Chromatographic carrier and purification method of antibody

Abstract

According to one embodiment, the present invention provides a chromatography carrier comprising: a base carrier containing porous particles; and a ligand immobilized on the base carrier, the ligand being represented by formula (1), or being a polyallylamine or a salt thereof in which a part of amino groups are modified,-NH-Z-X (1) (in formula (1), Z represents a divalent hydrophobic group; and X represents NH2 or a heterocyclic group containing a nitrogen atom). The ligand is immobilized on the base carrier via one or more active groups that are reactive with the ligand.

Description

TECHNICAL FIELD

[0001] The present application relates to a chromatography carrier and a method for purifying an antibody using the same. BACKGROUND

[0002] It is well known that purification of bio-pharmaceuticals is performed using chromatography techniques, which utilize various intermolecular interactions for separation of a target from impurities. As examples thereof, there are ion exchange chromatography utilizing electrostatic interaction, hydrophobic interaction chromatography utilizing hydrophobic interaction, protein A affinity chromatography utilizing affinity interaction for antibodies, and the like.

[0003] The ion exchange chromatography is most frequently used in the purification of bio-pharmaceuticals. In the ion exchange chromatography, in the case of a sample having a high conductivity of a treatment liquid, it is necessary to reduce the conductivity to, for example, 5 mS / cm by dilution or desalination before performing an adsorption treatment.

[0004] The hydrophobic interaction chromatography is widely used next to the ion exchange chromatography. In the hydrophobic interaction chromatography, in order to adsorb proteins and the like, it is necessary to contain a high concentration of, for example, 2.0 moles / liter or more of ammonium sulfate or sodium sulfate in an adsorption buffer. Therefore, in order to treat a large amount of culture solution, a large amount of salt is required, which becomes a factor of raising manufacturing costs.

[0005] In order to compensate for the disadvantages of such ion exchange chromatography and hydrophobic interaction chromatography, in recent years, development of a multimodal chromatography carrier is actively performed, which has two or more kinds of ligands capable of interacting with each other by different principles introduced on the same base carrier.

[0006] The multimodal chromatography carrier exhibits a different selectivity from a chromatography carrier on which a ligand having only an ion exchange group or only a hydrophobic group is supported.

[0007] For example, there is known a method of purifying an antibody using a carrier having an anion exchange group and an aromatic group in a flow-through mode (Patent Literature 1). In addition, there is also known a method of purifying an antibody using a carrier in which a part of a primary amino group is modified with a hydrophobic group (Patent Literature 2).

[0008] Further, there is also known a method of purifying a biological molecule using a chromatography carrier in which a ligand containing an anion exchange group, an alkyl group, and an aryl group or a heteroaryl group is bound to a base carrier via a spacer (Patent Literature 3). In particular, there is a view that a chromatography carrier having an amino group and a hydrophobic group adsorbs proteins under a high conductivity condition (Non-Patent Literature 1).

[0009] However, existing multi-mode chromatography carriers have insufficient separation properties between target proteins (e.g., antibodies) and impurity proteins (e.g., host cell proteins, HCPs), resulting in the adsorption of both impurity and target proteins simultaneously. Consequently, it is difficult to obtain the target protein with a high recovery rate.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: Japanese Patent No. 5064225

[0013] Patent Document 2: International Publication No. 2018 / 092691

[0014] Patent Document 3: International Publication No. 2021 / 046284

[0015] Non-patent literature

[0016] Non-patent literature 1: Journal of Chromatography A, 1016 (2003), 21-33 Summary of the Invention

[0017] The problem that the invention aims to solve

[0018] The purpose of this invention is to provide a chromatographic carrier capable of purifying target substances with high recovery rates, and a method for purifying antibodies using the carrier.

[0019] Technical means to solve the problem

[0020] Based on the aforementioned issues, the inventors conducted in-depth research and discovered that if a chromatographic carrier consisting of a ligand containing a hydrophobic group and an amino group and having a specific structure is immobilized on a base carrier, the target substance can be purified with a high recovery rate, thus completing the present invention.

[0021] The present invention is as described below, for example. [1]

[0023] A chromatographic carrier comprising:

[0024] A base carrier containing porous particles; and ligands immobilized on the base carrier.

[0025] The ligand is represented by the following formula (1), or is a polyallylamine or its salt with a partially amino-modified component.

[0026] -NH-ZX (1)

[0027] (In formula (1), Z represents a divalent hydrophobic group; X represents NH2, or a heterocyclic group containing a nitrogen atom)

[0028] The amino-modified polyallylamine or its salt comprises structural units represented by formula (2) and structural units represented by formula (3) or formula (3a), with a weight average molecular weight of 1,000 to 500,000.

[0029] [Chemistry 1]

[0030]

[0031] In formulas (3) and (3a), Y is independently selected from hydrogen atoms, alkyl groups, aryl groups, and -COR groups, respectively. 2 and -COOR 2 In the group formed (here, R) 2 [(alkyl, aryl, amino, or alkoxy)]

[0032] The ligand is immobilized on the base carrier via one or more active groups that are reactive to the ligand.

[0033] [2] According to the chromatographic carrier described in [1], wherein the ligand is represented by the formula (1).

[0034] [3] According to the chromatographic carrier of [1] or [2], wherein Z in the formula (1) represents alkylene, cycloalkylene, -NR-, -O-, arylene, -S- or a combination thereof, which may each have substituents (here, R represents hydrogen atom, alkyl, aryl, alkyl ester, aryl ester, alkyl ether or aryl ether).

[0035] [4] According to the chromatographic carrier described in [3], wherein Z in the formula (1) represents an alkylene group having 2 to 12 carbon atoms, a cycloalkylene group having 3 to 6 carbon atoms, -O-, a phenylene group, or a combination thereof.

[0036] [5] According to the chromatographic carrier of [1], wherein the ligand is a polyallylamine containing a portion of the structural unit represented by formula (2) and the structural unit represented by formula (3) and modified with amino groups, wherein in formula (3), one of Y is an alkyl, phenyl or -COR having 1 to 12 carbon atoms. 2 (Here, R) 2 One is an alkyl or phenyl group with 1 to 12 carbon atoms, and the other is a hydrogen atom.

[0037] [6] According to the chromatographic carrier of [1], wherein the ligand comprises a group derived from a compound:

[0038] The compound is selected from 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, 2-methyl-1,3-propanediamine, 1,5-diaminopentane, 1,3-diaminopentane, 1,6-diaminohexane, 3,3'-diaminodipropylamine, 3,3'-diamino-N-methyldipropylamine, 2-methyl-1,5-diaminopentane, 1,4-diaminocyclohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 4-(2-aminoethyl)cyclohexylamine, 1 The group consisting of one or more compounds including 9-diaminononane, 1,10-diaminodecane, 1,4-butanediol-bis(isopropylamine), 1,11-diaminoundecane, 1,12-diaminododecane, N,N-bis(3-aminopropyl)1,4-butanediamine, 6,6'-diaminodihexylamine, p-xylenediamine, 4-pyridinemethylamine, and polyallylamines that are modified with a portion of the amino groups of the structural units represented by formula (2') and the structural units represented by formula (3').

[0039] [Chemistry 2]

[0040]

[0041] [7] The chromatographic carrier according to any one of [1] to [6], wherein the active group is selected from the group consisting of epoxy group, formyl group and vinyl sulfone group.

[0042] [8] The chromatographic carrier according to any one of [1] to [7], wherein the porous particles are cross-linked cellulose.

[0043] [9] The chromatographic carrier according to any one of [1] to [8], wherein the average pore diameter of the porous particles is 200 nm or more.

[0044] [9-1] The chromatographic carrier according to any one of [1] to [9], wherein the volume average particle diameter of the porous particles is 30 μm to 1000 μm.

[0045] [9-2] The chromatographic carrier according to any one of [1] to [9-1], wherein the ligand density is 30 μmol / g to 1500 μmol / g.

[0046]

[10] A method for purifying an antibody, comprising: contacting a solution containing the antibody and a host-derived protein (HCP) with a chromatographic carrier according to any one of [1] to [9-2] to obtain an antibody solution with reduced HCP content.

[0047]

[11] According to the method of

[10] , the step of contacting the solution containing the antibody and host-derived protein (HCP) with the chromatographic carrier is performed by passing the solution through a container containing the chromatographic carrier in a flow-through mode.

[0048]

[12] The method according to

[10] or

[11] , wherein the antibody is a monoclonal antibody.

[0049]

[13] The method according to any one of

[10] to

[12] , wherein the HCP content in the antibody solution is less than 300 ppm.

[0050]

[14] The method according to any one of

[10] to

[13] , wherein the average pore size of the porous particles is 300 nm to 1500 nm, and the HCP content in the antibody solution is less than 100 ppm.

[0051]

[15] The antibody is purified by any one of

[10] to

[14] with a recovery rate of more than 85%.

[0052] The effects of the invention

[0053] This invention provides a chromatographic carrier capable of purifying target substances with high recovery rates, and a method for purifying antibodies using the carrier. Detailed Implementation

[0054] The embodiments of the present invention will now be described in detail. Furthermore, the materials, structures, etc., described below are not intended to limit the present invention, and various modifications can be made within the scope of the spirit of the present invention.

[0055] 1. Definition

[0056] Unless otherwise specified, the terminology used in this specification shall be used in the meanings commonly used by those skilled in the art.

[0057] In this specification, "Tris" refers to tris(hydroxymethyl)aminomethane.

[0058] In this specification, for example, when it is described as "20 mM Tris-HCl buffer (pH 7.0) + NaCl (6 mS / cm)", it refers to the following solution: each 1 L of the solution contains 2.42 g (20 mM) of tris(hydroxymethyl)aminomethane, and the pH is adjusted to 7.0 with hydrochloric acid (HCl) and the conductivity is adjusted to 6 mS / cm with sodium chloride (NaCl).

[0059] In this specification, "antibody" refers to an immunoglobulin molecule that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, or peptides through at least one antigen recognition site located in a variable region of the immunoglobulin molecule. As used in this specification, an antibody includes not only native (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments (Fab, Fab', F(ab')2, and Fv, etc.), single chains (single-chain variable fragments (scFv)), mutants thereof, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently bound modified antibodies, i.e., other modified configurations of immunoglobulin molecules containing antigen recognition sites of desired specificity. Antibodies include any class of antibodies, such as immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin G (IgG), immunoglobulin A (IgA), or immunoglobulin M (IgM) (or their subclasses). Antibodies do not need to be of any specific class. Immunoglobulins can be classified into different classes based on the amino acid sequence of the antibody invariant region of the heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0060] In this specification, "HCP" is a general term for proteins derived from the host cell.

[0061] In this specification, "removal" means the removal of substances other than the target substance from the solution before purification. For example, the term "HCP removed" refers to a state in which the amount of HCP is reduced compared to the solution before contact with the chromatographic carrier. This is because, for example, when a solution containing HCP is contacted with the chromatographic carrier, the HCP is retained on the chromatographic carrier, resulting in a reduction in the amount of HCP in the recovered solution.

[0062] In this specification, the term "binding-elution mode" refers to a purification method in which the target substance is temporarily bound to the chromatographic carrier, and then the target substance is eluted and recovered. For example, in the case of purifying antibodies, firstly, the antibody is bound to the chromatographic carrier, while impurities pass through the column without binding to the carrier. Subsequently, using a mobile phase with an appropriate salt concentration or pH, the antibody bound to the chromatographic carrier is eluted into the mobile phase and recovered. Examples of elution methods include: one-step elution, in which the antibody is eluted by passing a buffer solution with a specific salt concentration or pH that reduces the affinity between the antibody and the chromatographic carrier; stepwise elution, in which the antibody is eluted by changing the salt concentration or pH in stages; and gradient elution, in which the antibody is eluted by continuously changing the salt concentration or pH.

[0063] In this specification, "flow-through mode" refers to a purification method in which impurities bind to the chromatographic carrier while the target substance flows and is recovered without binding to the chromatographic carrier. For example, when the target substance is an antibody and the impurity is a host-derived protein (HCP), the HCP binds to the chromatographic carrier, while the antibody flows in the column without binding to the chromatographic carrier. In this case, the antibody may also bind to the chromatographic carrier to some extent, but the HCP binds to the chromatographic carrier more selectively, thereby purifying the antibody.

[0064] 2. Chromatographic carrier

[0065] A chromatographic support according to one embodiment of the present invention comprises: a base support containing porous particles; and a ligand immobilized on the base support.

[0066] The ligand is represented by the following formula (1),

[0067] -NH-ZX (1)

[0068] (In the formula, Z represents a divalent hydrophobic group; X represents NH2, or a heterocyclic group containing a nitrogen atom.)

[0069] The ligand is immobilized on the base carrier via one or more active groups that are reactive to the ligand.

[0070] Alternatively, in another embodiment, the ligand may also be a partially amino-modified polyallylamine or a salt thereof. The partially amino-modified polyallylamine or a salt thereof comprises a structural unit represented by formula (2) below, and a structural unit represented by formula (3) below or formula (3a) below, and has a weight average molecular weight of 1,000 to 500,000.

[0071] [Chemistry 3]

[0072]

[0073] In formulas (3) and (3a), Y is independently selected from hydrogen atoms, alkyl groups, aryl groups, and -COR groups, respectively. 2 and -COOR 2 In the group formed (here, R) 2 [(alkyl, aryl, amino, or alkoxy)]

[0074] The chromatographic support of the embodiment is a multimode chromatographic support having a hydrophobic group and an amino group on the ligand, exhibiting excellent separation ability for target substances and impurities. As a result, the target substance can be obtained with high recovery and purity; in other words, the impurity removal ability is excellent. Although the reason why this effect can be obtained by using a ligand with the structure described above is uncertain, it can be speculated that the amino group of the ligand can form hydrogen bonds in addition to participating in electrostatic interactions, and can also interact with impurities through the hydrophobic interaction of the hydrophobic group. Therefore, the impurities can be interacted with through multiple complementary interactions.

[0075] The target substance is not particularly limited, and examples include: antibodies and other proteins, peptides, nucleic acids, plasmids, viruses, virus-like particles, extracellular vesicles, etc. As defined above, antibodies can include: polyclonal antibodies, monoclonal antibodies, their antigen-binding fragments (Fab, Fab', F(ab')2, and Fv, etc.), single chains (scFv), their mutants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), as well as glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies, etc., preferably monoclonal antibodies or their antigen-binding fragments.

[0076] Impurities are not specifically limited but can include: antibody agglomerates, host-derived proteins (HCPs), host-derived deoxyribonucleic acid (DNA), exudate protein A, viruses, endotoxins, culture medium components, nutrients, etc. Among these, HCPs, being mixtures of various proteins, have attracted considerable attention in recent years. HCPs can not only be recognized as foreign antigens by the immune system, but they also contain substances with enzymatic activity that can break down target substances. Therefore, there is a need to effectively isolate HCPs to obtain target substances in low-concentration states. This is particularly beneficial in the field of antibody pharmaceuticals, where the effective removal of HCPs that can affect the quality or safety of pharmaceutical products is highly advantageous.

[0077] When the chromatographic carrier of the embodiments is used for antibody purification, antibodies can be obtained with high recovery rates, and the resulting antibody solution contains a low amount of HCP. In other words, it can be said that the chromatographic carrier of the embodiments has particularly excellent separation capabilities for antibodies and HCP.

[0078] (1) Basic carrier

[0079] Chromatographic supports generally have a structure in which ligands are bound to a base support. The base support used in the chromatographic supports of the embodiments contains porous particles, which, as described later, can be modified by an active group for introducing the ligands. The support used is not limited as long as it can be modified by an active group; preferred examples include: agarose, dextran, starch, cellulose, amylopectin, chitin, chitosan, cellulose triacetate, cellulose diacetate, and other polysaccharides and their derivatives; and organic polymers such as polyacrylamide, polymethacrylamide, polyacrylate, polymethyl methacrylate, polyalkyl vinyl ether, and polyvinyl alcohol. In terms of ensuring mechanical strength, the porous particles preferably have a cross-linked structure. More preferably, cross-linked cellulose particles are used, where the backbone of the cellulose particles is strengthened through a cross-linking reaction.

[0080] There are no particular restrictions on the cross-linked cellulose particles as long as they can be used as the base carrier for chromatography. The cellulose used as raw material can be crystalline cellulose or amorphous cellulose.

[0081] As a suitable cross-linked cellulose, a porous cellulose gel disclosed in Japanese Patent Application Publication No. 2009-242770 can be cited as an example. The porous cellulose gel disclosed in that publication can be obtained by a method comprising the following steps: continuously or partially adding, for more than 3 hours, a cross-linking agent in an amount of 4 to 12 times the molar amount of the cellulose monomer and an alkali in an amount of 0.1 to 1.5 times the molar amount of the cross-linking agent to a suspension of uncross-linked cellulose particles in the presence of at least one inorganic salt selected from the group consisting of hydrochloride, sulfate, phosphate, and borate, in an amount of 6 to 20 times the molar amount of the cellulose monomer. The cross-linked cellulose particles thus obtained have high mechanical strength, can be used under high-flow-rate chromatographic conditions, and can produce highly productive chromatographic carriers. Here, "cellulose monomer" refers to glucose units, which are the structural units of cellulose. Furthermore, the molar amount of cellulose monomer (i.e., the degree of polymerization) is calculated based on the amount obtained by subtracting water from the glucose units (i.e., the dry weight of cellulose) (with a molecular weight of 162 set as 1 mole). The crosslinking agent can be appropriately selected from those commonly used in this field, such as epichlorohydrin, epibromohydrin, dichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, glycerol polyglycidyl ether, etc., preferably epichlorohydrin, epibromohydrin or glycerol glycidyl ether.

[0082] The shape of the porous particles is not particularly limited, but spherical shapes are preferred in terms of creating a packed bed with high mechanical strength, excellent gel settling properties, and uniformity. In this case, the true sphericity of the porous particles is preferably 0.8 to 1.0. Here, "true sphericity" refers to the ratio of the minor axis to the major axis of the porous particles.

[0083] Spherical cellulose particles can be readily obtained, for example, by dissolving and regenerating crystalline cellulose or cellulose containing crystalline and amorphous regions. Examples of methods for manufacturing spherical cellulose particles include: methods using acetates as described in Japanese Patent Publication Nos. 55-39565, 55-40618, and 2023-037724; methods using a solution containing calcium thiocyanate as described in Japanese Patent Publication No. 63-62252; methods using a solution containing p-formaldehyde and dimethyl sulfoxide as described in Japanese Patent Publication No. 59-38203; and methods using a cellulose solution obtained by dissolving cellulose in an amide containing lithium chloride as described in Japanese Patent Publication No. 3663666. Furthermore, spherical cross-linked cellulose particles can be obtained by cross-linking spherical cellulose particles.

[0084] The particle size of the porous particles is preferably 10 μm to 500 μm, particularly preferably 50 μm to 150 μm. Furthermore, the average particle size is preferably 30 μm to 1000 μm, more preferably 40 μm to 200 μm, and particularly preferably 50 μm to 100 μm. Here, "particle size" refers to the measured value of the particle size of each porous particle, and "average particle size" refers to the average value calculated based on the particle size, particularly the volume average particle size.

[0085] In this specification, the particle diameter and average particle diameter of porous particles can be measured, for example, using a laser diffraction / scattering particle diameter distribution measuring device. The device irradiates a particle swarm with laser light and determines the particle size distribution based on the intensity distribution pattern of the emitted diffracted / scattered light. The particle diameter and average particle diameter are then calculated based on the particle size distribution. A specific measuring device such as the LA-960 laser diffraction / scattering particle diameter distribution measuring device (Horiba Manufacturing Co., Ltd.) can be used.

[0086] Alternatively, particle diameter can be determined using images taken with an optical microscope. Specifically, the particle diameter on the image is measured using a vernier or similar tool, and the original particle diameter is calculated based on the magnification. Then, the average particle diameter is calculated using the values ​​of each particle diameter obtained from the optical microscope image and the following formula.

[0087] Volume average particle diameter (MV) = Σ(nd4 ) / Σ(nd 3 )

[0088] [In the formula, d represents the particle diameter value of each particle obtained from the optical microscope image, and n represents the number of particles measured.]

[0089] The porosity of porous particles can be characterized by their pore size properties. One indicator of pore size is the retention time of a standard substance when porous particles are packed into a column. Here, retention time refers to the time required from sample injection into the column until the elution peak. Pore size affects the physical strength of the particles or the diffusivity of the target substance to be purified within the porous particles. Therefore, depending on the pore size, there will be differences in the flow rate of the liquid passing through the porous particles or the dynamic adsorption capacity of the porous particles. Therefore, it is necessary to design porous particles to achieve a pore size appropriate to the target.

[0090] The average pore diameter (d) of porous particles pore The average pore size is preferably 80 nm or more (e.g., 80 nm to 5000 nm, 80 nm to 3000 nm, 80 nm to 1500 nm), more preferably 200 nm or more (e.g., 200 nm to 5000 nm, 200 nm to 3000 nm, 200 nm to 1500 nm), further preferably 300 nm or more (e.g., 300 nm to 5000 nm, 300 nm to 3000 nm, 300 nm to 1500 nm), and particularly preferably 700 nm or more (e.g., 700 nm to 5000 nm, 700 nm to 3000 nm, 700 nm to 1500 nm). Alternatively, the average pore size may also be 500 nm or more (e.g., 500 nm to 5000 nm, 500 nm to 3000 nm, 500 nm to 1500 nm). Here, the average pore size refers to the value obtained based on the gel partition coefficient Kav calculated using standard polyethylene oxide, which can be specifically calculated using the method described in the examples below.

[0091] By using porous particles with an average pore size within the aforementioned range as the basic carrier, a high adsorption capacity is achieved for target substances or impurities with large molecular sizes, or aggregates of impurities. Cellulose particles with the aforementioned average pore size are particularly preferred, and cross-linked cellulose particles with the aforementioned average pore size are more preferred.

[0092] (2) Ligands

[0093] The chromatographic support of the embodiments comprises a ligand immobilized on a base support, and in one embodiment, the ligand has a structure represented by the following formula (1):

[0094] -NH-ZX (1)

[0095] (In the formula, Z represents a divalent hydrophobic group; X represents NH2, or a heterocyclic group containing a nitrogen atom).

[0096] In formula (1), the divalent hydrophobic group represented by Z is not particularly limited as long as it exhibits hydrophobicity. Z as a whole may exhibit hydrophobicity, or only a part of Z may exhibit hydrophobicity. Examples of such Z include alkylene, cycloalkylene, -NR-, -O-, arylene, -S-, or combinations thereof, which may each have substituents (here, R represents a hydrogen atom, alkyl, aryl, alkyl ester, aryl ester, alkyl ether, or aryl ether). More preferably, Z is an alkylene with 2 to 12 carbon atoms, a cycloalkylene with 3 to 6 carbon atoms, -O-, phenylene, or combinations thereof.

[0097] As for the substituents that Z can have, there are no limitations as long as they do not hinder the hydrophobicity of Z. Examples include: alkyl, cycloalkyl, aryl, alkyl ether, aryl ether, alkyl thioether, aryl thioether, etc.

[0098] Examples of heterocyclic groups containing nitrogen atoms in X in formula (1) include pyridyl, pyrrole, imidazolyl, indolyl, triazine, quinolinyl, etc. X is particularly preferred to be NH2.

[0099] In another embodiment, the ligand is a partially amino-modified polyallylamine or a salt thereof. The partially amino-modified polyallylamine or a salt thereof comprises a structural unit represented by formula (2) below, and a structural unit represented by formula (3) below or formula (3a):

[0100] [Chemistry 4]

[0101]

[0102] In formulas (3) and (3a), Y is independently selected from hydrogen atoms, alkyl groups, aryl groups, and -COR groups, respectively. 2 and -COOR 2 In the group formed (here, R) 2 (e.g., alkyl, aryl, amino, or alkoxy).

[0103] Y in formula (3) may further have substituents. There are no limitations on the substituents that Y may have; examples of substituents described above that are also possible substituents for Z in formula (1) are listed. Y is preferably a hydrogen atom, a phenyl group, an alkyl group having 1 to 12 carbon atoms, or -COR. 2 More preferably, it is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, or -COR. 2 (R)2 (It is an alkyl group having 1 to 4 carbon atoms).

[0104] A portion of amino-modified polyallylamines or their salts may be composed of structural units represented by formula (2), and structural units represented by formula (3) or formula (3a), or may contain other arbitrary structural units.

[0105] When the ligand is a salt of a partially amino-modified polyallylamine, the type of salt is not particularly limited; examples include hydrochloride, phosphate, sulfate, and acetate. The salt in formula (3a) is relative to N... + The anti-charged ion can be appropriately selected according to the structure of formula (3a). Examples of anti-charged ions include Cl... - SO4 2- HSO4 - PO4 3- HPO4 2- H2PO4 - COO - C6H5O7 3- C6H6O7 2- C6H7O7 - BO3 3- HBO3 2- H2BO3 - And so on, but not limited to these.

[0106] The weight average molecular weight of a portion of the amino-modified polyallylamine or its salt is 1,000 to 500,000, preferably 1,500 to 50,000, and more preferably 10,000 to 20,000.

[0107] As particularly preferred ligands, examples can be found derived from the compounds shown in Table 1 below.

[0108] [Table 1-1]

[0109] Table 1

[0110]

[0111] [Table 1-2]

[0112] Table 1 (continued)

[0113]

[0114] It is also preferable to use ligands derived from polyallylamine, for example preferably ligands derived from polyallylamine containing structural units represented by the following formula (2') and structural units represented by the following formula (3'), or partially amino-modified polyallylamine composed of these structural units.

[0115] [Chemistry 5]

[0116]

[0117] The method of binding the ligand to the base support is not particularly limited, and binding can be achieved through one or more active groups that are reactive to the amino group in the ligand. For example, the base support can be modified in advance using an active group, and then the ligand can be bound to it. Examples of such active groups include N-succinic acid hydroxyimide, epoxy, formyl, vinyl sulfone, acrylonitrile, etc., with epoxy, formyl, or vinyl sulfone being preferred.

[0118] The ligand density (the amount of ligand bound per 1 g of the base carrier) is preferably 30 μmol / g to 1500 μmol / g, more preferably 70 μmol / g to 750 μmol / g, and particularly preferably 140 μmol / g to 360 μmol / g or 170 μmol / g to 310 μmol / g. Alternatively, the ligand density may also be 140 μmol / g to 600 μmol / g or 150 μmol / g to 550 μmol / g. By setting this ligand density, a chromatographic carrier that maintains high adsorption capacity for target analytes or impurities while also exhibiting excellent cleanability and reusability can be obtained.

[0119] (3) Uses

[0120] The chromatographic carriers described in this embodiment can be used for the separation and purification of various target substances.

[0121] In purification, a chromatographic support is first packed into a column, but the packing morphology is not particularly limited. Then, a sample solution containing the target substance is brought into contact with the chromatographic support, thereby separating the target substance from impurities. Specifically, the chromatographic support is packed into the column, and a sample solution is flowed into it, selectively adsorbing either the target substance or the impurities onto the chromatographic support, thereby purifying the target substance. Alternatively, the target substance and impurities are adsorbed together onto the chromatographic support, and the elution conditions (e.g., salt concentration) are varied periodically or continuously, thereby utilizing differences in affinity for the chromatographic support to purify the target substance.

[0122] In setting chromatographic conditions, the differences in affinity between the target substance and impurities for the chromatographic carrier are utilized. For example, the conditions are set considering differences in carrier structure (ligand type, ligand density, ligand orientation, particle size, pore size, basic matrix composition, etc.) or the physicochemical properties of the target substance and impurities (isoelectric point, charge, hydrophobicity, molecular structure, stereostructure, etc.). The conditions can be adjusted to perform chromatography in binding-elution mode or flow-through mode.

[0123] The components of buffer solutions used in sample solutions, column cleaning, elution, etc., are not particularly limited as long as they have buffering capacity. Examples include: phosphates, citrates, acetates, succinates, maleates, borates, Tris (base), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-morpholinoethanesulfonic acid (MES), 1,4-piperazinediethanesulfonic acid (PIPES), 3-(N-morpholino)propanesulfonic acid (MOPS), Trisethanesulfonic acid (TES), Tris(hydroxymethyl)methylglycine, etc. The salt may also be used in combination with other salts such as sodium chloride, potassium chloride, calcium chloride, sodium citrate, sodium sulfate, and ammonium sulfate. Furthermore, the buffer solution may also contain amino acids such as glycine, alanine, arginine, serine, threonine, glutamic acid, aspartic acid, and histidine, sugars such as glucose, sucrose, lactose, and sialic acid, or derivatives thereof.

[0124] The pH of the buffer solution is preferably in the range of 2 to 9, and more preferably in the range of 3 to 8.

[0125] The linear velocity of the buffer solution is preferably in the range of 20 cm / h to 1000 cm / h.

[0126] By means of the method described herein, the target substance can be purified with a recovery rate preferably of 85% or more, more preferably 90% or more, and particularly preferably 95% or more. Here, the recovery rate refers to the ratio of the amount of the purified target substance recovered to the amount of the target substance loaded on the chromatographic carrier (i.e., the mass of the target substance in the sample solution before purification).

[0127] The chromatographic carrier described in this embodiment is preferably used for, for example, the purification of proteins, particularly antibodies. As mentioned above, due to its particularly excellent ability to separate antibodies from HCPs, an antibody solution with low HCP content can be obtained when used for antibody purification.

[0128] Therefore, according to one embodiment of the present invention, a method for purifying an antibody is provided, comprising: contacting a solution containing an antibody and a host-derived protein (HCP) with the chromatographic carrier to obtain an antibody solution with reduced HCP content. Here, the antibody is preferably a monoclonal antibody. The step of contacting the solution containing the antibody and the host-derived protein (HCP) with the chromatographic carrier is preferably performed by passing the sample solution through a container containing the chromatographic carrier in a flow-through mode.

[0129] The antibody loading per unit volume of the chromatographic carrier is, for example, 10 g / L to 2000 g / L, 100 g / L to 1500 g / L, or 200 g / L to 1500 g / L. When using the chromatographic carrier of the embodiment, even if the antibody loading per unit volume of the chromatographic carrier is 200 g / L or more, 500 g / L or more, or 1000 g / L or more, purified antibodies can be obtained with high recovery and / or low impurity levels (especially low HCP levels). That is, when using the chromatographic carrier of the embodiment, even if a large amount of antibody is loaded onto the chromatographic carrier at one time, purified antibodies can be obtained with high recovery and / or low impurity levels, thus enabling efficient and good antibody purification.

[0130] According to the method of the embodiment, the amount of HCP contained in the purified antibody solution (recovered fraction) is 300 ppm or less (0 ppm to 300 ppm), preferably 100 ppm or less (0 ppm to 100 ppm), more preferably 30 ppm or less (0 ppm to 30 ppm), particularly preferably 15 ppm or less (0 ppm to 15 ppm) or 10 ppm or less (0 ppm to 10 ppm). Furthermore, the amount of HCP is calculated according to the formula {amount of HCP in the purified antibody solution (ng) / amount of antibody in the purified antibody solution (mg)}.

[0131] The purification method described in this embodiment can also be combined with other purification methods. Other purification methods include those suitable for antibody purification, such as chromatography, activated carbon treatment, alcohol fractionation, precipitate removal, salting out, buffer exchange, concentration, dilution, filtration, virus inactivation, and virus removal. One or more of these methods can be selected and performed before or after the purification method described in this embodiment.

[0132] In addition, in the field of monoclonal antibody manufacturing, cation exchangers are generally used in binding-elution mode and anion exchangers are used in flow-through mode. However, as long as the chromatographic carrier of the embodiment is used, the method of using cation exchangers in flow-through mode can also be adapted.

[0133] In cases where other purification methods involve chromatography, the carriers or membranes used may include: heparin carriers and affinity carriers such as protein A, affinity membranes, cation exchange carriers, cation exchange membranes, anion exchange carriers, anion exchange membranes, gel filtration carriers, hydrophobic interaction carriers, reverse-phase carriers, hydroxyapatite carriers, fluorapatite carriers, sulfated cellulose carriers, sulfated agarose carriers, multimode carriers, etc.

[0134] Example

[0135] The present invention will be described in more detail below through embodiments, but the content of the present invention is not limited to the embodiments described.

[0136] <Example 1: Manufacturing of Cellulose Particles>

[0137] [Manufacturing of cross-linked cellulose particles (containing water) A]

[0138] (Granulation step)

[0139] (1) Add 6.4 g of crystalline cellulose (manufactured by Asahi Kasei Chemicals Co., Ltd., trade name: Ceolus PH101) to 100 g of 60% by weight aqueous solution of calcium thiocyanate, and heat to 110℃~120℃ to dissolve.

[0140] (2) Add 6 g of dehydrated sorbitol monooleate as a surfactant to the solution. Add it dropwise to 480 mL of o-dichlorobenzene preheated to 130℃~140℃ and stir at 200 rpm~300 rpm to obtain a dispersion.

[0141] (3) The dispersion was then cooled to 40°C. It was then injected into 190 mL of methanol to obtain a suspension of particles.

[0142] (4) The obtained suspension is filtered to separate and recover the particles, and the particles are washed with 190 mL of methanol. The washing operation is performed several times.

[0143] (5) The particles are further washed with a large amount of water to obtain spherical cellulose particles.

[0144] (6) The obtained spherical cellulose particles are passed through sieves with a mesh size of 125 μm and 53 μm to obtain spherical cellulose particles with a particle size of 53 μm to 125 μm.

[0145] (Cross-linking step)

[0146] (1) Add 121 g of pure water to 100 g of the obtained spherical cellulose particles (containing water) and heat while stirring. When the temperature reaches 30°C, add 3.3 g of 45% NaOH aqueous solution and 0.5 g of NaBH4, and then heat and stir. Here, the initial alkali concentration is 0.69% (w / w).

[0147] (2) After 30 minutes, add 60 g of Na2SO4 to the reaction solution and dissolve it. Starting from the point when the temperature of the mixture reaches 50°C, maintain the temperature at 50°C and continue stirring for 2 hours.

[0148] (3) While continuing to stir the mixture at 50°C, add 48 g of 45% NaOH aqueous solution and 50 g of epichlorohydrin in 24 equal portions every 15 minutes for about 6 hours.

[0149] (4) After the addition is complete, allow the mixture to react at 50°C for 16 hours.

[0150] (5) After cooling the reaction mixture to a temperature below 40°C, add 2.6 g of acetic acid for neutralization.

[0151] (6) The reaction mixture is filtered and the cellulose particles are recovered. The cellulose particles are then filtered and washed with pure water to obtain cross-linked cellulose particles A.

[0152] [Manufacturing of cross-linked cellulose particles (containing water) B]

[0153] (Granulation step)

[0154] (1) Add 100 g of cellulose acetate (L-20, manufactured by Daicel) to a mixed solvent of 391 g of benzyl alcohol, 276 g of 1-hexanol and 3.5 g of polypropylene glycol (Wako Pure Chemical Reagent, diol type, average molecular weight 1,000) and stir.

[0155] (2) Heat the mixture and stir it at 120°C for 4 hours to dissolve the cellulose acetate and obtain a transparent cellulose acetate solution.

[0156] (3) Add 2 g of polyvinyl alcohol (PVA) (JP-18E, manufactured by JAPAN VAM & POVAL) and 30 g of sodium carboxymethyl cellulose (CMC1140, manufactured by Daicel Miraizu) to 2300 g of pure water saturated with the mixed solvent, heat the mixture and stir and dissolve it at 80°C for more than 1 hour to obtain the dispersion medium.

[0157] (4) 770 g of the cellulose acetate solution was rapidly injected into 2300 g of the dispersion medium to obtain a dispersion system. Subsequently, the dispersion system was cooled to 35°C to obtain spherical cellulose acetate particles.

[0158] (5) After thoroughly washing the obtained cellulose acetate particles with a large amount of water and then with methanol, wash them again with pure water.

[0159] (Saponification step)

[0160] (1) 630 g of the obtained cellulose acetate particles (wet) (moisture content 6.86%) were added to a mixture of 1072 g of pure water and 242 g of methanol, and the mixture was stirred for 30 minutes after the temperature was set to 35°C. In addition, the moisture content of the cellulose particles was calculated by "wet weight of cellulose particles / dry weight".

[0161] (2) Add 201 g of 20% NaOH and stir at 35°C for 2 hours to allow it to react, thereby performing saponification.

[0162] (3) Cool to below 30°C, neutralize with acetic acid, and then wash thoroughly with pure water.

[0163] (4) The obtained saponified cellulose spherical particles are passed through sieves with a mesh size of 150 μm and 45 μm to obtain saponified cellulose particles with a particle size of 45 μm to 150 μm.

[0164] (Cross-linking step)

[0165] (1) After dispersing 96.4 g of the obtained saponified cellulose particles (moisture content 7.93%) in 194 g of pure water, 78.6 g of Na2SO4 was dissolved.

[0166] (2) After the temperature reaches 50℃, stir for 30 minutes, then add 4.0 g of 48% NaOH and 0.69 g of NaBH4, and let it dissolve and react for 30 minutes.

[0167] (3) After dissolving, add 36.4 mL of 48% NaOH solution and 62 g of epichlorohydrin in 8 equal portions every 30 minutes for about 4 hours.

[0168] (4) After the addition is complete, react at 50°C for 16 hours. Cool the temperature to below 30°C and add acetic acid for neutralization.

[0169] (5) The reaction mixture is filtered and the gel is recovered. The cross-linked porous particles are obtained by filtering and washing with pure water.

[0170] [Manufacturing of cross-linked cellulose particles (containing water) C]

[0171] The amount of polypropylene glycol used in the granulation step was changed to 7.0 g, and the particles were otherwise manufactured using the same method as the cross-linked cellulose particles B.

[0172] [Manufacturing of cross-linked cellulose particles (containing water) D]

[0173] The amount of polypropylene glycol used in the granulation step was changed to 10.5 g, and the particles were otherwise manufactured using the same method as the cross-linked cellulose particles B.

[0174] [Manufacturing of cross-linked cellulose particles (containing water) E]

[0175] The amount of polypropylene glycol used in the granulation step was changed to 14.0 g, and the particles were otherwise manufactured using the same method as the cross-linked cellulose particles B.

[0176] [Manufacturing of cross-linked cellulose particles (containing water) F]

[0177] The amount of polypropylene glycol used in the granulation step was changed to 8.5 g, and the particles were otherwise manufactured using the same method as the cross-linked cellulose particles B.

[0178] <Example 2: Analysis of Cross-linked Cellulose Particles>

[0179] The cross-linked cellulose particles A to F manufactured in Example 1 were analyzed as follows.

[0180] (Determination of average particle diameter)

[0181] The volume average particle diameter was measured using a laser diffraction / scattering particle diameter distribution measuring device LA-960 (manufactured by Horiba Manufacturing Co., Ltd.).

[0182] (Elution time of standard polyethylene oxide)

[0183] The cross-linked cellulose particles A to F obtained above, after being filled into a stainless steel tubular column (manufactured by Tosoh) with an inner diameter of 0.78 cm and a length of 30 cm, were used to measure the molecular weight of 4.42 × 10⁻⁶ cellulose particles. 4 ~5.8×10 5The holding time of TSKgel standard polyethylene oxide (SE-70, SE-30, SE-15, SE-8, SE-5, manufactured by Tosoh Corporation) was determined. Regarding the packing method, after dispersing the particles in pure water to form a slurry, it was packed into a column, and then pure water was circulated at a flow rate of 0.4 ml / min for at least 1 hour for compaction. Pure water was used as the mobile phase for the measurement, and 10 μL of polyethylene oxide prepared to a concentration of 5.0 mg / mL was injected. The elution peak was defined as the time when the refractive index (RI) detection intensity reached its maximum, and the holding time was defined as the time of this peak. The apparatus used for the measurement is described below.

[0184] Apparatus: 1260 Infinity high performance liquid chromatography (HPLC) system (manufactured by Agilent Technologies)

[0185] (average pore diameter d) pore (determination)

[0186] The average pore size can be calculated based on the gel partition coefficient K obtained using standard polyethylene oxide. av To calculate the relationship between the average pore size and the gel partition coefficient K av The relationship is expressed by the following formula (where the porosity within the particle = ε). p The viscosity radius of a standard polyethylene oxide molecule is r. m Average pore radius r pore ).

[0187] K av =ε p (1-r m / r pore ) 2

[0188] Gel partition coefficient K av It uses a weight-average molecular weight of 4.42 × 10⁻⁶. 4 Da ~ 5.8 × 10 5 The standard polyethylene oxide (SE-70, SE-30, SE-15, SE-8, SE-5, manufactured by Tosoh Corporation) was used as a sample, and the volume was calculated based on the relationship between its capacity and column volume using the following formula. Furthermore, pure water was used as the mobile phase.

[0189] K av =(V e -V0) / (V t -V0)

[0190] [In the formula, V] e V represents the sample retention capacity (mL). t V represents the volume of the empty column (mL), V0 represents the volume of the interparticle voids (mL), and the volume is calculated as 0.4 × V0. t And find out]

[0191] By comparing the viscosity radius of multiple standard polyethylene oxide molecules with K... av The measured values ​​to the power of 0.5 are plotted, and the average pore radius r is calculated based on the slope of the approximate curve. pore Let the value obtained by multiplying it by 2 be d. pore .

[0192] The analytical results of the cross-linked cellulose particles are shown in Table 2.

[0193] [Table 2]

[0194] Table 2

[0195]

[0196] <Example 3: Modification of cross-linked cellulose particles using active groups and ligands>

[0197] (1) Epoxidized cellulose particles

[0198] [Epoxidation of cellulose particles]

[0199] 3000 g of cross-linked cellulose particles A obtained in Example 1 and 1952 g of pure water were added to a 10 L stainless steel (SUS) container to prepare a slurry. Next, 1764 g of epichlorohydrin was added. After heating to 28°C, 1655 g of a 48.7% sodium hydroxide aqueous solution was added dropwise over 2 hours at a temperature not exceeding 30°C. After the addition was complete, the mixture was stirred at 30°C for 3 hours. Then, 145 g of acetic acid was added and stirred for 10 minutes. After the reaction was complete, the wet particles were filtered and recovered. The recovered wet particles were washed 16 times with 6 L of pure water to obtain the target epoxidized cellulose particles.

[0200] [Immobilization of ligands on epoxidized cellulose particles - 1]

[0201] Each ligand compound was dissolved in 16.5 mL of pure water, and the solution was heated to 30 °C. Then, 15.0 g of the epoxidized cellulose particles obtained above were added, and the mixture was stirred at 30 °C for 18 hours. After the reaction was complete, the wetted particles were filtered and recovered. The recovered particles were washed six times with 50 mL of pure water to obtain the target compounds (carriers 1 to 5). The densities of each ligand bound to the epoxidized cellulose particles are shown in Table 3. Here, ligand density (μmol / g) refers to the amount of ligand bound per 1 g of the base carrier.

[0202] [Table 3]

[0203] Table 3

[0204]

[0205] [Immobilization of ligands on epoxidized cellulose particles - 2]

[0206] The solvent for dissolving each ligand compound was changed to 13.1 g of methanol. After the reaction was completed, three washes were performed using 50 mL of methanol. Otherwise, the ligand immobilization reaction was carried out using the same method as described above to obtain the target compounds (carriers 6 to 9). The densities of each ligand bound to the epoxidized cellulose particles are shown in Table 4.

[0207] [Table 4]

[0208] Table 4

[0209]

[0210] [Ligand Immobilization on Epoxidized Cellulose Particles - 3]

[0211] Using the cross-linked cellulose particles E obtained in Example 1, except that epoxidized cellulose particles were obtained by the same method as those of the carrier 1.

[0212] 117.4 g of a 15.3% aqueous solution of polyallylamine PAA-15C (NITTOBO MEDICAL Co., Ltd.) with an average molecular weight of 15000 was mixed with 30.5 g of pure water. Next, 40.0 g of the epoxidized cellulose particles obtained above were added, and the mixture was heated to 45°C and stirred for 18 hours. After the reaction was complete, the wet particles were filtered and recovered. The recovered particles were washed six times with 120 mL of pure water to obtain an intermediate. 20.0 g of the obtained intermediate was suspended in 28.5 g of methanol, and 0.36 g of valeric anhydride was added. The mixture was stirred at 30°C for 16 hours (to obtain a ligand with a valeroyl group added to a portion of the amino group of PAA-15C). After the reaction was completed, the wetted particles were filtered and recovered. The recovered particles were then washed once with 60 mL of methanol, five times with 60 mL of pure water, once with 20 mL of 0.5 mol / L sodium hydroxide solution, and six times with 60 mL of pure water to obtain the target compound (support 23). The ligand density of support 23 is shown in Table 5. Furthermore, the ligand density of support 23 was calculated based on the molar amount of amino groups in the ligands.

[0213] [Table 5]

[0214] Table 5

[0215]

[0216] (2) Formylated cellulose particles

[0217] [Formylation of cellulose particles]

[0218] 100 g of cross-linked cellulose particles A obtained in Example 1 and 248 g of pure water were added to a glass container to prepare a slurry. After heating to 30°C, 0.57 g of sodium periodate was added, and the mixture was stirred at 30°C for 2 hours. After the reaction was completed, the wet particles were filtered and recovered. The recovered particles were washed 6 times with 200 mL of pure water to obtain the target formylated cellulose particles.

[0219] [Immobilization of ligands on formylated cellulose particles - 1]

[0220] 8.1 g of each ligand compound was dissolved in 11.2 g of pure water, and the mixture was heated to 30°C. Then, 15.0 g of the formylated cellulose particles obtained above were added, and the mixture was stirred at 30°C for 3 hours. Next, 0.06 g of sodium borohydride was added, and the mixture was stirred at 30°C for 2 hours. After the reaction was complete, the wetted particles were filtered and recovered. The recovered particles were washed six times with 50 mL of pure water to obtain the target compounds (carriers 10 and 11). The densities of each ligand bound to the formylated cellulose particles are shown in Table 6.

[0221] [Table 6]

[0222] Table 6

[0223]

[0224] [Immobilization of ligands on formylated cellulose particles - 2]

[0225] The solvent for dissolving the ligand compound was changed to 8.9 g of methanol. After the reaction was completed, three washes were performed using 50 mL of methanol. Otherwise, the ligand immobilization reaction was carried out using the same method as described above to obtain the target compounds (support 12 and support 13). The densities of the ligands bound to the formylated cellulose particles are shown in Table 7.

[0226] [Table 7]

[0227] Table 7

[0228]

[0229] (3) Vinylsulfonated cellulose particles

[0230] Vinylsulfonation of cellulose particles

[0231] 1.4 g of sodium carbonate was dissolved in 25.9 g of pure water, and 16.1 g of cross-linked cellulose particles A1 obtained in Example 1 were added to prepare a slurry. 1.5 g of divinyl sulfone was added while stirring at 25°C, and the mixture was stirred at 25°C for 1 hour. After the reaction was completed, the wet particles were filtered and recovered. The recovered particles were washed 6 times with 50 mL of pure water to obtain the target vinylsulfonated cellulose particles.

[0232] [Ligand Immobilization on Vinyl Sulfonated Cellulose Particles]

[0233] 5.8 g of the ligand compound was dissolved in 6.9 g of methanol, and the mixture was heated to 30 °C. Then, 10.0 g of the vinylsulfonated cellulose particles obtained above were added, and the mixture was stirred at 30 °C for 3 hours. After the reaction was complete, the wetted particles were filtered and recovered. The recovered particles were washed with 20 mL of methanol and then with 20 mL of pure water six times to obtain the target compound (support 14). The densities of the ligands bound to the vinylsulfonated cellulose particles are shown in Table 8.

[0234] Table 8

[0235] Table 8

[0236]

[0237] (4) Cellulose particles B ~ Cellulose particles F

[0238] In addition to using cross-linked cellulose particles B to F obtained in Example 1, carriers 15 to 20 and carrier 24 were manufactured using the same method as carrier 12, carrier 21 was manufactured using the same method as carrier 10, and carrier 22 was manufactured using the same method as carrier 11. Furthermore, in carrier 19, the amount of sodium periodate added during the formylation of the cellulose particles was changed to 0.23 g. The densities of ligands bound to the formylated cellulose particles are shown in Table 9.

[0239] [Table 9]

[0240] Table 9

[0241]

[0242] <Example 4: Antibody purification using a chromatographic carrier-1>

[0243] [Preparation of antibody solutions 1-9]

[0244] 471 mL of culture supernatant containing monoclonal antibody (IgG1) from Chinese hamster ovary (CHO) cells was purified using protein A carrier fibrin SPA-HC (manufactured by JNC Corporation) to obtain 50 mL of solution. 0.1 M hydrochloric acid was then added to this solution until the pH reached 3.4, and the solution was incubated at 25°C for 1 hour to inactivate the virus. 1 M trihydroxyaminomethane aqueous solution was added to the virus-inactivated solution until the pH reached 5.0. The solution was filtered using a 0.45 μm pore size filter due to turbidity. The concentration of monoclonal antibody in the filtrate was 19.6 mg / mL, which was diluted with ultrapure water. Subsequently, the pH was adjusted to 7.0 and the conductivity to 6 mS / cm using 1 M trihydroxyaminomethane aqueous solution and 5 M sodium chloride aqueous solution to obtain the antibody solution.

[0245] The concentration of the antibody solution was calculated by dividing the absorbance at 280 nm obtained using an absorbance meter by 1.40. The amount of HCP in the antibody solution was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cygnus F-550-1). Using the obtained HCP amount and the amount of monoclonal antibody calculated based on the absorbance at the measurement wavelength of 280 nm, the HCP amount (ppm) was calculated according to the formula {HCP amount in antibody solution (ng) / monoclonal antibody amount in antibody solution (mg)}. The obtained concentrations and HCP amounts of the antibody solutions are shown in Table 10.

[0246] [Table 10]

[0247] Table 10

[0248]

[0249] [Antibody purification using chromatographic carriers]

[0250] (Chromatographic carriers, equipment, etc.)

[0251] Carriers: Carriers 2 to 8, Carrier 12, Carriers 14 to 17

[0252] Tube string: inner diameter 0.5 cm, height 3 cm

[0253] System: AKTA avant 25

[0254] Buffer A: 20 mM Tris-HCl buffer (pH 7.0) + NaCl (6 mS / cm)

[0255] (process)

[0256] The carrier was packed into the column to a height of 1.5 cm. The column was connected to the system and equilibrated by passing 20 column volumes of buffer A through the column at a flow rate of 0.075 mL / min. Subsequent steps were also performed at a flow rate of 0.075 mL / min. Next, the antibody solution was passed through the column to achieve an antibody loading of 200 g / L per unit volume of the chromatographic carrier. Then, 10 column volumes of buffer A were passed through for washing. Following this, 5 column volumes of 0.1 M aqueous acetic acid were passed through. Next, 5 column volumes of 2 M sodium chloride solution were passed through. Following this, 5 column volumes of 0.5 M aqueous sodium hydroxide solution were passed through for washing. Finally, 20 column volumes of buffer A were passed through for reequilibration.

[0257] The total volume of 6 mL of column permeate during antibody solution passage and 3 mL of column permeate during unadsorbed material washing were combined as the recovery fraction. The recovery rate of the monoclonal antibody (the ratio of antibody recovery to the amount of antibody loaded on the chromatographic carrier) and the amount of HCP in the recovery fraction are shown in Table 11. The recovery rate of the monoclonal antibody was calculated using the method described below.

[0258] (Recovery rate of monoclonal antibody (%))

[0259] The absorbance of the antibody solution before purification and the recovered fraction (the purified antibody solution) was measured at a wavelength of 280 nm using a spectrophotometer. The antibody concentration was calculated by dividing the absorbance at 280 nm by 1.40, and the recovery rate (%) was calculated using the formula (amount of antibody in the recovered fraction / amount of antibody in the antibody solution before purification) × 100.

[0260] [Table 11]

[0261] Table 11

[0262]

[0263] As shown in Table 11, by using the chromatographic carriers of the embodiments, a high antibody recovery rate can be obtained, and the amount of impurities (HCP) in the recovered fraction is low. In particular, the amount of HCP in the chromatographic carriers (carriers 6, 12, 14 to 17) is reduced to a single-digit level.

[0264] <Example 5: Antibody purification using a chromatographic carrier - 2>

[0265] Carriers 12, 15-24 were used as chromatographic carriers, and antibody solutions 3, 4, 7-9 were used as antibody solutions. The antibody loading per unit volume of the chromatographic carrier was changed to 10¹⁰ g / L. Otherwise, the antibody was purified using the same method as in Example 4. The recovery rate of the monoclonal antibody and the amount of HCP in the recovered fraction are shown in Table 12.

[0266] Furthermore, in Table 12, the comparative example using Capto adhere as a carrier was used, and the antibody was purified using the same method as described above, except that Capto adhere (manufactured by Cytiva) was used. Additionally, the ligand compound of Capto adhere is a quaternary amine having a benzyl group in the substituent.

[0267] [Table 12]

[0268] Table 12

[0269]

[0270] As shown in Table 12, even with an increased antibody loading in the column, a higher antibody recovery rate can be achieved when using the chromatographic carriers described in this embodiment compared to using Capto adhere. Furthermore, the amount of HCP in the recovered fraction is significantly lower. A tendency was observed that a larger average pore size of the porous particles resulted in a lower amount of HCP in the recovered fraction; this was particularly evident when using carriers 17, 18, 22, 23, and 24.

[0271] Several embodiments of the present invention have been described, but these embodiments are provided as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments or variations thereof are included in the scope or spirit of the invention, and are included within the scope of the invention as described in the claims and its equivalents.

Claims

1. A chromatography carrier comprising: a base carrier containing porous particles; and a ligand immobilized on the base carrier, the ligand being represented by the following formula (1), or being a part-amino-modified polyallylamine or a salt thereof, -NH-Z-X (1) (in formula (1), Z represents a divalent hydrophobic group; X represents NH2, or represents a heterocyclic group containing a nitrogen atom) the part-amino-modified polyallylamine or the salt thereof containing a structural unit represented by the following formula (2), and a structural unit represented by the following formula (3) or formula (3a), and having a weight average molecular weight of 1000 to 500000, [Chemical 1] the ligand being immobilized on the base carrier via one or more active groups reactive to the ligand.

2. The chromatography carrier according to claim 1, wherein the ligand is represented by the formula (1).

3. The chromatography carrier according to claim 1 or 2, wherein Z in the formula (1) represents an alkylene group, a cycloalkylene group, -NR-, -0-, an arylene group, -S-, or a combination thereof, each of which can have a substituent (here, R represents a hydrogen atom, an alkyl group, an aryl group, an alkyl ester group, an aryl ester group, an alkyl ether group, or an aryl ether group).

4. The chromatography carrier according to any one of claims 1 to 3, wherein Z in the formula (1) represents an alkylene group having a carbon number of 2 to 12, a cycloalkylene group having a carbon number of 3 to 6, -0-, a phenylene group, or a combination thereof.

5. The chromatography carrier according to any one of claims 1 to 4, wherein the ligand contains a group derived from a compound selected from the group consisting of one or more of 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, 2-methyl-1,3-propanediamine, 1,5-diaminopentane, 1,3-diaminopentane, 1,6-diaminohexane, 3,3'-diaminodipropylamine, 3,3'-diamino-N-methyldipropylamine, 2-methyl-1,5-diaminopentane, 1,4-diaminocyclohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 4-(2-aminoethyl)cyclohexylamine, 1,9-diaminononane, 1,10-diaminodecane, 1,4-butanediol-bis(isopropylamine), 1,11-diaminoundecane, 1,12-diaminododecane, N,N-bis(3-aminopropyl)-1,4-butanediamine, 6,6'-diaminodihexylamine, p-xylylenediamine, 4-picolylamine, and a part-amino-modified polyallylamine containing a structural unit represented by the following formula (2), and a structural unit represented by the following formula (3'), [Chemical 2] (in formulae (2) and (3'), R represents a hydrogen atom, an alkyl group, an aryl group, an alkyl ester group, an aryl ester group, an alkyl ether group, or an aryl ether group).

6. The chromatography carrier according to any one of claims 1 to 5, wherein the active group is selected from the group consisting of an epoxy group, a formyl group, and a vinylsulfone group.

7. The chromatography carrier according to any one of claims 1 to 6, wherein the porous particles are crosslinked cellulose. [In formula (3) and formula (3a), Y is independently selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, -COR 2 , and -COOR 2 (where R 2 is an alkyl group, an aryl group, an amino group, or an alkoxy group)] 8. The chromatography carrier according to any one of claims 1 to 7, wherein the average pore diameter of the porous particles is 200 nm or more.

2. The chromatography support of claim 1, wherein, 9. The chromatography carrier according to any one of claims 1 to 8, wherein the base carrier contains a porous particle having a pore diameter of 100 nm or more and 1000 nm or less.

3. The chromatography support of claim 1 or 2, wherein, 10. A method for purifying an antibody, comprising the step of contacting a solution containing an antibody and a host-derived protein (host cell protein) with the chromatography carrier according to any one of claims 1 to 9, to obtain a solution of the antibody with a reduced content of the host cell protein.

4. The chromatography support of claim 3, wherein, 11. The method according to claim 10, wherein the step of contacting the solution containing the antibody and the host-derived protein (host cell protein) with the chromatography carrier is performed by passing the solution in a flow-through mode through a container containing the chromatography carrier.

5. The chromatography support of claim 1, wherein, The ligand is a polyallylamine in which a part of amino groups of a structure unit represented by the formula (2) and a structure unit represented by the formula (3) are modified, in the formula (3), one of Y is an alkyl group having 1 to 12 carbon atoms, a phenyl group or -COR 2 (where R 2 is an alkyl group having 1 to 12 carbon atoms or a phenyl group), and the other is a hydrogen atom.

6. The chromatography support of claim 1, wherein, 12. The method according to any one of claims 10 to 11, wherein the antibody is a monoclonal antibody.

13. The method according to any one of claims 10 to 12, wherein the host cell protein is a protein derived from a host cell used for producing the antibody. ​ 。 7. The chromatography support of any one of claims 1-6, wherein, ​ 8. The chromatography support of any one of claims 1-7, wherein, ​ 9. The chromatography support of any one of claims 1-8, wherein, ​ ​ 11. The method of claim 10, wherein, ​ 12. The method of claim 10 or 11, wherein, ​ 13. The method of any one of claims 10 to 12, wherein, The host cell protein content in the antibody solution is 300 ppm or less.

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