Method for purifying target substance using insoluble carrier

Abstract

An object of the present invention is to provide purification conditions under which a target substance can be purified with high efficiency.SOLUTION: A step of applying a sample containing a target substance to a column packed with an insoluble carrier to adsorb the target substance to the insoluble carrier, and a step of applying an eluent to the column to elute the target substance adsorbed to the insoluble carrier, wherein the insoluble carrier is a porous particle having a pore with a diameter of 150 nm or more and 1000 nm or less and having at least one of a functional group and a ligand capable of adsorbing the target substance on the surface of the carrier, the problem is solved by providing the method, wherein the linear speed at the time of applying the sample is 1200cm / h or more and 3500cm / h or less.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for purifying a target substance using an insoluble carrier. [Background technology]

[0002] In recent years, in the field of biopharmaceuticals, such as antibody drugs, remarkable progress has been made in the expression technology of target substances such as proteins, which has led to a demand for improved productivity in purification processes using chromatography, etc. In particular, there is a growing demand for highly accurate separation and purification methods that can be used for viral vectors and virus-like particles, which have large molecular sizes.

[0003] Generally, in purification using a column packed with porous particles, the larger the pore size, the less the back pressure of the column increases, enabling highly efficient purification under high linear velocity conditions. However, the larger the pore size, the smaller the surface area of ​​the porous particles, and the fewer functional groups there are on the particle surface, which raises concerns about a decrease in the amount of adsorption of the target substance.

[0004] Patent Document 1 discloses that exosomes and live influenza vaccines are detected using porous polymer particles with an average pore size of 30 nm to 500 nm as measured by mercury intrusion porosimetry.

[0005] However, there have been no reports on the purification of target substances using insoluble carriers with large pore sizes under high linear velocity conditions, and no investigations have been conducted into the purification conditions that improve the purification efficiency. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2021-115528 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide purification conditions that enable highly efficient purification of a target substance. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems and have found purification conditions that enable highly efficient purification of a target substance even when an insoluble carrier with a large pore size is used, thereby completing the present invention.

[0009] [1] A method for purifying a target substance, comprising the steps of: applying a sample containing the target substance to a column packed with an insoluble carrier to adsorb the substance to the insoluble carrier; and applying an eluate to the column to elute the substance adsorbed to the insoluble carrier, the insoluble carrier has pores with a diameter of 150 nm or more and 1000 nm or less, and has at least one of a functional group and a ligand capable of adsorbing the substance on the surface of the insoluble carrier; The linear velocity when the sample is applied is 1200 cm / h or more and 3500 cm / h or less. The method.

[0010] [2] The method according to [1] above, wherein the target substance is bovine serum albumin or adeno-associated virus. [Effects of the Invention]

[0011] According to the present invention, a method for purifying a target substance with high efficiency in a short time can be provided. [Brief explanation of the drawings]

[0012] [Figure 1] This figure compares the amount of bovine serum albumin (BSA) adsorbed to the insoluble carrier at different linear velocities when a sample containing BSA is applied to the column, depending on the pore diameter (average pore size) of the insoluble carrier packed in the column. The amount of BSA adsorbed is expressed as a relative value, with the amount of BSA adsorbed to particle 1 (average pore size 70 nm) at each linear velocity being set to 1. [Figure 2]This figure compares the amount of adeno-associated virus (AAV) adsorbed to insoluble carriers packed in a column depending on the diameter (average pore size) of the pores in the insoluble carriers. The amount of AAV adsorbed is expressed as a relative value, with the amount of AAV adsorbed by particle 1 being set to 1. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below.

[0014] In addition, regarding numerical ranges, the numbers on either side of "~" are considered to be included in the numerical range.

[0015] Examples of insoluble carriers used in embodiments of the present invention include inorganic carriers such as glass beads and silica gel, organic carriers made of synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene, and polysaccharides such as crystalline cellulose, cross-linked cellulose, cross-linked agarose, and cross-linked dextran, as well as composite carriers obtained by combining these.

[0016] Hydrophilic carriers are particularly preferred because they have relatively little nonspecific adsorption and good selectivity for the target substance used in this embodiment. Examples of hydrophilic carriers include polysaccharides such as cellulose, chitosan, and dextran, polyvinyl alcohol, saponified ethylene-vinyl acetate copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylic acid-grafted polyethylene, and polyacrylamide-grafted polyethylene.

[0017] The insoluble carrier is preferably a porous particle or a porous membrane. When the insoluble carrier is a porous particle or a porous membrane, the surface area is increased, and the processing capacity per unit time can be increased.

[0018] When the insoluble carrier is a porous particle or a porous membrane, the specific surface area is not particularly limited, but the specific surface area is preferably 1 m 2 / g~500m 2 / g, more preferably 1m 2 / g~100m 2 / g, more preferably 1m 2 / g~50m 2 / g, and even more preferably 1m 2 / g~45m 2 / g. If the specific surface area is within this range, the amount of antibody adsorption is improved. The specific surface area of ​​the insoluble carrier can be measured by mercury intrusion porosimetry using a mercury porosimeter.

[0019] The particle size of the insoluble carrier is not particularly limited, but is preferably 1500 μm or less, more preferably 1000 μm or less, even more preferably 300 μm or less, and even more preferably 100 μm or less. The particle size of the insoluble carrier is preferably 0.5 μm or more, more preferably 1 μm or more, and even more preferably 50 μm or more. If the particle size is within this range, the pressure loss when packed into a column and passed through the column is small, the liquid passing rate can be increased, the treatment efficiency is improved, and the adsorption capacity is improved. The particle size of the porous particles can be measured by known methods, for example, by using a particle size distribution measuring device.

[0020] When the insoluble carrier is a porous particle or a porous membrane, the pore size is not particularly limited, but is preferably 1000 nm or less, more preferably 900 nm or less, and even more preferably 800 nm or less. The pore size is preferably 150 nm or more, more preferably 200 nm or more, even more preferably 300 nm or more, and even more preferably 500 nm or more. When the pore size is within this range, the pressure loss when the column is filled with the carrier and a liquid is passed through it is small, the liquid passing rate can be increased, and the polymers to be separated can enter the pores, improving the processing efficiency. The pore size can be measured by a known method, for example, using a mercury porosimeter.

[0021] As used herein, an "insoluble carrier having a large pore size" refers to an insoluble carrier having a pore size of 150 nm or more, preferably 200 nm or more, more preferably 350 nm or more, and even more preferably 500 nm or more.

[0022] The pores of the insoluble carrier may be closed pores or interconnected pores, and the pores may include both closed pores and interconnected pores.

[0023] Commercially available insoluble carriers include, but are not limited to, the Sepharose series (manufactured by Cytiva), which is an agarose-based carrier; the Cellufine series (manufactured by JNC), which is a cellulose-based crosslinked carrier; the Sephacryl series (manufactured by Cytiva), which is a crosslinked polymer of allyl dextran and N,N'-methylenebisacrylamide; and the TOYOPEARL (registered trademark) HW series (manufactured by Tosoh Corporation), which is an acrylate-based carrier.

[0024] The surface of the insoluble carrier preferably has at least one of a functional group and a ligand capable of adsorbing the target substance.

[0025] The target substance may be, for example, a growth factor, hormone, cytokine, virus, viral protein, blood protein, enzyme, antigen, antibody (immunoglobulin), transcription factor, fluorescent protein, or a partial peptide or mutant thereof.

[0026] Hormones include insulin, glucagon, growth hormone, prolactin, leptin, calcitonin, and somatostatin.

[0027] Cytokines include interferon, interleukin (IL)-1, and IL-6.

[0028] Blood proteins include albumin, bovine serum albumin (BSA), globulins, fibrinogen, neutrophils, T lymphocytes, NK cells, and B lymphocytes.

[0029] Viral proteins include constituent proteins of virus-like particles (VLPs), including influenza virus, human immunodeficiency virus (HIV), human hepatitis C virus (HCV), human hepatitis B virus (HBV), adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), retrovirus, and coronavirus.

[0030] Antibodies (immunoglobulins) include complete antibodies, Fab, F(ab'), F(ab')2, Fc, Fc fusion proteins, heavy chains (H chains), light chains (L chains), single-chain Fvs (scFvs), sc(Fv)2, disulfide-linked Fvs (sdFvs), diabodies, VHH antibodies, chimeric antibodies, monoclonal antibodies, and albumin-binding proteins.

[0031] The functional group may be, for example, an N-hydroxysuccinimide (NHS) activated ester group, a hydroxy group, an epoxy group, an amino group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, or a haloacetamide group (such as an iodoacetamide group or a bromoacetamide group). A commercially available insoluble support already modified with the functional group may be purchased, or the functional group may be appropriately introduced onto the surface of the insoluble support. The method for introducing the functional group may be the same as that used to produce conventional particulate fillers. For example, the functional group may be introduced into the pores of the insoluble support under appropriate reaction conditions.

[0032] The ligand may be, for example, protein A, protein G, protein L, or functional variants thereof, various antibodies, lectins, adrenergic receptors, adenosine receptors, rhodopsin, nicotinic acetylcholine receptors, insulin receptors, LDL receptors, transferrin receptors, Fc receptors, viral receptors (such as AAV-binding proteins), or human chemokine receptor 5 (CCR5). The ligand may be immobilized on the surface of an appropriate insoluble carrier.

[0033] The method for immobilizing the ligand may be, for example, a method in which a polymerizable monomer capable of imparting a reactive functional group is incorporated into the crosslinked synthetic polymer particle in the form of copolymerization or the like, and then this reactive functional group is reacted directly with a functional group possessed by the ligand, or a method in which the ligand is bonded via a low molecular weight or high molecular weight compound having, in its molecule, at least one functional group capable of reacting with a functional group possessed by a constituent component of the crosslinked synthetic polymer and a functional group possessed by the ligand.

[0034] If the ligand is an AAV-binding protein, it may be, for example, a protein that contains at least the amino acid residues from serine (S) at position 312 to aspartic acid (D) at position 500, which is a region corresponding to extracellular domain 1 (PKD1) and domain 2 (PKD2) in the amino acid sequence of the native AAV-binding protein KIAA0319L (UniProt No. Q8IZA0) set forth in SEQ ID NO: 1, but which has an amino acid substitution at a specific position within the amino acid residues at positions 312 to 500.

[0035] In particular, the AAV binding protein may be selected from any of the following (i) to (iii): (i) a protein containing at least the amino acid residues from serine at position 312 to aspartic acid at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) a protein comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, with the proviso that one or more of the following occurs in one or more positions of the amino acid residues from positions 312 to 500: substitution, deletion, insertion, and addition of one or more amino acid residues, and the protein has AAV-binding activity; (iii) A protein comprising at least the amino acid residues from serine at position 312 to aspartic acid at position 500 of the amino acid sequence set forth in SEQ ID NO: 1, wherein the protein has 70% or more identity to the entire amino acid sequence consisting of amino acid residues at positions 312 to 500, and wherein the protein has AAV-binding activity.

[0036] In (ii), "one or several" varies depending on the position of the amino acid substitution in the three-dimensional structure of the AAV-binding protein and the type of amino acid residue, but, for example, means any of 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

[0037] The identity of the amino acid sequences in (iii) above may be 70% or more, but may be higher (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more). "Amino acid sequence identity" refers to identity with respect to the entire amino acid sequence. "Identity" between amino acid sequences refers to the proportion of amino acid residues in those amino acid sequences that have the same type of amino acid (Experimental Medicine, 31(3), Yodosha). Amino acid sequence identity can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0038] The AAV-binding protein may further have an oligopeptide attached to its N-terminus or C-terminus that is useful for separating it from a solution in the presence of contaminants. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. Furthermore, a cysteine-containing oligopeptide that is useful for immobilizing the AAV-binding protein of this embodiment on a solid phase such as a chromatographic support may further be attached to the N-terminus or C-terminus of the AAV-binding protein of this embodiment.

[0039] Furthermore, the AAV-binding protein may contain an additional sequence at the N-terminus, C-terminus, or both. The additional sequence is not particularly limited as long as it has AAV-binding activity. Examples of the additional sequence include a peptide tag, a signal peptide, and a protease recognition sequence. The additional sequence may be a single amino acid sequence or a combination of two or more amino acid sequences.

[0040] The method of this embodiment includes a step of applying a sample containing a target substance to a column packed with an insoluble carrier and adsorbing the substance to the insoluble carrier, and a step of applying an eluate to the column and eluting the substance adsorbed to the insoluble carrier (elution step).

[0041] A sample containing a target substance may be applied to the column using a liquid delivery means such as a pump.

[0042] The linear velocity at which the sample is applied is not particularly limited, but is preferably in the range of 1200 cm / h to 3500 cm / h, more preferably 1200 cm / h to 3000 cm / h, and even more preferably 1400 cm / h to 3000 cm / h. A linear velocity within this range improves the adsorption capacity. In this specification, linear velocity refers to the velocity at which a liquid passes through the cross section of the column, and may be calculated using the following formula:

[0043] [Number 1] Linear velocity (cm / h) = flow velocity (cm 3 / h) / Column cross-sectional area (cm 2 ) The separation performance of this embodiment is expressed by dynamic binding capacity (DBC). DBC can be determined by adding a standard substance solution of known concentration to a column, monitoring the absorbance of the eluate, and determining the amount of substance added at the point when 10% of the absorbance of the added sample breaks through (10% breakthrough time), and may be calculated using the following formula:

[0044] [Equation 2] DBC (mg / mL) = 10% breakthrough time (min) × flow rate (mL / min) × antibody concentration (mg / mL) / column volume (mL) [Example]

[0045] EXAMPLES Hereinafter, examples will be shown to explain the present invention in more detail, but the present invention is not limited to these examples.

[0046] Example 1 Preparation of insoluble carrier The insoluble carriers (porous particles) used in the following examples were prepared by radical polymerization of hydrophilic vinyl monomers. The diameters (average particle diameters) of the resulting carriers were measured using a particle size distribution analyzer MT3000II (MicrotracBEL), and the pore diameters (average pore diameters) and specific surface areas of the carriers were measured by mercury intrusion using a mercury porosimeter POREMASTER GT (Quantachrome Instruments).

[0047] The average particle size and average pore size of the prepared insoluble carrier, as well as the specific surface area per gram of the carrier, are shown in Table 1.

[0048] [Table 1]

[0049] Example 2 Purification of bovine serum albumin (BSA) contained in a sample (1) Preparation of an anion exchange column (1-1) Among the insoluble carriers (porous particles) prepared in Example 1, particles 1 (average pore diameter 70 nm), particles 3 (average pore diameter 370 nm), and particles 4 (average particle diameter 780 nm) were selected, and the hydroxyl groups present on the surface of each carrier were chemically modified to prepare insoluble carriers modified with quaternary ammonium groups (Q groups).

[0050] (1-2) 0.58 mL of the insoluble carrier modified with Q groups as functional groups, prepared in (1), was packed into an empty stainless steel column (diameter 4.6 mm, length 35 mm, manufactured by Tosoh Corporation) to prepare an anion exchange column for use in purifying BSA.

[0051] (2) BSA adsorption onto insoluble carrier (2-1) The columns prepared in (1) were each connected to an AKTA avant (Cytiva) and equilibrated with 50 mmol / L Tris (hydroxymethyl) aminomethane) hydrochloric acid buffer (pH 8.5) (hereinafter also referred to as "equilibration solution A").

[0052] (2-2) A solution of BSA (diameter approximately 5 nm) (Thermo Fisher Scientific) diluted to 0.3 mg / mL with equilibration solution A was applied at linear velocities of 480 cm / h, 950 cm / h, 1400 cm / h, 1800 cm / h, 2400 cm / h, and 2900 cm / h.

[0053] (2-3) The eluate from the column was monitored by absorbance at 280 nm. When the concentration of the eluate exceeded 0.03 mg / mL (10% of the added concentration), the application of the BSA solution was stopped, and the concentration of BSA contained in the eluate at that time was calculated as the adsorption amount.

[0054] The results are shown in Figure 1. The BSA adsorption amount is expressed as a relative value, with the result for particle 1 (average pore diameter 70 nm) at each linear velocity being set to 1. When the linear velocity was between 1200 cm / h and 3500 cm / h, the BSA adsorption amount for particles 3 and 4 was higher than that for particle 1.

[0055] When the linear velocity is slow (e.g., 950 cm / h or less), the decrease in functional groups (Q groups in this example) on the surface of the insoluble carrier due to the enlarged pore size directly leads to a decrease in the amount of adsorbed target substance (BSA in this example). On the other hand, when the linear velocity is fast (e.g., 1200 cm / h or more), the contribution of the increased opportunity for the target substance to come into contact with the functional groups on the surface of the insoluble carrier due to the increase in the applied amount per hour exceeds the contribution of the decrease in the functional groups, which is presumably why the amount of adsorbed target substance increases.

[0056] Example 3 Purification of adeno-associated virus (AAV) contained in a sample (1) Preparation of AAV vectors The AAV vector used as the AAV in this example was prepared by the following method.

[0057] (1-1) A nucleotide sequence (SEQ ID NO: 3) was designed in which a restriction enzyme EcoRI recognition sequence (GAATCC) was added to the 5'-end of a polynucleotide encoding EGFP (enhanced green fluorescent protein) consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a stop codon (TAG) and a BamHI recognition sequence (GGATTC) were added to the 3'-end.

[0058] (1-2) A polynucleotide consisting of the sequence set forth in SEQ ID NO: 3 was totally synthesized and cloned into a plasmid (commissioned to FASMAC, hereinafter also referred to as "pUC-EGFP"). Escherichia coli JM109 strain was transformed with pUC-EGFP, and the resulting transformant was cultured. pUC-EGFP was extracted from the culture medium using a QIAprep Spin Miniprep kit (Qiagen).

[0059] (1-3) The pUC-EGFP obtained in (1-2) was digested with the restriction enzymes EcoRI and BamHI and then ligated to the expression vector pAAV-CMV (manufactured by Takara Bio Inc.) which had been previously digested with the restriction enzymes EcoRI and BamHI. The ligation product (hereinafter also referred to as "pAAV-EGFP") was used to transform the Escherichia coli JM109 strain.

[0060] (1-4) The transformant obtained in (1-3) was cultured overnight at 37°C with shaking in a 5-L baffled flask containing 1 L of 2xYT medium (1.6% (w / v) Tryptone, 1% (w / v) Yeast Extract, 0.5% (w / v) sodium chloride) containing 100 μg / mL carbenicillin. After the culture was completed, the cells were collected by centrifugation. pAAV-EGFP was prepared in large quantities from the collected cells using a Plasmid Mega Kit (Qiagen).

[0061] (1-5) The Escherichia coli JM109 strain was transformed with a plasmid (hereinafter also referred to as "pRC8 Vector") containing a polynucleotide encoding the capsid of serotype 8 (AAV8) and the pHelper Vector (manufactured by Takara Bio Inc.) The resulting transformant was used in the same manner as in (1-4) to prepare large quantities of the pRC8 Vector and pHelper.

[0062] (1-6) HEK293T cells were cultured in ten T-225 flasks (Thermo Fisher Scientific) containing 45 mL of D-MEM medium (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) bovine serum. The pAAV-EGFP prepared in (1-4), the pRC8 Vector prepared in (1-5), pHelper, and polyethyleneimine (Polysciences) complex were added for gene transfection, and the cells were cultured statically for 3 days under conditions of 5% (v / v) carbon dioxide and 37°C. After culture, the cells were detached by centrifugation and collected. The cells obtained from each of the five T-225 flasks were stored frozen at -80°C.

[0063] (1-7) The frozen cells obtained in (1-6) were thawed and suspended in 10 mL of 20 mmol / L Tris-HCl buffer (pH 7.4) containing 150 mmol / L sodium chloride, 4 mmol / L magnesium chloride, and 0.01% (w / v) Tween® 20. A 1 / 2000 volume of Benzonase (Merck Millipore) was added, and the mixture was left to stand at 37°C for 1 hour. The mixture was then centrifuged at 13,000 × g and 4°C for 10 minutes to obtain a supernatant. Ammonium sulfate was added to the resulting supernatant to 15% saturation, and the mixture was centrifuged again under the same conditions. The resulting supernatant was passed through a 0.45 μm pore size filter to remove any floating material.

[0064] (1-8) The supernatant, from which the suspended matter had been removed, was applied to a 7 mL POROS AAVX column (manufactured by Thermo Fisher Scientific) that had been equilibrated in advance with 20 mmol / L Tris-HCl buffer (pH 8.0) containing 500 mmol / L sodium chloride (hereinafter also referred to as "equilibration solution B").

[0065] (1-9) After washing with equilibration solution B, the column was eluted with 100 mmol / L acetate buffer (pH 2.5) containing 500 mmol / L sodium chloride. The resulting eluate was neutralized by adding 1 / 4 volume of 1 mol / L Tris-HCl buffer (pH 8.5) containing 20 mmol / L magnesium chloride to obtain a solution of the AAV vector AAV8-EGFP (diameter 20 to 30 nm). The diameter of the AAV8-EGFP was measured by dynamic light scattering using a Zetasizer (Malvern Panalytical).

[0066] (2) Preparation of AAV-binding proteins (2-1) Escherichia coli strain BL21(DE3) was transformed with a plasmid containing a polynucleotide encoding an AAV-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. The resulting transformant was inoculated into 20 mL of 2xYT liquid medium containing 50 μg / mL kanamycin and precultured overnight at 37°C under aerobic shaking. In SEQ ID NO: 4, the AAV-binding protein AVR29c is located at the second serine (S) through the 190th aspartic acid (D), and the six C-terminal histidine (H) residues form a histidine tag.

[0067] AVR29c is a polypeptide of KIAA0319L (UniProt No. Q8IZA0) in which the following 29 amino acid substitutions have occurred in the amino acid residues 312 to 500 of SEQ ID NO: 1, which correspond to extracellular domain 1 (PKD1) and domain 2 (PKD2): The valine residue (V) at position 317 of SEQ ID NO: 1 (position 7 of SEQ ID NO: 4) is substituted with an aspartic acid residue (D). The asparagine residue (N) at position 324 of SEQ ID NO: 1 (position 14 of SEQ ID NO: 4) is substituted with a histidine residue (H). The valine residue (V) at position 326 of SEQ ID NO: 1 (position 16 of SEQ ID NO: 4) is replaced with an alanine residue (A). The asparagine residue (N) at position 329 of SEQ ID NO: 1 (position 19 of SEQ ID NO: 4) is substituted with a lysine residue (K). The alanine residue (A) at position 330 of SEQ ID NO: 1 (position 20 of SEQ ID NO: 4) is substituted with a valine residue (V). The glutamine residue (Q) at position 334 of SEQ ID NO: 1 (position 24 of SEQ ID NO: 4) is substituted with a leucine residue (L). The glutamic acid residue (E) at position 335 of SEQ ID NO: 1 (position 25 of SEQ ID NO: 4) is substituted with a valine residue (V). The threonine residue (T) at position 341 of SEQ ID NO: 1 (position 31 of SEQ ID NO: 4) is substituted with an alanine residue (A). The tyrosine residue (Y) at position 342 of SEQ ID NO: 1 (position 32 of SEQ ID NO: 4) is substituted with a serine residue (S). The lysine residue (K) at position 362 of SEQ ID NO: 1 (position 52 of SEQ ID NO: 4) is substituted with a glutamic acid residue (E). The isoleucine residue (I) at position 366 of SEQ ID NO: 1 (position 56 of SEQ ID NO: 4) is substituted with a phenylalanine residue (F). The lysine residue (K) at position 371 of SEQ ID NO: 1 (position 61 of SEQ ID NO: 4) is substituted with an asparagine residue (N). The phenylalanine residue (F) at position 379 of SEQ ID NO: 1 (position 69 of SEQ ID NO: 4) is replaced with a tyrosine residue (Y). The lysine residue (K) at position 380 of SEQ ID NO: 1 (position 70 of SEQ ID NO: 4) is substituted with an arginine residue (R). The valine residue (V) at position 381 of SEQ ID NO: 1 (position 71 of SEQ ID NO: 4) is substituted with an alanine residue (A). The isoleucine residue (I) at position 382 of SEQ ID NO: 1 (position 72 of SEQ ID NO: 4) is substituted with a valine residue (V) The glycine residue (G) at position 390 of SEQ ID NO: 1 (position 80 of SEQ ID NO: 4) is substituted with a serine residue (S). The valine residue (V) at position 394 of SEQ ID NO: 1 (position 84 of SEQ ID NO: 4) is substituted with an alanine residue (A). The lysine residue (K) at position 399 of SEQ ID NO: 1 (position 89 of SEQ ID NO: 4) is substituted with a glutamic acid residue (E). The glutamic acid residue (E) at position 401 of SEQ ID NO: 1 (position 91 of SEQ ID NO: 4) is replaced with a glycine residue (G) The threonine residue (T) at position 426 of SEQ ID NO: 1 (position 116 of SEQ ID NO: 4) is substituted with an alanine residue (A). The lysine residue (K) at position 455 of SEQ ID NO: 1 (position 145 of SEQ ID NO: 4) is substituted with an arginine residue (R). The alanine residue (A) at position 461 of SEQ ID NO: 1 (position 151 of SEQ ID NO: 4) is substituted with a proline residue (P) The lysine residue (K) at position 467 of SEQ ID NO: 1 (position 157 of SEQ ID NO: 4) is substituted with an asparagine residue (N). The serine residue (S) at position 476 of SEQ ID NO: 1 (position 166 of SEQ ID NO: 4) is substituted with an arginine residue (R). The serine residue (S) at position 482 of SEQ ID NO: 1 (position 172 of SEQ ID NO: 4) is substituted with a threonine residue (T). The asparagine residue (N) at position 487 of SEQ ID NO: 1 (position 177 of SEQ ID NO: 4) is substituted with an aspartic acid residue (D). The asparagine residue (N) at position 492 of SEQ ID NO: 1 (position 182 of SEQ ID NO: 4) is substituted with an aspartic acid residue (D). The lysine residue (K) at position 497 of SEQ ID NO: 1 (position 187 of SEQ ID NO: 4) is substituted with a glutamic acid residue (E). (2-2) In a 1 L baffled flask, 20 mL of the preculture solution from (2-1) was inoculated into 1000 mL of 2xYT liquid medium supplemented with 50 μg / mL of kanamycin, and the mixture was cultured aerobically at 37°C with shaking.

[0068] (2-3) After 2.0 hours of culturing, the mixture was cooled on ice, IPTG (IsoPropyl-β-D-ThioGalactopyranoside) was added to a final concentration of 0.1 mmol / L, and the mixture was subsequently cultured overnight at 25°C under aerobic shaking.

[0069] (2-4) After the culture was completed, the culture solution was centrifuged at 4°C and 8000 rpm for 20 minutes to collect the bacterial cells.

[0070] (2-5) The bacterial cells collected in (2-4) were suspended in 20 mmol / L Tris-HCl buffer (pH 7.4) containing 150 mmol / L sodium chloride and 20 mmol / L imidazole (hereinafter also referred to as "equilibration solution C") at a concentration of 5 mL / 1 g (bacterial cells), and then disrupted using an ultrasonic generator (Insonator 201M, Kubota Shoji Co., Ltd.) at an output of approximately 150 W for approximately 10 minutes at 8°C. The disrupted bacterial cell solution was centrifuged twice at 8,000 rpm for 20 minutes at 4°C, and the supernatant was collected.

[0071] (2-6) The supernatant obtained in (2-5) was applied to an XK26 / 20 column (Cytiva) packed with 20 mL of Ni Sepharose 6 Fast Flow (Cytiva) that had been previously equilibrated with equilibration solution C. After washing with equilibration solution C, the column was eluted with 20 mmol / L Tris-HCl buffer (pH 7.4) containing 500 mmol / L imidazole and 150 mmol / L sodium chloride.

[0072] (2-7) The eluate obtained in (2-6) was dialyzed against 20 mmol / L Tris buffer (pH 7.4) containing 150 mmol / L sodium chloride to prepare the amount of AAV binding protein (SEQ ID NO: 4) required for producing an AAV adsorbent.

[0073] (3) Preparation of AVR column (3-1) Hydroxy groups present on the surfaces of the insoluble carriers (particles 1 to 4) prepared in Example 1 were chemically modified to prepare carriers modified with iodoacetamide groups.

[0074] (3-2) To 3.5 g of the iodoacetamide group-modified insoluble support prepared in (3-1), 28 mg of the AAV binding protein (SEQ ID NO: 4) prepared in (2) and TCEP (Tris(2-CarboxyEthyl)Phosphine) as a reducing agent at a final concentration of 0.1 mmol / L were added, and the mixture was allowed to react by shaking at pH 7.4 and 4°C for 15 hours. This produced an insoluble support onto which the AAV binding protein was immobilized as a ligand (hereinafter also referred to as "AAV adsorbent").

[0075] (3-3) 0.58 mL of the AAV adsorbent prepared in (3-2) was packed into an empty stainless steel column (diameter 4.6 mm, length 35 mm, manufactured by Tosoh Corporation) to prepare a column (hereinafter also referred to as "AVR column").

[0076] (4) AAV adsorption onto the AVR column (4-1) The AVR column prepared in (3) was connected to an AKTA avant (Cytiva) and equilibrated with 50 mmol / L sodium acetate buffer (pH 6.0) containing 15 mmol / L glycine and 10 mmol / L magnesium chloride. The AAV8-EGFP solution obtained in (1) was then loaded at a linear velocity of 1800 cm / h.

[0077] (4-2) The eluate from the AVR column was monitored by absorbance at 280 nm. When the concentration of the eluate exceeded 10% of the concentration of the applied AAV8-EGFP solution, the application of the solution was stopped, and the concentration of AAV8-EGFP in the eluate at that time was calculated as the adsorption amount.

[0078] The results are shown in Figure 2. The amount of AAV (AAV8-EGFP in this example) adsorption is expressed as a relative value, with the result for particle 1 (average pore diameter 70 nm) set to 1. It can be seen that particles 2 (average pore diameter 210 nm), particle 3 (average pore diameter 370 nm), and particle 4 (average pore diameter 780 nm), which have smaller specific surface areas per particle than particle 1 (Table 1), adsorbed greater amounts of AAV.

[0079] Summarizing the results of Examples 2 and 3, when a sample containing a target substance is applied to a column packed with an insoluble carrier and the substance is adsorbed onto the insoluble carrier, it is found that the target substance contained in the sample can be efficiently adsorbed onto the insoluble carrier by using the insoluble carrier as porous particles with pores having a diameter of 150 nm or more and 1000 nm or less and by setting the linear velocity when applying the sample to 1200 cm / h or more and 3500 cm / h or less.

Claims

1. A method for purifying a target substance, comprising the steps of: applying a sample containing the target substance to a column packed with an insoluble carrier to adsorb the target substance to the insoluble carrier; and applying an eluate to the column to elute the target substance adsorbed to the insoluble carrier, the insoluble carrier is a porous particle having pores with a diameter of 150 nm or more and 1000 nm or less, and having at least one of a functional group and a ligand capable of adsorbing the target substance on the surface of the carrier, The linear velocity when the sample is applied is 1200 cm / h or more and 3500 cm / h or less. The method.

2. The method of claim 1, wherein the target substance is bovine serum albumin or adeno-associated virus.

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