Method for purifying lentiviral vectors

Abstract

To provide a technique for purifying lentiviral vectors (LVs) contained in a sample using affinity chromatography. [Solution] The above problem is solved by an affinity chromatography carrier comprising an insoluble carrier and at least one of the cysteine-rich domains 2 and 3 of a low-density lipoprotein receptor immobilized on the carrier, and a method for purifying LV comprising the steps of adsorbing LV contained in a sample onto the carrier and eluting the LV adsorbed onto the carrier using an eluent.

Description

Technical Field

[0001] The present invention relates to a method for purifying a lentiviral vector (LV). In particular, the present invention relates to a method capable of highly purifying LV contained in a sample with high recovery rate using affinity chromatography.

Background Art

[0002] A lentiviral vector (LV) refers to a modified enveloped virus classified into the family Retroviridae, genus Lentivirus, which is used as a gene transfer vector. Among them, LV in which the envelope of Human Immunodeficiency Virus (HIV) is replaced with the glycoprotein (VSVG) of Vesicular Stomatitis Indiana Virus (VSV) can infect a wide variety of cell types. For example, it is most widely used as a tool for producing CAR-T cells (chimeric antigen receptor T cells), which are one of the cell-based pharmaceuticals.

[0003] LV is generally cultured by a production system using HEK293 cells as a host, and then purified and manufactured from the culture solution by ultracentrifugation or anion exchange chromatography. Purification by anion exchange chromatography is easier to scale up compared to purification by ultracentrifugation, but there are problems such as low impurity removal rate and decrease in the infectivity titer of the purified LV. In recent years, purification of LV by affinity chromatography using an anti-VSVG antibody as a ligand has been disclosed (Non-Patent Document 1), but the low specificity is a problem.

[0004] Non-patent document 2 discloses the low-density lipoprotein receptor (LDLR) (UniProt No. P01130, SEQ ID NO: 1), which is the infection receptor for VSV, as another protein capable of specifically binding to LV. Non-patent document 2 also discloses that the cysteine-rich domain 2 (CR2) or 3 (CR3) of the LDLR is the one that binds to VSV. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] ASMoreira et al., International Journal of Molecular Sciences, Vol.24, 3354(2023) [Non-Patent Document 2] J. Nikolic et al., Nature Communications, Vol.9, 1029(2018) [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a technique for purifying lentiviral vectors contained in a sample using affinity chromatography. [Means for solving the problem]

[0007] As a result of diligent research, the inventors have solved the above problem by using a carrier for affinity chromatography for lentiviral vector (LV) purification that includes an insoluble carrier and at least one of the cysteine-rich domains 2 (CR2) and 3 (CR3) of the low-density lipoprotein receptor (LDLR) immobilized on the carrier. This led to the completion of the Ming Dynasty.

[0008] In other words, the present invention encompasses the following embodiments shown in [1] to

[14] . [1] A method for purifying a lentiviral vector (LV) containing the envelope of vesicular stomatitis virus (VSV) contained in a sample, The process of adsorbing the aforementioned LV onto a carrier for affinity chromatography, The process includes at least the step of eluting the LV adsorbed on the carrier using an eluent, A method wherein the carrier comprises an insoluble carrier and at least one of the cysteine-rich domains 2 (CR2) and 3 (CR3) of a low-density lipoprotein receptor (LDLR) immobilized on the carrier. [2] The method according to [1], wherein the affinity chromatography carrier comprises an insoluble carrier and at least CR3 of an LDLR immobilized on the carrier, wherein the CR3 is a polypeptide selected from any of (i) to (iii) below; (i) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, (ii) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions in the amino acid sequence, and which has VSV binding activity. (iii) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, provided that it has 70% or more identity with the entire amino acid sequence and has VSV binding activity. [3] The method according to [1] or [2], wherein the eluate contains at least magnesium ions. [4] The method according to any one of [1] to [3], wherein the pH of the eluate is 4.0 or more and 9.0 or less. [5] The method according to [3] or [4], wherein the magnesium ion concentration of the eluate is 300 mmol / L or more and 2500 mmol / L or less. [6] The method according to any one of [1] to [5], wherein the eluent is a buffer selected from the group consisting of MES buffer, bis-trispropane buffer, and PIPES buffer. [7] The method according to any one of [1] to [6], wherein the concentration of the surfactant contained in the eluate is 0.01% or less. [8] The method according to any one of [1] to [3] or [5], wherein the eluate contains acetate ions and the pH of the eluate is 3.0 or higher and 4.5 or lower. [9] The method according to any one of [1] to [3] or [5], wherein the eluate is a bis-trispropane buffer or a glycine buffer and has a pH of 8.0 or higher and 10.5 or lower.

[10] The method according to any one of [1] to [9], wherein the LV is a pseudotyped LV comprising the envelope of VSV and the capsid of human immunodeficiency virus.

[11] A carrier for affinity chromatography comprising an insoluble carrier and at least one of CR2 and CR3 of LDLR immobilized on the carrier.

[12] The affinity chromatography carrier according to

[11] , wherein the affinity chromatography carrier comprises an insoluble carrier and at least CR3 of an LDLR immobilized on the carrier, wherein the CR3 is a polypeptide selected from any of (i) to (iii) below; (i) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, (ii) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions in the amino acid sequence, and which has VSV binding activity. (iii) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, provided that it has 70% or more identity with the entire amino acid sequence and has VSV binding activity.

[13] The affinity chromatography carrier contains VSV in the sample. Affinity chromatography carrier according to

[11] or

[12] , used for the purification of LV containing rhubarb.

[14] The affinity chromatography carrier according to

[13] , wherein the LV is a pseudotyped LV comprising the envelope of VSV and the capsid of human immunodeficiency virus. [Effects of the Invention]

[0009] The present invention provides an affinity chromatography carrier comprising an insoluble carrier and at least one of the cysteine-rich domains 2 (CR2) and 3 (CR3) of a low-density lipoprotein receptor immobilized on the carrier, and a method for purifying lentiviral vectors (LV), which include the envelope of bullous stomatitis virus (VSV) contained in a sample, onto the affinity chromatography carrier, and elute the LV adsorbed on the carrier using an eluent. In a preferred embodiment of the present invention, a method for purifying the LV with a high recovery rate is provided by using an eluent containing at least magnesium ions or acetate ions. In another preferred embodiment of the present invention, a method for purifying the LV with a high recovery rate is provided by using an eluent such as tris-bispropane buffer or glycine buffer with a pH of 8.0 to 10.5. In a further preferred embodiment, a method for purifying the LV while suppressing a decrease in the infectivity titer is provided by selecting a mild buffer or a buffer with a low surfactant concentration as the eluent. [Brief explanation of the drawing]

[0010] [Figure 1] This figure shows the relative amounts of VSVG-LV contained in the eluted fraction (elution ratio of 0 mmol / L). (Example 8) [Figure 2] This figure shows the amount of infectious LV contained in the eluted fraction for each amount of Tween® added. (Example 14) [Modes for carrying out the invention]

[0011] The present invention will be described in detail below. In an embodiment of the present invention, the carrier for affinity chromatography comprises an insoluble carrier and at least one of cysteine-rich domain 2 (CR2) and domain 3 (CR3) of low density lipoprotein receptor (LDLR) immobilized on the carrier. Furthermore, in one aspect of the present invention, the carrier for affinity chromatography can be used for adsorption of a lentiviral vector (LV) containing an envelope of vesicular stomatitis virus (VSV). As described in the background art, LDLR is an infection receptor of VSV (Nat Commun 9, 1029: Non-Patent Document 2), and can be used as a ligand that specifically adsorbs a virus containing the envelope of VSV.

[0012] In this embodiment, the LV containing the envelope of VSV to be purified is not particularly limited as long as it is a LV pseudotyped with the envelope of VSV. As an example of a pseudotyped lentivirus, there can be exemplified a LV containing the envelope of VSV and the capsid of any one of herpes simplex virus type 1 (HSV-1), herpes simplex virus type ② (HSV-2), varicella-zoster virus (VZV), human immunodeficiency virus (HIV), rabies virus, influenza virus, hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, measles virus, and rubella virus. As a particularly preferred aspect of the LV, there is a LV containing the envelope of VSV and the capsid of HIV. <>

[0013] In this specification, the terms "lentivirus" and "lentiviral vector" may be used interchangeably, and by introducing a specific plasmid into a specific packaging cell Refers to the resulting lentiviral vector. The lentivirus or lentiviral vector disclosed herein may contain nucleic acids, such as transgenes. The transgene may be operably linked to a promoter sequence. The nucleic acid may contain one or more long terminal repeat (LTR) sequences. The LTR can facilitate the insertion of the transgene and the promoter into the host cell genome. The LTR sequence may contain the LTR sequence of wild-type lentivirus or a variant thereof. In some embodiments, the transgene is integrated into the chromosomal DNA of the target cell.

[0014] In this embodiment, the polypeptide containing at least one of CR2 and CR3 of LDLR used as a ligand for the carrier for affinity chromatography (hereinafter also referred to as "LV-binding protein") only needs to contain at least one of CR2 and CR3 of LDLR, may contain only CR2 of LDLR, may contain only CR3 of LDLR, or may contain both CR2 and CR3 of LDLR. An embodiment containing at least CR3 of LDLR (specifically, an embodiment containing CR3 of LDLR or both CR2 and CR3 of LDLR) is preferred. Further, it may contain all or part of CR1 or the signal sequence on the N-terminal side of CR2 of LDLR, or all or part of CR4, CR5, CR6, CR7 or the epidermal growth factor-like domain (EGF-like domain) on the C-terminal side of CR3 of LDLR.

[0015] As an example when the LV-binding protein is a polypeptide containing CR2 of LDLR, there is a polypeptide selected from any of the following (I) to (III); (I) A polypeptide containing at least the amino acid sequence set forth in SEQ ID NO: 2 (from serine at position 65 to proline at position 105 in SEQ ID NO: 1), (II) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 2, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions in the amino acid sequence, and which has VSV binding activity. (III) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 2, provided that it has 70% or more identity with the entire amino acid sequence and has VSV binding activity. An example of a polypeptide in which the LV-binding protein is a polypeptide containing CR3 of LDLR is a polypeptide selected from any of the following (i) to (iii): (i) A polypeptide comprising at least the amino acid sequence described in SEQ ID NO: 3 (from proline position 106 to proline position 144 in SEQ ID NO: 1), (ii) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions in the amino acid sequence, and which has VSV binding activity. (iii) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, provided that it has 70% or more identity with the entire amino acid sequence and has VSV binding activity.

[0016] In this specification, the VSV binding activity refers to the binding activity to the envelope of VSV, and in one embodiment of the present invention, it refers to the binding activity to pseudotyped LV on the envelope of VSV.

[0017] In (II) and (ii) above, "one or several" means one of the following, although it also depends on the position of amino acid substitutions and the type of amino acid residue in the three-dimensional structure of the LV-binding protein. For example, it means one to 15, one to 14, one to 13, one to 12, one to 11, one to 10, one to 9, one to 8, one to 7, one to 6, one to 5, one to 4, one to 3, one to 2, or one.

[0018] Furthermore, the "substitution of one or more amino acid residues" in (II) and (ii) above refers not only to amino acid substitutions at specific positions as described above, but also to physical properties and / or chemical properties. Conservative substitutions may occur between similar amino acids. In the case of conservative substitutions, it is generally known to those skilled in the art that the function of the protein is maintained between the substituted and unsubstituted parts. Examples of conservative substitutions include substitutions between glycine and alanine, serine and threonine, or glutamic acid and aspartic acid (Protein Structure and Function, Medical Science International, 9, 2005).

[0019] Furthermore, the "substitution, deletion, insertion, or addition of one or more amino acid residues" in (II) and (ii) above also includes naturally occurring mutations (mutants or variants) based on differences in the origin of LV-binding proteins or differences in species.

[0020] The amino acid sequence identity in (III) and (iii) above only needs to be 70% or higher, and may have a higher identity (e.g., 80% or higher, 85% or higher, 90% or higher, or 95% or higher). "Amino acid sequence identity" means identity of the entire amino acid sequence. "Identity" between amino acid sequences means the ratio of amino acid residues of the same type in those amino acid sequences (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.

[0021] The LV-binding protein may have an oligopeptide added to its N-terminus or C-terminus, which is useful for separating it from solutions containing impurities, such as the culture medium used during protein production. Examples of such oligopeptides include polyhistidine, polylysine, polyarginine, polyglutamic acid, and polyaspartic acid. Furthermore, an oligopeptide containing cysteine, which is useful for immobilization on an insoluble carrier, may be added to the N-terminus or C-terminus of the LV-binding protein.

[0022] Furthermore, a signal peptide may be added to the N-terminus of the LV-binding protein to promote efficient expression in the host during protein production. When the host is Escherichia coli, examples of signal peptides that induce protein secretion into the periplasm include PelB, OmpA, DsbA, DsbC, MalE, and TorT (Japanese Patent Publication No. 2011-097898).

[0023] The insoluble carrier is not particularly limited as long as it is insoluble in the sample containing LV including the VSV envelope or in the solution used for purification (eluent, equilibration solution, washing solution, etc.). Examples of insoluble carriers include carriers made from polysaccharides such as agarose, alginate (alginate salt), 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 carriers made from synthetic polymers are preferred as insoluble carriers. Examples of the preferred carriers include polymethacrylate gels with introduced hydroxyl groups such as Toyopal (manufactured by Tosoh Corporation), agarose gels such as Sepharose (manufactured by Cytiva), and cellulose gels such as Cellfine (manufactured by JNC Corporation). The shape of the insoluble carrier is not particularly limited and may be granular, monolithic, film-like, or fibrous, and may be porous or non-porous. A shape that can be packed into a column is preferred.

[0024] The LV-binding protein used in this embodiment may be immobilized on the aforementioned insoluble carrier by covalent bonding, for example. Specifically, the protein of the present invention can be immobilized on the insoluble carrier by covalent bonding between the protein of the present invention and the insoluble carrier via the active groups of the insoluble carrier. It can be converted. That is, the insoluble carrier may have active groups on its surface or elsewhere. Examples of the active groups include N-hydroxysuccinimide (NHS) activated ester group, epoxy group, carboxyl group, maleimide group, haloacetyl group, tresyl group, formyl group, and haloacetamide group. As for the insoluble carrier having active groups, for example, a commercially available insoluble carrier having active groups may be used as is, or an insoluble carrier may be used after introducing active groups. Examples of commercially available carriers containing active groups include TOYOPEARL® AF-Epoxy-650M, TOYOPEARL AF-Tresyl-650M, TOYOPEARL AF-Formyl-650M (all manufactured by Tosoh Corporation), HiTrap NHS-activated HP Columns, NHS-activated Sepharose 4 Fast Flow, Epoxy-activated Sepharose 6B (all manufactured by Cytiva), and SulfoLink Coupling Resin (manufactured by Thermo Fisher Scientific).

[0025] One example of a method for introducing active groups to a support surface is to react one of two or more active sites of a compound with hydroxyl groups, epoxy groups, carboxyl groups, amino groups, etc., present on the support surface.

[0026] Examples of compounds that introduce epoxy groups to hydroxyl or amino groups present on the support surface include epichlorohydrin, ethanediol diglycidyl ether, butanediol diglycidyl ether, and hexanediol diglycidyl ether.

[0027] Examples of compounds that introduce carboxyl groups to epoxy groups present on the support surface include 2-mercaptoacetic acid, 3-mercaptopropionic acid, 4-mercaptobutyric acid, 6-mercaptobutyric acid, glycine, 3-aminopropionic acid, 4-aminobutyric acid, and 6-aminohexanoic acid.

[0028] Furthermore, compounds that introduce maleimide groups to hydroxyl groups, epoxy groups, carboxyl groups, and amino groups present on the carrier 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-(maleimide)phenylisocyanate, 2-maleimidoacetic acid, and 3-maleimidopropionic acid. 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.

[0029] Examples of compounds that introduce haloacetyl groups to hydroxyl or amino groups present on the carrier surface 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-(iodoacetamide)acetic acid-N-hydroxysuccinimide, 3-(bromoacetamide)propionic acid-N-hydroxysuccinimide, and 4-(iodoacetyl)aminobenzoic acid-N-hydroxysuccinimide.

[0030] Another method for introducing active groups to the support surface is to react ω-alkenylalkaneglycidyl ether with hydroxyl groups or amino groups present on the support surface, and then activate the ω-alkenyl moiety by halogenating it with a halogenating agent. Examples of 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.

[0031] Another method for introducing active groups to the support surface is to introduce active groups to carboxyl groups present on the support surface using a condensing agent and an additive. Examples of condensing agents include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide, and carbonyldiimidazole. Examples of additives include N-hydroxysuccinimide (NHS), 4-nitrophenol, and 1-hydroxybenztriazole.

[0032] Immobilization of LV-binding proteins onto insoluble carriers 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 buffer, and borate buffer. The reaction temperature during immobilization can be appropriately set according to various conditions, such as the reactivity of the active group and the stability of the protein in the embodiment of the present invention. The reaction temperature during immobilization may be, for example, 5°C to 50°C, and preferably 10°C to 35°C.

[0033] The LV purification in this embodiment is (1) A step of adsorbing LV containing the VSV envelope present in the sample onto an affinity chromatography carrier using the aforementioned LV-binding protein as a ligand (adsorption step), (2) The process includes at least the step of eluting the virus adsorbed on the carrier using an eluent (elution step). The two steps described above will be explained in detail below. It is preferable to pack the affinity chromatography carrier into a column, as this simplifies these steps; therefore, the following explanation will describe the carrier in the form packed into a column (hereinafter simply referred to as "affinity chromatography column").

[0034] (1) Adsorption process Examples of samples containing LV with a VSV envelope include, but are not limited to, culture supernatant containing the LV obtained from cultured cells for LV preparation. Samples containing LV with a VSV envelope can be added (applied) to an affinity chromatography column using a liquid delivery means such as a pump. In this specification, adding liquid to a column is also referred to as "applying liquid to the column." Samples containing LV with a VSV envelope may be solvent-replaced beforehand using an appropriate buffer before being added to the affinity chromatography column. Alternatively, the column may be equilibrated using an appropriate buffer (equilibrium solution) before adding the sample to the affinity chromatography column (i.e., before the adsorption step). This equilibration can be expected to result in higher purity purification of LV with a VSV envelope.

[0035] Examples of buffers used for solvent substitution and equilibration include glycine buffer, phosphate buffer, acetate buffer, succinate buffer, citrate buffer, Tris buffer, HEPES buffer, and MES buffer, all of which have buffering capacity in the neutral range (referred to in this specification as the range of pH 4.0 to 9.0). Such buffers may also contain, for example, an inorganic salt such as sodium chloride in a concentration of 10 mmol / L to 500 mmol / L. The buffer used for solvent substitution and the equilibration solution may or may not be the same.

[0036] Prior to the elution step described later, it is advisable to wash the affinity chromatography column on which LV containing the VSV envelope has been adsorbed. This washing removes any LV remaining on the column that has not been adsorbed onto the insoluble carrier, as well as other impurities. The buffer solution can be used for washing.

[0037] (2) Elution process In this embodiment, a neutral buffer can be used as the eluate applied to the column. Examples of the neutral buffer include glycine buffer, phosphate buffer, acetate buffer, succinate buffer, citrate buffer, Tris buffer, HEPES buffer, MES buffer, bis-trispropane buffer, and PIPES (1,4-Piperazinediethanesulfonic acid) buffer. The neutral buffer is preferably MES buffer, bis-trispropane buffer, or PIPES buffer, and more preferably PIPES buffer.

[0038] Furthermore, magnesium ions can be added to the neutral buffer solution. Adding magnesium ions improves the recovery rate of the LV from the affinity chromatography column. The amount of magnesium ions added should be at least 30 mmol / L in final concentration, preferably between 300 mmol / L and 2500 mmol / L, and more preferably between 800 mmol / L and 1500 mmol / L.

[0039] The source of magnesium ions to be included in the eluate is not particularly limited, as long as it is one that a person skilled in the art would normally select in preparing a buffer solution, and examples include magnesium chloride, magnesium sulfate, magnesium carbonate, and magnesium hydroxide.

[0040] In this embodiment, an acidic buffer can be used as the eluate applied to the column. Examples of the acidic buffer include glycine buffer, phosphate buffer, acetate buffer, succinate buffer, and citrate buffer. The acidic buffer is preferably acetate buffer.

[0041] Furthermore, the acidic buffer solution may contain acetate ions. Including acetate ions improves the recovery rate of the LV from the affinity chromatography column.

[0042] The source of acetate ions to be included in the eluate is not particularly limited as long as it is one that a person skilled in the art would normally select in preparing a buffer solution, and examples include acetic acid, ammonium acetate, sodium acetate, potassium acetate, and calcium acetate.

[0043] In this embodiment, a basic buffer can be used as the eluate applied to the column. Examples of the basic buffer include glycine buffer, phosphate buffer, borate buffer, Tris buffer, and bis-trispropane buffer. The basic buffer is preferably glycine buffer, Tris buffer, or bis-trispropane buffer, and more preferably glycine buffer or bis-trispropane buffer.

[0044] There is no particular lower limit to the eluate flow rate, and the upper limit depends on the back pressure of the affinity chromatography column. Typically, a flow rate of 0.5 mL / min or more and 2.0 mL / min or less is preferred.

[0045] As described above, LV purification in this embodiment allows for the purification of a sample containing LV including a VSV envelope using affinity chromatography, by elution with the aforementioned eluent, under mild conditions with a high recovery rate.

[0046] Mild conditions refer to the pH of the buffer solution used as the eluent during the buffer preparation process. This means that at a normal temperature, preferably 25°C, the pH is in the range of 3.0 to 9.0, 3.5 to 8.5, 4.0 to 8.0, preferably 4.5 to 7.5, more preferably 5.0 to 7.0, and more preferably 6.0 to 7.0. It also means that the temperature of the buffer solution is in the range of 10°C to 40°C, preferably 15°C to 35°C, and more preferably 20°C to 30°C.

[0047] If the eluate is the neutral buffer containing magnesium ions, the pH of the neutral buffer is preferably in the range of pH 4.0 to 9.0, preferably pH 4.5 to 8.5, more preferably pH 5.0 to 8.0, more preferably pH 5.5 to 7.5, and more preferably pH 6.0 to 7.0 at the normal temperature during the buffer preparation process, preferably 25°C.

[0048] When the eluate is the acidic buffer containing acetate ions, the pH of the acidic buffer is preferably in the range of pH 3.0 to 4.5, more preferably pH 3.5 to 4.5, and more preferably pH 3.6 to 4.5, at the normal temperature during the buffer preparation process, preferably 25°C.

[0049] If the eluate is a basic buffer solution, the pH of the basic buffer solution is preferably in the range of pH 8.0 to 10.5, more preferably pH 8.5 to 10.0, and more preferably pH 9.0 to 9.5, at the normal temperature during the buffer solution preparation process, preferably 25°C.

[0050] As an example of a method for calculating the recovery rate, one can quantify the LV present in the sample containing the VSV envelope added to the affinity chromatography column, the eluate at the time of sample addition (pass-through fraction), the eluate from washing before the elution process (washing fraction), and the eluate from the washing fraction, and then calculate the recovery rate by dividing the amount of LV present in the eluate fraction by the amount of LV contained in the sample minus the amount of LV in the pass-through fraction and the washing fraction.

[0051] The quantification of LV is not particularly limited as long as it is a method usually chosen by those skilled in the art. For example, one method is to quantify it as the amount of p24 protein present in the LV capsid.

[0052] In this embodiment, the decrease in the infectivity titer of LV can be suppressed by reducing the surfactant concentration contained in the eluate. Preferably, when the eluate is the neutral buffer solution containing magnesium ions, the decrease in the infectivity titer of LV can be suppressed by reducing the surfactant concentration. The surfactant is not particularly limited, but examples include Triton® X-100, Tween® 20, NP-40, Brij®-35, and SDS (sodium dodecyl sulfate). The concentration of the surfactant in the eluate is preferably 0.01% or less, 0.005% or less, 0.001% or less, and more preferably 0%.

[0053] One method for quantifying the infectivity titer of LV is to measure the fluorescence of infected cells using flow cytometry, identify the cell population with high fluorescence intensity compared to uninfected cells as positive cells, and calculate the percentage of these positive cells. [Examples]

[0054] The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

[0055] Example 1: Cysteine-rich d of low-density lipoprotein receptor (LDLR) Preparation of omain 3 (CR3) expression vector (1) A polynucleotide encoding domain 3 (SEQ ID NO: 3, hereafter also referred to as "LDLR_CR3"), consisting of amino acid residues from proline (P) at position 106 to proline (P) at position 144, was synthesized from the cysteine-rich domain (CR) of Homo sapiens-derived LDLR (UniProt No. P01130, SEQ ID NO: 1), which consists of amino acid residues from glycine (G) at position 24 to cysteine ​​(C) at position 313. When synthesizing this polynucleotide sequence, the recognition sequence for restriction enzyme NcoI (CCATGG) was added to the 5' end, and an oligonucleotide encoding a tag peptide (6H3K) consisting of 6 histidine residues and 3 lysine residues, a stop codon (TGA), and the recognition sequence for restriction enzyme XhoI (CTCGAG) were added to the 3' end (SEQ ID NO: 4).

[0056] (2) The polynucleotides synthesized in (1) were treated with restriction enzymes NcoI and XhoI, and the size band of the target product was confirmed by agarose gel electrophoresis. After excising the target band, the polynucleotides were purified using the QIAquick Gel Extraction kit (QIAGEN).

[0057] (3) The polynucleotides obtained in (2) and plasmid pET-26b, which had been previously digested with restriction enzymes NcoI and XhoI, were ligated using a DNA Ligation Kit (Takara Bio Inc.). Escherichia coli BL21 (DE3) strain was transformed using the ligation product and cultured in LB (Luria-Bertani) plate medium containing 50 μg / mL kanamycin (37°C, 16 hours).

[0058] (4) The transformants (genetically modified Escherichia coli) obtained in (3) were selected and cultured in LB medium containing 50 μg / mL kanamycin. After purification using the QIAprep Spin Miniprep kit (QIAGEN), a vector capable of expressing LDLR_CR3 (named pET-LDLR_CR3) was obtained.

[0059] (5)(4) Of the expression vector pET-LDLR_CR3 obtained in (5)(4), the polynucleotide encoding LDLR_CR3 and its surrounding region were subjected to Big Dye Terminator Cycle Sequencing based on the chain terminator method. The DNA was subjected to a cycle sequencing reaction using a ready Reaction kit (Thermo Fisher Scientific), and the nucleotide sequence was analyzed using a fully automated DNA sequencer, the ABI Prism 3700 DNA analyzer (Thermo Fisher Scientific). For this analysis, oligonucleotides consisting of the sequences described in SEQ ID NO: 5 (5'-TAATACGACTCACTATAGGG-3') or SEQ ID NO: 6 (5'-TATGCTAGTTATTGCTCAG-3') were used as sequencing primers. Sequence analysis confirmed that the expression vector pET-LDLR_CR3 contained a polynucleotide consisting of the sequence described in SEQ ID NO: 4.

[0060] Example 2: Preparation of LDLR_CR3 (1) Recombinant Escherichia coli containing pET-LDLR_CR3 prepared in Example 1 was inoculated into 30 mL of 2×YT liquid medium (1.6% (w / v) tryptone, 1% (w / v) yeast extract, 0.5% (w / v) sodium chloride) containing 50 μg / mL kanamycin, and pre-cultured (37°C, 16 hours).

[0061] (2) Inoculate 10 mL of the culture medium after pre-culturing into 1 L of 2×YT liquid medium containing 50 μg / mL of kanamycin, and culture at 37°C for 2 to 3 hours, then add 200 μL of 0.5 mol / L of IPTG (Isopropyl-β-D-thiogalactopyranoside) The mixture was added and then incubated overnight with shaking at 20°C.

[0062] (3) The culture medium after incubation was centrifuged and the cultured cells were recovered (8 g by wet weight). The cultured cells were lysed using BugBuster® Protein Extraction Reagent (Merck Millipore), and the supernatant obtained by centrifugation was filtered and clarified.

[0063] (4) The supernatant clarified in (3) was added to a column packed with TALON® Metal Affinity Resin (manufactured by Takara Bio Inc.) which had been pre-equilibriumized with 20 mmol / L Tris hydrochloride buffer (pH 7.5) containing 500 mmol / L sodium chloride and 4 mmol / L imidazole (hereinafter also referred to as "equilibrium solution A").

[0064] (5) After washing the support packed in the column with equilibration solution A in an amount five times that of the support, 20 mmol / L Tris hydrochloride buffer (pH 7.5) containing 500 mmol / L sodium chloride and 250 mmol / L imidazole was passed through the column, and the fraction corresponding to LDLR_CR3 was recovered.

[0065] (6) The protein concentration of the recovered fraction was measured using Pierce 660nm Protein Assay Reagent (Thermo Fisher Scientific), and 3.18 mg of purified protein was confirmed per liter of bacterial culture medium. SDS-PAGE (SDS-polyacrylamide electrophoresis) was also performed, and a single band of LDLR_CR3 was confirmed.

[0066] Example 3: Preparation of an insoluble carrier with immobilized LDLR_CR3 (1) A suction-dried gel was prepared by suction filtration of a polymethacrylate gel slurry (Toyoparl, manufactured by Tosoh Corporation), an insoluble carrier having formyl groups as active groups on its surface, on a glass filter, followed by suction drying.

[0067] (2) The water content and bulk density were calculated using a portion of the suction dry gel prepared in (1).

[0068] (3) Based on the calculation results in (2), the suction dry gel prepared in (1) was measured onto a mini biospin column (BIORAD) so that the volume of the insoluble carrier was 50 μL. A solution containing LDLR_CR3 prepared in Example 2 was added to it, and the insoluble carrier and LDLR_CR3 were bound by Schiff base formation (hereinafter also referred to as "LDLR_CR3 immobilized gel").

[0069] Example 4: Preparation of a lentiviral vector (LV) containing the envelope of vesicular stomatitis virus (VSV) (1) Frozen stocks of Viral Production Cells (Thermo Fisher Scientific) were thawed and cultured in LV-MAX Production Medium (Thermo Fisher Scientific) at 37°C under 8% CO2 conditions.

[0070] (2) After subculturing the cells from (1) three times, place them in a 500 mL Erlenmeyer flask (Corning) with 4.8 × 10 8 The cells were transferred to form cells, and LV-MAX Production Medium (manufactured by Thermo Fisher Scientific) was added to a total volume of 100 mL.

[0071] (3) The cells from (2) were transiently transfused using the LV-MAX Transfection kit (Thermo Fisher Scientific) by adding 180 μL of ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific).

[0072] (4) The cells from (3) were cultured at 37°C for 48 hours under 8% CO2 conditions, and then the fraction containing LV (hereinafter referred to as "VSVG_LV") with the VSV glycoprotein (VSVG) as an envelope was collected as the culture supernatant by centrifugation.

[0073] (5) The culture supernatant containing VSVG_LV recovered in (4) was passed through a column packed with a CaptureSelect Lenti VSVG Affinity Matrix (manufactured by Thermo Fisher Scientific) that had been pre-equilibrated with 50 mmol / L HEPES (4-(2-HydroxyEthyl)-1-PiperazineEthaneSulfonic acid) buffer (pH 7.5) containing 500 mmol / L sodium chloride (hereinafter also referred to as "equilibrium solution B"). After washing with 10 times the volume of equilibrium solution of the Matrix, 50 mmol / L HEPES buffer (pH 7.5) containing 500 mmol / L sodium chloride and 800 mmol / L arginine was passed through the column, and the fraction corresponding to VSVG_LV (hereinafter also referred to as "VSVG_LV solution") was recovered.

[0074] (6) The VSVG_LV solution recovered in (5) was dialyzed to 20 mmol / L Tris hydrochloride buffer (pH 7.5), the presence of capsids was confirmed using Lenti-X GoStix Plus (Takara Bio), and it was confirmed that virus particles with a diameter of approximately 100 nm could be purified using a Zetasizer (Malvern Panalytical).

[0075] Example 5: Adsorption and desorption test of VSVG_LV using LDLR_CR3 immobilized gel (1) The spin column containing the LDLR_CR3 immobilized gel prepared in Example 3 (hereinafter also referred to as the "LDLR_CR3 column") was equilibrated by passing a 50 mmol / L MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH 6.0) (hereinafter also referred to as the "equilibrium solution C") containing 200 mmol / L sodium chloride, 2 mmol / L calcium chloride, and 0.05% (w / v) Tween 20 through the column.

[0076] (2) The VSVG_LV solution obtained in Example 4(5) was diluted 30-fold with equilibration solution C, and 250 μL of this was added to the LDLR_CR3 column. The column was then shaken at 25°C and 1000 rpm for 2 hours to adsorb the VSVG_LV onto the LDLR_CR3 immobilized gel.

[0077] (3) After washing the unadsorbed VSVG_LV by passing equilibration solution C through the LDLR_CR3 column, the VSVG_LV adsorbed on the LDLR_CR3 immobilized gel was eluted by passing various eluents through the column. Unless otherwise specified, the eluent was 50 mmol / L MES buffer (pH 6.0) containing 2000 mmol / L magnesium chloride and 0.05% (w / v) Tween 20.

[0078] (4) The amount of VSVG_LV present in the VSVG_LV solution added in (2) (hereinafter also referred to as the "added solution"), as well as in the pass-through fraction, washing fraction, and elution fraction, was quantified as the amount of p24 using the Lenti-X p24 Rapid Titer Kit (Single Wash) (manufactured by Takara Bio Inc.), which detects the p24 protein present in the LV capsid. The recovery rate (%) of VSVG_LV was then calculated by dividing the amount of VSVG_LV present in the elution fraction by the amount of VSVG_LV(p24) in the pass-through fraction and washing fraction by the amount of VSVG_LV(p24) in the added solution.

[0079] Example 6: Adsorption and desorption test of VSVG_LV using LDLR_CR3 immobilized gel Except for using either (a) or (b) below as the eluent passed through in Example 5(3), the recovery rate (%) of VSVG_LV was calculated in the same manner as in Example 5; (a) 50 mmol / L MES buffer (pH 6.0) containing 200 mmol / L sodium chloride, 2 mmol / L calcium chloride, 0.05% (w / v) Tween 20, and 100 mmol / L DTT (Dithiothreitol). (b) 50 mmol / L MES buffer (pH 6.0) containing 200 mmol / L sodium chloride, 2 mmol / L calcium chloride, 0.05% (w / v) Tween 20, and 1000 mmol / L L-arginine.

[0080] Example 7: Adsorption and desorption test of VSVG_LV using LDLR_CR3 immobilized gel. Except for using 70% (v / v) ethanol as the eluent passed through in Example 5(3), the recovery rate (%) of VSVG_LV was calculated using the same method as in Example 5. The results for Examples 5 and 6 are shown in Table 1, and the results for Example 7 are shown in Table 2. It was found that VSVG_LV could be recovered using buffer solutions in which the p24 value of the eluted fraction was greater than that of the washing fraction: buffer solution with magnesium chloride added (Example 5, recovery rate: 69.9%), buffer solution with arginine added (Example 6(b), recovery rate: 23.7%), or buffer solution with ethanol added (Example 7, recovery rate: 25.1%). Among these, the buffer solution with magnesium chloride added showed the best recovery rate.

[0081] From these results, it can be seen that by using the presented additive as the elution solution for eluting LV containing the VSV envelope adsorbed onto the LDLR_CR3 immobilized gel, LV containing the VSV envelope can be purified under mild conditions using a pH 6.0 buffer. Furthermore, it can be seen that the recovery rate of the LV into the elution fraction is significantly improved when a buffer containing magnesium ions is used.

[0082] [Table 1]

[0083] [Table 2]

[0084] Example 8: Investigation of magnesium ion concentration In Example 5, it was found that using a buffer containing magnesium ions was preferable for eluting the LV containing the VSV envelope adsorbed onto the LDLR_CR3 immobilized gel. The optimal concentration of magnesium ions to be included in the buffer solution was investigated.

[0085] Specifically, in Example 5(3), a 50 mmol / L MES buffer (pH 6.0) containing magnesium chloride at one of the concentrations shown in (a) to (f) below and 0.05% (w / v) Tween 20 was used as the eluate, and the amount of VSVG_LV contained in the eluate fraction was quantified in the same manner as in Example 5. (a) 0 mmol / L (without added magnesium chloride) (b) 10 mmol / L (c) 100 mmol / L (d) 500 mmol / L (e) 1000 mmol / L (f) 2000 mmol / L

[0086] Figure 1 shows the results based on an elution rate of 0 mmol / L. When magnesium ions were added to the buffer solution, the amount of VSVG_LV in the eluted fraction increased compared to when no magnesium ions were added (condition (a) above). When magnesium ions were added to the buffer solution in a concentration of 300 mmol / L to 2500 mmol / L (conditions (d) to (f) above), the amount of VSVG_LV recovered in the eluted fraction increased further. When magnesium ions were added to the buffer solution in a concentration of 800 mmol / L to 1500 mmol / L (condition (e) above), the amount of VSVG_LV recovered in the eluted fraction increased particularly significantly.

[0087] Example 9: Purification test of VSVG_LV from culture supernatant using LDLR_CR3 immobilized gel. (1) 500 μL of the culture supernatant containing VSVG_LV obtained in Example 4(4) was concentrated twice by ultrafiltration using Amicon Ultra-0.5 100KDa (Merck Millipore), and this solution was added to an LDLR_CR3 column equilibrated by the method described in Example 5(1). The column was then shaken at 25°C and 1000 rpm for 2 hours to adsorb VSVG_LV onto the LDLR_CR3 immobilized gel.

[0088] (2) After washing the unadsorbed VSVG_LV by passing equilibration solution C through the LDLR_CR3 column, 50 mmol / L MES buffer (pH 6.0) containing 1000 mmol / L magnesium chloride and 0.05% (w / v) Tween 20 was passed through the column as the eluent to elute the VSVG_LV adsorbed on the LDLR_CR3 immobilized gel.

[0089] (3) The amount and recovery rate of VSVG_LV present in the culture supernatant containing VSVG_LV added in (1), as well as in the pass-through fraction, washing fraction, and elution fraction, were determined by the method described in Example 5(4).

[0090] Example 10: Purification test of VSVG_LV from culture supernatant using LDLR_CR3 immobilized gel. Except for using a 50 mmol / L MES buffer (pH 6.0) containing 0.05% (w / v) Tween 20 as the eluate in Example 9(2), the recovery rate (%) of VSVG_LV was calculated using the same method as in Example 9.

[0091] The results for Examples 9 and 10 are shown in Table 3. When using a buffer solution with added magnesium chloride (Example 9) as the eluate, the recovery rate of VSVG_LV was 62.4%, which is an improvement over when using a buffer solution without magnesium chloride (Example 10, recovery rate: 19.5%). From these results, it can be seen that the purification method of the present invention can also be applied to the purification of culture supernatants containing many impurities.

[0092] [Table 3]

[0093] Example 11: Adsorption and desorption test of VSVG_LV using LDLR_CR3 immobilized gel We investigated whether elution was possible solely by a change in pH when no additives were used. Except for using (a) to (i) below as the eluate passed through in Example 5(3), the amount of VSVG_LV (p24 amount) contained in the additive solution and eluted fraction was calculated using the same method as in Example 5; (a) 100 mmol / L acetate buffer (pH 2.0) (b) 100 mmol / L acetate buffer (pH 4.0) (c) 100 mmol / L acetate buffer (pH 5.0) (d) 100 mmol / L glycine buffer (pH 2.0) (e) 100 mmol / L glycine buffer (pH 4.0) (f) 100 mmol / L bis-trispropane buffer (pH 4.0) (g) 100 mmol / L bis-trispropane buffer (pH 5.0) (h) 100 mmol / L bis-trispropane buffer (pH 6.0) (i) 100mmol / L MES buffer (pH6.0)

[0094] The results are shown in Table 4. A comparison of the acetate buffers (a) to (c) above shows that when the pH of the buffer is 4.0 (b) above, the amount of VSVG_LV recovered is increased compared to when the pH is 2.0 (a) above or 5.0 (c) above. Furthermore, even when the pH of the buffer is the same 4.0, the amount of VSVG_LV recovered is increased with the acetate buffer (b) above compared with the glycine buffer (e) above or the bis-trispropane buffer (f) above. From these results, it can be seen that when purifying VSVG_LV using an affinity chromatography carrier containing at least an insoluble carrier and CR3 of the LDLR immobilized on the carrier, eluting the VSVG_LV adsorbed on the carrier with an acetate buffer between pH 3.0 and pH 4.5 above allows for purification of the VSVG_LV with a high recovery rate.

[0095] [Table 4]

[0096] Example 12: Adsorption and desorption test of VSVG_LV using LDLR_CR3 immobilized gel. Following Example 11, we investigated whether elution was possible solely by changing the pH of the basic buffer. Except for using (a) through (f) below as the eluate passed through in Example 5(3), the amount of VSVG_LV (p24 amount) contained in the added solution and eluted fraction was calculated using the same method as in Example 5; (a) 50 mmol / L Tris buffer, 200 mmol / L sodium chloride, 2 mmol / L calcium chloride (pH 8.0) (b) 50 mmol / L Tris buffer (pH 8.0) (c) 50 mmol / L bis-trispropane buffer (pH 8.0) (d) 50 mmol / L bis-trispropane buffer (pH 9.0) (e) 50 mmol / L glycine buffer (pH 9.0) (f) 50 mmol / L glycine buffer (pH 10.0) The results are shown in Table 5. A comparison between the Tris buffer solutions (a and b) above shows that the amount of VSVG_LV recovered is greater when no salt is used. Furthermore, a comparison between the buffer solutions (b through f) above shows that the amount of VSVG_LV recovered is greater with the pH 9.0 buffer solution.

[0097] From the above results, it can be seen that when purifying VSVG_LV using an affinity chromatography carrier containing at least an insoluble carrier and CR3 of LDLR immobilized on the carrier, eluting the VSVG_LV adsorbed on the carrier with bis-trispropane buffer or glycine buffer with a pH of 8.5 to 9.5 allows for purification of the VSVG_LV with a high recovery rate.

[0098] [Table 5]

[0099] Example 13: Preparation of an infectious lentiviral vector (LV) containing the envelope of vesicular stomatitis virus (VSV) (1) Cells prepared according to the procedures in Example 4(1)-(2) were transiently transfused using the LV-MAX Transfection kit (Thermo Fisher Scientific) by adding 180 μL of ViraPower Lentiviral Packaging Mix (Thermo Fisher Scientific) and 120 μL of pLenti6.3 / V5-GW / EmGFP (Thermo Fisher Scientific) diluted to 1.0 μg / μL. (2) The supernatant fraction containing infectious LV was isolated from the cell culture medium of (1) using the procedure of Examples 4(4) to (6) and analyzed.

[0100] Example 14: Adsorption and desorption test of infectious LV using LDLR_CR3 immobilized gel. (1) From the results of Examples 8 and 9, it was found that VSVG_LV adsorbed on the LDLR_CR3 column could be recovered by using 1000 mmol / L magnesium chloride and 0.05% (w / v) Tween 20 as the eluate. However, surfactants such as Tween 20 have been reported to reduce the infectivity titer of LV. Therefore, the Tween 20 concentration was optimized using the culture supernatant containing infectious LV prepared in Example 13.

[0101] Specifically, the infectious LV was purified in the same manner as in Example 5, except that the sample added to the column in Example 5(2) was changed to 500 μL of the culture supernatant containing infectious LV prepared in Example 13, and the eluate passed through in Example 5(3) was 50 mmol / L MES buffer (pH 6.0) containing Tween 20 and 1000 mmol / L magnesium chloride at one of the concentrations shown in (a) to (f) below. (a) 0% (No Tween 20 added) (b) 0.01% (c) 0.1%

[0102] (2) The infectious LV solution present in the eluted fraction in (1) was replaced with 50 mmol / L HEPES buffer (pH 7.5) containing 20 mmol / L magnesium chloride by dialysis, and the p24 amount was quantified using the Lenti-X p24 Rapid Titer Kit (Single Wash) (Takara Bio Inc.). The amount of infectious LV between columns was then equalized by dilution with 50 mmol / L HEPES buffer (pH 7.5) containing 20 mmol / L magnesium chloride.

[0103] (3) The infectivity titer of the infectious LV prepared in (2) was measured using the following procedure. First, 7.0 × 10 4 HT1080 cells, prepared to cells / mL, were dispensed into 96-well cell culture plates to a concentration of 100 μL / well and incubated statically in a 37°C, 5% CO2 incubator for 4 hours. The supernatant was removed, and 100 μL / well of the infectious LV solution prepared in (2), diluted 10-fold in a medium containing 8 μg / mL of polyblen (DMEM / 10% FBS / PS), was added. The plates were then centrifuged at 25°C, 900 × g for 30 minutes, and incubated statically in a 37°C, 5% CO2 incubator for 16 hours.

[0104] (4) The culture supernatant from (3) was removed, and polyblen-free medium (DMEM / 10% FBS / PS) was added to 100 μL / well, and the culture was incubated statically for 72 hours at 37°C in a 5% CO2 incubator.

[0105] (5) Remove the culture supernatant and add cell eluent (75% TrypLE (Thermo Fisher Scientific), 25% phosphate-buffered saline (PBS)) to a volume of 150 μL / well. Incubate at 37°C in a 5% CO2 incubator for 15 minutes. After confirming that the cells had eluted, transfer the cells to a 1.5 mL tube and centrifuge at 25°C at 500 × g for 3 minutes, then remove the supernatant. The cell pellet was suspended in PBS and prepared as a sample for measurement.

[0106] (6)(5) Each sample prepared in (5) and HT1080 cells not infected with LV were prepared as a negative control, and the infectivity titer of LV purified in each eluate was measured by measuring the fluorescence of the cells using flow cytometry with Guava EasyCyte11 (Scitec).

[0107] The specific fluorescence measurement conditions involved detecting fluorescence from cells irradiated with a 488nm wavelength laser installed in the Guava EasyCyte11 (Scitec Corporation) using a 525nm wavelength filter. The group with higher fluorescence intensity than the negative control was defined as fluorescence-positive cells, and their percentage was calculated.

[0108] The infectivity titer (TU / mL) of LV was determined by applying the percentage of fluorescently positive cells to the following formula (1). (a × x × c) / (100 × b) (1)

[0109] In equation (1), x represents the percentage of fluorescently positive cells (%), a represents the number of HT1080 cells infected with LV, b represents the volume of culture medium per well containing HT1080 cells at the time of LV infection (mL), and c represents the dilution ratio of the pre-infection LV in the culture medium containing polyblen. In this example, a=7000, b=0.1, and c=10.

[0110] Infectious LV amount (TU) was calculated by multiplying the infectivity titer (TU / mL) of LV in the culture supernatant before purification and in the eluted fractions under each elution condition by the volume (mL) of each fraction. The amount of infectious LV in the culture supernatant before purification was also compared as a percentage, with the amount of infectious LV in the culture supernatant before purification set to 100%.

[0111] The results are shown in Figure 2. It can be seen that the infectivity titer of the recovered LV decreases as the Tween 20 concentration in the eluate increases.

[0112] Based on these results, it is considered optimal not to add Tween 20 to the eluent in order to gently purify LV using LDLR_CR3 immobilized gel.

[0113] Example 15: Adsorption and desorption test of infectious LV using LDLR_CR3 immobilized gel. From Example 14, it was found that infectious LV adsorbed to the LDLR_CR3 immobilized gel could be gently eluted by using a 50 mmol / L MES buffer (pH 6.0) containing 1000 mmol / L magnesium chloride as the eluent. On the other hand, various buffers other than MES have been reported for near-neutral pH (pH 6.0~8.0). Therefore, in addition to the elution buffer obtained in Example 14 (50 mmol / L MES buffer (pH 6.0) containing 1000 mmol / L magnesium chloride), buffers that could gently elute LV with a higher recovery rate at near-neutral pH were compared and investigated.

[0114] Specifically, the infectious LV amount (TU) was calculated in the same manner as in Example 14, except that the eluate passed through in Example 14(1) was one of the buffers shown in (a) to (f) below and a solution containing 1000 mmol / L magnesium chloride. (a) 50mmol / L MES buffer (pH6.0) (b) 50 mmol / L bis-trispropane buffer (pH 6.0) (c) 50 mmol / L bis-trispropane buffer (pH 7.0) (d) 50 mmol / L bis-trispropane buffer (pH 8.0) (e) 50mmol / L PIPES buffer (pH7.0) (f) 50mmol / L HEPES buffer (pH7.0)

[0115] The results are shown in Table 6. It can be seen that both the amount of infectious LV present in the eluted fraction and the infectivity titer are maximized in a 50 mmol / L PIPES buffer (pH 7.0) containing 1000 mmol / L magnesium chloride.

[0116] [Table 6]

[0117] Example 16: Adsorption and desorption test of infectious LV using LDLR_CR3 immobilized gel. From Example 15, it was found that infectious LV could be purified with high recovery efficiency and infectivity titer using a buffer with a pH near neutral, specifically a 50 mmol / L PIPES buffer (pH 7.0) containing 1000 mmol / L magnesium chloride as the eluate. PIPES has buffering capacity in the pH range of approximately 6.5 to 7.5, and it is thought that further improvements in the purification conditions for infectious LV can be made by examining the pH. Therefore, we investigated whether LV could be eluted more gently and with higher recovery rates using PIPES buffers at different pH levels.

[0118] Specifically, the infectious LV amount (TU) was calculated in the same manner as in Example 14, except that the eluate passed through in Example 14(1) was one of the buffers shown in (a) to (c) below and a solution containing 1000 mmol / L magnesium chloride. (a) 50mmol / L PIPES buffer (pH6.5) (b) 50mmol / L PIPES buffer (pH7.0) (c) 50mmol / L PIPES buffer (pH7.5)

[0119] The results are shown in Table 7. It can be seen that both the amount of infectious LV present in the eluted fraction and the infectivity titer are maximized in a 50 mmol / L PIPES buffer (pH 6.5) containing 1000 mmol / L magnesium chloride.

[0120] [Table 7]

[0121] Example 17: Adsorption and desorption test of infectious LV using LDLR_CR3 immobilized gel. From the results of Examples 11 to 16, it was found that the optimal eluate for LV adsorbed onto the LDLR_CR3 immobilized gel was an acetate buffer with a pH of 3.0 to 4.5 for acidic pH, a bis-trispropane buffer with a pH of 8.5 to 9.5 for basic pH, and a 50 mmol / L PIPES buffer (pH 6.5) containing 1000 mmol / L magnesium chloride for neutral pH. Therefore, the optimal combination of these three buffers and magnesium chloride was compared to determine the one that best resulted in the recovery rate and infectivity titer of infectious LV.

[0122] Specifically, the infectious LV amount (TU) was calculated in the same manner as in Example 14, except that one of the buffers shown in (a) to (f) below was used as the eluate passed through in Example 14(1). (a) 50mmol / L PIPES buffer (pH6.5) (b) 50mmol / L PIPES buffer, 1000mmol / L magnesium chloride (pH6.5) (c) 50 mmol / L acetate buffer (pH 4.0) (d) 50 mmol / L acetate buffer, 1000 mmol / L magnesium chloride (pH 4.0) (e) 50 mmol / L bis-trispropane buffer (pH 9.0) (f) 50 mmol / L bis-trispropane buffer, 500 mmol / L magnesium chloride (pH 9.0)

[0123] The results are shown in Table 8. It can be seen that both the amount of infectious LV present in the eluted fraction and the infectivity titer were higher in 50 mmol / L PIPES buffer (pH 6.5) containing 1000 mmol / L magnesium chloride compared to acidic and basic pH.

[0124] [Table 8]

[0125] Example 18: Adsorption and desorption test of infectious LV using LDLR_CR3 immobilized gel. The results of Example 5 showed that magnesium chloride is suitable as an eluate for LV adsorbed onto the LDLR_CR3 immobilized gel. Therefore, we compared and investigated which inorganic salt other than magnesium chloride was optimal in terms of the recovery rate and infectivity titer of infectious LV.

[0126] Specifically, the infectious LV amount (TU) was calculated in the same manner as in Example 14, except that one of the buffers shown in (a) to (d) below was used as the eluate passed through in Example 14(1). (a) 50mmol / L PIPES buffer (pH6.5) (b) 50mmol / L PIPES buffer, 1000mmol / L magnesium chloride (pH6.5) (c) 50mmol / L PIPES buffer, 1000mmol / L magnesium sulfate (pH6.5) (d) 50mmol / L PIPES buffer, 1000mmol / L sodium sulfate (pH6.5)

[0127] The results are shown in Table 9. It can be seen that both the amount of infectious LV present in the eluted fraction and the infectivity titer were high in 50 mmol / L PIPES buffer (pH 6.5) containing 1000 mmol / L magnesium chloride or magnesium sulfate. These values ​​were higher than those of 50 mmol / L PIPES buffer (pH 6.5) containing 1000 mmol / L sodium sulfate, indicating that magnesium ions are important for LV elution. [Table 9]

Claims

1. A method for purifying a lentiviral vector (LV) containing the envelope of vesicular stomatitis virus (VSV) contained in a sample, The process of adsorbing the LV onto a carrier for affinity chromatography, The process includes at least the step of eluting the LV adsorbed on the carrier using an eluent, A method wherein the carrier comprises an insoluble carrier and at least one of the low-density lipoprotein receptors (LDLRs) cysteine-rich domain 2 (CR2) and 3 (CR3) immobilized on the carrier.

2. The method according to claim 1, wherein the affinity chromatography carrier comprises at least an insoluble carrier and CR3 of LDLR immobilized on the carrier, and the CR3 is a polypeptide selected from any of (i) to (iii) below; (i) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, (ii) A polypeptide having VSV binding activity, wherein the amino acid sequence contains at least the amino acid sequence described in Sequence ID No. 3, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions in the amino acid sequence, (iii) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, provided that it has 70% or more identity with the entire amino acid sequence, and has VSV binding activity.

3. The method according to claim 2, wherein the eluate contains at least magnesium ions.

4. The method according to claim 3, wherein the pH of the eluent is 4.0 or higher and 9.0 or lower.

5. The method according to claim 4, wherein the magnesium ion concentration of the eluate is 300 mmol / L or more and 2500 mmol / L or less.

6. The method according to claim 5, wherein the eluent is a buffer selected from the group consisting of MES buffer, bis-trispropane buffer, and PIPES buffer.

7. The method according to claim 6, wherein the concentration of the surfactant contained in the eluate is 0.01% or less.

8. The method according to claim 2, wherein the eluent contains at least acetate ions and has a pH of 3.0 or higher and 4.5 or lower.

9. The method according to claim 2, wherein the eluate is a bis-trispropane buffer or a glycine buffer, and the pH is 8.0 or higher and 10.5 or lower.

10. The method according to any one of claims 1 to 9, wherein the LV is a pseudotyped LV comprising the envelope of VSV and the capsid of human immunodeficiency virus.

11. A carrier for affinity chromatography comprising an insoluble carrier and at least one of CR2 and CR3 of LDLR immobilized on the carrier.

12. The affinity chromatography carrier according to claim 11, wherein the affinity chromatography carrier comprises at least an insoluble carrier and CR3 of LDLR immobilized on the carrier, and the CR3 is a polypeptide selected from any of (i) to (iii) below; (i) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, (ii) A polypeptide having VSV binding activity, wherein the amino acid sequence contains at least the amino acid sequence described in Sequence ID No. 3, wherein one or more substitutions, deletions, insertions, and additions of one or more amino acid residues occur at one or more positions in the amino acid sequence, (iii) A polypeptide comprising at least the amino acid sequence described in Sequence ID No. 3, provided that it has 70% or more identity with the entire amino acid sequence, and has VSV binding activity.

13. The affinity chromatography carrier according to claim 11 or 12, wherein the affinity chromatography carrier is used for purifying LV containing the envelope of VSV contained in the sample.

14. The affinity chromatography carrier according to claim 13, wherein the LV is a pseudotyped LV comprising the envelope of VSV and the capsid of human immunodeficiency virus.

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