Method for purifying adeno-associated virus

Abstract

To provide a method capable of purifying an adeno-associated virus (AAV) contained in a sample by using affinity chromatography so that aggregates of the virus can be removed.SOLUTION: Provided is an AAV purification method, complying the following methods of: adding a sample containing AAV to a column packed with an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, and adsorbing the AAV on the adsorbent; eluting AAV adsorbed on the adsorbent by adding an acidic elution solution containing salt to the column; and eluting the AAV by a gradient that reduces the salt concentration and pH of the eluate in the elution process. The problem is solved by a method of removing AAV aggregates, which includes the process of collecting the fraction with the fastest elution time among the fractions obtained by the purification method.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a method for purifying adeno-associated virus (AAV), and in particular to a method for purifying AAV contained in a sample using affinity chromatography, and a method for removing AAV aggregates using the purification method. [Background technology]

[0002] Adeno-associated virus (AAV) is a non-enveloped virus classified in the Parvoviridae family and Dependovirus genus. The AAV envelope particle is composed of three proteins (VP1, VP2, and VP3), and approximately 60 protein molecules are mixed and assembled in a VP1:VP2:VP3 ratio of 1:1:10, forming a regular icosahedron with a diameter of 20 to 30 nm.

[0003] In nature, AAV lacks the ability to replicate autonomously and depends on helper viruses such as adenoviruses and herpesviruses for replication. In the presence of the helper virus, the AAV genome replicates in the host cell, forming complete AAV particles containing the AAV genome, which are then released from the host cell. On the other hand, in the absence of the helper virus, the AAV genome is maintained in an episome or integrated into the host chromosome (latent state).

[0004] AAV is capable of infecting cells of a wide range of species, including humans, and can also infect non-dividing cells that have completed differentiation, such as blood cells, muscles, and nerve cells. It is also non-pathogenic to humans, meaning there is little risk of side effects. Furthermore, the viral particles are physically and chemically stable. For these reasons, AAV is attracting attention as a potential vector for gene transfer aimed at treating congenital genetic diseases.

[0005] Recombinant AAV vectors (hereafter simply referred to as AAV vectors) are typically produced by introducing nucleic acids encoding elements essential for AAV particle formation into cells to generate cells capable of producing AAV (hereafter also referred to as AAV-producing cells), and then culturing these cells to express the elements essential for AAV particle formation. AAV is generally produced by introducing three types of plasmids into cells: a plasmid (vector plasmid) that delivers a recombinant AAV genome carrying a polynucleotide encoding the protein to be expressed, leaving the ITR (inverted terminal repeat) sequences at both ends; a plasmid (packaging plasmid) that delivers Rep proteins and capsid proteins (cosid proteins VP1, VP2, and VP3); and a plasmid (helper plasmid) that delivers only the adenovirus-derived elements essential for AAV particle formation. Alternatively, AAV vectors can be produced using host cells previously transfected with elements essential for AAV particle formation or using insect cells (e.g., Sf9 cells).

[0006] The produced AAV vectors are recovered from AAV-producing cells and purified into therapeutic AAV vector preparations. However, during this process, a certain amount of aggregates (AAV aggregates) may be generated. AAV aggregates have a reduced infectious titer compared to monomeric AAV molecules (Non-Patent Document 1). If AAV aggregates with a reduced infectious titer are contained in a therapeutic AAV vector preparation administered to a patient, this may lead to a reduced therapeutic effect due to reduced pharmacological efficacy or to the induction of severe side effects due to high dose burden. Therefore, to produce a high-quality therapeutic AAV vector preparation, it is necessary to purify the vector so that fractions containing AAV aggregates can be separated and removed.

[0007] A known method for purifying AAV is an affinity chromatography method using an AAV adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier (Patent Document 1). In the method disclosed in Patent Document 1, AAV adsorbed to the AAV adsorbent is eluted using a gradient that decreases the pH of the eluate. However, even when purified using the method disclosed in Patent Document 1, AAV aggregates could not be removed. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 2018-507707 [Non-patent literature]

[0009] [Non-Patent Document 1] Gerard A et al.,Pharm Res,36,29(2019) Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a method for purifying adeno-associated viruses contained in a sample using affinity chromatography so as to remove aggregates of the viruses. [Means for solving the problem]

[0011] As a result of extensive research, the inventors have found that the above-mentioned problems can be solved by using an affinity chromatography column packed with an adsorbent containing an insoluble carrier and an adeno-associated virus (AAV)-binding protein immobilized on the carrier, and optimizing the conditions for eluting AAV adsorbed to the adsorbent so that AAV aggregates are removed, thereby completing the present invention.

[0012] That is, the present invention includes the following inventions.

[0013] [1] A method for purifying AAV contained in a sample, comprising: (1) adding a sample containing AAV to a column packed with an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, thereby adsorbing the AAV to the adsorbent; and (2) adding an acidic elution solution containing salt to the column, thereby eluting the AAV adsorbed to the adsorbent, wherein in step (2), the AAV is eluted using a gradient that decreases the salt concentration and pH of the elution solution.

[0014] [2] A method for removing AAV aggregates contained in a sample, comprising: (1) adding a sample containing AAV to a column packed with an adsorbent comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier, thereby adsorbing the AAV to the adsorbent; (2) adding an acidic elution solution containing salt to the column, thereby eluting the AAV adsorbed to the adsorbent; and (3) recovering a fraction with an early elution time from the fractions containing AAV eluted in step (2), wherein in step (2), the AAV is eluted using a gradient that decreases the salt concentration and pH of the elution solution.

[0015] [3] The method according to [1] or [2], wherein the AAV-binding protein is a polypeptide selected from the group consisting of (i) and (iii) below: (i) a polypeptide 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 polypeptide having an amino acid sequence containing 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 the amino acid sequence further contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 500, and having AAV-binding activity; (iii) A polypeptide 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 polypeptide has 70% or more identity to the amino acid sequence from positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0016] The present invention will be described in detail below. However, the present invention is not limited to the following embodiments and can be implemented in any form without departing from the spirit of the present invention.

[0017] In the present invention, AAV contained in a sample is purified using a column packed with an adsorbent (hereinafter also referred to as "AAV adsorbent") comprising an insoluble carrier and an AAV-binding protein immobilized on the carrier. The AAV-binding protein is not particularly limited as long as it is a polypeptide capable of binding to AAV, and examples thereof include laminin receptors such as integrins, anti-AAV antibodies, and AAV receptors (AAVRs).

[0018] When the AAV binding protein is AAVR, preferred embodiments include polypeptides shown in any of (i) to (iii) below. (i) a polypeptide 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 polypeptide having an amino acid sequence containing 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 the amino acid sequence further contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions within the amino acid residues at positions 312 to 500, and having AAV-binding activity; (iii) A polypeptide 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 polypeptide has 70% or more identity to the amino acid sequence from positions 312 to 500, and wherein the polypeptide has AAV-binding activity.

[0019] The amino acid sequence set forth in SEQ ID NO: 1 is the amino acid sequence of KIAA0319L (official database: UniProt, accession number: Q8IZA0), which is one embodiment of AAVR, and the amino acid residues from serine (Ser) at position 312 to aspartic acid (Asp) at position 500 in the amino acid sequence set forth in SEQ ID NO: 1 correspond to the extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L.

[0020] The polypeptides described in any of (i) to (iii) above need only contain at least the regions corresponding to PKD1 and PKD2 of the aforementioned KIAA0319L, and may, for example, include all or part of the regions corresponding to other extracellular domains located on the C-terminal side of PKD2 (domain 3 (PKD3), domain 4 (PKD4), and domain 5 (PKD5)), or may include all or part of the region corresponding to a signal sequence or cysteine-rich region such as the MANSC (Motif At N terminus with Seven Cysteines) domain located on the N-terminal side of PKD1, or may include all or part of the transmembrane region and intracellular region located on the N-terminal and / or C-terminal side of the extracellular region.

[0021] In (ii), "one or several" varies depending on the position of the amino acid substitution in the three-dimensional structure of AAVR and the type of amino acid residue, but, as an example, means any of 1 to 50, 1 to 30, 1 to 20, 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.

[0022] An example of (ii) is a peptide 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, provided that the amino acid residues from position 312 to position 500 include any of the following: Gly390Ser (this notation indicates that glycine at position 390 of SEQ ID NO: 1 is substituted with serine, the same applies hereinafter), Ile319Phe, Leu321Pro, Pro322Leu, Asn324Asp, Asn324Ser, Gln327Arg, Asn329Asp, Asn329Tyr, Ala330Gly, Tyr331Cys, Val332Gln, Leu333Val, Leu333Pro, Gln334Arg, Pro337Leu, Lys338Arg, Lys338Glu, Glu340Gly, Thr341Ala, Tyr342Cys, Tyr342His, Tyr34 2Asn, Thr343Met, Tyr344His, Asp345Asn, Trp346Leu, Trp346Arg, Gln347Leu, Gln347Pro, Leu348Pro, Ile349Thr, Thr350Met, His351Leu, Pro352Leu, Arg353Cys, Asp354Gly, Tyr355His, Tyr355Asn, Tyr355Cys, Ser356Cys, Gly357Cys, His363Arg, His363Leu, Ser364Pro, Gln365Arg, Ile366Thr, Leu36 7Pro, Lys368Arg, Leu369Gln, Leu369Pro, Ser370Ala, Lys371Glu, Thr373Ala, Leu376Pro, Tyr377Cys, Phe379Ser, Val381Ala, Glu384Gly, Asn387Ser, His389Gln, His389Leu, His389Arg, Glu391Lys, Tyr393Cys, Val396Ala, Lys399Glu, Lys399Arg, Arg406Ser, Val412Ala, Gln415Leu, Phe416Ser, Gln44 1Leu, Tyr442Phe, Lys448Arg, Glu453Gly, Lys455Arg, Glu458Gly, Ala461Ser, Ser476Arg, Leu477Pro, Asn487Asp, Asn492Asp, Ile319Asn, Ile319Ser,Thr320Ile, Lys323Glu, Leu328Gln, Leu328Pro, Tyr331His, Val332Ala, Val332Glu, Pro336Gln, Pro337Gln, Lys338Asn, Tyr342 Arg, Thr350Ser, Ile366Phe, Val383Ala, Gln386Arg, His389Asp, Gly392Cys, Val394Ala, Ile409Val, Ser476Gly, Thr490Ser, Se r312Pro, Ala313Ser, Val317Asp, Gln318Pro, Thr320Ala, Lys323Arg, Val326Ala, Val326Glu, Gln327His, Gln327Leu, Asn329Hi s, Asn329Ile, Val332Ala, Glu335Gly, Glu335Val, Glu340Val, Thr341Pro, Thr343Ser, Tyr344Phe, Trp346Cys, Met359Leu, Glu3 60Lys, Glu360Val, Gly361Cys, Lys362Glu, Lys362Asn, Lys362Gly, Ser364Leu, Lys371Asn, Lys371Asp, Leu372Pro, Leu372Gln, Pro374Leu, Glu378Gly, Glu378Val, Phe379Tyr, Phe379Cys, Lys380Glu, Val381Asp, Ile382Val, Glu384Val, Val394Asp, Val394 Examples of such polypeptides include polypeptides in which at least one amino acid substitution has occurred among Ile, Asn395Ser, Thr397Ser, Glu401Val, Arg403His, Arg406His, Gln415Arg, Thr426Ala, Gln432Leu, Gln432Arg, Gln441Arg, His443Leu, Lys448Glu, Ile456Val, Asp483Asn, Ser488Leu, Val499Glu, and Val499Ile. Among these, Gly390Ser is an amino acid substitution that particularly improves stability against heat and acid. Therefore, the polypeptides contain 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, provided that in the amino acid residues at positions 312 to 500:A polypeptide in which at least the amino acid substitution Gly390Ser has occurred is a preferred embodiment of (ii) above.

[0023] The "substitution of one or several amino acid residues" in (ii) above may include not only the amino acid substitution at the specific position described above, but also conservative substitutions in which amino acids with similar physical and / or chemical properties are substituted. Those skilled in the art know that conservative substitutions generally maintain protein function between substituted and unsubstituted amino acids. Examples of conservative substitutions include substitutions between glycine and alanine, between serine and proline, or between glutamic acid and alanine (Protein Structure and Function, Medical Science International, 9, 2005). Furthermore, the "substitution, deletion, insertion, or addition of one or several amino acid residues" in (ii) above also includes naturally occurring mutations (mutants or variants) due to differences in the origin of AAVR or differences in species.

[0024] The amino acid sequence homology in (iii) above may be 70% or higher, but may also be higher (e.g., 80% or higher, 85% or higher, 90% or higher, or 95% or higher). As used herein, "homology" may refer to similarity or identity, and may particularly refer to identity. "Amino acid sequence homology" refers to homology across the entire amino acid sequence. "Identity" between amino acid sequences refers to the proportion of amino acid residues of the same type in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). "Similarity" between amino acid sequences refers to the sum of the proportion of amino acid residues of the same type and the proportion of amino acid residues with similar side chain properties in those amino acid sequences (Experimental Medicine, 31(3), Yodosha). Amino acid sequence homology can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) or FASTA.

[0025] The insoluble carrier, a component of the AAV adsorbent used in the present invention, is not particularly limited as long as it is insoluble in AAV-containing samples and solutions used for purification (e.g., elution solution, equilibration solution, and wash solution). Examples of insoluble carriers include carriers made from polysaccharides such as agarose, alginate (alginate salts), carrageenan, chitin, cellulose, dextrin, dextran, and starch; carriers made from synthetic polymers such as polyvinyl alcohol, polymethacrylate, poly(2-hydroxyethyl methacrylate), and polyurethane; and carriers made from ceramics such as silica. Among these, carriers made from polysaccharides and synthetic polymers are preferred. Examples of preferred carriers include polymethacrylate gels with hydroxyl groups, such as Toyopearl (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by GE Healthcare), and cellulose gels such as Cellufine (manufactured by JNC). The shape of the insoluble carrier is not particularly limited. The insoluble carrier may have any shape as long as it can be packed into a column, and may be, for example, granular, monolithic, membrane-like, or fibrous, and may be porous or non-porous.

[0026] When producing the AAV adsorbent used in the present invention, the AAV-binding protein may be immobilized on an insoluble carrier via, for example, a covalent bond. Specifically, for example, the AAV-binding protein may be immobilized on an insoluble carrier by covalently bonding the insoluble carrier via an active group possessed by the insoluble carrier, thereby producing the AAV adsorbent used in the present invention. Examples of the active group include an N-hydroxysuccinimide (NHS)-activated ester group, an epoxy group, a carboxy group, a maleimide group, a haloacetyl group, a tresyl group, a formyl group, and a haloacetamide group. As the insoluble carrier having an active group, for example, a commercially available insoluble carrier having an active group may be used as is, or an active group may be introduced into the insoluble carrier. Examples of commercially available supports having active groups include TOYOPEARL AF-Epoxy-650M and TOYOPEARL AF-Tresyl-650M (both manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, and Epoxy-activated Sepharose 6B (all manufactured by Cytiva), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0027] An example of a method for introducing active groups onto the support surface is to react one of a compound having two or more active sites with a hydroxy group, epoxy group, carboxy group, amino group, etc. present on the support surface.

[0028] Examples of compounds that introduce epoxy groups into hydroxy groups or amino groups present on the surface of a carrier include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

[0029] Examples of compounds that introduce carboxy groups into epoxy groups present on the surface of the carrier include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0030] Compounds that introduce maleimide groups into hydroxy groups, epoxy groups, carboxy groups, and amino groups present on the support surface include N-(ε-maleimidocaproic acid) hydrazide, N-(ε-maleimidopropionic acid) hydrazide, 4-(4-N-maleimidophenyl)acetic acid hydrazide, 2-aminomaleimide, 3-aminomaleimide, 4-aminomaleimide, 6-aminomaleimide, 1-(4-aminophenyl)maleimide, 1-(3-aminophenyl)maleimide, 4-(maleimido)phenyl isocyanate, 2-maleimidoacetic acid, 3-maleimidopropionic acid, and Examples include pionic acid, 4-maleimidobutyric acid, 6-maleimidohexanoic acid, N-(α-maleimidoacetoxy)succinimide ester, (m-maleimidobenzoyl)N-hydroxysuccinimide ester, succinimidyl-4-(maleimidomethyl)cyclohexane-1-carbonyl-(6-aminohexanoic acid), succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylic acid, (p-maleimidobenzoyl)N-hydroxysuccinimide ester, and (m-maleimidobenzoyl)N-hydroxysuccinimide ester.

[0031] Furthermore, examples of compounds that introduce haloacetyl groups into hydroxy groups or amino groups present on the surface of a carrier include chloroacetic acid, bromoacetic acid, iodoacetic acid, chloroacetic acid chloride, bromoacetic acid chloride, bromoacetic acid bromide, chloroacetic acid anhydride, bromoacetic acid anhydride, iodoacetic acid anhydride, 2-(iodoacetamido)acetic acid-N-hydroxysuccinimide ester, 3-(bromoacetamido)propionic acid-N-hydroxysuccinimide ester, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide ester.

[0032] Another example of a method for introducing active groups onto the support surface is to react hydroxyl or amino groups present on the support surface with an ω-alkenyl alkane glycidyl ether, followed by activation by halogenating the ω-alkenyl moiety with a halogenating agent. Examples of ω-alkenyl alkane glycidyl ethers include allyl glycidyl ether, 3-butenyl glycidyl ether, and 4-pentenyl glycidyl ether. Examples of halogenating agents include N-chlorosuccinimide, N-bromosuccinimide, and N-iodosuccinimide.

[0033] Another example of a method for introducing active groups onto the support surface is to use a condensing agent and an additive to introduce active groups into carboxy groups present on the support surface. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiamide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

[0034] Immobilization of an AAV-binding protein on an insoluble carrier can be carried out, for example, in a buffer solution. Examples of buffer solutions include acetate buffer, phosphate buffer, MES (2-morpholinoethanesulfonic acid) buffer, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Tris (Tris(hydroxymethyl)aminomethane) buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set depending on various conditions, such as the reactivity of the active group and the stability of the AAV-binding protein. The reaction temperature during immobilization may be, for example, 4°C to 50°C, and preferably 10°C to 35°C.

[0035] The purification method of the present invention comprises: (1) adding a sample containing AAV to a column packed with the AAV adsorbent, and allowing the AAV to be adsorbed onto the adsorbent; (2) adding an acidic elution solution containing a salt to the column to elute the AAV adsorbed to the adsorbent.

[0036] A sample containing AAV can be added to the column using a liquid delivery means such as a pump. Herein, adding a liquid to a column is also referred to as "delivering a liquid to the column." The AAV-containing sample may be solvent-substituted with an appropriate buffer solution before being added to the column. Furthermore, before loading the AAV-containing sample onto the column (i.e., before step (1) above), the column may be equilibrated with an appropriate buffer. This equilibration is expected to, for example, enable the purification of AAV with a higher purity. Examples of buffers used for solvent exchange and equilibration include phosphate buffer, acetate buffer, and MES buffer. Such buffers may further contain, for example, 10 mM to 100 mM of an inorganic salt, such as sodium chloride. The buffer used for solvent exchange and the buffer used for equilibration may or may not be the same. Furthermore, if components other than AAV, such as contaminants, remain on the column after the AAV-containing sample is passed through the column, these components may be removed from the column before eluting the AAV adsorbed to the AAV adsorbent (i.e., before step (2) above). Components other than AAV can be removed from the column using, for example, an appropriate buffer. The same descriptions regarding the buffers used for solvent exchange and equilibration can be applied mutatis mutandis to the buffers used for solvent exchange and equilibration.

[0037] The purification method of the present invention is characterized in that, when eluting AAV adsorbed to the AAV adsorbent (i.e., during step (2) above), the eluate is applied to the column in a gradient that decreases the salt concentration and pH of the eluate. By lowering the pH of the eluate (i.e., shifting it to the acidic side) and also decreasing the salt concentration of the eluate, AAV adsorbed to the AAV adsorbent can be separated into a fraction containing only AAV monomers and a fraction containing AAV aggregates. The gradient that decreases the salt concentration and pH of the eluate can be changed in two or more steps (stepwise) (step gradient) or can be changed linearly (linear gradient), but linear gradient elution is preferred. The eluate may contain salt and be more acidic than the buffer used for solvent exchange or equilibration. Examples of such buffers include citrate buffer, glycine-hydrochloric acid buffer, and acetate buffer. The pH and salt concentration of the eluate and the method for changing them can be determined appropriately depending on the properties of the AAV adsorbent. For example, when the eluate is an acetate buffer containing sodium chloride, step (2) can be performed using a linear gradient in which the sodium chloride concentration is decreased from 150 mM to 0 mM and the pH is decreased from 4.5 to 3.0.

[0038] According to the purification method of the present invention, AAV adsorbed to the AAV adsorbent is eluted in the order of the earliest elution time: a fraction containing only AAV monomers, and a fraction containing AAV aggregates. (3) recovering the fraction with the earliest elution time from the fractions containing AAV eluted in step (2); By adding the eluate, AAV aggregates contained in the sample can be removed. That is, a purified AAV monomer free of AAV aggregates can be produced by a method comprising steps (1) to (3) above. If three or more fractions are eluted in step (2) above, only the fraction with the earliest elution time may be collected, or all fractions except the fraction with the slowest elution time may be collected. Fractions containing only AAV monomers can be collected by, for example, conventional methods. Methods for collecting fractions containing only AAV monomers include replacing the collection container at regular intervals or at regular volume intervals, changing the collection container according to the shape of the chromatogram of the eluate, and collecting fractions using an automated fraction collector such as an autosampler. Furthermore, AAV can also be collected from fractions containing only AAV monomers. Collection of AAV monomers from fractions containing only AAV monomers can be performed, for example, by known methods used for protein purification. The purity of the AAV monomers in the collected fractions (the rate of contamination with AAV aggregates) can be confirmed, for example, by analyzing the particle size of the AAV in the fractions using a dynamic light scattering photometer. [Effects of the Invention]

[0039] The present invention comprises the steps of: adding a sample containing AAV to a column packed with an adsorbent comprising an insoluble carrier and an adeno-associated virus (AAV)-binding protein immobilized on the carrier, thereby adsorbing the AAV to the adsorbent; and adding an acidic eluent containing salt to the column to elute the AAV adsorbed to the adsorbent. In the elution step, the AAV is eluted using a gradient of decreasing salt concentration and pH of the eluent. The present invention enables separation of AAV contained in a sample into a fraction containing only AAV monomers and a fraction containing AAV aggregates based on the elution peaks obtained by liquid chromatography. Furthermore, AAV aggregates contained in the sample can be removed by collecting the fraction containing only AAV monomers, which has an early elution time. Because the present invention enables separation of AAV aggregates, which is difficult using conventional AAV purification methods, production and quality control in industrial AAV production can be facilitated. [Brief explanation of the drawings]

[0040] [Figure 1]This figure shows the purity results for the adeno-associated virus (AAV) vector solution obtained in Example 1 (5-5). In the figure, AP represents the analytical result of a 1000-fold dilution of the supernatant obtained in Example 1 (5-3), and E represents the analytical result of the AAV vector solution obtained in Example 1 (5-5). VP1 to VP3 represent bands corresponding to the three coat proteins that constitute AAV. [Figure 2] This figure shows the chromatography pattern when an AAV vector solution was purified using an AAV receptor (AAVR) column by the purification method of the present invention in Example 4. The fraction of the peak at an elution time of approximately 23 minutes (Fr23) and the fraction of the peak at an elution time of approximately 26 minutes (Fr26) were collected. [Figure 3] FIG. 1 shows the results of dynamic light scattering photometric analysis of the particle size distribution of AAV contained in the fractions (Fr23, Fr26) collected in Example 4. (a) shows the results for elution peak Fr23, and (b) shows the results for elution peak Fr26. [Figure 4] FIG. 1 shows the chromatography pattern when an AAV vector solution was purified using an AAVR column in Comparative Example 1. [Figure 5] FIG. 1 shows the chromatography pattern when an AAV vector solution was purified using an AAVR column in Comparative Example 2. [Example]

[0041] EXAMPLES Hereinafter, examples will be shown to explain the present invention in more detail, but the present invention is not limited to these examples.

[0042] Example 1 Preparation of Adeno-Associated Virus (AAV) Vectors (1) A nucleotide sequence (SEQ ID NO: 3) was designed in which a restriction enzyme EcoRI recognition sequence (GAATTC) was added to the 5' end of a polynucleotide encoding EGFP (enhanced green fluorescent protein), which consists 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.

[0043] (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, named 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).

[0044] (3) The pUC-EGFP obtained in (2) was digested with the restriction enzymes EcoRI and BamHI and then ligated to the expression vector pAAV-CMV (Takara Bio) previously digested with the restriction enzymes EcoRI and BamHI. The ligation product was used to transform Escherichia coli JM109. The resulting transformant was cultured in LB medium containing 100 μg / mL carbenicillin, and the EGFP-expressing vector pAAV-EGFP was extracted using a QIAprep Spin Miniprep kit (Qiagen).

[0045] (4) The transformant obtained in (3) was cultured overnight at 37°C with shaking in a 5-L baffled flask containing 1 L of 2YT 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).

[0046] (5) Preparation of AAV2-EGFP (5-1) Escherichia coli JM109 strain was transformed with the pRC2-mi342 Vector (Takara Bio Inc.) and pHelper Vector (Takara Bio Inc.). The resulting transformant was used in the same manner as in (4) to prepare large quantities of pRC2-mi342 and pHelper.

[0047] (5-2) 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 (4), the pRC2-mi342 prepared in (5-1), 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.

[0048] (5-3) The frozen cells obtained in (5-2) were thawed and suspended in 10 mL of 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M sodium chloride, 4 mM magnesium chloride, and 0.01% (w / v) Tween 20 (trade name). Benzonase (Merck Millipore) was added at a 1 / 2000 volume, 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 the 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.

[0049] (5-4) The supernatant from which the floating matter had been removed was applied to a 5 mL AVB Sepharose column (manufactured by Cytiva) that had been equilibrated in advance with 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M sodium chloride (hereinafter also referred to as "equilibration solution A").

[0050] (5-5) After washing with equilibration solution A, the column was eluted with 0.1 M acetate buffer (pH 2.5) containing 0.5 M sodium chloride. The resulting eluate was neutralized by adding 1 / 4 volume of 1 M Tris-HCl buffer (pH 8.5) containing 20 mM magnesium chloride to obtain a solution of the AAV vector, AAV2-EGFP.

[0051] (5-6) The AAV2-EGFP concentration in the solution obtained in (5-5) was quantified by qPCR using the AAVpro Titration Kit (Takara Bio Inc.). The solution was subjected to SDS-PAGE and silver stained using the Pierce Silver Stain Kit (Thermo Fisher Scientific) to confirm the purity of the AAV vector contained in the solution.

[0052] The purity results for (5-6) are shown in Figure 1. Only bands corresponding to VP1, VP2, and VP3, which constitute the AAV vector, were observed.

[0053] Example 2 Preparation of AAV binding proteins (1) Escherichia coli strain BL21(DE3) was transformed with the plasmid pET-AVR10s, which contains a polynucleotide (SEQ ID NO: 5) encoding an AAV-binding protein consisting of the amino acid sequence set forth in SEQ ID NO: 4. The resulting transformant capable of expressing the AAV-binding protein AVR10s was inoculated into 3 mL of 2YT liquid medium containing 50 μg / mL kanamycin and pre-cultured overnight at 37°C under aerobic shaking. In SEQ ID NO: 4, the portion from the 1st methionine (Met) to the 22nd alanine (Ala) represents the PelB signal peptide, the 25th serine (Ser) to the 213th aspartic acid (Asp) represents the AAV binding protein AVR10s (amino acid residues 312 to 500 of SEQ ID NO: 1, with the following amino acid substitutions occurring in the 312th to 500th amino acid residues: Val317Asp (this notation indicates that the 317th valine has been substituted with aspartic acid; the same applies below), Tyr342Ser, Lys362Glu, Lys371Asn, Val381Ala, Ile382Val, Gly390Ser, Lys399Glu, Ser476Arg, and Asn487Asp), and the 214th to 219th histidines (His) represent the tag sequence. In SEQ ID NO: 4, the aspartic acid of Val317Asp is at position 30, the serine of Tyr342Ser is at position 55, the glutamic acid of Lys362Glu is at position 75, the asparagine of Lys371Asn is at position 84, the alanine of Val381Ala is at position 94, the valine of Ile382Val is at position 95, the serine of Gly390Ser is at position 103, the glutamic acid of Lys399Glu is at position 112, the arginine of Ser476Arg is at position 189, and the aspartic acid of Asn487Asp is at position 200.

[0054] (2) 2 mL of the preculture solution from (1) was inoculated into 200 mL of 2YT liquid medium containing 50 μg / mL of kanamycin in a 1 L baffled flask, and the mixture was cultured aerobically at 37°C with shaking.

[0055] (3) After 2.0 hours from the start of the culture, the mixture was cooled on ice, IPTG was added to a final concentration of 0.1 mM, and the mixture was subsequently cultured overnight at 25°C under aerobic shaking.

[0056] (4) After the cultivation was completed, the culture medium was centrifuged at 4°C and 8000 rpm for 20 minutes to collect the bacterial cells.

[0057] (5) The cells recovered in (4) were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole at a concentration of 5 mL / 1 g (cells), and then disrupted using an ultrasonic generator (Insonator 201M [Kubota Shoji Co., Ltd.]) at 8°C for approximately 10 minutes at an output of approximately 150 W. The disrupted cell solution was centrifuged twice at 8,000 rpm for 20 minutes at 4°C, and the supernatant was recovered.

[0058] (6) The supernatant obtained in (5) was applied to an XK26 / 20 column (GE Healthcare) packed with 50 mL of Ni Sepharose 6 Fast Flow (GE Healthcare) that had been equilibrated with Tris-HCl buffer (pH 7.4) containing 150 mM sodium chloride and 20 mM imidazole (hereinafter also referred to as "equilibration solution B"). After washing with equilibration solution B, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) containing 0.5 M imidazole and 150 mM sodium chloride.

[0059] (7) The eluate obtained in (6) was dialyzed against 20 mM Tris buffer (pH 7.4) containing 150 mM sodium chloride to prepare the amount of AVR10s protein required for producing the AAV adsorbent.

[0060] Example 3 Preparation of AAV adsorbent (1) A hydrophilic vinyl polymer (Tosoh Corporation: Toyopearl) was chemically modified on the surface hydroxyl groups to introduce maleimide groups into the gel.

[0061] (2) To 3.5 g of the gel prepared in (1), 28 mg of AVR10s prepared in Example 2 and TCEP (Tris(2-CarboxyEthyl)Phosphine) as a reducing agent at a final concentration of 0.1 mM were added, and the mixture was allowed to react by shaking at pH 7.4 and 4°C for 15 hours. This resulted in the preparation of a gel in which AVR10s was immobilized (designated AAVR-immobilized gel).

[0062] Example 4 AAV purification using an AAVR column (1) 1.25 mL of the AAVR-immobilized gel prepared in Example 3 was packed into an empty stainless steel column (φ4.6 mm×75 mm, manufactured by Tosoh Corporation) to prepare an AAVR column.

[0063] (2) The prepared AAVR column was connected to an HPLC M40A (Shimadzu Corporation) and equilibrated with 50 mM acetate buffer (pH 4.5) containing 150 mM sodium chloride and 2 mM magnesium chloride (hereinafter also referred to as "equilibration solution C"). Then, the AAV2-EGFP solution obtained in Example 1 (5-5) (concentration: 2.8 × 10 11 0.5 mL of a solution of 1000 μg / mL of PEG was applied at a flow rate of 1 mL / min.

[0064] (3) After washing with equilibration solution C for 10 minutes, the pH was adjusted from 4.5 to 3.0 by adjusting the mixing ratio of 50 mM acetate buffer (pH 3.0) containing 2 mM magnesium chloride (hereinafter also referred to as "eluent A") to equilibration solution C. A linear gradient of sodium chloride concentration from 150 mM to 0 mM was then applied over 15 minutes. Elution solution A was then passed through for 5 minutes, and fractions expected to contain AAV2-EGFP were collected. AAV2-EGFP was detected by absorbance at 280 nm.

[0065] (4) The collected fractions were subjected to a dynamic light scattering spectrophotometer (Shoko Science Co., Ltd.) to obtain the particle size distribution of AAV contained in each fraction.

[0066] The resulting chromatographic pattern is shown in Figure 2. AAV2-EGFP peaks appeared at elution times of approximately 23 minutes (Fr23) and 26 minutes (Fr26), and the fractions corresponding to these peaks (Fr23 and Fr26) were collected. The dynamic light scattering analysis results for each fraction are shown in Figure 3. For Fr23, only a particle size peak near 20 nm, corresponding to the AAV monomer, was observed (Figure 3(a)). On the other hand, for Fr26, in addition to the particle size peak near 20 nm, corresponding to the AAV monomer, a particle size peak near 600 nm, corresponding to the AAV aggregates, was also observed (Figure 3(b)). These results demonstrate that when eluting AAV adsorbed to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, fractions containing AAV aggregates can be separated by eluting with a gradient of decreasing salt concentration and pH of the eluate, and that AAV aggregates can be removed by collecting fractions containing only AAV monomers.

[0067] Comparative Example 1 (1) The AAVR column prepared in Example 4(1) was connected to an HPLC M40A (Shimadzu Corporation), equilibrated with equilibration solution C, and then the AAV2-EGFP solution (concentration: 2.8 × 10) obtained in Example 1(5-5) was added. 11 0.5 mL of a solution of 1000 μg / mL of PEG was applied at a flow rate of 1 mL / min.

[0068] (2) After washing with equilibration solution C for 10 minutes, a linear gradient of sodium chloride from 150 mM to 0 mM was applied over 15 minutes by adjusting the mixing ratio of 50 mM acetate buffer (pH 4.5) containing 2 mM magnesium chloride (hereinafter referred to as "elution solution B") to equilibration solution C. Then, elution solution B was passed through for 5 minutes. AAV2-EGFP was detected by absorbance at 280 nm.

[0069] The resulting chromatographic pattern is shown in Figure 4. No elution peak for AAV2-EGFP was observed. This indicates that when eluting AAV adsorbed to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, AAV cannot be eluted using a gradient that decreases only the salt concentration of the eluate.

[0070] Comparative Example 2 (1) The AAVR column prepared in Example 4(1) was connected to an HPLC M40A (Shimadzu Corporation), equilibrated with equilibration solution C, and then the AAV2-EGFP solution (concentration: 2.8 × 10) obtained in Example 1(5-5) was added. 11 0.5 mL of a solution of 1000 μg / mL of PEG was applied at a flow rate of 1 mL / min.

[0071] (2) After washing with equilibration solution C for 10 minutes, a linear gradient was run over 15 minutes, changing the pH from 4.5 to 3.0 with 50 mM acetate buffer (pH 3.0) containing 150 mM sodium chloride and 2 mM magnesium chloride (hereinafter referred to as "elution solution C"), by adjusting the mixing ratio of the equilibration solution C to the equilibration solution C. Then, elution solution C was passed through for 5 minutes. AAV2-EGFP was detected by absorbance at 280 nm.

[0072] The resulting chromatographic pattern is shown in Figure 5. The elution peak of AAV2-EGFP was observed only around 25 minutes after elution. This indicates that when eluting AAV adsorbed to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier, AAV aggregates cannot be separated using a gradient that only decreases the pH of the eluate.

Claims

A method for purifying adeno-associated virus by affinity chromatography using a column packed with an adsorbent containing an insoluble carrier and an adeno-associated virus-binding protein immobilized on the carrier, comprising: (1) adding a sample containing adeno-associated virus to the column and adsorbing the virus to the adsorbent packed in the column; (2) adding an acidic eluent containing 150 mM sodium chloride to the column and eluting the adeno-associated virus adsorbed to the adsorbent; wherein the adeno-associated virus-binding protein is a polypeptide represented by any one of the following (i) to (iii); (i) a polypeptide comprising at least the amino acid residues from serine at position 312 to asparagine at position 500 in the amino acid sequence set forth in SEQ ID NO: 1; (ii) a polypeptide comprising at least the amino acid residues from serine at position 312 to asparagine at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, provided that the amino acid residues from position 312 to position 500 further contain substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, and having adeno-associated virus-binding activity; (iii) a polypeptide comprising at least the amino acid residues from serine at position 312 to asparagine at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, provided that it has 90% or more identity to the amino acid sequence from position 312 to position 500 and has adeno-associated virus-binding activity; and in the step (2), the virus is eluted by a gradient that decreases the pH together with the salt concentration of the eluent. A method for purifying adeno-associated virus. A method for removing adeno-associated virus aggregates by affinity chromatography using a column packed with an adsorbent containing an insoluble carrier and an adeno-associated virus-binding protein immobilized on the carrier, comprising: (1) adding a sample containing adeno-associated virus to the column and adsorbing the virus to the adsorbent packed in the column; (2) adding an acidic eluent containing 150 mM sodium chloride to the column and eluting the adeno-associated virus adsorbed to the adsorbent; (3) recovering the fraction with an earlier elution time from the fractions containing the adeno-associated virus eluted in the step (2). The adeno-associated virus-binding protein is a polypeptide shown in any one of the following (i) to (iii); (i) a polypeptide comprising at least the amino acid residues from serine at position 312 to asparagine at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, (ii) a polypeptide comprising at least the amino acid residues from serine at position 312 to asparagine at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, provided that in the amino acid residues from position 312 to position 500, the amino acid sequence further contains substitution, deletion, insertion, or addition of one or several amino acid residues at one or several positions, and has adeno-associated virus-binding activity, (iii) a polypeptide comprising at least the amino acid residues from serine at position 312 to asparagine at position 500 in the amino acid sequence set forth in SEQ ID NO: 1, provided that it has 90% or more identity to the amino acid sequence from position 312 to position 500, and has adeno-associated virus-binding activity, and a method for removing adeno-associated virus aggregates, wherein in the step (2), the virus is eluted by a gradient that lowers the pH together with the salt concentration of the eluate.

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