Solutions for antibody isolation

Abstract

To provide a solution for accurately separating IgG4 contained in a sample.SOLUTION: An equilibration liquid equilibrates a column filled with an insoluble carrier having Fc-binding protein immobilized thereto before and / or after adding an antibody sample, which can contain at least IgG4, to the column. The equilibration liquid has a composition that satisfies at least all the conditions of the following (a)-(c) formulae. Formula (a): electric conductivity [mS / cm]≤-0.81×buffer capacity value+12.5, formula (b): buffer capacity value>0, formula (c): electric conductivity [mS / cm]>0.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a solution for antibody separation. Specifically, the present invention relates to an equilibration solution used for accurately separating IgG4 contained in a sample. [Background technology]

[0002] In recent years, drugs containing antibodies (antibody drugs) have been used to treat cancer, immune diseases, and the like. The antibodies used in antibody drugs are produced by culturing cells capable of expressing the antibody (e.g., Chinese hamster ovary (CHO) cells, etc.) obtained by genetic engineering techniques, and then purifying them to a high degree using column chromatography or the like. However, recent research has revealed that the antibodies produced in this way are aggregates of various molecules due to modifications such as oxidation, reduction, isomerization, and glycosylation, and there are concerns about their impact on efficacy and safety. In particular, it has been reported that the sugar chain structure bound to the antibody has a significant effect on the activity, dynamics, and safety of antibody drugs, and detailed analysis of the sugar chain structure is important (Non-Patent Document 1). In addition, changes in the sugar chain structure attached to antibodies in the blood are known in diseases such as rheumatism (Non-Patent Documents 2 and 3), and it is possible to diagnose diseases by analyzing the sugar chain structure attached to the antibody.

[0003] As a method for analyzing the glycan structure of an antibody used in an antibody drug, LC-MS analysis including glycan excision (Patent Document 1 and Patent Document 2) is mainly performed. However, this analysis method involves very complicated operations and requires a lot of time. A simpler method for analyzing the molecular structure of an antibody is analysis by chromatography. Specifically, by using gel filtration chromatography to separate the antibody based on its molecular weight, it is possible to separate and quantify aggregates and decomposition products. In addition, ion exchange chromatography can separate the difference in the charge of the antibody molecule. However, the above-mentioned analysis by chromatography cannot distinguish minute structural changes of the antibody molecule, such as the glycan structure, so the analysis results obtained are limited.

[0004] On the other hand, it has been reported that the performance of an antibody can be measured and evaluated by an analysis based on the affinity between the antibody and an affinity ligand immobilized on an insoluble carrier (Patent Document 3). However, separation of antibodies based on differences in glycan structure, particularly separation of human-derived antibodies based on differences in glycan structure, has not been performed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2016-194500 A [Patent Document 2] JP 2016-099304 A [Patent Document 3] WO2013 / 120929 [Non-patent literature]

[0006] [Non-Patent Document 1] CHROMATOGRAPHY, 34(2), 83-88(2013) [Non-Patent Document 2] Science, 320, 373 (2008) [Non-Patent Document 3] Nature Communication, 7, 11205(2016) Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a solution for antibody separation. Specifically, an objective of the present invention is to provide an equilibration solution used for accurately separating IgG4 contained in a sample. [Means for solving the problem]

[0008] As a result of intensive investigations aimed at solving the above problems, the present inventors discovered that antibodies can be separated based on differences in their glycan structures by using an Fc-binding protein, and discovered a solution composition that can accurately separate IgG4 contained in a sample as an equilibration solution used when adsorbing an antibody to the Fc-binding protein, thereby completing the present invention.

[0009] That is, the present invention can be exemplified as follows.

[0010] [1] An equilibration solution for a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, comprising: An equilibration solution having a composition that satisfies at least all of the conditions in the following formulas (a) to (c). (a) Formula: Electrical conductivity [mS / cm] ≦-0.81 × buffer capacity value + 12.5 (b) Formula: Buffer capacity value > 0 (c) Formula: Electrical conductivity [mS / cm]>0 [2] The equilibration solution according to [1], wherein the equilibration solution has a composition that further satisfies the condition of the following formula (d): (d) Formula: Electrical conductivity [mS / cm]>0.24 [3] The equilibration solution according to [1] or [2], which is applied to the analysis of an antibody sample that may contain at least IgG4.

[0011] [4] The equilibration solution according to [3], wherein the antibody sample that may contain at least IgG4 is a blood-derived sample.

[0012] [5] The equilibration solution according to any one of [1] to [4], wherein the Fc-binding protein is any one of the following polypeptides (1) to (4): (1) A polypeptide comprising the amino acid residues 17 to 192 of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that, in the amino acid residues 17 to 192, at least the valine at position 176 is replaced with phenylalanine; (2) A polypeptide comprising the amino acid residues from position 17 to position 192 of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that, in the amino acid residues from position 17 to position 192, at least the valine at position 27 is replaced with glutamic acid, the phenylalanine at position 29 is replaced with isoleucine, the tyrosine at position 35 is replaced with asparagine, the glutamine at position 48 is replaced with arginine, the phenylalanine at position 75 is replaced with leucine, the asparagine at position 92 is replaced with serine, the valine at position 117 is replaced with glutamic acid, the glutamic acid at position 121 is replaced with glycine, the phenylalanine at position 171 is replaced with serine, and the valine at position 176 is replaced with phenylalanine; (3) A polypeptide comprising the amino acid residues from position 17 to position 192 of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that, in the amino acid residues from position 17 to position 192, at least the valine at position 27 is replaced with glutamic acid, the phenylalanine at position 29 is replaced with isoleucine, the tyrosine at position 35 is replaced with asparagine, the glutamine at position 48 is replaced with arginine, the phenylalanine at position 75 is replaced with leucine, the asparagine at position 92 is replaced with serine, the valine at position 117 is replaced with glutamic acid, the glutamic acid at position 121 is replaced with glycine, and the phenylalanine at position 171 is replaced with serine; (4) A polypeptide comprising the amino acid sequence of any one of the above polypeptides (1) to (3), provided that the amino acid sequence contains one or several amino acid mutations at positions other than the above substitutions.

[0013] [6] A method for separating IgG4 from an antibody sample containing at least IgG4, comprising the steps of: (1) adding an antibody sample containing IgG4 to a column packed with an insoluble carrier on which an Fc-binding protein has been immobilized, thereby allowing the antibody to be adsorbed onto the carrier; (2) adding the equilibration solution according to [1] or [2] before or after (1) to equilibrate the column; and (3) adding an eluate to the equilibrated column to elute the adsorbed antibody; A method comprising: Effect of the Invention

[0014] According to the present invention, it is possible to accurately separate IgG4, which is the detection target, from proteins including antibodies other than IgG4, and also to obtain a separation pattern of IgG4 based on the sugar chains bound to IgG4. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows the separation pattern when antibody-containing serum or human myeloma plasma-derived antibodies were analyzed on a column packed with an Fc-binding protein-immobilized gel using an equilibration buffer within the range of conditions of the present invention. [Diagram 2] FIG. 1 shows the separation pattern when antibody-containing serum or human myeloma plasma-derived antibodies were analyzed on a column packed with an Fc-binding protein-immobilized gel using an equilibration buffer outside the conditions of the present invention. [Diagram 3] This is a diagram showing classification of separation patterns when serum containing antibodies is analyzed using a column packed with Fc-binding protein-immobilized gel. [Figure 4] FIG. 1 shows a separation pattern of human albumin when analyzed on a column packed with an Fc-binding protein-immobilized gel using an equilibration buffer within the range of conditions of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention will be described in detail below.

[0017] The present invention provides an equilibration solution for separating IgG4 to be detected using a column packed with an insoluble carrier to which an Fc-binding protein has been immobilized (hereinafter, also simply referred to as an "Fc-binding protein column"). This solution is also referred to as the "equilibration solution of the present invention" or the "equilibration buffer solution of the present invention."

[0018] Specifically, the equilibration solution of the present invention may be an equilibration solution applied to a method for separating an antibody, the method comprising the following steps (1) to (3): (1) adding a solution containing an antibody to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, thereby adsorbing the antibody onto the carrier; (2) adding an equilibration solution to equilibrate the column; (3) A step of eluting the antibodies adsorbed on the carrier using an elution solution (hereinafter referred to as "elution buffer") to obtain an antibody separation pattern.

[0019] Steps (1) to (3) are also referred to as the "adsorption step", the "equilibration step" and the "elution step", respectively.

[0020] The present invention may provide an isolated antibody. That is, one embodiment of the present invention may be an equilibration solution for producing an isolated antibody by separating the antibody using an Fc-binding protein column. That is, one embodiment of the elution step may be a step of eluting the antibody adsorbed to the carrier with an elution solution to obtain the eluted antibody. In other words, one embodiment of the elution step may include a step of eluting the antibody adsorbed to the carrier with an elution solution and a step of obtaining the eluted antibody.

[0021] According to the present invention, data relating to the separation pattern of an antibody (hereinafter, also simply referred to as "separation data") may be obtained. That is, one aspect of the present invention may be used as an equilibration liquid for producing antibody separation data by separating the antibody using an Fc-binding protein column. That is, one aspect of the elution step may be a step of eluting the antibody adsorbed to the carrier with an elution liquid and obtaining antibody separation data. In other words, one aspect of the elution step may include a step of eluting the antibody adsorbed to the carrier with an elution liquid and a step of obtaining antibody separation data.

[0022] The antibody separation data may be used as an index to detect the presence or absence of a disease, the risk of developing a disease, the degree of progression of a disease, and / or the degree of aging in a subject. That is, the antibody separation data may be considered as data used as an index for detecting the presence or absence of a disease, the risk of developing a disease, the degree of progression of a disease, and / or the degree of aging in a subject. Specifically, the presence or absence of a disease, the risk of developing a disease, the degree of progression of a disease, and / or the degree of aging in a subject can be detected using separation data obtained by separating an antibody obtained from a subject using an Fc-binding protein column as an index. That is, the present invention may be provided as an equilibration solution for accurately detecting the presence or absence of a disease, the risk of developing a disease, the degree of progression of a disease, and / or the degree of aging in a subject using separation data obtained by separating an antibody obtained from a subject using an Fc-binding protein column as an index. The presence or absence of a disease, the risk of developing a disease, the degree of progression of a disease, and / or the degree of aging are collectively referred to as "risk". "Risk detection," "risk assessment," and "risk determination" may be used synonymously. <Adsorption process> The adsorption step is a step in which a solution containing an antibody is added to an Fc-binding protein column to adsorb the antibody onto the carrier.

[0023] "Antibody" means a molecule that includes an Fc region. An antibody may consist of an Fc region, or may include other regions in addition to the Fc region. An example of an Fc region is an Fc region of an immunoglobulin. An antibody may have a glycosylation. An antibody may have a glycosylation, for example, at least in its Fc region. An antibody may be a monoclonal antibody or a polyclonal antibody. The origin of an antibody is not particularly limited. An antibody may be derived from a single organism, or may be derived from a combination of two or more organisms. An antibody may be, for example, a chimeric antibody, a humanized antibody, a human antibody, or a variant thereof (e.g., an amino acid substitution). An example of an antibody is an immunoglobulin. An example of an immunoglobulin is IgG, IgM, IgA, IgD, or IgE. An example of an immunoglobulin is, in particular, IgG. An example of an IgG is IgG1, IgG2, IgG3, or IgG4. Further, the antibody may be an artificially structure-modified antibody such as a bispecific antibody, a fusion antibody of an Fc region with another protein, or an Fc region-drug complex (ADC). The antibody may be, for example, an antibody drug. Examples of the antibody drug include infliximab, an anti-TNF-α antibody, tocilizumab, an anti-IL-6 antibody, trastuzumab, an antibody against the cancer gene HER2, and nivolumab, an IgG4 antibody against PD-1. The antibody may be produced by a production cell such as a CHO (Chinese Hamster Ovary) cell, a mouse myeloma-derived cell (Sp2 / 0 cell), an NS0 cell, or a hybridoma cell.

[0024] Examples of glycans that may particularly contribute to antibody separation include G0, G0F, G1, G0F+GN, G1Fa, G1Fb, G1F+GN, G2, G2F, G1F+SA, G2F+SA, G2F+2SA, G2F+GN, G2+SA, G2+2SA, S1, S2, and S3.

[0025] Human-derived antibodies usually contain antibodies having sialic acid. The content of antibodies having sialic acid in human-derived antibodies can be, for example, about 0.1% to 20% by weight relative to the total antibody content. Human-derived antibodies often have two sialic acids bound to the sugar chain terminal. Furthermore, human-derived antibodies usually contain bisecting GlcNAc (denoted as "+GN") at about 1% to 20% by weight relative to the total antibody content. On the other hand, hamster- and mouse-derived antibodies usually do not contain bisecting GlcNAc, and the number of sialic acid bonds at the sugar chain terminal is 0 to 1.

[0026] The antibody subjected to the adsorption step may be a mixture containing multiple types of antibody molecules. Specifically, the antibody subjected to the adsorption step may be a mixture containing multiple types of antibody molecules having different glycan structures. More specifically, the antibody subjected to the adsorption step may be a mixture containing multiple types of antibody molecules having different glycan structures added to the Fc region.

[0027] When the purpose is to detect a risk, the equilibration solution of the present invention may be carried out using an antibody obtained from a subject.

[0028] The term "subject" simply means a human individual to be measured or a human individual to be detected for risk. The subject referred to here is also referred to as a "target subject" to distinguish it from a control subject described later. The subject is not particularly limited as long as an antibody sample derived therefrom can be used, that is, an antibody sample can be obtained or has already been obtained. The subject may be male or female. The subject may be an individual of any age, such as a child, a young person, a middle-aged person, or an elderly person. The subject may be a healthy individual or not. "Antibody sample" means a sample containing an antibody. Examples of antibody samples include blood samples such as blood (whole blood), diluted blood, serum, plasma, cerebrospinal fluid, umbilical cord blood, and blood component collection; samples that may contain blood-derived components such as urine, saliva, semen, feces, phlegm, amniotic fluid, and ascites (hereinafter also referred to as "blood-derived samples"); tissue fragments (tissue pieces) and cells such as liver, lung, spleen, kidney, skin, tumor, and lymph node; and antibodies separated therefrom. The antibody sample may be used in the adsorption step as is or after appropriate pretreatment. The pretreatment may be performed, for example, by a standard method. Examples of pretreatment include purification by centrifugation or column. Specifically, for example, gamma globulin may be purified and used in the adsorption step. The antibody sample is used in the adsorption step in the form of a solution containing an antibody. That is, the antibody sample may be appropriately prepared in the form of a solution containing an antibody and used in the adsorption step. For example, the antibody sample or a pretreated product thereof as exemplified above may be dissolved, suspended, dispersed, or solvent-exchanged in a liquid medium as appropriate, and used in the adsorption step as a solution containing the antibody. For such a liquid medium, the description of the equilibration liquid described below can be applied mutatis mutandis. The liquid medium may or may not be the same as the equilibration liquid.

[0029] The term "Fc-binding protein" refers to a protein having a binding ability to the Fc region of an antibody. The Fc-binding protein is not particularly limited as long as it can provide a desired antibody separation pattern. The antibody can be separated based on the difference in affinity with the Fc-binding protein based on the difference in the glycan structure of the antibody (e.g., the glycan structure of the Fc region). The affinity between the antibody and the Fc-binding protein (specifically, the glycan structure of the antibody) can be correlated with, for example, the function of the antibody, such as its efficacy. In addition, the affinity between the antibody and the Fc-binding protein (specifically, the glycan structure of the antibody) can be correlated with, for example, the risk of the subject. That is, the Fc-binding protein is preferably a protein that has a binding ability to the Fc region of an antibody and can recognize the difference in the glycan structure of the antibody (e.g., the glycan structure of the Fc region). Examples of Fc-binding proteins include human Fc-binding proteins. Examples of human Fc-binding proteins include Fc-binding proteins found in humans and variants thereof. Specific examples of human Fc-binding proteins include proteins containing the full-length or partial amino acid sequence of the extracellular region of human FcγRIIIa. In the case of native human FcγRIIIa, the amino acid sequence of the extracellular region of human FcγRIIIa includes the region from glycine at position 17 to glutamine at position 192 of the amino acid sequence set forth in SEQ ID NO: 1. Examples of partial sequences of the amino acid sequence of the extracellular region of human FcγRIIIa include the amino acid sequence of a region of the extracellular region of human FcγRIIIa that can express the function of binding to at least an Fc region (e.g., the Fc region of human IgG). Examples of human Fc-binding proteins include the following polypeptides (i) and (ii). (i) a polypeptide comprising at least amino acid residues 17 to 192 of the amino acid sequence set forth in SEQ ID NO:1; (ii) A polypeptide comprising at least amino acid residues 17 to 192 of the amino acid sequence set forth in SEQ ID NO:1, and including one or more substitutions, insertions, or deletions of amino acid residues within said amino acid residues.

[0030] An example of the above-mentioned (ii) is a polypeptide (JP 2015-086216 A) that contains the amino acid residues from the 17th to the 192nd in the amino acid sequence set forth in SEQ ID NO: 1, and in which at least one of the amino acid substitutions (1) to (40) below has occurred in the amino acid residues from the 17th to the 192nd: (1) Substitution of methionine at position 18 of SEQ ID NO:1 with arginine (2) Substitution of valine at position 27 of SEQ ID NO:1 with glutamic acid (3) Substitution of phenylalanine at position 29 of SEQ ID NO:1 with leucine or serine (4) Substitution of leucine at position 30 of SEQ ID NO:1 with glutamine (5) The 35th tyrosine in SEQ ID NO: 1 is substituted with aspartic acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, or histidine. (6) Substitution of lysine at position 46 of SEQ ID NO:1 with isoleucine or threonine (7) Substitution of glutamine at position 48 of SEQ ID NO:1 with histidine or leucine (8) Alanine at position 50 of SEQ ID NO:1 is substituted with histidine (9) Tyrosine at position 51 of SEQ ID NO:1 is substituted with aspartic acid or histidine (10) Substitution of glutamic acid at position 54 of SEQ ID NO:1 with aspartic acid or glycine (11) Substitution of asparagine at position 56 of SEQ ID NO:1 with threonine (12) Substitution of glutamine at position 59 of SEQ ID NO:1 with arginine (13) Substitution of phenylalanine at position 61 of SEQ ID NO:1 with tyrosine (14) Substitution of glutamic acid at position 64 of SEQ ID NO:1 with aspartic acid (15) Substitution of serine at position 65 of SEQ ID NO: 1 with arginine (16) Substitution of alanine at position 71 of SEQ ID NO:1 with aspartic acid (17) The 75th phenylalanine of SEQ ID NO: 1 is substituted with leucine, serine, or tyrosine (18) Substitution of aspartic acid at position 77 of SEQ ID NO:1 with asparagine (19) Substitution of alanine at position 78 of SEQ ID NO:1 with serine (20) Substitution of aspartic acid at position 82 of SEQ ID NO:1 with glutamic acid or valine (21) Substitution of glutamine at position 90 of SEQ ID NO:1 with arginine (22) Substitution of asparagine at position 92 of SEQ ID NO:1 with serine (23) Substitution of leucine at position 93 of SEQ ID NO:1 with arginine or methionine (24) Substitution of threonine at position 95 of SEQ ID NO:1 with alanine or serine (25) Substitution of leucine at position 110 of SEQ ID NO:1 with glutamine (26) Substitution of arginine at position 115 of SEQ ID NO:1 with glutamine (27) Tryptophan at position 116 of SEQ ID NO:1 is replaced with leucine (28) Substitution of phenylalanine at position 118 of SEQ ID NO:1 with tyrosine (29) Substitution of lysine at position 119 of SEQ ID NO:1 with glutamic acid (30) Substitution of glutamic acid at position 120 of SEQ ID NO:1 with valine (31) Substitution of glutamic acid at position 121 of SEQ ID NO:1 with aspartic acid or glycine (32) Substitution of phenylalanine at position 151 of SEQ ID NO: 1 with serine or tyrosine (33) Substitution of serine at position 155 of SEQ ID NO: 1 with threonine (34) Substitution of threonine at position 163 of SEQ ID NO:1 with serine (35) Serine at position 167 of SEQ ID NO:1 is replaced with glycine (36) Substitution of serine at position 169 of SEQ ID NO:1 with glycine (37) Substitution of phenylalanine at position 171 of SEQ ID NO:1 with tyrosine (38) Asparagine at position 180 of SEQ ID NO:1 is substituted with lysine, serine, or isoleucine (39) Substitution of threonine at position 185 of SEQ ID NO:1 with serine (40) Substitution of glutamine at position 192 of SEQ ID NO:1 with lysine Another embodiment of (ii) above is a polypeptide (JP 2016-169197 A) that contains the amino acid residues from the 17th to the 192nd in the amino acid sequence set forth in SEQ ID NO: 1, and in which at least one of the amino acid substitutions (41) to (57) below has occurred in the amino acid residues from the 17th to the 192nd: (41) Substitution of phenylalanine at position 29 of SEQ ID NO:1 with isoleucine or leucine (42) Substitution of glutamic acid at position 39 of SEQ ID NO:1 with glycine (43) Substitution of glutamine at position 48 of SEQ ID NO:1 with arginine (44) Tyrosine at position 51 of SEQ ID NO:1 is replaced with serine (45) Substitution of phenylalanine at position 61 of SEQ ID NO:1 with tyrosine (46) Aspartic acid at position 77 of SEQ ID NO:1 is replaced with glycine (47) Aspartic acid at position 82 of SEQ ID NO:1 is replaced with glutamic acid (48) Substitution of glutamine at position 90 of SEQ ID NO:1 with arginine (49) Substitution of glutamine at position 112 of SEQ ID NO:1 with leucine (50) Substitution of valine at position 117 of SEQ ID NO:1 with glutamic acid (51) Substitution of lysine at position 119 of SEQ ID NO:1 with asparagine or glutamic acid (52) Substitution of threonine at position 140 of SEQ ID NO:1 with isoleucine (53) Leucine at position 142 of SEQ ID NO:1 is replaced with glutamine (54) Substitution of phenylalanine at position 171 of SEQ ID NO:1 with serine (55) Substitution of leucine at position 175 of SEQ ID NO:1 with arginine (56) Substitution of asparagine at position 180 of SEQ ID NO:1 with serine (57) Isoleucine at position 188 of SEQ ID NO:1 is replaced with valine Furthermore, yet another embodiment of (ii) above is a polypeptide (JP 2016-169197 A) that contains amino acid residues 17 to 192 of the amino acid sequence set forth in SEQ ID NO: 1, and in which at least one of the following amino acid substitutions (58) to (61) has occurred in the amino acid residues 17 to 192: (58) Substitution of leucine at position 66 of SEQ ID NO:1 with histidine or arginine (59) Glycine at position 147 of SEQ ID NO:1 is replaced with aspartic acid (60) Tyrosine at position 158 of SEQ ID NO:1 is substituted with histidine (61) Valine at position 176 of SEQ ID NO:1 is replaced with phenylalanine Furthermore, yet another embodiment of (ii) above is a polypeptide comprising the 17th to 192nd amino acid residues of the amino acid sequence set forth in SEQ ID NO:1, in which at least one of the amino acid substitutions selected from (1) to (61) above has occurred in the 17th to 192nd amino acid residues.

[0031] The above-mentioned (ii) particularly includes the following polypeptides (ii-1) to (ii-3). (ii-1) a polypeptide comprising the amino acid residues from position 17 to position 192 of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that in the amino acid residues from position 17 to position 192, at least the valine at position 176 is substituted with phenylalanine; (ii-2) a polypeptide comprising the amino acid residues from position 17 to position 192 of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that in the amino acid residues from position 17 to position 192, at least the valine at position 27 is substituted with glutamic acid, the phenylalanine at position 29 is substituted with isoleucine, the tyrosine at position 35 is substituted with asparagine, the glutamine at position 48 is substituted with arginine, the phenylalanine at position 75 is substituted with leucine, the asparagine at position 92 is substituted with serine, the valine at position 117 is substituted with glutamic acid, the glutamic acid at position 121 is substituted with glycine, the phenylalanine at position 171 is substituted with serine, and the valine at position 176 is substituted with phenylalanine; (ii-3) A polypeptide comprising the amino acid residues from the 17th to the 192nd of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that in the 17th to the 192nd amino acid residues, at least the 27th valine is substituted with glutamic acid, the 29th phenylalanine is substituted with isoleucine, the 35th tyrosine is substituted with asparagine, the 48th glutamine is substituted with arginine, the 75th phenylalanine is substituted with leucine, the 92nd asparagine is substituted with serine, the 117th valine is substituted with glutamic acid, the 121st glutamic acid is substituted with glycine, and the 171st phenylalanine is substituted with serine.

[0032] The Fc-binding protein may be a polypeptide that contains "one or several" amino acid mutations (e.g., substitutions, insertions, or deletions) in the amino acid sequence of the above-exemplified Fc-binding protein (e.g., the polypeptide of (i) or (ii) above), so long as it has the function of binding to an Fc region (e.g., the Fc region of human IgG). "One or several" may be, for example, 1 to 50, preferably 1 to 40, more preferably 1 to 30, even more preferably 1 to 20, particularly preferably 1 to 10, and most preferably 1 to 5. "One or several" amino acid mutations may be made so that the amino acid substitutions of the above-exemplified Fc-binding proteins selected from the above amino acid substitutions (1) to (61) are preserved. In other words, "one or several" amino acid mutations may be made at positions other than the amino acid substitutions of the above-exemplified Fc-binding proteins selected from the above amino acid substitutions (1) to (61).

[0033] The Fc-binding protein may be a polypeptide comprising an amino acid sequence having high homology to the amino acid sequence of the above-exemplified Fc-binding protein (e.g., the polypeptide of (i) or (ii)) as long as it has a function of binding to an Fc region (e.g., the Fc region of human IgG). "High homology" may mean 70% or more, 80% or more, 90% or more, or 95% or more homology. "Homology" may mean similarity and / or identity. The homology of amino acid sequences can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool). For example, "identity of amino acid sequences" may mean identity between amino acid sequences calculated using blastp, specifically, identity between amino acid sequences calculated using blastp with default parameters. The above-mentioned changes in the amino acid sequence within the homology range may be made so as to preserve the amino acid substitutions in the above-mentioned exemplified Fc-binding proteins selected from the above amino acid substitutions (1) to (61). In other words, the above-mentioned changes in the amino acid sequence within the homology range may be made at positions other than the amino acid substitutions in the above-mentioned exemplified Fc-binding proteins selected from the above amino acid substitutions (1) to (61).

[0034] The Fc-binding protein can be produced, for example, by expressing a gene encoding the Fc-binding protein in a host having the gene. The gene encoding the Fc-binding protein can be obtained, for example, by cloning, chemical synthesis, mutagenesis, or a combination thereof. The host is not particularly limited as long as it can express the Fc-binding protein. Examples of the host include animal cells, insect cells, and microorganisms. Examples of the animal cells include COS cells, CHO cells, Hela cells, NIH3T3 cells, and HEK293 cells. Examples of the insect cells include Sf9 and BTI-TN-5B1-4. Examples of the microorganism include yeast and bacteria. Examples of the yeast include yeasts of the genus Saccharomyces such as Saccharomyces cerevisiae, yeasts of the genus Pichia such as Pichia pastoris, and yeasts of the genus Schizosaccharomyces such as Schizosaccharomyces pombe. Examples of the bacteria include bacteria of the genus Escherichia such as Escherichia coli. Examples of Escherichia coli include the W3110 strain, the JM109 strain, and the BL21(DE3) strain. In addition, the Fc-binding protein can also be produced, for example, by expressing a gene encoding the Fc-binding protein in a cell-free protein synthesis system.

[0035] In the present invention, the term "insoluble carrier" refers to a carrier that is insoluble in a liquid passed through a column (e.g., a liquid used for adsorbing or eluting an antibody, such as an equilibration liquid or an elution liquid). The insoluble carrier may have a functional group (e.g., a hydroxy group) for immobilizing an Fc-binding protein by covalent bonding. Examples of insoluble carriers include carriers derived from inorganic substances such as zirconia, zeolite, silica, and coated silica; carriers derived from natural organic polymeric substances such as cellulose, agarose, and dextran; and carriers derived from synthetic organic polymeric substances such as polyacrylic acid, polystyrene, polyacrylamide, polymethacrylamide, polymethacrylate, and vinyl polymers.

[0036] The Fc-binding protein can be immobilized on an insoluble carrier as appropriate. The Fc-binding protein can be immobilized on an insoluble carrier by covalent bonding, for example, by utilizing a functional group (e.g., a hydroxyl group) provided on the insoluble carrier for covalently immobilizing the Fc-binding protein. For example, when the insoluble carrier has a hydroxyl group on its surface, an activating agent can be used to form an activated group capable of covalently bonding with the Fc-binding protein from the hydroxyl group, and the activated group can be covalently bonded to the Fc-binding protein. Specific examples of activating agents for hydroxyl groups include epichlorohydrin (forming an epoxy group as an activated group), 1,4-butanediol diglycidyl ether (forming an epoxy group as an activated group), tresyl chloride (forming a tresyl group as an activated group), and vinyl bromide (forming a vinyl group as an activated group). In addition, the hydroxyl group can be converted to an amino group, a carboxyl group, or the like, and then activated by the action of an activating agent. Specific examples of activators for amino groups, carboxyl groups, etc. include N-succinimidyl 3-maleimidopropionate (which forms a maleimide group as the activating group), 1,1'-carbonyldiimidazole (which forms a carbonylimidazole group as the activating group), and halogenated acetic acid (which forms a halogenated acetyl group as the activating group).

[0037] The antibody can be adsorbed to the carrier by adding a solution containing the antibody to the Fc-binding protein column. The antibody-containing solution can be added to the column using a liquid-transporting means such as a pump. Adding a liquid to the column is also referred to as "transporting the liquid to the column". The conditions for carrying out the adsorption step, such as the amount of the antibody-containing solution added, the type of liquid phase, the liquid-phase transport rate, and the column temperature, are not particularly limited as long as the antibody is adsorbed to the carrier. The conditions for carrying out the adsorption step can be appropriately set depending on various conditions such as the type of antibody, the type of Fc-binding protein, the type of insoluble carrier, and the scale of the column. The liquid phase can be an equilibration liquid, which will be described later. The liquid transport rate can be, for example, 0.1 mL / min to 2.0 mL / min, 0.2 mL / min to 1.5 mL / min, or 0.4 mL / min to 1.2 mL / min when the inner diameter of the column is 4.6 mm. The liquid transport rate can be set, for example, to be proportional to the square of the inner diameter of the column. The column temperature may be, for example, 0°C to 50°C. <Equilibration process> The equilibration step is a step of equilibrating the column with the antibody adsorbed to the carrier using an equilibration solution (similarly referred to as "equilibration buffer"). Before the antibody-containing solution is added to the column, the column may be equilibrated using the equilibration solution. That is, the present invention may include a step of adding an equilibration solution to the column and equilibrating the column before the adsorption step.

[0038] By equilibration, antibodies that do not bind to the Fc-binding protein or that bind to the antibody-containing solution when added to the column but not to the equilibration buffer can be removed from the column. The fraction containing the antibody that does not bind to the carrier by equilibration is called the unadsorbed fraction, which refers to the fraction in the region until the peak detected after the antibody-containing solution is added to the column reaches its minimum value during equilibration. It is preferable that the unadsorbed fraction and the peak region detected after the addition of the eluent are separated in separation time from each other, since this increases the separation accuracy. In particular, it is preferable that the detection value between the unadsorbed fraction and the peak region detected after the addition of the eluent is a constant value, since this indicates that the unadsorbed fraction has been sufficiently removed from the column by the equilibration step. The constant value referred to here includes not only the same value but also a state in which the detection value changes with a constant slope.

[0039] The present invention relates to an equilibration solution that is a solution for accurately separating IgG4 contained in a body fluid as a detection target, and in particular to an equilibration solution that can accurately separate IgG4 from an unadsorbed fraction, and can accurately separate IgG4 even if it contains antibodies other than IgG4 (e.g., IgG1, IgG2, IgG3) that can bind to an Fc-binding protein. The IgG4 to be detected refers to IgG4 that is defined as being eluted at a time similar to the elution time of IgG4 derived from human myeloma plasma in the elution step described below, and does not include IgG4 contained in the unadsorbed fraction. In this specification, IgG4 does not include IgG4 contained in the unadsorbed fraction, unless otherwise noted.

[0040] The equilibration liquid may be an aqueous buffer solution. Specific examples of the equilibration liquid include weakly acidic to weakly alkaline buffer solutions with a pH of 5.0 to 8.0. The components of the buffer solution may be appropriately selected depending on various conditions such as the pH of the buffer solution. The components of the buffer solution may include phosphoric acid, acetic acid, formic acid, MES (2-Morpholinoethanesulfonic acid), MOPS (3-Morpholinopropanesulfonic acid), citric acid, succinic acid, glycine, and piperazine. In addition, a salt may be further added to the buffer solution, and is not particularly limited as long as it is a salt that can be easily imagined by a person skilled in the art, such as sodium chloride or potassium chloride.

[0041] Substances that are preferably separated from the IgG4 to be detected include IgG2 and proteins other than IgG, which hardly bind to Fc-binding proteins. IgG1 and IgG3 have high binding ability to Fc-binding proteins, and IgG4 has low binding ability compared to IgG1 and IgG3. Therefore, when a solution having a composition in which an antibody binds strongly to an Fc-binding protein is used as the equilibration liquid, IgG4 is adsorbed to the Fc-binding protein and is eluted by adding an elution liquid. On the other hand, when a solution having a composition in which an antibody binds weakly to an Fc-binding protein is used as the equilibration liquid, the IgG4 to be detected is eluted during equilibration and overlaps with the peak of the unadsorbed fraction containing proteins other than IgG2 and IgG. Therefore, the equilibration liquid needs to be adjusted so that the IgG4 to be detected is adsorbed, and in the present invention, it has been found that the equilibration liquid has a composition within the range of the following formulas (a) to (c). It is to be noted that, from formulas (a) to (c), the buffer capacity value <15.43 and the electrical conductivity [mS / cm] <12.5 can be further derived as the composition range of the equilibration liquid. (a) Formula: Electrical conductivity [mS / cm] ≦-0.81 × buffer capacity value + 12.5 (b) Formula: Buffer capacity value > 0 (c) Formula: Electrical conductivity [mS / cm]>0 Electrical conductivity is a value that is determined by the components of the buffer solution and the salt added, and reflects the amount of ions contained. Ions are involved in the electrostatic adsorption ability of Fc-binding proteins or carriers, and the higher the electrical conductivity, the lower the adsorption ability of antibodies to Fc-binding proteins or carriers. The buffer capacity value is the value obtained by dividing the number of moles of hydrochloric acid or sodium hydroxide dropped into the buffer solution by the pH that has changed after dropping (see Journal of Biological Chemistry, 52, 525 (1922)). In the present invention, the buffer capacity value is the value obtained by dividing the number of millimoles of hydrochloric acid dropped per 1 L of equilibration buffer solution when the pH of the equilibration buffer solution is changed by using hydrochloric acid in a range of 0.1 to 0.2 by the actually measured pH that has changed. The higher the buffer capacity value, the higher the buffer capacity. When the same buffer solution components are used, the higher the buffer capacity value, the higher the concentration of the buffer solution components, and the higher the electrical conductivity. Since the antibody adsorption capacity changes depending on the buffer capacity even when the electrical conductivity is the same, it is clear that the buffer capacity is also a factor that varies the adsorption capacity between an Fc-binding protein or a carrier and an antibody.

[0042] When measuring a sample containing a protein other than the antibody that is not the target of measurement as an antibody sample, the protein may be eluted after the elution step described below, approaching the elution peak of the target IgG4, which may result in a decrease in the separation ability of IgG4. It is presumed that if the electrical conductivity of the equilibration solution is low, the electrostatic interaction between the protein not to be measured and the Fc-binding protein becomes strong, and the protein continues to bind until the elution step described below. In order to suppress contamination of the protein after the elution step, it is preferable to use a composition that is within the range of the following formula (d) in addition to formulas (a) to (c), because this improves the separation accuracy of IgG4. (d) Formula: Electrical conductivity [mS / cm]>0.24 <Elution process> The elution step is a step in which the antibody adsorbed to the carrier is eluted with an elution solution.

[0043] That is, the antibody adsorbed on the carrier can be eluted by adding an elution solution to the column. The conditions for carrying out the elution step, such as the type of elution solution, the delivery format of the elution solution, the delivery rate of the liquid phase, and the column temperature, are not particularly limited as long as the antibody is separated in a desired manner, for example, as long as the separation data of the desired antibody is obtained. The conditions for carrying out the elution step can be appropriately set according to various conditions, such as the type of antibody, the type of Fc-binding protein, the type of insoluble carrier, and the scale of the column. The elution solution can be one that weakens the affinity between the antibody and the Fc-binding protein. The elution solution can be an aqueous buffer solution having a lower pH than the liquid phase before elution (e.g., the equilibration solution). Specific examples of the elution solution include acidic buffer solutions having a pH of 2.5 to 4.5. For example, when the liquid phase before elution (e.g., the equilibration solution) is a weakly acidic to weakly alkaline buffer solution having a pH of 5.0 to 8.0, the elution solution may be an acidic buffer solution having a pH of 2.5 to 4.5. The components of the buffer solution can be appropriately selected according to various conditions, such as the pH of the buffer solution. Components of the buffer solution include phosphoric acid, acetic acid, formic acid, MES (2-Morpholinoethanesulfonic acid), MOPS (3-Morpholinopropanesulfonic acid), citric acid, succinic acid, glycine, and piperazine.

[0044] The eluent may be fed in a gradient or isocratic manner. The eluent may be fed in a gradient manner. That is, the elution may be carried out by increasing the ratio of the eluent in the liquid phase. The gradient may be a linear gradient, a stepwise gradient, or a combination thereof. Specifically, the gradient may be set so that the ratio of the eluent in the liquid phase increases from 0% (v / v) to 100% (v / v) in 10 to 60 minutes, 15 to 50 minutes, or 20 to 40 minutes. The feed rate may be, for example, 0.1 mL / min to 2.0 mL / min, 0.2 mL / min to 1.5 mL / min, or 0.4 mL / min to 1.2 mL / min when the inner diameter of the column is 4.6 mm. The liquid delivery speed may be set, for example, so as to be proportional to the square of the inner diameter of the column. The column temperature may be, for example, 0°C to 50°C.

[0045] The elution step may provide a separated antibody. The separated antibody may be obtained, for example, as an elution fraction containing the antibody. That is, the separated antibody is obtained by collecting the elution fraction containing the separated antibody. The elution fraction can be collected, for example, by a conventional method. Specifically, the elution fraction can be collected, for example, by an automatic fraction collector such as an autosampler. Furthermore, the separated antibody may be recovered from the elution fraction. The separated antibody can be collected, for example, by a conventional method from the elution fraction. Specifically, the separated antibody can be collected, for example, by a known method used for separating and purifying proteins from the elution fraction.

[0046] The elution step may obtain antibody separation data (i.e., data related to the antibody separation pattern). The antibody separation data may be used as an index for detecting a risk in a subject, and is not particularly limited. Examples of the antibody separation data include characteristics of the antibody separation pattern. The characteristics of the antibody separation pattern are also simply referred to as "characteristics". That is, the step of obtaining separation data may include, for example, a step of obtaining an antibody separation pattern and a step of extracting the characteristics of the antibody separation pattern (i.e., extracting the characteristics from the antibody separation pattern). The antibody separation pattern is obtained by detecting the antibody with a detector. Examples of the detector include a UV detector and a mass detector. Examples of the antibody separation pattern include a chromatogram at the time of elution of the antibody. Examples of the characteristics include parameters obtained from the antibody separation pattern that are correlated with the risk in the subject. Specific examples of the characteristics include characteristics of the elution peak (i.e., the peak of the eluted antibody). Specific examples of the characteristics of the elution peak include a peak area, a peak elution time, a peak width, a number of peaks detected, and a peak height. An elution peak from which features such as peak area and peak height are extracted is also referred to as a "target peak." Elution peak features include peak area and peak height, among others. The antibody separation pattern may be used for feature extraction as is, or after appropriate correction such as baseline correction.

[0047] The feature may be an absolute value or a relative value. Examples of the relative value include a ratio or difference to the value of another elution peak (i.e., any elution peak other than the target peak) and a ratio or difference to the sum of the values ​​of all elution peaks (i.e., all elution peaks including the target peak). Examples of the relative value include, in particular, a ratio to the value of another elution peak and a ratio to the sum of the values ​​of all elution peaks. As the other elution peak, one elution peak may be used, or two or more elution peaks may be used in combination. For example, the peak area may specifically be peak area %. "Peak area %" means the ratio (%) of the area of ​​the target peak to the sum of the areas of all elution peaks. For example, the peak height may specifically be peak height %. "Peak height %" means the ratio (%) of the height of the target peak to the sum of the heights of all elution peaks. The feature may be corrected, such as a correction based on a peak obtained with an internal standard or a correction based on the properties of the subject. For example, the feature may be corrected based on the age of the subject. That is, for example, when the feature is affected by the age of the subject, the extracted feature may be corrected based on the age of the subject before being used in the detection step described below. The feature corrected based on the age of the subject may be used, for example, to detect the risk of a condition other than aging. <Detection process> The detection process is a process for detecting a risk in a subject (i.e., the presence or absence of a disease, the risk of developing a disease, the degree of progression of a disease, and / or the degree of progression of aging) using separation data (i.e., data related to the separation pattern of antibodies) as an indicator, and may be performed after the elution process described above.

[0048] The diseases include diseases affected by the activity (e.g., damaging or phagocytic) of immune cells, such as natural killer cells, monocytes, and macrophages.

[0049] Specific examples of the disease include cancer, autoimmune diseases, infectious diseases, allergies, and inflammatory diseases, and in particular, IgG4-related diseases, which are autoimmune diseases, can be mentioned since the present invention allows the detection and evaluation of IgG4. Any of these diseases can be diseases affected by the activity of immune cells.

[0050] Cancers include brain cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, esophageal cancer, stomach cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor (GIST), mesothelioma, head and neck cancer, kidney cancer, lung cancer, osteosarcoma, Ewing's sarcoma, chondrosarcoma, prostate cancer, testicular tumor, renal cell carcinoma, bladder cancer, rhabdomyosarcoma, skin cancer, and anal cancer. Cancers include pancreatic cancer, stomach cancer, breast cancer, colon cancer, and kidney cancer, among others.

[0051] Autoimmune diseases include Guillain-Barré syndrome, myasthenia gravis, multiple sclerosis, chronic gastritis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cholangitis, ulcerative colitis, Crohn's disease, primary biliary cholangitis, autoimmune pancreatitis, Takayasu's arteritis, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, type 1 diabetes, and chronic discoid erythrombocytopenia. psoriasis, localized scleroderma, pemphigus, pustular psoriasis, psoriasis vulgaris, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, Sutton's nevus, Harada's disease, autoimmune optic neuropathy, autoimmune inner ear disorder, idiopathic azoospermia, habitual abortion, rheumatism, systemic lupus erythematosus, antiphospholipid syndrome, polymyositis, dermatomyositis, scleroderma, Sjogren's syndrome, IgG4-related disease, vasculitis syndrome, mixed connective tissue disease. Autoimmune diseases include rheumatism and Sjogren's syndrome, in particular.

[0052] Infectious diseases include bacterial infections, fungal infections, parasitic protozoan infections, parasitic helminth infections, and viral infections. Bacterial infections include infections caused by various bacteria such as streptococci, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Listeria, Neisseria meningitidis, Neisseria gonorrhoeae, pathogenic Escherichia coli, Klebsiella, Proteus, Bordetella pertussis, Pseudomonas aeruginosa, Serratia, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridium, Rickettsia, and Chlamydia; tuberculosis, nontuberculous mycobacterial disease, cholera, plague, diphtheria, dysentery, scarlet fever, anthrax, syphilis, tetanus, leprosy, Legionnaires' disease, leptospirosis, Lyme disease, tularemia, and Q fever. Fungal infections include aspergillosis, candidiasis, cryptococcosis, tinea, histoplasmosis, and Pneumocystis carinii pneumonia. Parasitic protozoan infections include amebic dysentery, malaria, toxoplasmosis, leishmaniasis, and cryptosporidiosis. Parasitic helminth infections include echinococcosis, Japanese schistosomiasis, filariasis, ascariasis, and diphyllobothriasis. Examples of viral infections include influenza, viral hepatitis, viral meningitis, viral gastroenteritis, viral conjunctivitis, acquired immune deficiency syndrome (AIDS), adult T-cell leukemia, Ebola hemorrhagic fever, yellow fever, common cold syndrome, rabies, cytomegalovirus infection, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), acute respiratory disease (COVID-19), progressive multifocal leukoencephalopathy, chickenpox and shingles, herpes simplex, hand, foot and mouth disease, dengue fever, Japanese encephalitis, erythema infectiosum, infectious mononucleosis, smallpox, rubella, acute poliomyelitis (polio), measles, pharyngoconjunctival fever (pool fever), Marburg hemorrhagic fever, hemorrhagic fever with renal syndrome, Lassa fever, mumps, West Nile fever, herpangina, and chikungunya fever. The infection may be, for example, an opportunistic infection.

[0053] Allergies include anaphylactic shock, allergic rhinitis, conjunctivitis, bronchial asthma, urticaria, atopic dermatitis, hemolytic anemia, idiopathic thrombocytopenic purpura, drug-induced hemolytic anemia, granulocytopenia, thrombocytopenia, Goodpasture's syndrome, serum sickness, systemic lupus erythematosus (SLE), rheumatism, glomerulonephritis, hypersensitivity pneumonitis, allergic bronchopulmonary aspergillosis (ABPA), contact dermatitis, allergic encephalitis, transplant rejection, tuberculous cavities, and epithelioid cell granuloma.

[0054] Inflammatory diseases include diseases induced by inflammatory cytokines, such as IL-6 and TNF-α. Specific examples of inflammatory diseases include encephalitis, osteomyelitis, meningitis, neuritis, eye inflammation (dacryoadenitis, scleritis, episcleritis, keratitis, chorioretinitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, uveitis, etc.), ear inflammation (otitis externa, otitis media, otitis interna, etc.), mastitis, carditis (endocarditis, myocarditis, pericarditis, etc.), vasculitis (arteritis, phlebitis, capillaritis, etc.), respiratory inflammation (sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia, pleuritis, mediastinitis, etc.), oral inflammation (stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, siladenitis, parotitis, cheilitis, pulpitis, nasal inflammation, inflammation of the digestive tract (esophagitis, gastritis, gastroenteritis, enteritis, enteritis, colitis, duodenitis, ileitis, appendicitis, proctitis, etc.), dermatitis, cellulitis, hidradenitis, arthritis, dermatomyositis, myositis, synovitis, tendonitis, panniculitis, osteitis, osteomyelitis, periostitis, nephritis, ureteritis, cystitis, ureteritis, oophoritis, salpingitis, endometritis, cervicitis, vaginitis, vulvitis, orchitis, epididymitis, prostatitis, seminal vesicle cystitis, balanitis, presitis, chorioamnionitis, omphalitis, omphalitis, hepatitis, ascending cholangitis, cholecystitis, pancreatitis, peritonitis, hypophysitis, thyroiditis, parathyroiditis, adrenitis, lymphangitis, and lymphadenitis. Inflammatory diseases include, in particular, pancreatitis.

[0055] Examples of IgG4-related diseases include chronic inflammatory diseases in which blood IgG4 levels are high and infiltration of IgG4-positive plasma cells into diseased tissues is observed. Specific examples of IgG4-related diseases include Mikulicz disease, Kuttner tumor, dacryoadenitis, IgG4-related eye disease, IgG4-related respiratory disease, inflammatory pseudotumor, mediastinal fibrosis, enteritis, sclerosing cholangitis, IgG4-related liver disease, autoimmune pancreatitis, IgG4-related kidney disease, retroperitoneal fibrosis, prostatitis, autoimmune hypophysitis, thyroiditis, hypertrophic pachymeningitis, IgG4-related lymphadenopathy, arthritis, inflammatory abdominal aortic aneurysm, and periarteritis.

[0056] Specific examples of the disease include cachexia and age-related diseases. Age-related diseases include frailty, sarcopenia, and locomotive syndrome. Cachexia and these age-related diseases may also be inflammatory diseases.

[0057] Detecting risk in a subject includes determining whether the subject is at risk or not, or whether the subject is at high or low risk.

[0058] Examples of detection of the presence or absence of a disease include determining whether or not the subject is currently suffering from a disease, and determining whether or not the subject is highly likely to suffer from a disease. Examples of detection of the risk of developing a disease include determining whether or not the subject is likely to develop a disease in the future, or .... Examples of detection of the degree of progression of a disease include determining whether or not the degree of progression (e.g., severity) of a disease in a subject is currently high or low. Examples of detection of the degree of progression of aging include determining whether or not the degree of progression (e.g., severity) of aging in a subject is currently high or low.

[0059] That is, "the subject is at risk" may mean, for example, that the subject may currently have a disease, that the subject may develop a disease in the future, and / or that the subject may develop a severe disease if the subject develops a disease in the future. "The subject is not at risk" may mean, for example, that the subject is not currently at risk, that the subject is not likely to develop a disease in the future, and / or that the subject will not develop a severe disease if the subject develops a disease in the future. "The subject is at high risk" may mean, for example, that the subject is likely to currently have a disease, that the subject is likely to develop a disease in the future, that the subject will develop a severe disease if the subject develops a disease in the future, that the subject's current disease is progressing rapidly, and / or that the subject's current aging is progressing rapidly. "Low risk in a subject" may mean, for example, that the subject is less likely to currently have the disease, that the subject is less likely to develop the disease in the future, that the subject is less likely to have a severe disease if they do develop the disease in the future, that the subject is currently at a slower rate of disease progression, and / or that the subject is currently at a slower rate of aging.

[0060] The detection step can be carried out, for example, using the level of the value of the separated data (i.e., whether the value of the separated data is high or low) as an index. The level of the value of the separated data can be determined, for example, by comparing the value of the separated data with a predetermined threshold. In other words, the detection step can include, for example, a step of comparing the value of the separated data with a threshold. That is, "the value of the separated data is high" may mean, for example, that the value of the separated data is high based on the threshold. "The value of the separated data is high based on the threshold" may mean, for example, that the value of the separated data is equal to or higher than the threshold, that the value of the separated data is above the threshold, or that the value of the separated data is statistically significantly higher than the threshold. "The value of the separated data is high based on the threshold" may specifically mean, for example, that the value of the separated data is 1.01 times or more, 1.02 times or more, 1.03 times or more, 1.05 times or more, 1.07 times or more, 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.5 times or more, 1.7 times or more, 2 times or more, 2.5 times or more, or 3 times or more than the threshold. Furthermore, "the value of the separated data is low" may mean, for example, that the value of the separated data is low based on a threshold value. "The value of the separated data is low based on a threshold value" may mean, for example, that the value of the separated data is equal to or lower than the threshold value, that the value of the separated data is less than the threshold value, or that the value of the separated data is statistically significantly lower than the threshold value. "The value of the separated data is low based on a threshold value" may specifically mean, for example, that the value of the separated data is 0.99 times or less, 0.98 times or less, 0.97 times or less, 0.95 times or less, 0.93 times or less, 0.9 times or less, 0.85 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, or 0.3 times or less than the threshold value.

[0061] The value of the separated data may be divided into a risk range, for example, based on a threshold value. The value of the separated data may be divided into a non-risk range, for example, based on a threshold value. Specifically, the value of the separated data may be divided into a risk range and a non-risk range, for example, based on a threshold value. The "risk range" may mean a range in which the value of the separated data is likely to be at risk in the subject. The "non-risk range" may mean a range in which the value of the separated data is likely to be at no risk in the subject. That is, if the value of the separated data is in the risk range, it may be determined that the subject is at risk or at high risk. On the other hand, if the value of the separated data is in the non-risk range, it may be determined that the subject is at no risk or at low risk.

[0062] In addition, "determining that a subject is at risk, absent, high, or low when certain separated data meets a certain criterion (e.g., low or high, or within a certain range)" means that the subject is at risk, absent, high, or low at least in the range where the criterion is met, and does not require that the risk be determined in the subject in the range where the criterion is not met. However, in one embodiment, when "determining that a subject is at risk, absent, high, or low when certain separated data meets a certain criterion (e.g., low or high, or within a certain range)", the subject may be at risk, absent, absent, high, or high, respectively, in the range where the criterion is not met.

[0063] The threshold value can be appropriately set by a person skilled in the art depending on various conditions such as the type of separation data and the desired accuracy of determination. The threshold value may be set for each symptom to be determined such as disease or aging. There are no particular limitations on the means for determining the threshold value. The threshold value can be determined, for example, according to a known method used in data analysis for dividing a population into two groups.

[0064] The threshold value can be determined, for example, based on the value of separation data of an antibody sample obtained from a control subject. The separation data of an antibody sample obtained from a control subject is also referred to as "control separation data". The control separation data may be used in determining the threshold value, and thereby used in the detection step. Specifically, the control separation data may be used in determining the threshold value, and thereby used for comparison with the separation data. In other words, the detection step may include, for example, a step of comparing the separation data with the control separation data.

[0065] Examples of control subjects include positive controls and negative controls. The term "positive control" may refer to a subject who may be determined to be at risk or high. The term "negative control" may refer to a subject who may be determined to be at no risk or low. Examples of positive controls include individuals who are or have been affected by the above-mentioned diseases (particularly, the same disease as the disease to be detected for risk), individuals who have progressed in aging, and individuals with a combination of these characteristics. Examples of negative controls include individuals who are not affected by or have never been affected by the above-mentioned diseases (particularly, the same disease as the disease to be detected for risk), individuals who have not progressed in aging, and individuals with a combination of these characteristics. The threshold value may be determined based only on the value of the separation data measured for the positive control, may be determined only on the value of the separation data measured for the negative control, or may be determined based on the value of the separation data measured for both the positive control and the negative control. The threshold value may usually be determined based on the value of the separation data measured for both the positive control and the negative control. The number of positive controls and negative controls is not particularly limited as long as a threshold value that allows risk determination with a desired accuracy is obtained. The number of positive controls and negative controls may be one, two or more. The number of positive controls and negative controls may usually be more than one. The number of positive controls and negative controls may be, for example, 5 or more, 10 or more, 20 or more, or 50 or more. The number of positive controls and negative controls may be, for example, 10,000 or less, 1,000 or less, or 100 or less.

[0066] When the threshold is determined based only on the value of the separated data measured for the positive control, for example, a value selected from the range from the upper limit to the lower limit of the values ​​of the separated data measured for multiple positive control individuals, for example, an average value, may be set as the threshold. In addition, for example, the threshold may be determined so that a predetermined proportion of the positive control falls within the danger range in the distribution of the values ​​of the separated data measured for multiple positive control individuals. The predetermined proportion may be, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 100%.

[0067] When the threshold is determined based only on the value of the separation data measured for the negative control, for example, a value selected from the range from the upper limit to the lower limit of the values ​​of the separation data measured for multiple negative control individuals, for example, an average value, may be set as the threshold. In addition, for example, the threshold may be determined so that a predetermined proportion of the negative control falls within a non-risk range in the distribution of values ​​of the separation data measured for multiple negative control individuals. The predetermined proportion may be, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 100%.

[0068] When the threshold value is determined based on both the value of the separation data measured for the positive control and the value of the separation data measured for the negative control, the threshold value may be determined so that, for example, a predetermined proportion of the positive control falls within the risk range and a predetermined proportion of the negative control falls within the non-risk range. It is preferable that the proportion of the positive control that falls within the risk range and the proportion of the negative control that falls within the non-risk range are both high. These proportions may be, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 100%. When it is difficult to increase both of these proportions, the threshold value may be set so that one of the proportions is preferentially increased depending on various conditions such as the purpose of use of the determination result according to the present invention. For example, in order to reduce the false negative rate, the threshold value may be set so that the proportion of the positive control that falls within the risk range is preferentially increased.

[0069] The threshold value may be determined, for example, using software. For example, a threshold value that can statistically most appropriately distinguish between the negative control and the positive control may be determined using statistical analysis software. Examples of such software include statistical analysis software such as "R".

[0070] The control subject may also be the target subject itself. That is, for example, the risk in a subject may be determined using the fluctuation of the separation data in the subject as an index. "The value of the separation data is high" may include a case where the value of the separation data is increased. "The value of the separation data has increased" may specifically mean that the value of the separation data has increased compared to the past value. "The value of the separation data is low" may include a case where the value of the separation data has decreased. "The value of the separation data has decreased" may specifically mean that the value of the separation data has decreased compared to the past value. That is, the threshold may also include a past value. "Past value" means the value of the separation data of an antibody sample obtained from a target subject at a certain time in the past. The target subject at a certain time in the past may be, for example, a positive control or a negative control.

[0071] When the variation in the separation data in a subject is used as an index, the increase or decrease in the risk in the subject may be determined. "At risk or high risk" may include cases where the risk has increased. "Increased risk" may specifically mean that the risk has increased compared to a certain point in the past. "No risk or low risk" may include cases where the risk has decreased. "Reduced risk" may specifically mean that the risk has decreased compared to a certain point in the past.

[0072] In addition, "obtaining certain separation data and using it as an index for risk detection" is not limited to the case where the value of the separation data itself is obtained and used as an index for risk detection, but also includes obtaining other values ​​reflecting the value of the separation data and using them as an index for detection. For example, when the elution peaks are defined as the first to fourth peaks in the order of the earliest peaks from the start of elution, "obtaining the peak area% of the first peak and using it as an index for risk detection" is not limited to the case where the value of the peak area% of the first peak itself is obtained and used as an index for risk detection, but also includes obtaining other values ​​reflecting the value of the peak area% of the first peak, such as the total value of the peak area% of the second to fourth peaks, and using them as an index for detection. In either case, the values ​​of the measured values, threshold values, etc. used for risk detection are appropriately corrected according to the type of separation data. For example, when the elution peaks are composed of the first to fourth peaks, the relationship of "X=100%-Y" is established when the value of the peak area% of the first peak is X and the total value of the peak area% of the second to fourth peaks is Y. Therefore, when the total peak area percentage of the second to fourth peaks (i.e., "Y") is used as an index of detection instead of the peak area percentage of the first peak itself (i.e., "X"), "X satisfies a certain criterion (e.g., is low or high, or falls within a certain range)" should be read as "the corrected value of Y (i.e., "100%-Y") satisfies the criterion."

[0073] The detection result of the risk may be used as an index for determining whether to perform a treatment for reducing the risk (hereinafter, also referred to as "risk reduction treatment") on the subject. In other words, by carrying out the detection method of the present invention, an index for determining whether to perform a risk reduction treatment on the subject is obtained. That is, for example, when the detection method of the present invention determines that the subject has a risk or is at high risk, it may be determined that the subject is to be subjected to a risk reduction treatment. The detection method of the present invention may be used, for example, alone or in combination with other means, as an index for determining whether to perform a risk reduction treatment on the subject. For example, for a symptom determined to have a risk or is at high risk in the subject by the detection method of the present invention, a definitive diagnosis may be performed by other means, and then it may be determined that the subject is to be subjected to a risk reduction treatment. The risk reduction treatment may be a medical procedure or a non-medical procedure. Examples of the risk reduction treatment include the prevention and treatment of diseases and aging as exemplified above. That is, the present invention may provide, for example, a method for preventing or treating symptoms such as diseases and aging. The prevention or treatment method may be, for example, a method for preventing or treating symptoms such as disease or aging, including a step of carrying out the detection method of the present invention, and a step of carrying out prevention or treatment on the subject when the detection method of the present invention determines that the subject is at risk or at high risk. Specifically, prevention or treatment may be carried out for symptoms determined to be at risk or at high risk in the subject by the detection method of the present invention. Prevention or treatment can be carried out, for example, by general means for each symptom (e.g., medication or surgery).

[0074] According to the present invention, IgG4 to be detected can be accurately separated from proteins including antibodies other than IgG4. Also, a separation pattern of IgG4 based on glycans bound to IgG4 can be obtained. For example, separation data obtained by separating the antibody of a subject based on glycan structures characteristic of the risk of a specific disease or symptoms of aging, etc., can be accurately obtained by the separation pattern of IgG4 to be detected, and such risks can be easily detected by comparing with the reference separation data. In addition, by obtaining separation data before and / or after some treatment for a disease (medication, surgery, etc.) and comparing it with the reference separation data, the accuracy of monitoring the progress of treatment of the disease and determining the policy for the treatment can be improved. Examples of the reference separation data include separation data of a control subject. Specific examples of the reference separation data include separation data of the same subject at a different time point (for example, when healthy or when the same disease is developed), separation data of a healthy subject, separation data of different patients who have the same disease, and separation data that serves as a reference when dividing into two or more groups that have different efficacies to the treatment.

[0075] For example, by using the separation data of a healthy person (model) as model separation data and comparing the separation data of a subject with the model separation data, it is possible to evaluate the risk of having a certain disease or the risk of developing a disease. Furthermore, by separating the peak fraction that is the cause of the difference between the separation data of the model and the subject, the difference in the glycan pattern of the antibody between the healthy person (model) and the subject can be extracted. In addition, by using the separation data of the patient at a certain time point as model separation data and comparing the separation data of the same patient at a different time point with the model separation data, it is also possible to monitor the disease of the patient. When comparing two different sample groups, it is possible to evaluate whether the difference in the separation data obtained from the two different sample groups is significant using a statistical probability (P value). It is said that the evaluation result is significant when the P value is small, and when the P value is less than the significance level, it can be said that the evaluation result has a statistically significant difference. The significance level is generally 5%.

[0076] The strength of the affinity of an antibody for an Fc-binding protein affects the activities of immune cells such as natural killer cells, monocytes, and macrophages that have damaging or phagocytic effects on the binding substance to which the antibody binds. Therefore, by detecting the difference in such affinity, it becomes possible to evaluate the risk of developing diseases affected by the damage or phagocytic effects of natural killer cells, monocytes, and macrophages. Such diseases include cancer, autoimmune diseases, infectious diseases, allergies, inflammatory diseases, and IgG4-related diseases. Infectious diseases include opportunistic infections. Opportunistic infections are infections caused by pathogens that do not cause infections in a healthy state. Pathogens include viruses, bacteria, fungi, and protozoa. In addition, the effect on the activation of immune cells of antibodies acquired by vaccination or disease can also be evaluated from the strength of the affinity of the antibody for the Fc-binding protein. In addition to measuring the amount of the acquired antibody in the blood, by measuring the strength of the affinity of the antibody for the Fc-binding protein, it is also possible to accurately predict the risk of developing an infectious disease.

Example

[0077] Hereinafter, the present invention will be described more specifically with reference to non-limiting examples.

[0078] <Preparation of Fc-binding protein-immobilized gel> Example 1 Preparation of FcR9 amino acid substitution variant (FcR9_F) For FcR9 (SEQ ID NO: 2) prepared by the method described in WO2015 / 199154, an Fc-binding protein in which the 176th valine of the Fc-binding protein (the 176th valine in the amino acid number described in SEQ ID NO: 1) was substituted with phenylalanine was prepared. Specifically, for the plasmid pET-FcR9 (WO2015 / 199154) containing the polynucleotide encoding FcR9 (SEQ ID NO: 3), amino acid substitution was performed using PCR to prepare an Fc-binding protein in which the 176th valine of the Fc-binding protein in FcR9 was substituted with phenylalanine.

[0079] FcR9 (SEQ ID NO: 2) is an Fc-binding protein comprising the wild-type FcγRIII extracellular region shown in SEQ ID NO: 4, in which the 43rd valine is replaced with glutamic acid (corresponding to the 27th position in SEQ ID NO: 1), the 45th phenylalanine is replaced with isoleucine (corresponding to the 29th position in SEQ ID NO: 1), the 51st tyrosine is replaced with asparagine (corresponding to the 35th position in SEQ ID NO: 1), the 64th glutamine is replaced with arginine (corresponding to the 48th position in SEQ ID NO: 1), the 91st phenylalanine is replaced with leucine (corresponding to the 75th position in SEQ ID NO: 1), the 108th asparagine is replaced with serine (corresponding to the 92nd position in SEQ ID NO: 1), the 133rd valine is replaced with glutamic acid (corresponding to the 117th position in SEQ ID NO: 1), the 137th glutamic acid is replaced with glycine (corresponding to the 121st position in SEQ ID NO: 1), and the 187th phenylalanine is replaced with serine (corresponding to the 171st position in SEQ ID NO: 1).

[0080] The method for producing each Fc-binding protein is described in detail below. (1) Using plasmid pET-FcR9 (WO2015 / 199154) containing a polynucleotide (SEQ ID NO: 3) encoding FcR9 (SEQ ID NO: 2) as template DNA, an oligonucleotide consisting of the sequence described in SEQ ID NO: 5 (5'-TAATACGACTCACTATAGGG-3') as a forward primer, and an oligonucleotide consisting of the sequence described in SEQ ID NO: 6 (5'-CATTTTTGCTGCCGAACAGCCCACGGCAGG-3') as a reverse primer, a reaction solution having the composition shown in Table 1 was prepared, and the reaction solution was heat-treated at 95 ° C for 2 minutes, and 30 cycles of reaction were performed with 1 step at 95 ° C for 30 seconds, 2 step at 50 ° C for 30 seconds, and 3 step at 72 ° C for 90 seconds as one cycle, and finally heat-treated at 72 ° C for 7 minutes to perform PCR. The obtained PCR product was named V176p1.

[0081] [Table 1]

[0082] (2) Using plasmid pET-FcR9 (described in WO2015 / 199154) containing a polynucleotide (SEQ ID NO: 3) encoding FcR9 (SEQ ID NO: 2) as template DNA, an oligonucleotide consisting of the sequence described in SEQ ID NO: 7 (5'-TATGCTAGTTATTGCTCAG-3') as a reverse primer, and an oligonucleotide consisting of the sequence described in SEQ ID NO: 8 (5'-CCTGCCGTGGGCTGTTCGGCAGCAAAAATG-3') as a forward primer, a reaction solution having the composition shown in Table 1 was prepared, and the reaction solution was heat-treated at 95 ° C for 2 minutes, and 30 cycles of reaction were performed with 1 step at 95 ° C for 30 seconds, 2 step at 50 ° C for 30 seconds, and 3 step at 72 ° C for 90 seconds as one cycle, and finally heat-treated at 72 ° C for 7 minutes to perform PCR. The obtained PCR product was named V176p2. (3) A PCR product was prepared by mixing V176p1 obtained in (1) and V176p2 obtained in (2) in a 1:1 ratio, and a reaction solution having the composition shown in Table 2 was prepared. The reaction solution was heat-treated at 98°C for 5 minutes, and then PCR was carried out for 5 cycles, each cycle consisting of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute, to obtain the PCR product V176p in which V176p1 and V176p2 were linked.

[0083] [Table 2]

[0084] (4) A reaction solution having the composition shown in Table 3 was prepared using V176p obtained in (3) as template DNA, an oligonucleotide having the sequence shown in SEQ ID NO: 5 as a forward primer, and an oligonucleotide having the sequence shown in SEQ ID NO: 7 as a reverse primer. The reaction solution was heat-treated at 98°C for 5 minutes, and then PCR was carried out in which 30 cycles of reaction were performed, with the first step being at 98°C for 10 seconds, the second step being at 55°C for 5 seconds, and the third step being at 72°C for 1 minute. This resulted in a polynucleotide encoding an Fc-binding protein in which the 176th valine of the Fc-binding protein (FcR9) was replaced with phenylalanine. The resulting polynucleotide was designated V176p3.

[0085] [Table 3]

[0086] (5) After purification of the polynucleotide obtained in (4), it was digested with the restriction enzymes NcoI and HindIII and ligated into the expression vector pETMalE (JP Patent Publication No. 2011-206046) which had been previously digested with the restriction enzymes NcoI and HindIII, and used to transform the Escherichia coli BL21(DE3) strain (Nippon Gene). (6) The obtained transformant was cultured in LB medium supplemented with 50 μg / mL kanamycin. Plasmids were extracted from the collected bacterial cells (transformants). (7) The polynucleotide encoding human FcγRIIIa and the surrounding region of the obtained plasmid were subjected to cycle sequencing reaction using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) based on the chain terminator method, and the nucleotide sequence was analyzed using a fully automated DNA sequencer Applied Biosystems 3130 Genetic Analyzer (Life Technologies). In this analysis, an oligonucleotide consisting of the sequence described in SEQ ID NO: 5 (5'-TAATACGACTCACTATAGGG-3') or SEQ ID NO: 7 (5'-TATGCTAGTTATTGCTCAG-3') was used as a sequencing primer. As a result of the sequence analysis, a transformant was obtained that expresses the Fc binding protein FcR9_F (SEQ ID NO: 9) in which the 176th valine of the Fc binding protein was replaced with phenylalanine. The phenylalanine is located at the 192nd position in SEQ ID NO: 9.

[0087] Example 2: Preparation of a cysteine-tagged Fc-binding protein (FcR9_F_Cys) (1) The expression vector pET-FcR9_F containing the polynucleotide set forth in SEQ ID NO: 10 encoding the amino acid sequence set forth in FcR9_F (SEQ ID NO: 9) prepared in Example 1 was used as template DNA, an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 11 (5'-TAGCCATGGGCATGCGTACCGAAGATCTGCCGAAAGC-3') was used as a forward primer, and an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 12 (5'-CCCAAGCTTATCCGCAGGTATCGTTGCGGCACCCTTGGGTAATGGTAATATTCACGGTCTCGCTGC-3') was used as a reverse primer to prepare a reaction solution having the composition shown in Table 3. The reaction solution was heat-treated at 98°C for 5 minutes, and PCR was performed by repeating 30 cycles of a first step at 98°C for 10 seconds, a second step at 55°C for 5 seconds, and a third step at 72°C for 1 minute, which constitute one cycle. (2) The polynucleotide obtained in (1) was purified, digested with the restriction enzymes NcoI and HindIII, and then ligated to the expression vector pTrc-PelBV3 prepared by the method described in WO2015 / 199154, which had been previously digested with the restriction enzymes NcoI and HindIII. The ligation product was used to transform the E. coli W3110 strain. (3) The obtained transformant was cultured in LB medium containing 100 μg / mL carbenicillin, and then the expression vector pTrc-FcR9_F_Cys was obtained using a QIAprep Spin Miniprep kit (Qiagen). (4) Analysis of the nucleotide sequence of pTrc-FcR9_F_Cys was performed in the same manner as in Example 1(7), except that an oligonucleotide consisting of the sequence set forth in SEQ ID NO: 13 (5'-TGTGGTATGGCTGTGCAGG-3') or SEQ ID NO: 14 (5'-TCGGCATGGGGTCAGGTG-3') was used as a sequencing primer.

[0088] The amino acid sequence of the polypeptide FcR9_F_Cys expressed by the expression vector pTrc-FcR9_F_Cys is shown in SEQ ID NO: 15, and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 16. In SEQ ID NO: 15, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) is the improved PelB signal peptide, the portion from the 24th glycine (Gly) to the 199th glutamine (Gln) is the amino acid sequence of the Fc binding protein (corresponding to the region from the 17th to the 192nd in SEQ ID NO: 1), and the portion from the 200th glycine (Gly) to the 207th glycine (Gly) is the cysteine ​​tag sequence.

[0089] Example 3 Preparation of FcR9_F_Cys (1) The transformant expressing FcR9_F_Cys prepared in Example 2 was inoculated into 400 mL of 2YT liquid medium (peptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L) containing 100 μg / mL carbenicillin in a 2 L baffled flask, and pre-cultured overnight at 37° C. under aerobic shaking. (2) 1.8 L of liquid medium containing 10 g / L glucose, 20 g / L yeast extract, 3 g / L trisodium phosphate dodecahydrate, 9 g / L disodium hydrogen phosphate dodecahydrate, 1 g / L ammonium chloride, and 100 mg / L carbenicillin was inoculated with 180 mL of the culture solution from (1), and main culture was performed using a 3 L fermentation tank (Bio-T). The conditions were set to 30°C temperature, pH 6.9 to 7.1, aeration rate 1 VVM, and dissolved oxygen concentration 30% saturation concentration, and main culture was started. 50% phosphoric acid was used as acid and 14% ammonia water as alkali to control pH, and dissolved oxygen was controlled by changing the stirring speed, with the stirring speed set to a lower limit of 500 rpm and an upper limit of 1000 rpm. After the start of culture, when the glucose concentration could no longer be measured, a feed medium (glucose 248.9 g / L, yeast extract 83.3 g / L, magnesium sulfate heptahydrate 7.2 g / L) was added while controlling the dissolved oxygen (DO). (3) When the absorbance at 600 nm (OD600nm) reached approximately 150 as an indication of bacterial mass, the culture temperature was lowered to 25°C. After confirming that the set temperature had been reached, IPTG (IsoPropyl β-D-1-ThioGalactopyranoside) was added to a final concentration of 0.5 mM, and culture was continued at 25°C. (4) The culture was stopped about 48 hours after the initiation of the culture, and the culture medium was centrifuged at 8,000 rpm at 4° C. for 20 minutes to recover the bacterial cells. (5) The collected cells were suspended in 20 mM Tris-HCl buffer (pH 7.0) at 5 mL / 1 g (cells), and disrupted using an ultrasonic generator (Insonator 201M (product name), manufactured by Kubota Shoji) at 4° C. for about 10 minutes at an output of about 150 W. The disrupted cell solution was centrifuged twice at 8000 rpm for 20 minutes at 4° C., and the supernatant was collected. The supernatant obtained in (6)(5) was applied at a flow rate of 5 mL / min to a VL32×250 column (manufactured by Merck Millipore) filled with 140 mL of TOYOPEARL CM-650M (manufactured by Tosoh Corporation) pre-equilibrated with 20 mM phosphate buffer (8 mM sodium dihydrogen phosphate, 12 mM disodium hydrogen phosphate) (pH 7.0). After washing with the buffer used for equilibration, it was eluted with 20 mM phosphate buffer (pH 7.0) containing 0.5 M sodium chloride. (7) The eluate obtained in (6) was applied to an XK26 / 20 column (manufactured by GE Healthcare) filled with 90 mL of IgG Sepharose (manufactured by GE Healthcare) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride. After washing with the buffer used for equilibration, it was eluted with 0.1 M glycine-HCl buffer (pH 3.0). The pH of the eluate was adjusted back to near neutral by adding 1 / 4 volume of 1 M Tris-HCl buffer (pH 8.0) of the eluate volume. By the above purification, approximately 20 mg of high-purity FcR9_F_Cys was obtained.

[0090] Example 4 Preparation of Fc-binding protein (FcR9_F)-immobilized gel (1) After activating the hydroxyl groups on the surface of 2 mL of a hydrophilic vinyl polymer for separators (manufactured by Tosoh Corporation: packing material for liquid chromatography) with iodoacetyl groups, 4 mg of FcR9_F_Cys prepared in Example 3 was reacted to obtain an FcR9_F-immobilized gel. (2) 1.2 mL of the FcR9_F-immobilized gel prepared in (1) was packed into a stainless steel column of φ4.6 mm×75 mm to prepare an FcR9_F column.

[0091] <Antibody separation using Fc-binding protein-immobilized gel> Example 5 Separation of antibodies from serum containing antibodies and antibodies derived from human myeloma plasma using Fc-binding protein (FcR9_F)-immobilized gel (1) An equilibration buffer solution of 10 mM citrate buffer (pH 6.5) was prepared, and the electrical conductivity was measured using an electrical conductivity meter (Horiba, Ltd.). The pH of the equilibration buffer solution was changed by hydrochloric acid in the range of 0.1 to 0.2 using a pH meter (Horiba, Ltd.). The number of millimoles of hydrochloric acid added per 1 L of the equilibration buffer solution was divided by the actual pH value to be measured, and this value was used to measure the buffer capacity. (2) Blood was collected from subjects who had given informed consent and centrifuged to obtain serum, which was then diluted 10-fold with phosphate buffered saline (PBS) (pH 7.4) and passed through a 0.2 μm filter (Merck Millipore) to prepare a measurement sample. (3) The FcR9_F column prepared in Example 4 was connected to a high performance liquid chromatography apparatus (manufactured by Tosoh Corporation) and equilibrated with the equilibration buffer prepared in (1). 10 μL of the measurement sample (a) prepared in (2) and 0.5 mg / mL to 1 mg / mL of IgG4 human myeloma plasma-derived antibody (manufactured by Sigma Corporation) as measurement sample (b) were added at a flow rate of 1.0 mL / min. (4) After washing with the equilibration buffer for 5 minutes at a flow rate of 1.0 mL / min, the adsorbed gamma globulin was eluted with a pH gradient of elution buffer, 10 mM citrate buffer (pH 4.0) containing 500 mM sodium chloride (a gradient in which 10 mM citrate buffer (pH 4.0) containing 500 mM sodium chloride became 100% in 25 minutes), and a separation pattern was obtained.

[0092] Comparative Example 1 Separation of human myeloma plasma-derived antibodies using Fc binding protein (FcR9_F) immobilized gel A gamma globulin separation pattern was obtained in the same manner as in Example 1, except that 10 μL of human myeloma plasma-derived antibodies (Sigma) of (a) to (c) at 0.5 mg / mL to 1 mg / mL were added as measurement samples at a flow rate of 1.0 mL / min. (a) IgG1 (b) IgG2 (c) IgG3.

[0093] The results of Example 5 and Comparative Example 1 are shown in FIG. 1. The equilibration buffer used in Example 5 and Comparative Example 1 (electrical conductivity 2.4 mS / cm, buffer capacity value 3.2) is within the range of the equilibration buffer conditions of the present invention. For the serum, which is the measurement sample (a) of Example 5, elution was confirmed in the elution time range of 5 to 20 minutes. In addition, elution was confirmed in the elution time range of 5 to 20 minutes for IgG1, IgG3, and IgG4 (Comparative Examples 1(a) and (c), Example 5(b)), which shows that the elution peak of serum (Example 5(a)) is derived from IgG1, IgG3, and IgG4. On the other hand, for IgG2 (Comparative Example 1(b)), most of the elution (unadsorbed fraction) was confirmed in the elution time range of 0 to 5 minutes, where the column was washed with the equilibration buffer. From the results of IgG from human myeloma plasma, it was found that, by using the equilibration buffer, IgG4 was eluted in the intermediate region between the elution peaks of IgG1, IgG3, and IgG2, and furthermore, since IgG4 was eluted after the introduction of the elution buffer, it was found that IgG4 was adsorbed to the Fc-binding protein at the time of equilibration. Furthermore, it was found that the separation peak of IgG4 was separated into multiple peaks, similar to IgG1. Since it is known that the difference in binding ability to Fc-binding protein is mainly due to the difference in the glycan bound to IgG when the IgG is of the same class, it was also found that IgG4 was separated into multiple peaks due to the difference in the glycan bound to IgG4.

[0094] Comparative Example 2 Separation of antibody-containing serum and human myeloma plasma-derived antibodies using Fc binding protein (FcR9_F) immobilized gel A gamma globulin separation pattern was obtained in the same manner as in Example 5, except that 10 mM citrate buffer (pH 6.5) containing 100 mM sodium chloride was used as the equilibration buffer and (a) to (e) were used as the measurement samples. (a) Measurement sample prepared in the same manner as in Example 5(2) (b) IgG1 (c) IgG2 (d) IgG3 (e) IgG4.

[0095] The results of Comparative Example 2 are shown in FIG. 2. The equilibration buffer used in Comparative Example 2 (electrical conductivity 14.4 mS / cm, buffer capacity value 3.2) is outside the range of the equilibration buffer conditions of the present invention. For the serum, which is the measurement sample (a) of Comparative Example 2, elution was confirmed in the elution time region of 10 to 20 minutes. In addition, elution was confirmed in the elution time region of 10 to 20 minutes for IgG1 and IgG3 (Comparative Examples 2 (b) and (d)), so it can be seen that the elution peak of serum (Comparative Example 2 (a)) is derived from IgG1 and IgG3. On the other hand, for IgG2 and IgG4 (Comparative Examples 2 (c) and (e)), no peak was confirmed in the elution time region of 10 to 20 minutes, and most of the elution was confirmed in the elution time region of 0 to 5 minutes when the column was equilibrated with the equilibration buffer. Elution during equilibration with the equilibration buffer means that the binding force to the Fc-binding protein is weak. Therefore, it can be seen that IgG4 is eluted in the fraction not adsorbed to the column, and therefore cannot be separated with high accuracy. When the separation patterns of human myeloma plasma-derived IgG4 are compared between Example 5(b), which is within the range of equilibration buffer conditions of the present invention, and Comparative Example 2(d), which is outside the range, it can be seen that the peak derived from IgG4 in Example 5(b), which is within the range of conditions, is far away from the elution region of the unadsorbed fraction. It can be seen that the binding strength of IgG4 to Fc-binding proteins is improved by using an equilibration buffer within the range of conditions of the present invention, and that IgG4 can be separated with high accuracy from the unadsorbed fraction.

[0096] Example 6 Evaluation of separation behavior of serum containing antibodies depending on the composition of equilibration buffer (1) A gamma globulin separation pattern was obtained in the same manner as in Example 5, except that citrate buffer solutions (pH 6.5) having compositions (a) to (g) were used as the equilibration buffer solution, serum was used as the measurement sample, and the sample was further washed with the equilibration buffer solution for 10 minutes. (a) 1 mM citrate buffer (b) 1 mM citrate buffer containing 100 mM sodium chloride (c) 10 mM citrate buffer (d) 20 mM citrate buffer (e) 20 mM citrate buffer containing 1 mM sodium chloride (f) 20 mM citrate buffer containing 10 mM sodium chloride (g) 25 mM citrate buffer (2) Based on the obtained separation patterns, the samples were classified into ● and × based on the following criteria. ●: Conditions under which elution of IgG4-containing fractions is observed after the start of introduction of elution buffer ×: Conditions under which no elution of a fraction containing IgG4 was observed after the start of introduction of the elution buffer.

[0097] Comparative Example 3 Evaluation of separation behavior of serum containing antibodies due to differences in the composition of equilibration buffer The separation patterns of gamma globulins were classified in the same manner as in Example 6, except that citrate buffer solutions (pH 6.5) having compositions (a) to (n) were used as the equilibration buffer solutions. (a) 0.1 mM citrate buffer containing 150 mM sodium chloride (b) 1 mM citrate buffer containing 120 mM sodium chloride (c) 1 mM citrate buffer containing 150 mM sodium chloride (d) 1 mM citrate buffer containing 200 mM sodium chloride (e) 1 mM citrate buffer containing 300 mM sodium chloride (f) 10 mM citrate buffer containing 100 mM sodium chloride (g) 20 mM citrate buffer containing 50 mM sodium chloride (h) 20 mM citrate buffer containing 100 mM sodium chloride (i) 25 mM citrate buffer containing 100 mM sodium chloride (j) 30 mM citrate buffer (k) 30 mM citrate buffer containing 100 mM sodium chloride (l) 50 mM citrate buffer (m) 50 mM citrate buffer containing 100 mM sodium chloride (n) 80 mM citrate buffer.

[0098] The results of Example 6 and Comparative Example 3 are shown in Table 4 and Figure 3. From Table 4, which shows the electrical conductivity, buffer capacity, and classification of the equilibration buffers, it is clear that the equilibration buffer used in Example 6 is within the range of the equilibration buffer conditions of the present invention, while the equilibration buffer used in Comparative Example 3 is outside the range of the equilibration buffer conditions of the present invention.

[0099] [Table 4]

[0100] Figure 3 plots the classification based on electrical conductivity and buffer capacity. Figure 3 shows that there is a correlation between the classification and electrical conductivity and buffer capacity, and that the higher the electrical conductivity and buffer capacity, the more elution of only IgG1 or IgG3 is observed, and the lower the electrical conductivity and buffer capacity, the more elution of IgG4 is observed after introduction of the elution buffer. Based on these results, the boundary region between electrical conductivity and buffer capacity for accurate separation of IgG4 can be determined. Specifically, the line of the electrical conductivity and buffer capacity value of 1 mM citrate buffer containing 100 mM sodium chloride and 25 mM citrate buffer, which is the boundary between ● and ×, gives the formula (a) (electrical conductivity [mS / cm] ≦ -0.81 × buffer capacity value + 12.5) which shows the region of ●. Although not shown, because a compound with buffer capacity is added as the equilibration buffer, the formula (b) (buffer capacity value > 0) and formula (c) (electrical conductivity [mS / cm] > 0) were set.

[0101] By combining these equations, the region in which the separation pattern ● can be obtained is the solution that satisfies all of the conditions in equations (a) to (c). Example 7 Evaluation of albumin separation behavior depending on the composition of equilibration buffer A citrate buffer solution (pH 6.5) having the composition (a) to (c) was used as the equilibration buffer solution, and 10 μL of human albumin solution (catalog number: ab205808, manufactured by Abcam) diluted to 1 mg / mL with the equilibration buffer solution was added as the measurement sample at a flow rate of 1.0 mL / min, and a separation pattern of albumin was obtained in the same manner as in Example 5. (a) 1 mM citrate buffer (b) 3 mM citrate buffer (c) 5 mM citrate buffer.

[0102] The results of Example 7 are shown in Figure 4. From the results of Example 6, the equilibration buffers (a) to (c) of Example 7 are all within the range of the equilibration buffer conditions of the present invention as long as the citrate buffer is within the range of 1 mM to 20 mM. The separation pattern of human albumin is shown in Figure 4. When the elution of albumin after the peak formation due to the introduction of the eluent was evaluated, when 3 mM and 5 mM citrate buffers (Figures 4(b) and (c)) were used as the equilibration buffer, the elution of albumin after the start of the introduction of the eluent was greatly reduced compared to when 1 mM citrate buffer (Figure 4(a)) was used. When the citrate concentration was less than 3 mM, the effect of electrostatic interaction became strong, and it is presumed that some of the proteins other than the IgG to be measured bound to the Fc-binding protein immobilized gel. From this result, it can be seen that when evaluating measurement samples such as blood-derived samples containing albumin, the use of >1 mM citrate buffer (electrical conductivity [mS / cm] >0.24) can suppress the elution of albumin after the introduction of the eluent, and IgG4 can be measured with high accuracy.

Claims

1. 1. An equilibration solution for use in the analysis of a blood-derived sample containing at least IgG4 using a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, comprising: The equilibration solution comprises citric acid and sodium chloride; A composition that satisfies at least all of the following conditions (a) to (c): (a) Formula: Electrical conductivity [mS / cm]≦−0.81×buffer capacity value+12.5 (b) Formula: Buffer capacity value>0 (c) Formula: Electrical conductivity [mS / cm]>0 The buffer capacity value is calculated by dividing the number of millimoles (mmoles) of hydrochloric acid dropped per 1 L of the equilibration solution by the amount of change in pH due to the drop of hydrochloric acid when the pH of the equilibration solution is changed by 0.1 to 0.2 by adding hydrochloric acid to the equilibration solution, an equilibration solution, wherein the Fc binding protein is any one of the following polypeptides (1) or (2); (1) A polypeptide comprising the amino acid residues from the 17th to the 192nd of the amino acid sequence set forth in SEQ ID NO:1, with the proviso that in the amino acid residues from the 17th to the 192nd, the 27th valine is replaced with glutamic acid, the 29th phenylalanine with isoleucine, the 35th tyrosine with asparagine, the 48th glutamine with arginine, the 75th phenylalanine with leucine, the 92nd asparagine with serine, the 117th valine with glutamic acid, the 121st glutamic acid with glycine, the 171st phenylalanine with serine, and the 176th valine with phenylalanine; (2) A polypeptide comprising the amino acid sequence of the polypeptide of (1) above, except that the amino acid sequence contains 1 to 10 amino acid mutations at positions other than the above substitutions.

2. The equilibration solution according to claim 1, wherein the equilibration solution has a composition that further satisfies the condition of the following formula (d): (d) Formula: Electrical conductivity [mS / cm]>0.24

3. 1. A method for separating IgG4 from an antibody sample containing at least IgG4, comprising the steps of: (1) adding an antibody sample containing IgG4 to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon to adsorb the antibody to the carrier; (2) adding the equilibration solution according to claim 1 or 2 prior to the step (1) to equilibrate the column; and (3) adding an eluate to the equilibrated column to elute the adsorbed antibody; A method comprising:

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