Method for packing chromatography support into column, method for storing slurry, and slurry

Abstract

[Problem] To provide a technique for suppressing deterioration of liquid permeability and pressure resistance characteristics during liquid passage of a chromatography carrier when a solvent has been substituted with an aqueous solvent that does not contain a buffering agent.　[Solution] A method for filling a column with a chromatography carrier, said method comprising step 1 and step 2 below.　(Step 1) A substitution step for substituting, with an aqueous solvent that does not contain a buffering agent, a slurry containing a target substance capturing chromatography carrier, a buffering agent having an acid dissociation constant (pKa) within a range of ±1.0 of the isoelectric point of the buffering agent, and an aqueous solvent, the liquid-phase pH having been adjusted to be within a range of ±2.0 of the isoelectric point of the carrier. (Step 2) A filling step for filling a column with the slurry for which the solvent substitution has been carried out in step 1

Description

[Technical Field]

[0001] The present invention relates to a method for packing a chromatography support into a column, a method for storing a slurry, and the slurry. [Background technology]

[0002] In the field of biopharmaceuticals, such as antibody drugs, the expression technology for target substances such as proteins has advanced, and this has led to a demand for improved productivity in the purification process. The purification process for antibody drugs is being developed as a platform of purification techniques that combine unit operations of chromatography purification, and purification using a carrier bound to an affinity ligand (affinity chromatography carrier) is the most widely used unit operation (Patent Documents 1 to 3). Furthermore, after purification using an affinity chromatography carrier, ion exchange chromatography, hydrophobic interaction chromatography, etc. are often combined to remove impurities. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2020-2028 [Patent Document 2] International Publication No. 2015 / 199196 Brochure [Patent Document 3] International Publication No. 2017 / 090658 Brochure Summary of the Invention [Problem to be solved by the invention]

[0004] On the other hand, it has recently been discovered that when a chromatography support is packed into a column at a pH close to the support's isoelectric point, appropriate packing occurs, making it easier to obtain good liquid permeability. Therefore, in order to improve the liquid permeability, the present inventors have focused on incorporating a target substance-capturing chromatography carrier into a slurry and pre-adjusting the pH of the liquid phase of the slurry to near the isoelectric point of the carrier. Further investigation by the present inventors has revealed that when the pH of the liquid phase of the slurry is simply adjusted to near the isoelectric point of the carrier, the pH rises significantly when the solvent is replaced with an aqueous solvent containing no buffer to adjust the concentration or remove additives such as ethanol or impurities, resulting in insufficient liquid permeability and, further, insufficient pressure resistance characteristics during liquid passage.

[0005] Therefore, the problem to be solved by the present invention is to provide a technology for suppressing the deterioration of the liquid permeability and pressure resistance characteristics of a chromatography carrier when solvent is replaced with an aqueous solvent containing no buffer. [Means for solving the problem]

[0006] The above issues are addressed as follows: <1> ~ <9> was resolved by the following means. <1> A method for packing a chromatography support into a column, comprising the following steps 1 and 2: (Step 1) A substitution step in which a slurry containing a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and in which the pH of the liquid phase has been adjusted to within ±2.0 of the isoelectric point of the carrier, is substituted with an aqueous solvent that does not contain a buffer. (Step 2) A step of packing the slurry obtained by solvent replacement in Step 1 into a column.

[0007] <2> The pH of the liquid phase of the slurry after solvent replacement in step 1 is in the range of 3 to 10. <1> The method described below. <3> The electrical conductivity of the liquid phase of the slurry obtained by solvent substitution in step 1 is in the range of 0.006 mS / m or more and 200 mS / m or less; <1> or <2> The method described below. <4> The support is an affinity chromatography support or an ion exchange chromatography support. <1> ~ <3> A method according to any one of the preceding claims. <5> The buffer is selected from a compound having a plurality of acidic groups in the molecule and a salt thereof. <1> ~ <4> A method according to any one of the preceding claims. <6> The content of the buffering agent in the liquid phase of the slurry used in step 1 is 0.1 mM or more; <1> ~ <5> A method according to any one of the preceding claims.

[0008] <7> A method for storing a slurry that is used by replacing the aqueous solvent containing no buffer with an aqueous solvent that does not contain a buffer before filling a chromatography column, wherein the slurry contains a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within a range of ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and the pH of the liquid phase of the slurry is within a range of ±2.0 of the isoelectric point of the carrier.

[0009] <8> A slurry containing a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and the pH of the liquid phase is within ±2.0 of the isoelectric point of the carrier, which is used by replacing the aqueous solvent without buffer before filling the column. <9> The buffer is selected from a compound having a plurality of acidic groups in the molecule and a salt thereof. <8> The slurry according to claim 1. [Effects of the Invention]

[0010] According to the present invention, it is possible to suppress a decrease in the liquid permeability of a chromatography carrier and in its pressure resistance characteristics during liquid passage when the carrier is subjected to solvent substitution with an aqueous solvent containing no buffer. DETAILED DESCRIPTION OF THE INVENTION

[0011] <Method of packing chromatography support into a column> The packing method of the present invention is a method for packing a chromatography carrier into a column, and is characterized by comprising the following steps 1 and 2: (Step 1) A substitution step in which a slurry containing a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and in which the pH of the liquid phase has been adjusted to within ±2.0 of the isoelectric point of the carrier, is substituted with an aqueous solvent that does not contain a buffer. (Step 2) A step of packing the slurry obtained by solvent replacement in Step 1 into a column.

[0012] ((Step 1) Replacement step) Step 1 is a replacement step in which a slurry containing a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and in which the pH of the liquid phase has been adjusted to within ±2.0 of the isoelectric point of the carrier, is replaced with an aqueous solvent that does not contain a buffer.

[0013] -Water-based solvent- The slurry used in step 1 contains an aqueous solvent. Examples of aqueous solvents include water and mixtures of water and lower alcohols. Examples of lower alcohols include one or more selected from ethanol and isopropanol, with ethanol being preferred in order to maintain the storage state of the slurry. The content of the aqueous solvent in the slurry before substitution with an aqueous solvent not containing a buffer is preferably 20% by volume or more, more preferably 25% by volume or more, and particularly preferably 30% by volume or more to improve stability, and is preferably 80% by volume or less, more preferably 70% by volume or less, and particularly preferably 60% by volume or less to improve handleability. Specifically, the content in the slurry is preferably 20% by volume or more and 80% by volume or less, more preferably 25% by volume or more and 70% by volume or less, and particularly preferably 30% by volume or more and 60% by volume or less. By setting the content in this range, storage stability is particularly improved. The content of the aqueous solvent is calculated by subtracting the "content of the target substance-capturing chromatography carrier before replacement with the aqueous solvent not containing a buffer" described below and the content of other components in the slurry from the total volume of the slurry. When a mixture of water and a lower alcohol is used as the aqueous solvent, the content of the lower alcohol in the mixture is preferably 5% by volume or more and 25% by volume or less.

[0014] -Buffer- The buffer contained in the slurry has an acid dissociation constant (pKa) within ±1.0 of the isoelectric point of the target substance-capturing chromatography carrier ((isoelectric point - 1.0) ≦ pKa ≦ (isoelectric point + 1.0)), but if it has multiple acid dissociation constants (pKa), at least one of them should be within ±1.0 of the isoelectric point of the carrier. In this specification, the acid dissociation constant (pKa) is defined as the value at 25°C and an ionic strength of 0.1 mol dm -3 pKa is the negative common logarithm of the equilibrium constant Ka in the dissociation reaction in which hydrogen ions are released. In the present invention, pKa refers to the value listed in the Chemistry Handbook, Basic Edition, 6th Revised Edition (Maruzen Publishing). For substances not listed here, the published value from the manufacturer may be used. In order to improve the liquid permeability and pressure resistance characteristics during liquid passage, the acid dissociation constant (pKa) of the buffer is preferably in the range of (isoelectric point - 0.9) ≦ pKa ≦ (isoelectric point + 0.5), more preferably in the range of (isoelectric point - 0.8) ≦ pKa ≦ (isoelectric point ± 0), even more preferably in the range of (isoelectric point - 0.7) ≦ pKa ≦ (isoelectric point - 0.3), and particularly preferably in the range of (isoelectric point - 0.65) ≦ pKa ≦ (isoelectric point - 0.5). As the buffer, in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, a buffer selected from compounds having multiple acidic groups (such as carboxy groups) in the molecule and salts thereof is preferred, a buffer selected from compounds having 2 to 5 acidic groups in the molecule and salts thereof is more preferred, and a buffer selected from compounds having 3 acidic groups in the molecule and salts thereof is particularly preferred. When the carrier is an affinity chromatography carrier (isoelectric point ≈ 5.0) using an immunoglobulin-binding protein as a ligand, preferred buffers include citrate buffers selected from citric acid (pKa1 = 2.90, pKa2 = 4.35, pKa3 = 5.69) and its salts; phthalate buffers selected from phthalic acid (pKa1 = 2.75, pKa2 = 4.90) and its salts; tartaric acid buffers selected from meso-tartaric acid (pKa1 = 2.95, pKa2 = 4.46) and its salts; and acetate buffers selected from acetic acid (pKa = 4.57) and its salts. Of these, citrate buffers, phthalate buffers, and tartrate buffers are preferred, with citrate buffers being particularly preferred, in order to improve liquid permeability and pressure resistance characteristics during liquid flow. When the support is a weakly acidic cation exchange chromatography support with an isoelectric point of approximately 9.0, the buffer is preferably a CHES (N-cyclohexyl-2-amino ethanesulfonic acid) buffer selected from 2-(N-cyclohexylamino)ethanesulfonic acid (pKa=9.5) and its salts; a boric acid buffer selected from boric acid (pKa=8.98) and its salts; an ammonia buffer combining ammonia and an ammonium salt; or a Tris buffer selected from trometamol (pKa=8.2) and its salts. Of these, a CHES buffer is preferred. The pKa values ​​above, except for CHES, are those listed in the 6th revised edition of Maruzen Publishing Co., Ltd.'s Chemistry Handbook: Basics, and the CHES value is the value listed on the Merck website (https: / / www.sigmaaldrich.com / JP / ja / product / mm / 239779). Examples of salts of citrate, phthalate, tartrate, acetate, 2-(N-cyclohexylamino)ethanesulfonate, borate, and trometamol include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as magnesium salt and calcium salt.

[0015] Here, the concentration of the buffer in the slurry liquid phase before replacement with a buffer-free aqueous solvent is preferably 0.1 mM or more, more preferably 1 mM or more, even more preferably 5 mM or more, even more preferably 10 mM or more, even more preferably 20 mM or more, and particularly preferably 30 mM or more, and is preferably 1000 mM or less, more preferably 750 mM or less, and particularly preferably 500 mM or less, in the slurry liquid phase.

[0016] -Target substance capture chromatography carrier- The slurry used in step 1 contains a target substance-capturing chromatography carrier. Examples of target substances include antigens; antibodies such as monoclonal antibodies and polyclonal antibodies; cells (normal cells; cancer cells such as colon cancer cells and circulating cancer cells); nucleic acids such as DNA and RNA; and biologically relevant substances such as proteins, peptides, amino acids, sugars, polysaccharides, lipids, and vitamins, and may also be drugs that are targets for drug discovery or low-molecular-weight compounds such as biotin. The chromatography carrier may be any carrier capable of capturing a target substance, and examples thereof include affinity chromatography carriers, ion exchange chromatography carriers, and hydrophobic interaction chromatography carriers. Among these, the present invention is suitable for packing affinity chromatography carriers and ion exchange chromatography carriers, and is particularly suitable for packing affinity chromatography carriers.

[0017] The isoelectric point of the carrier is not particularly limited, but is preferably 3.5 or higher, more preferably 4 or higher, even more preferably 4.5 or higher, and particularly preferably 4.7 or higher, in order to suppress pH changes during substitution with an aqueous solvent. It is also preferably 10 or lower, and in the case of a target substance such as an antibody, in order to efficiently capture the substance, it is more preferably 7 or lower, even more preferably 6 or lower, and particularly preferably 5.5 or lower. Specifically, the isoelectric point is preferably 3.5 or higher and 10 or lower, more preferably 4 or higher and 7 or lower, even more preferably 4.5 or higher and 6 or lower, and particularly preferably 4.7 or higher and 5.5 or lower. Here, in this specification, the isoelectric point of a support refers to the pH value at which the surface zeta potential measured by the streaming potential method at 25°C becomes zero. During measurement, a sample is placed in a measurement cell so that the support to be measured is in contact with the electrolyte. The surface zeta potential is calculated by flowing the electrolyte through the measurement cell at various pressures, measuring the streaming potential at each pressure, and then using the following calculation formula. Specifically, the measurement can be performed according to the method described in the Examples below.

[0018]

number

[0019] (Here, ζ is the surface zeta potential, E is the streaming potential, ΔP is the pressure difference between the cells, η is the viscosity of the electrolyte, λ is the conductivity of the electrolyte, ε is the relative permittivity of the electrolyte, and ε0 is the permittivity of a vacuum.)

[0020] Examples of affinity chromatography carriers include those having a ligand and a support, with the ligand immobilized on the support. Examples of ion exchange chromatography supports include those having an ion exchange group in the molecule, but cation exchange chromatography supports are preferred, and weakly acidic cation exchange chromatography supports are more preferred. The support and ion exchange chromatography carrier may take the form of, for example, particles (beads), monoliths, plates, fibers, or membranes (including hollow fibers). Among these, particles are preferred, and porous particles are more preferred. Furthermore, the support and ion exchange chromatography carrier are preferably water-insoluble. Examples of porous particles include organic porous particles, inorganic porous particles, and organic-organic composite porous particles and organic-inorganic composite porous particles that are combinations of these, with organic porous particles being preferred, and polymer-containing porous particles being more preferred. These porous particles may be natural polymer porous particles composed of polysaccharides such as agarose, dextran, or cellulose, or synthetic polymer porous particles, but synthetic polymer porous particles are preferred in order to increase the dynamic binding capacity and improve the uniformity of particle size.

[0021] When the carrier is an affinity chromatography carrier, the ligand may be any molecule that binds to a target substance, including, for example, proteins such as protein A, protein G, and avidin; peptides such as insulin; nucleic acids such as DNA and RNA; enzymes; chelating compounds such as iminodiacetic acid; antibodies; antigens; hormones; carbohydrates such as heparin, Lewis X, and gangliosides; receptors; aptamers; vitamins such as biotin and its derivatives; metal ions; synthetic dyes; and low molecular weight compounds such as 2-aminophenylboronic acid, 4-aminobenzamidine, and glutathione. The ligands exemplified above may be used in their entirety, or fragments thereof obtained by recombinant or enzymatic treatment may also be used. Artificially synthesized peptides and peptide derivatives may also be used. Among the above-mentioned ligands, proteins, peptides, nucleic acids, enzymes, and chelating compounds are preferred, proteins and peptides are more preferred, and proteins are particularly preferred. In particular, among antibody affinity ligands targeting antibodies, immunoglobulin-binding proteins are preferred.

[0022] Antibody affinity ligands include peptide ligands, protein ligands, and chemically synthesized ligands (synthetic compounds), with peptide or protein ligands being preferred. Among these, Protein A, Protein G, Protein L, Protein H, Protein D, Protein Arp, Protein FcγR, antibody-binding synthetic peptide ligands, and analogs thereof are preferred, Protein A, Protein G, Protein L, and analogs thereof are more preferred, and Protein A and analogs thereof are particularly preferred.

[0023] The amount of the immobilized ligand is preferably 10 mg to 300 mg, more preferably 25 mg to 150 mg, per gram of dry weight of the support in order to increase the dynamic binding capacity.

[0024] When the carrier is an ion exchange chromatography carrier, the ion exchange group is preferably a cation exchange group, and more preferably a weakly acidic cation exchange group. - M + , -S - , -C(=O)OH, -C(=O)O - M + , -C(=O)O - , -S(=O)2OH, -S(=O)2O - M + , -S(=O)2O - , -OP(=O)(OH)2, -OP(=O)(O - M + )2, -OP(=O)(O - )2, -OP(=O)(OH)(O - M + ), and -OP(=O)(OH)(O - )(M above + and each represent a counter ion). + Examples of the counter ion represented by the formula (I) include alkali metal ions such as sodium ion and potassium ion; alkaline earth metal ions such as magnesium ion and calcium ion; ammonium ion; and organic ammonium ions.

[0025] Furthermore, examples of polymers contained in synthetic polymer porous particles include polymers having structural units derived from functional group-containing monomers. When the carrier is an affinity chromatography carrier, the functional group contained in the monomer is preferably one capable of immobilizing a ligand, and more preferably one capable of immobilizing a ligand and usable for additional chemical reactions (such as a reaction with a crosslinking agent). On the other hand, when the carrier is an ion exchange chromatography carrier, the functional group contained in the monomer may be an ion exchange group or one usable for chemical reactions of a compound having an ion exchange group. For example, when the structural unit derived from the functional group-containing monomer is a structural unit derived from the following cyclic ether group-containing monomer, -SH, -S can be obtained by reacting with mercapto alcohols such as thioglycerol. - M + and -S - An ion exchange group selected from the following can be introduced. Examples of the functional group contained in the monomer include functional groups selected from the group consisting of a cyclic ether group, a carboxy group, -C(=O)-OC(=O)-, a succinimideoxycarbonyl group, a formyl group, a hydroxyl group, and an isocyanate group. Among these, a cyclic ether group is preferred. Here, the "cyclic ether group" is preferably a cyclic ether group having 3 to 7 atoms constituting the ring. The cyclic ether group may have an alkyl group as a substituent. Specific examples of the cyclic ether group include cyclic ether groups represented by the following formulas (1) to (6), with the cyclic ether group represented by formula (1), (3) or (6) being preferred, and the cyclic ether group represented by formula (1) being more preferred.

[0026] [ka]

[0027] [In the formula, R 1 ~R 4 each independently represents a hydrogen atom or an alkyl group, and * represents a bond.

[0028] R 1~R 4 The number of carbon atoms in the alkyl group represented by the formula (I) is preferably 1 to 4, more preferably 1 or 2. The alkyl group may be linear or branched, and examples thereof include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, and a tert-butyl group. 1 ~R 4 is preferably a hydrogen atom.

[0029] The functional group-containing monomer is preferably a monomer having a functional group capable of immobilizing a ligand and a polymerizable unsaturated group. Examples of such monomers include glycidyl (meth)acrylate, 3-oxiranylpropyl (meth)acrylate, 4-oxiranylbutyl (meth)acrylate, 5-oxiranylpentyl (meth)acrylate, 6-oxiranylhexyl (meth)acrylate, 7-oxiranylheptyl (meth)acrylate, 8-oxiranyloctyl (meth)acrylate, (3-methyloxiranyl)methyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate glycidyl ether, glycerin mono(meth)acrylate glycidyl ether, 3,4-epoxycyclohexylmethyl (meth)acrylate, 3,4-epoxycyclohexylethyl (meth)acrylate, 3,4-epoxycyclohexylpropyl (meth)acrylate, α-(meth)acryl-ω-glycidyl polyethylene glycol, tetrahydrofurfuryl (meth)acrylate, and other monomers having a cyclic ether group ( Examples of suitable methacrylate monomers include aromatic vinyl monomers having a cyclic ether group, such as (vinylbenzyl)glycidyl ether, (isopropenylbenzyl)glycidyl ether, (vinylphenethyl)glycidyl ether, (vinylphenylbutyl)glycidyl ether, (vinylbenzyloxyethyl)glycidyl ether, (vinylphenyl)glycidyl ether, (isopropenylphenyl)glycidyl ether, and 1,2-epoxy-3-(4-vinylbenzyl)propane; allyl ether monomers having a cyclic ether group, such as allyl glycidyl ether; (meth)acrylate monomers having an isocyanate group, such as isocyanatoethyl (meth)acrylate; unsaturated dicarboxylic acid anhydride monomers, such as maleic anhydride, methylmaleic anhydride, and glutaconic anhydride; (meth)acrylic acid, 3,4-epoxy-1-butene, and 3,4-epoxy-3-methyl-1-butene. These monomers can be used alone or in combination of two or more. Among these monomers, (meth)acrylate monomers having a cyclic ether group are preferred, and glycidyl (meth)acrylate is particularly preferred.

[0030] The content of structural units derived from functional group-containing monomers is preferably 30% by mass or more, more preferably 50% by mass or more, and particularly preferably 70% by mass or more, based on all structural units in the polymer, and is preferably 99% by mass or less, more preferably 95% by mass or less, and particularly preferably 90% by mass or less, based on all structural units in the polymer.

[0031] Furthermore, the polymer contained in the synthetic polymer porous particles preferably has, in addition to the structural units derived from the functional group-containing monomer, structural units derived from a monomer other than the functional group-containing monomer (hereinafter also referred to as other monomers). The other monomers include polymerizable unsaturated group-containing monomers that do not have a functional group capable of immobilizing a ligand. The other monomers are broadly classified into non-crosslinkable monomers and crosslinkable monomers, and either one of these may be used alone or in combination.

[0032] Examples of the non-crosslinkable monomer include (meth)acrylate-based non-crosslinkable monomers, (meth)acrylamide-based non-crosslinkable monomers, aromatic vinyl-based non-crosslinkable monomers, vinyl ketone-based non-crosslinkable monomers, (meth)acrylonitrile-based non-crosslinkable monomers, N-vinyl amide-based non-crosslinkable monomers, etc. These may be used alone or in combination of two or more.

[0033] Examples of the (meth)acrylate non-crosslinkable monomer include methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 4-tert-butyl (meth)acrylate, isobutyl (meth)acrylate, n-octyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, cyclohexyl (meth)acrylate, methoxyethyl (meth)acrylate, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, glycerol mono(meth)acrylate, trimethylolethane mono(meth)acrylate, trimethylolpropane mono(meth)acrylate, butanetriol mono(meth)acrylate, polyethylene glycol mono(meth)acrylate, methoxypolyethylene glycol (meth)acrylate, pentaerythritol mono(meth)acrylate, dipentaerythritol mono(meth)acrylate, and inositol mono(meth)acrylate. These may be used alone or in combination of two or more.

[0034] Examples of the (meth)acrylamide-based non-crosslinkable monomer include (meth)acrylamide, dimethyl(meth)acrylamide, hydroxyethyl(meth)acrylamide, (meth)acryloylmorpholine, diacetone(meth)acrylamide, etc. These may be used alone or in combination of two or more.

[0035] Examples of the aromatic vinyl non-crosslinkable monomer include styrenes such as styrene, α-methylstyrene, halogenated styrene, 4-methylstyrene, 2,4-dimethylstyrene, 2,4,6-trimethylstyrene, ethylvinylbenzene, 4-isopropylstyrene, 4-n-butylstyrene, 4-isobutylstyrene, and 4-tert-butylstyrene; vinylnaphthalenes such as 1-vinylnaphthalene and 2-vinylnaphthalene; and the like. These may be used alone or in combination of two or more.

[0036] Examples of the vinyl ketone-based non-crosslinkable monomer include ethyl vinyl ketone, propyl vinyl ketone, isopropyl vinyl ketone, etc. These may be used alone or in combination of two or more. Furthermore, examples of the (meth)acrylonitrile-based non-crosslinkable monomer include acrylonitrile, methacrylonitrile, etc. These can be used alone or in combination of two or more. Examples of the N-vinylamide non-crosslinkable monomer include N-vinylacetamide, N-vinylpropionamide, etc. These can be used alone or in combination of two or more.

[0037] The content of structural units derived from non-crosslinkable monomers is preferably 0% by mass or more, more preferably 0.01% by mass or more, even more preferably 0.1% by mass or more, and particularly preferably 0.2% by mass or more, based on all structural units in the polymer, and is preferably 10% by mass or less, more preferably 5% by mass or less, and particularly preferably 3% by mass or less, based on all structural units in the polymer.

[0038] Examples of the crosslinkable monomer include (meth)acrylate crosslinkable monomers, aromatic vinyl crosslinkable monomers, and allyl crosslinkable monomers. These may be used alone or in combination of two or more. As the crosslinkable monomer, di- to penta-functional crosslinkable monomers are preferred, and di- or tri-functional crosslinkable monomers are more preferred. Among the crosslinkable monomers, (meth)acrylate crosslinkable monomers and aromatic vinyl crosslinkable monomers are preferred.

[0039] Examples of the (meth)acrylate crosslinkable monomer include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, tetraethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, dipropylene glycol di(meth)acrylate, tripropylene glycol di(meth)acrylate, tetrapropylene glycol di(meth)acrylate, polypropylene glycol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate, glycerin di(meth)acrylate, trimethylolethane di(meth)acrylate, trimethylolpropane di(meth)acrylate, and trimethylolpropane tri(meth)acrylate. acrylate, butanetriol di(meth)acrylate, pentaerythritol di(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, glucose di(meth)acrylate, glucose tri(meth)acrylate, glucose tetra(meth)acrylate, dipentaerythritol di(meth)acrylate, dipentaerythritol tri(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, inositol di(meth)acrylate, inositol tri(meth)acrylate, inositol tetra(meth)acrylate, mannitol di(meth)acrylate, mannitol tri(meth)acrylate, mannitol tetra(meth)acrylate, mannitol penta(meth)acrylate, etc. These can be used alone or in combination of two or more.

[0040] Examples of the aromatic vinyl crosslinkable monomer include divinylbenzene, trivinylbenzene, divinyltoluene, divinylxylene, divinylethylbenzene, divinylnaphthalene, etc. These may be used alone or in combination of two or more.

[0041] Examples of the allyl crosslinkable monomer include diallyl phthalate, diallyl isophthalate, diallyl terephthalate, diallyl maleate, diallyl fumarate, diallyl itaconate, diallyl trimellitate, triallyl trimellitate, triallyl cyanurate, diallyl isocyanurate, triallyl isocyanurate, etc. These may be used alone or in combination of two or more. Further, in addition to the above-mentioned crosslinkable monomers, examples thereof include dehydration condensation reaction products of amino alcohols such as diaminopropanol, trishydroxymethylaminomethane, and glucosamine with (meth)acrylic acid, and conjugated diolefins such as butadiene and isoprene.

[0042] The content of structural units derived from crosslinkable monomers is preferably 5% by mass or more, more preferably 10% by mass or more, and particularly preferably 15% by mass or more, based on all structural units in the polymer, and is preferably 50% by mass or less, more preferably 40% by mass or less, and particularly preferably 30% by mass or less, based on all structural units in the polymer.

[0043] Furthermore, when the support or ion exchange chromatography carrier is a porous particle, the porous particle may be one that has undergone the introduction of a crosslinked structure or surface modification as described in WO2019 / 039545, JP2011-252929A, WO2017 / 155105, JP62-25102A, etc. Examples of crosslinking agents that provide a crosslinked structure include oxalyl dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, 2,3-dihydroxysuccinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, pimelic acid dihydrazide, octanedioic acid dihydrazide, nonanedioic acid dihydrazide, sebacic acid dihydrazide, dodecanedioic acid dihydrazide, phthalic acid dihydrazide, and isophthalic acid dihydrazide. Dicarboxylic acid dihydrazides such as azide, terephthalic acid dihydrazide, and quinolinic acid dihydrazide; tricarboxylic acid trihydrazides such as cyclohexanetricarboxylic acid trihydrazide; (alkylenebisimino)bis(oxoalkanoic acids) such as N1,N1-(ethane-1,2-diyl)bis(succinic acid monoamide); halohydrins such as epichlorohydrin, epibromohydrin, and dichlorohydrin; Examples of suitable crosslinking agents include bisoxiranes such as resorcinol diglycidyl ether, neopentyl glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, hydrogenobisphenol A diglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane diglycidyl ether, diglycidyl terephthalate, diglycidyl orthophthalate, ethylene glycol diglycidyl ether, diethylene glycol diglycidyl ether, and propylene glycol diglycidyl ether; diamines such as 1,2-bis(2-aminoethoxy)ethane, m-xylylenediamine, 1,3-propanediamine, tetramethylenediamine, pentamethylenediamine, hexamethylenediamine, and 1,3-bis(aminomethyl)cyclohexane; and polyoxiranes with three or more functional groups. The crosslinking agent crosslinks functional group residues derived from functional group-containing monomers via a partial structure derived from the crosslinking agent.

[0044] The content of structural units derived from the crosslinking agent is preferably 0.5% by mass or more, more preferably 1% by mass or more, and particularly preferably 5% by mass or more, based on all structural units in the polymer, and is preferably 50% by mass or less, more preferably 40% by mass or less, and particularly preferably 35% by mass or less, based on all structural units in the polymer.

[0045] When the target substance-capturing chromatography carrier is a particulate carrier, the volume average particle diameter is preferably 40 to 150 μm, more preferably 50 to 100 μm, and the coefficient of variation of the volume average particle diameter is preferably 40% or less, more preferably 30% or less. The specific surface area of ​​the target substance-capturing chromatography carrier is preferably 1 to 500 m 2 / g, more preferably 10 to 300m 2 / g. The volume average pore diameter of the target substance-capturing chromatography carrier is preferably 10 to 300 nm. The volume average particle size, coefficient of variation, specific surface area, and volume average pore size can be measured by laser diffraction / scattering particle size distribution measurement or the like.

[0046] Target substance-capturing chromatography carriers can be produced according to known methods described in WO 2015 / 119255, WO 2015 / 041218, U.S. Pat. No. 6,399,750, Ljungquist C. et al., rEur. J. Biochem., 1989, Vol. 186, pp. 557-561, U.S. Pat. No. 5,260,373, JP 2010-133733 A, JP 2010-133734 A, JP 2011-256176 A, etc. For example, a mixed solution (monomer solution) containing a monomer composition and, if necessary, a porogen is suspended in an aqueous medium and thermally polymerized in the presence of a polymerization initiator, the resulting porous particles are reacted with a crosslinking agent, and ligands are immobilized or ion exchange groups are introduced into the resulting crosslinked porous particles. The porous particles to which the ligands are immobilized may be contacted with a thiol compound such as methanethiol or thioglycerol to open the ring of the unreacted functional groups. This treatment may be performed with reference to the description in WO 2015 / 119255. Furthermore, sieving may be performed before or after the ligand immobilization or the introduction of ion exchange groups.

[0047] The content of the target substance-capturing chromatography carrier in the slurry before replacement with a buffer-free aqueous solvent is preferably 20% by volume or more, more preferably 30% by volume or more, and particularly preferably 40% by volume or more, and is preferably 80% by volume or less, more preferably 75% by volume or less, and particularly preferably 70% by volume or less. Specific ranges are preferably 20% by volume or more and 80% by volume or less, more preferably 30% by volume or more and 75% by volume or less, and particularly preferably 40% by volume or more and 70% by volume or less. The volume percentage of the carrier in the slurry can be calculated by filling a 250 mL glass graduated cylinder (JIS R3505 Class A compliant) manufactured by Corning Incorporated with 200 mL of the slurry, leaving it to stand for 3 hours, and dividing the sediment volume by the volume of the slurry filled in the graduated cylinder.

[0048] The volume ratio of the target substance-capturing chromatography carrier to the aqueous solvent [(carrier) / (aqueous solvent)] is preferably 0.25 or more, more preferably 0.45 or more, particularly preferably 0.65 or more, and is preferably 5.5 or less, more preferably 4 or less, particularly preferably 3 or less. Specific ranges are preferably 0.25 or more and 5.5 or less, more preferably 0.45 or more and 4 or less, and particularly preferably 0.65 or more and 3 or less.

[0049] The slurry may also contain components other than the carrier, buffer, and aqueous solvent, such as preservatives.

[0050] - Slurry liquid phase pH (pH of the liquid phase of the slurry before replacement) - The slurry used in step 1 has a liquid phase pH adjusted to within ±2.0 of the isoelectric point of the target substance-capturing chromatography carrier ((isoelectric point - 2.0) ≦ slurry liquid phase pH ≦ (isoelectric point + 2.0)). By adjusting the liquid phase pH of the slurry to within ±2.0 of the isoelectric point of the carrier, the liquid permeability and pressure resistance characteristics of the chromatography carrier during liquid passage are less likely to decrease, even when solvent replacement is performed with an aqueous solvent that does not contain a buffer. In this specification, the pH of the liquid phase of the slurry refers to the hydrogen ion exponent that can be measured at 25°C using a commercially available pH meter (for example, a benchtop pH meter manufactured by Horiba, Ltd.). The pH of the liquid phase of the slurry (the pH of the liquid phase of the slurry before replacement) is preferably in the range of (isoelectric point - 1.5) ≦ slurry liquid phase pH ≦ (isoelectric point + 1.5) in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, more preferably in the range of (isoelectric point - 1.0) ≦ slurry liquid phase pH ≦ (isoelectric point + 1.0), and particularly preferably in the range of (isoelectric point - 0.5) ≦ slurry liquid phase pH ≦ (isoelectric point + 0.5). The pH of the liquid phase of the slurry is preferably 3 or more, more preferably 3.5 or more, and particularly preferably 4 or more in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, and is preferably 10 or less, more preferably 7 or less, and particularly preferably 6 or less in order to improve the liquid permeability and pressure resistance characteristics during liquid passage. Specifically, the pH range is preferably 3 or more and 10 or less, more preferably 3.5 or more and 7 or less, and particularly preferably 4 or more and 6 or less.

[0051] -Replacement- Step 1 is a substitution step in which the slurry is replaced with a buffer-free aqueous solvent. In this specification, "buffer-free aqueous solvent" encompasses not only aqueous solvents with a buffer concentration of 0 mM, but also those containing trace amounts of buffer or salt. It is sufficient for the aqueous solvent to contain at least one selected from buffers and salts at a concentration of 0 to 1 mM. An aqueous solvent with a buffer concentration of 0 to 0.01 mM is preferred, and an aqueous solvent with a buffer concentration of 0 mM is more preferred. Using such a buffer-free aqueous solvent eliminates the need for dissolving buffer or salt or adjusting the pH before packing, simplifying the process and preventing equipment corrosion and contamination due to salt precipitation. Step 1 replaces the slurry solvent with a buffer-free aqueous solvent. By including Step 1 in the packing method of the present invention, the concentration of the slurry can be easily adjusted and additives such as ethanol and impurities in the slurry can be removed, thereby suppressing convection and other problems that may occur within the column and achieving appropriate packing. Examples of aqueous solvents that do not contain a buffer include water and mixtures of water and lower alcohols. Examples of lower alcohols include one or more selected from ethanol and isopropanol. When a mixture of water and lower alcohols is used as the aqueous solvent that does not contain a buffer, the content of the lower alcohol in the mixture is preferably 5% by volume or more and 25% by volume or less. The replacement operation in step 1 may be carried out in the same manner as a conventional solvent replacement operation, except that the above-mentioned slurry is used. For example, the replacement operation may be carried out by combining removal of the solvent by natural precipitation, centrifugation, filtration, or the like with the addition of an aqueous solvent that does not contain a buffer, but the replacement operation using natural precipitation is preferred. In the case of a replacement operation using natural settling, step 1 preferably includes a settling step in which the carrier in the slurry is allowed to settle, a removal step in which 50% to 95% by volume of the slurry components other than the carrier are removed after the settling step, and an aqueous solvent addition step in which an aqueous solvent not containing a buffer is added in an amount of 70% to 150% by volume of the slurry components other than the carrier removed in the removal step, and more preferably includes a step in which a combination of the settling step, removal step, and aqueous solvent addition step is repeated two to ten times. By including such steps, it is possible to suppress variations in the physical properties of the slurry during replacement and to obtain a slurry that is prone to appropriate packing. The pH of the liquid phase of the slurry after solvent substitution in step 1 is preferably 3 or more, more preferably 3.5 or more, even more preferably 4 or more, and particularly preferably 4.5 or more in order to improve the liquid permeability and pressure resistance characteristics during liquid passage, and is preferably 10 or less, more preferably 7 or less, and particularly preferably 6 or less in order to improve the liquid permeability and pressure resistance characteristics during liquid passage. Specifically, the pH range is preferably 3 or more and 10 or less, more preferably 3.5 or more and 7 or less, even more preferably 4 or more and 6 or less, and particularly preferably 4.5 or more and 6 or less. The pH of the liquid phase of the post-replacement slurry, like the pH of the liquid phase of the pre-replacement slurry, refers to the hydrogen ion exponent that can be measured at 25°C using a commercially available pH meter (e.g., a benchtop pH meter manufactured by Horiba, Ltd.). The electrical conductivity of the liquid phase of the slurry after solvent substitution in step 1 is preferably 0.006 mS / m or more, more preferably 0.01 mS / m or more, even more preferably 0.1 mS / m or more, and particularly preferably 0.5 mS / m or more in order to improve liquid permeability and pressure resistance characteristics during liquid passage, and is preferably 200 mS / m or less, more preferably 150 mS / m or less, even more preferably 100 mS / m or less, even more preferably 50 mS / m or less, and particularly preferably 20 mS / m or less in order to suppress convection that may occur in the column and ensure appropriate packing. As a specific range, 0.006 mS / m or more and 200 mS / m or less is preferable, 0.01 mS / m or more and 150 mS / m or less is more preferable, 0.01 mS / m or more and 100 mS / m or less is even more preferable, 0.1 mS / m or more and 50 mS / m or less is even more preferable, and 0.5 mS / m or more and 20 mS / m or less is particularly preferable. The electrical conductivity of the liquid phase of the slurry after substitution can be measured using an electrical conductivity meter (for example, D-74SE manufactured by Horiba Ltd.) in accordance with JIS K0130 (general rules for measuring electrical conductivity).

[0052] ((Process 2) Filling process) Step 2 is a packing step in which the slurry obtained by solvent replacement in step 1 is packed into a column. The packing operation in step 2 may be carried out in the same manner as in conventional packing except that the slurry subjected to solvent substitution in step 1 is used. For example, the packing operation may be carried out by referring to the description in the packing protocol for Ampshere A3 manufactured by JSR Corporation.

[0053] The packing method of the present invention can suppress the deterioration of the liquid permeability and pressure resistance characteristics of the chromatography support when solvent replacement is performed with an aqueous solvent containing no buffer. The packing method of the present invention is also suitable for packing affinity chromatography supports and ion exchange chromatography supports, and is particularly suitable for packing affinity chromatography supports.

[0054] <Slurry storage method, slurry> The method for storing a slurry of the present invention is a method for storing a slurry that is used by replacing the aqueous solvent containing no buffer with an aqueous solvent before filling a chromatography column, characterized in that the slurry contains a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within a range of ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and the pH of the liquid phase of the slurry is within a range of ±2.0 of the isoelectric point of the carrier. The slurry of the present invention, which is used by replacing the aqueous solvent containing no buffer before packing into a column, is characterized in that it contains a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and the pH of the liquid phase is within ±2.0 of the isoelectric point of the carrier. The method for storing the slurry of the present invention may be carried out in the same manner as in the conventional method, except that the slurry contains a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and the pH of the liquid phase of the slurry is within ±2.0 of the isoelectric point of the carrier, and the slurry is to be stored for use by replacing it with an aqueous solvent that does not contain a buffer. The slurry of the present invention can be used for purifying a target substance, which can be purified by a conventional method. The method for storing the slurry of the present invention, the meaning of various terms in the slurry, the content and ratio of each component, etc. are the same as the meaning of various terms, the content and ratio of each component, etc. explained for the filling method of the present invention. [Example]

[0055] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0056] Example 1: Synthetic polymer-based affinity chromatography carrier slurry (1) 2.69 g of polyvinyl alcohol (PVA-217, manufactured by Kuraray Co., Ltd.) was added to 448 g of pure water and heated and stirred to dissolve the polyvinyl alcohol, preparing aqueous solution S. Separately, a monomer composition consisting of 3.99 g of divinylbenzene (manufactured by Wako Pure Chemical Industries, Ltd.) and 14.15 g of glycidyl methacrylate (manufactured by Mitsubishi Gas Chemical Company, Inc.) was dissolved in 29.38 g of 2-octanone (manufactured by Toyo Gosei Co., Ltd.) to prepare a monomer solution. The entire amount of aqueous solution S was then poured into a separable flask, which was then fitted with a thermometer, stirring blade, and condenser and placed in a hot water bath. Stirring was initiated under a nitrogen atmosphere. The entire amount of the monomer solution was poured into the separable flask and heated in the hot water bath until the internal temperature reached 85°C. 1.34 g of 2,2'-azobis(methyl isobutyrate) (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the internal temperature was raised to 86°C. (2) Thereafter, the mixture was stirred for 3 hours while maintaining the temperature at 86°C. The reaction solution was then cooled, filtered, and washed with pure water and ethanol. The washed particles were dispersed in pure water and decanted three times to remove small particles. The particles were then dispersed in pure water to a particle concentration of 10% by mass, yielding a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous solid support 1." (3) Then, 0.956 g of adipic acid dihydrazide (manufactured by Tokyo Chemical Industry Co., Ltd.) and 1.418 g of diisopropylethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) were added to 100 g of the porous solid support 1 dispersion, and the mixture was heated to 70°C and stirred for 8 hours while maintaining the temperature at 70°C. Next, the reaction solution was cooled, filtered, and washed with pure water and ethanol. The resulting particles were recovered with pure water to obtain a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous solid support 2." (4) The porous solid support 2 was then placed in a Sato-type vibrating sieve (Koei Sangyo Co., Ltd., 400DB-2S) equipped with a 77 μm mesh (Taiyo Wire Netting Co., Ltd.), and the sieve-passing fraction (liquid that passed through the sieve after wet classification) was collected. Next, the sieve-passing fraction was placed in the same Sato-type vibrating sieve, but with the wire mesh replaced with a 32 μm mesh (Taiyo Wire Netting Co., Ltd.), and the sieve-remaining fraction (porous particles remaining on the sieve after wet classification) was collected with pure water, yielding a porous particle dispersion. The porous particles contained in this dispersion are referred to as "porous solid support 3." (5) Next, 0.15 g of modified protein A (rSPA manufactured by Repligen) was dissolved in 40 mL of 1.2 M sodium sulfate / 0.1 M sodium phosphate buffer (pH 6.6) to obtain a protein A solution, and porous solid support 3 (equivalent to 1 g in terms of dry particle mass) was added to this protein A solution. This dispersion was shaken and stirred at 25°C for 10 hours to immobilize protein A on the particles. The resulting Protein A-immobilized particles were then dispersed in 40 mL of 1.0 M α-thioglycerol (Tokyo Chemical Industry Co., Ltd.) / 0.1 M sodium sulfate (pH 8.3) and stirred at 25 °C for 17 hours with shaking to open the unreacted epoxy groups. The resulting Protein A-immobilized particles with the unreacted epoxy groups open were then washed with 0.1 M sodium phosphate buffer (pH 6.6), 0.1 M aqueous sodium hydroxide, 0.1 M sodium citrate buffer (pH 3.2), and 0.1 M sodium phosphate buffer (pH 7.5) to obtain ligand-immobilized carrier V1. The resulting carrier was then substituted with 0.1 M sodium citrate buffer (pH 5.0) prepared using 16% ethanol (volume %) in aqueous solution to obtain ligand-immobilized carrier slurry W1 (solids content 50% volume %).

[0057] Example 2: Synthetic polymer-based affinity chromatography carrier slurry Ligand-immobilized support slurry W2 was obtained in the same manner as in Example 1, except that in step (5) of Example 1, the 0.1 M sodium citrate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution was changed to 0.1 M sodium citrate buffer (pH 3.5) prepared using 16 vol% aqueous ethanol solution.

[0058] Example 3: Synthetic polymer-based affinity chromatography carrier slurry Ligand-immobilized support slurry W3 was obtained in the same manner as in Example 1, except that in step (5) of Example 1, the 0.1 M sodium citrate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution was changed to 0.1 M sodium citrate buffer (pH 6.5) prepared using 16 vol% aqueous ethanol solution.

[0059] Example 4: Synthetic polymer-based affinity chromatography carrier slurry Ligand-immobilized support slurry W4 was obtained in the same manner as in Example 1, except that in step (5) of Example 1, the 0.1 M sodium citrate buffer (pH 5.0) prepared using 16% by volume aqueous ethanol solution was changed to 0.1 M sodium citrate buffer (pH 5.0).

[0060] Example 5: Synthetic polymer-based affinity chromatography carrier slurry Ligand-immobilized support slurry W5 was obtained in the same manner as in Example 1, except that in step (5) of Example 1, the 0.1 M sodium citrate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution was changed to 0.1 M sodium acetate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution.

[0061] Example 6: Synthetic polymer-based affinity chromatography carrier slurry Ligand-immobilized support slurry W6 was obtained in the same manner as in Example 1, except that in step (5) of Example 1, the 0.1 M sodium citrate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution was changed to 0.1 M sodium acetate buffer (pH 3.5) prepared using 16 vol% aqueous ethanol solution.

[0062] Example 7: Agarose-based affinity chromatography carrier slurry Mabselect SuRe (manufactured by Cytiva) was prepared, and the dispersion medium was replaced with 0.1 M sodium citrate buffer (pH 5.0) prepared using 16% by volume of aqueous ethanol solution to obtain ligand-immobilized support slurry W7 (solid content 50% by volume).

[0063] Example 8: Agarose-based affinity chromatography carrier slurry Mabselect SuRe (manufactured by Cytiva) was prepared, and the dispersion medium was replaced with 0.1 M sodium acetate buffer (pH 5.0) prepared using 16% by volume of aqueous ethanol solution to obtain ligand-immobilized support slurry W8 (solid content 50% by volume).

[0064] (Example 9 Ion Exchange Chromatography Support Slurry) To 100 g of a 10% by mass aqueous dispersion of the porous solid support 1 obtained in Example 1, 0.837 g of 1,2-bis(2-aminoethoxy)ethane (Tokyo Chemical Industry Co., Ltd.) and 0.452 g of diisopropylethylamine (Tokyo Chemical Industry Co., Ltd.) were added, heated to 70°C, and stirred for 8 hours while maintaining the temperature at 70°C. Subsequently, 12.215 g of α-thioglycerol (Tokyo Chemical Industry Co., Ltd.) was added, and the mixture was stirred at 70°C for 3 hours. The reaction solution was then cooled, filtered, and washed with pure water and ethanol. The resulting particles were recovered with pure water to obtain a weak ion-exchange ligand-immobilized support dispersion. The support contained in this dispersion is referred to as "ligand-immobilized support V2." Thereafter, the ligand-immobilized carrier V2 was classified and recovered in the same manner as in (4) of Example 1, and the recovered carrier was substituted with 0.1 M CHES (N-cyclohexyl-2-aminoethanesulfonic acid) buffer (pH 9.0) prepared using 16% by volume of aqueous ethanol solution, to obtain ligand-immobilized carrier slurry W9 (solid content 50% by volume).

[0065] Example 10: Synthetic polymer-based affinity chromatography carrier slurry Ligand-immobilized support slurry W10 was obtained in the same manner as in Example 1, except that in step (5) of Example 1, the 0.1 M sodium citrate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution was changed to 1 mM sodium citrate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution.

[0066] (Comparative Example 1: Synthetic Polymer-Based Affinity Chromatography Support Slurry) Ligand-immobilized support slurry Y1 was obtained in the same manner as in Example 1, except that in step (5) of Example 1, the 0.1 M sodium citrate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution was changed to 0.1 M sodium phosphate buffer (pH 6.5) prepared using 16 vol% aqueous ethanol solution.

[0067] (Comparative Example 2: Synthetic Polymer-Based Affinity Chromatography Support Slurry) Ligand-immobilized support slurry Y2 was obtained in the same manner as in Example 1, except that in step (5) of Example 1, the 0.1 M sodium citrate buffer (pH 5.0) prepared using 16 vol% aqueous ethanol solution was changed to 0.1 M sodium phosphate buffer (pH 7.5) prepared using 16 vol% aqueous ethanol solution.

[0068] (Comparative Example 3: Ion Exchange Chromatography Support Slurry) Ligand-immobilized support slurry Y3 was obtained in the same manner as in Example 9, except that the 0.1 M CHES buffer (pH 9.0) prepared using 16 vol.% ethanol aqueous solution in Example 9 was changed to 0.1 M sodium acetate buffer (pH 5.0) prepared using 16 vol.% ethanol aqueous solution.

[0069] (Test Example 1: Measurement of the isoelectric point of the support) The isoelectric points of the carriers in slurries W1 to W10 and Y1 to Y3 obtained in each Example and Comparative Example were measured using a zeta potential analyzer for solid surface analysis (SurPASS, manufactured by Anton Paar). Specifically, 0.5 mL of carrier was filled into a cylindrical cell and equilibrated with a 10 mM potassium chloride solution adjusted to pH 11 with sodium hydroxide. The zeta potential was then measured by the streaming potential method while varying the pH with 0.05 M hydrochloric acid. The pH at which the zeta potential became 0 mV was recorded as the isoelectric point of the carrier. The results are shown in Tables 1 to 3.

[0070] (Test Example 2: Evaluation of Liquid Permeability) For slurries W1 to W10 and Y1 to Y3 obtained in each Example and Comparative Example, the carrier was allowed to settle, 90% by volume of the supernatant of the slurry components other than the carrier was discarded, and an equal amount of pure water was added. This process was repeated five times to replace the dispersant, and the pH and electrical conductivity of the resulting substituted slurry were recorded. For slurry W1, the carrier was allowed to settle, 90% by volume of the supernatant of the slurry components other than the carrier was discarded, and an equal amount of pure water was added. This process was repeated twice to replace the dispersant, and the pH and electrical conductivity of the resulting substituted slurry were recorded as Example 11. For slurry W1, the carrier was allowed to settle, 90% by volume of the supernatant of the slurry components other than the carrier was discarded, and an equal amount of pure water was added. This process was repeated eight times to replace the dispersant, and the pH and electrical conductivity of the resulting substituted slurry were recorded as Example 12. The results are shown in Tables 1 to 3. Electrical conductivity was measured in accordance with JIS K0130 using a D-74SE manufactured by Horiba, Ltd. The above-mentioned displacement slurry was packed into two connected Hiscale 26 / 20 columns (manufactured by Cytiva) with an inner diameter of 26 mm and a packed height of 200 mm, and these columns were connected to a Cytiva AKTA AVANT 150. Next, pure water was passed through the column at a linear flow rate of 100 cm / hr for 30 minutes, after which the upper column was removed and the carrier was packed into the column by attaching an adapter to the carrier layer. The linear flow rate was then adjusted until a back pressure of 0.25 MPa was generated, and the linear flow rate at that time was recorded. The results are shown in Tables 1 to 3. The higher the linear flow rate, the better the liquid permeability.

[0071] (Test Example 3: Evaluation of pressure resistance characteristics during liquid passage) For the slurries W1 to W10 and Y1 to Y3 obtained in each example and comparative example, dispersion medium replacement and packing were carried out in the same manner as in Test Example 2, and then pure water was passed through at a linear flow rate of 800 cm / hr, and the back pressure and compressibility (rate of change in packed height) at that time were recorded. The compressibility was calculated using the following formula. The results are shown in Tables 1 to 3. It can be said that the smaller the back pressure and compressibility values, the better the pressure resistance characteristics.

[0072] (Compressibility) = 1-{(Filling height after passing liquid at 800 cm / h) / (Filling height before passing liquid at 800 cm / h)}

[0073] (Test Example 4: Evaluation of dynamic binding capacity) The DBC of the carrier in slurries W1-W8, W10, and Y1-Y3 was measured for a protein (human IgG antibody, Equitech Bio HGG-1000) at a linear flow rate of 300 cm / hr using a GE Healthcare AKTAprime plus. After dispersant replacement as in Test Example 2, a 4 mL column (5 mm diameter x 200 mm length) was used, and the protein was dissolved in 20 mM sodium phosphate / 150 mM sodium chloride aqueous solution (pH 7.5) at 5 mg / mL. The DBC was calculated from the protein capture amount at 10% breakthrough at the elution tip and the column volume. The results are shown in Tables 1-3.

[0074]

Table 1

[0075]

Table 2

[0076]

Table 3

Claims

1. A method for packing a chromatography support into a column, comprising the following steps 1 and 2: (Step 1) A substitution step in which a slurry containing a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within a range of ±1.0 of the isoelectric point of the carrier, and an aqueous solvent, and in which the pH of the liquid phase has been adjusted to within a range of ±2.0 of the isoelectric point of the carrier, is substituted with an aqueous solvent that does not contain a buffer. (Step 2) A step of packing the slurry obtained by solvent replacement in step 1 into a column.

2. The pH of the liquid phase of the slurry after solvent replacement in step 1 is in the range of 3 to 10. The method of claim 1.

3. The electrical conductivity of the liquid phase of the slurry after solvent substitution in step 1 is in the range of 0.006 mS / m or more and 200 mS / m or less. The method according to claim 1 or claim 2.

4. The support is an affinity chromatography support or an ion exchange chromatography support. The method according to any one of claims 1 to 3.

5. The buffer is selected from a compound having a plurality of acidic groups in the molecule and a salt thereof. The method according to any one of claims 1 to 4.

6. The content of the buffer agent in the liquid phase of the slurry used in step 1 is 0.1 mM or more; The method according to any one of claims 1 to 5.

7. A method for preserving a slurry to be used by replacing the slurry with a buffer-free aqueous solvent before packing the slurry into a chromatography column, comprising: the slurry contains a target substance-capturing chromatography carrier, a buffer having an acid dissociation constant (pKa) within a range of ±1.0 of the isoelectric point of the carrier, and an aqueous solvent; The pH of the liquid phase of the slurry is within the range of the isoelectric point of the support ±2.

0. How to store the slurry.

8. a target substance-capturing chromatography carrier; a buffer having an acid dissociation constant (pKa) within ±1.0 of the isoelectric point of the carrier; an aqueous solvent; and The pH of the liquid phase is within the range of the isoelectric point of the carrier ±2.

0. A slurry that is used by replacing the aqueous solvent with a buffer-free solvent before packing the column.

9. The buffer is selected from a compound having a plurality of acidic groups in the molecule and a salt thereof. The slurry of claim 8.

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