JP7577976B2 - Solutions for antibody isolation - Google Patents

Description

The present invention relates to a solution for antibody separation. More specifically, the present invention relates to an equilibration solution used to separate the antibody to be detected contained in a body fluid.

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 glycan structure bound to the antibody has a significant impact on the activity, dynamics, and safety of antibody drugs, and detailed analysis of the glycan structure is important (Non-Patent Document 1). In addition, changes in the glycan structure attached to antibodies in the blood are known in diseases such as rheumatism (Non-Patent Documents 2 and 3), and it may be possible to diagnose the disease by analyzing the glycan structure attached to the antibody.

LC-MS analysis including glycan excision is mainly used as a method for analyzing the glycan structure of antibodies used in antibody drugs (Patent Document 1 and Patent Document 2). However, this analysis method requires very complicated operations and takes a lot of time. A simpler method for analyzing the molecular structure of antibodies is analysis by chromatography. Specifically, it is possible to separate and quantify aggregates and decomposition products by separating antibodies based on their molecular weight using gel filtration chromatography. In addition, it is possible to separate differences in the electric charges possessed by antibody molecules by ion exchange chromatography. However, the above-mentioned chromatographic analysis cannot distinguish minute structural changes in antibody molecules such as glycan structure, so the analytical results obtained are limited.

On the other hand, it has been reported that it is possible to measure and evaluate the performance of an antibody by 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.

JP 2016-194500 A JP 2016-099304 A WO2013/120929 publication

CHROMATOGRAPHHY, 34(2), 83-88 (2013) Science, 320, 373 (2008) Nature Communication, 7, 11205 (2016)

The objective of the present invention is to provide a solution for antibody separation. Specifically, the objective of the present invention is to provide an equilibration solution used to separate the antibody to be detected contained in a body fluid.

As a result of intensive research conducted by the inventors to solve the above problems, they discovered that antibodies can be separated based on differences in glycan structure by using an Fc-binding protein, and discovered a solution composition that can accurately separate the target antibodies as an equilibration solution used when adsorbing the antibodies to the Fc-binding protein, thereby completing the present invention.

That is, the present invention can be exemplified as follows:

[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 following conditions (a) to (d) and (g):
(a) Formula: Electrical conductivity [mS/cm] < -0.78 x buffer capacity value + 36.5
(b) Formula: Electrical conductivity [mS/cm]>-0.81 x buffer capacity value + 12.5
(c) Formula: Buffer capacity value>0.16
(d) Formula: Electrical conductivity [mS/cm]>0
(g) Formula: 9>pH>5
[2]
The equilibration solution according to [1], wherein the equilibration solution has a composition that further satisfies the condition of formula (e).
(e) Formula: Electrical conductivity [mS/cm] < -0.84 x buffer capacity value + 30.5
[3]
The equilibration solution according to [1] or [2], wherein the equilibration solution has a composition that further satisfies the condition of formula (f).
(f) Formula: 0.32<buffer capacity value<16
[4]
The equilibration solution according to any one of [1] to [3], wherein the Fc-binding protein is any one of the following polypeptides (1) to (4):
(1) A polypeptide comprising the amino acid residues from the 17th to the 192nd positions 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 positions, at least the 176th valine is replaced with phenylalanine;
(2) 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, at least 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;
(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 amino acid residues from the 17th to the 192nd, at least 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, and the 171st phenylalanine 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 1 to 10 amino acid mutations at positions other than the above substitutions.

[5]
A method for equilibrating a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, comprising the steps of:
The equilibration solution for the column is the equilibration solution according to any one of [1] to [4],
A method wherein the analyte is an antibody-containing body fluid obtained from a subject.

[6]
1. A method for isolating IgG1 and/or IgG3 from an antibody-containing body fluid obtained from a subject, comprising the steps of:
(1) adding the body fluid to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon to adsorb the antibody onto the carrier;
(2) adding an equilibration solution according to any one of (1) to (4) before or after (1) to equilibrate the column; and (3) adding an elution solution to the equilibrated column to elute the adsorbed antibody.

The present invention makes it possible to accurately separate the antibodies to be detected, including IgG1 and/or IgG3, from antibodies other than IgG1 and/or IgG3.

FIG. 2 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. 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. FIG. 1 shows the separation pattern when antibody-containing serum was analyzed using a column packed with an Fc-binding protein-immobilized gel, using an equilibration buffer within or outside the conditions of the present invention. FIG. 1 is a diagram showing classification of separation patterns when antibody-containing serum is analyzed using a column packed with an Fc-binding protein-immobilized gel. FIG. 13 shows classification of separation patterns when antibody-containing serum is analyzed using a column packed with an Fc-binding protein-immobilized gel, and further shows CV values when the elution time of the first peak at each measurement point is measured 10 times. FIG. 1 shows the separation pattern when antibody-containing serum was analyzed using a column packed with an Fc-binding protein-immobilized gel and an equilibration buffer (pH 7.0) within the range of conditions of the present invention. FIG. 1 shows a plot of the first peak area percentage when gamma globulin derived from a cancer patient was analyzed using a column packed with an Fc-binding protein-immobilized gel.

The present invention will be described in detail below.

The present invention provides an equilibration solution for separating an antibody to be detected using an Fc-binding protein. This solution is also called the "equilibration solution of the present invention" or the "equilibration buffer of the present invention."

Specifically, the equilibration solution of the present invention may be an equilibration solution for separating an antibody, 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.

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

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 isolating the antibody using an Fc-binding protein. That is, one embodiment of the elution step may be a step of eluting the antibody adsorbed to the carrier using 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 using an elution solution and a step of obtaining the eluted antibody.

According to the present invention, data relating to the separation pattern of an antibody may be obtained. That is, one aspect of the present invention may be used as an equilibration liquid for producing data relating to the separation pattern of an antibody by separating the antibody using an Fc-binding protein. That is, one aspect of the elution step may be a step of eluting the antibody adsorbed to the carrier using an elution liquid and obtaining data relating to the separation pattern of the antibody. In other words, one aspect of the elution step may include a step of eluting the antibody adsorbed to the carrier using an elution liquid and a step of obtaining data relating to the separation pattern of the antibody.

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 in a subject may be detected using the separation data as an index. That is, the separation data may be regarded 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 progression 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 progression 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 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 progression of aging in a subject using separation data obtained by separating an antibody obtained from a subject using an Fc-binding protein 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 progression of aging are collectively referred to as "risk". "Risk detection,""riskassessment," 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 a column packed with an insoluble carrier to which an Fc-binding protein has been immobilized, and the antibody is adsorbed onto the carrier.

"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. Antibodies also include artificially structurally modified antibodies, such as bispecific antibodies, fusion antibodies of the Fc region with other proteins, and Fc region-drug complexes (ADCs). The antibody may be, for example, an antibody drug. Examples of antibody drugs include infliximab, an anti-TNF-α antibody, tocilizumab, an anti-IL-6 antibody, and trastuzumab, an antibody against the oncogene HER2. Antibodies can be produced by production cells, such as CHO cells, Sp2/0 cells, NS0 cells, and hybridoma cells.

Examples of glycans that may contribute particularly 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.

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 of the total antibody content. Human-derived antibodies often have two sialic acids bound to the glycan terminal. Furthermore, human-derived antibodies usually contain bisecting GlcNAc (denoted as "+GN") at about 1 to 20% by weight of 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 glycan terminal is 0 to 1.

The antibody subjected to the adsorption process may be a mixture containing multiple types of antibody molecules. Specifically, the antibody subjected to the adsorption process may be a mixture containing multiple types of antibody molecules with different glycan structures. More specifically, the antibody subjected to the adsorption process may be a mixture containing multiple types of antibody molecules with different glycan structures attached to the Fc region.

If the purpose is to detect risk, the equilibration solution of the present invention can be used with antibodies obtained from the subject.

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.
"Body fluid" means a sample that contains or may contain an antibody obtained from a subject (hereinafter, also referred to as "antibody sample"). Examples of body fluid 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; tissue fragments (tissue pieces) and cells such as liver, lung, spleen, kidney, skin, tumor, and lymph node; and samples that may contain antibodies separated therefrom or antibodies contained therein. Body fluids may be used in the adsorption step as they are or after appropriate pretreatment. 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 body fluid 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 an antibody. For such a liquid medium, for example, 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. In the present invention, the body fluid includes the sample subjected to the pretreatment and the solution containing the antibody, and the body fluid in the present invention is a sample that may substantially contain antibodies other than IgG1 and/or IgG3 in addition to IgG1 and/or IgG3. "Substantially containing the antibody" means that the antibody other than IgG1 and/or IgG3 is contained in an amount of 0.1% or more relative to IgG1 and/or IgG3.

"Fc binding protein" means a protein having binding ability to the Fc region of an antibody. The Fc binding protein is not particularly limited as long as it can obtain a desired antibody separation pattern. The antibody can be separated, for example, based on the difference in affinity with the Fc binding protein based on the difference in the sugar chain structure of the antibody (for example, the sugar chain structure of the Fc region). The affinity between the antibody and the Fc binding protein (specifically, the sugar chain structure of the antibody) can be correlated with, for example, the function of the antibody, such as the efficacy of the antibody. In addition, the affinity between the antibody and the Fc binding protein (specifically, the sugar chain structure of the antibody) can be correlated with, for example, the risk in the subject. That is, the Fc binding protein is preferably, for example, a protein that has binding ability to the Fc region of an antibody and can recognize the difference in the sugar chain structure of the antibody (for example, the sugar chain 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. Specifically, the human Fc-binding protein includes a protein containing the full length or partial sequence of the 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. A partial sequence of the amino acid sequence of the extracellular region of human FcγRIIIa includes 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 the 17th to 192nd amino acid residues 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 in said amino acid residues.

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) Methionine at position 18 of SEQ ID NO:1 is replaced by arginine (2) Valine at position 27 of SEQ ID NO:1 is replaced by glutamic acid (3) Phenylalanine at position 29 of SEQ ID NO:1 is replaced by leucine or serine (4) Leucine at position 30 of SEQ ID NO:1 is replaced by glutamine (5) Tyrosine at position 35 of SEQ ID NO:1 is replaced by aspartic acid, glycine, lysine, leucine, asparagine, proline, serine, threonine, or histidine (6) Lysine at position 46 of SEQ ID NO:1 is replaced by isoleucine or threonine (7) Glutamine at position 48 of SEQ ID NO:1 is replaced by histidine or leucine (8) Alanine at position 50 of SEQ ID NO:1 is replaced by histidine (9) Tyrosine at position 51 of SEQ ID NO:1 is replaced by 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) Substitution of phenylalanine at position 75 of SEQ ID NO:1 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) SEQ ID NO:1 (21) The 90th glutamine of SEQ ID NO:1 is replaced with arginine (22) The 92nd asparagine of SEQ ID NO:1 is replaced with serine (23) The 93rd leucine of SEQ ID NO:1 is replaced with arginine or methionine (24) The 95th threonine of SEQ ID NO:1 is replaced with alanine or serine (25) The 110th leucine of SEQ ID NO:1 is replaced with glutamine (26) The 115th arginine of SEQ ID NO:1 is replaced with glutamine (27) The 116th tryptophan of SEQ ID NO:1 is replaced with leucine (28) The 118th phenylalanine of SEQ ID NO:1 is replaced with tyrosine (29) The 119th lysine of SEQ ID NO:1 is replaced with glutamic acid (30) The 120th glutamine of SEQ ID NO:1 (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) Substitution of serine at position 167 of SEQ ID NO:1 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) Substitution of asparagine at position 180 of SEQ ID NO:1 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 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 following amino acid substitutions (41) to (57) has occurred in the 17th to 192nd amino acid residues (JP 2016-169197 A).
(41) Phenylalanine at position 29 of SEQ ID NO:1 is replaced with isoleucine or leucine (42) Glutamic acid at position 39 of SEQ ID NO:1 is replaced with glycine (43) Glutamine at position 48 of SEQ ID NO:1 is replaced with arginine (44) Tyrosine at position 51 of SEQ ID NO:1 is replaced with serine (45) Phenylalanine at position 61 of SEQ ID NO:1 is replaced 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) Glutamine at position 90 of SEQ ID NO:1 is replaced with arginine (49) (50) The 117th valine in SEQ ID NO:1 is replaced with glutamic acid (51) The 119th lysine in SEQ ID NO:1 is replaced with asparagine or glutamic acid (52) The 140th threonine in SEQ ID NO:1 is replaced with isoleucine (53) The 142nd leucine in SEQ ID NO:1 is replaced with glutamine (54) The 171st phenylalanine in SEQ ID NO:1 is replaced with serine (55) The 175th leucine in SEQ ID NO:1 is replaced with arginine (56) The 180th asparagine in SEQ ID NO:1 is replaced with serine (57) The 188th isoleucine in SEQ ID NO:1 is replaced with valine In addition, yet another embodiment of the above (ii) is a polypeptide (JP 2016-169197 A) that contains amino acid residues from the 17th to the 192nd amino acid residues in 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 from the 17th to the 192nd amino acid residues.
(58) Leucine at position 66 in SEQ ID NO:1 is replaced with histidine or arginine. (59) Glycine at position 147 in SEQ ID NO:1 is replaced with aspartic acid. (60) Tyrosine at position 158 in SEQ ID NO:1 is replaced with histidine. (61) Valine at position 176 in SEQ ID NO:1 is replaced with phenylalanine. Another embodiment of (ii) above is a polypeptide comprising 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 amino acid substitutions selected from (1) to (61) has occurred in the amino acid residues at positions 17 to 192.

The above-mentioned (ii) particularly includes the following polypeptides (ii-1) to (ii-3).
(ii-1) a polypeptide comprising the amino acid residues from the 17th to the 192nd positions 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 positions, at least the 176th valine is substituted with phenylalanine;
(ii-2) 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, at least the 27th valine is substituted 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;
(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 amino acid residues from the 17th to the 192nd, 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.

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 (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, and particularly preferably 1 to 10. "One or several" amino acid mutations may occur so that the amino acid substitutions of the above-exemplified Fc-binding protein selected from the amino acid substitutions (1) to (61) above are preserved. In other words, "one or several" amino acid mutations may occur at positions other than the amino acid substitutions of the above-exemplified Fc-binding protein selected from the amino acid substitutions (1) to (61) above.

The Fc-binding protein may be a polypeptide containing an amino acid sequence having high homology to the amino acid sequence of the above-exemplified Fc-binding protein (e.g., the polypeptide (i) or (ii)) as long as it has the 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 or identity. "Homology" may particularly mean identity. The homology of amino acid sequences can be determined using an alignment program such as BLAST. For example, "identity of amino acid sequences" may mean identity between amino acid sequences calculated using blastp, and specifically, may mean identity between amino acid sequences calculated using blastp with default parameters. The change in the amino acid sequence within the above-mentioned homology range may occur so as to preserve the amino acid substitutions in the above-mentioned exemplified Fc-binding proteins selected from the amino acid substitutions (1) to (61) above. In other words, the change in the amino acid sequence within the above-mentioned homology range may occur at a position other than the amino acid substitutions in the above-mentioned exemplified Fc-binding proteins selected from the amino acid substitutions (1) to (61) above.

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 microorganisms include yeast and bacteria. Examples of 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 bacteria include bacteria of the genus Escherichia such as Escherichia coli. Examples of Escherichia coli include the W3110 strain, JM109 strain, and 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.

In the present invention, the term "insoluble carrier" refers to a carrier that is insoluble in the liquid passed through the column (e.g., liquids used for adsorbing or eluting antibodies, such as equilibration liquid or elution liquid). The insoluble carrier may have a functional group (e.g., hydroxyl group) for immobilizing the 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.

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).

The antibody can be adsorbed to the carrier by adding a solution containing the antibody to a column packed with an insoluble carrier on which an Fc-binding protein is immobilized. The antibody-containing solution can be added to the column using a liquid-transporting means such as a pump. Adding a liquid to a 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 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 transport rate may 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 to 50°C.
<Equilibration step>
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.

By equilibration, it is possible to remove from the column 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. The fraction containing the antibody that does not bind to the carrier by equilibration is called the unadsorbed fraction, and 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 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 eluent is added is a constant value, since this indicates that the unadsorbed fraction has been sufficiently removed from the column by the equilibration process. The constant value here includes not only the same value but also a state in which the detection value changes with a constant slope.

The present invention relates to an equilibration solution that is a solution that accurately separates antibodies to be detected contained in a body fluid, and in particular to an equilibration solution that can accurately separate IgG1 and/or IgG3 from antibodies other than IgG1 and/or IgG3. The antibody to be detected refers to IgG1 or IgG3 that is defined as being eluted at a time similar to the elution time of IgG1 or IgG3 derived from human myeloma plasma in the elution step described below, and does not include IgG1 or IgG3 contained in the unadsorbed fraction. In this specification, IgG1 or IgG3 does not include IgG1 or IgG3 contained in the unadsorbed fraction unless otherwise specified. The antibody to be detected may be an antibody produced by the subject itself, may include a drug such as an antibody drug administered to the subject, or may be an antibody added later to the body fluid collected from the subject. In the risk assessment in this invention, the detection assessment is performed using the antibody to be detected, except when specified.

The equilibration liquid may be an aqueous buffer solution. The equilibration liquid is a weakly acidic to weakly alkaline buffer solution having a pH of more than 5.0 and less than 9.0, preferably a buffer solution having a pH of 5.2 or more and a pH of 8.0 or less, more preferably a buffer solution having a pH of 5.4 or more and a pH of 7.5 or less, and most preferably a buffer solution having a pH of 5.5 or more and a pH of 7.0 or less. 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 a person skilled in the art can easily imagine, such as sodium chloride or potassium chloride.

Substances that are preferably separated from the antibody to be detected include IgG2 and IgG4 that may bind to the Fc-binding protein. When a solution having a composition in which the antibody to be detected strongly binds to the Fc-binding protein is used as the equilibration liquid, IgG4 also adsorbs to the Fc-binding protein and is eluted by adding an elution liquid, so that the peak of the antibody to be detected overlaps with that of the antibody to be detected, and the separation accuracy decreases. On the other hand, when a solution having a composition in which the antibody to be detected weakly binds to the Fc-binding protein is used as the equilibration liquid, the antibody to be detected is eluted during equilibration and overlaps with the peak of the unadsorbed fraction containing IgG2 and IgG4. Therefore, the equilibration liquid needs to be adjusted so that the antibody to be detected is appropriately 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 (d) and (g).
(a) Formula: Electrical conductivity [mS/cm] < -0.78 x buffer capacity value + 36.5
(b) Formula: Electrical conductivity [mS/cm]>-0.81 x buffer capacity value + 12.5
(c) Formula: Buffer capacity value>0.16
(d) Formula: Electrical conductivity [mS/cm]>0
(g) Formula: 9>pH>5
Electrical conductivity is a value consisting of 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 with Fc-binding proteins or carriers, and the higher the electrical conductivity, the lower the adsorption ability of antibodies with 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 changed after dropping (see J. Biol. Chem., 52, 525 (1922)). In the present invention, the buffer capacity value is the value obtained by dividing the number of millimoles (mmoles) 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 of an antibody to an Fc-binding protein or carrier.

The equilibration solution in the present invention can also be said to have a solution composition that exhibits a separation pattern in which no elution of IgG1 or IgG3 is observed before the introduction of the eluent, and the unadsorbed fraction on the column is significantly separated from the elution region derived from IgG1 and IgG3, or in which elution derived from IgG1 or IgG3 is observed before the introduction of the eluent, but the peak top of the first peak associated with elution (the peak that is first detected after the start of elution derived from IgG1 or IgG3) is not observed during equilibration. The term "no elution of IgG1 or IgG3 is confirmed before the introduction of the eluent" means that the obtained separation pattern is corrected to a baseline of a line connecting the minimum measurement value during the equilibration step after the introduction of the measurement sample and the minimum measurement value in the region from when the eluent is introduced and the antibody begins to elute until the proportion of the eluent introduced into the column reaches 100%, and then the corrected value at the time of introduction of the eluent is 0.05 or less when the maximum measurement value in the region from when the eluent is introduced and the antibody begins to elute until the proportion of the elution buffer introduced into the column reaches 100% is corrected to 1. When a separation pattern is shown in which elution originating from IgG1 or IgG3 is confirmed before the introduction of the eluent, but the peak top of the first peak associated with the elution is not confirmed during equilibration, there is little overlap of elution with the unadsorbed fraction, and separation is performed with high accuracy, but when a baseline is set, the elution time serving as the reference showing the minimum measurement value during the equilibration step after the introduction of the measurement sample differs for each measurement, making the analysis complicated. Therefore, the composition of the equilibration solution is preferably such that no elution of IgG1 or IgG3 is observed before the introduction of the elution solution, and it is more preferable that the composition is within the range of the following formula (e) in addition to formulas (a) to (d) and (g).
(e) Formula: Electrical conductivity [mS/cm] < -0.84 x buffer capacity value + 30.5
It was also confirmed that the variation obtained by repeatedly measuring the same sample varies depending on the buffer capacity value of the equilibration solution. It was found that this variation increases when the buffer capacity value is low, and also increases when the buffer capacity value is high. It is presumed that a low buffer capacity value is likely to cause pH fluctuations, which causes variation, and a high buffer capacity value causes little pH fluctuation, but affects the adsorption of the antibody to the Fc-binding protein, which increases the variation. In this case, it is even more preferable that the composition of the equilibration solution that can suppress the variation is within the range of the following formula (f) in addition to the above formula.
(f) Formula: 0.32<buffer capacity value<16
<Elution step>
The elution step is a step in which the antibody adsorbed to the carrier is eluted with an elution solution.

That is, the antibody adsorbed to 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 desired separation data 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. As the elution solution, one that weakens the affinity between the antibody and the Fc-binding protein can be used. As the elution solution, an aqueous buffer solution having a lower pH than the liquid phase before elution (e.g., the equilibration solution) can be used. Specifically, as the elution solution, an acidic buffer solution having a pH of 2.5 to 4.5 can be used. 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. Examples of 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. The eluent may be, for example, gradient or isocratic. The eluent may be, in particular, gradient. That is, elution may be performed by increasing the ratio of the eluent in the liquid phase. The gradient may be, for example, a linear gradient, a stepwise gradient, or a combination thereof. The gradient may be specifically set so that the ratio of the eluent in the liquid phase increases from 0% (v/v) to 100% (v/v) in, for example, 10 to 60 minutes, 15 to 50 minutes, or 20 to 40 minutes. The liquid delivery 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 rate may 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 to 50°C.

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. Specifically, the separated antibody can be collected, for example, by a known method used for separating and purifying proteins.

The elution process may obtain separation data (i.e., data related to the antibody separation pattern). The separation data may be used as an index for detecting a risk in a subject, and is not particularly limited. Examples of the 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 process of obtaining separation data may include, for example, a process of obtaining an antibody separation pattern and a process 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. Specifically, examples of the characteristics include the characteristics of the elution peak (i.e., the peak of the eluted antibody). Specifically, examples of the characteristics of the elution peak include the peak area, peak elution time, peak width, number of peaks detected, and peak height. The elution peak from which features such as peak area and peak height are extracted is also called the "target peak." Elution peak features include peak area and peak height in particular. The antibody separation pattern may be used for feature extraction as is, or after appropriate correction such as baseline correction.

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 characteristics of the subject. For example, the feature may be corrected based on the age of the subject. That is, for example, if the features are affected by the age of the subject, the extracted features may be corrected based on the age of the subject before being used in the detection process. The features corrected based on the age of the subject may be used, for example, to detect the risk of symptoms other than aging.

The target peak can be appropriately selected according to various conditions such as the type of risk. Examples of the target peak include the first to fourth peaks. Examples of the target peak include, in particular, the first peak, the second peak, and the third peak. Examples of the target peak include, in particular, the first peak. The first peak can be preferably used, for example, to evaluate the risk of symptoms other than pancreatitis. The second peak can be preferably used, for example, to evaluate the risk of aging. The third peak can be preferably used, for example, to evaluate the risk of pancreatitis. Specifically, the third peak can be preferably used, for example, to distinguish between pancreatitis and pancreatic cancer. As the target peak, one elution peak may be used, or two or more elution peaks may be used in combination. The "first to fourth peaks" may mean the peaks that are eluted first to fourth after the start of elution (for example, after the start of the gradient), respectively, unless otherwise specified. In addition, the target peak may be particularly one having a peak area percentage of 1% or more. In other words, the "first to fourth peaks" may particularly mean peaks that are eluted first to fourth after the start of elution, respectively, and have a peak area percentage of 1% or more. In addition, when the elution step is performed by gradient elution, the "first peak" may mean, for example, a peak that is eluted first when the pH of the liquid phase becomes 5.4 or less, 5.2 or less, 5.0 or less, or 4.8 or less. In addition, when the elution step is performed by gradient elution, the "first peak" may mean, for example, a peak that is eluted during a period when the pH of the liquid phase is 5.4 to 4.4, 5.2 to 4.5, or 5.0 to 4.6. The pH of the liquid phase is calculated by the following formula (I) when the pH of the liquid phase (e.g., equilibration liquid) before the start of elution is X, the pH of the elution liquid is Y, and the ratio of the elution liquid in the liquid phase is Z%. In addition, the pH at which the peak is eluted is appropriately corrected taking into account the volume of the flow path, such as the volume of the column.

pH of liquid phase = X - ((X - Y) x Z%) (I)
<Detection step>
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 an antibody) as an indicator, and may be performed after the <elution process>.

Diseases include those affected by immune cell activity (e.g., damaging or phagocytic activity). Immune cells include natural killer cells, monocytes, and macrophages.

Specific examples of diseases include cancer, autoimmune diseases, infectious diseases, allergies, and inflammatory diseases. Any of these diseases may be affected by the activity of immune cells.

Cancers include brain tumors, 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 tumors, 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.

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. These include erythematosus, localized scleroderma, pemphigus, pustular psoriasis, psoriasis vulgaris, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo vulgaris, Sutton's acquired centrifugal 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, Sjögren's syndrome, IgG4-related disease, vasculitis syndrome, and mixed connective tissue disease. Autoimmune diseases include rheumatism and Sjögren's syndrome, among others.

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 diphyllobothrium latissimus. 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), progressive multifocal leukoencephalopathy, chickenpox/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.

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.

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, etc.), 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 in particular include pancreatitis.

Specific examples of diseases include cachexia and age-related diseases. Age-related diseases include frailty, sarcopenia, and locomotive syndrome. Cachexia and these age-related diseases can also be inflammatory diseases.

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

Examples of detecting the presence or absence of a disease include determining whether or not a subject is currently suffering from a disease, and determining whether or not the subject is likely to currently suffer from a disease. Examples of detecting the risk of developing a disease include determining whether or not a subject is likely to develop a disease in the future, or whether or not the disease will become severe if it does develop, and whether or not the subject is likely to develop a disease in the future, or whether or not the disease will become severe if it does develop. Examples of detecting the degree of progression of a disease include determining whether the current degree of progression (e.g., severity) of a disease in a subject is high or low. Examples of detecting the degree of progression of aging include determining whether the current degree of progression (e.g., severity) of aging in a subject is high or low.

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 have a severe disease if he or she 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 is not likely to have a severe disease if he or she 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 is likely to have a severe disease if he or she develops a disease in the future, that the subject's current disease is highly advanced, and/or that the subject's current aging is highly advanced. "Low risk in a subject" may mean, for example, that the subject is less likely to currently develop a disease, that the subject is less likely to develop a disease in the future, that the subject is less likely to develop a severe disease if they do develop a disease in the future, that the subject's current disease is less advanced, and/or that the subject's current aging is less advanced.

The detection process can be carried out, for example, using the value of the separated data (i.e., whether the value of the separated data is high or low) as an index. 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 process may include, for example, a process 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 of 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. "The value of the separated data is low based on a threshold" may mean, for example, that the value of the separated data is equal to or lower than the threshold, that the value of the separated data is less than the threshold, or that the value of the separated data is statistically significantly lower than the threshold. "The value of the separated data is low based on a threshold" 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 of the threshold.

The value of the separated data may be classified into a risk range, for example, based on a threshold value. The value of the separated data may be classified into a non-risk range, for example, based on a threshold value. Specifically, the value of the separated data may be classified 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 for 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 for the subject. In other words, 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.

For example, when the peak area (e.g., peak area%) and/or peak height (e.g., peak height%) of the first peak and/or the second peak are high, the subject may be determined to be at risk or at high risk. Specifically, when the peak area (e.g., peak area%) and/or peak height (e.g., peak height%) of the first peak and/or the second peak are high based on a threshold, the subject may be determined to be at risk or at high risk. In this case, the range that is considered high based on the threshold may be the risk range. More specifically, when the first peak area% is 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, or 30% or more, the subject may be determined to be at risk or at high risk. In addition, the higher the peak area (e.g., peak area %) and/or peak height (e.g., peak height %) of the first peak and/or the second peak, the higher the risk in the subject may be determined.

For example, when the peak area (e.g., peak area%) and/or peak height (e.g., peak height%) of the first peak and/or the second peak are low, the subject may be determined to have no risk or a low risk. Specifically, when the peak area (e.g., peak area%) and/or peak height (e.g., peak height%) of the first peak and/or the second peak are low based on a threshold, the subject may be determined to have no risk or a low risk. In this case, the range that is considered to be low based on the threshold may be a non-risk range. More specifically, when the first peak area% is 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, or 9% or less, the subject may be determined to have no risk or a low risk. In addition, the lower the value of the peak area (e.g., peak area %) and/or peak height (e.g., peak height %) of the first peak and/or the second peak, the lower the risk in the subject may be determined.

For example, if the value of the peak area (e.g., peak area %) and/or the peak height (e.g., peak height %) of the third peak is low, the subject may be determined to be at risk or at high risk. Specifically, for example, if the value of the peak area (e.g., peak area %) and/or the peak height (e.g., peak height %) of the third peak is low based on a threshold value, the subject may be determined to be at risk or at high risk. In this case, the range that is considered to be low based on the threshold value may be the danger range. Also, the lower the value of the peak area (e.g., peak area %) and/or the peak height (e.g., peak height %) of the third peak, the higher the risk may be determined to be.

For example, if the value of the peak area (e.g., peak area %) and/or the peak height (e.g., peak height %) of the third peak is high, the subject may be determined to have no risk or a low risk. Specifically, for example, if the value of the peak area (e.g., peak area %) and/or the peak height (e.g., peak height %) of the third peak is high based on a threshold value, the subject may be determined to have no risk or a low risk. In this case, the range that is considered high based on the threshold value may be a non-risk range. Also, the higher the value of the peak area (e.g., peak area %) and/or the peak height (e.g., peak height %) of the third peak, the lower the risk may be determined to be.

When the separated data having the first peak as the target peak is used as the index, the risk may be selected, for example, from the risk of symptoms other than pancreatitis. When the separated data having the second peak as the target peak is used as the index, the risk may be selected, for example, from the risk of aging. When the separated data having the third peak as the target peak is used as the index, the risk may be selected, for example, from the risk of pancreatitis.

Note that "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 to the extent that the criterion is met, and does not require that the risk be determined in the subject to the extent that 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 extent that the criterion is not met.

The threshold value can be set appropriately 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.

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 process. 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 process may include, for example, a step of comparing the separation data with the control separation data.

Examples of control subjects include positive controls and negative controls. A "positive control" may refer to a subject who may be determined to be at risk or high. A "negative control" may refer to a subject who may be determined to be at no risk or low. 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. 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 assessment with the desired accuracy is obtained. The number of positive controls and negative controls may each be one, two or more. The number of positive controls and negative controls may each usually be multiple. The number of positive controls and negative controls may each 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 each be, for example, 10,000 or less, 1,000 or less, or 100 or less.

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

When the threshold is determined based only on the values of the separation data measured for the negative control, the threshold may be set to, 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, such as the average value. 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 the 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%.

When the threshold value is determined based on both the values of the separation data measured for the positive control and the values of the separation data measured for the negative control, the threshold value may be determined, for example, so that a predetermined percentage of the positive control falls within the risk range and a predetermined percentage of the negative control falls within the non-risk range. It is preferable that the percentage of the positive control that falls within the risk range and the percentage of the negative control that falls within the non-risk range are both high. These percentages may be, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 100%. If it is difficult to increase both of these percentages, the threshold value may be set so that one of the percentages is preferentially high 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 percentage of the positive control that falls within the risk range is preferentially high.

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

Also, the control subject may 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 increases. "The value of the separation data has increased" may specifically mean that the value of the separation data has increased compared to a past value. Also, "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 a past value. That is, the threshold may also be 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.

When the variation in the separation data in a subject is used as an index, an increase or decrease in the risk in the subject may be determined. "Having or having a 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. Furthermore, "no 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.

Note that "obtaining certain separation data to use as an index for risk detection" is not limited to obtaining the value of the separation data itself to use as an index for risk detection, but also includes obtaining other values reflecting the value of the separation data to use as an index for detection. For example, when the elution peak consists of the first to fourth peaks, "obtaining the peak area % of the first peak to use as an index for risk detection" is not limited to obtaining the value of the peak area % of the first peak itself to use 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, to use as an index for detection. In either case, the values of the measured values, thresholds, etc. used for risk detection are appropriately corrected according to the type of separation data. For example, when the elution peak consists of the first to fourth peaks, the relationship "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 the detection indicator 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 interpreted as "the corrected value of Y (i.e., "100% - Y") satisfies the criterion."

The result of risk detection may be used as an index for determining whether to perform a treatment to reduce 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 is at risk or at high risk, it may be decided to perform a risk reduction treatment on the subject. 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, a definitive diagnosis may be performed by other means for a symptom determined to be at risk or at high risk in the subject by the detection method of the present invention, and then it may be decided to perform a risk reduction treatment on the subject. The risk reduction treatment may be a medical procedure or a non-medical procedure. Examples of 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).

The present invention allows accurate separation of antibodies to be detected, including IgG1 and/or IgG3, from antibodies other than IgG1 and/or IgG3. For example, separation data obtained by separating the antibodies of a subject based on a glycan structure characteristic of the risk of a particular disease or aging or other symptoms can be obtained accurately by the separation pattern of the antibody to be detected, and such risks can be easily detected by comparing the data 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 the data 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 reference separation data include separation data of a control subject. Examples of 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 the subject into two or more groups that have different efficacies to the treatment.

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 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 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%.

The strength of the affinity of an antibody to the Fc-binding protein according to the present invention affects the activity of immune cells such as natural killer cells, monocytes, and macrophages, which have the ability to damage or phagocytose the binding substance bound by the antibody, and therefore by detecting the difference in the affinity, it is possible to evaluate the risk of developing a disease that is affected by the damage or phagocytosis caused by natural killer cells, monocytes, and macrophages. Such diseases include cancer, autoimmune diseases, infectious diseases, allergies, and inflammatory diseases. Examples of infectious diseases include opportunistic infections. Opportunistic infections are infectious diseases caused by pathogens that do not cause infection in a healthy state. Examples of pathogens include viruses, bacteria, fungi, and protozoa. In addition, the effect of antibodies acquired by vaccination or disease on the activation of immune cells can also be evaluated from the strength of the affinity of the antibody to the Fc-binding protein. In addition to measuring the amount of the acquired antibody in the blood, the strength of the affinity of the antibody to the Fc-binding protein can also be measured to accurately predict the risk of developing an infectious disease.

The present invention will now be described in more detail with reference to the following non-limiting examples.

Preparation of Fc-binding protein immobilized gel Example 1 Preparation of FcR9 Val176 amino acid substitution product An Fc-binding protein was prepared by substituting valine at position 176 (Val at position 176 in the amino acid numbering of SEQ ID NO: 1) of FcR9 (SEQ ID NO: 2) prepared by the method described in WO2015/199154 with phenylalanine. Specifically, an amino acid substitution was performed using PCR on plasmid pET-FcR9 (WO2015/199154) containing a polynucleotide (SEQ ID NO: 3) encoding FcR9, to prepare an Fc-binding protein in which Val176 of FcR9 was substituted with phenylalanine.

FcR9 (SEQ ID NO: 2) is an Fc-binding protein containing the wild-type FcγRIII extracellular domain shown in SEQ ID NO: 4, in which the following amino acid substitutions have been made: Val at position 43 is replaced by Glu (corresponding to position 27 in SEQ ID NO: 1), Phe at position 45 is replaced by Ile (corresponding to position 29 in SEQ ID NO: 1), Tyr at position 51 is replaced by Asn (corresponding to position 35 in SEQ ID NO: 1), Gln at position 64 is replaced by Arg (corresponding to position 48 in SEQ ID NO: 1), Phe at position 91 is replaced by Leu (corresponding to position 75 in SEQ ID NO: 1), Asn at position 108 is replaced by Ser (corresponding to position 92 in SEQ ID NO: 1), Val at position 133 is replaced by Glu (corresponding to position 117 in SEQ ID NO: 1), Glu at position 137 is replaced by Gly (corresponding to position 121 in SEQ ID NO: 1), and Phe at position 187 is replaced by Ser (corresponding to position 171 in SEQ ID NO: 1).

The method for producing each Fc-binding protein is described in detail below.
(1) In order to replace the 176th valine (Val, the 176th amino acid in the amino acid numbering of SEQ ID NO: 1) of the Fc binding protein with phenylalanine, a plasmid pET-FcR9 (described in WO 2015/199154) containing a polynucleotide (SEQ ID NO: 3) encoding FcR9 (SEQ ID NO: 2) prepared by the method described in WO 2015/199154 was used as a template, and a polynucleotide sequence (SEQ ID NO: 5) (5'-TAATACGACTCACTATAGGG-3') and a polynucleotide sequence (SEQ ID NO: 6) were used. and an oligo primer consisting of the sequence shown in SEQ ID NO:6 (5'-CATTTTTGCTGCCGAACAGCCCACGGCAGG-3') were used to prepare a reaction solution similar to that shown in Table 1, and the reaction solution was heat-treated at 95°C for 2 minutes, and PCR was performed by carrying out 30 cycles of reaction consisting of a first step at 95°C for 30 seconds, a second step at 50°C for 30 seconds, and a third step at 72°C for 90 seconds, and finally heat-treated at 72°C for 7 minutes. The resulting PCR product was designated V176p1.

(2) Using the plasmid pET-FcR9 (described in WO2015/199154) containing the polynucleotide (SEQ ID NO: 3) encoding FcR9 (SEQ ID NO: 2) prepared by the method described in WO2015/199154 as a template, a reaction solution similar to that shown in Table 1 was prepared using oligo primers consisting of sequences described in SEQ ID NO: 7 (5'-TATGCTAGTTATTGCTCAG-3') and SEQ ID NO: 8 (5'-cctgccgtggggctgTTCGGCAGCAAAAATG-3'), the reaction solution was heat-treated at 95 ° C. for 2 minutes, and 30 cycles of reaction consisting of a first step at 95 ° C. for 30 seconds, a second step at 50 ° C. for 30 seconds, and a third step at 72 ° C. for 90 seconds were performed as one cycle, and finally, PCR was performed by heat-treating at 72 ° C. for 7 minutes. The obtained PCR product was designated V176p2.
(3) The two PCR products (V176p1, V176p2) obtained in (1) and (2) were mixed to prepare a reaction solution having the composition shown in Table 2. The reaction solution was heat-treated at 98° C. for 5 minutes, and then PCR was carried out in which one cycle consisted 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.

(4) PCR was performed using the PCR product V176p obtained in (3) as a template and oligonucleotides consisting of the sequences set forth in SEQ ID NOs: 5 and 7 as PCR primers. For PCR, a reaction solution having the composition shown in Table 3 was prepared, and the reaction solution was heat-treated at 98°C for 5 minutes, and 30 cycles of reaction were performed, 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. This resulted in a polynucleotide encoding an Fc-binding protein in which the 176th amino acid of the Fc-binding protein (FcR9) was replaced with phenylalanine. The resulting polynucleotide was designated V176p3.

(5) The polynucleotide obtained in (4) was purified, digested with the restriction enzymes NcoI and HindIII, and ligated into an expression vector pETMalE (JP Patent Publication No. 2011-206046) that had been previously digested with the restriction enzymes NcoI and HindIII, and used to transform Escherichia coli BL21 (DE3) strain (manufactured by Nippon Gene Co., Ltd.).
(6) The obtained transformant was cultured in LB medium supplemented with 50 μg/mL kanamycin, and plasmids were extracted from the collected 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 sequence analysis, a transformant was obtained that expresses an Fc binding protein (SEQ ID NO: 9) in which Val176 of the Fc binding protein FcR9 was substituted with Phe.

Example 2 Preparation of a cysteine-tagged Fc-binding protein (FcR9_F_Cys) (1) PCR was carried out using as a template the expression vector pET-FcR9_F containing the polynucleotide set forth in SEQ ID NO: 10 encoding the amino acid sequence set forth in SEQ ID NO: 9 prepared in Example 1. The primers used in the PCR were oligonucleotides consisting of the sequences set forth in SEQ ID NO: 11 (5'-TAGCCATGGGCATGCGTACCGAAGATCTGCCGAAAGC-3') and SEQ ID NO: 12 (5'-CCCAAGCTTATCCGCAGGTATCGTTGCGGCACCCTTGGGTAATGGTAATATTCACGGTCTCGCTGC-3'). PCR was performed by preparing a reaction solution having the composition shown in Table 3, heat-treating the reaction solution at 98°C for 5 minutes, and repeating 30 cycles of a reaction in which one cycle consisted 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.
(2) The polynucleotide obtained in (1) was purified, digested with restriction enzymes NcoI and HindIII, and then ligated to an expression vector pTrc-PelBV3 prepared by the method described in WO 2015/199154 that had been previously digested with restriction enzymes NcoI and HindIII, and the ligation product was used to transform Escherichia 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. The amino acid sequence of the polypeptide 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 an improved PelB signal peptide, the portion from the 24th glycine (Gly) to the 199th glutamine (Gln) is the amino acid sequence of an 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 a cysteine tag sequence.

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 of (1), and main culture was performed using a 3 L fermenter (manufactured by Biot). 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 (OD600 nm) reached approximately 150 as an indication of the bacterial mass, the culture temperature was lowered to 25° C., and after it was confirmed that the set temperature had been reached, IPTG 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 8,000 rpm for 20 minutes at 4° C., and the supernatant was collected.
(6) The supernatant obtained in (5) was applied at a flow rate of 5 mL/min to a VL32×250 column (Merck Millipore) packed with 140 mL of TOYOPEARL CM-650M (Tosoh) previously 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, elution was performed 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 (GE Healthcare) packed with 90 mL of IgG Sepharose (GE Healthcare) previously equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride. After washing with the buffer used for equilibration, elution was performed with 0.1 M glycine-HCl buffer (pH 3.0). The pH of the eluate was returned to near neutral by adding 1 M Tris-HCl buffer (pH 8.0) in an amount of 1/4 the amount of the eluate.

By this purification, approximately 20 mg of highly pure FcR9_F_Cys was obtained.

Example 4 Preparation of Fc-binding protein (FcR9_F) immobilized gel (1) The hydroxyl groups on the surface of 2 mL of hydrophilic vinyl polymer for separation medium (manufactured by Tosoh Corporation: packing material for liquid chromatography) were activated with iodoacetyl groups, and then 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 φ4.6 mm×75 mm stainless steel column to prepare an FcR9_F column.

Example 5 Separation of Antibody-Containing Serum Using Fc-Binding Protein (FcR9_F)-Immobilized Gel (1) An equilibration buffer of 10 mM citrate buffer (pH 6.5) containing 100 mM sodium chloride was prepared, and the electrical conductivity was measured using an electrical conductivity meter (Horiba, Ltd.). The pH of the equilibration buffer was changed by hydrochloric acid in the range of 0.1 to 0.2 from the pH of the equilibration buffer, and the number of millimoles (mmoles) of hydrochloric acid dropped per 1 L of equilibration buffer was divided by the actual measured pH value to measure the buffer capacity value using a pH meter (Horiba, Ltd.).
(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 prepared in (2) was added at a flow rate of 1.0 mL/min.
(4) After washing with the equilibration buffer for 5 minutes while maintaining the flow rate at 1.0 mL/min, the adsorbed gamma globulin was eluted with a pH gradient of an 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), to obtain a separation pattern.

Comparative Example 1 Separation of Human Myeloma Plasma-Derived Antibodies Using Fc-Binding Protein (FcR9_F) Immobilized Gel A separation pattern of gamma globulins was obtained in the same manner as in Example 5, except that 10 μL of 0.5 to 1 mg/mL human myeloma plasma-derived antibodies (manufactured by Sigma) of (a) to (d) were added as the measurement sample at a flow rate of 1.0 mL/min.
(a) IgG1
(b) IgG2
(c) IgG3
(d) IgG4
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 14.4 mS/cm, buffer capacity value 3.2) is within the range of the equilibration buffer conditions of the present invention. In Example 5, 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 1(a) and (c)), so it can be seen that the elution peak in Example 5 is derived from IgG1 and IgG3. On the other hand, no peak was confirmed in the elution time region of 10 to 20 minutes for IgG2 and IgG4 (Comparative Examples 1(b) and (d)), and most of the elution was confirmed in the elution time region of 0 to 5 minutes, when the column was washed with the equilibration buffer. The fact that it was eluted during washing with the equilibration buffer means that it has a weak binding force to the Fc-binding protein, and it can be seen that it can be clearly separated from IgG1 and IgG3, which are eluted only by introducing the elution buffer.

Comparative Example 2 Separation of antibody-containing serum using Fc-binding protein (FcR9_F)-immobilized gel A separation pattern of gamma globulin was obtained in the same manner as in Example 5, except that 10 mM citrate buffer (pH 6.5) was used as the equilibration buffer.

Comparative Example 3 Separation of human myeloma plasma-derived antibodies using Fc-binding protein (FcR9_F)-immobilized gel A separation pattern of gamma globulins was obtained in the same manner as in Comparative Example 1, except that 10 mM citrate buffer (pH 6.5) was used as the equilibration buffer.

The results of Comparative Examples 2 and 3 are shown in FIG. 2. The equilibration buffer used in Comparative Examples 2 and 3 (electrical conductivity 2.4 mS/cm, buffer capacity value 3.2) is outside the range of the equilibration buffer conditions of the present invention. In Comparative Example 2, 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), (d)), so it can be seen that the elution peak in Comparative Example 2 is derived from IgG1, IgG3, and IgG4. On the other hand, in IgG2 (Comparative Example 3(b)), most of the elution was confirmed in the elution time range of 0 to 5 minutes, where the column was washed with the equilibration buffer. Unlike the separation pattern of IgG4 in Comparative Example 1(d), it can be seen that the binding strength of IgG4 to the Fc-binding protein is improved under the equilibration buffer conditions in Comparative Example 3(d). On the other hand, because IgG4 is eluted after the introduction of the elution buffer, the accuracy of separation from IgG1 and IgG3 is reduced, and it can be seen that a clear separation is difficult, especially since the separation peaks partially overlap in the region around 15 minutes of elution time for IgG1 and IgG4.

Example 6 Evaluation of separation behavior of antibody-containing serum depending on the composition of the equilibration buffer (1) A measurement sample was added to the column in the same manner as in Example 5 (1) to (3), except that 1 mM citrate buffer (pH 6.5) containing 200 mM (a) or 250 mM (b) sodium chloride was used as the equilibration buffer.
(2) A separation pattern of gamma globulin was obtained in the same manner as in Example 5(4), except that the column was washed with the equilibration buffer for 10 minutes.
(3) The separation pattern obtained in (2) was corrected using as a baseline a line connecting the minimum measurement value during the equilibration step after the introduction of the measurement sample and the minimum measurement value in the region from when the elution buffer starts to elute the antibody until the percentage of the elution buffer introduced into the column reaches 100%, and then the maximum measurement value in the region from when the elution buffer starts to elute the antibody until the percentage of the elution buffer introduced into the column reaches 100% was corrected to 1.

Comparative Example 4 Evaluation of separation behavior of serum containing antibodies due to differences in the composition of the equilibration buffer A separation pattern of gamma globulins was obtained in the same manner as in Example 6, except that 1 mM citrate buffer (pH 6.5) containing 100 mM (a) or 300 mM (b) sodium chloride was used as the equilibration buffer.

The results of Example 6 and Comparative Example 4 are shown in Table 4 and Figure 3. From Table 4, which summarizes the electrical conductivity and buffer capacity values of the equilibration buffers, it can be seen that the equilibration buffer used in Example 6 is within the range of the equilibration buffer conditions of the present invention, and the equilibration buffer used in Comparative Example 4 is outside the range of the equilibration buffer conditions of the present invention.

From FIG. 3, in Example 6(a), elution of IgG1 or IgG3 was not confirmed before the introduction of the elution buffer, and the unadsorbed fraction on the column and the elution region derived from IgG1 and IgG3 were far apart, showing good separation, whereas in Example 6(b), elution derived from IgG1 or IgG3 was confirmed before the introduction of the elution buffer. However, in Example 6(b), the peak top of the first peak associated with elution (the peak first detected from the start of elution derived from IgG1 or IgG3) was not confirmed, and the unadsorbed fraction on the column and the elution derived from IgG1 or IgG3 did not overlap, so it can be seen that separation is possible, although with lower accuracy than in Example 6(a). On the other hand, in Comparative Example 4(a), the elution fraction derived from IgG4 was confirmed after the introduction of the elution buffer, and from the results of Comparative Examples 2 and 3, the elution fraction of IgG4 overlaps with the elution fraction of IgG1, so it can be seen that the separation accuracy between IgG1 and IgG3 has decreased. In addition, in Comparative Example 4(b), it was confirmed that the peak derived from IgG1 or IgG3 was eluted with a peak top before the introduction of the elution buffer, and the elution times of the fraction not adsorbed to the column and the peak derived from IgG1 or IgG3 were close to each other, which shows that the separation accuracy was greatly reduced. From this result, it can be seen that Example 6 can separate IgG1 and IgG3 with high accuracy compared to Comparative Example 4.

Example 7 Evaluation of separation behavior of serum containing antibodies depending on the composition of the equilibration buffer (1) A separation pattern of gamma globulins was obtained in the same manner as in Example 6, except that citrate buffers (pH 6.5) having the compositions (a) to (m) were used as the equilibration buffer.
(a) 1 mM citrate buffer containing 120 mM sodium chloride; (b) 1 mM citrate buffer containing 150 mM sodium chloride; (c) 1 mM citrate buffer containing 200 mM sodium chloride; (d) 10 mM citrate buffer containing 100 mM sodium chloride; (e) 20 mM citrate buffer containing 50 mM sodium chloride; (f) 20 mM citrate buffer containing 100 mM sodium chloride; (g) 100 mM sodium chloride (h) 30 mM citrate buffer (i) 30 mM citrate buffer containing 100 mM sodium chloride (j) 50 mM citrate buffer (k) 1 mM citrate buffer containing 250 mM sodium chloride (l) 40 mM citrate buffer containing 100 mM sodium chloride (m) 60 mM citrate buffer (2) Based on the obtained separation patterns, the solutions were classified into ●, ◆, ▲, × based on the following indicators.
●: Condition under which no elution of fractions containing IgG4 occurs after the start of introduction of elution buffer, and no elution of IgG1 and IgG3 is observed during equilibration. ◆: Condition under which elution of IgG1 and IgG3 is observed during equilibration, but the peak tops of the peaks derived from IgG1 and IgG3 occur after the start of elution. ▲: Condition under which elution of fractions containing IgG4 occurs after the start of introduction of elution buffer. ×: Condition under which the peak tops of the peaks derived from IgG1 and IgG3 are observed during equilibration. Comparative Example 5 Evaluation of separation behavior of antibody-containing serum due to differences in equilibration buffer composition The separation patterns of gamma globulins were classified in a manner similar to that of Example 7, except that citrate buffers (pH 6.5) with compositions (a) to (m) were used as the equilibration buffer.
(a) 0.5 mM citrate buffer containing 150 mM sodium chloride (b) 1 mM citrate buffer (c) 1 mM citrate buffer containing 100 mM sodium chloride (d) 10 mM citrate buffer (e) 20 mM citrate buffer (f) 20 mM citrate buffer containing 1 mM sodium chloride (g) 20 mM citrate buffer containing 10 mM sodium chloride (h) 25 mM citrate buffer (i) 0.1 mM citrate buffer containing 150 mM sodium chloride (j) 1 mM citrate buffer containing 300 mM sodium chloride (k) 50 mM citrate buffer containing 100 mM sodium chloride (l) 80 mM citrate buffer (m) 100 mM citrate buffer The results of Example 7 and Comparative Example 5 are summarized in Table 5 and FIG. 4. From Table 5, which summarizes the electrical conductivity, buffer capacity values, and classification of the equilibration buffers, it can be seen that the equilibration buffer used in Example 7 is within the range of conditions for the equilibration buffer of the present invention, while the equilibration buffer used in Comparative Example 5 is outside the range of conditions for the equilibration buffer of the present invention.

Figure 4 shows a plot of classification based on electrical conductivity and buffer capacity. From Figure 4, it can be seen that there is a correlation between classification and electrical conductivity and buffer capacity, and the higher the electrical conductivity and buffer capacity, the more elution of IgG1 or IgG3 is observed during equilibration, and the lower the electrical conductivity and buffer capacity, the more elution of IgG4 is observed after the introduction of the elution buffer. Based on this result, the boundary region of electrical conductivity and buffer capacity for accurate separation of IgG1 and IgG3 can be obtained. From the electrical conductivity and buffer capacity lines of 1 mM citrate buffer containing 300 mM sodium chloride and 50 mM citrate buffer containing 100 mM sodium chloride as the boundary between × and ◆, the formula (a) (electrical conductivity [mS/cm] < -0.78 x buffer capacity value + 36.5) showing the region of ◆ can be obtained, and the boundary between ◆ and ● can be obtained from the electrical conductivity and buffer capacity lines of 1 mM citrate buffer containing 250 mM sodium chloride and 60 mM citrate buffer. From the electric conductivity and buffer capacity value lines, formula (e) (electric conductivity [mS/cm] < -0.84 x buffer capacity value + 30.5) showing the region of ● can be obtained, and from the electric conductivity and buffer capacity value lines of 1 mM citrate buffer containing 100 mM sodium chloride and 25 mM citrate buffer as the boundary between ● and ▲, formula (b) (electric conductivity [mS/cm] > -0.81 x buffer capacity value + 12.5) showing the region of ● can be obtained. Also, a diagram of the buffer capacity value in FIG. 4(a) with the scale reduced is shown in FIG. 4(b). From this diagram, as the boundary between ● and ▲, 0.5 mM citrate buffer containing 150 mM sodium chloride is classified as ▲, and 1 mM citrate buffer containing 150 mM sodium chloride is classified as ●, so formula (c) (buffer capacity value > 0.16) showing the region of ● can be obtained. Although not shown in the figure, a compound with buffering capacity is added as the equilibration buffer, so formula (d) (electrical conductivity [mS/cm]>0) was set.

By combining these equations, the area in which the ● and ◆ separation patterns can be obtained is a solution that satisfies all the conditions in equations (a) to (d), and the area in which the ● separation pattern can be obtained is a solution that satisfies all the conditions in equations (a) to (e).

Example 8 Accuracy evaluation of repeated measurements of separation behavior of serum containing antibodies due to differences in the composition of the equilibration buffer (1) A citrate buffer (pH 6.5) having the composition of Examples 7(a) to (j) classified as ● in Example 7 was used as the equilibration buffer, and a gamma globulin separation pattern was obtained in the same manner as in Example 7(1), except that the same measurement sample was measured 10 times.
(2) The separation pattern obtained in (1) was defined as a first peak, a second peak, and a third peak in order of the peaks having the earliest elution times after the start of the pH gradient application, and the CV value of the elution time of the first peak in 10 repeated measurements was calculated.

The results of Example 8 are shown in Table 6 and Figure 5. In the region of buffer capacity values between 0.32 and 16 (equation (f): 0.32 < buffer capacity value < 16), the CV value is 0.4 to 0.7%, which is lower than the CV value (0.9% or higher) at low buffer capacity values (0.32) or high buffer capacity values (16), demonstrating high accuracy in obtaining the same separation pattern in repeated measurements.

Example 9 Evaluation of separation behavior of serum containing antibodies depending on difference in pH of equilibration buffer A separation pattern of gamma globulins was obtained in the same manner as in Example 6, except that a mixed solution of 30 mM phosphoric acid and 30 mM citric acid (pH 7.0) was used as the equilibration buffer.

The results of Example 9 are shown in Figure 6. The equilibration buffer used in Example 9 (electrical conductivity 5.4 mS/cm, buffer capacity value 24.2) is within the range of the equilibration buffer conditions of the present invention. As shown in Figure 6, no elution of IgG1 or IgG3 was confirmed before the introduction of the elution buffer, and the non-adsorbed fraction on the column was far from the elution region derived from IgG1 and IgG3, indicating good separation. From these results, it can be seen that good separation is also observed when the equilibration buffer has a pH of 7.0, and furthermore, good separation is also observed in a mixed buffer of phosphate and citric acid.

Example 10 Separation of gamma globulin from a cancer patient (1) Electrical conductivity and buffer capacity were measured in the same manner as in Example 5 (1), except that a 20 mM acetate buffer (pH 5.5) containing 50 mM sodium chloride was used as the equilibration buffer.
(2) Blood samples were collected from healthy subjects and patients with pancreatic cancer, gastric cancer, and breast cancer who had given informed consent. The measurement samples were loaded onto the column in the same manner as in Example 5 (2) and (3), except that the measurement samples were loaded onto the column at 0.6 mL/min.
(3) As for the elution conditions, after washing with the equilibration buffer for 5 minutes at a flow rate of 0.6 mL/min, the adsorbed gamma globulin was eluted with a pH gradient of 10 mM glycine hydrochloride buffer (pH 3.0) (a gradient in which 10 mM glycine hydrochloride buffer (pH 3.0) became 100% in 30 minutes), to obtain a separation pattern.
(4) The separation pattern obtained in (3) was defined as a first peak, a second peak, a third peak, and a fourth peak in order of the peaks having the earliest elution times from the start of application of the pH gradient, and the value obtained by dividing the area value of the first peak by the total area value from the start to the end of application of the pH gradient was defined as the first peak area %.

The results of Example 10 are shown in Figure 7. Compared to healthy subjects, the values of the first peak area% were significantly increased in patients with pancreatic cancer (panel a in Figure 7), gastric cancer (panel b in Figure 7), and breast cancer (panel c in Figure 7). It was also found that the value of the first peak area% increased depending on the stage of the disease. The disease stage is based on the disease stage classification established by the Union for International Cancer Control (UICC). This result means that immune activity related to Fc-binding proteins is reduced in cancer diseases, and it can be seen that immune activity is greatly reduced, especially in cancer diseases, where the first peak area% is significantly increased. In cancer diseases, it is thought that the reduction in the immune activity is a factor in the onset of the disease, or that the onset of the disease causes the reduction in the immune activity. Since the values of the first peak area% of healthy subjects and specimens differ from those of disease specimens, it can be used to diagnose diseases, and since the value of the first peak area% changes depending on the stage of the disease in cancer diseases, it can also be used to evaluate the progression and malignancy of cancer. These results show that the separation pattern of antibodies including IgG1 and IgG3 is useful in diagnosing diseases.

The equilibration buffer used in Example 10 (electrical conductivity 7.8 mS/cm, buffer capacity value 8.5) is within the range of the equilibration buffer conditions of the present invention. Even when this buffer is used, elution of IgG1 or IgG3 was not confirmed before the introduction of the elution buffer, and good separation was demonstrated because the unadsorbed fraction on the column was far from the elution region derived from IgG1 and IgG3. From these results, it can be seen that good separation is also demonstrated when the equilibration buffer has a pH of 5.5, and furthermore, similarly good separation is also demonstrated when the acetate buffer is used.

Example 11 Separation of human-derived antibodies using different Fc-binding protein (FcR9_V) immobilized gels (1) A cysteine-tagged Fc-binding protein (FcR9_V_Cys) was prepared in the same manner as in Example 3, except that an expression vector was used in which the amino acid sequence of the expressed polypeptide is shown in SEQ ID NO: 17 and the sequence of the polynucleotide encoding the polypeptide is shown in SEQ ID NO: 18.
(2) An FcR9_V column was prepared in the same manner as in Example 4, except that the Fc-binding protein prepared in (1) was used.
(3) A gamma globulin separation pattern was obtained in the same manner as in Example 10(1) and (3), except that blood was collected from healthy individuals who gave informed consent and an FcR9_V column was used.

The equilibration buffer used in Example 11 (electrical conductivity 7.8 mS/cm, buffer capacity value 8.5) is within the range of the equilibration buffer conditions of the present invention. Even when this buffer is used, elution of IgG1 or IgG3 was not confirmed before the introduction of the elution buffer, and good separation was observed because the unadsorbed fraction on the column was far from the elution region derived from IgG1 and IgG3. From this result, it can be seen that good separation is also observed when a column (FcR9_V column) packed with an insoluble carrier on which an Fc-binding protein with an amino acid sequence different from that of Example 10 is immobilized is used.

Example 12 Evaluation of separation behavior of serum containing antibodies depending on difference in pH of equilibration buffer A separation pattern of gamma globulins was obtained in the same manner as in Example 6, except that a buffer solution (pH 8.0) containing 100 mM sodium chloride, which was a mixed solution of 30 mM phosphoric acid and 30 mM citric acid, was used as the equilibration buffer.

Comparative Example 6 Evaluation of separation behavior of serum containing antibodies depending on pH of equilibration buffer A separation pattern of gamma globulin was obtained in the same manner as in Example 6, except that 30 mM citrate buffer (pH 5.0) was used as the equilibration buffer.

The equilibration buffer used in Example 9 (electrical conductivity 16.0 mS/cm, buffer capacity value 4.3) and the equilibration buffer used in Comparative Example 6 (electrical conductivity 4.56 mS/cm, buffer capacity value 20.1) are within the range of the equilibration buffer conditions of the present invention excluding formula (g). In Example 12, elution of IgG1 and IgG3 was observed during equilibration, but the peak top positions of the peaks derived from IgG1 and IgG3 were separated after the start of elution, whereas in Comparative Example 6, the peak tops of the peaks derived from IgG1 and IgG3 were observed during equilibration. From this result, it can be seen that if the pH is low, it becomes difficult to continue to hold gamma globulin in the column during equilibration, and it is eluted. In addition, when Example 9 and Example 12 were compared, it was confirmed that when the equilibration buffer was changed from pH 7.0 to pH 8.0, a part of the gamma globulin was retained but was eluted during equilibration. Based on these results, the pH condition of the equilibration buffer was set to (g) formula (9>pH>5).

Claims (4)

When analyzing antibody-containing body fluids obtained from a subject,
A method for equilibrating a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, comprising the steps of:
The column equilibration solution has a composition that satisfies at least all of the following conditions (a) to (d) and (g):
(a) Formula: Electrical conductivity [mS/cm] < -0.78 x buffer capacity value + 36.5
(b) Formula: Electrical conductivity [mS/cm]>-0.81 x buffer capacity value + 12.5
(c) Formula: Buffer capacity value>0.16
(d) Formula: Electrical conductivity [mS/cm]>0
(g) Formula: 9>pH>5
The Fc-binding protein is any one of the following polypeptides (1) to (3):
(1) A polypeptide comprising the amino acid residues from the 17th to the 192nd positions 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 positions, at least the 176th valine is replaced with phenylalanine;
(2) 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, at least 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;
(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 amino acid residues from the 17th to the 192nd, at least 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, and the 171st phenylalanine with serine;
That is, the method. The method according to claim 1 , wherein the equilibration solution has a composition that further satisfies the condition of formula (e).
(e) Formula: Electrical conductivity [mS/cm] < -0.84 x buffer capacity value + 30.5 The method according to claim 1 or 2, wherein the equilibration solution has a composition that further satisfies the condition of formula (f).
(f) Formula: 0.32<buffer capacity value<16 1. A method for isolating IgG1 and/or IgG3 from an antibody-containing body fluid obtained from a subject, comprising the steps of:
(1) adding the body fluid to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon to adsorb the antibody onto the carrier;
(2) a step including the equilibration method according to any one of claims 1 to 3 before and after (1), and (3) a step of adding an elution solution to the equilibrated column to elute the adsorbed antibody.