Containers and methods for preserving, pretreating and analyzing biological materials

Abstract

To provide an excellent storage container for biological samples, which prevents trace amounts of biomarkers contained in the biological sample from adhering to the surface of the storage container, reduces sample loss, and enables accurate and highly precise analysis. Provided is a biological sample storage container for reducing sample loss of biomarkers contained in the biological sample, the biological sample storage container having, on at least a portion of the surface of the container, a coating containing a copolymer including a repeating unit containing a group represented by the following formula (a), a repeating unit containing a group represented by the following formula (b), and optionally a repeating unit containing a group represented by the following formula (c): (In the formula, U a1 , U a2 , U a3 , U b1 , U b2 , U b3 , An - and R c is as described in the present specification and claims.)

Description

[Technical field]

[0001] The present invention relates to a storage container for a biological sample for reducing sample loss of biomarkers contained in the biological sample, and in particular to a storage container for a biological sample that is provided with a coating that inhibits adhesion of trace amounts of biomarkers contained in the biological sample and reduces sample loss. [Background technology]

[0002] Biomarkers are biological substances such as peptides, proteins, endogenous metabolites, or genes contained in biological samples such as blood and urine that correlate with and serve as indicators of changes in disease or responses to treatment. Biomarkers are known to include diagnostic markers, prognostic markers, predictive markers, monitoring markers, and safety markers, depending on their purpose. In recent years, particularly in the diagnosis and treatment of cancer, efforts have been made to measure the amount of tumor markers as one of the indicators of the presence and progression of cancer and the effectiveness of treatment.

[0003] Although biomarkers contained in biological samples are generally present in trace amounts, the amount increases or decreases in correlation with the presence or progression of a disease and response to treatment. Therefore, in order to properly evaluate biomarkers, it is necessary to measure them accurately and with high precision using an instrument that enables highly sensitive analysis of trace components, such as a liquid chromatography / mass spectrometry (LC-MS). For such measurements, in addition to the requirements for the instrument and method for measuring the biological sample, requirements regarding the quality of the sample to be measured are also important. For example, biological samples collected from subjects are usually stored in storage containers such as glass or plastic tubes until they are subjected to measurement, but there is a problem in that biomarker proteins and endogenous metabolites are adsorbed to the inner surface of the container, causing sample loss and affecting proper evaluation of the biomarkers.

[0004] To solve these problems, containers for storing biological samples have been proposed in which the inner surface is modified to be hydrophilic by plasma treatment or corona discharge treatment, or by surface treatment with a photocrosslinkable superhydrophilic polymer, thereby suppressing adsorption of proteins including biomarkers and endogenous metabolites and reducing sample loss.

[0005] The present inventors have focused on polymers having phosphate ester groups, which are expected to be coating materials capable of inhibiting adhesion of various biological substances, and have conducted extensive research. As a result, they have reported that a coating agent containing a copolymer containing a specific anionic group and a cationic group can be firmly attached to any type of substrate, and that after adhesion, the coating film has excellent resistance to aqueous solvents and exhibits excellent adhesion inhibition ability for biological substances (e.g., platelets, fibrinogen, etc.) (see, for example, Patent Documents 1 and 2). They have also reported that a cell culture vessel coated with the same coating agent has excellent cell adhesion inhibition ability and excellent resistance to solvents and radiation (see, for example, Patent Document 3). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2014 / 196650 [Patent Document 2] International Publication No. 2016 / 093293 [Patent Document 3] International Publication No. 2014 / 196652 Summary of the Invention [Problem to be solved by the invention]

[0007] It is desirable for a coating applied to a biological sample storage container to have not only the ability to inhibit cell adhesion and resistance to aqueous solvents and radiation, but also the ability to inhibit adhesion of trace amounts of biomarkers contained in the biological sample, such as proteins and endogenous metabolites, in order to reduce sample loss and enable accurate and highly precise analysis. However, no biological sample storage containers equipped with a coating that has all of these properties have been reported. [Means for solving the problem]

[0008] The present inventors have discovered that a container for storing biological samples, which has a coating on at least a portion of the surface thereof comprising a copolymer having a specific anionic structure, a specific cationic structure, and optionally a specific hydrophobic structure, not only has excellent cell adhesion-inhibiting ability and resistance to aqueous solvents and radiation, but also has excellent adhesion-inhibiting ability against trace amounts of biomarkers contained in biological samples, thereby reducing sample loss and enabling appropriate evaluation of biomarkers, and have completed the present invention. The present invention is as follows.

[0009] [1] A biological sample storage container for reducing sample loss of a biomarker contained in the biological sample, comprising: A copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): [ka] (In the formula, U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. A container for storing a biological sample, comprising a coating comprising the compound on at least a portion of a surface of the container.

[0010] [2] The copolymer further comprises the following formula (c): [ka] [In the formula, R c represents a linear or branched alkyl group having 1 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, or an aryloxyalkyl group having 7 to 15 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom) The container according to [1], comprising a repeating unit having a group represented by the following formula:

[0011] [3] The container described in [1] or [2], wherein the biomarker is an antibody or an amyloid.

[0012] [4] A container for storing a peptide, comprising: A copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): [ka] (In the formula, U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. A container for storing a peptide, comprising a coating comprising the compound on at least a portion of a surface of the container.

[0013] [5] A container for storing a peptide according to [4], wherein the peptide is contained in a biological sample to be subjected to mass spectrometry.

[0014] [6] A container for fragmenting a protein and / or peptide, comprising a copolymer comprising a repeating unit having a group represented by the following formula (a) and a repeating unit having a group represented by the following formula (b): [ka] (In the formula, U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. A container for fragmenting proteins and / or peptides, comprising a coating comprising the above on at least a portion of the surface of the container.

[0015] [7] A method for analyzing fragmented proteins and / or peptides, comprising: Providing a sample containing the proteins and / or peptides to be analyzed; A copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): [ka] (In the formula, U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An -represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. carrying out a fragmentation treatment of proteins and / or peptides in the sample in a container having, on at least a part of its surface, a coating comprising subjecting the fragmented proteins and / or peptides in the sample to a measuring device; A method comprising:

[0016] [8] A method for measuring the absolute amount of a biomarker in a solution, comprising: A copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): [ka] (In the formula, U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. A method comprising the steps of: containing and / or storing a solution containing a biomarker contained in a biological sample in a container for storing a biological sample, the container having a coating comprising the coating on at least a portion of a surface of the container; and then measuring the absolute amount of the biomarker.

[0017] [9] The method according to [7], wherein the biomarker is an antibody or an amyloid. Effect of the Invention

[0018] The biological sample storage container of the present invention has an excellent adhesion suppression ability against trace amounts of biomarkers contained in biological samples, such as proteins and endogenous metabolites, and can reduce sample loss, and is provided with a coating containing a copolymer containing an anionic group represented by formula (a), a cationic group represented by formula (b), and optionally a hydrophobic group represented by formula (c) on at least a part of the surface of the container, preferably the entire surface of the container, thereby suppressing adhesion of trace amounts of biomarkers contained in biological samples to the container. In addition, the cationic group and the anionic group in the coating form an ionic bond (ion complex), which allows the coating to be fixed to any type of container base material, such as glass, fiber, inorganic particles, or resin (synthetic resin and natural resin), and after fixing, the coating has excellent resistance to aqueous solvents (water, phosphate buffer solution (PBS), alcohol, etc.). In other words, the present invention can provide an excellent biological sample storage container that suppresses adhesion of trace amounts of biomarkers contained in biological samples to the surface of the storage container during storage, reduces sample loss, and enables accurate and highly precise analysis. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a graph showing the results of comparing the sums of the areas of mass chromatograms of eight specific peptides in the samples as a result of HPLC measurement in Test Example 1, Test Example in which the PP tubes and vials coated in Example 1 were used as containers for frozen storage of biological samples, containers for pretreatment for HPLC measurement, and vials for HPLC measurement, Comparative Example 1 in which uncoated PP tubes and vials were used, and Comparative Example 2 in which commercially available coated tubes and vials (Proteosave (registered trademark)) were used. [Diagram 2]FIG. 1 is a graph showing the results of comparing the sums of the areas of mass chromatograms of eight specific peptides in the samples as a result of HPLC measurement in Test Example 1, Test Example in which the PP tubes and vials coated in Example 1 were used as the containers for frozen storage of biological samples, the containers for pretreatment for HPLC measurement, and the vials for HPLC measurement; Comparative Example 3 in which uncoated PP tubes and vials were used other than the containers for frozen storage of biological samples; and Comparative Example 4 in which commercially available coated tubes and vials (Proteosave (registered trademark)) were used other than the containers for frozen storage of biological samples. [Diagram 3] FIG. 13 is a graph showing the results of comparing the sums of the mass chromatogram areas of three specific peptides in the samples as a result of HPLC measurement in Test Example 2, which uses a PP tube coated in Example 1 as a container for cryopreserving biological samples of various concentrations, Comparative Example 5, which uses an uncoated PP tube, and Comparative Example 6, which uses a commercially available coated tube (Proteosave (registered trademark)). [Figure 4] FIG. 1 is a graph comparing the area values ​​of Aβ42 in the samples as a result of HPLC-mass spectrometry (SIM measurement) for Test Example 3, in which the PP vial obtained in Example 2 was used as the dilution container in the dilution procedure of Test Example 3 (10-fold dilution three times), and Comparative Example 7, in which an uncoated PP vial was used. [Diagram 5] FIG. 1 is a graph comparing the area values ​​of Aβ42 in the samples as a result of HPLC-mass spectrometry (SIM measurement) for Test Example 3, in which the PP vial obtained in Example 2 was used as the dilution container in the dilution procedure of Test Example 3 (10-fold dilution five times), and Comparative Example 7, in which an uncoated PP vial was used. [Figure 6] This is a graph comparing the area values ​​of Aβ42 in the samples after 6 and 15 hours of storage at 4°C after the dilution operation of Test Example 3 (10-fold dilution five times) in Test Example 3, in which the PP vial obtained in Example 2 was used as the dilution container, and Comparative Example 7, in which an uncoated PP vial was used. [Figure 7]FIG. 13 is a graph comparing the area values ​​of Aβ42 in the samples as a result of HPLC-mass spectrometry (SIM measurement) for Test Example 4, in which the PP tube and PP vial obtained in Example 2 were used as the dilution container and the HPLC measurement vial; Comparative Example 8, in which the PP tube obtained in Example 2 was used as the dilution container and an uncoated PP vial was used as the HPLC measurement vial; Comparative Example 9, in which an uncoated PP tube was used as the dilution container and the PP vial obtained in Example 2 was used as the HPLC measurement vial; and Comparative Example 10, in which an uncoated PP tube and PP vial were used as the dilution container and the HPLC measurement vial. [Figure 8] This is a graph comparing the area values ​​of Aβ40 in the samples obtained as a result of HPLC-mass spectrometry (SIM measurement) in Test Example 5, in which the PP tube and PP vial obtained in Example 2 were used as the dilution container and HPLC measurement vial, with the area values ​​of Aβ40 in the samples obtained as a result of HPLC-mass spectrometry (SIM measurement) in Comparative Example 11, in which a low-adsorption PP vial manufactured by Shimadzu GLC was used as the HPLC measurement vial; Comparative Example 12, in which an uncoated PP vial was used; and Comparative Example 13, in which a PP vial manufactured by Waters was used. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] <Terminology explanation> The terms used in the present invention have the following definitions unless otherwise specified.

[0021] In the present invention, a "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

[0022] In the present invention, the term "alkyl group" refers to a monovalent group of a linear or branched saturated aliphatic hydrocarbon. Examples of the linear or branched alkyl group having 1 to 5 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and 1-ethylpropyl. Examples of the linear or branched alkyl group having 1 to 18 carbon atoms include, in addition to the linear or branched alkyl group having 1 to 5 carbon atoms, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl groups, and isomers thereof. Similarly, examples of the "linear or branched alkyl group having 1 to 10 carbon atoms" include, in addition to the examples of the "linear or branched alkyl group having 1 to 5 carbon atoms", a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, or isomers thereof.

[0023] In the present invention, "a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom" means the linear or branched alkyl group having 1 to 5 carbon atoms as described above, or the linear or branched alkyl group having 1 to 5 carbon atoms as described above which is substituted with one or more halogen atoms as described above. Examples of the "linear or branched alkyl group having 1 to 5 carbon atoms" are as described above. On the other hand, "a linear or branched alkyl group having 1 to 5 carbon atoms which is substituted with one or more halogen atoms" means the linear or branched alkyl group having 1 to 5 carbon atoms as described above in which one or more arbitrary hydrogen atoms are replaced with a halogen atom, and examples thereof include a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a chloromethyl group, a dichloromethyl group, a trichloromethyl group, a bromomethyl group, an iodomethyl group, a 2,2,2-trifluoroethyl group, a 2,2,2-trichloroethyl group, a perfluoroethyl group, a perfluorobutyl group, and a perfluoropentyl group.

[0024] In the present invention, an "ester bond" means -C(=O)-O- or -OC(=O)-, an "amide bond" means -NHC(=O)- or -C(=O)NH-, and an "ether bond" means -O-.

[0025] In the present invention, "a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with one or more halogen atoms" means a linear or branched alkylene group having 1 to 10 carbon atoms, or a linear or branched alkylene group having 1 to 10 carbon atoms which is substituted with one or more halogen atoms. Here, the "alkylene group" means a divalent organic group corresponding to the above alkyl group. Examples of the "linear or branched alkylene group having 1 to 10 carbon atoms" include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, a 1-ethyl-trimethylene group, a hexamethylene group, an octamethylene group, and a decamethylene group. Of these, an ethylene group, a propylene group, an octamethylene group, and a decamethylene group are preferred, and a linear or branched alkylene group having 1 to 5 carbon atoms, such as an ethylene group, a propylene group, a trimethylene group, or a tetramethylene group, is more preferred, and an ethylene group or a propylene group is particularly preferred. The "linear or branched alkylene group having 1 to 10 carbon atoms substituted with one or more halogen atoms" means the above-mentioned alkylene group in which any one or more hydrogen atoms are replaced with halogen atoms, and in particular, an ethylene group or a propylene group in which some or all of the hydrogen atoms are replaced with halogen atoms is preferred.

[0026] In the present invention, the term "alicyclic hydrocarbon group having 3 to 10 carbon atoms" refers to a monocyclic or polycyclic, saturated or partially unsaturated, aliphatic hydrocarbon monovalent group having 3 to 10 carbon atoms. Among these, a monovalent, monocyclic or bicyclic, saturated aliphatic hydrocarbon group having 3 to 10 carbon atoms is preferred, and examples thereof include cycloalkyl groups having 3 to 10 carbon atoms, such as a cyclopropyl group, a cyclobutyl group, or a cyclohexyl group, or a bicycloalkyl group having 4 to 10 carbon atoms, such as a bicyclo[3.2.1]octyl group, a bornyl group, or an isobornyl group.

[0027] In the present invention, the term "aryl group having 6 to 10 carbon atoms" refers to a monovalent monocyclic or polycyclic aromatic hydrocarbon group having 6 to 10 carbon atoms, such as a phenyl group, a naphthyl group, an anthryl group, etc. The "aryl group having 6 to 10 carbon atoms" may be substituted with one or more of the above-mentioned "linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom".

[0028] In the present invention, the "aralkyl group having 7 to 15 carbon atoms" refers to a group -R-R' (wherein R represents the above-mentioned "linear or branched alkylene group having 1 to 5 carbon atoms" and R' represents the above-mentioned "aryl group having 6 to 10 carbon atoms"), and examples thereof include a benzyl group, a phenethyl group, an α-methylbenzyl group, etc. The aryl portion of the "aralkyl group having 7 to 15 carbon atoms" may be substituted with one or more of the above-mentioned "linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom".

[0029] In the present invention, "aryloxyalkyl group having 7 to 15 carbon atoms" means a group -RO-R' (wherein R represents the above-mentioned "linear or branched alkylene group having 1 to 5 carbon atoms" and R' represents the above-mentioned "aryl group having 6 to 10 carbon atoms"), and examples thereof include a phenoxymethyl group, a phenoxyethyl group, and a phenoxypropyl group. The aryl portion of the "aryloxyalkyl group having 7 to 15 carbon atoms" may be substituted with one or more of the above-mentioned "linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom".

[0030] In the present invention, the term "halide ion" means a fluoride ion, a chloride ion, a bromide ion, or an iodide ion. In the present invention, the term "inorganic acid ion" means a carbonate ion, a sulfate ion, a phosphate ion, a hydrogen phosphate ion, a dihydrogen phosphate ion, a nitrate ion, a perchlorate ion, or a borate ion. The above An - Preferred as the cation are halide ions, sulfate ions, phosphate ions, hydroxide ions and isothiocyanate ions, and particularly preferred are halide ions.

[0031] In the present invention, the (meth)acrylate compound means both an acrylate compound and a methacrylate compound. For example, (meth)acrylic acid means acrylic acid and methacrylic acid. In the present invention, the term "anion" or "anionic group" refers to an anion or an anionic group, including those that can dissociate in water to become an anion or an anionic group. Similarly, in the present invention, the term "cation" or "cationic group" refers to a cation or a cationic group, including those that can dissociate in water to become a cation or a cationic group.

[0032] Description of the invention The biological sample storage container of the present invention is provided with a coating that inhibits adhesion of trace amounts of biomarkers contained in the biological sample and reduces sample loss.

[0033] <Biosamples> The biological sample in the present invention is not particularly limited as long as it is collected from a human, animal, or plant and includes urine, blood (whole blood, serum, plasma, etc.), tissue (normal tissue, tumor tissue, pathological tissue, etc.), cerebrospinal fluid, synovial fluid, etc., and contains biological substances such as peptides, proteins, endogenous metabolites, or genes that can serve as biomarkers.

[0034] <Biomarkers> In the present invention, a biomarker refers to a biological substance, such as a peptide, a protein, an endogenous metabolite, or a gene, contained in a biological sample in a normal or pathological state, which correlates with and serves as an indicator of changes in a disease or a response to a treatment, for example, an antigen such as a tumor marker, a pathogenic peptide or protein such as an amyloid or prion, or an antibody. Thanks for reading Aβ16, Aβ28, Aβ38, Aβ40, Aβ42, Aβ4 3, APP, α‐Synuclein, a2-Macroglobulin, Acrp‐30, Ang‐1, Ang‐2, Apo A-1, Apo B-100, AR, BAFF, BCA-1 (CXCL13), b-NGF, BDNF, BD-2, BMP-9, Cathepsin-D, CA 19-9, CA-125, Cathepsin S, CD-14, CD40L, CEA, c-MET, Clusterin, CNTF, COX-2, C-peptide, CRP, Eotaxin / CXCL11, E-Cadherin, EGF, EGFR, ENA-78, Endoglin, Eotaxin, Eotaxin 3. ER (Epiregulin), ErbB2 (Her2), E-Selectin, FasL, FGF basic, Fibrinogen, Fibronectin, G-CSF, GDNF, GFAP, GM-CSF, gp130 (IL-6ST) GROa、GROg、HB-EGF、HE4 / WFDC2、HGF、HGH、HIV p24, I-309, ICAM-1, IFNα, IFNγ, IGFBP-1, IGFBP-2, IGFBP-3, IgE, IL-1a, IL-1b, IL-1ra, IL-2, IL-2Ra, IL-2Rg, IL -3, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17A, IL-17E, IL-18, IL-22 (Total) IL-23 IL-28A IL-33 IL-36β Insulin IP-10 ITAC KGF Leptin LIF L-Selectin Lymp hotactin, MCP-1, MCP-2, MCP-3, MCP-4, M-CSF, MDC, MIF, MIG, MIP-1a, MIP-1b, MIP-3a, MIP-3b, MIP-4 (PARC) MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13, MPO, NAP -2, pNF-heavy, NF-light(free-range), NGAL, NSE, NT3, NT-proBNP, OPG, OPN, PAI-1 Active、PAI-1Total, PAPP-A, PD-1, PD-L1, PDGF-AA, PDGF-AB, PDGF-BB, PEDF, PlGF, PLGF, Prolactin, PSA, P-Selectin, RAGE, RANK L, RANTES, RBP4, Resistin, SAA, SCF, SDF-1, TARC, Tau, P-Tau 231, TDP-43, TFF-3, TGFα, TGFβ, Tie-2, TIM-1, TIMP-1, TIMP-2, TNFα, TNFβ, TNF-RI, TNF-RII, TRAIL, Troponin-I , TSLP, TSP-1, TSP-2, TWEAK, UCH-L1, VCAM-1, VEGF-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2, and VEGF-R3.

[0035] In the present invention, "reducing sample loss of biomarkers contained in a biological sample" means that, in HPLC measurement performed by the method described in the Examples described later (e.g., Test Example 1), the percentage of sample loss (%) ((sum of areas of coated containers) / (sum of areas of uncoated containers)×100) when comparing the sum of the areas of mass chromatograms of predetermined components contained in a biological sample stored in a storage container having a coating according to the present invention with the sum of the areas of mass chromatograms of predetermined components contained in a biological sample stored in an uncoated storage container as shown in Comparative Example 1 is 50% or less, preferably 30% or less, and more preferably 20% or less.

[0036] <Coating> The polymer contained in the coating of the biological sample storage container of the present invention provided on at least a portion of the surface of the container is a copolymer containing a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b):

[0037] [ka]

[0038] [In the formula, U a1 , Ua2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion.

[0039] The copolymer may further comprise a compound represented by the following formula (c): [ka] [In the formula, R c represents a linear or branched alkyl group having 1 to 18 carbon atoms, an alicyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, or an aryloxyalkyl group having 7 to 15 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom) The repeating unit may contain a group represented by the following formula:

[0040] The copolymer related to the biological sample storage container of the present invention is not particularly limited as long as it contains a repeating unit containing a group represented by the above formula (a), a repeating unit containing a group represented by the above formula (b), and optionally a repeating unit containing a group represented by the above formula (c). In the present invention, the repeating unit containing a group represented by the above formula (c) is different from the repeating unit containing a group represented by the above formula (a) and the repeating unit containing a group represented by the above formula (b). The copolymer is preferably obtained by radical polymerization of a monomer containing a group represented by the above formula (a), a monomer containing a group represented by the above formula (b), and optionally a monomer containing a group represented by the above formula (c), but it can also be used by polycondensation or polyaddition reaction. Examples of copolymers include vinyl polymerization polymers reacted with olefins, polyamides, polyesters, polycarbonates, polyurethanes, etc., among which vinyl polymerization polymers reacted with olefins or (meth)acrylic polymers polymerized with (meth)acrylate compounds are particularly preferable.

[0041] The proportion of the repeating unit containing the group represented by formula (a) in the copolymer is 3 mol % to 80 mol %. The copolymer may contain two or more kinds of repeating units containing the group represented by formula (a).

[0042] The proportion of the repeating unit containing the group represented by formula (b) in the copolymer is 3 mol % to 80 mol %. The copolymer may contain two or more kinds of repeating units containing the group represented by formula (b).

[0043] The proportion of the repeating units containing the group represented by formula (c) in the copolymer may be the remainder after subtracting the repeating units of formulas (a) and (b) from the total copolymer, and is, for example, 0 mol % to 90 mol %. The copolymer may contain repeating units containing two or more types of groups represented by formula (c).

[0044] A preferred embodiment of the copolymer for use in a biological sample storage container of the present invention is a copolymer containing repeating units of the following formulae (a1) and (b1).

[0045] [ka]

[0046] In the formula, T a and T b each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Q a and Q b each independently represents a single bond, an ester bond, or an amide bond; R a and R b each independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom; U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion; and m represents an integer of 0 to 6.

[0047] The copolymer may further contain a repeating unit of the following formula (c1):

[0048] [ka]

[0049] In the formula, T c represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Q c represents a single bond, an ether bond or an ester bond; R crepresents a linear or branched alkyl group having 1 to 18 carbon atoms, an alicyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, or an aryloxyalkyl group having 7 to 15 carbon atoms (wherein the aryl moiety is optionally substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom).

[0050] In formula (a1), m represents an integer of 0 to 6, preferably an integer of 1 to 6, more preferably an integer of 1 to 5, and particularly preferably 1.

[0051] The proportion of the repeating unit represented by formula (a1) contained in the copolymer is 3 mol % to 80 mol %. The copolymer may contain two or more kinds of repeating units represented by formula (a1).

[0052] The proportion of the repeating unit represented by formula (b1) contained in the copolymer is 3 mol % to 80 mol %. The copolymer may contain two or more kinds of repeating units represented by formula (b1).

[0053] The proportion of the repeating unit represented by formula (c1) contained in the copolymer may be the remainder after subtracting the repeating units represented by formula (a1) and formula (b1) from the total copolymer, and is, for example, 0 mol % to 90 mol %. The copolymer may contain two or more kinds of repeating units represented by formula (c1).

[0054] Another preferred embodiment of the copolymer for the biological sample storage container of the present invention is represented by the following formulae (A) and (B):

[0055] [ka]

[0056] [In the formula, T a and T beach independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Q a and Q b each independently represents a single bond, an ester bond, or an amide bond; R a and R b each independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom; U a1 , U a2 , U b1 , U b2 and U b3 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An - represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion; m represents an integer from 0 to 6. It is a copolymer obtained by reacting (polymerizing) a monomer mixture containing a compound represented by the following formula (1) in a solvent.

[0057] The copolymer may further comprise the following formula (C):

[0058] [ka]

[0059] [In the formula, T c represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Q c represents a single bond, an ether bond or an ester bond; R crepresents a linear or branched alkyl group having 1 to 18 carbon atoms, an alicyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, or an aryloxyalkyl group having 7 to 15 carbon atoms (wherein the aryl moiety may be substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom) The copolymer may be a copolymer obtained from a monomer mixture containing a compound represented by the following formula:

[0060] T a , T b and T c is preferably a hydrogen atom, a methyl group, or an ethyl group, and more preferably a hydrogen atom or a methyl group. a , Q b and Q c R is preferably a single bond or an ester bond, and more preferably an ester bond. a and R b R is preferably a linear or branched alkylene group having 1 to 5 carbon atoms, and more preferably a methylene group, an ethylene group, or a propylene group. c As U, a linear or branched alkyl group having 4 to 18 carbon atoms or a cycloalkyl group having 3 to 10 carbon atoms is preferable, and a butyl group, a pentyl group, a hexyl group or an isomer thereof, or a cyclohexyl group is more preferable. a1 , U a2 , U b1 , U b2 and U b3 is preferably a hydrogen atom, a methyl group, an ethyl group or a t-butyl group, and U in formula (a) a1 and U a2 As U in formula (b), a hydrogen atom is more preferable. b1 , U b2 and U b3 is more preferably a hydrogen atom, a methyl group, an ethyl group or a t-butyl group.

[0061] Specific examples of the above formula (A) include vinylphosphonic acid, acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, acid phosphooxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, acid phosphooxypolyoxypropylene glycol mono(meth)acrylate, and the like. Among these, vinyl phosphonic acid, acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate) or acid phosphooxypolyoxyethylene glycol monomethacrylate is preferably used, and acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate) is most preferably used.

[0062] The structural formulas of vinylphosphonic acid, acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), acid phosphooxypolyoxyethylene glycol monomethacrylate, and acid phosphooxypolyoxypropylene glycol monomethacrylate are represented by the following formulas (A-1) to (A-4), respectively.

[0063] [ka]

[0064] During synthesis, these compounds may contain a (meth)acrylate compound having two functional groups, such as those represented by general formula (D) or (E) described below.

[0065] Specific examples of the above formula (B) include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, methacryloylcholine chloride, and the like. Among these, dimethylaminoethyl (meth)acrylate, methacryloylcholine chloride, and 2-(t-butylamino)ethyl (meth)acrylate are preferably used, and dimethylaminoethyl (meth)acrylate is most preferably used.

[0066] The structural formulas of dimethylaminoethyl acrylate (=2-(dimethylamino)ethyl acrylate), diethylaminoethyl methacrylate (=2-(diethylamino)ethyl methacrylate), dimethylaminoethyl methacrylate (=2-(dimethylamino)ethyl methacrylate), methacryloylcholine chloride and 2-(t-butylamino)ethyl methacrylate (=2-(t-butylamino)ethyl methacrylate) are respectively represented by the following formulas (B-1) to (B-5).

[0067] [ka]

[0068] Specific examples of the above formula (C) include linear or branched alkyl esters of (meth)acrylic acid such as butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, lauryl (meth)acrylate, and stearyl (meth)acrylate; cyclic alkyl esters of (meth)acrylic acid such as cyclohexyl (meth)acrylate and isobornyl (meth)acrylate; aralkyl esters of (meth)acrylic acid such as benzyl (meth)acrylate and phenethyl (meth)acrylate; styrene-based monomers such as styrene, methylstyrene, and chloromethylstyrene; vinyl ether-based monomers such as methyl vinyl ether and butyl vinyl ether; and vinyl ester-based monomers such as vinyl acetate and vinyl propionate. Among these, butyl (meth)acrylate or cyclohexyl (meth)acrylate is preferably used.

[0069] The structural formulas of butyl methacrylate (=butyl methacrylate) and cyclohexyl methacrylate (=cyclohexyl methacrylate) are represented by the following formulas (C-1) and (C-2), respectively.

[0070] [ka]

[0071] In another embodiment of the copolymer according to the present invention, in addition to the compounds represented by the above formulae (A), (B) and optionally (C), an arbitrary fourth component may be copolymerized. For example, a (meth)acrylate compound having two or more functional groups may be copolymerized as the fourth component, and a part of the polymer may be partially three-dimensionally crosslinked. As such a fourth component, for example, a compound represented by the following formula (D) or (E):

[0072] [ka]

[0073] [In the formula, T d , T e and U e each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R d and R e each independently represents a linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom; and n represents an integer of 1 to 6. In other words, the copolymer according to the present invention preferably contains a crosslinked structure derived from such a bifunctional monomer.

[0074] In formulas (D) and (E), T d and T e are preferably each independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably each independently a hydrogen atom or a methyl group.

[0075] In formula (E), U e is preferably a hydrogen atom, a methyl group or an ethyl group, and more preferably a hydrogen atom.

[0076] In formula (D), R d are preferably linear or branched alkylene groups having 1 to 3 carbon atoms which may be substituted with a halogen atom, more preferably each independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group. In formula (D), n preferably represents an integer of 1 to 5, and particularly preferably 1.

[0077] In formula (E), R e are preferably linear or branched alkylene groups having 1 to 3 carbon atoms which may be substituted with a halogen atom, more preferably each independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group. In formula (E), n preferably represents an integer of 1 to 5, and particularly preferably 1.

[0078] Preferred examples of the bifunctional monomer represented by formula (D) include ethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, and propylene glycol di(meth)acrylate. The bifunctional monomer represented by formula (E) is preferably bis(methacryloyloxymethyl) phosphate, bis[(2-methacryloyloxy)ethyl] phosphate, bis[3-(methacryloyloxy)propyl] phosphate, or a bifunctional monomer derived from the above formula (A-3) or (A-4). Of the formulae (D) and (E), the bifunctional monomer represented by formula (E) is more preferably used.

[0079] Moreover, an example of the trifunctional (meth)acrylate compound is phosphinylidinedri(oxy-2,1-ethanediyl) triacrylate.

[0080] Among these fourth components, particularly preferred are ethylene glycol dimethacrylate, dimethacrylates having repeating units of ethylene glycol and propylene glycol among the bifunctional monomers derived from the above formulae (A-3) and (A-4), bis[2-(methacryloyloxy)ethyl]phosphate, and dimethacrylates having repeating units of ethylene glycol and propylene glycol via a phosphate ester group among the bifunctional monomers derived from the above formulae (A-3) and (A-4), whose structural formulas are represented by the following formulae (D-1) to (D-3) and formulae (E-1) to (E-3), respectively.

[0081] [ka]

[0082] Among these, formulae (E-1) to (E-3) are particularly preferable.

[0083] The copolymer may contain one or more of these fourth components. The proportion of the fourth component in the copolymer, for example, the crosslinked structure derived from the bifunctional monomer represented by the above formula (D) or (E), is 0 mol % to 50 mol %, preferably 5 mol % to 45 mol %, and most preferably 10 mol % to 40 mol %.

[0084] The proportion of the compound represented by formula (A) to the total monomers forming the copolymer is 3 mol % to 80 mol %. The compound represented by formula (A) may be of two or more kinds. The proportion of the compound represented by formula (B) to the total monomers forming the copolymer is 3 mol % to 80 mol %. The compound represented by formula (B) may be of two or more kinds.

[0085] The ratio of the compound represented by formula (C) to the total monomers forming the copolymer may be the remainder after subtracting the ratios of the compounds represented by formulas (A) and (B), and may be, for example, 0 mol % to 90 mol %. The compound represented by formula (C) may be of two or more kinds.

[0086] The copolymer according to the present invention may further include an ethylenically unsaturated monomer, or a polysaccharide or a derivative thereof as an optional fifth component. Examples of the ethylenically unsaturated monomer include one or more ethylenically unsaturated monomers selected from the group consisting of (meth)acrylic acid and its esters, vinyl acetate, vinylpyrrolidone, ethylene, vinyl alcohol, and hydrophilic functional derivatives thereof. Examples of polysaccharides or derivatives thereof include cellulose polymers such as hydroxyalkylcellulose (e.g., hydroxyethylcellulose or hydroxypropylcellulose), starch, dextran, and curdlan.

[0087] Examples of the hydrophilic functional group of the hydrophilic functional derivative include phosphoric acid, phosphonic acid and their ester structures; betaine structures; amide structures; alkylene glycol residues; amino groups; and sulfinyl groups.

[0088] Here, the phosphoric acid and its ester structure are represented by the following formula: [ka] [Where, R 11 , R 12 and R 13 are each independently a hydrogen atom or an organic group (e.g., a linear or branched alkyl group having 1 to 5 carbon atoms, etc.). The phosphonic acid and its ester structure are represented by the following formula: [ka] [Where, R 14 and R 15are each independently a hydrogen atom or an organic group (e.g., a linear or branched alkyl group having 1 to 5 carbon atoms, etc.). Examples of ethylenically unsaturated monomers having such a structure include acid phosphooxyethyl (meth)acrylate and vinyl phosphonic acid.

[0089] The betaine structure means a monovalent or divalent group of a compound having an amphoteric center of a quaternary ammonium type cation structure and an acidic anion structure, for example, a phosphorylcholine group: [ka] An example of an ethylenically unsaturated monomer having such a structure is 2-methacryloyloxyethyl phosphorylcholine (MPC).

[0090] The amide structure has the following formula: [ka] [Where, R 16 , R 17 and R 18 are each independently a hydrogen atom or an organic group (e.g., a methyl group, a hydroxymethyl group, a hydroxyethyl group, etc.). It means a group represented by the following formula: Examples of ethylenically unsaturated monomers having such a structure include (meth)acrylamide, N-(hydroxymethyl)(meth)acrylamide, etc. Furthermore, monomers or polymers having such a structure are disclosed, for example, in JP-A-2010-169604 and the like.

[0091] The alkylene glycol residue means an alkyleneoxy group (-Alk-O-) remaining after one or both terminal hydroxyl groups of an alkylene glycol (HO-Alk-OH; where Alk is a linear or branched alkylene group having 1 to 10 carbon atoms) undergoes a condensation reaction with another compound, and also includes a poly(alkyleneoxy) group in which alkyleneoxy units are repeated. Examples of ethylenically unsaturated monomers having such a structure include 2-hydroxyethyl (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, etc. Furthermore, monomers or polymers having such a structure are disclosed, for example, in JP 2008-533489 A.

[0092] The amino group has the formula: -NH2, -NHR 19 or -NR 20 R 21 [Where, R 19 , R 20 and R 21 are each independently an organic group (e.g., a straight-chain or branched alkyl group having 1 to 5 carbon atoms, etc.). In this specification, the amino group includes quaternized or salified amino groups. Examples of ethylenically unsaturated monomers having such a structure include dimethylaminoethyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, and methacryloylcholine chloride.

[0093] The sulfinyl group has the formula: [ka] [Where, R 22 is an organic group (e.g., an organic group having 1 to 10 carbon atoms, preferably an alkyl group having 1 to 10 carbon atoms having one or more hydroxyl groups, etc.). An example of a polymer having such a structure is a copolymer disclosed in JP-A-2014-48278.

[0094] <Method of producing copolymer> The copolymer according to the present invention can be synthesized by a method such as radical polymerization, anionic polymerization, or cationic polymerization, which is a general method for synthesizing acrylic polymers or methacrylic polymers, etc. The form of the polymerization can be various methods such as solution polymerization, suspension polymerization, emulsion polymerization, and bulk polymerization.

[0095] The solvent in the polymerization reaction may be water, a phosphate buffer solution, an alcohol such as ethanol, or a mixed solvent of these, but preferably contains water or ethanol. It is more preferable that the solvent contains 10% by mass or more and 100% by mass or less of water or ethanol. It is more preferable that the solvent contains 50% by mass or more and 100% by mass or less of water or ethanol. It is more preferable that the solvent contains 80% by mass or more and 100% by mass or less of water or ethanol. It is more preferable that the solvent contains 90% by mass or more and 100% by mass or less of water or ethanol. Preferably, the total of water and ethanol is 100% by mass.

[0096] As for the reaction concentration, for example, the concentration of the compound represented by the above formula (A) or formula (B) in the reaction solvent is preferably 0.01% by mass to 4% by mass. If the concentration exceeds 4% by mass, for example, the strong association of the phosphate group represented by formula (A) may cause the copolymer to gel in the reaction solvent. If the concentration is less than 0.01% by mass, the concentration of the obtained varnish is too low, making it difficult to prepare a coating agent for obtaining a coating film with a sufficient thickness. It is more preferable that the concentration is 0.01% by mass to 3% by mass, for example, 3%, 2% by mass, or 1% by mass.

[0097] In the synthesis of the copolymer according to the present invention, for example, the salt represented by the following formula (1) may be converted and then polymerized together with a compound represented by formula (C) to prepare the copolymer.

[0098] [ka]

[0099] Since the phosphate group-containing monomer is a monomer that easily associates, it may be added dropwise to the reaction solvent in small amounts so that it can be dispersed quickly when added dropwise to the reaction system.

[0100] Additionally, the reaction solvent may be heated (eg, 40° C. to 100° C.) to increase the solubility of the monomers and polymers.

[0101] In order to efficiently advance the polymerization reaction, it is desirable to use a polymerization initiator. Examples of polymerization initiators include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile) (manufactured by Wako Pure Chemical Industries, Ltd.; VA-065, 10-hour half-life temperature; 51°C), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile), 1-[( 1-cyano-1-methylethyl)azo]formamide, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide] (Wako Pure Chemical Industries, Ltd.; VA-086, 10-hour half-life temperature; 86°C), benzoyl peroxide (BPO), 2,2'-azobis[N-(2-carboxyethyl)-2-methyl azobis[2-(2-imidazolin-2-yl)propane]n-hydrate (Wako Pure Chemical Industries, Ltd.; VA-057, 10-hour half-life temperature; 57°C), 4,4'-azobis(4-cyanopentanoic acid) (Wako Pure Chemical Industries, Ltd.; V-501), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (Wako Pure Chemical Industries, Ltd.; VA-044, 10-hour half-life temperature; 44°C), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfide Examples of compounds that can be used include fert dihydrate (Wako Pure Chemical Industries, Ltd.; VA-046B, 10-hour half-life temperature: 46°C), 2,2'-azobis[2-(2-imidazolin-2-yl)propane] (Wako Pure Chemical Industries, Ltd.; VA-061, 10-hour half-life temperature: 61°C), 2,2'-azobis(2-amidinopropane) dihydrochloride (Wako Pure Chemical Industries, Ltd.; V-50, 10-hour half-life temperature: 56°C), peroxodisulfuric acid, and t-butyl hydroperoxide. In consideration of solubility in water, ion balance, and interaction with the monomer, it is preferable to select from 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n-hydrate, 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] disulfate dihydrate, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis(2-amidinopropane) dihydrochloride, and peroxodisulfuric acid. Considering the solubility in organic solvents, ion balance, and interaction with monomers, it is desirable to use 2,2'-azobis(2,4-dimethylvaleronitrile) or 2,2'-azobis(isobutyronitrile).

[0102] The amount of the polymerization initiator added is 0.05% by mass to 10% by mass based on the total weight of the monomers used in the polymerization.

[0103] The reaction conditions are as follows: the reaction vessel is heated to 50° C. to 200° C. in an oil bath or the like, and the mixture is stirred for 1 to 48 hours, more preferably at 80° C. to 150° C., for 5 to 30 hours, whereby the polymerization reaction proceeds to obtain the copolymer of the present invention. The reaction atmosphere is preferably a nitrogen atmosphere.

[0104] As for the reaction procedure, all the reactants may be placed in a reaction solvent at room temperature and then heated to the above temperature to polymerize, or the whole or part of the mixture of reactants may be added dropwise in small portions to a pre-heated solvent.

[0105] According to the latter reaction procedure, the copolymer of the present invention can be prepared by a production method including a step of dropping a mixture containing the compounds represented by the above formulas (A), (B) and, optionally, (C), a solvent and a polymerization initiator into a solvent kept at a temperature higher than the 10-hour half-life temperature of the polymerization initiator, and reacting (polymerizing) them.

[0106] The molecular weight of the copolymer according to the present invention may be several thousand to several million, preferably 5,000 to 5,000,000. More preferably, it is 10,000 to 2,000,000, and most preferably 5,000 to 1,000,000. In addition, it may be any of random copolymers, block copolymers, and graft copolymers, and the copolymerization reaction for producing the copolymer is not particularly limited, and known methods of synthesis in a solution such as polymerization using radical polymerization, ionic polymerization, photopolymerization, and emulsion polymerization can be used. Depending on the intended use, any of the copolymers of the present invention may be used alone, or multiple copolymers may be mixed and used at different ratios.

[0107] The coating agent used to form a coating on the biological sample storage container according to the present invention may be prepared by diluting the desired copolymer, optionally with a desired solvent, to a desired concentration.

[0108] Examples of such a solvent include water, phosphate buffered saline (PBS), and alcohols, such as alcohols having 2 to 6 carbon atoms, for example, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (=neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (=t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, and 3,3-dimethyl. Examples of the solvent include cyclohexanol, ...

[0109] Furthermore, the coating agent may be prepared from a copolymer-containing varnish. The copolymer-containing varnish can be prepared, for example, by a production method including a step of reacting (polymerizing) the compounds represented by the above formulas (A), (B) and, optionally, (C) in a solvent at a total compound concentration of 0.01% by mass to 20% by mass.

[0110] The concentration of solids in the coating agent is preferably 0.01 to 50% by mass in order to form a uniform coating film. The concentration of the copolymer in the coating agent is preferably 0.01 to 4% by mass, more preferably 0.01 to 3% by mass, particularly preferably 0.01 to 2% by mass, and even more preferably 0.01 to 1% by mass. If the concentration of the copolymer is less than 0.01% by mass, the concentration of the copolymer in the coating agent is too low to form a coating film with a sufficient thickness, and if it exceeds 4% by mass, the storage stability of the coating agent is deteriorated, and precipitation or gelation of the dissolved material may occur.

[0111] In addition to the copolymer and the solvent, other substances may be added to the coating agent as necessary within the range that does not impair the performance of the resulting coating film. Examples of other substances include preservatives, surfactants, primers that improve adhesion to the substrate, antifungal agents, and sugars.

[0112] In order to adjust the ion balance of the copolymer in the coating agent, the method may further include a step of adjusting the pH of the coating agent in advance. The pH adjustment may be performed, for example, by adding a pH adjuster to a composition containing the copolymer and a solvent, and adjusting the pH of the composition to 2 or more, preferably 2 to 9, more preferably 2.5 to 8.5, and even more preferably 3 to 8. The type and amount of the pH adjuster that can be used are appropriately selected depending on the concentration of the copolymer, the abundance ratio of its anion and cation, and the like. Examples of pH adjusters include organic amines such as ammonia, diethanolamine, pyridine, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane; alkali metal hydroxides such as potassium hydroxide and sodium hydroxide; alkali metal halides such as potassium chloride and sodium chloride; inorganic acids or alkali metal salts thereof such as sulfuric acid, phosphoric acid, hydrochloric acid, and carbonic acid; quaternary ammonium cations such as choline, or mixtures thereof (e.g., buffer solutions such as phosphate buffered saline). Among these, ammonia, diethanolamine, sodium hydroxide, choline, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane are preferred, and ammonia, diethanolamine, sodium hydroxide, and choline are particularly preferred.

[0113] The biological sample storage container of the present invention has a coating formed from the above coating agent on at least a portion of the surface of the storage container, specifically, on at least a portion of the inner and / or outer surface of the container that may come into contact with a biological sample containing a biomarker.

[0114] <Container> The container coated with the copolymer constituting the biological sample storage container of the present invention may be any type of container that can be used in the art, and examples thereof include dishes / petri dishes such as Petri dishes, tissue culture dishes, and multi-dishes that are commonly used for storing biological samples, flasks such as cell culture flasks and spinner flasks, bags such as plastic bags, Teflon (registered trademark) bags, and culture bags, vials such as screw vials, tubes such as culture tubes, centrifuge tubes, and microtubes, trays, roller bottles, etc. Preferred are vials and tubes.

[0115] Examples of the material of the container include glass, metal, metal-containing compound or metalloid-containing compound, activated carbon, and resin. Examples of the metal include typical metals (alkali metals: Li, Na, K, Rb, Cs; alkaline earth metals: Ca, Sr, Ba, Ra), magnesium group elements: Be, Mg, Zn, Cd, Hg, aluminum group elements: Al, Ga, In, rare earth elements: Y, La, Ce, Pr, Nd, Sm, Eu, tin group elements: Ti, Zr, Sn, Hf, Pb, Th, iron group elements: Fe, Co, Ni, earth elements: V, Nb, Ta, chromium group elements: Cr, Mo, W, U, manganese group elements: Mn, Re, precious metals: Cu, Ag, Au, platinum group elements: Ru, Rh, Pd, Os, Ir, Pt, etc. Examples of metal-containing compounds or metalloid-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and are hardened by heat treatment at high temperatures; semiconductors such as silicon, metal oxides or metalloid oxides (silicon oxide, alumina, etc.), metal carbides or metalloid carbides, metal nitrides or metalloid nitrides (silicon nitride, etc.), metal borides or metalloid borides, and other inorganic solid materials; aluminum, nickel titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).

[0116] The resin may be a natural resin or a derivative thereof, or a synthetic resin. Examples of the natural resin or a derivative thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose with immobilized dextran sulfate. Examples of the synthetic resin include polyacrylonitrile (PAN), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polycarbonate (PC), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), and Teflon (registered trademark). In the manufacture of the biological sample storage container of the present invention, since no high temperature treatment is required when coating the copolymer so that it is present on at least a portion of the surface of the container, resins with low heat resistance can also be used.

[0117] The container may be made of one material or a combination of two or more materials. Among these materials, glass, silicon, silicon oxide, polystyrene (PS), polypropylene (PP), polyethersulfone (PES), polyethylene terephthalate (PET), polycarbonate (PC), polyvinyl chloride (PVC), Teflon (registered trademark), cycloolefin polymer (COP), polydimethylsiloxane (PDMS), or stainless steel (SUS304, SUS316, SUS316L, etc.) alone or in combination is preferred, and glass, polystyrene (PS), polypropylene (PP), or stainless steel (SUS304, SUS316, SUS316L, etc.) is particularly preferred.

[0118] <Method of manufacturing container for storing biological samples> The present invention relates to a method for producing a biological sample storage container having a coating on at least a part of the surface of the container, comprising a step of contacting at least a part of the surface of the container with the coating agent as described above. There is no particular limitation on the contact between the coating agent and the surface of the container, and methods such as immersing the container in the coating agent, adding the coating agent to the container and leaving it for a predetermined time, or applying the coating agent to the surface of the container are used, but a method of adding the coating agent to the container and leaving it for a predetermined time is preferred. The addition can be performed, for example, by adding the coating agent in an amount of 0.5 to 1 times the total volume of the container using a syringe or the like. The leaving is performed by appropriately selecting the time and temperature depending on the material of the container and the type of coating agent, and is performed, for example, for 5 minutes to 24 hours, preferably 30 minutes to 3 hours, at 10 to 35°C, preferably 20 to 30°C, and most preferably 25°C. This makes it possible to produce a biological sample storage container having a coating on at least a part, preferably the entire surface of the container.

[0119] Furthermore, the coating on the surface of the container obtained by this method can be used as a container for storing biological samples after the step of contacting the coating agent with at least a portion of the surface of the container, preferably after the step of adding the coating agent and leaving it for a predetermined period of time, and if any excess coating agent remains, it can be removed from the container and used as is without a drying step, or after washing with water or the medium of the sample to be stored (e.g., water, a buffer solution, a culture medium, etc.) as a container for storing biological samples. That is, after the step of contacting at least a portion of the surface of the container, preferably after the step of adding a coating agent and leaving it for a predetermined period of time, within 48 hours, preferably within 24 hours, more preferably within 12 hours, more preferably within 6 hours, more preferably within 3 hours, and even more preferably within 1 hour, if any excess coating agent remains, it is removed from the container, and the container can be used as a storage container for biological samples without a drying step, or after washing with water or the medium of the sample to be stored (e.g., water, a buffer solution, a culture medium, etc., particularly preferably a culture medium (e.g., DMEM medium (Dulbecco's modified Eagle's medium)).

[0120] The container may be subjected to a drying step, which is carried out under atmospheric pressure or under vacuum, preferably at a temperature within the range of −200° C. to less than 200° C. The drying step removes the solvent in the coating agent, and also causes the copolymer of the present invention represented by formula (a) and formula (b) to form ionic bonds with each other, thereby completely adhering the copolymer to the substrate. The coating can be formed by drying at room temperature (10°C to 35°C, preferably 20°C to 30°C, e.g., 25°C), but in order to form the coating more quickly, drying at, for example, 40°C to 50°C may be used. A drying process at extremely low or low temperatures (around -200°C to -30°C) using the freeze-drying method may also be used. Freeze-drying is called vacuum freeze-drying, and is a method in which the material to be dried is usually cooled with a refrigerant and the solvent is removed by sublimation in a vacuum state. Common refrigerants used in freeze-drying include a mixture of dry ice and methanol (-78°C), liquid nitrogen (-196°C), etc.

[0121] If the drying temperature is less than -200°C, an uncommon refrigerant must be used, resulting in lack of versatility, and drying takes a long time due to solvent sublimation, resulting in poor efficiency. If the drying temperature is 200°C or higher, the ionic bond reaction on the coating surface proceeds too much, causing the surface to lose hydrophilicity, and the biomarker adhesion inhibitory function is not exerted. The more preferred drying temperature is 10°C to 180°C, and the more preferred drying temperature is 25°C to 150°C. The coating of the present invention is produced through the above-mentioned simple steps.

[0122] In addition, in order to remove impurities remaining in the coating, unreacted monomers, etc., and further to adjust the ion balance of the copolymer in the coating, a step of washing with at least one solvent selected from water and an aqueous solution containing an electrolyte may be carried out. Washing is preferably performed with running water or ultrasonic washing. The aqueous solution containing water and an electrolyte may be heated, for example, to a temperature in the range of 40°C to 95°C. The aqueous solution containing an electrolyte is preferably PBS, saline (containing only sodium chloride), Dulbecco's phosphate buffered saline, Tris buffered saline, HEPES buffered saline, and Veronal buffered saline, and PBS is particularly preferred. After adhesion, the coating film remains firmly attached to the substrate without being eluted even when washed with water, PBS, alcohol, etc. The formed coating can be easily removed by washing with water, etc. even if cells or proteins are attached to the formed coating, and the surface of the container on which the coating of the present invention is formed has the ability to inhibit adhesion of biomarkers.

[0123] The thickness of the coating applied to the surface of the container of the present invention can be adjusted appropriately depending on the shape of the container, the type of sample, etc., and may be approximately uniform over the entire surface of the container or may be partially uneven; however, there is no particular limitation and the thickness is preferably 10 to 1000 Å, more preferably 10 to 500 Å, and most preferably 10 to 300 Å.

[0124] <Other embodiments> The present invention also relates to a storage container for a peptide, the surface of which is provided with a coating containing a copolymer containing a repeating unit containing a group represented by formula (a), a repeating unit containing a group represented by formula (b), and optionally a repeating unit containing a group represented by formula (c) on at least a part of the surface of the container. The meanings of the copolymer, coating, container, etc. in the storage container are as described above. In addition, the peptide is preferably contained in a biological sample to be subjected to mass spectrometry.

[0125] The present invention also relates to a container for fragmenting a protein and / or peptide, the container having a coating on at least a part of the surface thereof, the coating comprising a copolymer comprising a repeating unit comprising a group represented by formula (a), a repeating unit comprising a group represented by formula (b), and optionally a repeating unit comprising a group represented by formula (c). The meanings of the copolymer, coating, container, etc. in such a processing container are as described above. The fragmentation process means treating a protein or peptide with a proteolytic enzyme (protease) such as trypsin to break it down into fragments (=peptides) with smaller molecular weights. A protein and / or peptide sample is fragmented with a protease, and the resulting sample containing many peptides can be subjected to mass spectrometry.

[0126] The present invention also relates to a method for analyzing fragmented proteins and / or peptides, comprising: preparing a sample containing a protein and / or peptide to be analyzed; fragmenting the protein and / or peptide in the sample in a container having, on at least a part of its surface, a coating containing a copolymer containing a repeating unit containing a group represented by formula (a), a repeating unit containing a group represented by formula (b), and optionally a repeating unit containing a group represented by formula (c); and providing the fragmented protein and / or peptide in the sample to a measuring device. The meanings of the copolymer, coating, container, etc. in this method are as described above. In the method for analyzing fragmented proteins and / or peptides of the present invention, fragmenting the protein and / or peptide to be analyzed in a container having a specific coating suppresses adhesion of the fragmented peptides to the surface of the processing container, reduces sample loss, and enables accurate and highly precise analysis, thereby improving the sequence match rate.

[0127] The present invention also relates to a method for measuring the absolute amount of a biomarker in a solution, comprising the steps of preparing a sample containing the biomarker to be analyzed, storing and / or preserving a solution containing the biomarker contained in the biological sample in a biological sample storage container having, on at least a part of its surface, a coating containing a copolymer comprising a repeating unit containing a group represented by formula (a), a repeating unit containing a group represented by formula (b), and optionally a repeating unit containing a group represented by formula (c), and then measuring the absolute amount of the biomarker. The meanings of the copolymer, coating, container, etc. in this method are as described above. When the sample is subjected to a pretreatment such as dilution before measurement, the pretreatment container also preferably has a coating containing the copolymer on at least a part of its surface. When the dilution treatment is performed multiple times, it is preferable that all of the pretreatment containers have a coating containing the copolymer on at least a part of their surface. The above method can suppress the loss of the biomarker in a sample containing the biomarker, and can accurately measure the absolute amount of the biomarker in the sample. When a plurality of biomarkers are present in the sample, the content ratio of each can be accurately measured by measuring the absolute amount of each biomarker.

[0128] The present invention also relates to a method for reducing loss of a biomarker contained in a biological sample, the method comprising: preparing a biological sample containing a biomarker to be analyzed; and containing and / or storing a solution containing the biological sample in a storage container for the biological sample, the storage container having, on at least a part of its surface, a coating comprising a copolymer comprising a repeating unit comprising a group represented by formula (a), a repeating unit comprising a group represented by formula (b), and optionally a repeating unit comprising a group represented by formula (c). In this method, the meanings of the terms biological sample, copolymer, coating, container, etc. are as described above.

[0129] The present invention also relates to the use of a coating comprising a copolymer comprising a repeating unit comprising a group represented by formula (a), a repeating unit comprising a group represented by formula (b), and optionally a repeating unit comprising a group represented by formula (c) in a container for containing and / or storing a solution containing a biological sample, in order to reduce loss of a biomarker contained in the biological sample. In such a use, at least a portion of the surface of the container, preferably at least a portion or all of the internal and / or external surfaces of the container that may come into contact with the biological sample, has a coating comprising the copolymer. EXAMPLES

[0130] The present invention will be described in more detail below based on Synthesis Examples, Examples, Test Examples, etc., but the present invention is not limited to these.

[0131] <Synthesis Example 1> 260 g of acid phosphooxyethyl methacrylate (product name: Hosmer M, manufactured by Unichemical Co., Ltd., non-volatile content at 100°C for 1 hour: 91.8%, mixture of acid phosphooxyethyl methacrylate (44.2% by mass), bis[2-(methacryloyloxy)ethyl]phosphate (28.6% by mass), and other substances (27.2% by mass) was added to 390 g of ethanol, and while cooling to below 35°C, 310 g of choline (48-50% aqueous solution: manufactured by Tokyo Chemical Industry Co., Ltd.) was added and stirred until homogenous. 220 g of methacryloylcholine chloride 80% aqueous solution (manufactured by Tokyo Chemical Industry Co., Ltd.) and 300 g of butyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) were added to this mixture, and an additional 260 g of ethanol was added and stirred. Furthermore, an aqueous solution of 22 g of 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate (product name: VA-057, manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 230 g of pure water was added to the above solution while maintaining the temperature at 35 ° C or lower, and the mixture containing all of the above was introduced into a three-necked flask via a dropping pump after thorough stirring until it became homogenous. Meanwhile, 650 g of pure water and 980 g of ethanol were separately added to a three-necked flask equipped with a cooling tube, and nitrogen was flowed and the temperature was raised to the reflux temperature while stirring. While maintaining this state, the above mixture was dropped into a boiling liquid of pure water and ethanol over a period of 1.5 hours using a dropping pump connected to a Teflon tube. After dropping, the mixture was heated and stirred while maintaining the above environment for 2 hours. After 2 hours, the mixture was cooled to obtain 3610 g of a copolymer-containing varnish with a solid content of about 24.20 mass%. The weight average molecular weight of the resulting liquid in GFC was about 23,225.

[0132] <Preparation Example 1> A coating agent was prepared by adding 157 g of 1 mol / L hydrochloric acid (1N) (Kanto Chemical Co., Ltd.), 1697 g of pure water, and 4456 g of ethanol to 568 g of the copolymer-containing varnish obtained in Synthesis Example 1, and thoroughly stirring the mixture. The pH was 2.6.

[0133] <Example 1> The coating agent obtained in Preparation Example 1 was placed in a polypropylene (PP) jacket tube (FCR&Bio, #JRDS-0M) at 1.0 mL, in a PP microtube at 1.5 mL, and in a PP screw vial (YMC, #XRAV1103-P) at 300 μL, and left to stand at 25° C. for 0.5 hours. After removing the coating agent, the tube and vial were dried at 25° C. for 3 hours. After that, the tube and vial were thoroughly washed with pure water to obtain a PP tube and vial on which a coating film was formed.

[0134] <Test Example 1> Immunoglobulin G (IgG, Sigma-Aldrich, #I4506) was dissolved in phosphate buffered saline (PBS) to prepare a solution with a concentration of 2 μg / mL, and 1 mL of the solution was dispensed into the PP jacketed tube with the coating film formed thereon obtained in Example 1. As a comparative example, the IgG solution of the same concentration was dispensed into an uncoated PP jacketed tube or a ProteoSave (registered trademark) SS 1.5 mL slim tube (manufactured by Sumitomo Bakelite Co., Ltd., #MS-4202X) (Comparative Example 1 and Comparative Example 2, respectively). The dispensed samples were stored frozen (-80°C) for 5 days. The samples after storage were left to stand at room temperature for 30 minutes, then vortexed and transferred to a pretreatment tube (a PP microtube with the coating film obtained in Example 1). For Comparative Example 1 and Comparative Example 2, the samples were transferred to an uncoated PP microtube or a ProteoSave (registered trademark) SS 1.5 mL microtube (manufactured by Sumitomo Bakelite Co., Ltd., #MS-4215M), respectively. Furthermore, for Comparative Example 3 and Comparative Example 4, the samples stored in the coated tubes were transferred to an uncoated PP microtube or a ProteoSave (registered trademark) SS 1.5 mL microtube (manufactured by Sumitomo Bakelite Co., Ltd., #MS-4215M), respectively.

[0135] [Table 1]

[0136] For each of the above samples, 800 μL of cold acetone (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the protein was allowed to stand at -20 ° C for 1 hour or more. After that, the protein was precipitated by centrifugation at 4 ° C and 16000 × g for 10 minutes, and the supernatant was removed, and then 100 μL of Tris-HCl buffer (pH 8.3) was added to redissolve it. 2.1 μL of 0.5 mol / L dithiothreitol (DTT, manufactured by Thermo Fisher Scientific Co., Ltd.) and 11.5 μL of 0.5 mol / L iodoacetamide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and after stirring well, the mixture was allowed to stand in the dark for 20 minutes. Then, 460 μL of cold acetone (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after stirring well, the mixture was allowed to stand at -20 ° C for 1 hour. After stirring well, the mixture was centrifuged at 4 ° C and 16000 × g for 5 minutes, and the supernatant was removed. Next, 50 μL of 90% cold acetone was added, and after thorough stirring, the mixture was centrifuged again at 4°C and 16000×g for 5 minutes, and the supernatant was removed. Furthermore, 100 μL of 50 mmol / L ammonium bicarbonate solution (pH 8.0) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to redissolve the protein pellet. 2 μL of 0.1 mg / mL trypsin (manufactured by Sigma) dissolved in 50 mmol / L acetic acid solution was added, and the mixture was shaken overnight at 37°C. Then, 10 μL of 20% trifluoroacetic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to prepare a sample, which was then transferred to an HPLC vial (a PP screw vial with the coating film obtained in Example 1). For comparative examples, the sample was transferred to an uncoated PP screw vial or an ultra-low adsorption 0.3 mL vial ProteoSave (manufactured by AMR, #PSVial). As described above, the samples transferred to HPLC vials were subjected to measurement by HPLC-mass spectrometry under the following conditions.

[0137] HPLC conditions HPLC equipment: Nexera X2 (Shimadzu Corporation) Mass spectrometer: Orbitrap Fusion (Thermo Fisher Scientific) Column: PLRP-S (300 Å, 5 μm, 150 × 2.1 mm, Agilent Technologies) Eluent: 0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile Gradient: 90 / 10 → 50 / 50 (0-10 min), 50 / 50 → 5 / 95 (10-12 min), 5 / 95 (12-17 min) Flow rate: 0.25mL / mL Column temperature: 55℃ Injection volume: 5μL

[0138] After HPLC measurement, the mass chromatograms of each of the eight peptides (VVSVLTVLHQDWLNGK, TPEVTCVVVDVSHEDPEVK, FNWYVDGVEVHNAK, GPSVFPLAPSSK, DTLMISR, VVSVLTVVHQDWLNGK, GPSVFPLAPCSR, STSESTAALGCLVK) were extracted, the areas were calculated, and the sum was compared with Comparative Example 1 and Comparative Example 2. The results are shown in FIG. 1. As a result, it was revealed that the area value was significantly larger when the coating was performed in all three steps than in the comparative examples. In addition, the results of comparison with Comparative Example 3 and Comparative Example 4 are shown in FIG. 2. As a result, it was revealed that the area value was significantly larger when the coating was performed even in the steps of the pretreatment container and HPLC vial alone than in the comparative examples.

[0139] <Test Example 2> Bovine serum albumin (BSA, Sigma-Aldrich, #A9647) was dissolved in phosphate buffered saline (PBS) to prepare solutions with concentrations of 0.4 μg / mL, 2 μg / mL, and 10 μg / mL, and 1 mL of each solution was dispensed into the PP jacketed tube with the coating film formed thereon obtained in Example 1. As a comparative example, an IgG solution of the same concentration was dispensed into an uncoated PP jacketed tube or a ProteoSave (registered trademark) SS 1.5 mL slim tube (manufactured by Sumitomo Bakelite Co., Ltd., #MS-4202X). The dispensed samples were stored frozen (-80°C) for 12 days (referred to as Comparative Example 5 and Comparative Example 6, respectively). The sample after storage was left to stand at room temperature for 30 minutes, then vortexed and transferred to a PP microtube (200 μL). The same procedures were carried out for the comparative example. For each of the above samples, 800 μL of cold acetone (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the protein was allowed to stand at -20 ° C for 1 hour or more. After that, the protein was precipitated by centrifugation at 4 ° C and 16000 × g for 10 minutes, and after removing the supernatant, 100 μL of Tris-HCl buffer (pH 8.3) was added to redissolve it. 2.1 μL of 0.5 mol / L dithiothreitol (DTT, manufactured by Thermo Fisher Scientific Co., Ltd.) and 11.5 μL of 0.5 mol / L iodoacetamide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added, and after stirring well, the mixture was allowed to stand in the dark for 20 minutes. Then, 460 μL of cold acetone (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and after stirring well, the mixture was allowed to stand at -20 ° C for 1 hour. After stirring well, the mixture was centrifuged at 4 ° C and 16000 × g for 5 minutes, and the supernatant was removed. Next, 50 μL of 90% cold acetone was added, and after thorough stirring, the mixture was centrifuged again at 4 ° C. and 16000 × g for 5 minutes, and the supernatant was removed. Furthermore, 100 μL of 50 mmol / L ammonium bicarbonate solution (pH 8.0) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to redissolve the protein pellet. 2 μL of 0.1 mg / mL trypsin (Sigma) dissolved in 50 mmol / L acetic acid solution was added, and the mixture was shaken overnight at 37 ° C., and then 10 μL of 20% trifluoroacetic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and the solvent was removed with a centrifugal concentrator (EZ-2 Elite, Genavac). 100 μL of 0.1% formic acid aqueous solution: acetonitrile mixture (50:50) was added to this to prepare a sample, which was then transferred to an HPLC vial (a PP screw vial). As described above, the samples transferred to HPLC vials were subjected to measurement by HPLC-mass spectrometry under the following conditions.

[0140] HPLC conditions HPLC equipment: Nexera X2 (Shimadzu Corporation) Mass spectrometer: Orbitrap Fusion (Thermo Fisher Scientific) Column: PLRP-S (300 Å, 5 μm, 150 × 2.1 mm, Agilent Technologies) Eluent: 0.1% formic acid aqueous solution / 0.1% formic acid acetonitrile Gradient: 90 / 10 → 50 / 50 (0-10 min), 50 / 50 → 5 / 95 (10-12 min), 5 / 95 (12-17 min) Flow rate: 0.25mL / mL Column temperature: 55℃ Injection volume: 10μL

[0141] After the HPLC measurement, the mass chromatograms of the three peptides (LGEYGFQNALIVR, LVNELTEFAK, KVPQVSTPTLVEVSR) were extracted, and the areas were calculated. The results of comparing each peptide with Comparative Example 5 and Comparative Example 6 are shown in Figure 3. As a result, it was revealed that the area values ​​were significantly larger when the coating was performed during the storage process than in the comparative examples. Furthermore, the percentage of identified peptides in the amino acid sequence of BSA was calculated in each measurement and the results are shown in Table 2. The percentage of identified peptides was significantly higher with this coating than with the comparative example, and it was clear that this coating was also superior in terms of qualitative analysis.

[0142] [Table 2]

[0143] <Example 2> The coating agent obtained in Preparation Example 1 was placed in polypropylene (PP) vials (Shimadzu GLC Corporation, #GLCTV-PP) in an amount of 300 μL each, and left to stand at 25° C. for 0.5 hours. After removing the coating agent from the vials, they were dried at 25° C. for 3 hours. The vials were then thoroughly washed with pure water to obtain PP vials on which a coating film had been formed. The coating agent obtained in Preparation Example 1 was placed in 1.5 mL PP tubes (Thermo Fisher Scientific or Sarstedt) in 1.5 mL portions and allowed to stand at 25° C. for 30 minutes. After removing the coating agent, the tubes were dried at 25° C. for 3 hours. After that, the tubes were thoroughly washed with pure water to obtain 1.5 mL PP tubes on which a coating film had been formed.

[0144] <Test Example 3> Amyloid β protein fragment 1-42 (Aβ42, Peptide Institute, #4349-v) was dissolved in a 1:1 (volume ratio) mixed solution of 0.3% ammonia water and acetonitrile to prepare a solution with a concentration of 1000 ppm. Using the PP vial on which the coating film obtained in Example 2 was formed, the solution was diluted 10-fold three or five times with a 1:1 (volume ratio) mixed solution of 0.3% ammonia water and acetonitrile. As a comparative example, a similar 10-fold dilution of Aβ42 was performed three or five times to prepare a diluted solution using an uncoated PP vial (Comparative Example 7). As described above, the sample in the PP HPLC vial was subjected to measurement by HPLC-mass spectrometry under the following conditions.

[0145] HPLC conditions HPLC equipment: LC-20AD (Shimadzu Corporation) Mass spectrometer: LTQ-Orbitrap (Thermo Fisher Scientific) Column: ACQUITY UPLC CSH C18 (2.1x150 mm, 1.7μm, manufactured by Nihon Waters, Inc.) Eluent: 0.3% aqueous ammonia solution / acetonitrile Gradient: 90 / 10 (0-1 min), 90 / 10 → 45 / 55 (1-6.5 min), 45 / 55 (6.5-6.7 min), 45 / 55 → 10 / 90 (6.7-7 min), 10 / 90 (7-9 min) Flow rate: 0.20mL / min Column temperature: 55℃ Injection volume: 5μL Ionization method: ESI Observation mode: Posi tSIM(m / z):1129.32 Scan width(m / z):2

[0146] The area of ​​the tetravalent ion at m / z=1129.32 was calculated by SIM measurement (selective ion monitoring), and the results compared with Comparative Example 7 are shown in Table 3 (results of 3 x 10 dilutions) and Table 4 (results of 5 x 10 dilutions) (N=2). The results are also shown in Figure 4 (results of 3 x 10 dilutions) and Figure 5 (results of 5 x 10 dilutions). As a result, it was clear that the area values ​​of the samples with the coating were significantly larger than those of the Comparative Example in both concentrations.

[0147] [Table 3]

[0148] [Table 4]

[0149] In addition, samples of 10-fold dilution 5 times were prepared in the same manner as above, stored at 4°C, and the area of ​​the tetravalent ion of m / z=1129.32 was calculated by SIM measurement after 6 hours and 15 hours, and the results compared with Comparative Example 7 are shown in Table 5 and Figure 6 (N=2). As a result, it was revealed that the area value was significantly larger when the coating was performed for both 6 hours and 15 hours than in the Comparative Example. Furthermore, the change from 6 hours to 15 hours ((average area value for 6 hours-average area value for 15 hours) x 100 / average area value for 6 hours) was 18.7% in Test Example 3 and 34.3% in Comparative Example 7, and it was found that the change was also significantly lower when the coating was performed than in the Comparative Example.

[0150] [Table 5]

[0151] <Test Example 4> Amyloid β protein fragment 1-42 (Aβ42, Peptide Institute, #4349-v) was dissolved in a mixed solution of 0.3% ammonia water and acetonitrile at a volume ratio of 9:1 to prepare a solution with a concentration of 1000 ppm. Using a 1.5 mL PP tube on which the coating film obtained in Example 2 was formed, the solution was diluted 10-fold four times with a mixed solution of 0.3% ammonia water and acetonitrile at a volume ratio of 9:1. The resulting solution was transferred to a PP vial on which the coating film obtained in Example 2 was formed, and measurements were performed by HPLC-mass spectrometry under the following conditions. The following three comparative examples were performed. In Comparative Example 8, a similar 10-fold diluted Aβ42 solution was prepared using a 1.5 mL tube made of PP on which a coating film was formed, the resulting solution was transferred to an uncoated PP vial, and the measurement was performed by HPLC-mass spectrometry under the following conditions. In Comparative Example 9, a similar 10-fold diluted Aβ42 solution was prepared using a 1.5 mL tube made of PP on which a coating film was formed obtained in Example 2, and the measurement was performed by HPLC-mass spectrometry under the following conditions. In Comparative Example 10, a similar 10-fold diluted Aβ42 solution was prepared using a 1.5 mL tube made of PP on which a coating film was formed, and the measurement was performed by HPLC-mass spectrometry under the following conditions.

[0152] [Table 6]

[0153] HPLC conditions HPLC equipment: LC-20AD (Shimadzu Corporation) Mass spectrometer: LTQ-Orbitrap (Thermo Fisher Scientific) Column: ACQUITY UPLC CSH C18 (2.1x150mm, 1.7μm, Japan Waters) Eluent: 0.3% aqueous ammonia / acetonitrile = 80 / 20 (0-13 min) Flow rate: 0.20mL / min Column temperature: 55℃ Injection volume: 5μL Ionization method: ESI Observation mode: Posi tSIM(m / z):1129.32 Scan width(m / z):2

[0154] The area of ​​the tetravalent ion at m / z=1129.32 was calculated by SIM measurement (selective ion monitoring), and the results of comparing Test Example 4 with Comparative Examples 8, 9, and 10 are shown in Table 7 and Figure 7. As a result, it was revealed that the area value was significantly larger when the coating was applied to both the dilution container and the HPLC vial.

[0155] [Table 7]

[0156] <Test Example 5> Amyloid β protein fragment 1-40 (Aβ40, Peptide Institute, #4307-v) was dissolved in a mixed solution of 0.3% ammonia water and acetonitrile at a volume ratio of 9:1 to prepare a solution with a concentration of 1000 ppm. Using a 1.5 mL PP tube on which the coating film obtained in Example 2 was formed, 10-fold dilution was performed four times with a mixed solution of 0.3% ammonia water and acetonitrile at a volume ratio of 9:1. The resulting solution was transferred to a PP vial on which the coating film obtained in Example 2 was formed, and measurement was performed by HPLC-mass spectrometry under the following conditions. Measurement was performed twice: immediately after transfer to the vial (0 h) and after 4 hours had passed in an autosampler kept at 4°C (4 h). The following three comparative examples were performed. In Comparative Example 11, a low-adsorption PP vial TORAST-H Bio Vial (Shimadzu GLC Corporation, #GLCTV-H-BIO) was used as the analytical vial, and the other conditions were the same as those of Test Example 5. In Comparative Example 12, an uncoated PP vial was used as the analytical vial, and the other conditions were the same as those of Test Example 5. In Comparative Example 13, a low-adsorption PP vial QuanRecovery with MaxPeak HPS 300 μL vials (Waters Corporation, #186009186) was used as the analytical vial, and the other conditions were the same as those of Test Example 5.

[0157] HPLC conditions HPLC equipment: LC-20AD (Shimadzu Corporation) Mass spectrometer: LTQ-Orbitrap (Thermo Fisher Scientific) Column: ACQUITY UPLC CSH C18 (2.1x150 mm, 1.7μm, Japan Waters) Eluent: 0.3% aqueous ammonia / acetonitrile = 80 / 20 (0-13 min) Flow rate: 0.20mL / min Column temperature: 55℃ Injection volume: 5μL Ionization method: ESI Observation mode: Posi tSIM(m / z):1083.29 Scan width(m / z):2

[0158] The area of ​​the tetravalent ion at m / z=1083.29 was calculated by SIM measurement (selective ion monitoring), and the results of comparing Test Example 5 with Comparative Examples 11, 12, and 13 are shown in Table 8 and Figure 8. As a result, it was revealed that the area value was significantly larger when the coating was applied to the HPLC vial. Furthermore, the area value significantly decreased after 4 hours in Comparative Examples 11, 12, and 13, while the area value in Test Example 5 was maintained at the same level.

[0159] [Table 8]

[0160] Therefore, when analyzing extremely small amounts of peptides known to be easily adsorbed, such as Aβ42 and Aβ40, it was confirmed that the coating suppresses adsorption, thereby reducing sample loss and enabling accurate and precise analysis.

Claims

1. A container for measuring a protein and / or peptide fragmented by a protease, comprising: A copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): 【Chemistry 1】 (In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An － represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. A container comprising, on at least a portion of a surface of the container, a coating comprising:

2. The copolymer further comprises a compound represented by the following formula (c): 【Chemistry 2】 [In the formula, R ｃ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, or an aryloxyalkyl group having 7 to 15 carbon atoms (wherein the aryl moiety is optionally substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom) The container according to claim 1 , comprising a repeating unit containing a group represented by:

3. The container according to claim 1 or 2, wherein the fragmented proteins and / or peptides are contained in a biological sample to be subjected to mass spectrometry.

4. A container for fragmenting a protein and / or peptide with a protease, comprising a copolymer comprising a repeating unit having a group represented by the following formula (a) and a repeating unit having a group represented by the following formula (b): 【Chemistry 3】 (In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An － represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. A container comprising, on at least a portion of a surface of the container, a coating comprising:

5. The copolymer further comprises a compound represented by the following formula (c): 【Chemistry 4】 [In the formula, R ｃ represents a linear or branched alkyl group having 1 to 18 carbon atoms, a cyclic hydrocarbon group having 3 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, or an aryloxyalkyl group having 7 to 15 carbon atoms (wherein the aryl moiety is optionally substituted with a linear or branched alkyl group having 1 to 5 carbon atoms which may be substituted with a halogen atom) The container according to claim 4 , comprising a repeating unit containing a group represented by:

6. A method for analyzing proteins and / or peptides fragmented by a protease, comprising the steps of: Providing a sample containing the proteins and / or peptides to be analyzed; A copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): 【Chemistry 5】 (In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An － represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. carrying out a proteolytic enzyme fragmentation treatment of proteins and / or peptides in the sample in a container having a coating comprising the compound on at least a part of its surface; and subjecting the fragmented proteins and / or peptides in the sample to a measuring device; A method comprising:

7. 1. A method for measuring the absolute amount of proteins and / or peptides fragmented by a proteolytic enzyme in a solution, comprising: A copolymer comprising a repeating unit containing a group represented by the following formula (a) and a repeating unit containing a group represented by the following formula (b): 【Chemistry 6】 (In the formula, U ａ１ , U ａ２ , U ｂ１ , U ｂ２ and U ｂ３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An － represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion. and then measuring the absolute amount of the fragmented protein and / or peptide.

8. The method of claim 7, wherein the protein and / or peptide is an antibody or an amyloid.

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