Glycan preparation method and glycan analysis method

Abstract

To provide a glycan preparation method for preparing labeled glycans quickly from glycoproteins.SOLUTION: A glycan preparation method comprises: a releasing step in which a glycosidase is allowed to act on a glycoprotein immobilized on a solid phase in a container to obtain a released product containing glycans; and a labeling step in which a labeling reagent is added to the released product in the container to obtain a labeled product containing labeled glycans, wherein the glycoprotein is an antibody, the solid phase has on its surface a ligand selected from the group consisting of protein A, protein G, protein L, protein H, protein D, and protein Arp, and the labeling reagent comprises a reducer, a solution and at least one selected from the group consisting of 8-aminopyrene-1,3,6-trisulfonic acid, a sodium salt thereof, and 9-aminopyrene-1,4,6-trisulfonic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a sugar chain and a method for analyzing a sugar chain. This application claims priority based on Japanese Patent Application No. 2023-169184 filed on September 29, 2023, Japanese Patent Application No. 2024-072974 filed on April 26, 2024, Japanese Patent Application No. 2024-113192 filed on July 16, 2024, Japanese Patent Application No. 2023-168984 filed on September 29, 2023, Japanese Patent Application No. 2024-072852 filed on April 26, 2024, and Japanese Patent Application No. 2024-113238 filed on July 16, 2024, the contents of which are incorporated herein by reference. [Background technology]

[0002] Many proteins that make up the body are glycosylated and exist as glycoproteins with attached sugar chains. The structure and distribution of sugar chains on glycoproteins are involved in the function of proteins, and it is known that sugar chain structures change with the onset and progression of many diseases.

[0003] Therefore, structural analysis of glycoproteins is expected to play an important role in various technological fields such as life science, medicine, and drug discovery, elucidating the mechanisms of pathogenesis of various diseases accompanied by changes in glycan structure, and developing treatment and diagnostic techniques for diseases. Generally, analysis of glycans bound to glycoproteins is performed after releasing the glycans from the glycoprotein, and then the released glycans obtained are analyzed.

[0004] A method for preparing released glycans from glycoproteins is known in which the glycans released from the glycoprotein are captured on a carrier that specifically binds to the glycans and then recovered (see, for example, Patent Document 1). The method described in Patent Document 1 involves releasing the glycans from the glycoprotein retained in an electrophoresis gel used in electrophoresis, capturing the released glycans on a capture carrier, and then re-releasing the glycans bound to the capture carrier. In this method, the re-release of the glycans is carried out by an exchange reaction with a labeling reagent.

[0005] Furthermore, the present inventors have proposed a method for releasing O-linked glycans from glycoproteins using a basic catalyst in an aqueous solution in the presence of hydroxylamine, as a method for preparing free glycans by releasing O-linked glycans from proteins in a short processing time using safe and inexpensive chemicals and without using a special system (see, for example, Patent Document 2). Patent Document 1 also describes a method for further labeling the obtained free glycans. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-156587 [Patent Document 2] International Publication No. 2018 / 062167 Summary of the Invention [Problem to be solved by the invention]

[0007] When electrophoresis is used as in Patent Document 1, in order to detect glycoproteins separated by electrophoresis, a step is required in which the glycoprotein bands are visualized on the gel by isotope labeling or staining, or by transferring the glycoproteins onto a membrane and then visualizing them by antigen-antibody reaction or staining. Furthermore, in order to obtain the sugar chains, it is necessary to separate the components used for the visualization. Furthermore, when analyzing the obtained sugar chains, a labeling step is required to modify the sugar chains with a labeling group suitable for analysis.

[0008] Thus, analyzing sugar chains obtained from glycoproteins requires many steps and is extremely time-consuming.

[0009] Although solid phases capable of binding and immobilizing glycoproteins (e.g., Protein A Sepharose, which specifically captures antibodies) are known, such solid phases are primarily used for glycoprotein purification. That is, such solid phases are used to capture glycoproteins, separate them from contaminants, and then release the captured glycoproteins from the solid phase. The process of releasing glycans from glycoproteins is performed on glycoproteins purified in this way, so obtaining the glycans still requires time.

[0010] On the other hand, there is a constant demand for faster sugar chain preparation from glycoproteins.

[0011] Furthermore, until now, there has been no method for analyzing O-linked glycans using capillary electrophoresis or the like.

[0012] The present invention has been made in view of the above circumstances, and aims to provide a method for preparing a sugar chain that rapidly prepares a labeled sugar chain from a glycoprotein. Another aim is to provide a sugar chain preparation method that enables analysis of O-linked sugar chains by capillary electrophoresis or the like. Another aim is to provide a sugar chain analysis method that analyzes sugar chains contained in a prepared sample. [Means for solving the problem]

[0013] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0014] [1] A method for preparing a sugar chain, comprising: a releasing step in which a sugar chain-releasing enzyme is allowed to act on a glycoprotein immobilized on a solid phase in a container to obtain a released product containing a sugar chain; and a labeling step in which a labeling reaction reagent is added to the released product in the container to obtain a labeled product containing a labeled form of the sugar chain, wherein the glycoprotein is an antibody, the solid phase has on its surface a ligand selected from the group consisting of protein A, protein G, protein L, protein H, protein D, and protein Arp, and the labeling reaction reagent comprises at least one selected from the group consisting of 8-aminopyrene-1,3,6-trisulfonic acid, sodium salt of 8-aminopyrene-1,3,6-trisulfonic acid, and 9-aminopyrene-1,4,6-trisulfonic acid, a reducing agent, and a solution.

[0015] [2] A method for preparing a sugar chain, comprising: a releasing step in which a sugar chain-releasing enzyme is allowed to act on a glycoprotein immobilized on a solid phase in a container to obtain a released product containing a sugar chain; and a labeling step in which a labeling reaction reagent is added to the released product in the container to obtain a labeled product containing a labeled form of the sugar chain, wherein the glycoprotein is an antibody, the solid phase has on its surface a ligand selected from the group consisting of protein A, protein G, protein L, protein H, protein D, and protein Arp, and the labeling reaction reagent comprises at least one selected from the group consisting of 2-aminobenzoic acid and 3-aminobenzoic acid, a reducing agent, and a solution.

[0016] [3] The method for preparing a sugar chain according to [1] or [2], wherein the reducing agent is picoline borane.

[0017] [4] The method for preparing a sugar chain according to any one of [1] to [3], wherein the release step is carried out in an open system under heated conditions.

[0018] [5] The method for preparing a sugar chain according to any one of [1] to [4], wherein the solution contains an organic acid.

[0019] [6] The method for preparing a sugar chain according to [5], wherein the solution further contains a polar solvent different from the organic acid.

[0020] [7] The method for preparing a sugar chain according to any one of [1] to [6], further comprising a separation step of obtaining a separation liquid containing the released product by solid-liquid separation after the release step.

[0021] [8] The method for preparing a sugar chain according to any one of [1] to [6], further comprising a separation step, after the labeling step, of obtaining a separation solution containing the labeled sugar chain by solid-liquid separation.

[0022] [9] The method for preparing a sugar chain according to [8], further comprising a purification step of passing the separation solution through a solid phase for purification to capture the labeled entity on the solid phase for purification, washing the solid phase for purification with a mixed solvent, and then re-eluting the captured labeled entity, wherein the mixed solvent comprises an organic solvent and water.

[0023]

[10] The method for preparing a sugar chain according to [9], wherein the volume ratio of the mixed solvent is acetonitrile:alcohol:water=20-98:0-60:2-15 (total 100% by volume).

[0024]

[11] The method for preparing a sugar chain according to [9] or

[10] , wherein in the purification step, the labeled product obtained by removing the solvent from the separation solution is dissolved in an ionic aqueous solution to reconstitute the separation solution, and the reconstituted separation solution is passed through the solid phase for purification.

[0025]

[12] The method for preparing a sugar chain according to any one of [1] to

[11] , wherein the release step is carried out in the presence of a deglycosylation promoter containing an acid-derived anionic surfactant.

[0026]

[13] A method for analyzing sugar chains, comprising an analysis step of analyzing a sample prepared by the sugar chain preparation method according to any one of [1] to

[12] .

[0027]

[14] The method for analyzing sugar chains according to

[13] , wherein the analysis step comprises the following steps (1) to (3): (1) A step of filling the capillary used for separation with the sample, and then filling the capillary with a preceding electrolyte solution from the downstream end of the capillary. (2) A step of concentrating the plurality of labels contained in the sample on the upstream side of the capillary by applying a voltage to the capillary with both ends immersed in the electrophoresis solution, with the downstream side of the capillary being positive and the upstream side being negative. (3) separating the concentrated plurality of labeled entities by capillary electrophoresis;

[0028]

[15] The method for analyzing sugar chains according to

[14] , wherein the labeled substance is analyzed by laser-excited fluorescence in the analysis step.

[0029]

[16] The method for analyzing sugar chains according to any one of

[12] to

[14] , wherein the migration speed of the electrolyte contained in the preceding electrolyte solution is faster than the migration speed of the label.

[0030] Furthermore, one aspect of the present invention also includes the following aspects.

[0031]

[17] The method includes a release step in which a glycan-releasing reagent is allowed to act on a sample containing glycoprotein to obtain a released product containing a glycan; an adsorption step in which a purification agent is brought into contact with a mixture containing the released product to adsorb the glycan to the purification agent; an elution step in which the glycan is eluted from the purification agent; and a labeling step in which the eluate obtained is reacted with a labeling reaction reagent to obtain a labeled product containing a labeled form of the glycan, wherein the labeling reaction reagent is selected from the group consisting of 8-aminopyrene-1,3,6-trisulfonic acid, sodium salt of 8-aminopyrene-1,3,6-trisulfonic acid, and 9-aminopyrene-1,4,6-trisulfonic acid. a first reagent containing at least one selected from the group consisting of an organic acid and an aqueous solution of an organic acid; and a second reagent containing a reducing agent and a solvent, wherein the labeling step comprises a first step of adding the first reagent to the reaction solution obtained in the first step and heating the mixture, and a second step of adding the second reagent to the reaction solution obtained in the first step and heating the mixture.

[0032]

[18] A method for preparing a sugar chain, comprising: a release step in which a sugar chain releasing reagent is allowed to act on a sample containing a glycoprotein to obtain a released product containing a sugar chain; an adsorption step in which a purification agent is brought into contact with a mixture containing the released product to adsorb the sugar chain to the purification agent; an elution step in which the sugar chain is eluted from the purification agent; and a labeling step in which the obtained eluate is reacted with a labeling reaction reagent to obtain a labeled product containing a labeled form of the sugar chain, wherein the labeling reaction reagent contains at least one acid selected from the group consisting of 2-aminobenzoic acid and 3-aminobenzoic acid, a reducing agent, and a solution, the solution being a mixed solution of alcohol, organic acid, and ultrapure water, and the concentration of the reducing agent in the labeling reaction reagent is 0.1 mmol / L or more and 20 mmol / L or less.

[0033]

[19] The method for preparing a sugar chain according to

[17] or

[18] , wherein the content of the organic acid in the labeling reaction reagent solution is 10% by volume or less.

[0034]

[20] The method for preparing a sugar chain according to any one of

[17] to

[19] , wherein the purification agent is at least one of a polymer having a betaine structure and a complex having the polymer and a support carrying the polymer.

[0035]

[21] The method for preparing a sugar chain according to any one of

[17] to

[20] , wherein the sugar chain releasing reagent comprises a hydroxylamine compound and a basic reagent, the hydroxylamine compound being at least one selected from the group consisting of hydroxylamine, a salt of hydroxylamine, an O-substituted hydroxylamine, and a salt of an O-substituted hydroxylamine, and the basic reagent being at least one selected from the group consisting of an alkali metal hydroxide, a weak alkali metal acid salt, an alkaline earth metal hydroxide, a salt of an alkaline earth metal dissolved in an aqueous ammonia solution, and an organic base.

[0036]

[22] The method for preparing a sugar chain according to any one of

[17] to

[21] , further comprising a purification step of passing the separation solution through a solid phase for purification to capture the labeled entity on the solid phase for purification, washing the solid phase for purification with a mixed solvent, and then re-eluting the captured labeled entity, wherein the mixed solvent contains an organic solvent and water.

[0037]

[23] The method for preparing a sugar chain according to

[22] , wherein the volume ratio of the mixed solvent is acetonitrile:alcohol:water=20-98:0-60:2-15 (total 100% by volume).

[0038]

[24] The method for preparing a sugar chain according to

[22] or

[23] , wherein in the purification step, the labeled product obtained by removing the solvent from the sample solution is dissolved in an ionic aqueous solution to reconstitute the sample solution, and the reconstituted sample solution is passed through the solid phase for purification.

[0039]

[25] A method for analyzing sugar chains, comprising an analysis step of analyzing a sample prepared by the sugar chain preparation method according to any one of

[17] to

[24] .

[0040]

[26] The method for analyzing sugar chains according to

[25] , wherein the analysis step comprises the following steps (1) to (3): (1) A step of filling the capillary used for separation with the sample, and then filling the capillary with a preceding electrolyte solution from the downstream end of the capillary. (2) A step of concentrating the plurality of labels contained in the sample on the upstream side of the capillary by applying a voltage to the capillary with both ends immersed in the electrophoresis solution, with the downstream side of the capillary being positive and the upstream side being negative. (3) separating the concentrated plurality of labeled entities by capillary electrophoresis;

[0041]

[27] The method for analyzing sugar chains according to

[26] , wherein the labeled substance is analyzed by laser-excited fluorescence in the analysis step.

[0042]

[28] The method for analyzing sugar chains according to

[26] or

[27] , wherein the migration speed of the electrolyte contained in the preceding electrolyte solution is faster than the migration speed of the label. [Effects of the Invention]

[0043] According to the present invention, a sugar chain preparation method for rapidly preparing labeled sugar chains from glycoproteins can be provided. It is also possible to provide a sugar chain preparation method that enables analysis of O-linked sugar chains by capillary electrophoresis or the like. It is also possible to provide a sugar chain analysis method for analyzing sugar chains contained in a prepared sample. [Brief explanation of the drawings]

[0044] [Figure 1] FIG. 1 is a schematic diagram illustrating an example of an apparatus for carrying out the sugar chain preparation method of the first embodiment. [Figure 2] FIG. 2 is a schematic diagram showing an example of a purification agent used in the purification method of the second embodiment. [Figure 3] FIG. 3 is an LC chart showing the results of the HPLC analysis of Example 1-1. [Figure 4] FIG. 4 is a chart obtained by capillary electrophoresis analysis in Example 1-2. [Figure 5] FIG. 5 is a chart obtained by capillary electrophoresis analysis in Examples 1-3. [Figure 6] FIG. 6 is a chart obtained by capillary electrophoresis analysis in Examples 1-4. [Figure 7] FIG. 7 is a chart obtained by capillary electrophoresis analysis in Reference Example 1-1. [Figure 8] FIG. 8 is a chart obtained by capillary electrophoresis analysis in Examples 1-5. [Figure 9] FIG. 9 is a chart obtained by capillary electrophoresis analysis in Reference Example 1-2. [Figure 10] FIG. 10 is a graph showing the area values ​​of unreacted labeled compounds detected in Examples 1-7 and Reference Examples 1-3. [Figure 11]FIG. 11 is a graph showing the area values ​​of sugar chain labels detected in Examples 1-7 and Reference Examples 1-3. [Figure 12] FIG. 12 is a graph showing the area values ​​of unreacted labeled compounds detected in Examples 1-7, 1-8, 1-9, 1-10, 1-11, and 1-12 and Reference Example 1-3. [Figure 13] FIG. 13 is an LC chart showing the results of HPLC analysis of Example 2-1 and Comparative Example 2-1. [Figure 14] FIG. 14 is a graph showing the total peak area of ​​the major O-glycans of fetuin determined from the LC chart. [Figure 15] FIG. 15 is an LC chart showing the results of HPLC analysis of Example 2-2 and Comparative Examples 2-2 and 2-3. [Figure 16] FIG. 16 is a graph showing the total peak area of ​​the major O-glycans of fetuin determined from the LC chart. [Figure 17] FIG. 17 is a chart obtained by capillary electrophoresis analysis in Example 2-3. [Figure 18] FIG. 18 is a chart obtained by capillary electrophoresis analysis in Reference Example 2-1. [Figure 19] FIG. 19 is a chart obtained by capillary electrophoresis analysis in Example 2-4. [Figure 20] FIG. 20 is a chart obtained by capillary electrophoresis analysis in Reference Example 2-2. DETAILED DESCRIPTION OF THE INVENTION

[0045] [First embodiment] <Glycan preparation method> The glycan preparation method of this embodiment includes a release step in which a glycan-releasing enzyme is allowed to act on a glycoprotein immobilized on a solid phase in a container to obtain a released product containing a glycan, and a labeling step in which a labeling reaction reagent is added to the released product in the container to obtain a labeled product containing a labeled form of the glycan.

[0046] According to the method of this embodiment, the glycans are released on the solid phase without eluting the glycoprotein immobilized on the solid phase, and the labeling reaction reagent is added in layers to the resulting released product without separating it, thereby enabling the extremely rapid preparation of labeled glycans from the glycoprotein.

[0047] In the method of this embodiment, the glycoprotein to be subjected to the release step is immobilized on a solid phase. The manner of immobilization in this case includes non-covalent bonding (hydrogen bonding and ionic bonding) through specific binding, and covalent bonding, but does not include modes in which the glycoprotein is merely retained by, for example, application to a migration gel or transfer to a blotting membrane. The following explains each in order.

[0048] [Release process] In the release step, a glycan-releasing enzyme is allowed to act on the glycoprotein immobilized on the solid phase to release the glycans, thereby obtaining a released product. Preferably, the glycan-releasing enzyme is allowed to act in the presence of a deglycosylation promoter. This step does not substantially include a step of fragmenting the protein by chemical fragmentation, enzymatic fragmentation, or the like.

[0049] (Solid-phase immobilized glycoprotein) Glycoproteins In the first embodiment, the glycoprotein may be any protein containing at least an N-glycan (N-linked glycan) as a complex component. That is, the glycoprotein is composed of a "glycan portion" containing an N-glycan and a "protein portion."

[0050] In the first embodiment, a "glycoprotein" refers to a protein having at least one N-glycan bound to its amino acid sequence. The glycoprotein to be used in the method for preparing a glycan from a glycoprotein is not particularly limited, and may be naturally occurring or synthetic.

[0051] In the first embodiment, "glycan" includes N-glycans, and any N-glycans can be prepared from glycoproteins. N-glycans refer to glycans that bind to the nitrogen atom of an amide group in the side chain of an asparagine residue in a protein. N-glycans include those that form branches with mannose as the base, such as di-branched, tri-branched, and tetra-branched. Furthermore, N-glycans can be classified into basic, high-mannose, hybrid, complex, and other types based on their structure.

[0052] It should be noted that the term "sugar chain" includes monosaccharides.

[0053] (Example of glycoprotein structure) In glycoproteins, the sugar chain portion may have a natural structure or may be artificially modified. Furthermore, the sugar chain portion may be a neutral sugar chain or an acidic sugar chain.

[0054] The protein portion of a glycoprotein may be folded before denaturation so as to incorporate the sugar chain portion into its interior. The molecular weight of such a protein portion may be, for example, 1 kDa or more, or 10 kDa or more. The upper limit of the molecular weight range of the protein portion is not particularly limited and may be, for example, 1,000 kDa.

[0055] Furthermore, the site in the protein portion of the glycoprotein to which the sugar chain moiety is bound (sugar chain binding site) may be the same site as in the natural product, or may be a site to which no sugar chain is bound in the natural product.

[0056] Specific examples of glycoproteins include physiologically active substances selected from the group consisting of antibodies, hormones, enzymes, and complexes containing these. Examples of complexes include antigen-antibody complexes, hormone-receptor complexes, and enzyme-substrate complexes. Since these glycoproteins are physiologically active substances prepared by cell culture engineering, the resulting glycan moieties are heterogeneous, making it particularly important to shorten the time required for glycan analysis.

[0057] Furthermore, when the glycoprotein contains an antibody, glycan analysis is particularly important, as it allows rapid release of glycans that affect the activity of the antibody.

[0058] As antibodies, Immunoglobulins such as IgG, IgM, IgA, IgD, and IgE; low molecular weight antibodies such as Fab, F(ab'), F(ab')2, single chain antibodies (scFv), and bispecific antibodies (diabodies); Fc-containing molecules such as Fc fusion proteins or peptides constructed by fusing the Fc region with other functional proteins or peptides; Examples include chemically modified antibodies to which chemical modifying groups such as radioisotope-coordinating chelates and polyethylene glycol have been added. Furthermore, the antibody may be a monoclonal antibody or a polyclonal antibody.

[0059] The antibody may also be an antibody drug candidate or antibody drug. Antibody drug candidates are substances in the development stage of antibody drugs, and are used to evaluate their activity and safety as antibody drugs. When glycan release is performed from antibody drug candidates, the development of antibody drugs can be expedited, and when glycan release is performed from antibody drugs, the quality control of antibody drugs can be expedited.

[0060] 《Solid phase》 In the method of this embodiment, the glycoprotein is immobilized on a solid phase. The manner of immobilization includes non-covalent binding (hydrogen bond and ionic bond) through specific binding, and covalent binding, but does not include a manner in which the glycoprotein is simply held by, for example, application to a migration gel or transfer to a blotting membrane. When the glycoprotein is immobilized by non-covalent binding, the binding rate constant ka (unit: M -1 s -1 ) but for example, 10 3 or more, for example, 10 4 or more, for example, 10 3 ~10 5 , e.g. 104 ~10 5 It is preferred that the affinity of

[0061] The solid phase on which the glycoprotein is immobilized is not particularly limited as long as it is a carrier having on its surface a linker that can be non-covalently or covalently linked to the protein portion of the glycoprotein.

[0062] Linker Examples of linkers that the carrier has on its surface include ligands that can capture the protein portion of glycoproteins. Examples include (i) molecules that have affinity for the protein portion of glycoproteins (hereinafter, sometimes simply referred to as molecules that have affinity for glycoproteins), (ii) ion exchange groups, and (iii) hydrophobic groups.

[0063] (i) Molecules with affinity for glycoproteins The molecule having affinity for glycoproteins is not particularly limited and can be easily determined by those skilled in the art depending on the glycoprotein to be captured. Examples include peptide or protein ligands, aptamers (synthetic DNA, synthetic RNA, or peptides capable of specifically binding to glycoproteins), and chemically synthesized ligands (thiazole derivatives, etc.).

[0064] For example, when the glycoprotein is an antibody, the molecule having affinity for the glycoprotein may be one that specifically binds to the antibody or an Fc-containing molecule, which is the constant region of the antibody.

[0065] More specifically, as peptide or protein ligands, microbial-derived ligands such as protein A, protein G, protein L, protein H, protein D, and protein Arp; functional variants (analogs) obtained by recombinant expression of those ligands; Examples include recombinant proteins such as antibody Fc receptors. This enables high-throughput preparation and analysis of glycan samples for antibodies, for which glycan analysis is particularly important. Among these, microbial-derived ligands are preferred.

[0066] (ii) Ion exchange groups The ion exchange group is not particularly limited as long as it is a functional group that can capture glycoproteins by its ion exchange function and can release glycoproteins in an ionic strength-dependent manner by a counter ion. Preferred examples include cation exchange groups such as carboxyl groups (more specifically, carboxymethyl groups, etc.) and sulfonic acid groups (more specifically, sulfoethyl groups, sulfopropyl groups, etc.), and may also be anion exchange groups such as quaternary amino groups.

[0067] (iii) Hydrophobic group Examples of the hydrophobic group include an alkyl group having 2 to 8 carbon atoms and an aryl group. More specifically, examples thereof include a butyl group, a phenyl group, and an octyl group, and these groups may be used alone or in combination of two or more.

[0068] In addition to the above, the linker on the surface of the support may be a linking group covalently bonded to the C-terminus of the C-terminal amino acid residue, which is a constituent of the protein portion of the glycoprotein. Examples of such linking groups include linking groups derived from amino group-containing compounds, which are solid-phase surface modification reagents used in solid-phase peptide synthesis.

[0069] Carrier The carrier is not particularly limited as long as it is a water-insoluble substrate on which the linker can be immobilized, and examples thereof include organic carriers, inorganic carriers, and composite carriers thereof. Examples of organic carriers include carriers made of synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene; and polysaccharides such as cross-linked sepharose, crystalline cellulose, cross-linked cellulose, cross-linked amylose, cross-linked agarose, and cross-linked dextran. These may be used alone or in combination of two or more.

[0070] Examples of inorganic carriers include glass beads, silica gel, and monolithic silica.

[0071] While organic carriers tend to absorb water easily, inorganic carriers do not absorb water easily. In the method of this embodiment, since various reactions are carried out on the solid phase, it is preferable to use an inorganic carrier that does not absorb water easily. This is preferable because the enzymes and / or reagents used in the treatment are not diluted by water seeping out from the solid phase, and the effects of the enzymes and reagents are not reduced. Preventing the dilution of the effects of the enzymes and / or reagents contributes to preventing the detection of unnecessary signals in the analysis.

[0072] Furthermore, if the carrier is inorganic, for example, a portion of the carrier will not be liberated by a glycan-releasing enzyme, and when a sugar-derived resin is used, sugars that remain on the resin from the beginning will not be eluted, which makes it easier to suppress the appearance of unnecessary signals in the analysis of liberated sugar chains.

[0073] The shape of the carrier is not particularly limited, and may be particulate or non-particulate. In the case of a particulate carrier (beads), it may be a porous carrier. In the case of a particulate carrier, the average particle diameter may be, for example, 1 to 100 μm. It is preferable that the average particle diameter is equal to or greater than the above lower limit in terms of liquid permeability, and it is preferable that the average particle diameter is equal to or less than the above upper limit in terms of preventing a decrease in the theoretical limit.

[0074] Examples of non-particulate carriers include monolithic silica gel and membranes. Monolithic silica gel is a bulk silica gel having micrometer-sized three-dimensional network pores (macropores) and nanometer-sized pores (mesopores). The diameter of the macropores may be, for example, 1 to 100 μm, 1 to 50 μm, 1 to 30 μm, or 1 to 20 μm. It is preferable that the macropores be equal to or greater than the above lower limit in terms of liquid permeability, and it is preferable that the macropores be equal to or less than the above upper limit in terms of preventing a decrease in theoretical yield. The diameter of the mesopores may be, for example, 1 to 100 nm or 1 to 50 nm. This allows for efficient capture of sugars.

[0075] The volume of the carrier used (in the case of a particulate carrier, the volume of the carrier itself includes the volume of voids when packed, and in the case of a non-particulate carrier, the volume of the carrier itself includes the volume of mesopores and macropores) is, for example, 0.001 to 0.1 cm 3 For example, 0.001 to 0.01 cm 3 It may be. A volume equal to or greater than the above lower limit is preferred in terms of preventing a decrease in the theoretical cutoff, and a volume equal to or less than the above upper limit is preferred in terms of liquid permeability. In addition, by having the volume within the above range, it becomes easy to obtain a separated solution after elution at a concentration suitable for HPLC analysis.

[0076] The solid phase may be used in a state packed in a container such as a column, each well of a multi-well plate, each well of a filter plate, or a microtube.

[0077] (Preparation of solid-phase-immobilized glycoproteins) By contacting a sample containing glycoproteins (hereinafter sometimes abbreviated as "sample") with the above-mentioned solid phase, the glycoproteins contained in the sample can be captured (immobilized) on the solid phase.

[0078] From the viewpoint of rapid glycosylation preparation, the glycoprotein-containing sample to be contacted with the solid phase may not have undergone glycoprotein purification (i.e., separation of the glycoprotein from its contaminants). Examples of such samples include body fluids such as blood (e.g., serum, plasma), lymph, peritoneal exudate, interstitial fluid, cerebrospinal fluid, and ascites; culture supernatants of antibody-producing cells such as B cells, hybridomas, and CHO cells; and ascites of animals transplanted with antibody-producing cells. The sample may also be a mixture of glycoprotein variations in which the protein portion is homogeneous and the glycan portion is heterogeneous, such as glycoprotein preparations prepared by cell culture engineering, such as culture supernatants.

[0079] The solid phase on which a glycoprotein is immobilized may be a complex in which a glycoprotein is captured on the solid phase, or may be a product obtained by solid-phase synthesis of a glycoprotein, i.e., a complex in which a glycoprotein obtained by solid-phase synthesis is bound to a solid phase used for solid-phase synthesis.

[0080] In a sample containing glycoprotein, the concentration of glycoprotein is not particularly limited and may be, for example, 0.1 μg / mL to 50 mg / mL. A concentration equal to or higher than the above lower limit is preferable from the viewpoint of detection, and a concentration equal to or lower than the above upper limit is preferable from the viewpoint of quantitativeness.

[0081] The amount of glycoprotein to be contacted with the solid phase may be 0.001 μg to 100 mg per container, or may be 0.001 μg to 5 mg. It is preferable from the viewpoint of detection that the amount of glycoprotein is equal to or greater than the lower limit. The method of this embodiment has a small number of steps and very little sample loss, making it particularly useful when the glycoprotein is on a small scale (particularly 0.001 to 500 μg). It is preferable from the viewpoint of quantitation that the amount of glycoprotein is equal to or less than the upper limit.

[0082] The glycoprotein immobilized on the solid phase may be prepared in a state in which the glycoprotein immobilized on the solid phase is dispersed in a liquid component, or in a state in which the liquid component is separated.

[0083] Furthermore, it is believed that contaminants will be attached to the solid phase on which the glycoprotein is immobilized when the sample is brought into contact with the solid phase and capture of the glycoprotein is completed, or when solid-phase synthesis is completed. Examples of contaminants include components contained in the sample before immobilization on the solid phase, reagents used in solid-phase synthesis of the glycoprotein, etc. More specific examples of contaminants include salts, low-molecular-weight compounds, proteins (proteins that do not bind to the solid phase), and other biomolecules.

[0084] Therefore, the solid phase on which the glycoprotein is immobilized may be washed after the capture of the glycoprotein or after the solid-phase synthesis is completed. This allows contaminants to be removed while the glycoprotein remains immobilized on the solid phase. Washing can be performed by passing a washing solution through the solid phase. Methods for passing the solution include gravity, suction, pressure, and centrifugation.

[0085] The washing solution can be appropriately selected by those skilled in the art from a solution with a liquid property and composition that does not cleave the bond between the protein portion of the glycoprotein and the linker on the solid phase surface. Specifically, it may be a buffer solution or other aqueous solution, or water. When an aqueous solution is used, it is preferable that the pH is 5 to 10. If the pH of the aqueous solution is within this range, it is easy to maintain the activity of the glycan-releasing enzyme used in the subsequent step. Furthermore, when the glycoprotein is immobilized on the solid phase by a non-covalent bond, it is easy to prevent the glycoprotein from being released.

[0086] When a buffer solution is used as the washing solution, examples of the buffering agent include ammonium salts such as ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, diammonium hydrogen citrate, and ammonium carbamate; Tris buffers such as trishydroxymethylammonium; and phosphates.

[0087] (container) The solid phase on which the glycoprotein is immobilized is prepared in a container. It is efficient and preferable to prepare the solid phase on which the glycoprotein is immobilized in the container. The container is not particularly limited as long as it is capable of holding a liquid and a solid phase and separating (passing) the liquid while holding the solid phase, and examples thereof include a column, each well of a multiwell plate, each well of a filter plate, a microtube, etc.

[0088] (glycan-releasing enzyme) Examples of sugar chain-releasing enzymes that act on glycoproteins immobilized on a solid phase include peptide N-glycanase (PNGase F, PNGase A) and endo-β-N-acetylglucosaminidase (Endo-H, Endo-F, Endo-A, Endo-M).

[0089] The glycan-releasing enzyme may be prepared in a state dispersed in water or a buffer solution. When a buffer solution is used, examples of the buffering agent include ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, diammonium hydrogen citrate, and ammonium carbamate. The buffer solution preferably has a pH of 5 to 10. When the pH of the buffer solution is within this range, the activity of the glycan-releasing enzyme is easily maintained. The water or buffer solution may contain components such as salts such as metal salts, and protein stabilizers such as glycerol.

[0090] (Deglycosylation promoter) The release step may be carried out in the presence of a deglycosylation promoter, which can promote the release of glycans from glycoproteins and improve the recovery rate of glycans.

[0091] The deglycosylation promoter preferably contains an acid-derived anionic surfactant. The acid-derived anionic surfactant denatures the protein portion of the glycoprotein, changing its tertiary structure and making it easier for the glycan-releasing enzyme to act on the degradation target site (e.g., the glycan-binding site). This facilitates decomposition of the degradation target site, releasing the glycan.

[0092] Acid-derived anionic surfactants are anionic surfactants derived from organic acids. Examples include carboxylic acid-type anionic surfactants, sulfonic acid-type anionic surfactants, sulfate ester-type anionic surfactants, and phosphate ester-type anionic surfactants. Among these, carboxylic acid-type anionic surfactants are preferred. When the acid-derived anionic surfactant is a carboxylic acid-type anionic surfactant, it is thought that it will denature the protein portion of glycoproteins but will not denature glycan-releasing enzymes as easily.

[0093] As the deglycosylation promoter, compounds known as surfactants that are used together with the sugar chain-releasing enzyme to promote the release of sugar chains can be used appropriately.

[0094] <Composition of deglycosylation promoter> The deglycosylation promoter may be prepared in a state in which an acid-derived anionic surfactant is dissolved or dispersed in water or a buffer solution.

[0095] The buffering agent contained in the buffer solution can be the same compound as the buffering agent used as the washing solution. The buffer solution preferably has a pH of 5 to 10. When the pH of the buffer solution is within this range, the activity of the glycan-releasing enzyme is easily maintained.

[0096] In the deglycosylation promoter, components other than the acid-derived anionic surfactant contained in the water or buffer solution include salts such as metal salts other than surfactants.

[0097] (Operation and reaction conditions for the release step) In the release step, a release reaction solution containing a glycoprotein and a glycan-releasing enzyme is prepared, which satisfies the optimal conditions (temperature and pH) for the glycan-releasing enzyme.

[0098] When a deglycosylation promoter is used, it is sufficient to prepare a release reaction solution containing a glycoprotein, an acid-derived anionic surfactant, and a glycan-releasing enzyme that satisfies the optimal conditions (temperature and pH) for the glycan-releasing enzyme. Therefore, when a deglycosylation promoter is used, the solid phase on which the glycoprotein is immobilized, the deglycosylation promoter, and the glycan-releasing enzyme may be mixed using any procedure.

[0099] For example, a release reaction solution may be prepared by simultaneously mixing a solid phase on which a glycoprotein is immobilized, a deglycosylation promoter, and a glycan-releasing enzyme. Alternatively, a release reaction solution may be prepared by first adding a deglycosylation promoter and then adding a glycan-releasing enzyme. Furthermore, when the glycoprotein immobilized on the solid phase has been obtained through a pretreatment described below, and the deglycosylation promoter and the surfactant used in the pretreatment are the same substance, an amount of surfactant equivalent to the deglycosylation promoter may be added first to an amount of surfactant equivalent to the pretreatment agent during the pretreatment, and then only the glycan-releasing enzyme may be added in the subsequent release step (since the deglycosylation promoter is already present).

[0100] Specifically, all components are mixed to prepare a release reaction solution, which is then set at an optimal temperature to carry out the reaction of releasing the sugar chain from the glycoprotein. In this case, the reaction time may be, for example, 5 seconds to 24 hours.

[0101] When a deglycosylation promoter is used, the solid phase on which the glycoprotein is immobilized may be first mixed with an acid-derived anionic surfactant to denature the protein portion of the glycoprotein, and then mixed with the glycan-releasing enzyme.

[0102] In this case, the denaturation time of the protein portion may be, for example, 5 seconds to 24 hours. The reaction time between the glycan-releasing enzyme and the glycoprotein may be, for example, 5 seconds to 24 hours.

[0103] In the release reaction solution, the concentration of the glycoprotein may be, for example, 0.1 μg / mL to 100 mg / mL, or may be, for example, 1 μg / mL to 10 mg / mL. It is preferable that the concentration of the glycoprotein in the release reaction solution is equal to or higher than the above lower limit in terms of detectability, and it is preferable that the concentration is equal to or lower than the above upper limit in terms of quantitation.

[0104] When a deglycosylation promoter is used, the concentration of the acid-derived anionic surfactant in the release reaction solution may be, for example, 0.01 to 30% by mass, for example, 0.2 to 1.0% by mass, for example, 0.2 to 0.3% by mass, or for example, 0.22 to 0.27% by mass. Alternatively, the acid-derived anionic surfactant may be used in an amount of 0.001 μg to 100 mg or less per μg of glycoprotein.

[0105] Setting the amount of the acid-derived anionic surfactant used within the above range is favorable in terms of maintaining the activity of the glycan-releasing enzyme and recovering the amount of liberated glycans (hereinafter referred to as "released glycans"), and is also favorable in terms of stabilizing the amount recovered. Furthermore, when purifying the released glycans using a solid phase carrier, for example, it is also preferable in terms of preventing lengthy drying times.

[0106] In the release reaction solution, the concentration of the glycan-releasing enzyme may be, for example, 0.001 μU / mL to 1000 mU / mL, or may be, for example, 0.01 μU / mL to 100 mU / mL. Alternatively, the glycan-releasing enzyme may be used at 0.001 μU to 1000 mU per μg of glycoprotein. Setting the amount of glycan-releasing enzyme used within the above range enables efficient glycan release.

[0107] The reaction pH may be adjusted to the optimum pH of the glycan-releasing enzyme, and may be, for example, 5 to 10. The reaction temperature may also be adjusted to the optimum temperature of the glycan-releasing enzyme, and may be, for example, 4 to 90°C.

[0108] The release step is preferably carried out under heating conditions that make the reaction system open and evaporate the solvent in the solution. The heating temperature may be, for example, 40°C or higher, e.g., 45°C or higher. This allows the solvent in the solution to evaporate during the release step, gradually increasing the concentration of the reaction solution, making it easy to achieve a concentration at which glycan release proceeds efficiently, regardless of the scale of the glycoprotein used in the method of this embodiment. Furthermore, since the solvent in the solution is removed simultaneously with the release reaction, the time required for performing a solvent removal step in the solution separately from the release step is shortened or eliminated, enabling even more rapid glycan preparation. The upper limit of the heating temperature range may be, for example, 80°C, from the viewpoint of preventing denaturation of the glycan-releasing enzyme.

[0109] (free product) The released product obtained by the release step includes a released glycan and a protein bound to the solid phase. The released glycan corresponds to the glycan portion of the glycoprotein, and the protein bound to the solid phase corresponds to the protein portion of the glycoprotein.

[0110] In the protein bound to the solid phase, the peptide bonds between amino acid residues in the protein portion that constituted the glycoprotein are not cleaved. The released product may be obtained in a state containing an organic solvent, or, particularly when the release step is performed in an open system under heating conditions, may be obtained in the form of an evaporated dry product from which the organic solvent has completely evaporated.

[0111] [Separation process 1] In the method of this embodiment, the sugar chains are released while the protein portion remains fixed to the solid phase, so the protein portion can be removed simply by separating the solid phase. Therefore, after the release step, a separation step (separation step 1) may be included in which the solid phase is separated by solid-liquid separation to obtain a separation liquid containing the released product (released sugar chains).

[0112] The separated solution obtained by separating the solid phase is a mixture in which the surfactant used in the pretreatment step (described below) and the deglycosylation promoter used in the release step are dissolved together with the released glycans. Depending on the glycan analysis method, the glycans may be subjected to analysis in the form of a mixture in which they coexist with the surfactants described above, but when analyzing by mass spectrometry, for example, it is preferable to purify the glycans from the mixture before analysis.

[0113] When purifying glycans, for example, a polymer having a hydrazide group can be used as a solid support for purification, and the separation solution can be brought into contact with the solid support. In the separation solution, free glycans are in equilibrium between cyclic hemiacetal forms and acyclic aldehyde forms. When the separation solution is brought into contact with the solid support for purification, the aldehyde group -CHO of the free glycan reacts specifically with the hydrazide group -NH-NH2 of the solid support for purification, forming a stable bond -C=N-NH-. This allows the free glycan to be captured by the solid support for purification.

[0114] The sugar chains captured on the solid support for purification may be re-released. Examples of re-release methods include contacting a mixed solvent of an acid and an organic solvent, or a mixed solvent of an acid, water, and an organic solvent, with the solid support to cause a reaction. The acidity of the mixed solvent may be, for example, pH 2 to 9, pH 2 to 7, or pH 2 to 6. Reactions at weakly acidic to near-neutral pH are preferred because they can suppress hydrolysis of sugar chains, such as elimination of sialic acid residues. However, strongly acidic conditions with even lower pHs are also acceptable.

[0115] As described below, free sugar chains can be modified with low molecular weight compounds (labeling compounds). The low molecular weight compounds can be selected appropriately depending on the analytical method. Low molecular weight compounds are distinguished from high molecular weight compounds that constitute solid phase supports, and are preferably compounds that are soluble in water, buffer solutions, or organic solvents.

[0116] When the separation step 1 is carried out, the separation step 2 described below is not necessary.

[0117] [Pretreatment process] The method of this embodiment may further include a pretreatment step prior to the release step. This facilitates the release of glycans from glycoproteins without decomposing the protein portion. As a result, the time required for the glycan release process can be significantly reduced.

[0118] In the pretreatment step, a pretreatment agent containing a surfactant is contacted with the glycoprotein immobilized on a solid phase. The pretreatment step may be performed after the sample is brought into contact with the solid phase and glycoprotein capture is complete, or after solid-phase synthesis is completed or after a further washing step is performed, but before contact with the glycan-releasing enzyme. By performing the pretreatment step, the surfactant denatures the protein portion of the glycoprotein and changes its tertiary structure. This makes it easier for the glycan-releasing enzyme to act on the degradation target site (e.g., glycan-binding site), facilitating the action of the glycan-releasing enzyme on the glycoprotein in the release step.

[0119] The surfactant contained in the pretreatment agent may be any of anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants.

[0120] The anionic surfactant is not particularly limited, and examples thereof include salts of fatty acids such as soap, alkylbenzenesulfonates, higher alcohol sulfates, polyoxyethylene alkyl ether sulfates, α-sulfofatty acid esters, α-olefinsulfonates, monoalkyl phosphates, and alkylsulfonates. However, anionic surfactants that can be used as deglycosylation promoters in the release step described above are preferred (in this specification, anionic surfactants that can also be used as deglycosylation promoters are particularly referred to as acid-derived anionic surfactants). When an acid-derived anionic surfactant is used in the pretreatment step, it may be the same surfactant as the surfactants listed as deglycosylation promoters used in the release step, or it may be a different surfactant.

[0121] Cationic surfactants are not particularly limited, and examples thereof include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkyldimethylbenzylammonium salts, and amine salts. Amphoteric surfactants are not particularly limited, and examples thereof include alkylamino fatty acid salts, alkylbetaines, and alkylamine oxides. Nonionic surfactants are not particularly limited, and examples thereof include polyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, alkylglucosides, polyoxyethylene fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, fatty acid alkanolamides, and polyoxyethylene-polyoxypropylene block copolymers.

[0122] The pretreatment agent may be used in a state in which the surfactant is dissolved in water or a buffer solution. The buffer solution may be the same as the buffer solution that the deglycosylation promoter described above may contain.

[0123] The concentration of the surfactant in the pretreatment agent may be, for example, 0.01 to 30% by mass, for example, 0.2 to 1.0% by mass, for example, 0.2 to 0.3% by mass, or for example, 0.22 to 0.27% by mass. When the concentration is equal to or greater than the above-mentioned lower limit and equal to or less than the above-mentioned upper limit, the sugar chains released in the above-mentioned release step can be obtained with a good recovery rate.

[0124] After contacting the pretreatment agent with the solid phase, it is separated from the glycoprotein immobilized on the solid phase. Separation may be carried out all at once after placing the entire predetermined amount into a container, or may be carried out each time portions of the predetermined amount are added in portions. Separation of the pretreatment agent can be carried out by reducing pressure, centrifugation, or the like.

[0125] After the pretreatment step, the solid phase on which the glycoprotein is immobilized can be subjected to the release step described below without being washed, from the viewpoint of rapid preparation. Alternatively, the solid phase on which the glycoprotein is immobilized may be washed after the pretreatment step and before the release step. The washing solution described above can be used for washing.

[0126] [Labeling process] In the labeling step, a labeling reaction reagent (labeling reaction solution) containing a labeling compound is added to the released product in the container in which the release step was performed, to obtain a labeled product containing a labeled glycan (hereinafter sometimes referred to as a labeled glycan).

[0127] (labeled compound) The labeling compound has a group reactive to a sugar chain and a modifying group to be attached to the sugar chain. In this embodiment, each labeling compound has an amino group as the reactive group and an aromatic group as the modifying group. When a labeling compound having an amino group and an aromatic group is used, the sugar chain is modified by reductive amination.

[0128] In the reductive amination modification, the amino group reacts with the aldehyde group formed at the reducing end of the glycan to form a Schiff base. The formed Schiff base is reduced by a separately added reducing agent to form a peptide bond at the reducing end of the glycan. This allows the labeling compound to introduce a modifying group into the glycan via the reactive amino group, enabling efficient labeling.

[0129] Aromatic groups are preferred in that they have ultraviolet-visible absorption properties or fluorescent properties, thereby improving the detection sensitivity in UV detection or fluorescent detection.

[0130] Specifically, the labeling compound used in this embodiment is at least one selected from the group consisting of 8-aminopyrene-1,3,6-trisulfonic acid, the sodium salt of 8-aminopyrene-1,3,6-trisulfonic acid, and 9-aminopyrene-1,4,6-trisulfonic acid. The sodium salt is preferably the trisodium salt (8-aminopyrene-1,3,6-trisulfonic acid trisodium, APTS). These labeling compounds contain a sulfate group (sulfonic acid group).

[0131] Alternatively, the labeling compound may be at least one selected from the group consisting of 2-aminobenzoic acid (2-AA) and 3-aminobenzoic acid (3-AA). These labeling compounds contain a carboxy group.

[0132] (Labeling reaction reagent) The labeling reaction reagent contains a labeling compound, a solution for dissolving the labeling compound, and a reducing agent.

[0133] The solution may be water, a buffer solution and / or an organic solvent. The buffer solution may be an aqueous solution of the same buffer as that used in the release step described above.

[0134] Examples of organic solvents include aprotic polar organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N-methylpyrrolidone (NMP), etc., protic polar organic solvents such as organic acids (formic acid, acetic acid, propionic acid, butyric acid, etc.) and alcohols (methanol, ethanol, propanol, etc.), and aprotic nonpolar solvents such as hexane, etc. These solvents may be used alone or in combination of two or more.

[0135] Examples of the reducing agent include sodium cyanoborohydride, sodium triacetoxyborohydride, methylamine borane, dimethylamine borane, trimethylamine borane, picoline borane, and pyridine borane.

[0136] Among these, picoline borane (2-picoline borane) is preferably used from the viewpoints of both safety and reactivity.

[0137] (Operation and reaction conditions for labeling process) In the labeling step, a labeling reaction reagent is added to the released product. In the labeling step, the container in which the release step was performed is subsequently used, but when adding the labeling reaction reagent, the released product is not subjected to any treatment, such as washing, that would change its relative composition (ratio of components other than the solvent). It is permissible to add water, a buffer solution, and / or an organic solvent to dissolve or dilute the released product.

[0138] The labeling reaction system is constructed in a state where a labeling reaction reagent containing a sugar chain and a labeling compound is mixed with the residue from the release step in water, a buffer solution, and / or an organic solvent. If the above-mentioned separation step 1 is not performed, the labeling reaction system contains a protein immobilized on a solid phase.

[0139] In the labeling step, the labeling reaction reagent may be used in a volume that is, for example, 0.1 to 10 times the volume of the carrier used, or may be used in a volume that is, for example, 0.5 to 5 times. The concentration of the labeling compound in the labeling reaction reagent may be, for example, 0.1 to 5.0 mol / L, or may be, for example, 0.1 to 1.0 mol / L. It is preferable that the amount of the labeling compound is equal to or greater than the above-mentioned lower limit in terms of quantitative labeling, and it is preferable that the amount is equal to or less than the above-mentioned upper limit in terms of easy removal of excess reagent.

[0140] The concentration of the reducing agent in the labeling reaction reagent may be, for example, 0.1 to 5.0 mol / L, or may be, for example, 0.1 to 1.5 mol / L. It is preferable that the amount of the reducing agent is equal to or greater than the above lower limit, since labeling is easily carried out quantitatively, and it is preferable that the amount is equal to or less than the above upper limit, since excess reagent can be easily removed.

[0141] The amount of the solution in the labeling reaction reagent may be 0.5 to 10 times, for example 1 to 5 times the volume of the carrier used. It is preferable that the amount of the solvent is equal to or greater than the above lower limit in terms of solubility, and it is preferable that the amount is equal to or less than the above upper limit in terms of quantitative labeling.

[0142] The reaction temperature of the labeling reaction reagent may be, for example, 4 to 80°C, or may be, for example, 25 to 70°C. A reaction temperature equal to or greater than the above lower limit is preferred in that the reaction time is short, and a reaction temperature equal to or less than the above upper limit is preferred in that partial decomposition of the sugar chain due to high temperature is suppressed. The reaction time of the labeling reaction reagent may be, for example, 5 to 600 minutes, or may be, for example, 30 to 300 minutes. A reaction time equal to or greater than the above lower limit is preferred in that partial decomposition of the sugar chain is suppressed.

[0143] Since the labeling reaction proceeds rapidly at room temperature, the labeling compound acts to produce a labeled glycan from the moment the labeling reaction reagent is added. Therefore, after adding the labeling reaction reagent, the separation step described below can be performed at any time, regardless of whether the reaction is complete or not.

[0144] Hereinafter, the case where picoline borane is used as the reducing agent will be described. When picoline borane is used as the reducing agent, it is preferable that the labeling reaction reagent contains an organic acid as a solution. This allows the labeling compound and picoline borane to be dissolved at high concentrations, thereby shortening the time required for the labeling step.

[0145] From the viewpoint of more favorably shortening the time required for the labeling step, the organic acid is preferably formic acid, acetic acid, propionic acid, butyric acid, etc. Furthermore, the organic acid is preferably a liquid in the labeling reaction system. Among these, from the viewpoint of ease of operation, the organic acid is preferably citric acid. When the organic acid is solid at room temperature, it may be used as an aqueous solution.

[0146] The concentration of the organic acid in the solvent may be, for example, 40 to 100% by volume. This allows for good labeling efficiency. From the viewpoint of obtaining even better labeling efficiency, the concentration of the organic acid in the solution may be 50 to 100% by volume or less, or may be 75 to 100% by volume.

[0147] In addition to the organic acid, a polar solvent different from the organic acid may be used in combination. This improves the solubility of the reducing agent, thereby enabling the production of a sugar chain-labeled product in good yield.

[0148] Examples of the polar solvent include alcohols such as methanol, dimethyl sulfoxide, and the like.

[0149] When a polar solvent is used in combination, it may be 4% by volume or more but less than 100% by volume, or may be 4 to 70% by volume, of the organic acid. An amount of polar solvent equal to or greater than the above lower limit is preferred because it makes it easier to slow down the evaporation rate of the organic acid, while an amount equal to or less than the above upper limit is preferred because it makes it easier to obtain the effect of the organic acid (the effect of improving the solubility and reactivity of the labeling compound and the reducing agent).

[0150] When picoline borane is used as the reducing agent, it is most preferable to use a mixed solution of an aqueous citric acid solution and methanol, or a mixed solution of acetic acid, methanol and water as the solvent.

[0151] When picoline borane is used as the reducing agent, the concentration of the labeling compound in the labeling reaction reagent may be 0.1 to 5.0 mol / L, or may be 0.1 to 1.5 mol / L. It is preferable that the concentration of the labeling compound is equal to or greater than the above lower limit in terms of shortening the time required for the labeling step, and it is preferable that the concentration is equal to or less than the above upper limit in terms of facilitating removal of excess reagent.

[0152] The amount of picoline borane in the labeling reaction reagent may be, for example, 0.05 to 2.5 mol / L, or may be, for example, 0.05 to 0.75 mol / L. It is preferable that the amount of picoline borane is not less than the above lower limit in terms of shortening the time for the labeling step, and it is preferable that the amount is not more than the above upper limit in terms of facilitating removal of excess reagent.

[0153] When picoline borane is used as the reducing agent, the amount of the solvent may be 0.1 to 10 times, or 0.5 to 5 times, the volume of the carrier used. It is preferable that the amount of the solvent is equal to or greater than the above lower limit in terms of solubility, and it is preferable that the amount of the solvent is equal to or less than the above upper limit in terms of shortening the time required for the labeling step.

[0154] The reaction temperature of the labeling reaction reagent may be, for example, 4 to 80°C, or may be, for example, 25 to 70°C. A reaction temperature equal to or greater than the above lower limit is preferred in that the reaction time is short, and a reaction temperature equal to or less than the above upper limit is preferred in that partial decomposition of the sugar chain due to high temperature is suppressed. The reaction time of the labeling reaction reagent may be, for example, 30 to 600 minutes, or may be, for example, 90 to 300 minutes. A reaction time equal to or greater than the above lower limit is preferred in that partial decomposition of the sugar chain is suppressed.

[0155] (labeled product) After the labeling step, a sugar chain-labeled product is present in the container. Furthermore, if the above separation step 1 is not performed, a protein bound to the solid phase is present in the container after the labeling step. Therefore, it can also be said that the labeled product obtained by the labeling step contains a sugar chain-labeled product and a protein bound to the solid phase. In the protein bound to the solid phase, the peptide bonds between amino acid residues in the protein portion that constituted the glycoprotein are still not cleaved. The labeled product may be contained in water, a buffer solution, and / or an organic solvent.

[0156] [Separation process 2] (Elution of sugar chain label) If the above separation step 1 is not performed, a separation step (separation step 2) may be performed after the labeling step, in which a separation solution containing labeled glycans is obtained from the labeled product by solid-liquid separation. This allows for easy separation of the glycan-labeled product. For example, the glycan-labeled product can be eluted by passing an eluent through the labeled product. The eluent used in this case may be an aqueous solution such as water, an aqueous solution, or a colloidal solution. The eluent selected may have the ability to cleave the bond between the solid phase and the protein moiety (e.g., when the analysis of the glycan-labeled product is performed by chromatography), or may not have such a property (e.g., when the analysis of the glycan-labeled product is performed by mass spectrometry). This allows for the production of a separation solution containing labeled glycans.

[0157] In addition to the glycoconjugate, the separation solution contains unwanted substances such as excess labeled compound used in the labeling step and, if a deglycosylation promoter is used in the release step, an acid-derived anionic surfactant. If an eluent having the ability to cleave the bond between the solid phase and the protein moiety is selected, proteins will also be mixed into the separation solution. If an eluent not having the ability to cleave the bond between the solid phase and the protein moiety is selected, proteins will not be substantially contained in the separation solution.

[0158] (purification process) Depending on the glycan analysis technique, the glycan-labeled substance may be purified by removing unnecessary substances from the separation solution, which may be achieved by passing the separation solution through a solid phase for purification to capture the glycan-labeled substance, and then re-eluting the captured glycan-labeled substance.

[0159] (Solid phase for purification) Examples of solid phases for purification include those that capture sugar chain labels by non-covalent bonding, such as silica gel columns, amino columns, and other normal phase solid phases.

[0160] As such a solid phase for purification, a clean-up column attached to a commercially available antibody sugar chain analysis kit (manufactured by Sumitomo Bakelite, product number: BS-X4410) can be used.

[0161] Another example of a solid phase for purification is a solid phase that captures a sugar chain label by a covalent bond. This allows for improved purification of glycan-labeled products when proteins are present. Specifically, a polymer containing a hydrazide group can be used as a solid support for purification. In the separation solution, free glycans are in equilibrium between cyclic hemiacetal and acyclic aldehyde groups, and this aldehyde group -CHO reacts specifically with the hydrazide group -NH-NH2 to form a stable bond -C=N-NH-. This allows the free glycans to be captured on the solid support for purification.

[0162] (Re-preparation of separation solution) When capturing a glycoconjugate on a solid phase for purification, an ionic aqueous solution may be added to a mixture containing the glycoconjugate and excess labeled compound to prepare a new separation solution, which facilitates the removal of excess labeled compound contained in the separation solution.

[0163] The pH of the ionic aqueous solution can be, for example, from pH 1 to 13. Examples of ionic aqueous solutions include an ammonium chloride aqueous solution (pH about 5), a sodium chloride aqueous solution (pH about 7), an ammonium bicarbonate aqueous solution (pH about 8), an ammonia aqueous solution (pH about 11), a sodium carbonate aqueous solution (pH about 11), and a sodium hydroxide aqueous solution (pH about 13).

[0164] When a pH of 6.5 or more and 7.5 or less is defined as neutral, the ionic aqueous solution may be acidic with a pH of 1 or more and 6.5 or less, neutral with a pH of 6.5 or more and 7.5 or less, or basic with a pH of 7.5 or more and 13 or less. When the ionic aqueous solution is basic, the pH may be 12 or less, 11 or less, or 10 or less.

[0165] When an ammonium bicarbonate aqueous solution is used as the ionic aqueous solution, the concentration of the aqueous solution is preferably 50 mmol / L or more, more preferably 150 mmol / L or more, and even more preferably 200 mmol / L or more. Furthermore, a sufficient effect can be obtained when the concentration of the aqueous solution is 500 mmol / L or less, and it may be 400 mmol / L or less. The upper and lower limits of the concentration of the aqueous solution can be arbitrarily combined.

[0166] When an aqueous ammonium hydrogen carbonate solution is used as the ionic aqueous solution, the concentration can be, for example, 200 mmol / L or more and 400 mmol / L or less.

[0167] The inventors compared the separation liquid containing excess APTS when it did not contain ionic compounds (condition 1) with when it contained ionic compounds (condition 2), and confirmed that condition 2 clearly reduced the impurity (APTS) after the purification process compared to condition 1.

[0168] Furthermore, the inventors have confirmed that when a separation solution containing excess APTS but not containing ionic compounds is passed through a solid phase for purification, a yellow solid precipitates on the top surface of the solid phase. On the other hand, when a separation solution containing ionic compounds is passed through the solid phase for purification, no yellow solid precipitate is observed. This solid is assumed to be excess APTS.

[0169] As described above, the labeled compound used in this embodiment has an acid group (sulfonic acid group, carboxy group). When a mixture containing such a labeled compound is dissolved in an ionic aqueous solution, it is thought that the labeled compound will be more likely to remain dissolved in the solution. Therefore, when a separation solution containing added ionic aqueous solution is passed through a solid phase for purification, the labeled compound passes through the solid phase for purification, while the glycan label to be captured is captured by the solid phase. This is thought to enable efficient separation (purification) of the labeled compound and the glycan label.

[0170] (Washing) In the above purification, after capturing the sugar chain-labeled product on the solid support for purification, the solid support for purification may be washed with a solvent. The solvent used here is preferably a mixed solvent containing an organic solvent and water, more preferably a mixed solvent of acetonitrile, water, and alcohol. Either methanol or ethanol, or both, may be used as the alcohol.

[0171] In purification, washing is preferably carried out using a mixed solvent prepared by mixing the above three solvents with different hydrophobicities in a volume ratio of acetonitrile:alcohol:water=20-98:0-60:2-15 (total 100% by volume).

[0172] In the mixed solvent, acetonitrile is contained in an amount of preferably 30% by volume or more and 70% by volume or less, and more preferably 40% by volume or more and 65% by volume or less.

[0173] The mixed solvent preferably contains 30% by volume or more and 60% by volume or less, and more preferably 30% by volume or more and 55% by volume or less of the alcohol. The alcohol used is preferably ethanol.

[0174] The mixed solvent preferably contains water in an amount of 3% by volume or more and 15% by volume or less, and more preferably 2% by volume or more and 10% by volume or less.

[0175] An example of the mixed solvent is a mixed solvent of acetonitrile, alcohol, and water in a ratio of 50:40:10 (total 100% by volume).

[0176] After passing the mixed solvent through the solid phase carrier for purification, the solid phase carrier for purification can be washed by centrifuging to remove the solvent from the solid phase carrier for purification. This washing operation can remove the unreacted labeled compound as well as the above-mentioned unnecessary substances. The washing operation may be performed only once or may be performed two or more times. Repeated washing operations can remove unnecessary substances, but may also wash away glycan-labeled substances adhering to the solid phase carrier for purification, raising the concern that the yield of glycan-labeled substances may decrease. Therefore, it is recommended that the washing operation be performed three times or less.

[0177] In the re-release, a mixed solvent of an acid and an organic solvent or a mixed solvent of an acid, water, and an organic solvent can be brought into contact with the solid support to cause a reaction. The acidity of the mixed solvent may be, for example, pH 2 to 9, pH 2 to 7, or pH 2 to 6. A weakly acidic to near-neutral reaction is preferred because it can suppress hydrolysis of sugar chains, such as the elimination of sialic acid residues. However, strongly acidic conditions with even lower pH are also acceptable.

[0178] <Glycan analysis method> The glycan-labeled compounds prepared by the method of this embodiment can be analyzed qualitatively and / or quantitatively by known methods such as mass spectrometry (e.g., MALDI-TOF MS), chromatography (e.g., high-performance liquid chromatography or HPAE-PAD chromatography), and electrophoresis (e.g., capillary electrophoresis). Various databases (e.g., GlycoMod, Glycosuite, SimGlycan (registered trademark), etc.) can be used in glycan analysis. The glycan-labeled compounds prepared by the method of this embodiment are particularly suitable for capillary electrophoresis.

[0179] Such glycoprotein glycan analysis enables rapid analysis of glycosylation of antibody drugs, which is carried out during research and development, manufacturing, and quality assurance of antibody drugs; analysis of glycoproteins in samples such as serum, which is carried out during research to search for glycobiomassicators; glycoanalysis of stem cells; glycoanalysis of electrophoresis gel bands; and glycoanalysis of plant tissues.

[0180] When the amount of sugar chain-labeled substance obtained by the above-mentioned method is small, the analysis step may be carried out in the order of the following steps (1) to (3) using a known capillary electrophoresis apparatus. (1) A step of filling a sample into a capillary used for separation, and then filling the capillary with a leading electrolyte solution from the downstream end of the capillary. (2) A step of concentrating multiple glycan labels contained in the sample on the upstream side of the capillary by applying a voltage to the downstream side of the capillary with both ends immersed in the electrophoresis solution, with the voltage being positive on the downstream side and negative on the upstream side. (3) Separating the concentrated glycoconjugates by capillary electrophoresis.

[0181] That is, in the analysis step, the sugar chain-labeled compounds contained in the sample may be concentrated in steps (1) and (2), and then separated in step (3).

[0182] (Process (1)) The leading electrolyte solution contains an electrolyte. The electrophoretic speed of the electrolyte contained in the leading electrolyte solution is faster than that of the sugar chain label when compared under the same application conditions. Examples of such a leading electrolyte solution include Cl. - An electrolyte solution containing ions is preferred, for example, an aqueous solution of sodium chloride is preferred as the preceding electrolyte solution.

[0183] The concentration of the preceding electrolyte solution may be 0.5 mmol / L or more and 300 mmol / L or less, or 5 mmol / L or more and 100 mmol / L or less.

[0184] That is, the preceding electrolyte solution is preferably an aqueous sodium chloride solution having a concentration of 0.5 mmol / L or more and 300 mmol / L or less.

[0185] The capillary is filled with the sample and the preceding electrolyte solution by injecting each liquid into the end of the capillary. The preceding electrolyte solution is filled by controlling the pressure and the time of application of pressure when filling the capillary. The amount of the preceding electrolyte solution filled is preferably 0.05% to 20% of the total length of the capillary.

[0186] (Process (2)) In step (2), for example, both ends of the capillary are immersed in a vial filled with an electrophoresis buffer, and a voltage is applied to the capillary, which causes the preceding electrolyte solution filled on the downstream side of the capillary to migrate to the upstream side of the capillary, and the glycan-labeled compounds in the sample also migrate to the upstream side of the capillary and are concentrated.

[0187] (Step (3)) In step (3), the sugar chain-labeled products are separated by a known capillary electrophoresis method.

[0188] The separated sugar chain labels are then detected. In the analysis step, any detection method can be used as long as it is used in the technical field of the present invention and is capable of detecting the target substance. Examples of detection methods that can be used include laser-excited (induced) fluorescence detection and mass spectrometry. Among these, in this embodiment, it is preferable to detect sugar chain labels using laser-excited fluorescence detection.

[0189] By the above analytical method, sugar chain labels can be suitably detected from low-concentration samples.

[0190] [kit] In this embodiment, a kit for preparing a glycan of a glycoprotein is provided, which includes a solid phase for immobilizing a glycoprotein, a container for holding the solid phase and releasing and labeling the glycan, and a glycan-releasing enzyme.

[0191] The kit of this embodiment is for carrying out the above-mentioned method for preparing a sugar chain of a glycoprotein. The kit of this embodiment may include protocol information for using the kit. The protocol information for using the kit may be a printed document showing the above-mentioned method for preparing a sugar chain of a glycoprotein of the present invention, or may be access information that enables access to information on the web showing the method.

[0192] Furthermore, the kit of this embodiment may further include any one or all of a pretreatment agent containing a surfactant, a deglycosylation promoter containing an acid-derived anionic surfactant, a labeling reaction reagent, a cleanup solid phase, and a container for filling the cleanup solid phase.

[0193] Here, the surfactant contained in the pretreatment agent and the acid-derived anionic surfactant contained in the deglycosylation promoter may be the same compound. In this case, the pretreatment agent and the deglycosylation promoter may be contained in the same container without being distinguished from each other.

[0194] The container for holding the solid phase for immobilizing glycoproteins and for releasing and labeling glycans, or the container for packing the cleanup solid phase, may be a column, a multiwell plate, a filter plate, a microtube, or the like, but is preferably a spin column. The spin column may further include a collection tube for collecting the separated liquid obtained by solid-liquid separation by centrifugation. The container may be included in the kit in a state where it is packed with the solid phase, or may be included as an item separate from the solid phase.

[0195] The solid phase for immobilizing glycoproteins is a solid phase having on its surface binding functional groups, such as non-covalent groups (hydrogen-bonding groups and ionic-bonding groups) and covalent groups, that are capable of specifically binding to glycoproteins. Examples of solid phases include cation exchange carriers, hydrophobic interaction carriers, and inorganic carriers, but do not include solid phases that simply retain glycoproteins, such as electrophoresis gels or transfer membranes.

[0196] The solid phase may be an inorganic support. When the support is an inorganic support, for example, a portion of the support will not be released by a glycan-releasing enzyme. Therefore, it is easy to suppress the appearance of unnecessary signals in the analysis of released glycans.

[0197] When the glycoprotein is an antibody, the solid phase may have on its surface a ligand selected from the group consisting of protein A, protein G, protein L, protein H, protein D, and protein Arp. This enables high-throughput preparation and analysis of glycan samples for antibodies, for which glycan analysis is particularly important.

[0198] The labeling reaction reagent may contain a labeling compound, a reducing agent, and a solvent. Alternatively, the labeling compound, the reducing agent, and the solvent may be contained in separate containers and mixed at the time of use.

[0199] The kit of this embodiment enables the release of glycans from glycoproteins without decomposing the protein portion. Therefore, the time required for the glycan release treatment can be significantly reduced. Furthermore, the glycan release enzyme can be made more effective in the glycan release treatment.

[0200] Furthermore, when the kit contains a labeling reaction reagent, the labeling reaction reagent can be added in layers without separating the released product, allowing the glycan to be prepared from the glycoprotein in an analytical sample (in a labeled form) extremely quickly.

[0201] [Device] 1 is a schematic diagram illustrating an example of an apparatus for carrying out the sugar chain preparation method of this embodiment. The apparatus 100 includes a holding unit 20 for holding a container 15 containing a glycoprotein immobilized on a solid phase 10, and an introduction unit 30 for introducing a reagent into the container 15.

[0202] The holder 20 is for holding a container 15 that is to contain a glycoprotein immobilized on the solid phase 10. The manner in which the holder 20 holds the container 15 is not particularly limited, and examples include a manner in which the container is held by fitting most of the container into a holding hole or a holding aperture of the holder 20. Other examples include a manner in which the container is held by engaging an engaging protrusion (engaging recess) of the holder with an engaging recess (engaging protrusion) of the container, and a manner in which the container is clamped and held by the clamping portion of the holder.

[0203] The introduction unit 30 is used to introduce liquids into the container 15 held in the holding unit 20. In the example of FIG. 1, the introduction unit 30 is equipped with a tank 34 that contains a glycan-releasing enzyme 31, a labeling reaction reagent 32, and a pretreatment agent / deglycosylation promoter 33, a liquid delivery pipe 35a that delivers each of the reagents contained in the tank 34, valves (36, 37, 38) that control the delivery of each of the reagents, and a nozzle 35 that introduces each of the reagents into the container 15.

[0204] The introduction section 30 includes at least a glycan-releasing enzyme introduction section for introducing a glycan-releasing enzyme 31 used in the release step, and a labeling reaction reagent introduction section for introducing a labeling reaction reagent 32 used in the labeling step.

[0205] The introduction unit 30 adds a glycan-releasing enzyme 31 and a labeling reaction reagent 32 to the same container 15. The manner in which the introduction unit 30 introduces a liquid into the container 15 is not particularly limited, and examples include a manner in which the liquid to be delivered is delivered from a tank 34 in which the liquid is stored to the container 15 via a tubular member. Other examples include a manner in which the liquid collected in a tubular member is poured into the container.

[0206] In the introduction unit 30, the glycan-releasing enzyme introduction unit and the labeling reaction reagent introduction unit may be configured as separate, independent components. In this case, the glycan-releasing enzyme 31 and the labeling reaction reagent 32 may be introduced sequentially in this order, or may be introduced at the same time. The labeling reaction reagent introduction unit may be automatically controlled, and when both reagents are introduced sequentially, the timing of operation of the labeling reaction reagent introduction unit may be controlled based on the reaction time required for the release step, etc.

[0207] Alternatively, the glycan-releasing enzyme introduction section and the labeling reaction reagent introduction section may be configured as the same component in the introduction section 30. In this case, the glycan-releasing enzyme 31 and the labeling reaction reagent 32 may be introduced in a mixed state, or may be introduced sequentially in that order. When both reagents are introduced sequentially, the timing at which the labeling reaction reagent is sent, i.e., the timing at which the introduction section 30 is made to function as the labeling reaction reagent introduction section, may be controlled based on the reaction time required for the release step, etc.

[0208] The apparatus 100 may further include a separation unit 40 that separates the contents of the container 15 into solid and liquid. When the apparatus 100 includes the separation unit 40, the separation unit 40 separates solids and liquids from the contents contained in the container 15. The solids are those that remain in the container 15, and are essentially the solid phase 10 and the substances fixed thereto. In this case, the container 15 is equipped with a filter capable of solid-liquid separation (e.g., a spin column, a microplate with a filter, etc.). Furthermore, the container 15 may be used with a collection container 16 (e.g., a collection tube, a collection plate, etc.) attached thereto. Furthermore, in this case, the holding unit 20 may be configured to include a collection holding unit that holds the collection container 16 attached to the container 15. In the example of FIG. 1, the collection holding unit and the holding unit 20 are configured from the same member.

[0209] The specific separation type of the separation unit 40 is not particularly limited, and may be any of centrifugal filtration, vacuum filtration, and pressure filtration. In the example of Fig. 1, the separation type of the separation unit 40 is centrifugal filtration. The separation unit 40 includes a rack 41 that holds the container 15 (or 16), a drive shaft 42, and a motor 43.

[0210] 1, the separation unit 40 may be configured as a component independent of the holding unit 20 in which the releasing step and the labeling step are performed. In this case, the device 100 may include a transfer unit 50 that automatically transfers the container 15 (and 16) from the holding unit 20 to the separation unit 40.

[0211] The transfer unit 50 may be configured to transfer only the container 15 (and 16), or may be configured to transfer the container 15 with the collection container 16 attached. The transfer unit 50 may be configured to include an arm that operates to grip, release, and move the container 15 directly or indirectly (i.e., via the collection container 16), and an arm control unit that controls the operation of the arm.

[0212] By operating the separation unit 40, the liquid is collected in the collection container 16. Therefore, for example, a separated liquid containing a glycoconjugate can be collected in the collection container 16 by vacuum filtration, pressure filtration, centrifugation, or the like from the reaction product in the reaction container (i.e., the contents of the reaction container after the reaction) obtained by introducing the glycan-releasing enzyme 31 and the labeling reaction reagent 32. Furthermore, for example, when preparing a glycoprotein immobilized on the solid phase 10, the glycoprotein immobilized on the solid phase 10 can be left in the container 15 from the preparation obtained by bringing the sample into contact with the solid phase, and the remainder of the sample after removing the glycoprotein can be discarded in the collection container 16.

[0213] Furthermore, when the device 100 has the separation unit 40, the introduction unit 30 may be configured to be able to further introduce a cleaning liquid into the container 15. This allows the cleaning liquid to pass through the container 15.

[0214] The apparatus 100 may further include a temperature control unit 60 that controls the temperature of the contents of the container 15. When the apparatus 100 includes the temperature control unit 60, the temperature control unit 60 only needs to have at least a heater function. The temperature control unit 60 heats the container 15 to the temperatures required for the release step and the labeling step.

[0215] Furthermore, the apparatus 100 may be configured to provide an open space that communicates with the space inside the reaction vessel. This allows the solvent in the vessel 15 to evaporate when the release step is performed in an open system, making it easy to provide a concentration at which glycan release proceeds efficiently, regardless of the amount of glycoprotein. Furthermore, because the solvent removal is performed simultaneously with the release reaction, there is no need for a solvent removal step separate from the release step, enabling even more rapid glycan preparation.

[0216] The apparatus 100 may include a liquid transfer unit (not shown) that automatically transfers the separation liquid containing the sugar chain-labeled substance recovered in the recovery container by solid-liquid separation after the labeling step to a purification column containing a solid phase for purification. The purification column may be installed in the separation unit 40 described above.

[0217] In the apparatus 100, at least one, preferably all, of the operable components (e.g., the introduction section 30, the separation section 40, the transfer section 50, the temperature control section 60, and the liquid transfer section) may be automatically controlled, thereby enabling the preparation of sugar chains of glycoproteins to be carried out more quickly.

[0218] According to the above configuration, it is possible to provide a sugar chain preparation method for quickly preparing a labeled sugar chain from a glycoprotein.

[0219] Furthermore, according to the sugar chain analysis method described above, even if the amount of sugar chain used for analysis is small, analysis can be performed suitably.

[0220] [Second embodiment] <Glycan preparation method> The glycan preparation method of this embodiment includes a release step in which a glycan-releasing reagent is applied to a sample containing a glycoprotein to obtain a released product containing a glycan; an adsorption step in which a purification agent is brought into contact with a mixture containing the released product to adsorb the glycan to the purification agent; an elution step in which the glycan is eluted from the purification agent; and a labeling step in which the obtained eluate is reacted with a labeling reaction reagent to obtain a labeled product containing a labeled form of the glycan.

[0221] As will be described in more detail below, in this embodiment, a labeling reaction reagent for labeling glycans is used, which contains at least one labeling compound selected from the group consisting of 8-aminopyrene-1,3,6-trisulfonic acid, the sodium salt of 8-aminopyrene-1,3,6-trisulfonic acid, and 9-aminopyrene-1,4,6-trisulfonic acid.

[0222] According to the method of this embodiment, a sugar chain preparation method can be provided that allows for rapid preparation of labeled sugar chains from glycoproteins. The following explains each in order.

[0223] [Release process] In the release step, a glycan-releasing enzyme is allowed to act on a sample containing glycoproteins (hereinafter sometimes simply referred to as the sample) to release glycans and obtain a released product. This step does not substantially include a step of fragmenting proteins by chemical fragmentation, enzymatic fragmentation, or the like.

[0224] Glycoproteins In the second embodiment, the glycoprotein may be any protein containing at least an O-glycan (O-linked glycan) as a complex component. That is, the glycoprotein is composed of a "glycan portion" containing an O-glycan and a "protein portion."

[0225] In the second embodiment, the term "glycoprotein" refers to a protein having at least one O-glycan bound to its amino acid sequence. The glycoprotein to be used in the method for preparing a glycan from a glycoprotein is not particularly limited, and may be naturally occurring or synthetic.

[0226] Furthermore, in the second embodiment, "glycan" includes O-glycans, and any O-glycan can be prepared from a glycoprotein. An O-glycan is a structure in which a glycan is bound to a serine (Ser) or threonine (Thr) amino acid residue in a protein via an -OH group contained in each amino acid side chain. Furthermore, O-glycans are classified into 1 to 8 types depending on the core structure.

[0227] It should be noted that the term "sugar chain" includes monosaccharides.

[0228] Examples of the structure and glycoprotein of the glycoprotein of the second embodiment are the same as those shown in (Glycoprotein structure and examples) of the first embodiment.

[0229] <Glycan-releasing reagent> The sugar chain releasing reagent acts on the sugar chain binding site in the glycoprotein to release the sugar chain from the glycoprotein. The sugar chain releasing reagent contains a hydroxylamine compound and a basic reagent.

[0230] The sugar chain releasing reagent may be used as long as it ultimately brings the glycoprotein, hydroxylamines, and basic reagent into contact with each other. For example, any of the following (i) to (iii) may be used. In order to suppress the decomposition (peeling) of O-glycans, (i) is preferred. (i) Addition of hydroxylamines to glycoproteins followed by addition of a basic reagent. (ii) Addition of a basic reagent to the glycoprotein followed by addition of hydroxylamines. (iii) Hydroxylamines and a basic reagent are mixed and then added to the glycoprotein.

[0231] Hydroxylamines The hydroxylamines include at least one selected from the group consisting of hydroxylamine, salts of hydroxylamine, O-substituted hydroxylamines, and salts of O-substituted hydroxylamines.

[0232] Specifically, aqueous hydroxylamine; Hydroxylamine hydrochloride, hydroxylamine sulfate, hydroxylamine phosphate; O-(tetrahydro-2H-pyran-2-yl)hydroxylamine, O-tertiarybutyldimethylsilylhydroxylamine, O-trimethylsilylhydroxylamine; O-methylhydroxylamine hydrochloride, O-ethylhydroxylamine hydrochloride, nitrobenzylhydroxylamine hydrochloride; At least one compound selected from the group consisting of:

[0233] The hydroxylamine is preferably an aqueous hydroxylamine solution.

[0234] The final concentration of hydroxylamines in the mixture (reaction solution) of the glycoprotein and the glycan-releasing reagent may be 2% to 70% by volume, 5% to 70% by volume, or 10% to 60% by volume. However, the concentration is not limited to the above ranges, and a person skilled in the art can appropriately adjust the concentration depending on the type of glycoprotein of interest, other components (amines, basic reagents, other additives), contact conditions (time, temperature, etc.), etc.

[0235] Basic reagents The basic reagent may include at least one compound selected from the group consisting of alkali metal hydroxides, weak alkali metal acid salts, alkaline earth metal hydroxides, alkaline earth metal salts dissolved in aqueous ammonia, and organic bases.

[0236] Examples of alkali metal hydroxides include, but are not limited to, lithium hydroxide, sodium hydroxide, and potassium hydroxide.

[0237] Furthermore, examples of weak acid salts of alkali metals include, but are not limited to, sodium bicarbonate and sodium carbonate.

[0238] The hydroxides of alkaline earth metals include, but are not limited to, calcium hydroxide, barium hydroxide, and strontium hydroxide.

[0239] The alkaline earth metal salt dissolved in aqueous ammonia may include, but is not limited to, calcium acetate, calcium chloride, barium acetate, and magnesium acetate.

[0240] Of these, lithium hydroxide is particularly preferred.

[0241] Examples of organic bases include, but are not limited to, DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene TBD: 1,5,7-triazabicyclo[4.4.0]dec-5-ene, MTBD: 7-Methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene, TMG: 1,1,3,3-Tetramethylguanidine t-BuTMG: 2-tert-butyl-1,1,3,3-tetramethylguanidine, DBN: 1,5-diazabicyclo[4.3.0]non-5-ene, CTAH: cetyltrimethylammonium hydroxide The following can be mentioned:

[0242] The compounds listed as organic bases may be used alone or in combination of two or more.

[0243] The organic base is preferably a strong organic base (pKa of 12 or more), and specific examples thereof include DBU, TBD, MTBD, TMG, and CTAH. When DBU, TMG, TBD, MTBD, or CTAH is used as the organic base, it is preferable because the base can be more easily removed after the reaction by washing with an organic solvent after the release step.

[0244] The final concentration of the basic reagent in the reaction solution can be, for example, in the range of 2 mmol / L to 10 mol / L. However, the concentration is not limited to the above range, and those skilled in the art can appropriately adjust the concentration depending on the type of glycoprotein of interest, other components in the reaction solution (hydroxylamines, other additives), reaction conditions (time, temperature, etc.), etc. For example, when lithium hydroxide is used as the basic reagent, the concentration can be 6 mmol / L to 8 mol / L.

[0245] By setting the concentration of the basic reagent to the above lower limit or more, the reaction time can be shortened, and by setting the concentration of the basic reagent to the above upper limit or less, the influence of the remaining basic reagent on the next step can be suppressed.

[0246] The molar ratio of hydroxylamines to the basic reagent is preferably 1:2 or more and 300:1 or less, and more preferably 1:1 or more and 100:1 or less. By setting the molar ratio of hydroxylamines to the basic reagent within the above range, the decomposition (peeling) reaction of the released sugar chains can be suppressed and the yield of sugar chains can be improved.

[0247] The conditions for contacting the glycoprotein with the glycan-releasing reagent, such as the temperature (reaction temperature) and time (reaction time), are not particularly limited as long as the glycan can be released from the target protein, and can be appropriately determined by a person skilled in the art depending on the conditions, such as the type and concentration of the target glycoprotein, hydroxylamines, and basic reagent.

[0248] The reaction temperature can be, for example, from room temperature to 80°C. Lowering the reaction temperature can suppress the rate of glycosylation (peeling). In particular, N-glycolyl groups and the like are easily decomposed when reacted at high temperatures, so when targeting glycoproteins with unknown glycosylation, the temperature is preferably 50°C or lower, for example, about 37°C.

[0249] The reaction time can be, for example, about 5 minutes to 16 hours.

[0250] Amines Amines may be further added to the sugar chain releasing reagent, including, but not limited to, at least one compound selected from the group consisting of aqueous ammonia, aqueous methylamine solution, aqueous dimethylamine solution, ethylamine, diethylamine, ethanolamine, ethylenediamine, butylamine, morpholine, DABCO, and anthranilic acid.

[0251] The amines are preferably aqueous ammonia, morpholine, DABCO, or anthranilic acid. By using aqueous ammonia, morpholine, DABCO, or anthranilic acid as the amine, peeling, isomerization, and decomposition of amides are suppressed.

[0252] The final concentration of amines in the reaction solution can be, for example, in the range of 40 mmol / L to 15 mol / L, but is not limited to the above-mentioned concentration range, and can be appropriately adjusted by a person skilled in the art depending on the type of glycoprotein of interest, other components in the reaction solution (hydroxylamines, basic reagents, other additives), reaction conditions (time, temperature, etc.), etc.

[0253] When aqueous ammonia is used as the amine, the final concentration of ammonia in the reaction solution can be 2% by volume or more and 25% by volume or less, preferably 10% by volume or more and 20% by volume or less, and more preferably 20% by volume.

[0254] (Step of removing hydroxylamines) As described above, it is preferable that the final concentration of hydroxylamines be as high as possible, especially when liberating O-linked glycans. However, if the mixture of glycoprotein and reaction solution contains a high concentration of hydroxylamines, unreacted hydroxylamines may remain in the mixture after liberation of the glycans. Unreacted hydroxylamines inhibit the labeling reaction when labeling and analyzing glycans, so they are preferably removed.

[0255] Therefore, the releasing step may further include a step of removing unreacted hydroxylamines after releasing the sugar chains from the glycoprotein. In the removing step, a ketone, an aldehyde, or an acid anhydride is added to the reaction solution from which the sugar chains have been released.

[0256] By adding a ketone to the reaction solution, the hydroxylamine and the ketone can be reacted to convert them into ketoximes. Examples of ketones that can be used include acetone, methyl ethyl ketone, methyl isobutyl ketone, and 4-hydroxybutanone.

[0257] By adding an aldehyde to the reaction mixture, the hydroxylamine and the aldehyde can be reacted to convert them into aldoximes. Examples of aldehydes that can be used include salicylaldehyde, benzaldehyde, and 4-hydroxybenzaldehyde.

[0258] By adding an acid anhydride to the reaction solution, the hydroxylamine can be reacted with the acid anhydride to convert it into an amide. Examples of the acid anhydride that can be used include acetic anhydride and succinic anhydride.

[0259] (free product) The released product obtained by the release step is a mixture containing released sugar chains corresponding to the sugar chain moieties of the glycoprotein and proteins corresponding to the protein moieties of the glycoprotein.

[0260] [Adsorption process] In the adsorption step, a purification agent is brought into contact with the mixture containing the liberated product, and the sugar chains (liberated sugar chains) are adsorbed onto the purification agent.

[0261] Refiner The purification agent is a solid phase that has an affinity for glycans. When the purification agent comes into contact with the mixture, it can adsorb the glycans contained in the mixture.

[0262] Examples of such purification agents include hydrophilic carriers such as graphite carbon, crystalline cellulose, silica, and monolithic silica. Monolithic silica is a filter-like porous continuous silica with a three-dimensional network structure, and has advantages such as better liquid permeability and less dead volume than conventional particulate silica. The monolithic silica may be fixed in a column-shaped container, such as a multi-well plate.

[0263] The pore size of the monolithic silica is preferably 1 to 100 μm, more preferably 1 to 50 μm, further preferably 1 to 30 μm, and particularly preferably 1 to 20 μm, in terms of diameter of interconnected pores (through pores).

[0264] The purification agent is preferably at least one of the following (A) and (B): (A) Polymer having a betaine structure (B) A complex having a polymer having a betaine structure and a support supporting the polymer.

[0265] The term "betaine structure" refers to a molecular structure that satisfies the following requirements (a) to (c): (a) Positive and negative charges are present in non-adjacent positions within the same molecular structure. (b) The positively charged atom has no dissociable hydrogen atoms bonded to it. (c) The molecular structure as a whole does not have an electric charge.

[0266] Specifically, the betaine structure is either of the structures of the following formula (1) or formula (2): A compound having the structure of either of the following formula (1) or formula (2) in the molecule has a betaine structure at the molecular terminal. -ZLA...(1) -ALZ...(2) In the formulae (1) and (2), Z represents a cationic group selected from the group consisting of a secondary amino group, a tertiary amino group, a quaternary ammonium group, an imino group, and an iminium group. L represents an alkylene group having 1 to 10 carbon atoms. A represents an anionic group selected from the group consisting of a phosphate group, a carboxyl group, a phosphonate group, a phosphinate group, a sulfonic acid group, a sulfinate group, a sulfene group, a hydroxyl group, a thiol group, and a boronic acid group.

[0267] That is, the betaine structure in the purification agent has an anionic group, a cationic group, and a linker connecting the anionic group and the cationic group.

[0268] The cationic groups include secondary amino groups (-NHR), tertiary amino groups (-NR2), and quaternary ammonium groups (-N + R3), imino group (-C(=NR)-) and iminium group (-C(=N + R2)-) are included, but are not limited to these.

[0269] R in the cationic group is an alkyl group or an aryl group, and an alkyl group is preferred. The alkyl group may be linear or branched. When the cationic group has multiple R, the multiple R may be different or the same. R is preferably, for example, an alkyl group having 1 to 3 carbon atoms.

[0270] The cationic group is preferably a quaternary ammonium group, and in the above formula (2), the cationic group is more preferably a trimethylammonium group.

[0271] The cationic group may bond ionically with an anion to form a salt. Even in such a cationic group, the anion is released in the mixed solution and the cationic group functions as a cationic group. Examples of anions that bond ionically with the cationic group include fluoride ions, chloride ions, bromide ions, iodide ions, hydrochloride ions, acetate ions, sulfate ions, hydrofluoric acid ions, and carbonate ions.

[0272] In formulas (1) and (2), the group represented by L is a linker that connects a cationic group and an anionic group. The alkylene group that is the linker may be linear, cyclic, or branched, but is preferably linear. The alkylene group preferably has 2 to 5 carbon atoms.

[0273] Examples of anionic groups include, but are not limited to, phosphate groups (-OP(=O)(OH)), carboxyl groups (-COOH), phosphonic acid groups (-P(=O)(OH)), phosphinic acid groups (-P(=O)R(OH)), sulfonic acid groups (-SOH), sulfine groups (-S(=O)OH), sulfene groups (-SOH), hydroxyl groups (-OH), thiol groups (-SH), and boronic acid groups (-B(OH)).

[0274] As the anionic group, a phosphate group, a phosphonate group, a sulfonic acid group and a carboxyl group are preferred, and a phosphate group and a carboxyl group are more preferred.

[0275] The anionic group may bond with a cation to form a salt. Even in the case of such an anionic group, the cation is released in the mixed solution and the anionic group functions as an anionic group. Examples of the cation that bonds with the anionic group include alkali metal ions such as sodium ions and potassium ions, and alkaline earth metal ions such as calcium ions.

[0276] In the betaine structure of the purification agent, the combination of the cationic group and the anionic group is not particularly limited. A preferred combination is one in which the anionic group is a group selected from the group consisting of a phosphate group, a phosphonate group, a carboxyl group, and a sulfonic acid group, the cationic group is a quaternary ammonium group, and the linker is an alkylene group having 1 to 4 carbon atoms.

[0277] In addition, in the betaine structure of the purifying agent, the anion group is more preferably a phosphate group. Furthermore, in the betaine structure of the purification agent, the moiety exhibiting the betaine structure is preferably a phosphorylcholine group.

[0278] ((A) Polymer having a betaine structure) The purification agent may be a polymer (macromolecule) having a betaine structure represented by the above formula (1) or (2). In the following description, the "polymer having a betaine structure" may be referred to as "polymer A."

[0279] The polymer A may be linear or may have a branched structure.

[0280] When polymer A has a branched structure, the main chain may have a betaine structure, or the side chain may have a betaine structure.When the side chain has a betaine structure, polymer A may have all of the side chains have a betaine structure, or only a part of the side chains have a betaine structure.The side chains that do not have a betaine structure may or may not have a charge.

[0281] There are no particular restrictions on the main chain of polymer A. The main chain preferably contains a repeating unit derived from a (meth)acrylic compound, and more preferably contains a repeating unit derived from a (meth)acrylic acid ester or a derivative of a (meth)acrylic acid ester.

[0282] The main chain may further contain repeating units copolymerizable with the above repeating units, such as repeating units derived from a vinyl group, an allyl group, an α-alkoxymethylacryloyl group, a maleic acid residue, a fumaric acid residue, an itaconic acid residue, a crotonic acid residue, an isocrotonic acid residue, and a citraconic acid residue.

[0283] Polymer A is preferably a polymer of a polymerizable monomer having a betaine structure. Known monomers can be used as the polymerizable monomer having a betaine structure. Examples include (i) phosphobetaine monomers having a phosphobetaine group, (ii) carboxybetaine monomers having a carboxybetaine group, and (iii) sulfobetaine monomers having a sulfobetaine group.

[0284] (i) As the phosphobetaine monomer, a polymerizable monomer having a phosphorylcholine group is preferred. For example, 2-(meth)acryloyloxyethyl phosphorylcholine, 2-(meth)acryloyloxyethoxyethyl phosphorylcholine, 6-(meth)acryloyloxyhexylphosphorylcholine, 10-(meth)acryloyloxyethoxynonylphosphorylcholine, 2-(meth)acryloyloxypropylphosphorylcholine, and 2-(meth)acryloyloxybutylphosphorylcholine.The above monomers have a betaine structure in which A is a phosphate group, Z is a quaternary ammonium group, and L is an ethylene group in formula (2).

[0285] Among these, 2-(meth)acryloyloxyethyl phosphorylcholine is preferred as the phosphobetaine monomer because of its easy availability, and 2-methacryloyloxyethyl phosphorylcholine (MPC) is more preferred.

[0286] Furthermore, as the phosphobetaine monomer, a monomer having a betaine structure represented by the above formula (1) can also be used. For example, Dimethyl(2-methacryloyloxyethyl)(phosphonatomethyl)aminium, dimethyl(2-acryloyloxyethyl)(phosphonatomethyl)aminium (A in the above formula (1) is a phosphonic acid group, Z is a quaternary ammonium group, and L is a methylene group) Dimethyl(2-methacryloyloxyethyl)(2-phosphonatoethyl)aminium, dimethyl(2-acryloyloxyethyl)(2-phosphonatoethyl)aminium (where A in the above formula (1) is a phosphonic acid group, Z is a quaternary ammonium group, and L is an ethylene group) Dimethyl(2-methacryloyloxyethyl)(3-phosphonatopropyl)aminium, dimethyl(2-acryloyloxyethyl)(3-phosphonatopropyl)aminium (A in the above formula (1) is a phosphonic acid group, Z is a quaternary ammonium group, and L is a propylene group) Dimethyl(2-methacryloyloxyethyl)(4-phosphonatobutyl)aminium, dimethyl(2-acryloyloxyethyl)(4-phosphonatobutyl)aminium (where A in the above formula (1) is a phosphonic acid group, Z is a quaternary ammonium group, and L is a butylene group) Examples include:

[0287] As the carboxybetaine monomer, a monomer having a betaine structure represented by the above formula (1) can be used. For example, Dimethyl(2-methacryloyloxyethyl)(carboxylatomethyl)aminium, dimethyl(2-acryloyloxyethyl)(carboxylatomethyl)aminium (in the above formula (1), A is a carboxyl group, Z is a quaternary ammonium group, and L is a methylene group) Dimethyl(2-methacryloyloxyethyl)(2-carboxylatoethyl)aminium, dimethyl(2-acryloyloxyethyl)(2-carboxylatoethyl)aminium (in the above formula (1), A is a carboxyl group, Z is a quaternary ammonium group, and L is an ethylene group) Dimethyl(2-methacryloyloxyethyl)(3-carboxylatopropyl)aminium, dimethyl(2-acryloyloxyethyl)(3-carboxylatopropyl)aminium (A in the above formula (1) is a carboxyl group, Z is a quaternary ammonium group, and L is a propylene group) Dimethyl(2-methacryloyloxyethyl)(4-carboxylatobutyl)aminium, dimethyl(2-acryloyloxyethyl)(4-carboxylatobutyl)aminium (A in the above formula (1) is a carboxyl group, Z is a quaternary ammonium group, and L is a butylene group) Examples include:

[0288] As the sulfobetaine monomer, a monomer having a betaine structure represented by the above formula (1) can be used. For example, Dimethyl(2-methacryloyloxyethyl)(sulfonatomethyl)aminium, dimethyl(2-acryloyloxyethyl)(sulfonatomethyl)aminium (in the above formula (1), A is a sulfonic acid group, Z is a quaternary ammonium group, and L is a methylene group) Dimethyl(2-methacryloyloxyethyl)(2-sulfonatoethyl)aminium, dimethyl(2-acryloyloxyethyl)(2-sulfonatoethyl)aminium (A in the above formula (1) is a sulfonic acid group, Z is a quaternary ammonium group, and L is an ethylene group) Dimethyl(2-methacryloyloxyethyl)(3-sulfonatopropyl)aminium, dimethyl(2-acryloyloxyethyl)(3-sulfonatopropyl)aminium (A in the above formula (1) is a sulfonic acid group, Z is a quaternary ammonium group, and L is a propylene group) Dimethyl(2-methacryloyloxyethyl)(4-sulfonatobutyl)aminium, dimethyl(2-acryloyloxyethyl)(4-sulfonatobutyl)aminium (where A in the above formula (1) is a sulfonic acid group, Z is a quaternary ammonium group, and L is a butylene group) Examples include:

[0289] As these polymerizable monomers having a betaine structure, phosphobetaine monomers are preferred, and phosphobetaine monomers having a phosphorylcholine group are more preferred.

[0290] ((B) Complex Having a Polymer Having a Betaine Structure and a Support Carrying the Polymer) As the purification agent, the above polymer A may be used alone, or may be used as a complex in which the polymer A is supported on an insoluble support. The complex in which the above polymer A is supported on an insoluble support is preferred because it can be easily separated from the mixture after adsorption of the sugar chain, and the purification procedure becomes simple.

[0291] Fig. 2 is a schematic diagram showing an example of a purification agent used in the purification method of this embodiment. As shown in Fig. 2, purification agent 1 is a complex having a polymer having a betaine structure (polymer A) and a support 2 supporting the polymer. In purification agent 1 shown in Fig. 2, support 2 is spherical (particulate), and the polymer having a betaine structure is provided in the form of a layer on the surface of support 2. In Fig. 2, the layer of polymer provided in the form of a layer is indicated by reference numeral 3, and the polymer having a betaine structure that constitutes layer 3 is indicated by reference numeral 3a.

[0292] (Support) 2, the support 2 is shown as being spherical, but is not limited to this. For example, the shape of the support may be a plate such as a substrate or a multiwell plate, a membrane such as a sheet, film, or membrane, or a fiber.

[0293] When the support 2 is spherical and the purification agent 1 is spherical, the average particle size of the purification agent 1 is preferably 0.5 μm to 100 μm, more preferably 1 μm to 50 μm, even more preferably 1 μm to 10 μm, and particularly preferably 3 μm to 10 μm. It is preferable that the average particle size of the purification agent 1 is equal to or greater than the lower limit, as this facilitates handling. It is also preferable that the average particle size of the purification agent 1 is equal to or less than the upper limit, as this facilitates good contact between the glycans and the purification agent 1 in the mixed solution prepared in the release step, and facilitates adsorption of the glycans to the purification agent 1.

[0294] The average particle size of the purification agent 1 can be measured, for example, with a particle size distribution analyzer.

[0295] The purification agent 1 may be used in a state where it is filled in a container such as a filter cup of a spin column or the like, each well of a multi-well plate, each well of a filter plate, or a microtube.

[0296] In addition, in FIG. 2, the polymer 3a covers the entire surface of the support 2, but this is not limiting, and the surface of the support 2 may be exposed.

[0297] The material of the support 2 is a base material that is insoluble in water and organic solvents used in the purification process of the sugar chains and that can support the polymer having the betaine structure (polymer A). The material of such a support 2 may be an inorganic material, an organic material, or a composite material of an inorganic material and an organic material.

[0298] Examples of inorganic materials include glass, iron oxides (ferrite, magnetite, etc.), oxides such as silica, alumina, titania, and zirconia, metals such as iron, copper, gold, silver, platinum, cobalt, aluminum, palladium, iridium, and rhodium, and alloys thereof, and carbon materials such as graphite. These materials may be used alone or in combination of two or more.

[0299] Examples of organic materials include synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene, and polysaccharides such as cross-linked sepharose, crystalline cellulose, cross-linked cellulose, cross-linked amylose, cross-linked agarose, and cross-linked dextran. These materials may be used alone or in combination of two or more.

[0300] It is preferable to use an inorganic material as the support 2. Organic materials that can be used as the support 2 have a specific gravity of around 1, which is small in difference from the specific gravity of the mixed solution used in purification. Therefore, if an organic material is used as the material for the support 2, solid-liquid separation may become complicated. On the other hand, if an inorganic material is used as the material for the support 2, solid-liquid separation of the mixed solution and the purification agent can be easily and simply performed. This can contribute to improving work efficiency. As the material for the support 2, silica is particularly preferred.

[0301] The support 2 may be porous. By using a porous support 2, it is possible to increase the amount of polymer A having a betaine structure fixed to the surface of the support 2. This can contribute to improving work efficiency.

[0302] The specific gravity of the entire purification agent can also be adjusted by using a support having pores in combination with a support having no pores, or by adjusting the amount of pores in a porous support.

[0303] The method for supporting polymer A on the surface of the support may be either physical adsorption or chemical bonding. Chemical bonding is preferred as the method for supporting polymer A because polymer A is unlikely to be released from the support during the sugar chain purification process.

[0304] For example, the polymer can be bonded to and supported on the surface of a support by polymerizing the polymerizable monomer in the presence of a support having reactive sites such as hydroxyl groups on the surface. Alternatively, polymer A can be supported on the surface of a support by a known method described in WO2019 / 088167.

[0305] In such a purification agent 1, the weight of polymer A bound to support 2 is determined based on the unit surface area (m 2 The polymer weight per unit surface area is preferably 0.5 mg to 1.5 mg, more preferably 0.6 mg to 1.3 mg, and even more preferably 0.7 mg to 1.2 mg. When the polymer weight per unit surface area is within the above range, polymer A can be easily grown from support 2 during polymerization of polymer A, resulting in good handling during polymer synthesis. Furthermore, in purification agent 1, polymer A can be easily brought into contact with sugar chains, allowing efficient adsorption of sugar chains.

[0306] The weight of polymer A bound to support 2 can be determined from the weight loss rate determined by thermogravimetric analysis of the purification agent and the BET specific surface area of ​​support 2 determined by nitrogen adsorption method.

[0307] The specific gravity of such a purification agent is preferably 1.05 to 3.00, more preferably 1.1 to 2.7, and even more preferably 1.5 to 2.5. When the specific gravity of the purification agent is equal to or greater than the lower limit, the purification agent is easily precipitated, facilitating the solid-liquid separation process. Furthermore, when the specific gravity of the purification agent is equal to or less than the upper limit, the purification agent is easily dispersed in the mixture prepared in the release step. This makes it easier to bring the purification agent into contact with the glycans in the mixture prepared in the release step, and to adsorb the glycans to the purification agent.

[0308] Such a purification agent is contacted with a mixed solution containing the liberated product. The mixed solution may be treated by a known method, such as desalting. The purification agent adsorbs the sugar chains contained in the mixed solution. Furthermore, proteins, peptide fragments, and the like present in the mixed solution are not adsorbed to the purification agent and remain in a free state.

[0309] The solvent for the mixed solution can be an organic solvent or a mixed solvent of an organic solvent and water. The solvent can be selected appropriately depending on the type of sugar chain to be concentrated, etc. The organic solvent is not particularly limited as long as it can dissolve the sugar chain, and examples include acetonitrile, tetrahydrofuran, acetone, dioxane, pyridine, methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol. Organic solvents such as 1-butanol and ethanol are preferably used. Various buffer solutions can be used to adjust the pH. When a mixed solvent of an organic solvent and water is used, the mixing ratio of the organic solvent to the water is, for example, 3:1 or more and 1000:1 or less by volume.

[0310] If necessary, the purification agent to which the sugar chains have been adsorbed can be washed, which allows impurities other than the sugar chains adsorbed to the purification agent, such as proteins, peptide fragments, lipids, salts, etc., to be removed.

[0311] The washing solution may be, for example, the solvents described above. Acetonitrile is preferably used as the solvent, since it is possible to elute impurities while suppressing the elution of sugar chains.

[0312] [Elution process] In the elution step, the glycans are eluted from the purifying agent to which they have been adsorbed. An organic solvent or a mixed solvent of an organic solvent and water can be used as the eluent for eluting the glycans from the purifying agent, and the eluent can be appropriately selected depending on the type of glycans to be eluted and the type of purifying agent.

[0313] As the organic solvent, those mentioned above can be used.

[0314] In the elution step, the sugar chains can be eluted efficiently by using a solvent with enhanced hydrophilicity. For example, water alone can be used without using an organic solvent, or a mixed solvent of organic solvent and water can be used. When using a mixed solvent, the organic solvent should be used in a volume ratio of 3 times or less to water, but it is particularly preferable to use only water without using an organic solvent.

[0315] Ultrapure water is particularly preferred as the eluent for eluting sugar chains from the purification agent. Note that "ultrapure water" refers to ultrapure water prepared using, for example, an ultrapure water production system (model number: MilliQ EQ7000, manufactured by Merck) or water equivalent thereto.

[0316] In this embodiment, some or all of the adsorption and elution steps can be performed by a batch method, a spin column method, etc. The batch method and the spin column method will be described in detail below, and the reagents and reaction conditions are as described above.

[0317] (batch method) In the batch method, first, the released product obtained in the release step is brought into contact with the above-mentioned purification agent in an appropriate container (e.g., a microtube, a centrifuge tube, a microplate, etc.), and the glycans are adsorbed onto the purification agent (adsorption step).

[0318] Preferably, the purification agent is immobilized on an insoluble support.

[0319] Next, the purification agent with adsorbed glycans is subjected to solid-liquid separation to remove the liquid phase containing impurities such as proteins, peptide fragments, lipids, salts, etc., and recover the purification agent with adsorbed glycans. Solid-liquid separation can be performed by known methods, such as natural settling due to gravity, centrifugation, filtration, or magnetic force (if the support for the purification agent is made of a magnetic material).

[0320] Next, the purification agent with adsorbed glycans is washed. This washing process allows the removal of contaminants such as proteins and peptide fragments other than the glycans adsorbed to the purification agent. Washing can be performed by immersing the purification agent with adsorbed glycans in a washing solution in an appropriate container and repeatedly replacing the washing solution.

[0321] After washing, the sugar chains are eluted from the purification agent to which they have been adsorbed (elution step).

[0322] Glycans can be eluted by immersing the purification agent to which the glycans have been adsorbed in the elution solution. For example, after thoroughly removing the washing solution, an appropriate amount of the elution solution is added to the carrier to which the glycans have been adsorbed, and the mixture is shaken or stirred. The carrier is then recovered by solid-liquid separation, and the eluate is collected in a new appropriate container (e.g., a collection tube or collection plate), and the eluate can be evaporated as needed to concentrate the glycans.

[0323] (Spin column method) The spin column method can be carried out using a container with a built-in filter, such as a filter cup, etc. The filter cup can have openings at the top and bottom, with the bottom opening covered with a filter, for example.

[0324] When a filter cup is used, the liberated product obtained in the liberation step is first introduced into the filter cup filled with the purification agent and passed through the filter cup to bring the purification agent into contact with the liberated product (adsorption step).

[0325] Preferably, the purification agent is immobilized on an insoluble support.

[0326] The solution may be passed through the purification agent by gravity, by centrifugation, or under reduced pressure or pressure. After passing the solution through the purification agent, the effluent containing free proteins, peptide fragments, lipids, salts, etc. is removed.

[0327] Next, the purification agent with adsorbed glycans is washed by passing a washing solution through the purification agent in the filter cup, allowing washing to be performed continuously from the adsorption of glycans.

[0328] After washing, the sugar chains are eluted from the purification agent that has adsorbed the sugar chains (elution step).

[0329] Glycans can be eluted by passing the eluate through the purification agent in the filter cup, and this can be done continuously through the glycan adsorption and washing procedures. After passing the eluate through the purification agent, the eluate is collected in an appropriate container (e.g., collection tube or collection plate). If necessary, the glycans can be concentrated by distilling off the eluate.

[0330] [Labeling process] In the labeling step, the eluate obtained in the elution step is reacted with a labeling reaction reagent containing a labeling compound to obtain a labeled product containing a labeled glycan (hereinafter sometimes referred to as a labeled glycan).

[0331] (labeled compound) The labeling compound has a group reactive to a sugar chain and a modifying group to be attached to the sugar chain. In this embodiment, each labeling compound has an amino group as the reactive group and an aromatic group as the modifying group. When a labeling compound having an amino group and an aromatic group is used, the sugar chain is modified by reductive amination.

[0332] In the reductive amination modification, the amino group reacts with the aldehyde group formed at the reducing end of the glycan to form a Schiff base. The formed Schiff base is reduced by a separately added reducing agent to form a peptide bond at the reducing end of the glycan. This allows the labeling compound to introduce a modifying group into the glycan via the reactive amino group, enabling efficient labeling.

[0333] Aromatic groups are preferred in that they have ultraviolet-visible absorption properties or fluorescent properties, thereby improving the detection sensitivity in UV detection or fluorescent detection.

[0334] As described above, the labeled compound used in this embodiment is at least one selected from the group consisting of 8-aminopyrene-1,3,6-trisulfonic acid, the sodium salt of 8-aminopyrene-1,3,6-trisulfonic acid, and 9-aminopyrene-1,4,6-trisulfonic acid. The sodium salt is preferably the trisodium salt (8-Aminopyrene-1,3,6-trisulfonic acid trisodium, APTS). These labeled compounds contain a sulfate group (sulfonic acid group). In the following description, these labeled compounds are referred to as "labeled compound 1."

[0335] (Labeling reaction reagent) The labeling reaction reagent used in this embodiment includes, in addition to the labeling compound 1, a solution for dissolving the labeling compound 1, and a reducing agent.

[0336] ·solution The solution in the labeling reaction reagent is a mixture of alcohol, organic acid, and ultrapure water.

[0337] Examples of alcohols include methanol, ethanol, and propanol, which may be used singly or in combination of two or more.

[0338] Examples of organic acids include monocarboxylic acids such as formic acid, acetic acid, propionic acid, and butyric acid, dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, and phthalic acid, and tricarboxylic acids such as citric acid. These may be used alone or in combination of two or more. Among these, it is preferable to use citric acid, which is a tricarboxylic acid.

[0339] Examples of the reducing agent include sodium cyanoborohydride, sodium triacetoxyborohydride, methylamine borane, dimethylamine borane, trimethylamine borane, picoline borane, and pyridine borane.

[0340] Among these, picoline borane (2-picoline borane) is preferably used from the viewpoints of both safety and reactivity.

[0341] The labeling reaction reagent may contain a buffer solution. When a buffer solution is contained, examples of the buffering agent include ammonium carbonate, ammonium hydrogen carbonate, ammonium chloride, diammonium hydrogen citrate, ammonium carbamate, etc. There are no particular limitations on the pH of the buffer solution, but a pH of 5 to 10 is preferred.

[0342] In this embodiment, the labeling reaction reagents are divided into a first reagent containing labeled compound 1 and an aqueous solution of an organic acid, and a second reagent containing a reducing agent and a solvent. Specifically, the labeling step is divided into the following first and second steps, and the first and second reagents are added in each step.

[0343] ·1st process In the first step, the eluate obtained in the elution step is dried to solid, and then the first reagent is added and heated.

[0344] The method for drying the eluate can be a known method, specifically, the solvent contained in the eluate is removed in any environment such as drying (low humidity), heating, reduced pressure, air blowing, or a combination thereof.

[0345] The concentration of labeled compound 1 in the first reagent can be, for example, 100 mmol / L or more and 500 mmol / L or less.

[0346] The concentration of the organic acid in the first reagent can be, for example, 100 mmol / L or more and 1000 mmol / L or less.

[0347] The first reagent may contain labeled compound 1, an organic acid, water, and an alcohol.

[0348] The heating temperature in the first step can be, for example, 40°C or higher and 80°C or lower. The heating time in the first step can be, for example, 10 minutes or more and 24 hours or less.

[0349] ·Second process In the second step, a second reagent is added to the reaction mixture obtained in the first step and heated.

[0350] The concentration of the reducing agent in the second reagent can be 10 mmol / L or more and 500 mmol / L or less.

[0351] The solvent in the second reagent includes an alcohol.

[0352] The heating temperature in the second step can be, for example, 40°C or higher and 80°C or lower. The heating time in the second step can be, for example, 1 hour or more and 24 hours or less.

[0353] When labeling compound 1 is used, going through the above-mentioned steps 1 and 2 makes it easier to label glycans compared to using a labeling reaction reagent in which the first and second reagents are mixed from the beginning. It is assumed that heating under acidic conditions in step 1 facilitates the reaction between labeling compound 1 and glycans, and as a result, reductive amination by the reduction reaction in step 2 proceeds more easily. By the labeling step, a reaction solution containing a sugar chain-labeled product is obtained.

[0354] (purification process) The resulting reaction solution contains not only the glycoconjugate but also unwanted substances such as the excess labeled compound 1 used in the labeling step. Therefore, the glycoconjugate may be purified by removing unwanted substances from the reaction solution (sample solution containing the labeled product). The removal of unwanted substances may be performed by passing the sample solution through a solid phase for purification to capture the glycoconjugate, and then re-eluting the captured glycoconjugate.

[0355] In the purification step of the second embodiment, the same solid phase for purification as that shown in (Solid phase for purification) of the first embodiment can be used as the solid phase for purification.

[0356] In the purification step of the second embodiment, the separated liquid may be prepared again by adding an ionic aqueous solution to the sample solution in the same manner as in the method shown in (repreparation of separated liquid) of the first embodiment.

[0357] In the above purification, after capturing the sugar chain-labeled product on the solid support for purification, the solid support for purification may be washed with a solvent. The solvent used here is preferably a mixed solvent containing an organic solvent and water, more preferably a mixed solvent of acetonitrile, water, and alcohol. Either methanol or ethanol, or both, may be used as the alcohol.

[0358] In the purification, washing is carried out using a mixed solvent prepared by mixing the above three solvents with different hydrophobicities in a volume ratio of acetonitrile:alcohol:water=20-98:0-60:2-15 (total 100% by volume).

[0359] The mixed solvent preferably contains 45% by volume or more and 98% by volume or less of acetonitrile, and more preferably 70% by volume or more and 98% by volume or less.

[0360] The mixed solvent preferably contains 0% to 40% by volume of alcohol, more preferably 0% to 20% by volume. When alcohol is used, ethanol is preferred.

[0361] The mixed solvent preferably contains water in an amount of 3% by volume or more and 15% by volume or less, and more preferably 2% by volume or more and 10% by volume or less.

[0362] An example of the mixed solvent is a mixed solvent of acetonitrile, alcohol, and water in a ratio of 50:40:10 (total 100% by volume).

[0363] After passing the mixed solvent through the solid phase carrier for purification, the solid phase carrier for purification can be washed by centrifuging to remove the solvent from the solid phase carrier for purification. This washing operation can remove the unreacted labeled compound as well as the above-mentioned unnecessary substances. The washing operation may be performed only once or may be performed two or more times. Repeated washing operations can remove unnecessary substances, but may also wash away glycan-labeled substances adhering to the solid phase carrier for purification, raising the concern that the yield of glycan-labeled substances may decrease. Therefore, it is recommended that the washing operation be performed three times or less.

[0364] <Glycan analysis method> The glycan-labeled substance prepared by the method of this embodiment can be analyzed by the method described in the "Glycan Analysis Method" of Embodiment 1. Therefore, the glycan-labeled substance can be suitably detected from a low-concentration sample.

[0365] According to the sugar chain preparation method configured as described above, labeled sugar chains can be rapidly prepared from glycoproteins.

[0366] Furthermore, according to the sugar chain analysis method described above, even if the amount of sugar chain used for analysis is small, analysis can be performed suitably.

[0367] [Third embodiment] The sugar chain preparation method of this embodiment shares the release step and adsorption step with the sugar chain preparation method of the second embodiment, but differs from it in the elution step and labeling step. Therefore, in the following explanation, the steps common to the second embodiment will be omitted, and the different steps will be described in detail.

[0368] [Elution process] In the elution step, the glycans are eluted from the purifying agent to which they have been adsorbed. The eluent for eluting the glycans from the purifying agent can be any of the above-mentioned organic solvents or a mixed solution of an organic solvent and water, and can be appropriately selected depending on the type of glycans to be eluted and the type of purifying agent.

[0369] In the elution step of this embodiment, the labeling reaction reagent used in the labeling step described below can be used as the eluent. By performing the elution step using the labeling reaction reagent, the operation is simplified compared to when elution is performed using a separate eluent.

[0370] Furthermore, compared to the case where glycans are eluted using an eluent other than the labeling reaction reagent and the labeling step is performed by adding the labeling reaction reagent to the obtained eluate, the labeling step is performed at a relatively high concentration by eluting the glycans using the labeling reaction reagent, which makes it easier for the free glycans to react with the labeling reaction reagent.

[0371] Furthermore, compared to the case where glycans are eluted using an eluent other than the labeling reaction reagent, the solvent is removed from the resulting eluate to concentrate the glycans, and then the labeling step is carried out, eluting the glycans using the labeling reaction reagent simplifies the procedure.

[0372] [Labeling process] In the labeling step of this embodiment, at least one labeled compound selected from the group consisting of 2-aminobenzoic acid (2-AA) and 3-aminobenzoic acid is used as the labeled compound contained in the labeling reaction reagent. These labeled compounds contain a carboxy group. In the following description, these labeled compounds will be referred to as "labeled compound 2."

[0373] The concentration of the reducing agent in the labeling reaction reagent is 0.1 mmol / L or more and 20 mmol / L or less.

[0374] As an example, the content of organic acid in the solvent of the labeling reaction reagent solution can be 1% by volume or more and 15% by volume or less, and the content of organic acid in the labeling reaction reagent solution is preferably 10% by volume or less.

[0375] The alcohol used is preferably methanol, and the reducing agent used is preferably picoline borane.

[0376] Even with the sugar chain preparation method configured as above, labeled sugar chains can be rapidly prepared from glycoproteins.

[0377] [kit] The purification kit according to this embodiment comprises a glycan releasing reagent that releases glycans from glycoproteins, a purification agent that purifies the released glycans, and a labeling reaction reagent that labels the glycans.

[0378] The sugar chain releasing reagent and purification agent used are the same as those described in the above embodiment.

[0379] In the kit for carrying out the sugar chain preparation method of the first embodiment, the above-mentioned first and second reagents are used as labeling reaction reagents.

[0380] In the kit for carrying out the sugar chain preparation method of the third embodiment, a labeling reaction reagent containing labeling compound 2 is used as the labeling reaction reagent.

[0381] According to the kit of this embodiment, the above-mentioned sugar chain preparation method can be easily carried out, and sugar chain labels can be rapidly prepared from glycoproteins.

[0382] While the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to these examples. The shapes and combinations of the components shown in the above examples are merely examples, and various modifications can be made based on the design, specifications, etc., without departing from the spirit of the present invention. [Example]

[0383] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0384] <N-glycan analysis> In the examples relating to N-glycan analysis, a commercially available antibody glycan analysis kit (Sumitomo Bakelite, product number: BS-X4410) was used. The kit contained the following components used in the examples. Column 1: Antibody Capturing Column Reagent 2: Antibody Capturing Solution Reagent 3: Washing Buffer Reagent 4: Glycan Release Enhancer (enhancing solution, deglycosylation enhancer) Reagent 5A: PNGase F solution (glycan-releasing enzyme) Reagent 5B: Diluting Buffer

[0385] The column 1 corresponds to the "solid phase" of the present invention. The column 1 has the ligand described in the above embodiment on its surface.

[0386] [Example 1-1: APTS labeling. Confirmation by HPLC analysis] (Release process) Column 1 was inserted into a 2.0 mL tube and centrifuged at 500 × g for 3 minutes. Next, 600 μL of Reagent 2 was added to Column 1, and the tube was centrifuged at 500 × g for 3 minutes.

[0387] 40 μg of antibody (human IgG (Sigma-Aldrich, product number: I4506)) was diluted with Reagent 2 to prepare a 40 μg / 600 μL antibody-containing solution. The resulting antibody-containing solution was added to Column 1 and centrifuged at 500 × g for 3 minutes to capture the antibody on Column 1. Furthermore, Reagent 3 was added to Column 1 and washed by centrifugation at 500 × g for 3 minutes to prepare a "glycoprotein immobilized on a solid phase."

[0388] 100 μL of Reagent 4 was added to Column 1, and the column was centrifuged at a centrifugal force of 3000×g for 1 minute. Then, Column 1 was inserted into a 1.5 mL tube and heated at 50° C. for 30 minutes (pretreatment step).

[0389] Thereafter, 3 μL of a mixture of Reagent 5A and Reagent 5B (volume ratio 1:1) was impregnated into the carrier of the heated Column 1, and the column was heated at 50° C. for 30 minutes (release step).

[0390] (labeling process) 8-Aminopyrene-1,3,6-trisulfonic acid trisodium (trisodium salt, APTS) (manufactured by BIOSYNTH, product number: FA45174) and a 500 mmol / L aqueous citric acid solution were mixed to prepare a 400 mmol / L labeled compound solution 1. Citric acid corresponds to the "organic acid" of the present invention.

[0391] Furthermore, 2-picoline borane (manufactured by Junsei Chemical Co., Ltd., product number: 59096-1610) was dissolved in methanol to prepare a reducing agent solution 1 with a concentration of 600 mmol / L. Methanol corresponds to the "polar solvent different from organic acids" in the present invention.

[0392] After the release step, 10 μL of labeled compound solution 1 and 10 μL of reducing agent solution 1 were added to column 1 to prepare a labeling reaction reagent, and then the column was centrifuged at a centrifugal force of 3000×g for 1 minute.

[0393] The resulting solution was heated at 50°C for 3 hours with the tube lid open (open system) to obtain a labeled product.

[0394] After heating, 40 μL of ultrapure water was added to the dried contents of the tube to prepare a diluted sample solution. The ultrapure water was prepared using an ultrapure water production system (model number: MilliQ EQ7000, manufactured by Merck).

[0395] Using the obtained sample solution, HPLC analysis was carried out under the conditions shown in the following Table 1. Figure 3 is an LC chart showing the results of the HPLC analysis of Example 1-1.

[0396] [Table 1]

[0397] As a result of the evaluation, it was confirmed that the peak of the major N-glycan of APTS-labeled human IgG could be detected.

[0398] [Example 1-2: Confirmation by capillary electrophoresis analysis 1] APTS was mixed with a 500 mmol / L aqueous citric acid solution to prepare labeled compound solution 2 with a concentration of 250 mmol / L.

[0399] Reducing Reagent (manufactured by Sumitomo Bakelite, product number: attached to BS-X4410) (Reagent 7) was dissolved in 800 μL of methanol to prepare Reducing Reagent Solution 2.

[0400] (Labeling process, separation process) After the above (release step), labeled compound solution 2 was added to column 1, which was then centrifuged at a centrifugal force of 3000×g for 1 minute. Next, reducing agent solution 2 was added to column 1, which was then centrifuged at a centrifugal force of 3000×g for 1 minute.

[0401] The resulting solution was heated at 50° C. for 2 hours with the tube cap open (open system).

[0402] (purification process) After heating, 30 μL of ultrapure water was added to the sample, followed by 600 μL of acetonitrile. The entire volume of the resulting solution was added to a cleanup column (manufactured by Sumitomo Bakelite, product number: BS-X4410) to capture the sugar chain label on the cleanup column, which was then centrifuged at 500 × g for 1 minute to remove the solution. The cleanup column corresponds to the solid phase for purification in this invention.

[0403] Then, 600 μL of a 95% acetonitrile / water mixture was added, and the cleanup column was centrifuged at 500 × g for 1 minute to remove the solution. 600 μL of a 95% acetonitrile / water mixture was then added again, and the cleanup column was centrifuged at 3,000 × g for 1 minute to remove the solution.

[0404] 50 μL of ultrapure water was added to the cleanup column to elute the glycan-labeled substance captured on the cleanup column, followed by centrifugation, and the aqueous solution containing the glycan-labeled substance (sample solution) was collected in a microtube.

[0405] The obtained sample solution was diluted two-fold with ultrapure water and subjected to capillary electrophoresis (CE) analysis under the following conditions: The CE analysis under the following conditions is capillary gel electrophoresis analysis. (conditions) CE device: P / ACE MDQ Plus (AB Sciex) Capillary: N-CHO capillary (AB Sciex, part number: 477601) Buffer solution: N-Linked Carbohydrate Separation Gel Buffer (AB Sciex, product number: 477623) Fluorescence detection: Excitation wavelength 488nm, Fluorescence wavelength 520nm

[0406] The sample was injected into the capillary for 3 seconds at 0.5 psi (where 1 psi = 6894.76 Pa). Separation was carried out for 13 minutes using a voltage of 30 kV.

[0407] [Example 1-3: Confirmation by capillary electrophoresis analysis 2] The analysis was performed in the same manner as in Example 1-2, except that the buffer used in the capillary electrophoresis analysis was 50 mmol / L HEPES (pH 7.25) and the sample was injected into the capillary at 0.3 psi for 3 seconds. The CE analysis in Example 1-3 was a capillary zone electrophoresis analysis.

[0408] HEPES stands for 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0409] Figure 4 is a chart obtained by capillary electrophoresis analysis in Example 1-2, and Figure 5 is a chart obtained by capillary electrophoresis analysis in Example 1-3. As a result of the evaluation, it was confirmed that the peak of the major N-glycan of APTS-labeled human IgG was detected in both cases.

[0410] The results of Examples 1-1 to 1-3 confirmed that the sugar chain preparation method of the present invention enables APTS labeling of N-glycans, and that the resulting labeled products can be suitably analyzed.

[0411] [Example 1-4: Confirmation by capillary electrophoresis analysis 3] In Examples 1-4, model experiments were carried out using APTS-labeled commercially available products. Purchased NA2F Glycan, APTS Labeled (manufactured by Ludger, product number: CAPTS-NA2F-01) was redissolved in pure water to prepare a 0.25 pmol / μL model sample.

[0412] Using the obtained model sample, capillary electrophoresis (CE) analysis was carried out under the following conditions. (conditions) CE device: P / ACE MDQ Plus (AB Sciex) Capillary: N-CHO capillary (AB Sciex, part number: 477601) Buffer: 50mmol / L HEPES (pH7.25) Preliminary electrolyte solution: 12mmol / L sodium chloride aqueous solution Fluorescence detection: Excitation wavelength 488nm, Fluorescence wavelength 520nm

[0413] The sample was injected into the capillary at 20 psi for 30 seconds, followed by a pre-electrolyte solution at 0.5 psi for 20 seconds from the downstream side of the capillary. Then, a voltage of 30 kV was applied to the downstream side of the capillary, with the downstream side being positive and the upstream side being negative, to concentrate the sample for 22 minutes, followed by capillary electrophoresis for 18 minutes.

[0414] [Reference example 1-1] The model sample prepared in Example 1-4 was subjected to capillary electrophoresis analysis under the conditions of Example 1-3.

[0415] Figure 6 is a chart obtained by capillary electrophoresis analysis in Example 1-4, and Figure 7 is a chart obtained by capillary electrophoresis analysis in Reference Example 1-1. As a result of the evaluation, the peak area value of NA2F glycan in Example 1-4 was improved by about 400 times compared to the result in Reference Example 1-1, confirming that highly sensitive analysis was possible under the conditions of Example 1-4.

[0416] Based on the results confirmed in Examples 1-4, it can be inferred that highly sensitive analysis is possible in both the analysis of N-type glycans and the analysis of O-type glycans.

[0417] [Example 1-5: Confirmation by capillary electrophoresis analysis 4] (Labeling process, separation process) After the above (release step), labeled compound solution 2 was added to column 1, which was then centrifuged at a centrifugal force of 3000×g for 1 minute. Next, reducing agent solution 2 was added to column 1, which was then centrifuged at a centrifugal force of 3000×g for 1 minute.

[0418] The resulting solution was heated at 50° C. for 2 hours with the tube cap open (open system).

[0419] (purification process) After heating, 60 μL of ultrapure water was added to the sample, followed by 600 μL of ethanol. The resulting solution was loaded entirely onto a cleanup column (Sumitomo Bakelite, part number BS-X4410), which was then centrifuged at 500 × g for 1 minute to remove the solution. Next, 600 μL of a 95% ethanol / water mixture was added, and the cleanup column was centrifuged at 500 × g for 1 minute to remove the solution. Another 600 μL of a 95% ethanol / water mixture was added, and the cleanup column was centrifuged at 3000 × g for 1 minute to remove the solution.

[0420] 100 μL of ultrapure water was added to the cleanup column to elute the glycan-labeled substance captured on the cleanup column, followed by centrifugation, and the aqueous solution containing the glycan-labeled substance (sample solution) was collected in a microtube.

[0421] The obtained sample solution was diluted 20 times with ultrapure water, and capillary electrophoresis analysis was carried out under the same conditions as in Example 1-4, except that the concentration of the preceding electrolyte was set to 10 mmol / L.

[0422] [Reference example 1-2] The model sample prepared in Example 1-5 was subjected to capillary electrophoresis analysis under the conditions of Example 1-3.

[0423] Figure 8 is a chart obtained by the capillary electrophoresis analysis of Example 1-5, and Figure 9 is a chart obtained by the capillary electrophoresis analysis of Reference Example 1-2. As a result of the evaluation, the total peak area value of the N-glycans of human IgG in Example 1-5 was improved by about 400 times compared to the results of Reference Example 1-2, confirming that highly sensitive analysis was possible under the conditions of Example 1-4.

[0424] [Example 1-6: Confirmation of cleaning effect with mixed solvent] (Preparation of glycan-labeled compounds) As in Example 1-2, labeled compound solution 2 was added to column 1 after the above (release step), and the column was centrifuged at a centrifugal force of 3000 × g for 1 minute. Next, reducing agent solution 2 was added to column 1, and the column was centrifuged at a centrifugal force of 3000 × g for 1 minute. The resulting solution was heated at 50°C for 2 hours with the tube lid open (open system), to obtain a reaction mixture. The resulting reaction mixture contains unreacted labeled compound in addition to the sugar chain-labeled product.

[0425] (Purification process - Glycan analysis, preliminary experiments) To the reaction mixture, 30 μL of ultrapure water was added, followed by 600 μL of ethanol. The entire volume of the resulting solution was loaded onto a cleanup column (Sumitomo Bakelite, part number BS-X4410), which was then centrifuged at 500 × g for 1 minute to remove the solution. Subsequently, 600 μL of a mixed solution containing an organic solvent and water was added, and the cleanup column was centrifuged at 500 × g for 1 minute to remove the solution. The mixing ratio of the mixed solution is shown in Table 2. Furthermore, 600 μL of the mixed solution shown in Table 2 below was added again, and the cleanup column was centrifuged at 3000 × g for 1 minute to remove the solution.

[0426] 50 μL of ultrapure water was added to the cleanup column to elute the glycoconjugates captured on the cleanup column, followed by centrifugation, and the aqueous solution containing the glycoconjugates was collected in a microtube. The resulting sample solution was subjected to capillary electrophoresis analysis in the same manner as in Example 1-2, and the amounts of unreacted labeled compound and glycoconjugates contained in the resulting sample solution were measured. The results are shown in Table 3.

[0427] [Table 2]

[0428] [Table 3]

[0429] In Table 3, the area values ​​of the unreacted labeled compounds in Preliminary Experiment 1 are approximate values. The "residual rate" in Table 3 indicates the percentage when the result of Preliminary Experiment 1 (acetonitrile and water mixed solvent) is set to 100. As shown in Table 3, when the mixed solvent used during washing was changed from an acetonitrile and water mixed solvent to an alcohol and water mixed solvent, it was confirmed that the amount of unreacted labeled compound was significantly reduced. On the other hand, it was also confirmed that the amount of the target glycan label was also significantly reduced. To enable the measurement of trace amounts of glycans, washing conditions that reduce the amount of unreacted labeled compound in the obtained sample solution and leave a large amount of glycan label are preferred.

[0430] (purification process to sugar chain analysis) A sample solution was prepared in the same manner as in the preliminary experiment above, except that 60 μL of ultrapure water was added to the reaction mixture obtained in the same manner as in the above (preparation of sugar chain-labeled compound), followed by the addition of 600 μL of ethanol, and that the mixed solvent shown in Table 4 below was used for washing. The results are shown in Table 5.

[0431] [Table 4]

[0432] [Table 5]

[0433] The "residual rate" in Table 5 indicates the percentage when the amount (area value) of unreacted labeled compound or sugar chain label contained in the sample solution without purification is taken as 100.

[0434] As a result of the evaluation, it was confirmed that, compared to unpurified sample solutions, washing the glycan-labeled compound with a mixed solvent of organic solvent and water in the purification process reduced the amount of unreacted labeled compound under all conditions.

[0435] In particular, washing with mixed solvents Nos. 1, 3, 4, 5, 6, 8, 9, 11, 12, 16, and 17 resulted in a high purification effect, with the remaining rate of unreacted labeled compound being 17% or less and the remaining rate of glycan labels being 75% or more. In each of the sample solutions Nos. 1, 3, 4, 5, 6, 8, 9, 11, 12, 16, and 17, the amount of unreacted labeled compound was reduced compared to the sample solution without purification, and sufficient glycan labels remained, indicating that accurate analysis is possible.

[0436] [Example 1-7: Confirmation of purification effect by adding ionic aqueous solution] (Preparation of glycan-labeled compounds) As in Example 1-2, labeled compound solution 2 was added to column 1 after the above (release step), and the column was centrifuged at a centrifugal force of 3000 × g for 1 minute. Next, reducing agent solution 2 was added to column 1, and the column was centrifuged at a centrifugal force of 3000 × g for 1 minute. The resulting solution was heated at 50°C for 2 hours with the tube lid open (open system), to obtain a reaction mixture. The resulting reaction mixture contained unreacted labeled compound in addition to the glycoconjugate.

[0437] (purification process) To the reaction mixture, 60 μL of an aqueous ammonium bicarbonate solution (pH approximately 8) was added, followed by 600 μL of acetonitrile. The resulting solution was loaded entirely onto a cleanup column (manufactured by Sumitomo Bakelite, product number: BS-X4410), and the column was centrifuged at 500 × g for 1 minute to remove the solution. The "aqueous ammonium bicarbonate solution" corresponds to the ionic aqueous solution of the present invention.

[0438] Aqueous ammonium bicarbonate solutions of 0, 10, 50, 100, 250, and 400 mmol / L were prepared for addition to the reaction mixture, and the same procedure was carried out for each solution, and the aqueous solution containing the sugar chain label (sample solution) was collected in a microtube.

[0439] After centrifugation, 600 μL of acetonitrile / ethanol / water (50 / 40 / 10 volume ratio) was added to the cleanup column, and the column was centrifuged at 500 × g for 1 minute to remove the solution. Another 600 μL of acetonitrile / ethanol / water (50 / 40 / 10 volume ratio) was then added, and the column was centrifuged at 3000 × g for 1 minute to remove the solution.

[0440] 100 μL of ultrapure water was added to the cleanup column to elute the glycan-labeled substance captured on the cleanup column, followed by centrifugation, and the aqueous solution containing the glycan-labeled substance (sample solution) was collected in a microtube.

[0441] A number of different concentrations of aqueous ammonium bicarbonate solutions were prepared for addition to the reaction mixture, and the same procedure was carried out for each solution, with the sample solution then being collected in a microtube.

[0442] [Examples 1-8] The sample solution was collected in a microtube in the same manner as in Example 1-7, except that 60 μL of a 250 mmol / L aqueous ammonium chloride solution (pH about 5) was added to the reaction mixture.

[0443] [Examples 1-9] The sample solution was collected in a microtube in the same manner as in Example 1-7, except that 60 μL of a 250 mmol / L aqueous sodium chloride solution (pH about 7) was added to the reaction mixture.

[0444] [Examples 1-10] The sample solution was collected in a microtube in the same manner as in Example 1-7, except that 60 μL of a 250 mmol / L aqueous ammonia solution (pH about 11) was added to the reaction mixture.

[0445] [Examples 1-11] The sample solution was collected in a microtube in the same manner as in Example 1-7, except that 60 μL of a 250 mmol / L aqueous sodium carbonate solution (pH about 11) was added to the reaction mixture.

[0446] [Examples 1-12] The sample solution was collected in a microtube in the same manner as in Example 1-7, except that 60 μL of a 250 mmol / L aqueous sodium hydroxide solution (pH about 13) was added to the reaction mixture.

[0447] [Reference example 1-3] The sample solution was collected in a microtube in the same manner as in Example 1-7, except that 60 μL of pure water was added to the reaction mixture.

[0448] For each of the sample solutions of Examples 1-7, 1-8, 1-9, 1-10, 1-11, and 1-12 and Reference Example 1-3, capillary electrophoresis analysis was performed in the same manner as in Example 1-2, and the amount of unreacted labeled compound and the amount of glycan-labeled product contained in the obtained sample solution were measured.

[0449] FIG. 10 is a graph showing the area values ​​of unreacted labeled compounds detected in Examples 1-7 and Reference Example 1-3. FIG. 11 is a graph showing the area values ​​of sugar chain labels detected in Examples 1-7 and Reference Example 1-3. In FIGS. 10 and 11, the horizontal axis represents the concentration of the aqueous ammonium bicarbonate solution, and the vertical axis represents the detected peak area values. The results for an ammonium bicarbonate concentration of 0 mmol / L correspond to the results of Reference Example 1-3. FIG. 12 is a graph showing the area values ​​of unreacted labeled compounds detected in Examples 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, and Reference Example 1-3. In FIG. 12, the vertical axis represents the detected peak area values.

[0450] 10 and 12, the amount of unreacted labeled compound in the sample solution was significantly reduced in Examples 1-7, 1-8, 1-9, 1-10, 1-11, and 1-12, which used an ionic aqueous solution, compared to Reference Example 1-3, which used pure water in the purification process. Furthermore, as shown in Figure 11, in Example 1-7, the amount of recovered glycan-labeled compound was greater than in Reference Example 1-3, despite the reduction in the amount of unreacted labeled compound in the sample solution.

[0451] In Examples 1-7, 1-8, 1-9, 1-10, 1-11, and 1-12, the effect of adding an ionic solution was confirmed in the separation and purification of N-glycan-labeled glycans and unreacted labeled compounds (APTS), but it is believed that the type of glycan does not matter in terms of separating and removing unreacted labeled compounds. Therefore, based on the results confirmed in Example 1-7, it can be inferred that the same effect can also be obtained in the separation and purification of O-glycan-labeled glycans and unreacted labeled compounds.

[0452] <O-glycan analysis> In the examples relating to O-type glycan analysis, a commercially available antibody glycan analysis kit (Sumitomo Bakelite, product number: BS-41601) was used. The kit contained the following components used in the examples. Reagent 1: Glycan Reagent A (a solution containing 50% hydroxylamine solution) Reagent 2: Glycan Reagent B (a solution containing diazabicycloundecene (an organic base)) Purification Agent: Glycan Capturing Beads Column 1: Filter column Column 2: Cleanup column Reagent 7: Reducing Reagent (Picoline Borane)

[0453] The glycan releasing reagent was prepared by mixing Reagent 1 and Reagent 2 in a volume ratio of 5:2.

[0454] The purification agent corresponds to the purification agent described in the above embodiment with reference to FIG.

[0455] The ultrapure water used in the examples was prepared using an ultrapure water production system (model number: MilliQ EQ7000, manufactured by Merck).

[0456] [Example 2-1: APTS labeling. Confirmation by HPLC analysis] (Release process) Bovine fetuin (Sigma-Aldrich, product number F3004) was dissolved in ultrapure water to prepare a 10 mg / mL fetuin solution. 10 μL of this solution was added to a 1.5 mL tube, followed by the addition of 15 μL of the glycan release solution and mixing. The resulting mixture was heated at 37°C for 75 minutes.

[0457] (Adsorption process) After the release step, 1000 μL of acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product code: 018-19853, purity 99.9%) was added to the mixed solution and mixed well.

[0458] The purification agent was added to the resulting mixed solution and suspended, and the resulting suspension was then added to a spin column (filter column). The filter column was then centrifuged at 3000 × g for 1 minute using a tabletop centrifuge to separate the purification agent from the solution and remove the solution. The same centrifugation conditions were used in the following description.

[0459] Next, 200 μL of acetonitrile was added to the filter column, and the solution was removed by centrifugation. After that, 200 μL of acetonitrile was added to the filter column again, and the solution was removed by centrifugation.

[0460] (Elution process) 50 μL of ultrapure water was added to the solid (purification agent) remaining in the column to elute the glycans from the purification agent, and the eluate (glycan solution) from which the glycans had been eluted was collected in a PCR tube by centrifugation.

[0461] (labeling process) 8-Aminopyrene-1,3,6-trisulfonic acid trisodium (trisodium salt, APTS) (manufactured by BIOSYNTH, product number: FA45174) and a 500 mmol / L aqueous citric acid solution were mixed to prepare a first reagent with a concentration of 250 mmol / L. Citric acid corresponds to the "organic acid" of the present invention.

[0462] Furthermore, 1200 μL of methanol was added to Reagent 7 (reducing agent) included in the kit to prepare a second reagent with a concentration of 330 mmol / L.

[0463] The eluate collected in the PCR tube in the elution step was frozen and then dried in a centrifugal dryer for 1 hour. Next, 5 μL of the first reagent was added to the PCR tube, and the tube was heated at 50°C for 1 hour (first step).

[0464] After heating, 5 μL of the second reagent was added to the PCR tube, which was then heated at 70°C for 3 hours to obtain a labeled product (step 2). 40 μL of ultrapure water was added to the contents of the heated tube to prepare a diluted sample solution.

[0465] [Comparative Example 2-1] A sample solution was prepared in the same manner as in Example 2-1, except that in the labeling step, the first and second reagents were added to and mixed with the PCR tube in which the eluate had been dried, and the mixture was left at 70°C for 3 hours to obtain a labeled product.

[0466] Using the sample solutions prepared in Example 2-1 and Comparative Example 2-1, HPLC analysis was carried out under the conditions shown in Table 6. Figure 13 is an LC chart showing the results of the HPLC analysis of Example 2-1 and Comparative Example 2-1.

[0467] [Table 6]

[0468] As a result of the evaluation, it was confirmed that the peak intensity of the major O-type sugar chain of bovine fetal fetuin was clearly stronger in Example 2-1 than in Comparative Example 2-1.

[0469] Figure 14 is a graph showing the total peak area values ​​of the major O-glycans of fetuin determined from the LC chart. The values ​​shown in Figure 14 correspond to the glycan yield of each sample. As a result of the evaluation, it was confirmed that the yield of O-glycans was higher in Example 2-1 than in Comparative Example 2-1.

[0470] That is, it was confirmed that the glycan preparation method of the present invention enables APTS labeling of O-glycans and that the resulting labeled product can be suitably analyzed. Furthermore, it was shown that the glycan yield can be significantly increased by dividing the labeling process into Step 1 and Step 2 and carrying out the reaction stepwise.

[0471] [Example 2-2: 2AA labeling. Confirmation by HPLC analysis] A 2 mg / mL fetuin solution was prepared by dissolving bovine fetuin (Sigma-Aldrich, product number F3004) in ultrapure water. The prepared fetuin solution was used to carry out the release step and adsorption step in the same manner as in Example 2-1.

[0472] (Elution process) 2-Aminobenzoic acid (2-AA) (Tokyo Chemical Industry Co., Ltd., product number: A0497) was dissolved in a mixed solution of methanol:acetic acid:ultrapure water=49:2:49 to prepare solution 1 with a concentration of 300 mmol / L.

[0473] Then, 1 mL of a mixed solution of methanol:acetic acid:ultrapure water = 49:2:49 was added to Reagent 7 (reducing agent) included in the kit to prepare Solution 2. 15 μL of Solution 2 was dissolved in 1 mL of the above Solution 1 to prepare a labeling reaction reagent. The concentration of the reducing agent in the labeling reaction reagent was 5 mmol / L.

[0474] 50 μL of the labeling reaction reagent was added to the purification agent remaining in the filter column after the adsorption step to elute the glycans into the labeling reaction reagent.Then, the solution (labeling reaction reagent from which the glycans were eluted) was collected in a microtube by centrifugation.

[0475] (labeling process) The solution collected in the microtube was heated at 50°C for 3 hours to obtain a labeled product. After heating, 1 mL of acetonitrile was added to the microtube and stirred.

[0476] (purification process) The entire volume of the resulting solution was added to column 2 (cleanup column) to capture the glycans (glycan labels) on the cleanup column, followed by centrifugation at 500 × g for 1 minute to remove the solution. Next, 600 μL of acetonitrile was added to the cleanup column, and the column was centrifuged at 500 × g for 1 minute to remove the solution. Thereafter, 600 μL of acetonitrile was added again to the cleanup column, and the column was centrifuged at 3000 × g for 1 minute to remove the solution. The cleanup column corresponds to the solid phase for purification in the present invention.

[0477] 50 μL of ultrapure water was added to the cleanup column to elute the glycans from the cleanup column, which was then centrifuged to collect the aqueous solution containing the labeled glycans (sample solution) in a microtube.

[0478] [Comparative Example 2-2] An aqueous solution containing a sugar chain label (sample solution) was prepared in the same manner as in Example 2-2, except that the concentration of the reducing agent in the labeling reaction reagent was 25 mmol / L.

[0479] [Comparative Example 2-3] An aqueous solution containing a sugar chain label (sample solution) was prepared in the same manner as in Example 2-2, except that the concentration of the reducing agent in the labeling reaction reagent was 0.05 mmol / L.

[0480] Using the sample solutions prepared in Example 2-2 and Comparative Examples 2-2 and 2-3, HPLC analysis was carried out under the conditions shown in Table 7. Figure 15 is an LC chart showing the results of the HPLC analysis of Example 2-2 and Comparative Examples 2-2 and 2-3.

[0481] [Table 7]

[0482] The concentration gradient conditions indicate that the concentration of mobile phase B was changed continuously at a constant rate to 100% mobile phase A over 50 minutes.

[0483] As a result of the evaluation, it was confirmed that the peak intensity of the major O-type sugar chain of bovine fetal fetuin was clearly stronger in Example 2-2 than in Comparative Examples 2-2 and 2-3.

[0484] Figure 16 is a graph showing the total peak area values ​​of the major O-glycans of fetuin obtained from the LC chart. The values ​​shown in Figure 16 correspond to the glycan yield of each sample. As a result of the evaluation, it was confirmed that the yield of O-glycans was higher in Example 2-2 than in Comparative Examples 2-2 and 2-3.

[0485] [Example 2-3: Confirmation by capillary electrophoresis analysis 1] The sample solution of Example 2-1 was diluted two-fold with ultrapure water and subjected to capillary electrophoresis (CE) analysis under the following conditions: The CE analysis under the following conditions is capillary gel electrophoresis analysis. (conditions) CE device: P / ACE MDQ Plus (AB Sciex) Capillary: N-CHO capillary (AB Sciex, part number: 477601) Buffer solution: N-Linked Carbohydrate Separation Gel Buffer (AB Sciex, product number: 477623) Fluorescence detection: Excitation wavelength 488nm, Fluorescence wavelength 520nm

[0486] The sample was injected into the capillary for 3 seconds at 0.5 psi (where 1 psi = 6894.76 Pa). Separation was carried out for 13 minutes using a voltage of 30 kV.

[0487] [Reference example 2-1] 10 μg of commercially available O-glycan sample, Sialylated Core 1 O Glycan (manufactured by Ludger, product number: CO-C1(S3)1-20U), was dissolved in 5 μL of the first reagent described above and transferred to a PCR tube. 5 μL of the second reagent was added to the PCR tube, and the mixture was heated at 70°C for 3 hours to obtain a labeled product. 40 μL of ultrapure water was added to the heated contents of the tube to prepare a diluted sample solution.

[0488] The obtained sample solution was subjected to CE analysis in the same manner as in Example 2-3.

[0489] Fig. 17 is a chart obtained by capillary electrophoresis analysis in Example 2-3, and Fig. 18 is a chart obtained by capillary electrophoresis analysis in Reference Example 2-1. As a result of the evaluation, a peak was confirmed in Example 2-3 at the same position as the elution time of Sialylated Core 1 in Reference Example 2-1.

[0490] [Example 2-4: Confirmation by capillary electrophoresis analysis 2] The analysis was performed in the same manner as in Example 2-3, except that the buffer used in the capillary electrophoresis analysis was 50 mmol / L HEPES (pH 7.25) and the sample was injected into the capillary at 0.3 psi for 3 seconds. The CE analysis in Example 2-4 was a capillary zone electrophoresis analysis.

[0491] [Reference example 2-2] The analysis was performed in the same manner as in Reference Example 2-1, except that the buffer used in the capillary electrophoresis analysis was 50 mmol / L HEPES (pH 7.25) and the sample was injected into the capillary at 0.3 psi for 3 seconds. The CE analysis in Reference Example 2-2 was a capillary zone electrophoresis analysis.

[0492] Figure 19 is a chart obtained by capillary electrophoresis analysis in Example 2-4, and Figure 20 is a chart obtained by capillary electrophoresis analysis in Reference Example 2-2. As a result of the evaluation, a peak was confirmed in Example 2-4 at the same position as the elution time of Sialylated Core 1 in Reference Example 2-2.

[0493] From the results of Examples 2-3 and 2-4 and Reference Examples 2-1 and 2-2, it was confirmed that the major O-type sugar chain of bovine fetal fetuin was labeled by the sugar chain preparation method of the present invention and could be analyzed by CE analysis.

[0494] In other words, it was confirmed that the glycan preparation method of the present invention enables APTS labeling of O-glycans and that the resulting labeled product can be suitably analyzed. Furthermore, it was shown that the glycan yield can be significantly increased by setting the reducing agent concentration in the labeling reaction reagent in the 2AA labeling step to 0.1 mmol / L or more and 20 mmol / L or less.

[0495] [Example 2-5: Confirmation of cleaning effect with mixed solvent] (Preparation of glycan-labeled compounds) A reaction mixture containing a labeled product was obtained in the same manner as in Example 2-1. The reaction mixture thus obtained contained unreacted labeled compound in addition to the sugar chain-labeled product.

[0496] (purification process to sugar chain analysis) To the reaction mixture, 60 μL of ultrapure water was added, followed by 600 μL of ethanol. The entire solution obtained was added to a cleanup column (manufactured by Sumitomo Bakelite, product number: BS-X4410), and the cleanup column was centrifuged to remove the solution. 600 μL of the mixed solution shown in Table 8 below was then added, and the cleanup column was centrifuged to remove the solution. Another 600 μL of the mixed solution shown in Table 8 below was then added, and the cleanup column was centrifuged to remove the solution.

[0497] 50 μL of ultrapure water was added to the cleanup column, followed by centrifugation, and the aqueous solution containing the glycan-labeled product was collected in a microtube. The resulting sample solution was subjected to capillary electrophoresis analysis in the same manner as in Example 2-3, and the amounts of unreacted labeled compound and Sialylated Core 1 glycan-labeled product contained in the resulting sample solution were measured. The results are shown in Table 9.

[0498] [Table 8]

[0499] [Table 9]

[0500] The "residual rate" in Table 9 indicates the percentage when the amount (area value) of unreacted labeled compound or sugar chain label contained in the sample solution without purification is taken as 100.

[0501] As a result of the evaluation, it was confirmed that, compared to unpurified sample solutions, washing the glycan-labeled compound with a mixed solvent of organic solvent and water in the purification process reduced the amount of unreacted labeled compound under all conditions.

[0502] In particular, washing with the mixed solvents No. 1 to 3 resulted in a high purification effect, with the remaining rate of unreacted labeled compounds being 15% or less and the remaining rate of glycan labels being 40% or more. In each of the sample solutions No. 1 to 3, the amount of unreacted labeled compounds was reduced compared to the sample solution without purification, and sufficient glycan labels remained, so it can be evaluated that accurate analysis is possible.

[0503] From the above results, it was confirmed that the present invention is useful. [Explanation of symbols]

[0504] 1...purification agent, 2...support, 3...polymer layer, 3a...polymer, 10...solid phase, 15...container, 16...recovery container, 20...holding section, 30...introduction section, 31...glycan-releasing enzyme, 32...labeling reaction reagent, 33...pretreatment agent / deglycosylation promoter, 34...tank, 35...nozzle, 35a...liquid transfer pipe, 36, 37, 38...valve, 40...solid-liquid separation section, 41...rack, 42...drive shaft, 43...motor, 50...container transfer section (liquid transfer section), 60...temperature control section, 100...apparatus for preparing glycans of glycoproteins

Claims

1. a releasing step in which a glycan-releasing reagent is allowed to act on a sample containing a glycoprotein to obtain a released product containing a glycan; an adsorption step of contacting a purification agent with the mixture containing the liberated product to adsorb the sugar chain onto the purification agent; an elution step of eluting the sugar chain from the purification agent; a labeling step of reacting the obtained eluate with a labeling reaction reagent to obtain a labeled product containing a labeled form of the sugar chain, The labeling reaction reagent comprises a first reagent containing at least one selected from the group consisting of 8-aminopyrene-1,3,6-trisulfonic acid, sodium salt of 8-aminopyrene-1,3,6-trisulfonic acid, and 9-aminopyrene-1,4,6-trisulfonic acid, and an aqueous solution of an organic acid; a second reagent including a reducing agent and a solvent; The labeling step includes a first step of drying the eluate, adding the first reagent, and heating the mixture; a second step of adding the second reagent to the reaction solution obtained in the first step and heating the mixture.

2. a releasing step in which a glycan-releasing reagent is allowed to act on a sample containing a glycoprotein to obtain a released product containing a glycan; an adsorption step of contacting a purification agent with the mixture containing the liberated product to adsorb the sugar chain onto the purification agent; an elution step of eluting the sugar chain from the purification agent; a labeling step of reacting the obtained eluate with a labeling reaction reagent to obtain a labeled product containing a labeled form of the sugar chain, the labeling reaction reagent includes at least one selected from the group consisting of 2-aminobenzoic acid and 3-aminobenzoic acid, a reducing agent, and a solution; the solution is a mixed solution of alcohol, an organic acid, and ultrapure water, A method for preparing a sugar chain, wherein the concentration of the reducing agent in the labeling reaction reagent is 0.1 mmol / L or more and 20 mmol / L or less.

3. 3. The method for preparing a sugar chain according to claim 1, wherein the content of the organic acid in the labeling reaction reagent solution is 10% by volume or less.

4. 3. The method for preparing a sugar chain according to claim 1, wherein the purification agent is at least one of a polymer having a betaine structure and a complex having the polymer and a support supporting the polymer.

5. the sugar chain releasing reagent contains a hydroxylamine compound and a basic reagent, the hydroxylamine compound is at least one selected from the group consisting of hydroxylamine, a salt of hydroxylamine, an O-substituted hydroxylamine, and a salt of an O-substituted hydroxylamine; The method for preparing a sugar chain according to claim 1 or 2, wherein the basic reagent is at least one selected from the group consisting of alkali metal hydroxides, weak alkali metal acid salts, alkaline earth metal hydroxides, alkaline earth metal salts dissolved in aqueous ammonia solution, and organic bases.

6. a purification step of passing a sample solution containing the labeled product through a solid phase for purification to capture the labeled product on the solid phase for purification, washing the solid phase for purification with a mixed solvent, and then re-eluting the captured labeled product, The method for preparing a sugar chain according to claim 1 or 2, wherein the mixed solvent contains an organic solvent and water.

7. 7. The method for preparing a sugar chain according to claim 6, wherein the volume ratio of the mixed solvent is acetonitrile: alcohol: water = 20-98: 0-60: 2-15 (total 100% by volume).

8. In the purification step, the labeled product obtained by removing the solvent from the sample solution is dissolved in an ionic aqueous solution to reconstitute the sample solution; The method for preparing a sugar chain according to claim 6, wherein the reconstituted sample solution is passed through the solid phase for purification.

9. A method for analyzing a sugar chain, comprising an analysis step of analyzing a sample prepared by the sugar chain preparation method according to claim 1 or 2.

10. The method for analyzing sugar chains according to claim 9, wherein the analysis step comprises the following steps (1) to (3): (1) A step of filling the capillary used for separation with the sample, and then filling the capillary with a preceding electrolyte solution from the downstream end of the capillary. (2) A step of concentrating the plurality of labels contained in the sample on the upstream side of the capillary by applying a voltage to the capillary with both ends immersed in the electrophoresis solution, with the downstream side of the capillary being positive and the upstream side being negative. (3) Separating the concentrated plurality of labeled entities by capillary electrophoresis.

11. The method for analyzing sugar chains according to claim 10, wherein the labeled substance is analyzed by laser-induced fluorescence in the analyzing step.

12. The method for analyzing sugar chains according to claim 10, wherein the migration speed of the electrolyte contained in the preceding electrolyte solution is faster than the migration speed of the label.

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