JP7602715B2 - Antibody Analysis Methods - Google Patents

Description

The present invention relates to a method for analyzing antibodies contained in a blood-derived sample. In particular, the present invention relates to a method for analyzing the antibodies with high accuracy.

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 purity 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 glycan structure bound to the antibody has a significant effect 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 detect the disease by analyzing the glycan structure attached to the antibody.

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

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 glycan structure bound to the Fc region of the antibody affects the affinity of the antibody with the Fc-binding protein, and the glycan structure can be determined based on the peak area in the resulting separation pattern (elution pattern, chromatogram) (Patent Document 3).

However, when the above analysis is performed on a blood-derived sample, the antibodies in the sample are subject to various glycosylation modifications, and as shown in the lower part of Figure 1 (measurement sample), for example, the boundaries between regions in the antibody separation pattern become unclear. This makes it difficult to accurately identify the peak regions and, ultimately, to accurately determine the glycostructure of the antibody.

JP 2016-194500 A JP 2016-099304 A WO2019/244901

CHROMATOGRAPHHY, 34(2), 83-88 (2013) Science, 320, 373-376 (2008) Nature Communication, 7, 11205 (2016)

The objective of the present invention is to provide a method for accurately identifying peak regions from the separation pattern of antibodies obtained when analyzing antibodies contained in a blood-derived sample by affinity chromatography using an insoluble carrier on which an Fc-binding protein is immobilized.

The inventors of the present invention 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 further, 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 affinity chromatography (Figure 1). In other words, the inventors have found that, by using the clear separation pattern for rituximab as an indicator, peak regions can be identified with high accuracy from the unclear separation pattern obtained when analyzing antibodies contained in a blood-derived sample, and have completed the present invention.

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) a step of 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) 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): identifying the structure of the glycan bound to the antibody based on the peak area identified by the method (5) (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 the following (1) to (3):
(1) A polypeptide comprising a sequence consisting of amino acid residues 17 to 192 of SEQ ID NO: 1, in which at least the 176th valine of SEQ ID NO: 1 is replaced 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 having substitutions, deletions, insertions and/or additions 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 with 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.

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 in 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 using 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 identified in this way.

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. 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.

The present invention relates to a method for analyzing an antibody 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) identifying peak regions in the separation pattern obtained in (1) based on the elution times identified in (3);

(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 blood (whole blood), diluted blood, serum, plasma, cerebrospinal fluid, umbilical cord blood, and blood components;
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.

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. Pretreatment may be appropriately selected from standard methods. Standard methods include centrifugation, purification using a column, etc.

The "subject" from which the blood-derived sample is isolated and which is the subject of the present invention refers to an individual who is the subject of 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 (cows, 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.

The antibody contained in the blood-derived sample in the present invention is also called gamma globulin or immunoglobulin, and may contain at least an Fc region with a sugar chain attached. 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.

(Rituximab)
In the method of the present invention, "rituximab" is used as an indicator for accurately identifying peak regions from the unclear separation pattern obtained when analyzing antibodies contained in the above-mentioned blood-derived sample. This is a monoclonal antibody consisting of an anti-human CD20 human-mouse chimeric antibody, and is an antibody 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.

There are no particular limitations on the sample containing rituximab that is subjected to the method of the present invention, but from the viewpoint of efficient binding to an insoluble carrier on which an Fc-binding protein is immobilized, a sample containing rituximab that has been substituted or diluted with the equilibration solution described below is preferred.

Fc-binding proteins
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 receptors, which are receptors for human immunoglobulin G (IgG), human Fcα receptors, which are receptors for human immunoglobulin A (IgA), human Fcδ receptors, which are receptors for human immunoglobulin D (IgD), and human Fcε receptors, which are receptors for human immunoglobulin E (IgE), and any of these receptors can be used as the human Fc binding protein in the present invention.

Specific examples of human Fcγ receptors include polypeptides that contain 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 some of the amino acid residues constituting the polypeptide have been substituted, deleted, inserted and/or added. Among these, polypeptides that contain at least a partial sequence of the extracellular domain of human FcγRIIIa and polypeptides in which some of the amino acid residues constituting the polypeptide have been substituted, deleted, inserted and/or added are preferred as human Fcγ receptors used as human Fc-binding proteins in the present invention.

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 replaced with phenylalanine;
(2) A polypeptide comprising the amino acid residues from position 17 to position 192 of the amino acid sequence set forth in SEQ ID NO:1, in which at least the valine at position 176 is replaced by phenylalanine, and further comprising substitution, deletion, insertion and/or addition of one or several amino acid residues at one or several positions other than position 176, and having antibody binding activity;
(3) A polypeptide having an amino acid sequence having 70% or more homology with 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.

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

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.

In (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).

In the present invention, the "number" of each amino acid residue refers to the order in which the first methionine is number 1 in the amino acid sequence described in each SEQ ID NO. Therefore, "number 176" in the present invention refers to the 176th amino acid in the amino acid sequence described in SEQ ID NO:1. Furthermore, "an amino acid residue corresponding to valine at number 176 in SEQ ID NO:1" refers to an amino acid residue in an amino acid sequence having 70% or more homology, which is arranged in the same position as valine at number 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 described in SEQ ID NO:1.

(Insoluble Carrier)
In the present invention, in order to separate the antibody from the blood-derived sample, the Fc-binding protein is immobilized on an insoluble carrier before use. The term "insoluble carrier" refers to a carrier that is insoluble in a liquid (e.g., a liquid used for antibody adsorption or elution, such as an equilibration liquid or an elution liquid) that is 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 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 polymer.

The immobilization of the Fc-binding protein to 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 the activated group), 1,4-butanediol diglycidyl ether (forming an epoxy group as the activated group), tresyl chloride (forming a tresyl group as the activated group), and vinyl bromide (forming a vinyl group as the 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).

(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.

In these steps (1) and (2), the separated patterns can be obtained by carrying out the following steps (a) and (b).
(a) adding a sample to a column packed with an insoluble carrier having an Fc-binding protein immobilized thereon, 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").

Each step is explained in detail below.

(a) Adsorption Step This step is a step in which an antibody-containing sample 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.

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.

The sample may be added to a column filled 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 the adsorption process, 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 the adsorption process 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 can be set appropriately within the range of 0°C to 50°C.

Before and/or after this step, an equilibration step of adding (delivering) an equilibration solution to the column may be performed. 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 the 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 higher separation accuracy. In particular, if the detection value between the unadsorbed fraction and the peak region detected after the addition of the eluent is a constant value, this is preferable because it 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.

An example of an equilibration solution is an aqueous buffer solution. Specifically, a weakly acidic to weakly alkaline buffer solution with a pH of 5.0 to 8.0 can be mentioned. 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. A salt such as sodium chloride or potassium chloride may be further added to the buffer solution. The salt is not particularly limited as long as it is a salt that can be easily imagined by a person skilled in the art.

(b) Separation step This step is a step of eluting the antibody adsorbed to the insoluble carrier in the step (a) using an eluent to obtain a separation pattern of the antibody. That is, the antibody adsorbed to the carrier can be eluted by adding (delivering) the eluent to the column. The conditions for carrying out the elution step, such as the type of eluent, the delivery format of the eluent, 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 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 eluent, it is sufficient to use one that weakens the affinity between the antibody and the Fc-binding protein, and for example, 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 when the equilibration step is performed). As a specific example, when the liquid phase before elution (for example, an equilibration solution) 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 a linear gradient elution in which the ratio of the eluent in the liquid phase is changed continuously to elute, a 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.

The antibody separation pattern is obtained by detecting the antibody eluted from the column using a detector. Examples of the detector include a UV detector and a mass detector. An example of the antibody separation pattern is a chromatogram at the time of antibody elution. The measurement data obtained by the detector can be acquired at any time interval, but since a longer time interval reduces the accuracy in reflecting the original characteristics of the antibody separation pattern in the chromatogram, the time interval is preferably 1 minute or less, more preferably 10 seconds or less, and even more preferably 2 seconds or less.

By this process, the antibody contained in the sample is 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, from the elution fraction by a conventional method. Specifically, the separated antibody can be collected, for example, from the elution fraction by a known method used for separating and purifying proteins.

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

More specifically, the method of the present invention (3) identifies the elution times that are the boundaries between the peak regions from the separation pattern obtained in (2) above, and (4) identifies the peak regions in the separation pattern obtained in (1) above based on the elution times identified in (3) above, thereby analyzing the antibodies contained in the blood-derived sample.

Specific examples of the peaks in the separation pattern in the present invention include peak area, peak elution time, peak width, number of peaks detected, and peak height. In particular, examples of the peaks identified in the present invention include peak elution time, peak width, and number of peaks detected. The antibody separation pattern may be used for peak extraction as is, or after appropriate correction such as baseline correction. The peaks may be absolute values or relative values. As a relative value, any elution time may be used as an arbitrary value, for example, the time when elution begins 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.

In the present invention, the "first to third peaks" may mean the peaks that elute first to third after the start of elution (e.g., after the start of the gradient), respectively, unless otherwise specified. The peaks may particularly have a peak area percentage of 1% or more. In other words, the "first to third peaks" may particularly mean the peaks that elute first to third after the start of elution, respectively, and have a peak area percentage of 1% or more. "Peak area percentage" means the value (percentage) obtained by dividing the area of each peak (peak region) by the area of all peaks.

In the present invention, the "peak region" 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 (hereinafter also referred to as the boundary time) that is the boundary between the first and second peaks 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 filled 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 performing baseline correction or may be the end time of elution, and is not particularly limited. However, it is preferable to set the arbitrary value from the time after a separation peak derived from an antibody bound to an insoluble carrier on which an Fc-binding protein packed in the column is immobilized to the time when a change in the detection value that occurs when the column is washed with an elution solution and then switched to an equilibration solution to equilibrate the column is obtained.

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 is at a minimum in the region between each peak top, or the elution time at which the sign of the derivative obtained by differentiating the chromatogram showing the detection value in the region between each peak top changes, which allows the definition of the peak region to be automated and also suppresses differences between operators, is particularly preferred.

The peak of the antibody can be identified by applying the boundary time between the peak regions used to determine the peak region of rituximab to the separation pattern of the antibody contained in the blood-derived sample. 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 process 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 with the elution time corrected can be used as a fixed value, and it is particularly preferable because it is not necessary to measure the separation pattern of rituximab each time in accordance with the measurement of the antibody. 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.

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

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.

<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. 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 exception that it has the amino acid substitutions shown in (I) to (X) below:
(I) Valine (Val) at position 27 of SEQ ID NO:1 (position 34 in SEQ ID NO:2) is replaced with glutamic acid (Glu); (II) Phenylalanine (Phe) at position 29 of SEQ ID NO:1 (position 36 in SEQ ID NO:2) is replaced with isoleucine (Ile); (III) Tyrosine (Tyr) at position 35 of SEQ ID NO:1 (position 42 in SEQ ID NO:2) is replaced with asparagine (Asn); (IV) Glutamine (Gln) at position 48 of SEQ ID NO:1 (position 55 in SEQ ID NO:2) is replaced with arginine (Arg); (V) Phenylalanine (Phe) at position 75 of SEQ ID NO:1 (position 82 in SEQ ID NO:2) is replaced with leucine (Leu); (VI) (VII) Substitution of valine (Val) at position 117 (position 124 in SEQ ID NO: 2) of SEQ ID NO: 1 with glutamic acid (Glu); (VIII) Substitution of glutamic acid (Glu) at position 121 (position 128 in SEQ ID NO: 2) of SEQ ID NO: 1 with glycine (Gly); (IX) Substitution of phenylalanine (Phe) at position 171 (position 178 in SEQ ID NO: 2) of SEQ ID NO: 1 with serine (Ser); (X) Substitution of valine (Val) at position 176 (position 183 in SEQ ID NO: 1) with phenylalanine (Phe).

(1) The hydroxyl groups on the surface of 2 mL of hydrophilic vinyl polymer for separation (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.

(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.

(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 a serum sample.

(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.

(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 "eluate") to obtain a separation pattern.

(4) After analyzing with the standard substance, the same procedure as in (2) and (3) was 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.

(5) The separation patterns obtained in (3) and (4) were baseline corrected so that the detection values at the time when the pH gradient started (7 minutes after the start of elution) and the time when the pH gradient ended (i.e., the eluate reached 100%) (18 minutes after the start of elution) were both 0.

(6) From the baseline-corrected separation pattern of the standard substance, the peak regions were determined as the regions divided by two elution times where 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 is zero. The first peak region was defined as the range from 7 minutes after the start of elution to the elution time where the derivative of the valley region between the first and second peaks is zero (boundary time between the first and second peaks), the second peak region was defined as the range from the elution time where the derivative of the valley region between the first and second peaks is zero to the elution time where the derivative of the valley region between the second and third peaks is zero (boundary time between the second and third peaks), and the third peak region was defined as the range from the elution time where the derivative of the valley region between the second and third peaks is 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.

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

(Comparative Example 1) <Peak identification of measurement sample when human myeloma plasma-derived IgG1 is used as 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.

As shown in Figure 1, when vertical dotted lines were drawn through the boundary times between the peak regions obtained from the separation pattern of rituximab, the dotted lines passed through points close to the boundary times of the peaks in the measured sample, revealing that the boundaries between the peak regions of rituximab and the antibodies contained in the blood-derived sample were almost identical.

On the other hand, as shown in Figure 2, when vertical dotted lines were drawn between the peak regions obtained from the separation pattern of human myeloma plasma-derived IgG1, the dotted lines passed through points different from the boundary times of the peaks of the measured sample, revealing that compared to rituximab, the boundaries between the peak regions of human myeloma plasma-derived IgG1 did not coincide with those of the antibodies contained in the blood-derived sample.

The results of the boundary times for Example 1 and Comparative Example 1 are summarized in Table 1 below.

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 regions of antibodies contained in blood-derived samples can be accurately identified by using each boundary time obtained in the analysis results of rituximab as an index.

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 medicine, such as predicting the activity (drug efficacy) of antibodies in the body and testing for diseases.

Claims (2)

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 elution times that are boundaries between peak regions from the separation pattern obtained in (2); and (4) identifying peak regions in the separation pattern obtained in (1) based on the elution times 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 . The method according to claim 1, further comprising the following step (5): (5) identifying the structure of the glycan bound to the antibody based on the peak area identified in (4).