditions were the same as in Experiment 3. The mixed sample was divided into four concentration points, and five independent analyses were performed for each concentration (N=5). These conditions meet FDA standards.

[0097] The ratio of cps of other antibodies to cps of trastuzumab (i.e., the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody) was shown to be constant regardless of the antibody concentration (Figures 7 to 9). Using this ratio (constant), it is possible to quantify antibodies other than trastuzumab using a calibration curve generated with trastuzumab. The cps ratio of one antibody to another antibody can be the average or median value of multiple experiments.

[0098] <Experiment 5> Antibodies (target antibodies) contained in clinical samples were quantified based on a calibration curve prepared using trastuzumab (reference antibody). The clinical samples were serum samples from humans administered ipilimumab (N = 71) and pembrolizumab (N = 95). Quantification was performed using the same method as described in Experiment 3. The calibration curve for trastuzumab was prepared using a dilution series method (trastuzumab was added to human serum to a concentration of 1000 μg / mL, and this was used as the calibration curve sample through a two-fold dilution series). The calibration curve for trastuzumab was corrected based on the ratio of the detection intensity of trastuzumab to that of ipilimumab calculated in Experiment 4. The concentration of ipilimumab was determined based on the corrected calibration curve. Using the same method, a calibration curve prepared using trastuzumab was corrected based on the ratio of the detection intensity of trastuzumab to the detection intensity of pembrolizumab, and the concentration of pembrolizumab was determined.

[0099] To confirm the reproducibility of the quantitative values, ipilimumab in the same clinical samples was quantified based on a standard curve prepared using ipilimumab. The ipilimumab standard curve was prepared using a dilution series method (ipilimumab was added to human serum to a concentration of 1000 μg / mL, and this was used as the standard curve sample through a two-fold dilution series). Pembrolizumab in clinical samples was also quantified based on a standard curve prepared using pembrolizumab using the same method.

[0100] The concentrations of ipilimumab quantified based on the trastuzumab calibration curve were compared with those quantified based on the ipilimumab calibration curve. Pearson product-moment correlation analysis of the values ​​obtained by the two methods yielded R 2 = 0.81, indicating that both methods have high reproducibility (Figure 10). Similarly, the concentration of pembrolizumab quantified based on the trastuzumab calibration curve was compared with the concentration of pembrolizumab quantified based on the pembrolizumab calibration curve. Pearson's product-moment correlation analysis of the values ​​obtained by the two methods yielded R 2 = 0.86, demonstrating high reproducibility for both (Figure 11). It was found that the concentrations of multiple target antibodies contained in clinical samples can be determined using the ratio of the detection intensity of the reference antibody to the detection intensity of the target antibody, relative to a single reference antibody.

[0101] <Experiment 6> Based on the results of Experiment 5, we investigated whether antibodies other than trastuzumab and Fc fusion proteins could be used as reference proteins (i.e., biological proteins). Specifically, antibodies (target antibodies) contained in clinical samples were quantified based on a calibration curve prepared using one of the following reference proteins. The clinical samples were serum samples from humans administered ipilimumab (N = 71) and pembrolizumab (N = 74). Quantification was performed using the same method as described in Experiment 3. A calibration curve for the reference protein was prepared using a dilution series method (collected human serum was spiked with the reference protein to a concentration of 1000 μg / mL, and this was used as the calibration curve sample through a two-fold dilution series). The calibration curve for the reference protein was corrected based on the ratio of the detection intensity of the reference protein to the detection intensity of ipilimumab, calculated using the same method as in Experiment 4. The concentration of ipilimumab was determined based on the corrected calibration curve. Using the same method, a calibration curve prepared using a reference protein was corrected based on the ratio of the detection intensity of the reference protein to the detection intensity of pembrolizumab, and the concentration of pembrolizumab was determined.

[0102] (Reference proteins used) Trastuzumab (Tra), Brentuximab Vedotin (Bre), Cetuximab (Cet), Rituximab (Rit), Infliximab (Ifx), Atezolizumab (Atz), Bevacizumab (Bev), Pembrolizumab (Pem), Eculizumab (Ecu), Mepolizumab (Mep), Tocilizumab (Toc), Avelumab (Ave), Durvalumab (Dyr), Ipilimumab (Ipi), Nivolumab (Niv), Ramucirumab (Ram), Adalimumab (Ada), Golimumab (Gol), Abatacept (Abt) and Etanercept (Etn).

[0103] To confirm the reproducibility of the quantitative values, ipilimumab in the same clinical samples was quantified based on a standard curve prepared using ipilimumab. The ipilimumab standard curve was prepared using a dilution series method (ipilimumab was added to human serum to a concentration of 1000 μg / mL, and this was used as the standard curve sample through a two-fold dilution series). Pembrolizumab in clinical samples was also quantified based on a standard curve prepared using pembrolizumab using the same method.

[0104] The concentrations of ipilimumab quantified based on the standard curve of the reference protein were compared with the concentrations of ipilimumab quantified based on the standard curve of ipilimumab (Figures 12, 14A, and 14B). Pearson product-moment correlation analysis of the values ​​obtained by the two methods revealed a significant difference in R for both reference proteins. 2 The values ​​were 0.99 or higher, demonstrating high reproducibility (Table 2).

[0105] [Table 2]

[0106] Similarly, the concentrations of pembrolizumab quantified based on the calibration curve of the reference protein were compared with the concentrations of pembrolizumab quantified based on the calibration curve of pembrolizumab (Figures 13, 15A, and 15B). Pearson's product-moment correlation analysis of the values ​​obtained by the two methods showed that R 2 The values ​​were 0.99 or higher, demonstrating high reproducibility (Table 3). These results demonstrate that the concentrations of multiple target antibodies contained in clinical samples can be determined using the ratio of the detection intensity of the reference protein to the detection intensity of the target antibody, relative to a single reference protein.

[0107] [Table 3]

[0108] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0109] (Section 1) In one embodiment, a method for analyzing an antibody comprises: digesting the biological protein and the target antibody with a protease; Mass spectrometry is performed on the digested peptide fragments of the biological protein and the target antibody; quantifying the target antibody based on a ratio between the detection intensity of the biological protein obtained by the mass spectrometry and the detection intensity of the target antibody; The digestion with the protease comprises: Immobilizing the biological protein and the target antibody within the pores of a carrier; and bringing the carrier into close proximity to microparticles having a diameter larger than the diameter of the pores and having the protease immobilized on the surface thereof, The biological protein has a domain that can be immobilized in the pore.

[0110] According to the method for analyzing an antibody described in item 1, the antibody can be quantified by mass spectrometry without preparing a calibration curve for the antibody to be measured.

[0111] (Section 2) 2. The method for analyzing an antibody according to claim 1, comprising: performing mass spectrometry on the biological protein having a predetermined concentration and the target antibody having a predetermined concentration; The method further includes determining in advance a ratio between the detection intensity of the biological protein and the detection intensity of the target antibody based on the results of the mass spectrometry.

[0112] If the ratio between the detection intensity of a biological protein (for example, a reference antibody) and the detection intensity of a target antibody is determined in advance, the target antibody can be quantified easily.

[0113] (Section 3) In the method of analyzing an antibody according to paragraph 1 or 2, the biological protein comprises a reference antibody or an Fc fusion protein.

[0114] Reference antibodies and Fc fusion proteins are suitable as biological proteins because they are widely available and readily available.

[0115] (Section 4) In the method of analyzing an antibody described in paragraph 3, the reference antibody is a monoclonal antibody.

[0116] Monoclonal antibodies are suitable as reference antibodies because they are readily available and their concentrations and amino acid sequences can be easily identified.

[0117] (Section 5) In the method of analyzing an antibody according to paragraph 3, the reference antibody is selected from the group consisting of trastuzumab, bevacizumab, cetuximab, rituximab, nivolumab, brentuximab, pembrolizumab, ipilimumab, mogamulizumab, ramucirumab, atezolizumab, durvalumab, avelumab, infliximab, tocilizumab, adalimumab, golimumab, mepolizumab, ustekinumab, and eculizumab.

[0118] The above antibodies are suitable as reference antibodies because they are highly versatile and readily available. (Section 6) In the method of analyzing an antibody according to paragraph 3, the Fc fusion protein is selected from the group consisting of abatacept, aflibercept and etanercept.

[0119] The above-mentioned Fc fusion proteins are suitable as biological proteins because they are highly versatile and easily available.

[0120] (Section 7) In the method for analyzing an antibody according to any one of items 1 to 6, the target antibody is an antibody contained in a biological sample.

[0121] According to the method for analyzing an antibody described in item 7, it is possible to quantify the target antibody contained in a biological sample collected from a subject.

[0122] (Section 8) In the method for analyzing an antibody according to any one of items 1 to 7, the target antibody comprises at least two types of antibodies.

[0123] The method for analyzing antibodies described in item 8 allows for the quantification of multiple target antibodies using one biological protein (e.g., a reference antibody) as a standard. Two or more types of antibodies can be quantified simultaneously.

[0124] (Section 9) In the method for analyzing an antibody according to any one of items 1 to 8, the target antibody includes an antibody produced in vivo.

[0125] According to the method for analyzing an antibody described in item 9, it is possible to quantify even an antibody produced in vivo, for which it is difficult to prepare a calibration curve.

[0126] (Section 10) In one embodiment, a method for analyzing an antibody comprises: digesting a biological protein and a sample containing at least two target antibodies with a protease; Mass spectrometry is performed on the digested peptide fragments of the biological protein and the target antibody; quantifying the target antibody based on a ratio between the detection intensity of the biological protein obtained by the mass spectrometry and the detection intensity of the target antibody; The biological protein is a monoclonal antibody.

[0127] According to the method for analyzing an antibody described in item 10, the antibody can be quantified by mass spectrometry without preparing a calibration curve using the antibody to be measured.

[0128] (Section 11) The kit according to one embodiment comprises: A kit for use in the method for analyzing an antibody according to any one of items 1 to 10, The biological protein; microparticles having the protease immobilized on their surfaces; and a carrier having the pores formed therein.

[0129] The kit described in item 11 allows the method described in item 1 to be carried out simply and easily. (Section 12) A program according to one aspect includes: A program used in the method for analyzing an antibody according to any one of items 1 to 10, The program receiving a mass spectrum output from a mass spectrometer; reading out from a storage unit a ratio between the detection intensity of the biological protein and the detection intensity of the target antibody; correcting the detected amount of the biological protein calculated from the mass spectrum; The processing device executes a process for calculating the amount of the target antibody.

[0130] According to the program described in item 12, the process of quantifying the target antibody can be easily carried out based on the ratio of the detection intensity of the biological protein to the detection intensity of the target antibody. [Explanation of symbols]

[0131] 10 Microparticle, 11 Spacer, 15 Protease, 20 Carrier, 21 Linker, 25 Antibody, 29 Pore.

Claims

1. digesting the biological protein and the target antibody with a protease; Mass spectrometry is performed on the digested peptide fragments of the biological protein and the target antibody; quantifying the target antibody based on a ratio between the detection intensity of the biological protein obtained by the mass spectrometry and the detection intensity of the target antibody; The digestion with the protease comprises: Immobilizing the biological protein and the target antibody within the pores of a carrier; and bringing the carrier into close proximity to microparticles having a diameter larger than the diameter of the pores and having the protease immobilized on the surface thereof, the biological protein has a domain that can be immobilized in the pore; The biological protein has a specified concentration, The targeting antibody comprises at least two antibodies; Methods for analyzing antibodies.

2. mass spectrometry of the biological protein having a predetermined concentration and the target antibody having a predetermined concentration; and determining a ratio of the detection intensity of the biological protein to the detection intensity of the target antibody based on the results of the mass spectrometry. The method for analyzing an antibody according to claim 1 , wherein the predetermined concentration of the biological protein is the same as or different from the predetermined concentration of the target antibody.

3. The method for analyzing an antibody according to claim 1 or claim 2, wherein the biological protein comprises a reference antibody or an Fc fusion protein.

4. The method for analyzing an antibody according to claim 3 , wherein the reference antibody is a monoclonal antibody.

5. 4. The method of claim 3, wherein the reference antibody is selected from the group consisting of trastuzumab, bevacizumab, cetuximab, rituximab, nivolumab, brentuximab, pembrolizumab, ipilimumab, mogamulizumab, ramucirumab, atezolizumab, durvalumab, avelumab, infliximab, tocilizumab, adalimumab, golimumab, mepolizumab, ustekinumab, and eculizumab.

6. The method for analyzing an antibody according to claim 3, wherein the Fc fusion protein is selected from the group consisting of abatacept, aflibercept and etanercept.

7. The method for analyzing an antibody according to claim 1 or 2, wherein the target antibody is an antibody contained in a biological sample.

8. The method for analyzing an antibody according to claim 1 or claim 2, wherein the target antibody comprises an antibody produced in vivo.

9. digesting the biological protein and the at least two target antibodies with a protease; Mass spectrometry is performed on the digested peptide fragments of the biological protein and the target antibody; quantifying the target antibody based on a ratio between the detection intensity of the biological protein obtained by the mass spectrometry and the detection intensity of the target antibody; The biological protein has a specified concentration, The biological protein is a monoclonal antibody. Methods for analyzing antibodies.

10. A program used in the method for analyzing an antibody according to claim 1 or 2, The program receiving a mass spectrum output from a mass spectrometer; reading out from a storage unit a ratio between the detection intensity of the biological protein and the detection intensity of the target antibody; correcting the detected amount of the biological protein calculated from the mass spectrum; A program that causes a processing device to execute a process for calculating the amount of the target antibody.

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