Method for analyzing antibody

Abstract

Provided is a novel antibody analysis method that does not require capture or immobilization through an Fc structure. Specifically provided is a method for preparing a peptide fragment, the method comprising: a substrate immobilization step in which a target substrate antibody 125 in a biological sample is immobilized in a pore 129 of a porous body 120 that has affinity for a Fab domain of the substrate antibody 125; and a digestion step in which the porous body 120 on which the substrate antibody 125 has been immobilized is brought into contact with fine particles 110 each having a surface on which a protease 115 has been immobilized, whereby the digestion of the substrate antibody 125 with the protease is performed and a peptide fragment of the Fab domain is produced. In the substrate immobilization step, the Fab domain of the substrate antibody 125 is immobilized in the pore 129 of the porous body 120, and the average particle diameter D11 of the fine particles 110 is larger than the average pore diameter D12 of the porous body 120.

Description

Antibody analysis methods

[0001] The present invention relates to a method for preparing peptide fragments by regioselectively cleaving antibody proteins using a protease, and an analytical method for detecting and quantifying antibodies by analyzing the peptide fragments prepared by this method using mass spectrometry.

[0002] Pharmacokinetics of pharmaceuticals is one of the most fundamental indicators of drug efficacy. Understanding its overall picture provides indicators of drug efficacy and toxicity, supporting efficient drug development.

[0003] The purpose of therapeutic drug monitoring, particularly concentration monitoring (therapeutic drug monitoring, or TDM), is to individually adjust drug doses according to each patient's pharmacokinetic characteristics. TDM enables the drug to reach effective concentrations more rapidly while minimizing drug toxicity. The main prerequisites for TDM are the large individual differences in exposure, the defined relationship between concentration and efficacy, and the availability of reliable and clinically feasible measurement methods. TDM is applied to small molecule drugs such as antibiotics, antiepileptic drugs, immunosuppressants, and anticancer drugs. Monoclonal antibodies, which are now widely used as antibody drugs, dramatically improve clinical outcomes in inflammatory and malignant diseases. Furthermore, in anticancer treatment, numerous studies have reported a correlation between higher circulating concentrations of monoclonal antibodies and better clinical outcomes, suggesting that TDM may be useful in treatments using monoclonal antibodies.

[0004] ELISA (Enzyme-Linked ImmunoSorbent Assay) has been the dominant method for quantifying proteins such as antibodies. However, ELISA has drawbacks, including the need to prepare specific antibodies for each protein to be detected and the inability to detect multiple proteins simultaneously.

[0005] Our group of inventors has discovered that by immobilizing both the monoclonal antibody to be measured and the protease capable of digesting it as a substrate on a solid phase, regioselective solid-phase-solid reaction of protease digestion of monoclonal antibodies is possible, and has succeeded in obtaining peptide fragments specific to individual monoclonal antibodies (Patent Documents 1-2 and Non-Patent Documents 1-8). This method is a mass spectrometry pretreatment method that performs selective protease digestion of monoclonal antibodies by contacting a porous body in which monoclonal antibodies are immobilized in the pores with nanoparticles on which proteases are immobilized in a liquid, and is a groundbreaking technology that allows for effective detection and quantification of the obtained peptide fragments by liquid chromatography-mass spectrometry (LC-MS). We have named this method the "nano-surface and molecular-orientation limited proteolysis (nSMOL) method."

[0006] International Publication No. 2015 / 033479, International Publication No. 2016 / 194114

[0007] 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.Iwamoto N et al., Development of the validated LCMS bioanalysis of Trastuzumab in human plasma using selective detection method for complementarity-determining regions of monoclonal antibody: nano-surface and molecular-orientation limited (nSMOL) proteolysis, Anal. Methods, 2015; 21: 9177-9183.Iwamoto N et al., Fully validated LCMS bioanalysis of Bevacizumab in human plasma using nano-surface and molecular-orientation limited (nSMOL) proteolysis, Drug Metabolism and Pharmacokinetics, 2016; 31: 46-50.Iwamoto N et al., Application of nano-surface and molecular-orientation limited proteolysis to LC-MS bioanalysis of cetuximab, Bioanalysis. 2016; 8(10):1009-20. doi: 10.4155. bio-2016-0018.Iwamoto N et al., Validated LC / MS Bioanalysis of Rituximab CDR Peptides Using Nano-surface and Molecular-Orientation Limited (nSMOL) Proteolysis., Biol Pharm Bull, 2016;39(7):1187-94. doi: 10.1248 / bpb.b16-00230.Iwamoto N et al. Validated LC-MS / MS analysis of immune checkpoint inhibitor Nivolumab in human plasma using a Fab peptide-selective quantitation method: nano-surface and molecular-orientation limited (nSMOL) proteolysis, J Chromatogr B Analyt Technol Biomed Life Sci; 2016; 1023-1024:9-16. doi: 10.1016 / j.jchromb.2016.04.038.Iwamoto N et al., Multiplex LCMS Bioanalysis of Brentuximab Vedotin, Rituximab and Cetuximab towards Therapeutic Drug Monitoring Application by Combined Calibration Curve Using Fab-Selective Limited Proteolysis nSMOL, Clin Pharmacol Biopharm 2016; 5:164. doi:10.4172 / 2167-065X.1000164.Iwamoto N et al., A rapid and universal liquid chromatograph-mass spectrometry-based platform, refmAb-Q nSMOL, for monitoring monoclonal antibody therapeutics, Analyst.2022 Sep 26;147(19):4275-4284. doi: 10.1039 / d2an01032a.

[0008] Thus, the inventors have established a highly reliable and clinically applicable measurement method for quantifying antibody drugs (monoclonal antibodies) in the blood by combining pretreatment techniques using the nSMOL method (i.e., preparation of peptide fragments specific to monoclonal antibodies) with LC-MS / MS analysis.

[0009] Bioanalysis of antibody drugs using LC-MS / MS analysis is attracting attention as an alternative quantitative method to ELISA. In particular, the nSMOL method is excellent in quantitative accuracy and reliability, and can be applied to most antibody drugs (those with an IgG structure), so it is used for the quantification of many IgG-like antibodies.

[0010] Our nSMOL method allows for the immobilization of antibody drugs within the pores of porous beads using protein A, which has affinity for the Fc region, a common structure of immunoglobulin (IgG)-like monoclonal antibodies. By digesting only the variable region of IgG using nanobeads immobilized with trypsin, and performing LC-MS / MS analysis of trypsin-digested peptides with sequences specific to each antibody drug, highly accurate quantification can be achieved.

[0011] In recent years, development has been progressing on next-generation novel antibody drugs that can replace IgG-like antibodies, namely antibody drugs that lack the Fc region, a common structural feature of IgG (fragment antibodies). These antibody drugs are smaller in molecular size than IgG-like antibody drugs and possess various structures aimed at achieving higher functionality. Examples of novel antibody drug structures include scFv (single-chain variable fragment), Fab, and F(ab'). 2 、 Examples include BiTEs (bi-specific T cell engagers), and both the structure and function as antibody drugs are becoming increasingly complex. With this diversification of antibody drugs, there is an urgent need to develop new measurement methods for antibody drugs that are difficult to measure using the known nSMOL method based on capture and immobilization by Fc structures.

[0012] Therefore, the object of the present invention is to provide a novel pretreatment method and analytical method that does not require capture and immobilization by an Fc structure.

[0013] One embodiment of the present invention is a method for preparing peptide fragments comprising: a substrate immobilization step of immobilizing a target substrate antibody in a biological sample within the pores of a porous material having affinity for the Fab domain of the substrate antibody; and a digestion step of contacting the porous material on which the substrate antibody is immobilized with fine particles on which a protease is immobilized on its surface to perform protease digestion of the substrate antibody and obtain peptide fragments of the Fab domain, wherein in the substrate immobilization step, the Fab domain of the substrate antibody is immobilized within the pores of the porous material, and the average particle size of the fine particles is larger than the average pore diameter of the porous material.

[0014] One embodiment of the present invention is a method for analyzing an antibody, comprising: a substrate immobilization step of immobilizing a target substrate antibody in a biological sample within the pores of a porous material having affinity for the Fab domain of the substrate antibody; a digestion step of contacting the porous material on which the substrate antibody is immobilized with fine particles on which a protease is immobilized on its surface to perform protease digestion of the substrate antibody and obtain a peptide fragment of the Fab domain; and an analysis step of analyzing the obtained peptide fragment of the Fab domain of the substrate antibody by mass spectrometry, wherein in the substrate immobilization step, the Fab domain of the substrate antibody is immobilized within the pores of the porous material, and the average particle size of the fine particles is larger than the average pore diameter of the porous material.

[0015] One embodiment of the present invention is a peptide fragment preparation kit for use in a method for preparing the above-mentioned peptide fragment or a method for analyzing the above-mentioned antibody, comprising: a porous body having pores on which the Fab domain of the substrate antibody can be immobilized; and fine particles on which the protease can be immobilized on the surface, wherein the average particle size of the fine particles is larger than the pore diameter of the porous body.

[0016] According to the present invention, antibody immobilization is performed without the need for capture and immobilization using an Fc structure, and the preparation of peptide fragments of the antibody is achieved. This makes it possible to analyze antibodies other than IgG-like antibodies that do not have an Fc region (fragment antibodies). In particular, it can contribute to pharmacokinetic analysis and therapeutic drug monitoring (TDM) of novel antibody drugs. Furthermore, it can contribute to pharmacokinetic analysis and concentration monitoring of novel antibody drug candidates during the development stage of novel antibody drugs.

[0017] Figure 1 is a schematic diagram illustrating the structure of an IgG-like antibody. Figure 2 is a schematic diagram illustrating an example of the structure of a fragment antibody lacking an Fc region. Figure 3 is a conceptual diagram illustrating the principle of regioselective cleavage during protease digestion of a fragment antibody lacking an Fc region. Figure 4 is a graph showing the LC-MS / MS analysis results of the monitor peptide QRPGQGLEWIGYINPSR (SEQ ID NO: 6) of the antibody drug Blinatumomab in Experimental Example 1. The peak intensity (vertical axis) of the monitor peptide QRPGQGLEWIGYINPSR (SEQ ID NO: 6) is shown for each of the following pretreatment methods: when Blinatumomab spiked in serum is reacted with Protein A beads and then immobilized with Protein L beads ("Protein A_L"), when immobilized with Protein L beads ("Protein L"), and when immobilized with Protein A beads ("Protein A"). Figure 5 shows the LC-MS / MS analysis results for the monitor peptide LLIYLASTLASGVPSR (SEQ ID NO: 1) of the antibody drug Brolucizumab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody spike (0 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 6 shows the LC-MS / MS analysis results for the monitor peptide LLIYLASTLASGVPSR (SEQ ID NO: 1) of the antibody drug Brolucizumab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 7 shows the LC-MS / MS analysis results for the monitor peptide LLIYLASTLASGVPSR (SEQ ID NO: 1) of the antibody drug Brolucizumab in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL).Figure 8 shows the LC-MS / MS analysis results for the monitor peptide GLEWVGFIDPDDDPYYATWAK (SEQ ID NO: 2) of the antibody drug Brolucizumab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody drug spike (0 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 9 shows the LC-MS / MS analysis results for the monitor peptide GLEWVGFIDPDDDPYYATWAK (SEQ ID NO: 2) of the antibody drug Brolucizumab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody drug spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 10 shows the LC-MS / MS analysis results for the monitor peptide GLEWVGFIDPDDDPYYATWAK (SEQ ID NO: 2) of the antibody drug Brolucizumab in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL). Figure 11 shows the LC-MS / MS analysis results for the monitor peptide FTFSLDTSK (SEQ ID NO: 3) of the antibody drug Certolizumab pegol in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody drug spike (0 μg / mL). The vertical axis represents peak intensity and the horizontal axis represents retention time (min). Figure 12 shows the LC-MS / MS analysis results for the monitor peptide FTFSLDTSK (SEQ ID NO: 3) of the antibody drug Certolizumab pegol in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody drug spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 13 shows the LC-MS / MS analysis results for the monitor peptide FTFSLDTSK (SEQ ID NO: 3) of the antibody drug Certolizumab pegol in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL).Figure 14 shows the LC-MS / MS analysis results for the monitor peptide LLIYDASNLVSGIPPR (SEQ ID NO: 4) of the antibody drug Blinatumomab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody drug spike (0 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 15 shows the LC-MS / MS analysis results for the monitor peptide LLIYDASNLVSGIPPR (SEQ ID NO: 4) of the antibody drug Blinatumomab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody drug spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 16 shows the LC-MS / MS analysis results for the monitor peptide LLIYDASNLVSGIPPR (SEQ ID NO: 4) of the antibody drug Blinatumomab in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL). Figure 17 shows the LC-MS / MS analysis results for the monitor peptide FSGSGSGTDFTLNIHPVEK (SEQ ID NO: 5) of the antibody drug Blinatumomab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody drug spike (0 μg / mL). The vertical axis represents peak intensity and the horizontal axis represents retention time (min). Figure 18 shows the LC-MS / MS analysis results for the monitor peptide FSGSGSGTDFTLNIHPVEK (SEQ ID NO: 5) of the antibody drug Blinatumomab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody drug spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 19 shows the LC-MS / MS analysis results for the monitor peptide FSGSGSGTDFTLNIHPVEK (SEQ ID NO: 5) of the antibody drug Blinatumomab in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL).Figure 20 shows the LC-MS / MS analysis results for the monitor peptide QRPGQGLEWIGYINPSR (SEQ ID NO: 6) of the antibody drug Blinatumomab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody spike (0 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 21 shows the LC-MS / MS analysis results for the monitor peptide QRPGQGLEWIGYINPSR (SEQ ID NO: 6) of the antibody drug Blinatumomab in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 22 shows the LC-MS / MS analysis results for the monitor peptide QRPGQGLEWIGYINPSR (SEQ ID NO: 6) of the antibody drug Blinatumomab in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL). Figure 23 shows the LC-MS / MS analysis results for the monitor peptide LIYYSWAQGDFQK (SEQ ID NO: 7) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody reagent spike (0 μg / mL). The vertical axis represents peak intensity and the horizontal axis represents retention time (min). Figure 24 shows the LC-MS / MS analysis results for the monitor peptide LIYYSWAQGDFQK (SEQ ID NO: 7) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody drug spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 25 shows the LC-MS / MS analysis results for the monitor peptide LIYYSWAQGDFQK (SEQ ID NO: 7) of the antibody drug Tebentafusp in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL).Figure 26 shows the LC-MS / MS analysis results for the monitor peptide GDIAEGYSVSR (SEQ ID NO: 8) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody spike (0 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 27 shows the LC-MS / MS analysis results for the monitor peptide GDIAEGYSVSR (SEQ ID NO: 8) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 28 shows the LC-MS / MS analysis results for the monitor peptide GDIAEGYSVSR (SEQ ID NO: 8) of the antibody drug Tebentafusp in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL). Figure 29 shows the LC-MS / MS analysis results for the monitor peptide VSATFWQDPR (SEQ ID NO: 9) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody drug spike (0 μg / mL). The vertical axis represents peak intensity and the horizontal axis represents retention time (min). Figure 30 shows the LC-MS / MS analysis results for the monitor peptide VSATFWQDPR (SEQ ID NO: 9) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody drug spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 31 shows the LC-MS / MS analysis results for the monitor peptide VSATFWQDPR (SEQ ID NO: 9) of the antibody drug Tebentafusp in Experimental Example 2, and shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL). Figure 32 shows the LC-MS / MS analysis results for the monitor peptide AKPVTQIVSAEAWGR (SEQ ID NO: 10) of the antibody drug Tebentafusp in Experimental Example 2, and is an MRM chromatogram of the monitor peptide with no spike in the antibody drug (0 μg / mL).The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 33 shows the LC-MS / MS analysis results for the monitor peptide AKPVTQIVSAEAWGR (SEQ ID NO: 10) of the antibody drug Tebentafusp in Experimental Example 2, and is an MRM chromatogram of the monitor peptide with an antibody drug spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 34 shows the LC-MS / MS analysis results for the monitor peptide AKPVTQIVSAEAWGR (SEQ ID NO: 10) of the antibody drug Tebentafusp in Experimental Example 2, and shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL). Figure 35 shows the LC-MS / MS analysis results for the monitor peptide GLVHLILIR (SEQ ID NO: 11) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody spike (0 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 36 shows the LC-MS / MS analysis results for the monitor peptide GLVHLILIR (SEQ ID NO: 11) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with an antibody spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 37 shows the LC-MS / MS analysis results for the monitor peptide GLVHLILIR (SEQ ID NO: 11) of the antibody drug Tebentafusp in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL). Figure 38 shows the LC-MS / MS analysis results for the monitor peptide SSSLLITASR (SEQ ID NO: 12) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with no antibody drug spike (0 μg / mL). The vertical axis represents peak intensity and the horizontal axis represents retention time (min).Figure 39 shows the LC-MS / MS analysis results for the monitor peptide SSSLLITASR (SEQ ID NO: 12) of the antibody drug Tebentafusp in Experimental Example 2. It is an MRM chromatogram of the monitor peptide with a spike in the antibody drug (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 40 shows the LC-MS / MS analysis results for the monitor peptide SSSLLITASR (SEQ ID NO: 12) of the antibody drug Tebentafusp in Experimental Example 2. It shows a calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL).

[0018] The method for preparing peptide fragments according to this embodiment comprises: a substrate immobilization step in which a target substrate antibody in a biological sample is immobilized in the pores of a porous material having affinity for the Fab domain of the substrate antibody; and a digestion step in which the porous material on which the substrate antibody is immobilized is brought into contact with microparticles on which a protease is immobilized on the surface, and the substrate antibody is protease digested to obtain peptide fragments of the Fab domain, wherein in the substrate immobilization step the Fab domain of the substrate antibody is immobilized in the pores of the porous material, and the average particle size of the microparticles is larger than the average pore diameter of the porous material.

[0019] Furthermore, the antibody analysis method according to this embodiment includes: a substrate immobilization step of immobilizing a target substrate antibody in a biological sample within the pores of a porous material having affinity for the Fab domain of the substrate antibody; a digestion step of bringing the porous material on which the substrate antibody is immobilized into contact with microparticles on which a protease is immobilized on the surface, thereby performing protease digestion of the substrate antibody to obtain a peptide fragment of the Fab domain; and an analysis step of analyzing the obtained peptide fragment of the Fab domain of the substrate antibody by mass spectrometry, wherein in the substrate immobilization step, the Fab domain of the substrate antibody is immobilized within the pores of the porous material, and the average particle size of the microparticles is larger than the average pore diameter of the porous material.

[0020] [Substrate Antibody] In this specification, the substrate antibody is the target of cleavage / analysis, from which peptide fragments are prepared. While typical antibodies have an Fc region, which is a common structure of immunoglobulin IgG, in this embodiment, fragment antibodies lacking an Fc region are primarily intended.

[0021] Here, we will explain the structure of the antibody with reference to the diagram. Figure 1 is a schematic diagram illustrating the structure of an IgG-like antibody.

[0022] IgG-like antibodies have two heavy chains (H chains) and two light chains (L chains). One light chain and one heavy chain are linked by a disulfide bond to form a heterodimer, and these two heterodimers are further linked by two disulfide (S-S) bonds to form a "Y" shaped heterotetramer (see Figure 1). IgG-like antibodies have one Fc (Fragment, crystallizable) domain consisting of the heavy chain and two Fab (Fragment, antigen binding) domains consisting of the heavy and light chains, and the Fc domain and Fab domain are connected via a hinge.

[0023] The Fc domain of IgG-like antibodies primarily functions as an effector, inducing reactions after the antibody binds to the antigen. Most antibodies derived from the same species share a common amino acid sequence in the Fc domain. On the other hand, the Fab domain has the function of binding to the antigen at its tip (N-terminal side). The N-terminal portion of the Fab domain exhibits diverse amino acid sequence variations to enable binding to a variety of antigens. This region is called the variable region (V region), with the variable region of the light chain being called the VL region and the variable region of the heavy chain being called the VH region. The Fab domain and Fc domain other than the V region are regions with little change in amino acid sequence and are called the constant region (C region). The constant region of the light chain is called the CL region, and the constant region of the heavy chain is called the CH region. The CH region is further divided into three regions: CH1, CH2, and CH3. The heavy chain's Fab domain consists of a VH region and a CH1 region, while the heavy chain's Fc domain consists of CH2 and CH3. The hinge region is located between CH1 and CH2.

[0024] The specificity of an antibody (i.e., its ability to specifically bind to an antigen) is determined by the combination of amino acid sequences in the V region. Both the light and heavy chains have three complementarity-determining regions (CDRs) within the V region of the Fab domain. CDRs, also known as hypervariable regions, have different amino acid sequences for each type of antibody. Because antibodies have three CDRs in both the light and heavy chains (a total of six types of CDRs), they exhibit versatility, allowing them to bind to various antigens. In other words, CDRs are characteristic regions of an antibody, and by identifying the amino acid sequence of its CDR, the antibody can be identified.

[0025] As mentioned above, the Fab domain and Fc domain of an antibody are connected via a hinge. Papain, a type of protease, cleaves this hinge, so digestion of the antibody with papain produces two Fab domains and one Fc domain. Pepsin, another type of protease, cleaves the Fc domain side (C-terminal side) of the two disulfide bonds in the hinge, so digestion with pepsin produces an F(ab') domain where the two Fab domains are joined. 2 A domain and numerous Fc domain fragments are produced.

[0026] In contrast to IgG-like antibodies that possess an Fc region, fragment antibodies lacking an Fc region have been developed. Figure 2 is a schematic diagram illustrating an example of the structure of a fragment antibody lacking an Fc region.

[0027] Figure 2 shows the structure of scFv (single-chain variable fragment) as an example of a fragment antibody structure that does not have an Fc region. This antibody is mainly composed of only the variable region (V region) of the Fab domain, with the N-terminal side of the variable region VL of one light chain and the C-terminal side of the variable region VH of one heavy chain linked together. The specificity of the antibody (i.e., its specific binding ability to the antigen) is determined by the combination of amino acid sequences in the V region. Both the light chain and the heavy chain have three complementarity-determining regions (CDRs) within the variable region VL of the light chain and the variable region VH of the heavy chain, respectively. CDRs are also called hypervariable regions, and their amino acid sequences differ for each type of antibody. Because an antibody has three CDRs each in the light chain VL and heavy chain VH (a total of six types of CDRs), it exhibits diversity that allows it to bind to various antigens. In other words, CDRs are regions that characterize antibodies, and by identifying the amino acid sequence of the antibody's CDRs, the antibody can be identified.

[0028] Examples of fragment antibody structures include scFv (single-chain variable fragment), Fab, F(ab'), and F(ab'). 2 、 BiTEs (bi-specific T cell engagers), bsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), DNL- (Fab) 3 (dock-and-lock trivalent Fab), sdAb (single-domain antibody), bssAb (bispecific single-domain antibody), Bispecific F (mab') 2 These are some examples. Such fragment antibodies are being developed as antibody drugs with enhanced functionality for the treatment of various diseases.

[0029] The type of the matrix antibody (fragment antibody) is not particularly limited, but from the viewpoint of performing site-selective cleavage, those having a larger molecular diameter than proteases are preferred. Note that the matrix antibody may be a protein complex. The molecular diameter can be obtained from various documents and databases, etc., as numerical values determined based on structural analysis by X-ray, NMR, etc. Also, the molecular diameter can be determined by small-angle X-ray scattering, etc., and can also be approximately determined from the molecular weight. There are various structures for fragment antibodies that do not have an Fc region, and their molecular sizes are diverse. Note that the molecular size of an IgG-like antibody having an Fc region is, for example, about 14.5 nm.

[0030] Figure 3 is a conceptual diagram for explaining the principle of site-selective cleavage during protease digestion of a fragment antibody that does not have an Fc region.

[0031] Referring to Figure 3, as an example of the matrix antibody 125 to be cleaved, a single-chain scFv antibody that does not have an Fc region is depicted. The single-chain scFv antibody is immobilized in the pores 129 of the protein L porous body 120 by the Fab region (Kappa light chain), and the porous body 120 on which the scFv antibody is immobilized and the fine particles 110 (for example, FG beads Trypsin) on which the protease 115 is immobilized on the surface are brought into contact with each other in a liquid. At that time, the protease 115 (for example, trypsin) is immobilized on the surface of the fine particles 110 via the spacer 111. The porous body 120 has a large number of pores 129 (only two pores are depicted in the figure), and the matrix antibody 125 is immobilized in the pores 129 via the linker molecule 121 by the Fab region (Kappa light chain). Thus, in this embodiment, both the protease 115 and the matrix antibody 125 are immobilized on the solid phase in a minute region, and protease digestion is performed by the contact between the solid phases.

[0032] The average particle size D11 of the microparticles 110 is larger than the average pore diameter D12 of the porous body 120. Therefore, the microparticles 110 can access the vicinity of the surface layer of the pores 129, but cannot access the deep part of the pores 129. Along with this, the protease 125 immobilized on the surface of the microparticles 110 also cannot access the deep part of the pores 129. In FIG. 3, the dotted line near the pores 129 represents the boundary of the region accessible to the protease 115.

[0033] Thus, the access of the protease 115 to the substrate antibody 125 in the pores 129 is position-specifically restricted, and the relative access probability to the opening side of the pores 129 is increased. Thereby, the substrate antibody 125 can be position-selectively protease-digested to obtain peptide fragments.

[0034] In this embodiment, an example of the substrate antibody is a monoclonal antibody. Generally, the heavy chain and light chain of a monoclonal antibody each consist of a constant region and a variable region. The constant region has an amino acid sequence common to most antibodies derived from the same species. On the other hand, in the variable region, there are three complementarity-determining regions (CDRs) each (CDR1, CDR2, CDR3). The three-dimensional structure defined by these regions is involved in the specific binding to the antigen, and thereby an antigen-antibody complex is formed.

[0035] As a result of the three-dimensional structure analysis of the antibody, it has been confirmed that the CDR regions involved in the specific binding to the antigen are located almost on the outside of the antibody molecule. Among them, the CDR2 region is located the most outside and is optimal as the target for the restricted protease digestion of this embodiment. For antibodies clinically used, their entire amino acid sequence and the sequence of the CDRs are disclosed. Also, those skilled in the art can identify the CDR regions based on the amino acid sequence information of the antibody.

[0036] In this embodiment, as the antibody to be analyzed, antibodies with an IgG-like structure are not excluded, but antibodies without an IgG-like structure, that is, fragment antibodies without an Fc region, are mainly intended. Examples of the structure of the fragment antibody include scFv (single-chain variable fragment), Fab, F(ab’), F(ab’)2 、 BiTEs (bi-specific T cell engagers), bsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), DNL- (Fab) 3 (dock-and-lock trivalent Fab), sdAb (single-domain antibody), bssAb (bispecific single-domain antibody), Bispecific F (mab') 2 Examples include brolucizumab, certolizumab pegol, blinatumomab, and teventafusp. However, these are merely examples, and this embodiment can also be applied to newly developed monoclonal fragment antibodies. Depending on the type of fragment antibody to be analyzed, it is advisable to appropriately select a monitor peptide from the peptide fragments obtained by protease digestion.

[0037] The antibodies exemplified above are used as antibody drugs (molecular targeted therapies), and quantitative measurement of antibody concentrations in the blood is required in clinical trials and actual treatments.

[0038] Blood samples include whole blood, plasma, and serum. Blood samples can be prepared by processing whole blood collected from a subject as appropriate. There are no particular limitations on the processing performed when preparing blood samples from collected whole blood; any clinically acceptable processing may be performed. For example, centrifugation may be performed. In addition, blood samples used in the measurement process may be stored at low temperatures, such as by freezing, at an intermediate or final stage of the preparation process.

[0039] In addition to blood samples, other biological samples such as cerebrospinal fluid (CSF), urine, body secretions, saliva, and sputum, as well as feces, may be used depending on the purpose and the site of the disease. In this embodiment, biological samples such as blood samples are discarded without being returned to the original subject. In the development stage of novel antibody drugs, biological samples may include not only human samples but also animal samples.

[0040] According to the method of this embodiment, the Fab domain of a monoclonal fragment antibody, particularly the CDR2 region, can be regioselectively digested with protease. By detecting the resulting peptide fragment of the CDR2 region by mass spectrometry, the antibody can be identified or quantitatively detected. Of course, the monitor peptide is not limited to peptide fragments of the CDR2 region. As described above, the monitor peptide should be appropriately selected from the peptide fragments obtained by protease digestion, depending on the type of fragment antibody to be analyzed.

[0041] In biological samples such as blood samples, antibodies with an Fc structure (IgG-like antibodies) are present in addition to the target substrate antibody. When such endogenous antibodies are present, it may be impossible to distinguish whether the obtained peptide fragment originates from the target substrate antibody or from the endogenous antibodies. Therefore, it is recommended to perform an endogenous antibody removal step before the substrate fixation step to remove antibodies with an Fc structure other than the substrate antibody (IgG-like antibodies) present in the biological sample.

[0042] The endogenous antibody removal step may involve, for example, capturing and removing endogenous IgG-like antibodies in a biological sample such as a blood sample using protein A beads based on their Fc structure. The target substrate antibody contained in the sample solution after the endogenous antibody has been removed may be reacted with protein L beads to immobilize the substrate antibody onto the protein L beads.

[0043] [Porous material] In this embodiment, the porous material is not particularly limited as long as it is a carrier that has affinity for the Fab domain of the substrate antibody and has a large number of pores. Activated carbon, porous membranes, porous resin beads, metal particles, etc., can be used. Among these, materials capable of site-specific binding of the antibody are preferred.

[0044] The shape of the pores is not particularly limited. Furthermore, materials with pores penetrating the porous body, such as porous membranes, can also be used. The size of the pores in the porous body is not particularly limited, and it is preferable to determine it considering the molecular size of the antibody, etc., so that when the antibody is immobilized, the sites to be selectively digested are located near the surface of the pores. The average pore diameter D12 of the porous body is appropriately set, for example, in a range of about 10 nm to 200 nm, and smaller than the average particle size D11 of the fine particles. For example, the average pore diameter D12 of the porous body is preferably about 20 nm to 200 nm, more preferably in the ranges of 30 nm to 150 nm, 40 nm to 120 nm, and 50 nm to 100 nm, and particularly preferably about 100 nm.

[0045] In this embodiment, a porous material is preferably used in which linker molecules that interact site-specifically with the antibody are immobilized within the pores of the porous material. Preferably, the linker molecule is protein L, which binds site-specifically to the Fab domain fragment of the antibody. By using a porous material in which these linker molecules are immobilized within the pores, the Fab domain of the antibody is immobilized within the pores, and the variable region of the Fab domain tends to be located near the surface of the pores. In this way, the orientation of the antibody within the pores is controlled, enabling site-selective digestion of the Fab domain, particularly CDR2, by proteases.

[0046] The size of the linker molecule is selected so that the selective cleavage site of the antibody is located near the surface of the pore. The molecular size of the linker molecule when bound to the antibody is preferably about 0.5 to 1.5 times the pore diameter of the porous material, more preferably about 0.6 to 1.2 times, even more preferably about 0.7 to 1.1 times, and particularly preferably about 0.8 to 1 time. When the linker molecule is not fixed to the porous material and the antibody is directly bound to the pore, it is preferable that the molecular diameter of the antibody and the pore diameter of the porous material satisfy the above relationship.

[0047] In this embodiment, specifically, Protein L beads (manufactured by Tosoh Biosciences) and KaapaFabSelect resin (manufactured by Cytiva) can be used as materials with affinity for the κ light chain of Fab, and LambdaFabSelect resin (manufactured by Cytiva) can be used as materials with affinity for the λ light chain of Fab. Of course, various other carriers with affinity for the Fab domain of the substrate antibody can also be used.

[0048] [Immobilization of Antibodies into Porous Materials] In this embodiment, the method for immobilizing substrate antibodies into the pores of a porous material is not particularly limited, and an appropriate method can be adopted depending on the characteristics of the antibody, the porous material, or the linker molecule. For example, when immobilizing antibodies into a porous material in which protein L is immobilized in the pores, the antibodies can be easily immobilized into the pores by mixing a suspension of the porous material with a solution containing the antibody.

[0049] The ratio of porous material to substrate antibody can be appropriately set depending on the purpose. For example, when performing quantitative analysis of antibodies, it is desirable that almost the entire amount of target antibody in the sample be immobilized on the porous material. Therefore, it is preferable to set the ratio so that the amount of porous material is in excess of the estimated antibody content in the sample.

[0050] It is preferable that the substrate antibody binds to the Fab domain in a site-specific manner within the pores of the porous material. Binding via a linker molecule allows the substrate antibody to bind site-specifically. Site-specific binding of the substrate antibody makes the areas other than the binding site more easily digested by proteases.

[0051] After immobilizing the substrate antibody onto the porous material, it is preferable to perform a washing operation as appropriate so that mainly only the substrate antibody adheres to the protease digestion in the next step. The washing and purification operations may be carried out by methods known to those skilled in the art.

[0052] [Protease] In this embodiment, the protease recognizes the amino acid sequence of the substrate antibody and selectively cleaves specific bonds in specific sequences. The protease cleaves the substrate antibody Fab domain, which is immobilized within the pores of the porous material, at specific amino acid sequence sites to obtain peptide fragments of the Fab domain.

[0053] Examples of proteases are not particularly limited, but include trypsin (cleaves peptides at the C-terminus of basic amino acid residues (Arg and Lys)), lysyl endopeptidase (cleaves peptides at the C-terminus of Lys residues), arginine endopeptidase (cleaves peptides at the C-terminus of Arg residues), chymotrypsin (cleaves peptides at the C-terminus of aromatic amino acid residues (Phe, Tyr, and Trp)), V8 protease (cleaves peptides at the C-terminus of Glu residues), aspartate N-terminal protease (cleaves peptides at the N-terminus of Asp), pepsin, papain, etc. These can be appropriately selected depending on the substrate antibody. For example, proteases having the functions of trypsin, Lys-C, Asp-N, and Glu-C can be preferably selected. Two or more proteases can also be used in combination.

[0054] When using peptide fragments of substrate antibodies after protease digestion as sample material for mass spectrometry, it is preferable to use a protease that exhibits minimal autodigestion and high selectivity for the cleavage sequence. When using commercially available proteases, it is preferable to use mass spectrometry grade or sequencing grade proteases. For example, native trypsins derived from living organisms are known to have low specificity of the cleavage site because they produce pseudotrypsins that exhibit chymotrypsin-like activity through autodigestion. Therefore, mass spectrometry grade trypsins are commercially available in which the lysine residues of the trypsin have been reduced-methylated to increase resistance to autodigestion.

[0055] To enhance the regioselectivity of substrate antibody digestion by protease, it is important to limit the region accessible by the protease to the substrate antibody. Therefore, the molecular diameter of the protease is preferably smaller than that of the substrate antibody. More specifically, the molecular diameter of the protease is preferably 10 nm or less, more preferably 8 nm or less, even more preferably 6 nm or less, and particularly preferably 5 nm or less. For reference, proteins with a molecular weight of about 30 kDa, such as trypsin and lysyl endopeptidase, have a molecular diameter of about 4 nm.

[0056] Among the proteases mentioned above, trypsin is particularly preferred in this embodiment. As described above, trypsin has a small molecular diameter and its active site is located inside the molecule. Therefore, the region in which the active site can access the substrate antibody is limited, thereby improving the regioselectivity of protease digestion.

[0057] In proteomic analysis research, mixed digestion with trypsin and lysyl endopeptidase has recently attracted attention as a technique to improve the recovery rate of peptide fragments. This is thought to be because trypsin has the characteristic of progressively degrading the reaction from the outside of the three-dimensional structure, while lysyl endopeptidase mainly cleaves the hinge region of the antibody first. In contrast, to suppress the cleavage of the hinge region of the antibody and selectively cleave the Fab domain (more preferably the V region of the Fab domain), in this embodiment, it is preferable to use trypsin alone or, when used in combination with lysyl endopeptidase, etc., to ensure that the amount of trypsin in the total protease is 90% or more.

[0058] [Microparticles] In this embodiment, microparticles are used to control the access of proteases to antibodies immobilized in the pores of the porous material by immobilizing proteases on their surface. Therefore, the average particle size D11 of the microparticles is made larger than the average pore diameter D12 of the porous material so that they do not penetrate deep into the pores of the porous material.

[0059] The shape of the microparticles is not particularly limited, but spherical microparticles are preferred from the viewpoint of uniformly facilitating protease access to the pores of the porous material. Furthermore, it is preferable that the microparticles have a uniform average particle size.

[0060] The average particle size D11 of the fine particles is preferably in the range of 50 nm to 500 nm, more preferably 1.2 times or more the average pore diameter D12 of the porous material, even more preferably 1.5 times or more, and particularly preferably 1.8 times or more, for example, about 2 times. When the average pore diameter D12 of the porous material is about 30 to 150 nm, the average particle size D11 of the fine particles is preferably 100 nm or more, and more preferably 150 nm or more. When the average pore diameter D12 of the porous material is about 50 nm to 100 nm, the average particle size D11 of the fine particles is preferably 120 nm or more, more preferably 150 nm or more, and particularly preferably 170 nm or more. From the viewpoint of improving the digestion efficiency by protease, the upper limit of the average particle size D11 of the fine particles is preferably 500 nm or less, and even more preferably 300 nm or less.

[0061] As long as the microparticles can immobilize the above protease on their surface, the material is not particularly limited, and metals, resins, etc. are appropriately used. Further, those in which the metal surface is coated with resin, those in which the resin surface is coated with metal, etc. can also be used.

[0062] As the type of microparticles, magnetic microparticles that can be dispersed or suspended in an aqueous medium and can be easily recovered from the dispersion or suspension by applying a magnetic field are preferable. Further, magnetic microparticles whose surface is coated with an organic polymer are more preferable in that aggregation hardly occurs. Examples of the base material of the magnetic microparticles include ferromagnetic alloys such as iron oxide [magnetite (Fe 3 O 4 ), maghemite (γ-Fe 2 O 3 )], ferrite (Fe / M 3 O 4 ), etc. In ferrite (Fe / M 3 O 4 ), M means a metal ion that can form a magnetic metal oxide together with iron ions, and typically Co 2+ , Ni 2+ , Mn 2+ , Mg 2+ , Cu 2+ , Ni 2+ , etc. are used. Examples of the organic polymer for coating the magnetic microparticles include polyglycidyl methacrylate (polyGMA), a copolymer of GMA and styrene, polymethyl methacrylate (PMMA), polyacrylate methyl (PMA), etc. Specific examples of the magnetic nanobeads coated with an organic polymer include FG beads, SG beads, Adembeads, nanomag, etc. As a commercially available product, for example, FG beads manufactured by Tamagawa Seiki Co., Ltd. (polymer magnetic microparticles with a particle size of about 200 nm in which ferrite particles are coated with polyglycidyl methacrylate (polyGMA)) are preferably used.

[0063] The above-mentioned microparticles are preferably modified with a spacer molecule capable of binding to proteases in order to suppress the adsorption of nonspecific proteins and to selectively immobilize proteases. By immobilizing proteases via the spacer molecule, the detachment of proteases from the microparticle surface is suppressed, and the regioselectivity of protease digestion is enhanced. Furthermore, by adjusting the molecular size of the spacer, it is possible to selectively allow proteases to access desired positions on the antibody, thereby enhancing regioselectivity.

[0064] The spacer is preferably capable of binding to the protease and not inactivating it. From the viewpoint of controlling the access range of the protease immobilized on the surface of the microparticles, the spacer is preferably small in 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.

[0065] Spacer molecules capable of immobilizing proteases at the above molecular diameter are preferably non-protein molecules, and preferably molecules having functional groups such as amino groups, carboxyl groups, ester groups, epoxy groups, tosyl groups, hydroxyl groups, thiol groups, aldehyde groups, maleimide groups, succinimide groups, azide groups, biotin, avidin, or chelates at their terminals. For example, for the immobilization of trypsin, spacer molecules having activated ester groups are preferred. Furthermore, among the spacer molecules, hydrophilic molecules such as polyethylene glycol and its derivatives, polypropylene glycol and its derivatives, polyacrylamide and its derivatives, polyethyleneimine and its derivatives, poly(ethylene oxide) and its derivatives, and poly(ethylene terephthalic acid) and its derivatives can be used for the spacer arm portion other than the functional groups mentioned above.

[0066] Microparticles surface-modified with such spacer molecules are also commercially available and can be used. For example, microparticles modified with spacer molecules having an ester group activated with N-hydroxysucciimide (active ester group) are commercially available under the trade name "FG beads NHS" (Tamagawa Seiki Co., Ltd.). The particle size of FG beads NHS is approximately 200 nm ± 20 nm, making them very homogeneous as microparticles.

[0067] [Immobilization of Protease onto Microparticles] The method for immobilizing protease on the surface of microparticles is not particularly limited, and an appropriate method can be adopted depending on the characteristics of the protease and the microparticles (or the spacer molecule that modifies the surface of the microparticles). For example, when immobilizing protease on the surface of microparticles modified with spacers, the protease can be immobilized on the surface of the microparticles by mixing a suspension of microparticles with a solution containing protease. An amine coupling method between the microparticles and the protease via the functional group of the above-mentioned spacer molecule is preferred. For example, the carboxyl group surface-modified on the microparticles can be esterified with N-hydroxysuccinimide (NHS) to form an activated ester group, to which the amino group of the protease can be bonded. In this coupling reaction, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), N,N'-dicyclohexylcarbodiimide (DCC), and bis(2,6-diisopropylphenyl) This can be carried out in the presence of a carbodiimide coupling agent such as carbodiimide (DIPC). Alternatively, the amino groups of a protease may be attached to the surface-modified amino groups of the fine particles using crosslinking agents such as glutaraldehyde, bifunctional succinimide, bis(sulfosuccinimidyl)sverate (BS3), sulfonyl chloride, maleimide, or pyridyl disulfide.

[0068] The coupling method between microparticles and proteases via the functional groups of spacer molecules can be carried out in a simple operation by adding a protease solution to a suspension of microparticles and mixing and stirring under certain conditions.

[0069] It is preferable to immobilize the protease on the surface of the microparticles and then inactivate the active portion of the protease that is not bound to the microparticle surface.

[0070] In this embodiment, commercially available beads with trypsin immobilized on the phase may be used. For example, Trypsin-immobilized FG beads, Trypsin-immobilized beads (Sepharose beads, agarose beads, etc.) may be used.

[0071] [Protease Digestion] In this embodiment, the antibody Fab domain is protease-digested by contacting a porous body on which a substrate antibody is immobilized with fine particles on which a protease is immobilized on its surface, thereby producing peptide fragments. The contact is preferably carried out in a liquid. Here, "liquid" means that the substrate (solid phase) and the enzyme (solid phase) are in contact in a liquid phase, and also refers to an aqueous medium suitable for the protease digestion reaction.

[0072] The conditions for protease digestion in this embodiment are not particularly limited, and conditions similar to those for general protease digestion can be appropriately adopted. For example, it is preferable to incubate the protease in a buffer solution adjusted to near the optimal pH of the protease at a temperature of approximately 37°C for about 4 to 20 hours.

[0073] The mixing ratio of the porous body on which the substrate antibody is immobilized and the microparticles on which the protease is immobilized on the surface is not particularly limited and should be set so that the amount of protease corresponds to the amount of substrate antibody. In this embodiment, since the access between the substrate antibody and the protease is physically restricted by the combination of the porous body and the microparticles, it is preferable to use a larger amount of protease compared to general protease digestion. For example, a substrate antibody:protease ratio of approximately 30:1 to 3:1 is preferred, approximately 15:1 to 4:1 is more preferred, and approximately 10:1 to 5:1 is even more preferred.

[0074] In the method of this embodiment, protease digestion is performed while the antibody remains immobilized on the porous material. Since the peptide fragments produced by protease digestion are present in the liquid phase, the target peptide fragments can be obtained regioselectively without antibody elution or denaturation. According to the method of this embodiment, peptide fragments can be recovered regioselectively with simpler operations compared to conventional methods.

[0075] Next, the target peptide fragment obtained by protease digestion is detected. For detection, it is preferable to remove porous materials and fine particles. This can be achieved by performing operations such as filtration, centrifugation, magnetic separation, and dialysis on the sample after protease digestion. For example, porous materials and fine particles can be easily removed by filtering using a polyvinylidene fluoride (PVDF) filtration membrane (Low-binding hydrophilic PVDF, pore size 0.2 μm, manufactured by Millipore).

[0076] [Analysis] The sample containing the Fab peptide fragment obtained above can be analyzed by chromatography or mass spectrometry to identify and quantify the substrate antibody. In this invention, since the substrate antibody is regioselectively treated with protease, the number of types of peptide fragments contained in the sample is reduced. Therefore, it is possible to easily set the analytical conditions by mass spectrometry, etc.

[0077] To ensure more reliable separation of peptide fragments and improve analytical accuracy, the sample may be separated and concentrated by liquid chromatography (LC) before being subjected to mass spectrometry. When separating the sample by LC, the eluate from the LC may be directly ionized and subjected to mass spectrometry (LC-MS). Combinations of LC and tandem mass spectrometry, such as LC / MS / MS or LC / MS, are also available. ｎ Analysis can also be performed using this method. Alternatively, the eluate from LC may be separated and then subjected to mass spectrometry. The LC column is not particularly limited, and hydrophobic columns such as C30, C18, C8, and C4, which are commonly used for peptide analysis, or hydrophilic affinity chromatography supports can be appropriately selected and used.

[0078] Mass spectrometry allows for the determination of amino acid sequences, making it possible to distinguish whether a peptide fragment originates from a specific substrate antibody. Furthermore, the concentration of peptide fragments in a sample can be determined based on peak intensity. For analysis, the sample may be subjected to desalting, solubilization, extraction, concentration, drying, or other treatments as needed before being used for analysis.

[0079] The ionization method used in mass spectrometry is not particularly limited, and methods such as electron ionization (EI), chemical ionization (CI), field desorption (FD), fast atomic collision (FAB), matrix-assisted laser desorption / ionization (MALDI), and electrospray ionization (ESI) can be employed. The analytical method for the ionized sample is also not particularly limited, and methods such as magnetic field deflection, quadrupole (Q), ion trap (IT), time-of-flight (TOF), and Fourier transform ion cyclotron resonance (FT-ICR) can be appropriately determined depending on the ionization method. Furthermore, MS / MS analysis or MS can be performed using a triple quadrupole mass spectrometer or similar device. ３ The above multi-stage mass spectrometry can also be performed.

[0080] Suitable mass spectrometers for the method of this embodiment include, but are not limited to, liquid chromatograph-triple quadrupole mass spectrometers such as LCMS-8030, LCMS-8040, and LCMS-8050 (all manufactured by Shimadzu Corporation), and structural analysis mass spectrometers for precise mass analysis such as LCMS-IT-TOF and LCMS-Q-TOF (both manufactured by Shimadzu Corporation).

[0081] The antibody can be identified by determining the amino acid sequence of a peptide fragment that contains at least a portion of the amino acid sequence of a complementarity-determining region (CDR), such as the CDR2 region, which has an amino acid sequence specific to the substrate antibody.

[0082] Existing databases can be used to identify antibodies based on mass spectrometry results. Alternatively, antibodies can be identified by determining the amino acid sequence of peptide fragments through multi-stage mass spectrometry.

[0083] Furthermore, when identifying or quantifying substrate antibodies based on the detection results, the peptide to be detected preferably has about 5 to 30 amino acid residues, and more preferably about 7 to 25.

[0084] When quantifying the concentration of a substrate antibody, the amount of antibody can be calculated based on the peak area and peak intensity of the detected peptide fragment ions (or, in the case of multi-stage MS, the fragment ions obtained by the cleavage of the parent ion). For example, the concentration of peptide fragments in the sample can be calculated by correlating the peak area with a pre-determined calibration curve, or by correlating the peak area derived from an internal standard added to the sample with the peak area derived from the sample. Based on the peptide fragment concentration, the amount and concentration of the antibody can then be calculated.

[0085] In mass spectrometry, high-sensitivity analysis of specific peptides of antibody drugs can be achieved by performing purification using, for example, cation exchange resins (WCX and MCX) following protease digestion.

[0086] [Peptides containing amino acids in the CDR2 region] The peptide fragment to be detected in the method of this embodiment has an amino acid sequence that includes amino acids derived from the CDR2 region of the heavy chain or light chain of a monoclonal antibody such as an antibody drug.

[0087] Monoclonal antibodies intended for use as antibody drugs have publicly available amino acid sequence information, making it possible to obtain information such as the amino acid sequences of the heavy and light chains, the Fab domain, the Fc domain (not intended in this specification), the CDR region, and disulfide bonds. Furthermore, because the contact between the antibody and the protease is limited by the method of this embodiment, the types of peptides obtained by selective protease digestion are also limited. Therefore, those skilled in the art can predict the amino acid sequence of peptide fragments digested by a specific protease.

[0088] Therefore, the method of this embodiment makes it possible to detect and quantify antibodies by simultaneously detecting multiple predicted peptide fragments in parallel. However, for simpler measurement, and to reduce the time and cost required for measurement, it is preferable to select the optimal peptide fragment (quantification peptide, monitor peptide) for each antibody. If the optimal peptide fragment is known, it is possible to determine mass spectrometry conditions suitable only for the detection of that peptide fragment, and to provide such information.

[0089] Accordingly, the method of this embodiment includes, in one embodiment, selecting a peptide fragment containing amino acids in a CDR region, such as the CDR2 region, that is suitable for the quantification of individual monoclonal antibodies. Another embodiment provides a method that includes detecting a peptide fragment containing amino acids in a CDR region, such as the CDR2 region, that has been selected as suitable for the quantification of individual monoclonal antibodies.

[0090] [Kit] Furthermore, this embodiment also relates to a peptide fragment preparation kit for use in the peptide fragment preparation method or antibody analysis method described above, comprising: a porous body having pores on which the Fab domain of the substrate antibody can be immobilized; and fine particles on which the protease can be immobilized on the surface, wherein the average particle size of the fine particles is larger than the pore diameter of the porous body.

[0091] In this embodiment, the fine particles may have the protease immobilized on their surface. Furthermore, optimizations or additional kit components may be added to allow for various modifications of the above method.

[0092] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the amounts of substances indicated in percentage below are given on a weight basis if the substance is solid, and on a volume basis if it is liquid.

[0093] [Experimental Procedure: Analysis of Substrate Antibodies] An example of the experimental procedure for analyzing substrate antibody drugs is shown below.

[0094] 1) Preparation of samples for quantitative determination of substrate antibody drug concentrations in serum or plasma 1-1) Spike each substrate antibody in human serum or human plasma to create a dilution series (0.98 to 250 μg / mL). 1-2) Store the serum or plasma spiked with each substrate antibody at -80°C or -30°C until use.

[0095] 2) Pretreatment for analysis of substrate antibody drugs by LC-MS / MS 2-1) To purify the substrate antibody spiked in serum or plasma, the large amount of IgG inherent in the serum or plasma is removed with Protein A beads, and then the substrate antibody in the sample is reacted with Protein L beads to immobilize it on the Protein L beads. 2-2) Trypsin-immobilized FG beads are reacted with the substrate antibody immobilized on the Protein L beads and the reaction is carried out at 50°C for 4 to 5 hours. After the reaction, 10% FA is added to a final concentration of 0.5% FA, the Protein L beads and FG beads are filtered, and the sample is then subjected to LC-MS / MS analysis.

[0096] 3) Development of analytical methods using LC-MS / MS 3-1) Trypsin digestion peptide fragments of substrate antibody drugs were prepared and analyzed using Mikros-LCMS-9050 (QTOF). Among the detected trypsin digestion peptide fragments derived from antibody drugs, sequences common to IgG sequences with affinity for Protein L were excluded from the peptides used for quantification by LC-MS / MS. 3-2) The MRM analysis method was optimized for all candidate peptides for quantification of substrate antibody drugs using LCMS-8050 or LCMS-8060. 3-3) The substrate antibody drug samples pre-treated in 2) were measured using the optimized method. 3-4) Peptides with strong peak intensity and low noise during blank measurement (noise of 20% or less of the peak intensity at the lower limit of quantification) that allow for accurate calibration curve construction were selected and set as the quantification peptide (monitor peptide) specific to the substrate antibody.

[0097] The following antibody drugs (biosimilar antibodies) were used as substrate antibody drugs: Brolucizumab (Syd Labs), Certolizumab pegol (GLPBIO), Blinatumomab (ichorbio), and Tebentafusp (Antibody System).

[0098] The reagents used for the analysis of the substrate antibody drug are as follows: Trypsin DART FG beads (Shimadzu), Trypsin DART (Shimadzu), 25 mM Tris-HCl (pH 8.5) (Sigma), Human serum (Sigma; filtered through a 5 μm filter, then filtered through a 0.8 μm filter), Human plasma (Sigma; filtered through a 5 μm filter, then filtered through a 0.8 μm filter), Tris(2-carboxyethyl)phosphine Hydrochloride (TCEP) (Sigma), Iodoacetamide (Sigma), Protein A beads (TOSOH BIOSCIENCE), Protein L beads (TOSOH BIOSCIENCE).

[0099] The conditions for liquid chromatography (LC) and mass spectrometry (MS) in LC-MS / MS were as follows:

[0100] [LC conditions] Solvent A: 0.1% formic acid in H 2 Solvent B: 0.1% formic acid in acetonitrile Autosampler washing: Ultrapure water Flow rate: 0.4 mL / min Column: Shim-pack GISS 2.1 x 50 mm, 1.9 μm, 20 nm Column oven: 50°C Sample cooler: 8°C

[0101] [MS Conditions] Equipment: LCMS-8050, LCMS-8060 (triple quadruple mass spectrometer) Interface conditions: Interface voltage: 1.5kV Nebulizer gas flow rate: 3L / min Heating gas flow rate: 10L / min Drying gas flow rate: 10L / min Interface temperature: 350℃ DL temperature: 250℃ Heat block temperature: 400℃

[0102] [Experimental Example 1: Confirmation of Binding of Antibody Drugs to Protein L] Samples spiked with antibody drugs in Serum or Plasma were reacted with Protein A beads and / or Protein L beads, and LC-MS / MS analysis was performed under the conditions described above.

[0103] Blinatumomab was used as the substrate antibody. QRPGQGLEWIGYINPSR (SEQ ID NO: 6) was used as the monitor peptide for LC-MS / MS analysis.

[0104] After reacting serum spiked with blinatumomab with Protein A beads, pretreatment was performed using Protein L beads (denoted as "Protein A_L"), pretreatment using Protein L beads (denoted as "Protein L"), and pretreatment using Protein A beads (denoted as "Protein A"). LC-MS / MS analysis was performed for each of these methods to obtain the peak intensity of QRPGQGLEWIGYINPSR (SEQ ID NO: 6). Figure 4 shows the peak intensity for each case.

[0105] Thus, using samples in which the antibody drug blinatumomab was spiked in serum, we confirmed that the antibody drug blinatumomab can be specifically retained by Protein L beads.

[0106] Similarly, when using samples in which plasma was spiked with blinatumomab, it was confirmed that the antibody drug blinatumomab could be specifically retained by Protein L beads.

[0107] [Experimental Example 2: Analysis of Antibody Drugs] Antibody drugs were analyzed according to the experimental procedure described above. Brolucizumab, certolizumab pegol, blinatumomab, and teventafusp were used as antibody drugs. All of these are fragment antibody drugs that do not have an Fc chain structure.

[0108] Tables 1 to 4 show the monitor peptides for each antibody drug and the MRM (Multiple Reaction Monitoring) conditions for each monitor peptide using a triple quadrupole mass spectrometer (Triple Q MS).

[0109]

[0110]

[0111]

[0112]

[0113] Using samples in which each of the above antibody drugs was spiked in serum, LC-MS / MS analysis was performed according to the experimental procedure described above, and a calibration curve was created using the obtained quantitative values. The monitor peptides used for quantification for each antibody drug are shown in Table 5.

[0114]

[0115] Figures 5-40 show the analysis results for each monitor peptide of each antibody drug. Figures 5-7 show the LC-MS / MS analysis results for the monitor peptide LLIYLASTLASGVPSR (SEQ ID NO: 1) of the antibody drug Brolucizumab. Figure 5 is the MRM chromatogram of the monitor peptide with no antibody drug spike (0 μg / mL), and Figure 6 is the MRM chromatogram of the monitor peptide with antibody drug spike (250 μg / mL). The vertical axis represents peak intensity, and the horizontal axis represents retention time (min). Figure 7 shows the calibration curve for the concentration of the antibody drug, with the vertical axis representing peak intensity and the horizontal axis representing the concentration of the antibody drug (μg / mL). Similarly, Figures 8-40 show the analysis results for each monitor peptide of each antibody drug.

[0116] Figures 5-40 show that the monitor peptide could be detected and quantified for all four antibody drugs that lacked an Fc chain structure, which were used as examples.

[0117] In the MRM chromatogram, the upper left corner of each chart shows the MRM transition and the corresponding ionic intensity of each monitor peptide.

[0118] Thus, according to this embodiment, fragment antibody drugs that do not have an Fc structure can be immobilized on beads, and the preparation of peptide fragments derived from the antibody drug is achieved. This makes it possible to analyze fragment antibody drugs other than IgG-like antibodies that do not have an Fc structure. In particular, it can contribute to pharmacokinetic analysis and therapeutic drug monitoring (TDM) of novel antibody drugs. Furthermore, it can contribute to pharmacokinetic analysis and concentration monitoring of novel fragment antibody drug candidates during the development stage of novel fragment antibody drugs.

[0119] [Aspects] The above-described exemplary embodiments and examples will be understood by those skilled in the art to be specific examples of the following aspects.

[0120] (1) A method for preparing a peptide fragment comprising: a substrate immobilization step of immobilizing a target substrate antibody in a biological sample within the pores of a porous material having affinity for the Fab domain of the substrate antibody; and a digestion step of contacting the porous material on which the substrate antibody is immobilized with fine particles on which a protease is immobilized on the surface, thereby performing protease digestion of the substrate antibody to obtain a peptide fragment of the Fab domain, wherein in the substrate immobilization step, the Fab domain of the substrate antibody is immobilized within the pores of the porous material, and the average particle size of the fine particles is larger than the average pore diameter of the porous material.

[0121] (2) The method for preparing a peptide fragment according to (1), wherein in the digestion step, the Fab domain of the substrate antibody is regioselectively cleaved by the protease.

[0122] (3) The method for preparing a peptide fragment according to (1) or (2) above, wherein a linker molecule that site-specifically interacts with the Fab domain of the substrate antibody is immobilized in the pores of the porous body, and in the substrate immobilization step, the Fab domain of the substrate antibody is immobilized in the pores of the porous body via the linker molecule.

[0123] (4) The method for preparing a peptide fragment according to (3) above, wherein, after the substrate immobilization step, the molecular size of the linker molecule and the substrate antibody bound together is 0.5 to 1.5 times the pore diameter of the porous material.

[0124] (5) The pores of the porous body have affinity for the light chain or heavy chain of the Fab domain of the substrate antibody, a method for preparing a peptide fragment according to any one of (1) to (4) above.

[0125] (6) The method for preparing a peptide fragment according to any one of (1) to (5) above, wherein the substrate antibody does not have an Fc structure.

[0126] (7) The substrate antibody is Fab, F(ab'), F(ab') 2 , scFv, and BiTEs (bi-specific T cell engagers), bsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), DNL- (Fab) 3 (dock-and-lock trivalent Fab), sdAb (single-domain antibody), bssAb (bispecific single-domain antibody), and Bispecific F (mab') 2 A method for preparing a peptide fragment according to any of (1) to (6) above, selected from the group consisting of structures.

[0127] (8) A method for preparing a peptide fragment according to any one of (1) to (7) above, wherein the substrate antibody described in 117 is a monoclonal antibody.

[0128] (9) A method for preparing a peptide fragment according to any one of (1) to (8) above, wherein the surface of the fine particles is modified with a spacer molecule capable of binding to the protease, and the protease is immobilized on the surface of the fine particles via the spacer molecule.

[0129] (10) A method for preparing a peptide fragment according to any one of (1) to (9) above, wherein the protease is selected from the group consisting of trypsin, pepsin, papain, lysyl endopeptidase, arginine endopeptidase, chymotrypsin, V8 protease, and aspartate N-terminal protease.

[0130] (11) A method for preparing peptide fragments according to any one of (1) to (10) above, wherein the average pore size of the porous body is 30 to 150 nm and the average particle size of the fine particles is 100 nm or more.

[0131] (12) A method for preparing a peptide fragment according to any one of (1) to (11) above, further comprising an endogenous antibody removal step of removing antibodies having an Fc structure other than the substrate antibody (IgG-like antibody) inherent in the biological sample before the substrate immobilization step.

[0132] (13) A method for preparing peptide fragments according to any one of (1) to (12) above, wherein the biological sample is selected from the group consisting of blood, cerebrospinal fluid, urine, body secretions, feces, saliva, and sputum.

[0133] (14) The substrate antibody is an antibody drug or a candidate antibody drug, a method for preparing a peptide fragment according to any one of (1) to (13) above.

[0134] (15) A method for analyzing an antibody, comprising analyzing a peptide fragment of the Fab domain of the substrate antibody prepared by any of the methods described in (1) to (14) above, by mass spectrometry.

[0135] (16) The method for analyzing an antibody according to (15), wherein the peptide fragment of the Fab domain of the substrate antibody to be analyzed is a peptide fragment that includes at least a portion of the amino acid sequence of the complementarity-determining region (CDR) of the antibody.

[0136] (17) A peptide fragment preparation kit for use in any of the methods described in (1) to (14) above, or in any of the methods described in (15) to (16) above, comprising: a porous body having pores on which the Fab domain of the substrate antibody can be immobilized; and fine particles on which the protease can be immobilized on the surface, wherein the average particle size of the fine particles is larger than the pore diameter of the porous body.

[0137] (18) The peptide fragment preparation kit according to (17) above, wherein the fine particles have the protease immobilized on their surface.

[0138] All patent and non-patent documents by the inventors cited herein are incorporated by reference into this specification.

[0139] 110 Microparticles 111 Spacer 115 Protease 120 Porous material 121 Linker molecule 125 Substrate antibody 129 Pore D11 Particle size of microparticle 110 D12 Pore diameter of porous material 120

Claims

1. A method for preparing a peptide fragment comprising: a substrate immobilization step of immobilizing a target substrate antibody in a biological sample within the pores of a porous material having affinity for the Fab domain of the substrate antibody; and a digestion step of contacting the porous material on which the substrate antibody is immobilized with fine particles on which a protease is immobilized on its surface to perform protease digestion of the substrate antibody and obtain a peptide fragment of the Fab domain, wherein in the substrate immobilization step, the Fab domain of the substrate antibody is immobilized within the pores of the porous material, and the average particle size of the fine particles is larger than the average pore diameter of the porous material.

2. The method for preparing a peptide fragment according to claim 1, wherein in the digestion step, the Fab domain of the substrate antibody is regioselectively cleaved by the protease.

3. A method for preparing a peptide fragment according to claim 1, wherein a linker molecule that site-specifically interacts with the Fab domain of the substrate antibody is immobilized within the pores of the porous body, and in the substrate immobilization step, the Fab domain of the substrate antibody is immobilized within the pores of the porous body via the linker molecule.

4. The method for preparing a peptide fragment according to claim 3, wherein, after the substrate immobilization step, the molecular size of the linker molecule and the substrate antibody bound together is 0.5 to 1.5 times the pore diameter of the porous body.

5. The method for preparing a peptide fragment according to claim 1, wherein the pores of the porous body have affinity for the light chain or heavy chain of the Fab domain of the substrate antibody.

6. The method for preparing a peptide fragment according to claim 1, wherein the substrate antibody does not have an Fc structure.

7. The substrate antibody is Fab, F(ab'), F(ab') 2 , scFv, and BiTEs (bi-specific T cell engagers), bsDb (bispecific diabody), scBsDb (single-chain bispecific diabody), scBsTaFv (single-chain bispecific tandem variable domain), DNL- (Fab) 3 (dock-and-lock trivalent Fab), sdAb (single-domain antibody), bssAb (bispecific single-domain antibody), and Bispecific F (mab') 2 A method for preparing a peptide fragment according to claim 1, selected from the group consisting of structures.

8. The method for preparing a peptide fragment according to claim 1, wherein the substrate antibody is a monoclonal antibody.

9. The method for preparing a peptide fragment according to claim 1, wherein the surface of the fine particles is modified with a spacer molecule capable of binding to the protease, and the protease is immobilized on the surface of the fine particles via the spacer molecule.

10. The method for preparing a peptide fragment according to claim 1, wherein the protease is selected from the group consisting of trypsin, pepsin, papain, lysyl endopeptidase, arginine endopeptidase, chymotrypsin, V8 protease, and aspartate N-terminal protease.

11. The method for preparing peptide fragments according to claim 1, wherein the average pore diameter of the porous body is 30 to 150 nm, and the average particle size of the fine particles is 100 nm or more.

12. The method for preparing a peptide fragment according to claim 1, further comprising an endogenous antibody removal step, which removes antibodies having an Fc structure other than the substrate antibody (IgG-like antibodies) inherent in the biological sample, prior to the substrate immobilization step.

13. The method for preparing peptide fragments according to claim 1, wherein the biological sample is selected from the group consisting of blood, cerebrospinal fluid, urine, body secretions, feces, saliva, and sputum.

14. The method for preparing a peptide fragment according to claim 1, wherein the substrate antibody is an antibody drug or a candidate antibody drug.

15. A method for analyzing an antibody, comprising analyzing a peptide fragment of the Fab domain of a substrate antibody prepared by the method described in claim 1, by mass spectrometry.

16. The method for analyzing an antibody according to claim 15, wherein the peptide fragment of the Fab domain of the substrate antibody to be analyzed is a peptide fragment containing at least a portion of the amino acid sequence of the complementarity-determining region (CDR) of the antibody.

17. A peptide fragment preparation kit for use in the method described in claim 1, comprising: a porous body having pores on which the Fab domain of the substrate antibody can be immobilized; and fine particles on which the protease can be immobilized on the surface, wherein the average particle size of the fine particles is larger than the pore diameter of the porous body.

18. The peptide fragment preparation kit according to claim 17, wherein the fine particles have the protease immobilized on their surface.

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