Antibody analysis method

Abstract

To provide a method for accurately identifying peak regions of antibodies contained in a blood-derived sample, when analyzing by affinity chromatography using an Fc-binding protein-immobilized insoluble carrier, from the separation pattern of antibodies obtained in the analysis.SOLUTION: Disclosed is a method for analyzing antibodies contained in a blood-derived sample, comprising the steps of: (1) applying a blood-derived sample to a column filled with an Fc-binding protein-immobilized insoluble carrier to obtain a separation pattern of the antibodies; (2) applying a sample containing rituximab to the column to obtain a separation pattern of the rituximab; (3) identifying an elution time at each circumscription between peak regions from the separation pattern obtained in (2); and (4) identifying the peak regions in the separation pattern obtained in (1) on the basis of the elution times identified in (3).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for analyzing an antibody contained in a blood-derived sample, and in particular to a method for analyzing said antibody with high accuracy. [Background technology]

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

[0003] As a method for analyzing the sugar chain structure of an antibody, LC-MS analysis including cleavage of the sugar chain is mainly carried out (Patent Documents 1 and 2). However, this analysis method involves very complicated operations and requires a lot of time.

[0004] A simpler method for analyzing the molecular structure of an antibody is affinity chromatography analysis based on the affinity between an antibody and an Fc-binding protein immobilized on an insoluble carrier. In this method, the sugar chain structure bound to the Fc region of an antibody affects the affinity of the antibody with the Fc-binding protein, and the sugar chain structure can be determined based on the peak area in the resulting separation pattern (elution pattern, chromatogram) (Patent Document 3).

[0005] However, when the above analysis is performed on a blood-derived sample, the antibodies in the sample are subjected to various sugar chain modifications, and therefore the boundaries between regions in the antibody separation pattern become unclear, as shown in the lower part of Figure 1 (measurement sample). Therefore, it is difficult to accurately identify the peak regions and, by extension, to accurately determine the sugar chain structure of the antibody. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2016-194500 A [Patent Document 2] JP 2016-099304 A [Patent Document 3] WO2019 / 244901 [Non-patent literature]

[0007] [Non-Patent Document 1] CHROMATOGRAPHY, 34(2), 83-88(2013) [Non-Patent Document 2] Science, 320, 373-376 (2008) [Non-Patent Document 3] Nature Communication, 7, 11205(2016) Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for accurately identifying peak regions from the obtained separation pattern of antibodies when antibodies contained in a blood-derived sample are analyzed by affinity chromatography using an insoluble carrier onto which an Fc-binding protein is immobilized. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and have found that when anti-human CD20 human-mouse chimeric antibody (rituximab) is analyzed by affinity chromatography using an insoluble carrier on which an Fc-binding protein is immobilized, a clear separation pattern can be obtained, and furthermore, that the elution times (boundary times) at the boundaries between peaks are almost identical to those obtained when antibodies in a blood-derived sample are analyzed by the above affinity chromatography (Figure 1). In other words, the inventors have found that, by using the clear separation pattern of rituximab as an indicator, it is possible to identify the peak region with high accuracy from the unclear separation pattern obtained when analyzing antibodies contained in a blood-derived sample, and have completed the present invention.

[0010] That is, the present invention includes the following aspects [1] to [4]. [1] A method for analyzing antibodies contained in a blood-derived sample, comprising the following steps (1) to (4): (1) subjecting a blood-derived sample to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, and separating antibodies contained in the sample to obtain a separation pattern of the antibodies; (2) subjecting a sample containing rituximab to the column and separating rituximab contained in the sample to obtain a separation pattern of the rituximab; (3) identifying elution times that are boundaries between the peak regions from the separation pattern obtained in (2); (4) A step of identifying peak regions in the separation pattern obtained in (1) based on the elution times identified in (3). [2] The method according to [1], further comprising the following step (5): (5) A step of identifying the structure of the glycan bound to the antibody based on the peak region identified in (4). [3] The method according to [1] or [2], wherein the Fc binding protein is FcγRIIIa. [4] The method according to [1] or [2], wherein the Fc-binding protein is a polypeptide selected from any one of the following (1) to (3): (1) A polypeptide comprising a sequence consisting of amino acid residues 17 to 192 set forth in SEQ ID NO: 1, in which at least valine at position 176 set forth in SEQ ID NO: 1 is substituted with phenylalanine; (2) A polypeptide comprising a sequence consisting of amino acid residues from positions 17 to 192 of SEQ ID NO: 1, in which at least the valine at position 176 of SEQ ID NO: 1 is replaced with phenylalanine, and further comprising substitution, deletion, insertion and / or addition of one or several amino acid residues at one or several positions other than the 176th position, and having antibody-binding activity; (3) A polypeptide having an amino acid sequence having 70% or more homology to the sequence consisting of amino acid residues 17 to 192 of SEQ ID NO: 1, in which the amino acid residue corresponding to valine at position 176 of SEQ ID NO: 1 is replaced with phenylalanine, and having antibody-binding activity. Effect of the Invention

[0011] According to the present invention, when an antibody contained in a blood-derived sample is analyzed by affinity chromatography using an insoluble carrier on which an Fc-binding protein is immobilized, it is possible to accurately identify a peak region from the separation pattern of the antibody obtained by the analysis. This makes it possible to automate the identification of the peak of the antibody contained in the blood-derived sample, and since objectivity can be ensured by mechanically identifying the peak by a program, it is possible to obtain the same results regardless of the measuring device, location, operator, etc. Furthermore, it is possible to identify the structure of the glycan bound to the antibody in the blood-derived sample based on the peak region thus identified. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows a separation pattern obtained by analyzing rituximab using a column packed with an Fc-binding protein-immobilized gel, and the separation patterns of antibodies contained in a blood-derived sample, and shows the results of determining the peak regions of the antibodies based on the separation pattern of rituximab. [Diagram 2] FIG. 1 shows a separation pattern obtained by analyzing human myeloma plasma-derived IgG1 using a column packed with an Fc-binding protein-immobilized gel, and the separation patterns of antibodies contained in a blood-derived sample, and shows the results of determining the peak regions of the antibodies based on the separation pattern of human myeloma plasma-derived IgG1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention relates to a method for analyzing antibodies contained in a blood-derived sample, the method comprising the following steps (1) to (4): (1) subjecting a blood-derived sample to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, and separating antibodies contained in the sample to obtain a separation pattern of the antibodies; (2) subjecting a sample containing rituximab to the column and separating rituximab contained in the sample to obtain a separation pattern of the rituximab; (3) identifying elution times that are boundaries between the peak regions from the separation pattern obtained in (2); (4) A step of identifying peak regions in the separation pattern obtained in (1) based on the elution times identified in (3).

[0014] (Blood-derived samples) In the present invention, the term "blood-derived sample" refers to a solution that contains blood (whole blood) or a component thereof and that contains or may contain an antibody. The form of the solution may be a body fluid itself, such as blood or urine, as described below. Blood samples such as whole blood, diluted blood, serum, plasma, cerebrospinal fluid, cord blood, and apheresis; Specimens that may contain blood-derived components, such as urine, saliva, semen, feces, sputum, amniotic fluid, and ascites. Furthermore, the protein may be in the form of a buffer solution that can contain the protein separated from these body fluids or the protein contained therein.

[0015] These blood-derived samples may be directly used for evaluation in the method of the present invention described below, or may be used after appropriate pretreatment. The pretreatment may be appropriately selected from conventional methods. Conventional methods include centrifugation, purification using a column, etc.

[0016] The "subject" from which the blood-derived sample is isolated and which is the subject of the present invention refers to an individual subject to the method of the present invention, and is not particularly limited, and may be not only a human but also a non-human animal. Such subjects include, for example, vertebrates, preferably mammals, more preferably primates (humans, monkeys, chimpanzees, orangutans, gorillas, etc.), ungulates (cattle, horses, sheep, goats, etc.), and rodents (mice, rats, guinea pigs, etc.), but are usually humans. The subject may be either male or female. It may also be an individual of any age, such as a child, a young person, a middle-aged person, or an elderly person. Furthermore, it may be either a healthy individual or not.

[0017] The antibody contained in the blood-derived sample in the present invention is also called gamma globulin or immunoglobulin, and may contain at least a sugar chain-added Fc region. The antibody may be any of IgG, IgM, IgA, IgD, and IgE. However, when an Fc receptor is used as the Fc-binding protein used to separate the antibody described below, the antibody must be one that corresponds to the receptor. For example, when a human Fcγ receptor is used as the Fc-binding protein, the target antibody is human IgG. The IgG may be any of IgG1, IgG2, IgG3, and IgG4.

[0018] (Rituximab) In the method of the present invention, "rituximab" is used as an indicator to accurately identify peak regions from the unclear separation pattern obtained when analyzing antibodies contained in the above-mentioned blood-derived sample. It is a monoclonal antibody consisting of an anti-human CD20 human-mouse chimeric antibody and is identified by the following number. CAS Number: 174722-31-7 ATC Code: L01XC02 DrugBank:DB00073 UNII:4F4X42SYQ6 KEGG:D02994 ChEMBL:CHEMBL1201576. Rituximab is sold as a pharmaceutical (for example, by Zenyaku Kogyo and Chugai Pharmaceutical under the trade name Rituxan), and can be obtained by purchasing it.

[0019] The sample containing rituximab to be subjected to the method of the present invention is not particularly limited, but from the viewpoint of efficient binding to an insoluble carrier on which an Fc-binding protein is immobilized, a sample containing rituximab substituted or diluted with an equilibration solution described below is preferred.

[0020] (Fc binding protein) In the method of the present invention, the "Fc binding protein" used to separate the antibody from the blood-derived sample is not particularly limited as long as it is a polypeptide that has the ability to bind to the Fc region of the antibody contained in the sample and can recognize the difference in the sugar chain structure of the antibody (for example, the sugar chain structure of the Fc region). For example, when the antibody is an antibody derived from a human, the Fc binding protein can be a human Fc binding protein. A preferred example of the human Fc binding protein is a human Fc receptor. Examples of human Fc receptors include human Fcγ receptor, which is a receptor for human immunoglobulin G (IgG), human Fcα receptor, which is a receptor for human immunoglobulin A (IgA), human Fcδ receptor, which is a receptor for human immunoglobulin D (IgD), and human Fcε receptor, which is a receptor for human immunoglobulin E (IgE), and any of these receptors can be used as the human Fc binding protein in the present invention.

[0021] Specific examples of human Fcγ receptors include polypeptides comprising at least a partial sequence of the extracellular domain of human FcγRI (CD64), human FcγRIIa (CD32a), human FcγRIIb (CD32b), human FcγRIIc (CD32c), human FcγRIIIa (CD16a) or human FcγRIIIb (CD16b), as well as polypeptides in which a portion of the amino acid residues constituting the polypeptide has been substituted, deleted, inserted and / or added. Among these, polypeptides comprising at least a partial sequence of the extracellular domain of human FcγRIIIa and polypeptides in which a portion of the amino acid residues constituting the polypeptide has been substituted, deleted, inserted and / or added are preferred as human Fcγ receptors used as human Fc-binding proteins in the present invention.

[0022] Specific examples of polypeptides comprising at least a partial sequence of the extracellular region of human FcγRIIIa, or polypeptides in which some of the amino acid residues constituting the polypeptide have been substituted, deleted, inserted and / or added, include the polypeptides described in (1) to (3) below. (1) A polypeptide comprising amino acid residues 17 to 192 of the amino acid sequence set forth in SEQ ID NO:1, in which at least the 176th valine is substituted with phenylalanine; (2) A polypeptide comprising amino acid residues from position 17 to position 192 of the amino acid sequence set forth in SEQ ID NO:1, in which at least valine at position 176 is replaced by phenylalanine and further has substitutions, deletions, insertions and / or additions of one or several amino acid residues at one or several positions other than position 176, and which has antibody-binding activity; (3) A polypeptide having an amino acid sequence having 70% or more homology with a sequence consisting of amino acid residues 17 to 192 of SEQ ID NO: 1, in which the amino acid residue corresponding to valine at position 176 of SEQ ID NO: 1 is replaced with phenylalanine, and having antibody-binding activity.

[0023] An example of the polypeptide described in (1) above is a polypeptide containing amino acid residues 24 to 199 of the amino acid sequence described in SEQ ID NO: 2, and a polypeptide (Fc binding protein) disclosed in JP 2018-197224 A.

[0024] Examples of the substitution, deletion, insertion or addition described in (2) above include the substitutions of amino acid residues disclosed in JP 2015-086216 A, JP 2016-169167 A, and JP 2017-118871 A.

[0025] In the above (3), homology means similarity or identity, and can be determined using an alignment program such as BLAST (Basic Local Alignment Search Tool). For example, "identity of amino acid sequences" may mean identity between amino acid sequences calculated using blastp, specifically, identity between amino acid sequences calculated using blastp with default parameters. The homology may be 70% or more, and may be 80% or more, 85% or more, 90% or more, or 95% or more (e.g., 96% or more, 97% or more, 98% or more, or 99% or more).

[0026] In the present invention, the "position" of each amino acid residue refers to the order in which the first methionine is placed as the first in the amino acid sequence of each SEQ ID NO. Therefore, "position 176" in the present invention refers to the 176th position in the amino acid sequence of SEQ ID NO: 1. Furthermore, "an amino acid residue corresponding to valine at position 176 in SEQ ID NO: 1" refers to an amino acid residue in an amino acid sequence having a homology of 70% or more, which is arranged at the same position as valine at position 176 in the amino acid sequence shown in SEQ ID NO: 1, when the amino acid sequence is aligned with a sequence consisting of amino acid residues 17 to 192 of SEQ ID NO: 1.

[0027] (Insoluble Carrier) In the present invention, in order to separate antibodies from the above-mentioned blood-derived sample, the above-mentioned Fc-binding protein is used after immobilization on an insoluble carrier. The term "insoluble carrier" refers to a carrier that is insoluble in a liquid (e.g., a liquid used for adsorbing or eluting antibodies, such as an equilibration liquid or an elution liquid) passed through a column packed with the carrier, and it is preferable that the carrier has a functional group (e.g., a hydroxyl group) for immobilizing the Fc-binding protein by a covalent bond. 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.

[0028] The immobilization of the Fc-binding protein on the insoluble carrier can be achieved, for example, by utilizing a functional group on the surface of the carrier that can be immobilized by covalent bonding with the Fc-binding protein. More specifically, when the surface of the insoluble carrier has a hydroxyl group, an activating agent can be used to form an activated group capable of covalent bonding with the Fc-binding protein from the hydroxyl group, thereby covalently bonding the activated group 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, carboxy 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).

[0029] (Method for analyzing antibodies contained in blood-derived samples) The method of the present invention involves the steps of: (1) subjecting a blood-derived sample to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, and separating the antibodies contained in the sample to obtain a separation pattern of the antibodies; and (2) subjecting a sample containing rituximab to the column, and separating the rituximab contained in the sample to obtain a separation pattern of the rituximab.

[0030] In these steps (1) and (2), the separated pattern can be obtained by carrying out the following steps (a) and (b). (a) adding a sample to a column packed with an insoluble carrier onto which an Fc-binding protein has been immobilized, and allowing the antibody contained in the sample to be adsorbed onto the carrier (hereinafter also referred to as the "adsorption step"); (b) A step of eluting the antibody adsorbed on the carrier with an eluent to obtain an antibody separation pattern (hereinafter also referred to as a "separation step").

[0031] Each step will be described in detail below.

[0032] (a) Adsorption process This step involves adding an antibody-containing sample to a column packed with an insoluble carrier to which an Fc-binding protein has been immobilized, thereby allowing the antibody to be adsorbed onto the carrier.

[0033] The sample to be subjected to this step is the above-mentioned blood-derived sample or a sample containing rituximab. The sample may be added to the column after being dissolved, suspended, dispersed, or solvent-exchanged in a suitable liquid medium. An example of the liquid medium is the equilibration liquid described below.

[0034] The sample may be added to a column packed with an insoluble carrier on which an Fc-binding protein is immobilized, for example, by using a liquid delivery means such as a pump (hereinafter, in this specification, adding a liquid to a column is also referred to as "delivering a liquid to a column"). The conditions for carrying out the adsorption step, such as the amount of sample added (delivery), the type of liquid phase, the liquid phase delivery 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. An example of the liquid phase is the equilibration liquid described below. The 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 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 appropriately set within the range of 0°C to 50°C.

[0035] An equilibration step of adding (delivering) an equilibration solution to the column may be performed before and / or after this step. In particular, performing an equilibration step after this step is preferable in that it can remove from the column antibodies that do not bind to the Fc-binding protein and antibodies that bind to the protein in the sample atmosphere but not in the equilibration buffer atmosphere (the fraction containing the removed antibodies is also referred to as the "unadsorbed fraction" in this specification). The unadsorbed fraction refers to a fraction in the region until the peak detected after the sample is added (delivered) to the column reaches its minimum value during the equilibration step in the separation pattern described below. It is preferable that the unadsorbed fraction and the peak detected after the addition of the eluent in the separation step described below are separated in elution time from each other, since this results in high separation accuracy. In particular, it is preferable that the detection value between the unadsorbed fraction and the peak region detected after the addition of the eluent is a constant value, since this indicates that the unadsorbed fraction has been sufficiently removed from the column by the equilibration step. The constant value referred to here includes not only the same value but also a state in which the detection value changes with a constant slope.

[0036] An example of the equilibration solution is an aqueous buffer solution. Specifically, a weakly acidic to weakly alkaline buffer solution having a pH of 5.0 to 8.0 can be exemplified. The components of the buffer solution can be appropriately selected depending on various conditions such as the pH of the buffer solution. Examples of the 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 buffer solution may further contain a salt such as sodium chloride or potassium chloride. The salt is not particularly limited as long as it is a salt that a person skilled in the art can easily imagine.

[0037] (b) Separation process This step is a step of eluting the antibody adsorbed to the insoluble carrier in the step (a) with an elution solution to obtain a separation pattern of the antibody. That is, the antibody adsorbed to the carrier can be eluted by adding (delivering) the 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 a desired separation pattern is obtained. The conditions for carrying out the elution 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. As the elution solution, one that weakens the affinity between the antibody and the Fc-binding protein may be used, and examples thereof include an aqueous buffer solution having a pH that is more acidic than the liquid phase before elution (more specifically, the equilibration solution used in the equilibration step if an equilibration step is performed). As a specific example, when the liquid phase before elution (e.g., equilibration liquid) is a weakly acidic to weakly alkaline buffer solution having a pH of 5.0 to 8.0, an acidic buffer solution having a pH of 2.5 to 4.5 may be used as the eluent. The components of the buffer solution can be appropriately selected according to various conditions such as the pH of the buffer solution. Examples of the 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 delivery method of the eluent may be linear gradient elution in which the ratio of the eluent in the liquid phase is changed continuously to elute, or stepwise elution in which the ratio is changed stepwise to elute, or a combination thereof. The gradient may be set, for example, so that the ratio of the eluent in the liquid phase increases from 0% (v / v) to 100% (v / v) in 10 to 60 minutes, 15 to 50 minutes, or 20 to 40 minutes. The 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, so as to be proportional to the square of the inner diameter of the column. The column temperature may be set, for example, appropriately in the range of 0°C to 50°C.

[0038] The antibody eluted from the column is detected using a detector to obtain a separation pattern of the antibody. Examples of the detector include a UV detector and a mass detector. Examples of the separation pattern of the antibody include a chromatogram at the time of elution of the antibody. The measurement data obtained by the detector may be acquired at any time interval, but since the accuracy of reflecting the original characteristics of the antibody separation pattern in the chromatogram decreases as the time interval increases, the time interval is preferably 1 minute or less, more preferably 10 seconds or less, and particularly preferably 2 seconds or less.

[0039] This step allows the antibody contained in the sample to be obtained in a separated form. The separated antibody may be obtained, for example, as an elution fraction containing the antibody. That is, the separated antibody is obtained by collecting the elution fraction containing the separated antibody. The elution fraction can be collected, for example, by a conventional method. Specifically, the elution fraction can be collected, for example, by an automatic fraction collector such as an autosampler. Furthermore, the separated antibody may be recovered from the elution fraction. The separated antibody can be collected, for example, by a conventional method from the elution fraction. Specifically, the separated antibody can be collected, for example, from the elution fraction by a known method used for separating and purifying proteins.

[0040] The analytical method of the present invention is characterized in that it identifies a peak region of the separation pattern of antibodies contained in a blood-derived sample based on the separation pattern of rituximab obtained in the step (b).

[0041] More specifically, the method of the present invention analyzes antibodies contained in a blood-derived sample by (3) identifying elution times that are boundaries between each peak region from the separation pattern obtained in (2) above, and (4) identifying peak regions in the separation pattern obtained in (1) above based on the elution times identified in (3).

[0042] Specifically, the peak of the separation pattern in the present invention includes a peak area, a peak elution time, a peak width, the number of peaks detected, and a peak height. In particular, the peaks identified in the present invention include a peak elution time, a peak width, and the number of peaks detected. The separation pattern of the antibody may be used for peak extraction as it is, or after appropriate correction such as baseline correction. The peak may be an absolute value or a relative value. As the relative value, any elution time may be used as an arbitrary value, for example, the time when elution starts may be set to 0, or it may be a ratio or difference with respect to the total time for which the eluent was introduced.

[0043] In the present invention, the "first to third peaks" may mean the peaks which are eluted first to third after the start of elution (for example, after the start of gradient), respectively, unless otherwise specified. The peaks may particularly have a peak area% of 1% or more. In other words, the "first to third peaks" may particularly mean the peaks which are eluted first to third after the start of elution, respectively, and have a peak area% of 1% or more. The "peak area%" means the value (percentage) obtained by dividing the area of ​​each peak (peak region) by the area of ​​all peaks.

[0044] The "peak region" in the present invention refers to a range of detection values ​​having the above-mentioned peak and sandwiched between a specific elution time. For example, the first peak region refers to a detection value region between the elution time that is the boundary between the first and second peaks (hereinafter also referred to as the boundary time) and an arbitrary value of the elution time before the boundary time. The arbitrary value of the elution time may be a reference point when performing baseline correction or may be the start time of elution, and is not particularly limited, but is preferably set between the peak of the non-adsorbed fraction that does not bind to the insoluble carrier on which the Fc-binding protein packed in the column is immobilized and the separation peak derived from the antibody bound to the carrier is obtained. In addition, the second peak region refers to a detection value region between the boundary time of the first and second peaks and the boundary time of the second and third peaks, and the third peak region refers to a detection value region between the boundary time of the second and third peaks and an arbitrary value of the elution time after the boundary time. The arbitrary value of the elution time may be a reference point when baseline correction is performed or may be the end time of elution, and is not particularly limited. However, it is preferable to set the arbitrary value of the elution time from the time after a separation peak derived from the antibody bound to the insoluble carrier on which the Fc-binding protein packed in the column is immobilized to the column, until the time when a change in the detection value that occurs when the column is washed with the elution solution and then switched to an equilibration solution to equilibrate the column is obtained.

[0045] In the present invention, the method for determining the peak region of rituximab may be a conventional method, for example, the boundary time of the peak region may be determined visually. However, a method in which the boundary time of each peak is determined as the elution time at which the detection value in the region between each peak top shows a minimum value, or a method in which the boundary time is determined as the elution time at which the sign of the derivative obtained by differentiating a chromatogram showing the detection value in the region between each peak top changes, which can identify the boundary time of the peak region using a program language, is particularly preferred, since it enables the definition of the peak region to be automated and also suppresses differences between operators.

[0046] The boundary time between the peak regions used in determining the peak region of rituximab can be applied to the separation pattern of the antibody contained in the blood-derived sample to identify the peak of the antibody. The separation pattern of rituximab may be measured before or after the measurement of the antibody, and it is preferable to identify the peak based on the value measured immediately before or immediately after the measurement of the antibody, since the peak of the antibody can be identified with high accuracy. In addition, if the separation pattern of the antibody is corrected for elution time based on a standard substance and then the step of identifying the peak of the antibody contained in the blood-derived sample is performed, the boundary time of the peak obtained from the separation pattern of rituximab whose elution time has been corrected can be used as a fixed value, and it is not necessary to measure the separation pattern of rituximab each time in accordance with the measurement of the antibody, which is particularly preferable. By correcting the elution time, the peak boundary time of rituximab determined separately can be used even between column lots, between devices, and between different compositions of the equilibration solution and / or elution solution, so that the peak region of the antibody contained in the blood-derived sample can be easily identified.

[0047] The peak regions thus identified can distinguish the differences in the glycan structures of N-linked glycans bound to antibodies contained in blood-derived samples (Patent Document: WO2019 / 244901). Examples of the identifiable glycan structures include sialic acid, galactose, mannose, N-acetylglucosamine, and fucose that constitute N-linked glycans, and examples of the glycan structures 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. EXAMPLES

[0048] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0049] <Preparation of affinity chromatography column (FcR9_F column)> The Fc-binding protein FcR9_F_Cys obtained by the method of JP 2018-197224 A was immobilized on a gel by the method shown below to prepare an FcR9_F column. In FcR9_F_Cys (amino acid sequence described in SEQ ID NO: 2), the 1st methionine (Met) to the 22nd alanine (Ala) are the improved PelB signal peptide, the 24th glycine (Gly) to the 199th glutamine (Gln) are the amino acid sequence of the Fc-binding protein FcR9_F (JP 2018-197224 A) (corresponding to the region from the 17th to the 192nd in SEQ ID NO: 1), and the 200th glycine (Gly) to the 207th glycine (Gly) are the cysteine ​​tag sequence. Furthermore, the FcR9_F is a polypeptide consisting of amino acid residues from position 17 to position 192 of native FcγRIIIa shown in SEQ ID NO: 1, with the following amino acid substitutions (I) to (X): (I) Substitution of valine (Val) at position 27 of SEQ ID NO: 1 (position 34 of SEQ ID NO: 2) with glutamic acid (Glu) (II) Substitution of phenylalanine (Phe) at position 29 of SEQ ID NO: 1 (position 36 of SEQ ID NO: 2) with isoleucine (Ile) (III) Substitution of tyrosine (Tyr) at position 35 of SEQ ID NO: 1 (position 42 of SEQ ID NO: 2) with asparagine (Asn) (IV) Substitution of glutamine (Gln) at position 48 of SEQ ID NO: 1 (position 55 of SEQ ID NO: 2) with arginine (Arg) (V) Substitution of phenylalanine (Phe) at position 75 of SEQ ID NO: 1 (position 82 of SEQ ID NO: 2) with leucine (Leu) (VI) Substitution of asparagine (Asn) at position 92 of SEQ ID NO: 1 (position 101 of SEQ ID NO: 2) with serine (Ser) (VII) Substitution of valine (Val) at position 117 of SEQ ID NO: 1 (position 124 of SEQ ID NO: 2) with glutamic acid (Glu) (VIII) Substitution of glutamic acid (Glu) at position 121 of SEQ ID NO: 1 (position 128 of SEQ ID NO: 2) with glycine (Gly) (IX) Substitution of phenylalanine (Phe) at position 171 of SEQ ID NO: 1 (position 178 of SEQ ID NO: 2) with serine (Ser) (X) Substitution of valine (Val) at position 176 of SEQ ID NO: 1 (position 183 of SEQ ID NO: 2) with phenylalanine (Phe).

[0050] (1) The hydroxyl groups on the surface of 2 mL of hydrophilic vinyl polymer for separation agent (manufactured by Tosoh Corporation: packing material for liquid chromatography) were activated with iodoacetyl groups, and then 4 mg of FcR9_F_Cys obtained by the method disclosed in JP 2018-197224 A was reacted to obtain an FcR9_F immobilized gel.

[0051] (2) 1.2 mL of the FcR9_F immobilized gel prepared in (1) was packed into a φ4.6 mm × 50 mm stainless steel column to prepare an FcR9_F column.

[0052] (Example 1) <Peak identification of measurement sample when rituximab is used as standard substance> (1) Blood was collected from healthy subjects who had given informed consent and centrifuged to obtain serum. The serum was diluted 20-fold with phosphate buffered saline (PBS) (pH 7.4) and then passed through a 0.2 μm filter (Merck Millipore) to prepare serum samples.

[0053] (2) The FcR9_F column prepared above was connected to a high-performance liquid chromatography device (manufactured by Tosoh Corporation) and equilibrated with 10 mM citrate buffer (pH 6.5) containing 100 mM sodium chloride (hereinafter also referred to as "equilibration solution"), after which 10 μL of a rituximab antibody solution containing rituximab (manufactured by Zenyaku Kogyo Co., Ltd., trade name: Rituxan Infusion 100 mg) as a standard substance, prepared in the equilibration solution to a rituximab concentration of 1 mg / mL, was added at a flow rate of 1.2 mL / min. The measurement interval by the detector was set to obtain data every 1 / 5 seconds.

[0054] (3) After washing with the equilibration solution for 7 minutes at a flow rate of 1.2 mL / min, the adsorbed antibodies were eluted with a pH gradient (a gradient in which the eluate reached 100% in 11 minutes) using 10 mM citrate buffer (pH 4.5) containing 500 mM sodium chloride (hereinafter also referred to as the "elution solution") to obtain a separation pattern.

[0055] (4) After analysis with the standard substance, the same procedures as in (2) and (3) were used to analyze the serum sample prepared in (1) by adding 10 μL as the measurement sample, and the separation pattern of the antibodies contained in the serum sample was obtained.

[0056] (5) The separation patterns obtained in (3) and (4) were subjected to baseline correction so that the detection values ​​at the start of the pH gradient (7 min after the start of elution) and the end of the pH gradient (i.e., the eluate reached 100%) (18 min after the start of elution) were both 0.

[0057] (6) From the baseline-corrected separation pattern of the standard substance, the peak regions were determined as the regions divided by two elution times at which the derivative of the valley region between the three peaks (peaks 1, 2, and 3 from the shortest elution time) detected between 7 and 18 minutes after the start of elution was zero. The first peak region was defined as the range from 7 minutes after the start of elution to the elution time at which the derivative of the valley region between the first and second peaks was zero (boundary time between the first and second peaks), the second peak region was defined as the range from the elution time at which the derivative of the valley region between the first and second peaks was zero to the elution time at which the derivative of the valley region between the second and third peaks was zero (boundary time between the second and third peaks), and the third peak region was defined as the range from the elution time at which the derivative of the valley region between the second and third peaks was zero to 18 minutes after the start of elution. The peak regions of the sample were identified by applying the peak regions defined for the standard substance to those of the serum sample measured immediately after the analysis of the standard substance.

[0058] (7) On the other hand, based only on the separation pattern of the baseline-corrected measured sample, the elution times at the valleys of the peaks were estimated from the slope of the increase and decrease in the detection value 7 minutes after the start of elution, and the boundary times between the first and second peaks, and the boundary times between the second and third peaks were determined in order from the shortest elution time. The results are shown in Figure 1.

[0059] (Comparative Example 1) <Peak identification of measurement sample when human myeloma plasma-derived IgG1 is used as the standard substance> Except for using human myeloma plasma-derived IgG1 (Sigma) as the standard substance, the boundary times of the peaks in the serum samples were determined in the same manner as in Example 1. The results obtained are shown in FIG.

[0060] As shown in FIG. 1, when a vertical dotted line was drawn through the boundary times between each peak region obtained from the separation pattern of rituximab, the dotted line passed through a point close to the boundary times of each peak of the measurement sample, revealing that the boundaries between each peak region of rituximab and the antibodies contained in the blood-derived sample almost coincided.

[0061] On the other hand, as shown in Figure 2, when a vertical dotted line was drawn between each peak region obtained from the separation pattern of human myeloma plasma-derived IgG1, the dotted line passed through a point different from the boundary time of each peak of the measurement sample, revealing that, compared with rituximab, the boundaries between each peak region of human myeloma plasma-derived IgG1 did not coincide with those of the antibodies contained in the blood-derived sample.

[0062] The boundary times of Example 1 and Comparative Example 1 are summarized in Table 1 below.

[0063] [Table 1]

[0064] As shown in Table 1, the boundary times between the first and second peak regions obtained by analyzing the measurement sample were much closer in Example 1 (rituximab) than in Comparative Example 1 (human myeloma plasma-derived IgG1). Therefore, from this result, it is clear that the peak region of the antibody contained in the blood-derived sample can be accurately identified by using each boundary time obtained in the analysis result of rituximab as an index. [Industrial Applicability]

[0065] As described above, according to the present invention, it is possible to accurately identify the peak region of an antibody contained in a blood-derived sample. The peak region thus identified also makes it possible to identify the glycan structure bound to the antibody in the blood. It is known that the glycan structure bound to an antibody has a significant effect on the activity, dynamics, and safety of the antibody (antibody drug, etc.). Furthermore, diseases can be detected by analyzing the glycan structure attached to the antibody. Therefore, the present invention is useful in the fields of pharmaceuticals and medical care, such as prediction of the activity (drug efficacy) of an antibody in the body and testing for diseases.

Claims

1. A method for analyzing an antibody contained in a blood-derived sample, comprising the steps of: (1) subjecting a blood-derived sample to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, and separating antibodies contained in the sample to obtain a separation pattern of the antibodies; (2) subjecting a sample containing rituximab to the column and separating rituximab contained in the sample to obtain a separation pattern of the rituximab; (3) identifying the elution times that are the boundaries between the peak regions from the separation pattern obtained in (2); and (4) identifying a peak region in the separation pattern obtained in (1) based on the elution time identified in (3), The Fc-binding protein is a polypeptide comprising an amino acid sequence consisting of amino acid residues 17 to 192 of SEQ ID NO: 1, into which any of the following amino acid substitutions (I) to (X) have been introduced, and having antibody-binding activity. (I) Substitution of valine at position 27 of SEQ ID NO:1 with glutamic acid (II) Substitution of phenylalanine at position 29 of SEQ ID NO:1 with isoleucine (III) Substitution of tyrosine at position 35 of SEQ ID NO:1 with asparagine (IV) Substitution of arginine for glutamine at position 48 of SEQ ID NO:1 (V) Substitution of phenylalanine at position 75 of SEQ ID NO:1 with leucine (VI) Substitution of asparagine at position 92 of SEQ ID NO: 1 with serine (VII) Substitution of valine at position 117 of SEQ ID NO: 1 with glutamic acid (VIII) Substitution of glutamic acid at position 121 of SEQ ID NO:1 with glycine (IX) Substitution of phenylalanine at position 171 of SEQ ID NO: 1 with serine (X) The 176th valine in SEQ ID NO: 1 is replaced with phenylalanine.

2. The method according to claim 1, further comprising the following step (5): (5) A step of identifying the structure of the sugar chain bound to the antibody based on the peak area identified in (4).

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