WO2020032039A1 - Method for purifying virus-like particles - Google Patents

Method for purifying virus-like particles Download PDF

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WO2020032039A1
WO2020032039A1 PCT/JP2019/030937 JP2019030937W WO2020032039A1 WO 2020032039 A1 WO2020032039 A1 WO 2020032039A1 JP 2019030937 W JP2019030937 W JP 2019030937W WO 2020032039 A1 WO2020032039 A1 WO 2020032039A1
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particles
envelope
column
virus
type virus
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PCT/JP2019/030937
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French (fr)
Japanese (ja)
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謙 小木戸
青木　裕史
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昭和電工株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/02—Hepadnaviridae, e.g. hepatitis B virus

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  • the present invention relates to a method for purifying enveloped virus-like particles.
  • virus-like particles In biopharmaceuticals, the use of virus-like particles (VLPs) for treatment in the medical field is rapidly expanding. For example, application as a new vaccine or gene therapy as a virus vector / drug delivery carrier. The market for virus-like particles is expected to expand further in the future.
  • virus-like particles are produced by cell culture using cell lines derived from bacterial cells, yeast cells, insect cells, plant cells, and animals including humans. Virus-like particles are built by mimicking the unique shell structure of a virus. Virus-like particles have different properties depending on the virus species. For this reason, there is no one that can be said to be a common skeleton, and it is necessary to improve the expression level of each individual and study the purification process.
  • virus-like particles may be decomposed or have no complete structure due to structural instability, and may become impurities. It is feared that these impurities have a detrimental effect on pharmaceuticals, such as reduced drug efficacy and immunogenicity. Therefore, it is desired that impurities be removed to the extent that harmful effects are eliminated.
  • Ultracentrifugal purification methods have been studied to remove impurities from virus-like particles. However, the ultracentrifugation purification method had poor production efficiency. Many purification methods by chromatography have been reported for the purpose of improving production efficiency. These are purified by using anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, etc., alone or in combination, or by using mixed mode chromatography in which they are mixed.
  • the envelope-type virus-like particles generally have a structure in which a protein is inserted into a phospholipid membrane.
  • the hydrophobic interaction between the lipid membrane and the chromatography carrier is used to separate impurities contained in the culture solution in a large amount.
  • Non-Patent Document 1 describes a comparison of binding capacities in a process in which various porous polymer carriers having various ion exchange groups immobilized thereon and various pH values of an eluent are combined.
  • a particulate porous polymer carrier having anion-exchange groups immobilized thereon has a higher binding capacity in a wider pH range than a particulate porous polymer carrier having immobilized cation exchange groups. It describes that excellent concentration and purification are possible.
  • Non-Patent Document 2 describes a method for purifying enveloped virus-like particles by a column using a packing composed of a porous polymer carrier to which sulfate groups are fixed.
  • Non-Patent Document 3 describes a method for purifying envelope-type virus-like particles using a monolithic column having a hydrophobic surface or a column composed of a particulate porous polymer carrier to which butyl groups are fixed. Have been. In this method, an appropriate pretreatment or washing method is required to prevent the column from being clogged or contaminated by the contaminating lipid.
  • the present inventors have conducted intensive studies on a method for purifying enveloped virus-like particles. As a result, under the conditions in which a monolith-type column having a fixed cation exchange group and an eluent / sample buffer having a pH higher than the isoelectric point of the outer region of the inserted protein envelope and a pH of 10.0 or less were used. The present inventors have found that envelope-type virus-like particles can be efficiently and highly purified.
  • the present invention is as follows. [1] a step of passing a liquid containing enveloped virus-like particles through a monolithic column to which a cation exchange group is fixed to adsorb the enveloped virus-like particles to the column, and an enveloped virus adsorbed to the column Comprising the step of selectively eluting like particles, A method for purifying enveloped virus-like particles, wherein the pH of the solution containing the enveloped virus-like particles is higher than the isoelectric point of the outer region of the envelope of the protein inserted into the envelope and is pH 10.0 or less.
  • the monolithic column is a monolithic column composed of a monolithic carrier having a cation exchange group fixed to a copolymer containing a monomer unit derived from glycidyl methacrylate and a monomer unit derived from ethylene glycol dimethacrylate.
  • the purification method according to 1. [3] The purification method according to any one of [1] and [2], wherein the cation exchange group is a sulfonic acid group. [4] The purification method according to any one of the above items 1 to 3, wherein the protein inserted into the enveloped virus-like particle contains a hepatitis B virus antigen (HBsAg).
  • HBsAg hepatitis B virus antigen
  • enveloped virus-like particles useful as raw materials for pharmaceuticals and the like can be efficiently and highly purified.
  • FIG. 1 is an electron micrograph of the envelope-type virus-like particles of the fraction collected in Example 2.
  • Envelope type virus-like particles are composed of a phospholipid membrane. Further, the isoelectric point of the outer region of the protein envelope inserted on the phospholipid surface is on the acidic side. For this reason, the envelope-type virus-like particles are negatively charged as anions near neutrality. For these reasons, enveloped virus-like particles have been mainly purified by a method combining an eluent having a neutral or higher pH with anion exchange chromatography. In this method, an envelope-type virus-like particle is bound according to the binding capacity of the anion exchange carrier, and then a purification method of eluting is used. However, in this method, the activity of the resulting enveloped virus-like particles after purification may not be sufficient.
  • the present inventors adjusted the solution containing the envelope-type virus-like particles to a pH higher than the isoelectric point of the outer region of the envelope of the protein inserted into the envelope, and then fixed the cation exchange group to a monolithic column. To bind the envelope-type virus-like particles and then elute the envelope-type virus-like particles, thereby finding that the envelope-type virus-like particles can be purified in a highly active state.
  • the liquid containing the envelope-type virus-like particles is a liquid containing the envelope-type virus-like particles and impurities to be separated, and specifically, a liquid obtained in the production of the envelope-type virus-like particles.
  • Envelope-type virus-like particles can be produced by a method of culturing cell lines derived from bacterial cells, yeast cells, insect cells, plant cells, and animals including humans. Although the obtained culture solution contains impurities, envelope-type virus-like particles can be separated and purified from the culture solution by the purification method of the present invention.
  • the impurities to be separated include, for example, contaminants, proteins, and phospholipids derived from host cultured cells and the like.
  • the culture solution can be passed through the column as it is, but usually the culture supernatant separated from the culture solution is used. By using the culture supernatant, column contamination can be suppressed.
  • the pH Before passing the liquid containing the envelope-type virus-like particles through the column, the pH can be adjusted or a buffer can be added.
  • the envelope-type virus-like particles applicable to the purification method of the present invention are not particularly limited as long as they are artificial particles that mimic the outer-shell structure of an envelope-type virus in which a protein is inserted into a phospholipid membrane.
  • Envelope viruses include, for example, herpesviridae, poxviridae, hepadnaviridae, flaviviridae, togaviridae, coronaviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, bunyaviridae, Viruses belonging to the family Filoviridae, Retroviridae and the like can be mentioned.
  • the phospholipids contained in the enveloped virus-like particles include compounds such as phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol.
  • the protein inserted into the phospholipid is a transmembrane protein or a surface antigen protein.
  • a virus-like particle derived from hepatitis B virus has a hepatitis B virus surface antigen (HBsAg) having a pI of 7.3 inserted into a phospholipid, and has a ratio of 5 to 10 times by mass of the phospholipid. Included in.
  • Monolith type column is a column holding a monolith carrier.
  • the monolithic carrier is a monolithic porous body having a characteristic structure in which a network structure of a micrometer order is connected.
  • the monolithic carrier is characterized in that many uniform through holes are provided throughout. These monolith carriers have a mechanical strength capable of self-supporting their shape when left standing. That is, the physical structure is different from that of the particulate porous polymer carrier or porous membrane.
  • the monolithic carrier according to the present invention preferably comprises an organic polymer.
  • the monomer unit for constituting the monolithic carrier include a monomer unit having no functional group and a monomer unit having a functional group serving as a cation exchange group. These monomer units may contain two or more kinds.
  • Monomers include cross-linking monomers.
  • a crosslinking monomer is a monomer having two or more polymerizable sites.
  • the organic polymer as a monolithic carrier is preferably a copolymer of a monomer and a crosslinking monomer.
  • Examples of the monomer include styrene and substituted styrene (provided that the substituent is a chloromethyl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a t-butyloxycarbonyl group, a halogen group, a nitro group, an amino group, (Including protected hydroxyl or amino groups), vinyl naphthalenes, acrylates, methacrylates such as glycidyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, vinyl acetate and pyrrolidone, and mixtures thereof.
  • the substituent is a chloromethyl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a t-butyloxycarbonyl group, a halogen group, a nitro group, an amino group, (Including protected hydroxyl or amino groups), vinyl naphthalenes, acrylates,
  • crosslinking monomer divinylbenzene, divinylnaphthalene, divinylpyridine, methacrylates, acrylates, vinyl esters, vinyl ethers such as divinyl ether, alkylene bisacrylamides such as ethylene bisacrylamide and propylene bisacrylamide or methacrylamides and And mixtures thereof.
  • Examples of the methacrylates as the crosslinking monomer include alkylene dimethacrylates such as ethylene glycol dimethacrylate and propylene glycol dimethacrylate, pentaerythritol di-, tri- or tetramethacrylate, and trimethylolpropane trimethacrylate.
  • Examples of the acrylates as the crosslinking monomer include ethylene glycol diacrylates, pentaerythritol di-, tri- and tetraacrylates.
  • the monomer is preferably appropriately selected from methacrylates and acrylates. More preferably, it is a combination of glycidyl methacrylate and alkylene dimethacrylates, and still more preferably, a combination of glycidyl methacrylate and ethylene glycol dimethacrylate.
  • Preferred monolithic carriers are those obtained by derivatizing a copolymer obtained from the monomer mixture with a compound having a cation exchange group.
  • a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate is produced from a mixture of glycidyl methacrylate and ethylene glycol dimethacrylate in the presence of a porogen and a polymerization initiator.
  • a porogen is an additive substance having no carbon-carbon double bond for forming porosity, and includes aliphatic hydrocarbons, aromatic hydrocarbons, esters, alcohols, ketones, ethers, Soluble polymer solutions and mixtures thereof can be used. Preferably, it is normal hexane.
  • a soluble polymer may be added as a porogen. When a soluble polymer is added, more pore structures are formed. The amount of the soluble polymer is preferably 10 to 40% by mass based on the total mass of the copolymer.
  • a free radical generation initiator can be used.
  • azo compounds such as azobisisobutyronitrile and 2,2'-azobis (isobutylamide) dihydrate, and peroxides such as benzoyl peroxide and dipropyl dicarboxylate can be used.
  • peroxides such as benzoyl peroxide and dipropyl dicarboxylate.
  • pore structures having different shapes can be formed.
  • the amount of the polymerization initiator is preferably 0.5 to 4% by mass based on the mass of the monomer.
  • the mixture of glycidyl methacrylate and ethylene glycol dimethacrylate containing a porogen and a polymerization initiator is preferably degassed with an inert gas such as nitrogen or argon before being put into a mold.
  • the mold is preferably sealed to prevent air contamination.
  • the polymerization can be carried out, for example, by heating to a temperature of 50 ° C. to 90 ° C. for 40 to 50 hours.
  • the solvent and the soluble polymer used as the porogen are removed by washing.
  • methanol, ethanol, benzene, toluene, acetone, tetrahydrofuran and the like can be used.
  • the washing step may be repeated a plurality of times.
  • the cation exchange group is an ion exchange group that exhibits acidity.
  • Specific examples of the cation exchange group include a sulfate group, a sulfonic acid group (—SO 3 H), a carboxylic acid group, and a phosphoric acid group. Of these, a sulfonic acid group and a phosphoric acid group are preferred, and a sulfonic acid group is more preferred.
  • the sulfonic acid groups are selected from the group consisting of bromoethyl sulfonic acid, chlorohydroxypropane sulfonic acid, 1,3-propane sultone, 1,4-butane sultone, sodium 3-hydroxypropane sulphonate, sulfurous acid and sodium sulphite.
  • a sulfite such as potassium sulfite or the like, it can be introduced into the monolithic carrier.
  • the copolymer when the copolymer has an epoxy group, sodium sulfite, potassium sulfite and the like can be reacted to obtain a copolymer having a sulfonic acid group, and when the copolymer has a hydroxyl group, an appropriate base can be used. At the same time, bromoethylsulfonic acid, chlorohydroxypropanesulfonic acid, 1,4-butanesultone and the like are reacted to obtain a copolymer having a sulfonic acid group. The amount of sulfonic acid groups introduced into the copolymer is appropriately adjusted according to the buffer actually used.
  • the monolithic column with fixed cation exchange group is one in which a monolithic carrier with fixed cation exchange group is housed in a column container.
  • the size (volume) of the column is not particularly limited, and is appropriately adjusted according to the amount of impurities such as enveloped virus-like particles and aggregates to be bound.
  • the shape of the monolithic carrier has an integral structure such as a plate shape, a tubular shape, and a cylindrical shape.
  • the thickness of the monolith carrier in the flow direction is preferably from 1 mm to 100 mm, more preferably from 2 mm to 70 mm, further preferably from 3 mm to 60 mm, and most preferably from 3 mm to 50 mm.
  • the monolith carrier By setting the thickness of the monolith carrier to 1 mm or more, the monolith carrier has sufficient mechanical strength. By setting the thickness of the monolithic carrier to 100 mm or less, a pressure at which the monolithic carrier is not broken can be maintained, and an increase in pump pressure can be prevented.
  • columns include CIMac (registered trademark) SO3-0.1 Analytical column, CIM (registered trademark) SO3 DISK, CIM (registered trademark) SO3-1 Tube Monolithic column, and CIM (registered trademark) SO3-8f Tube Monolithic column, CIM (registered trademark) SO3-80 Tube Monolithic column, CIM (registered trademark) SO3-800 Tube Monolithic column, CIM (registered trademark) SO3-8000 ⁇ Tublic Co., Ltd.
  • CIMmultus (climbing Trademarks) SO3-8 Advanced Composite column CIMmultus (registered trademark) SO3-80 Advanced Advanced Composite column, CIMmultus (registered trademark) SO3-800 Advanced Advanced Composite trademark, and registered trademark of CIMMult CM-0.1 Analytical column, CIMac (registered trademark) COOH-0.1 Analytical column, CIM (registered trademark) COOH DISK, CIM (registered trademark) COOH-1 Tube Monolithic column, CIM (registered trademark) OOH-8f Tube Monolithic column, CIM (registered trademark) COOH-80 Tube Monolithic column, CIM (registered trademark) COOH-800 Tube Monolithic column, CIM (registered trademark) COOH-Mutum Columbine, TMIM-registered trademark COIM-Molco-Tumol-Tol-Molco-Tumol-Molco-Tony.
  • the monolithic carriers in these columns are all glycidyl methacrylate and ethylene dimethacrylate copolymers with cation exchange groups introduced.
  • the column is preferably equilibrated through a buffer before use.
  • the buffer used for equilibration of the column is preferably one in which the salt therein does not react with the solution containing the envelope-type virus-like particles or the salt in the culture supernatant to cause precipitation. Further, it is preferable to select a buffer so that the salt concentrations of the two do not greatly differ. Therefore, the buffer is selected in consideration of the type and concentration of the salt in the solution containing the envelope-type virus-like particles or the culture supernatant.
  • the buffer used for equilibration of the column has a pH higher than the isoelectric point of the outer region of the envelope of the protein inserted in the envelope.
  • the buffer is not particularly limited as long as it is generally used.
  • a phosphate buffer, a citrate buffer, and a trishydroxymethylaminomethane buffer (hereinafter, referred to as Tris buffer).
  • Tris buffer An acetate buffer, a borate buffer, etc., and containing an inorganic salt such as sodium chloride.
  • a phosphate buffer, a citrate buffer, and a Tris buffer are preferable as the buffer from the viewpoint of a pH range having a buffer capacity.
  • the amount of buffer required for column equilibration is not particularly limited, but is preferably 1 CV (column volume multiple) or more, more preferably 3 CV or more, and even more preferably 5 CV or more.
  • the concentration of the buffer is not particularly limited, but is preferably 1 mM to 100 mM, more preferably 2 mM to 50 mM, and still more preferably 5 mM to 30 mM.
  • the culture supernatant is usually a liquid having a buffering capacity, and a buffer can be added thereto.
  • the buffer is not particularly limited as long as it is generally used.For example, based on a phosphate buffer, a citrate buffer, a Tris buffer, an acetate buffer, a borate buffer, and the like, Those containing an inorganic salt such as sodium chloride are used. Among them, a buffer containing an inorganic salt in a phosphate buffer, a citrate buffer, or a Tris buffer is preferable as the buffer in view of a usable pH range having a buffering capacity.
  • the pH of the liquid containing the envelope-type virus-like particles can be adjusted before being injected into the column.
  • the pH is adjusted to a range higher than the isoelectric point of the outer region of the envelope of the protein inserted in the envelope, so that the envelope-type virus-like particles can be adsorbed.
  • the pH of the buffer is adjusted to a range above 5.6.
  • the pH is too high, there is a possibility that the decomposition of proteins and the like in the envelope-type virus-like particles may occur, which is not preferable.
  • the upper limit is pH 10.0, preferably pH 8.5, and more preferably 7.5.
  • the pH is a value measured at 25 ° C.
  • the isoelectric point of the outer region of the envelope of the protein inserted into the envelope can be determined by calculation. Specifically, first, the amino acid sequence of the protein is examined using an online database or the like, and the sequence of a site to be arranged outside the envelope is specified from the sequence. From this sequence, the isoelectric point is calculated using commercially available software or an online database. For example, in the case of hepatitis B antigen, it is composed of PreS1-PreS2-S, and a part of S is inserted into the envelope. Therefore, the isoelectric point can be obtained from GESYX (manufactured by Genetics) from a partial sequence of PreS1-PreS2 and S. The calculation may be performed under initial conditions.
  • the pKa values of charged amino acids set under initial conditions are 12.5 for arginine, 6.0 for histidine, 10.5 for lysine, 3.9 for aspartic acid, 8.3 for cysteine, and 8.3 for glutamic acid.
  • tyrosine is 10.1
  • the isoelectric point can be similarly calculated in an online database such as ExPASy.
  • the concentration of the buffer in the solution containing the envelope-type virus-like particles is not particularly limited, but is preferably 1 mM to 100 mM, more preferably 2 mM to 50 mM, and still more preferably 5 mM to 30 mM.
  • the concentration of the inorganic salt is not limited as long as it is bound to the column, but is preferably 1 mM to 200 mM, more preferably 2 mM to 100 mM, still more preferably 5 mM to 50 mM.
  • the electric conductivity of the whole culture solution is not limited in the range of being bound to the column, but is preferably 1 mS / cm to 20 mS / cm, and more preferably 2 mS / cm to 15 mS / cm. More preferably, it is more preferably from 3 mS / cm to 10 mS / cm.
  • the concentration of the enveloped virus-like particles in the liquid containing the enveloped virus-like particles is preferably 0.01 mg / mL to 10 mg / mL, more preferably 0.1 mg / mL to 5 mg / mL, More preferably, it is 0.2 mg / mL to 3 mg / mL.
  • the flow rate of the liquid containing the envelope-type virus-like particles in the column is not particularly limited as long as the protein can be adsorbed to the column, but is preferably 2 CV / min to 12.5 CV / min, and more preferably 2.5 CV / min. More preferably, it is 5 CV / min.
  • the temperature of the column and the liquid containing the envelope-type virus-like particles is not particularly limited, but is preferably 2 ° C to 50 ° C, and preferably 4 ° C to 40 ° C. Is more preferable, and the temperature is more preferably 8 ° C to 30 ° C.
  • the envelope-type virus-like particles are selectively eluted from the monolith-type column to which the envelope-type virus-like particles are adsorbed.
  • a buffer solution used for equilibrating the column and an inorganic salt buffer solution in which an inorganic salt is dissolved are mixed at an appropriate ratio, and a mixed solution is used.
  • the envelope-type virus-like particles are selectively eluted.
  • Selective elution is performed by passing a mobile phase consisting of a mixture of a buffer solution having a pH higher than the isoelectric point in the outer region of the envelope of the protein inserted in the envelope and an inorganic salt buffer solution. Can be.
  • the inorganic salt used for the inorganic salt buffer is not limited as long as it can selectively elute envelope-type virus-like particles, and ammonium sulfate and sodium chloride are preferable.
  • the concentration of the inorganic salt in the mixture of the buffer and the inorganic salt buffer is preferably 50 mM to 500 mM, more preferably 75 to 400 mM, and still more preferably 100 to 300 mM.
  • the type, concentration and pH of the buffer for dissolving the inorganic salt may be the same as or different from the buffer contained in the solution containing the enveloped virus-like particles.
  • the flow rate of the mobile phase is not particularly limited as long as the envelope-type virus-like particles adsorbed on the column can be selectively eluted, but is preferably 2 CV / min or more, preferably 2 CV / min to 12.5 CV / min. Is more preferable, and more preferably 2.5 CV / min to 5 CV / min.
  • the temperature of the column and the mobile phase is not particularly limited, but is preferably 2 ° C to 50 ° C, more preferably 4 ° C to 40 ° C, and 8 ° C to 30 ° C. It is more preferred that there be.
  • the above-mentioned inorganic salt buffer alone is passed through the column as a mobile phase to elute contaminants derived from host culture cells and the like which are still adsorbed to the column. be able to. After elution of them, the column can be regenerated by passing the buffer used for equilibration through the column again.
  • envelope-type virus-like particles In the elution step, if a gradient based on the salt concentration is used, it is possible to separately elute envelope-type virus-like particles, host culture cells, and other contaminants. Envelope type virus-like particles, by collecting fractions in the gradient according to the salt concentration from the column to which contaminants derived from host culture cells are adsorbed, if the fraction containing the envelope type virus-like particles is collected, Envelope type virus-like particles with high purity can be obtained at high yield and high concentration at high speed.
  • the envelope-type virus-like particles thus obtained are useful as raw materials for pharmaceuticals and the like.
  • the purification method of the present invention is industrially and economically excellent because the column can be easily regenerated. Furthermore, the method according to the present invention uses a monolithic carrier to which a cation exchange group is immobilized, so that adsorption of anionic contaminants in the liquid can be prevented.
  • the monolithic column to which the cation exchange group is fixed can purify enveloped virus-like particles with high yield and high purity even at a pH higher than that of a conventional particulate porous polymer carrier. Also, due to the monolithic shape characteristics, even a substance such as an enveloped virus-like particle has a sufficient immobilization capacity, so that the carrier capacity required for purification can be reduced. Alternatively, in the case of the same carrier capacity, the amount of one treatment can be increased.
  • Total protein yield The ratio of the amount of purified total protein to the total amount of total protein loaded on the column was evaluated as total protein yield. The amount of total protein was quantified by a Bio-Rad Quick Start protein assay.
  • HBsAg-VLP yield The ratio of the amount of purified HBsAg-VLP to the total amount of hepatitis B virus antigen virus-like particles (hereinafter abbreviated as “HBsAg-VLP”) loaded on the column was evaluated as the HBsAg-VLP yield.
  • the amount of HBsAg-VLP was determined using an HBS antigen quantification kit manufactured by Vehicle.
  • Purification factor (PF) The ratio of the specific activity of HBsAg-VLP before purification to the specific activity of HBsAg-VLP after purification on the column was evaluated as Purification factor (PF).
  • the specific activity is a ratio of the amount of HBsAg-VLP to the amount of the total protein.
  • Non-Patent Document 2 envelope-type virus-like particles were expressed in yeast cells, and a culture supernatant was prepared. Specifically, recombinant yeast S. cerevisiae was cultured for 72 hours. The obtained culture solution was centrifuged, and the precipitated yeast was mixed with 7.5 M urea, 0.1 M Tris, 50 mM disodium hydrogen phosphate, 15 mM EDTA, 0.1% (v / v) Tween 80, and 4 mM PMSF mixed solution (pH 7.0). The suspension was suspended in 4) and crushed with 0.5 mm glass beads. The crushed extract was precipitated and dialyzed against PBS containing 1 mM EDTA.
  • the dialysate was incubated at 70 ° C. for 20 minutes and immediately placed on ice. Insoluble matter was removed by centrifugation, and the obtained supernatant was filtered with a 0.45 ⁇ m filter.
  • the theoretical value of the isoelectric point of the region outside the envelope of the inserted protein was 5.6.
  • the calculation of the theoretical value of the isoelectric point is performed by the analysis software GENETYX Ver. The calculation was performed at 10.
  • the carrier of CIMmultus (registered trademark) SO3-1 Advanced Composite column is a modified product of a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, and is a monolithic type in which a sulfonic acid group is retained in some glycidyl methacrylates. It is a column.
  • a sample solution was prepared by adjusting the pH of the culture supernatant to 8.5 with an aqueous sodium hydroxide solution.
  • the electric conductivity of the sample solution was about 10 mS / cm. 20 mL of this sample solution was injected into the column equilibrated in (1), and the envelope-type virus-like particles contained in the sample solution were adsorbed on the column.
  • the flow rate of the sample solution was 5 CV / min.
  • Example 2 The procedure was performed in the same manner as in Example 1 except that the pH of the culture supernatant was adjusted to 7.5 and the buffer was changed to a phosphate buffer of 20 mM and pH 7.5.
  • FIG. 1 shows the results of electron microscopic observation of the collected fraction.
  • HiTrap SP FF is a column composed of cross-linked agarose and packed with a gel having a particle size of 45-165 ⁇ m and modified with sulfonic acid groups. The unit volume is 1 mL.
  • Example 2 The operation was performed in the same manner as in Example 2 except that HiTrap (registered trademark) SP FF manufactured by GE Healthcare was used as the column, and the flow rate of the sample solution and the buffer solution was 1 CV / min.
  • HiTrap registered trademark
  • Example 3 The procedure was performed in the same manner as in Example 1 except that CIMmultus (registered trademark) QA-1 Advanced Composite column manufactured by BIA separations was used as a column.
  • the CIMmultus QA-1 Advanced Composite column is a monolithic column in which a quaternary ammonium group is modified on the same base material as that of the CIMmultus SO3-1 Advanced column.
  • the unit volume is 1 mL.

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Abstract

The present invention is a method for purifying envelope-type virus-like particles, comprising: a step in which a liquid containing envelope-type virus-like particles is passed through a monolithic column having a cation-exchange group immobilized thereon, causing the envelope-type virus-like particles to be adsorbed to the column; and a step in which the envelope-type virus-like particles adsorbed to the column are selectively eluted. The pH of the liquid containing the envelope-type virus-like particles is 10.0 or less, which is higher than the isoelectric point in a region outside the envelope having a protein inserted therein. The present invention makes it possible to purify, in a highly active state, envelope-type virus-like particles which are useful as a material for medicinal drugs etc., such purification being economical on an industrial scale.

Description

ウイルス様粒子の精製法Purification of virus-like particles
　本発明は、エンベロープ型ウイルス様粒子の精製方法に関する。 The present invention relates to a method for purifying enveloped virus-like particles.
　近年、バイオ医薬品の市場が拡大している。バイオ医薬品に用いられる医薬用タンパク質は、夾雑物を多量に含む細胞培養液から高度に精製される必要がある。このため、クロマトグラフィーや超遠心等の方法が一般に用いられている。医薬用タンパク質の代表として、抗体医薬品がよく知られている。抗体医薬品として、抗体の基本骨格をベースとした動物細胞での発現量向上の研究が広く為され、さらには抗体の基本骨格をベースとした精製プロセスが盛んに研究されてきた。 In recent years, the biopharmaceutical market has been expanding. Pharmaceutical proteins used in biopharmaceuticals need to be highly purified from cell cultures containing large amounts of contaminants. For this reason, methods such as chromatography and ultracentrifugation are generally used. As a representative of pharmaceutical proteins, antibody drugs are well known. As an antibody drug, research on improving the expression level in animal cells based on the basic skeleton of an antibody has been widely conducted, and furthermore, a purification process based on the basic skeleton of the antibody has been actively studied.
　バイオ医薬品において、ウイルス様粒子（ＶＬＰ：ｖｉｒｕｓ－ｌｉｋｅ　ｐａｒｔｉｃｌｅｓ）を医療分野で治療に用いる方法が急速に拡大している。例えば、新たなワクチンとしての応用やウイルスベクター・ドラッグデリバリー担体としての遺伝子治療等である。ウイルス様粒子の市場は、今後さらに拡大していくことが期待されている。 In biopharmaceuticals, the use of virus-like particles (VLPs) for treatment in the medical field is rapidly expanding. For example, application as a new vaccine or gene therapy as a virus vector / drug delivery carrier. The market for virus-like particles is expected to expand further in the future.
　一般に、ウイルス様粒子は細菌細胞や酵母細胞、昆虫細胞、植物細胞、ヒトを含む動物由来の細胞株を用いた細胞培養によって生産される。ウイルス様粒子はウイルスの特有な外殻構造を模倣して構築される。ウイルス様粒子は、ウイルス種に応じて異なる性状を有する。このため、共通の骨格と言えるものはなく、個々の発現量向上や精製プロセスの研究が必要である。 ウ イ ル ス In general, virus-like particles are produced by cell culture using cell lines derived from bacterial cells, yeast cells, insect cells, plant cells, and animals including humans. Virus-like particles are built by mimicking the unique shell structure of a virus. Virus-like particles have different properties depending on the virus species. For this reason, there is no one that can be said to be a common skeleton, and it is necessary to improve the expression level of each individual and study the purification process.
　ウイルス様粒子は、凝集物に加えて、構造の不安定さから、分解物や、完全な構造を有さない物も発生し、不純物となることがある。それらの不純物は、薬効の低下や免疫原性の発現等、医薬品にとって有害な影響をもたらすことが懸念されている。そのため、有害な影響がなくなる程度まで、不純物が除去されることが望まれている。 In addition to aggregates, virus-like particles may be decomposed or have no complete structure due to structural instability, and may become impurities. It is feared that these impurities have a detrimental effect on pharmaceuticals, such as reduced drug efficacy and immunogenicity. Therefore, it is desired that impurities be removed to the extent that harmful effects are eliminated.
　ウイルス様粒子における、不純物の除去には、超遠心による精製法が研究されている。しかし超遠心精製手法は、生産効率が悪かった。生産効率の向上を目的として、クロマトグラフィーによる精製方法が多数報告されている。これらは、陰イオン交換クロマトグラフィー、陽イオン交換クロマトグラフィー、疎水性クロマトグラフィー等を単独もしくは複合して用いる、あるいはそれらを混合した混合モードクロマトグラフィーを用いる等により精製するものである。 超 Ultracentrifugal purification methods have been studied to remove impurities from virus-like particles. However, the ultracentrifugation purification method had poor production efficiency. Many purification methods by chromatography have been reported for the purpose of improving production efficiency. These are purified by using anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, etc., alone or in combination, or by using mixed mode chromatography in which they are mixed.
　ウイルス様粒子の内、エンベロープ型ウイルス様粒子は、一般的にはリン脂質膜にタンパク質が挿入された構造を取る。一般的には、その脂質膜とクロマトグラフィー担体との疎水的相互作用を利用し、培養液中に多量に含まれる夾雑物との分離を行う。 Of the virus-like particles, the envelope-type virus-like particles generally have a structure in which a protein is inserted into a phospholipid membrane. In general, the hydrophobic interaction between the lipid membrane and the chromatography carrier is used to separate impurities contained in the culture solution in a large amount.
　エンベロープ型ウイルス様粒子の精製方法についても多数報告されている。例えば非特許文献１では、様々なイオン交換基が固定された粒子状の多孔質高分子担体と様々な溶離液ｐＨを組み合わせたプロセスにおける結合容量の比較が記載されている。文献１によると、カチオン交換基が固定された粒子状の多孔質高分子担体よりもアニオン交換基が固定された粒子状の多孔質高分子担体の方が、広いｐＨ範囲で、結合容量が高く、優れた濃縮・精製が可能であることが記載されている。 多数 A number of methods for purifying enveloped virus-like particles have been reported. For example, Non-Patent Document 1 describes a comparison of binding capacities in a process in which various porous polymer carriers having various ion exchange groups immobilized thereon and various pH values of an eluent are combined. According to Document 1, a particulate porous polymer carrier having anion-exchange groups immobilized thereon has a higher binding capacity in a wider pH range than a particulate porous polymer carrier having immobilized cation exchange groups. It describes that excellent concentration and purification are possible.
　また、非特許文献２では、硫酸エステル基が固定された多孔質高分子担体からなる充填剤を用いたカラムによるエンベロープ型ウイルス様粒子の精製方法が記載されている。 Non-Patent Document 2 describes a method for purifying enveloped virus-like particles by a column using a packing composed of a porous polymer carrier to which sulfate groups are fixed.
　さらに、非特許文献３には、疎水性の表面を有するモノリス型カラム、あるいはブチル基が固定された粒子状の多孔質高分子担体からなるカラムを用いたエンベロープ型ウイルス様粒子の精製方法が記載されている。本方法では、混入する脂質による、カラムの閉塞や汚染を防ぐために適切な前処理や洗浄方法が必要となる。 Further, Non-Patent Document 3 describes a method for purifying envelope-type virus-like particles using a monolithic column having a hydrophobic surface or a column composed of a particulate porous polymer carrier to which butyl groups are fixed. Have been. In this method, an appropriate pretreatment or washing method is required to prevent the column from being clogged or contaminated by the contaminating lipid.
　これまでの種々の試みにもかかわらず、依然として、医薬品等の原料として有用なエンベロープ型ウイルス様粒子の効率の良い精製法が望まれている。 に も か か わ ら ず Despite various attempts so far, there is still a need for a method for efficiently purifying enveloped virus-like particles useful as a raw material for pharmaceuticals and the like.
　本発明者らは、エンベロープ型ウイルス様粒子の精製方法について鋭意検討を行なった。その結果、カチオン交換基が固定されたモノリス型カラムと、挿入されているタンパク質のエンベロープの外側領域の等電点よりも高く、ｐＨ１０．０以下の溶離液・サンプルバッファーを組み合わせて使用した条件において、エンベロープ型ウイルス様粒子を効率よく、高活性に精製できることを見出し、本発明を完成した。 The present inventors have conducted intensive studies on a method for purifying enveloped virus-like particles. As a result, under the conditions in which a monolith-type column having a fixed cation exchange group and an eluent / sample buffer having a pH higher than the isoelectric point of the outer region of the inserted protein envelope and a pH of 10.0 or less were used. The present inventors have found that envelope-type virus-like particles can be efficiently and highly purified.
　すなわち、本発明は以下のとおりである。
［１］カチオン交換基が固定されたモノリス型カラムに、エンベロープ型ウイルス様粒子を含む液を通液してエンベロープ型ウイルス様粒子を前記カラムに吸着させる工程、および前記カラムに吸着したエンベロープ型ウイルス様粒子を選択的に溶出させる工程を含み、
　前記のエンベロープ型ウイルス様粒子を含む液のｐＨが、エンベロープに挿入されているタンパク質のエンベロープの外側領域の等電点より高く、ｐＨ１０．０以下である、エンベロープ型ウイルス様粒子の精製方法。
［２］前記モノリス型カラムが、グリシジルメタクリレート由来のモノマー単位とエチレングリコールジメタクリレート由来のモノマー単位を含む共重合体にカチオン交換基が固定されたモノリス担体から構成されるモノリス型カラムである、前記１に記載の精製方法。
［３］前記カチオン交換基がスルホン酸基である、前記１あるいは２のいずれかに記載の精製方法。
［４］前記エンベロープ型ウイルス様粒子に挿入されているタンパク質がＢ型肝炎ウイルス抗原（ＨＢｓＡｇ）を含む、前記１～３のいずれか１項に記載の精製方法。
［５］前記エンベロープ型ウイルス様粒子が酵母細胞により調製されたものである、前記１～４のいずれか１項に記載の精製方法。
［６］前記モノリス型カラムの通液方向の厚さが１～１００ｍｍである、前記１～５のいずれか１項に記載の精製方法。 That is, the present invention is as follows.
[1] a step of passing a liquid containing enveloped virus-like particles through a monolithic column to which a cation exchange group is fixed to adsorb the enveloped virus-like particles to the column, and an enveloped virus adsorbed to the column Comprising the step of selectively eluting like particles,
A method for purifying enveloped virus-like particles, wherein the pH of the solution containing the enveloped virus-like particles is higher than the isoelectric point of the outer region of the envelope of the protein inserted into the envelope and is pH 10.0 or less.
[2] The monolithic column is a monolithic column composed of a monolithic carrier having a cation exchange group fixed to a copolymer containing a monomer unit derived from glycidyl methacrylate and a monomer unit derived from ethylene glycol dimethacrylate. 2. The purification method according to 1.
[3] The purification method according to any one of [1] and [2], wherein the cation exchange group is a sulfonic acid group.
[4] The purification method according to any one of the above items 1 to 3, wherein the protein inserted into the enveloped virus-like particle contains a hepatitis B virus antigen (HBsAg).
[5] The purification method according to any one of [1] to [4], wherein the envelope-type virus-like particles are prepared by yeast cells.
[6] The purification method according to any one of [1] to [5], wherein the thickness of the monolith type column in the liquid flow direction is 1 to 100 mm.
　本発明のエンベロープ型ウイルス様粒子の精製方法によれば、医薬品等の原料として有用なエンベロープ型ウイルス様粒子を効率よく、高活性に精製できる。 According to the method for purifying enveloped virus-like particles of the present invention, enveloped virus-like particles useful as raw materials for pharmaceuticals and the like can be efficiently and highly purified.
図１は実施例２にて回収された画分の、エンベロープ型ウイルス様粒子の電子顕微鏡写真である。FIG. 1 is an electron micrograph of the envelope-type virus-like particles of the fraction collected in Example 2.
　エンベロープ型ウイルス様粒子は、リン脂質膜で構成される。また、リン脂質表面に挿入されているタンパク質のエンベロープの外側領域の等電点が酸性側にある。このため、エンベロープ型ウイルス様粒子は、中性付近ではアニオンとして、マイナスの電荷を帯びる。こうした理由により、エンベロープ型ウイルス様粒子は、中性以上のｐＨの溶離液とアニオン交換クロマトグラフィーを組み合わせた方法により、主に精製されてきた。この方法では、アニオン交換担体の結合容量に応じて、エンベロープ型ウイルス様粒子を結合させ、その後に溶出する精製方法が取られる。しかし、この方法では、得られたエンベロープ型ウイルス様粒子の精製後の活性が十分でないことがあった。 Envelope type virus-like particles are composed of a phospholipid membrane. Further, the isoelectric point of the outer region of the protein envelope inserted on the phospholipid surface is on the acidic side. For this reason, the envelope-type virus-like particles are negatively charged as anions near neutrality. For these reasons, enveloped virus-like particles have been mainly purified by a method combining an eluent having a neutral or higher pH with anion exchange chromatography. In this method, an envelope-type virus-like particle is bound according to the binding capacity of the anion exchange carrier, and then a purification method of eluting is used. However, in this method, the activity of the resulting enveloped virus-like particles after purification may not be sufficient.
　本発明者らは、エンベロープ型ウイルス様粒子を含む液を、エンベロープに挿入されているタンパク質のエンベロープの外側領域の等電点より高いｐＨに調節した後、カチオン交換基が固定されたモノリス型カラムに通液し、エンベロープ型ウイルス様粒子を結合させ、次いで、エンベロープ型ウイルス様粒子を溶出させることにより、高活性な状態でエンベロープ型ウイルス様粒子を精製できることを見出した。 The present inventors adjusted the solution containing the envelope-type virus-like particles to a pH higher than the isoelectric point of the outer region of the envelope of the protein inserted into the envelope, and then fixed the cation exchange group to a monolithic column. To bind the envelope-type virus-like particles and then elute the envelope-type virus-like particles, thereby finding that the envelope-type virus-like particles can be purified in a highly active state.
　本反応が予想に反して好結果を与える理由は不明である。一つの解釈として次のように考えることができる。すなわち、エンベロープ型ウイルス様粒子は、タンパク質のエンベロープの外側領域の等電点より高いｐＨに調節した溶液中では、リン脂質の部分が、マイナスの電荷を持つ。これに対し、エンベロープに挿入されているタンパク質の部分では、マイナスの電荷は相対的に少ない。このため、タンパク質の部分を中心に、モノリス型カラムの基材との間に、疎水性相互作用が働く。複数の箇所でこうした疎水性相互作用が働くことで、エンベロープ型ウイルス様粒子全体は、疎水性相互作用により、カラムに吸着する。このとき、リン脂質のみからなる不純物は、カラムに吸着することが無いので、効果的に高活性な目的物を精製することができる。以上は、仮説であり、本発明の理解を容易にするためのものであり、本発明の範囲を限定する意図はない。
　以下に、本発明における各要件について、詳細に説明する。 It is unclear why this reaction gives unexpectedly good results. One interpretation can be considered as follows. That is, in the envelope-type virus-like particles, the phospholipid portion has a negative charge in a solution adjusted to a pH higher than the isoelectric point of the outer region of the protein envelope. In contrast, the portion of the protein inserted into the envelope has relatively little negative charge. For this reason, hydrophobic interaction acts between the protein portion and the substrate of the monolith type column. When such hydrophobic interaction acts at a plurality of locations, the entire enveloped virus-like particle is adsorbed to the column by the hydrophobic interaction. At this time, impurities consisting only of phospholipids are not adsorbed on the column, so that a highly active target substance can be effectively purified. The above is a hypothesis, and is intended to facilitate understanding of the present invention, and is not intended to limit the scope of the present invention.
Hereinafter, each requirement in the present invention will be described in detail.
＜エンベロープ型ウイルス様粒子を含む液＞
　本発明においてエンベロープ型ウイルス様粒子を含む液とは、エンベロープ型ウイルス様粒子及び分離すべき不純物を含む液であり、具体的には、エンベロープ型ウイルス様粒子の製造にて得られる液である。 <Liquid containing envelope-type virus-like particles>
In the present invention, the liquid containing the envelope-type virus-like particles is a liquid containing the envelope-type virus-like particles and impurities to be separated, and specifically, a liquid obtained in the production of the envelope-type virus-like particles.
　エンベロープ型ウイルス様粒子は、細菌細胞や酵母細胞、昆虫細胞、植物細胞、ヒトを含む動物由来の細胞株を培養する方法等によって製造することができる。得られた培養液には不純物が含まれるが、本発明の精製方法によりこの培養液からエンベロープ型ウイルス様粒子を分離精製することができる。分離される不純物は、例えば宿主培養細胞等由来の夾雑物、タンパク質、リン脂質等が挙げられる。 Envelope-type virus-like particles can be produced by a method of culturing cell lines derived from bacterial cells, yeast cells, insect cells, plant cells, and animals including humans. Although the obtained culture solution contains impurities, envelope-type virus-like particles can be separated and purified from the culture solution by the purification method of the present invention. The impurities to be separated include, for example, contaminants, proteins, and phospholipids derived from host cultured cells and the like.
　培養液はそのままカラムに通すことができるが、通常は培養液から分離した培養上清を用いる。培養上清を用いることによりカラム汚染を抑制することができる。 The culture solution can be passed through the column as it is, but usually the culture supernatant separated from the culture solution is used. By using the culture supernatant, column contamination can be suppressed.
　エンベロープ型ウイルス様粒子を含む液は、カラムに通す前にｐＨを調整したり、緩衝剤を添加したり、することができる。 (4) Before passing the liquid containing the envelope-type virus-like particles through the column, the pH can be adjusted or a buffer can be added.
　本発明の精製方法に適用できるエンベロープ型ウイルス様粒子は、リン脂質膜にタンパク質が挿入されたエンベロープ型ウイルスの外殻構造を模倣した人工粒子であれば、特に制限されない。エンベロープ型ウイルスとは、例えば、ヘルペスウイルス科、ポックスウイルス科、ヘパドナウイルス科、フラビウイルス科、トガウイルス科、コロナウイルス科、オルトミクソウイルス科、パラミクソウイルス科、ラブドウイルス科、ブニヤウイルス科、フィロウイルス科、レトロウイルス科等に属するウイルスが挙げられる。 エ ン The envelope-type virus-like particles applicable to the purification method of the present invention are not particularly limited as long as they are artificial particles that mimic the outer-shell structure of an envelope-type virus in which a protein is inserted into a phospholipid membrane. Envelope viruses include, for example, herpesviridae, poxviridae, hepadnaviridae, flaviviridae, togaviridae, coronaviridae, orthomyxoviridae, paramyxoviridae, rhabdoviridae, bunyaviridae, Viruses belonging to the family Filoviridae, Retroviridae and the like can be mentioned.
　エンベロープ型ウイルス様粒子に含まれるリン脂質には、ホスファチジルコリン、ホスファチジルセリン、ホスファチジルグリセロールなどの化合物が含まれる。前記リン脂質に挿入されたタンパク質は、膜貫通タンパク質や表面抗原タンパク質である。例えばＢ型肝炎ウイルスに由来するウイルス様粒子は、リン脂質に、ｐＩ７．３のＢ型肝炎ウイルス表面抗原（ＨＢｓＡｇ）が挿入されており、リン脂質に対し、５倍から１０倍質量％の割合で含まれている。 リ ン The phospholipids contained in the enveloped virus-like particles include compounds such as phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. The protein inserted into the phospholipid is a transmembrane protein or a surface antigen protein. For example, a virus-like particle derived from hepatitis B virus has a hepatitis B virus surface antigen (HBsAg) having a pI of 7.3 inserted into a phospholipid, and has a ratio of 5 to 10 times by mass of the phospholipid. Included in.
＜カチオン交換基が固定されたモノリス型カラム＞
（１）モノリス型カラム
　モノリス型カラムとは、モノリス担体が保持されたカラムである。モノリス担体とは、マイクロメートルオーダーの網目状の骨格が繋がった特徴的な構造をもつ一体型の多孔質体である。モノリス担体は、全体にわたって均一な貫通孔が多数設けられていることを特徴とする。これらのモノリス担体は、静置したとき、その形状を自己支持できる機械的強度を有する。すなわち、粒子状の多孔質高分子担体や、多孔膜とは物理的構造が異なる。 <Monolith column with fixed cation exchange group>
(1) Monolith type column A monolith type column is a column holding a monolith carrier. The monolithic carrier is a monolithic porous body having a characteristic structure in which a network structure of a micrometer order is connected. The monolithic carrier is characterized in that many uniform through holes are provided throughout. These monolith carriers have a mechanical strength capable of self-supporting their shape when left standing. That is, the physical structure is different from that of the particulate porous polymer carrier or porous membrane.
　本発明によるモノリス担体としては、一つの態様として、有機高分子からなることが好ましい。モノリス担体を構成するためのモノマー単位としては、官能基を持たないモノマー単位、カチオン交換基の基となる官能基を有するモノマー単位等が挙げられる。これらモノマー単位は２種以上含んでいても良い。 モ ノ In one embodiment, the monolithic carrier according to the present invention preferably comprises an organic polymer. Examples of the monomer unit for constituting the monolithic carrier include a monomer unit having no functional group and a monomer unit having a functional group serving as a cation exchange group. These monomer units may contain two or more kinds.
　モノマーには、架橋モノマーが含まれる。架橋モノマーとは重合性部位を２つ以上有するモノマーである。 Monomers include cross-linking monomers. A crosslinking monomer is a monomer having two or more polymerizable sites.
　モノリス担体としての前記有機高分子は、モノマーと架橋モノマーとの共重合体が好ましい。 The organic polymer as a monolithic carrier is preferably a copolymer of a monomer and a crosslinking monomer.
　前記モノマーとしては、例えば、スチレン、置換スチレン（但し、置換基は、クロロメチル基、炭素原子数１～１８のアルキル基、水酸基、ｔ－ブチルオキシカルボニル基、ハロゲン基、ニトロ基、アミノ基、保護水酸基またはアミノ基を包含する）、ビニルナフタレン、アクリル酸エステル類、グリシジルメタクリレート、ヒドロキシエチルメタクリレート、ヒドロキシプロピルメタクリレートなどのメタクリル酸エステル類、酢酸ビニルおよびピロリドン、並びに、これらの混合物が挙げられる。 Examples of the monomer include styrene and substituted styrene (provided that the substituent is a chloromethyl group, an alkyl group having 1 to 18 carbon atoms, a hydroxyl group, a t-butyloxycarbonyl group, a halogen group, a nitro group, an amino group, (Including protected hydroxyl or amino groups), vinyl naphthalenes, acrylates, methacrylates such as glycidyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, vinyl acetate and pyrrolidone, and mixtures thereof.
　前記架橋モノマーとしては、ジビニルベンゼン、ジビニルナフタレン、ジビニルピリジン、メタクリレート類、アクリレート類、ビニルエステル類、ジビニルエーテルなどのビニルエーテル類、エチレンビスアクリルアミドやプロピレンビスアクリルアミドなどのアルキレンビスアクリルアミド類またはメタクリルアミド類及びそれらの混合物が挙げられる。 Examples of the crosslinking monomer, divinylbenzene, divinylnaphthalene, divinylpyridine, methacrylates, acrylates, vinyl esters, vinyl ethers such as divinyl ether, alkylene bisacrylamides such as ethylene bisacrylamide and propylene bisacrylamide or methacrylamides and And mixtures thereof.
　架橋モノマーとしてのメタクリレート類としては、エチレングリコールジメタクリレートやプロピレングリコールジメタクリレートなどのアルキレンジメタクリレート類、ペンタエリトリトールジ－、トリ－若しくはテトラメタクリレート、トリメチロールプロパントリメタクリレートが挙げられる。架橋モノマーとしてのアクリレート類としては、エチレングリコールジアクリレート類、ペンタエリトリトールジ－、トリ－若しくはテトラアクリレートが挙げられる。 Examples of the methacrylates as the crosslinking monomer include alkylene dimethacrylates such as ethylene glycol dimethacrylate and propylene glycol dimethacrylate, pentaerythritol di-, tri- or tetramethacrylate, and trimethylolpropane trimethacrylate. Examples of the acrylates as the crosslinking monomer include ethylene glycol diacrylates, pentaerythritol di-, tri- and tetraacrylates.
　モノマーは、好ましくは、メタクリレート類あるいはアクリレート類から適宜選択される。より好ましくはグリシジルメタクリレートとアルキレンジメタクリレート類の組み合わせ、さらに好ましくはグリシジルメタクリレートとエチレングリコールジメタクリレートの組み合わせである。
　好ましいモノリス担体は、前記モノマー混合物から得られた共重合体を、カチオン交換基を有する化合物で誘導体化したものである。 The monomer is preferably appropriately selected from methacrylates and acrylates. More preferably, it is a combination of glycidyl methacrylate and alkylene dimethacrylates, and still more preferably, a combination of glycidyl methacrylate and ethylene glycol dimethacrylate.
Preferred monolithic carriers are those obtained by derivatizing a copolymer obtained from the monomer mixture with a compound having a cation exchange group.
　グリシジルメタクリレートとエチレングリコールジメタクリレートの共重合体は、ポロゲンと重合開始剤の存在下で、グリシジルメタクリレートとエチレングリコールジメタクリレートの混合物から製造される。ポロゲンとは、多孔を形成するための、炭素－炭素二重結合をもたない添加物質であり、脂肪族炭化水素類、芳香族炭化水素類、エステル類、アルコール類、ケトン類、エーテル類、可溶性高分子溶液、およびそれらの混合物などが使用できる。好ましくはノルマルヘキサンである。ポロゲンとして可溶性高分子が添加されていてもよい。可溶性高分子が添加されていると、孔構造がより多く形成される。可溶性高分子の量は、共重合体全体の質量に対して、好ましくは１０～４０質量％の量である。 (4) A copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate is produced from a mixture of glycidyl methacrylate and ethylene glycol dimethacrylate in the presence of a porogen and a polymerization initiator. A porogen is an additive substance having no carbon-carbon double bond for forming porosity, and includes aliphatic hydrocarbons, aromatic hydrocarbons, esters, alcohols, ketones, ethers, Soluble polymer solutions and mixtures thereof can be used. Preferably, it is normal hexane. A soluble polymer may be added as a porogen. When a soluble polymer is added, more pore structures are formed. The amount of the soluble polymer is preferably 10 to 40% by mass based on the total mass of the copolymer.
　重合開始剤としては、フリーラジカル発生開始剤が使用できる。具体的にはアゾビスイソブチロニトリルや２，２’－アゾビス（イソブチルアミド）ジヒドレートなどのアゾ化合物、過酸化ベンゾイルや過酸化ジプロピル二カルボン酸エステルなどの過酸化物が使用できる。種類の異なる重合開始剤を用いると、形の異なる孔構造を形成することができる。重合開始剤の量は、モノマー質量に対して好ましくは０．５～４質量％である。 フ リ ー As the polymerization initiator, a free radical generation initiator can be used. Specifically, azo compounds such as azobisisobutyronitrile and 2,2'-azobis (isobutylamide) dihydrate, and peroxides such as benzoyl peroxide and dipropyl dicarboxylate can be used. When different types of polymerization initiators are used, pore structures having different shapes can be formed. The amount of the polymerization initiator is preferably 0.5 to 4% by mass based on the mass of the monomer.
　ポロゲンや重合開始剤を含むグリシジルメタクリレートとエチレングリコールジメタクリレートの混合物は、成形型の中に入れる前に、窒素又はアルゴンのような不活性ガスを用いて脱気することが好ましい。成形型は、空気汚染を防ぐために密封することが好ましい。重合は、例えば、４０～５０時間、５０℃～９０℃の温度に加熱して行うことができる。重合後、洗浄により、ポロゲンとして用いた溶媒や可溶性高分子を除去する。洗浄のための溶媒としては、メタノール、エタノール、ベンゼン、トルエン、アセトン、テトラヒドロフランなどが使用できる。洗浄工程は複数回繰り返してもよい。 The mixture of glycidyl methacrylate and ethylene glycol dimethacrylate containing a porogen and a polymerization initiator is preferably degassed with an inert gas such as nitrogen or argon before being put into a mold. The mold is preferably sealed to prevent air contamination. The polymerization can be carried out, for example, by heating to a temperature of 50 ° C. to 90 ° C. for 40 to 50 hours. After the polymerization, the solvent and the soluble polymer used as the porogen are removed by washing. As a solvent for washing, methanol, ethanol, benzene, toluene, acetone, tetrahydrofuran and the like can be used. The washing step may be repeated a plurality of times.
（２）カチオン交換基
　カチオン交換基とは、酸性を示すイオン交換基である。カチオン交換基として、具体的に、硫酸基、スルホン酸基（－ＳＯ3Ｈ）、カルボン酸基及びリン酸基が挙げられる。このうち、スルホン酸基及びリン酸基が好ましく、スルホン酸基がより好ましい。スルホン酸基は、適当な共重合体に対し、ブロモエチルスルホン酸、クロロヒドロキシプロパンスルホン酸、１，３－プロパンスルトン、１，４－ブタンスルトン、３－ヒドロキシプロパンスルホン酸ナトリウム、亜硫酸、および亜硫酸ナトリウム、亜硫酸カリウム等の亜硫酸塩を反応させることにより、モノリス担体に導入することができる。例えば、共重合体がエポキシ基を有する場合、亜硫酸ナトリウム、亜硫酸カリウムなどを反応させてスルホン酸基を有する共重合体を得ることができるし、共重合体が水酸基を有する場合は、適当な塩基と共に、ブロモエチルスルホン酸、クロロヒドロキシプロパンスルホン酸、１，４－ブタンスルトンなどを反応させてスルホン酸基を有する共重合体を得ることができる。共重合体に導入されるスルホン酸基の量は、実際に使用する緩衝液に応じて、適宜調節される。 (2) Cation exchange group The cation exchange group is an ion exchange group that exhibits acidity. Specific examples of the cation exchange group include a sulfate group, a sulfonic acid group (—SO 3 H), a carboxylic acid group, and a phosphoric acid group. Of these, a sulfonic acid group and a phosphoric acid group are preferred, and a sulfonic acid group is more preferred. The sulfonic acid groups are selected from the group consisting of bromoethyl sulfonic acid, chlorohydroxypropane sulfonic acid, 1,3-propane sultone, 1,4-butane sultone, sodium 3-hydroxypropane sulphonate, sulfurous acid and sodium sulphite. By reacting with a sulfite such as potassium sulfite or the like, it can be introduced into the monolithic carrier. For example, when the copolymer has an epoxy group, sodium sulfite, potassium sulfite and the like can be reacted to obtain a copolymer having a sulfonic acid group, and when the copolymer has a hydroxyl group, an appropriate base can be used. At the same time, bromoethylsulfonic acid, chlorohydroxypropanesulfonic acid, 1,4-butanesultone and the like are reacted to obtain a copolymer having a sulfonic acid group. The amount of sulfonic acid groups introduced into the copolymer is appropriately adjusted according to the buffer actually used.
（３）カチオン交換基が固定されたモノリス型カラム
　カチオン交換基が固定されたモノリス型カラムは、カチオン交換基が固定されたモノリス担体がカラム容器に収納されたものである。カラムの大きさ（体積）は、特に制限されず、結合させるエンベロープ型ウイルス様粒子およびその凝集体等の不純物の量に応じて適宜調整される。モノリス担体の形状は、板状、管状、円筒状等の一体構造をなしている。モノリス担体の通液方向の厚さは、１ｍｍ～１００ｍｍであることが好ましく、２ｍｍ～７０ｍｍであることがより好ましく、３ｍｍ～６０ｍｍであることがさらに好ましく、３ｍｍ～５０ｍｍであることが最も好ましい。モノリス担体の厚さを１ｍｍ以上とすることにより、モノリス担体は充分な機械的強度を持つ。モノリス担体の厚さを１００ｍｍ以下とすることにより、モノリス担体が破壊しない圧力を保ち、ポンプ圧の上昇を防ぐことができる。 (3) Monolithic Column with Fixed Cation Exchange Group The monolithic column with fixed cation exchange group is one in which a monolithic carrier with fixed cation exchange group is housed in a column container. The size (volume) of the column is not particularly limited, and is appropriately adjusted according to the amount of impurities such as enveloped virus-like particles and aggregates to be bound. The shape of the monolithic carrier has an integral structure such as a plate shape, a tubular shape, and a cylindrical shape. The thickness of the monolith carrier in the flow direction is preferably from 1 mm to 100 mm, more preferably from 2 mm to 70 mm, further preferably from 3 mm to 60 mm, and most preferably from 3 mm to 50 mm. By setting the thickness of the monolith carrier to 1 mm or more, the monolith carrier has sufficient mechanical strength. By setting the thickness of the monolithic carrier to 100 mm or less, a pressure at which the monolithic carrier is not broken can be maintained, and an increase in pump pressure can be prevented.
　カラムとしては、具体的には、ＣＩＭａｃ（登録商標）　ＳＯ３－０．１　Ａｎａｌｙｔｉｃａｌ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＳＯ３　ＤＩＳＫ、ＣＩＭ（登録商標）　ＳＯ３－１　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＳＯ３－８ｆ　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＳＯ３－８０　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＳＯ３－８００　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＳＯ３－８０００　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＳＯ３－１　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＳＯ３－８　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＳＯ３－８０　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＳＯ３－８００　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＳＯ３－８０００　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭａｃ（登録商標）　ＣＭ－０．１　Ａｎａｌｙｔｉｃａｌ　ｃｏｌｕｍｎ、ＣＩＭａｃ（登録商標）　ＣＯＯＨ－０．１　Ａｎａｌｙｔｉｃａｌ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＣＯＯＨ　ＤＩＳＫ、ＣＩＭ（登録商標）　ＣＯＯＨ－１　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＣＯＯＨ－８ｆ　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＣＯＯＨ－８０　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＣＯＯＨ－８００　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭ（登録商標）　ＣＯＯＨ－８０００　Ｔｕｂｅ　Ｍｏｎｏｌｉｔｈｉｃ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＣＯＯＨ－１　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＣＯＯＨ－８　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＣＯＯＨ－８０　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＣＯＯＨ－８００　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＣＯＯＨ－８０００　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ等が挙げられる。 Specific examples of the columns include CIMac (registered trademark) SO3-0.1 Analytical column, CIM (registered trademark) SO3 DISK, CIM (registered trademark) SO3-1 Tube Monolithic column, and CIM (registered trademark) SO3-8f Tube Monolithic column, CIM (registered trademark) SO3-80 Tube Monolithic column, CIM (registered trademark) SO3-800 Tube Monolithic column, CIM (registered trademark) SO3-8000 {Tublic Co., Ltd. column, CIMmultus (climbing Trademarks) SO3-8 Advanced Composite column, CIMmultus (registered trademark) SO3-80 Advanced Advanced Composite column, CIMmultus (registered trademark) SO3-800 Advanced Advanced Composite trademark, and registered trademark of CIMMult CM-0.1 Analytical column, CIMac (registered trademark) COOH-0.1 Analytical column, CIM (registered trademark) COOH DISK, CIM (registered trademark) COOH-1 Tube Monolithic column, CIM (registered trademark) OOH-8f Tube Monolithic column, CIM (registered trademark) COOH-80 Tube Monolithic column, CIM (registered trademark) COOH-800 Tube Monolithic column, CIM (registered trademark) COOH-Mutum Columbine, TMIM-registered trademark COIM-Molco-Tumol-Tol-Molco-Tumol-Molco-Tony. 1 Advanced Composite column, CIMmultitus (registered trademark) COOH-8 Advanced Advanced Composite column, CIMmultitus (registered trademark) COOH-80 Advanced Composite column, registered trademark of CIMmultus-800 nsed \ Composite \ column, CIMmultus (registered trademark) \ COOH-8000 \ Advanced \ Composite \ column and the like.
　これらカラムのモノリス担体は全てグリシジルメタクリレートとエチレンジメタクリレートの共重合体にカチオン交換基が導入されたものである。 モ ノ The monolithic carriers in these columns are all glycidyl methacrylate and ethylene dimethacrylate copolymers with cation exchange groups introduced.
＜吸着工程＞
　カラムは、使用前に緩衝液を通して平衡化しておくことが好ましい。
　カラムの平衡化に用いられる緩衝液は、その中の塩が、エンベロープ型ウイルス様粒子を含む液あるいは培養上清中の塩と反応して沈殿が生じないものが好ましい。また、両者の塩濃度が大きく異ならないように緩衝液を選択することが好ましい。そのため、前記緩衝液はエンベロープ型ウイルス様粒子を含む液あるいは培養上清中の塩の種類及び濃度を考慮して選択される。
　カラムの平衡化に用いられる緩衝液は、ｐＨがエンベロープに挿入されているタンパク質のエンベロープの外側領域の等電点より高いものが用いられる。緩衝液としては、一般的に使用されているものであれば特に制限されないが、例えば、リン酸緩衝液、クエン酸緩衝液、トリスヒドロキシメチルアミノメタン緩衝液（以下、トリス緩衝液と称する。）、酢酸緩衝液、ホウ酸緩衝液等をベースとし、塩化ナトリウムなどの無機塩を含むものが用いられる。これらの中で、緩衝液としては緩衝能を有するｐＨ範囲の観点から、リン酸緩衝液、クエン酸緩衝液、トリス緩衝液が好ましい。 <Adsorption process>
The column is preferably equilibrated through a buffer before use.
The buffer used for equilibration of the column is preferably one in which the salt therein does not react with the solution containing the envelope-type virus-like particles or the salt in the culture supernatant to cause precipitation. Further, it is preferable to select a buffer so that the salt concentrations of the two do not greatly differ. Therefore, the buffer is selected in consideration of the type and concentration of the salt in the solution containing the envelope-type virus-like particles or the culture supernatant.
The buffer used for equilibration of the column has a pH higher than the isoelectric point of the outer region of the envelope of the protein inserted in the envelope. The buffer is not particularly limited as long as it is generally used. For example, a phosphate buffer, a citrate buffer, and a trishydroxymethylaminomethane buffer (hereinafter, referred to as Tris buffer). , An acetate buffer, a borate buffer, etc., and containing an inorganic salt such as sodium chloride. Among these, a phosphate buffer, a citrate buffer, and a Tris buffer are preferable as the buffer from the viewpoint of a pH range having a buffer capacity.
　カラムの平衡化に要する緩衝液の量は、特に制限されないが、１ＣＶ（カラム容積倍数）以上であることが好ましく、３ＣＶ以上であることがより好ましく、５ＣＶ以上であることがさらに好ましい。緩衝液の濃度は、特に制限されないが、１ｍＭ～１００ｍＭであることが好ましく、２ｍＭ～５０ｍＭであることがより好ましく、５ｍＭ～３０ｍＭであることがさらに好ましい。 量 The amount of buffer required for column equilibration is not particularly limited, but is preferably 1 CV (column volume multiple) or more, more preferably 3 CV or more, and even more preferably 5 CV or more. The concentration of the buffer is not particularly limited, but is preferably 1 mM to 100 mM, more preferably 2 mM to 50 mM, and still more preferably 5 mM to 30 mM.
　カラムの平衡化の後、エンベロープ型ウイルス様粒子を含む液がカラムに通液される。 の 後 After equilibration of the column, a liquid containing enveloped virus-like particles is passed through the column.
　前記液として培養上清を用いる場合、通常、培養上清は緩衝能をもつ液になっているが、それに緩衝液を加えることができる。緩衝液としては、一般的に使用されているものであれば特に制限されないが、例えば、リン酸緩衝液、クエン酸緩衝液、トリス緩衝液、酢酸緩衝液、ホウ酸緩衝液等をベースとし、塩化ナトリウムなどの無機塩を含むものが用いられる。これらの中で、緩衝液としては緩衝能を有する使用ｐＨ範囲の点から、リン酸緩衝液、クエン酸緩衝液、トリス緩衝液に無機塩を含むものが好ましい。 培養 When a culture supernatant is used as the liquid, the culture supernatant is usually a liquid having a buffering capacity, and a buffer can be added thereto. The buffer is not particularly limited as long as it is generally used.For example, based on a phosphate buffer, a citrate buffer, a Tris buffer, an acetate buffer, a borate buffer, and the like, Those containing an inorganic salt such as sodium chloride are used. Among them, a buffer containing an inorganic salt in a phosphate buffer, a citrate buffer, or a Tris buffer is preferable as the buffer in view of a usable pH range having a buffering capacity.
　エンベロープ型ウイルス様粒子を含む液は、カラムに注入される前にｐＨを調整することができる。前記ｐＨは、エンベロープに挿入されているタンパク質のエンベロープの外側領域の等電点より高く、エンベロープ型ウイルス様粒子を吸着できる範囲に調整する。例えば、タンパク質のエンベロープの外側領域の等電点が５．６の場合、緩衝液のｐＨは、５．６より高い範囲に調整される。ただし、高すぎるｐＨでは、エンベロープ型ウイルス様粒子中のタンパク質などの分解が起こる可能性があるので、好ましくない。通常上限は、ｐＨ１０．０、好ましくは、ｐＨ８．５、より好ましくは、７．５である。前記ｐＨは２５℃にて測定された値である。 (4) The pH of the liquid containing the envelope-type virus-like particles can be adjusted before being injected into the column. The pH is adjusted to a range higher than the isoelectric point of the outer region of the envelope of the protein inserted in the envelope, so that the envelope-type virus-like particles can be adsorbed. For example, if the isoelectric point of the outer region of the protein envelope is 5.6, the pH of the buffer is adjusted to a range above 5.6. However, if the pH is too high, there is a possibility that the decomposition of proteins and the like in the envelope-type virus-like particles may occur, which is not preferable. Usually, the upper limit is pH 10.0, preferably pH 8.5, and more preferably 7.5. The pH is a value measured at 25 ° C.
　エンベロープに挿入されたタンパク質のエンベロープの外側領域の等電点は、計算により求めることができる。具体的には、まず、オンライン上のデータベースなどを用いてタンパク質のアミノ酸配列を調べ、その配列から、エンベロープの外側に配置する部位の配列を特定する。この配列から、市販のソフトや、オンライン上のデータベースなどを用いて等電点を計算する。例えば、Ｂ型肝炎の抗原の場合は、ＰｒｅＳ１－ＰｒｅＳ２－Ｓで構成されており、Ｓの一部が、エンベロープに挿入されている。そこで、ＰｒｅＳ１－ＰｒｅＳ２とＳの一部の配列から、ＧＥＮＥＴＹＸ（ゼネティックス社製）を用いて等電点を求めることができる。計算は、初期条件で行えばよい。例えば、初期条件設定された、電荷をもつアミノ酸のｐＫａ値は、アルギニンが１２．５、ヒスチジンが６．０、リジンが１０．５、アスパラギン酸が３．９、システインが８．３、グルタミン酸が４．３、チロシンが１０．１となる。等電点は、ＥｘＰＡＳｙなどのオンライン上のデータベースでも同様に計算することができる。 等 The isoelectric point of the outer region of the envelope of the protein inserted into the envelope can be determined by calculation. Specifically, first, the amino acid sequence of the protein is examined using an online database or the like, and the sequence of a site to be arranged outside the envelope is specified from the sequence. From this sequence, the isoelectric point is calculated using commercially available software or an online database. For example, in the case of hepatitis B antigen, it is composed of PreS1-PreS2-S, and a part of S is inserted into the envelope. Therefore, the isoelectric point can be obtained from GESYX (manufactured by Genetics) from a partial sequence of PreS1-PreS2 and S. The calculation may be performed under initial conditions. For example, the pKa values of charged amino acids set under initial conditions are 12.5 for arginine, 6.0 for histidine, 10.5 for lysine, 3.9 for aspartic acid, 8.3 for cysteine, and 8.3 for glutamic acid. 4.3, tyrosine is 10.1 The isoelectric point can be similarly calculated in an online database such as ExPASy.
　エンベロープ型ウイルス様粒子を含む液中の緩衝液の濃度は、特に制限されないが、１ｍＭ～１００ｍＭであることが好ましく、２ｍＭ～５０ｍＭであることがより好ましく、５ｍＭ～３０ｍＭであることがさらに好ましい。無機塩の濃度は、カラムに結合される範囲においては制限されないが、１ｍＭ～２００ｍＭであることが好ましく、２ｍＭ～１００ｍＭであることがより好ましく、５ｍＭ～５０ｍＭであることがさらに好ましい。
　以上を踏まえ、培養液全体としての電気伝導度は、カラムに結合される範囲においては制限されないが、１ｍＳ／ｃｍ～２０ｍＳ／ｃｍであることが好ましく、２ｍＳ／ｃｍ～１５ｍＳ／ｃｍであることがより好ましく、３ｍＳ／ｃｍ～１０ｍＳ／ｃｍであることがさらに好ましい。 The concentration of the buffer in the solution containing the envelope-type virus-like particles is not particularly limited, but is preferably 1 mM to 100 mM, more preferably 2 mM to 50 mM, and still more preferably 5 mM to 30 mM. The concentration of the inorganic salt is not limited as long as it is bound to the column, but is preferably 1 mM to 200 mM, more preferably 2 mM to 100 mM, still more preferably 5 mM to 50 mM.
Based on the above, the electric conductivity of the whole culture solution is not limited in the range of being bound to the column, but is preferably 1 mS / cm to 20 mS / cm, and more preferably 2 mS / cm to 15 mS / cm. More preferably, it is more preferably from 3 mS / cm to 10 mS / cm.
　エンベロープ型ウイルス様粒子を含む液中におけるエンベロープ型ウイルス様粒子の濃度は、０．０１ｍｇ／ｍＬ～１０ｍｇ／ｍＬであることが好ましく、０．１ｍｇ／ｍＬ～５ｍｇ／ｍＬであることがより好ましく、０．２ｍｇ／ｍＬ～３ｍｇ／ｍＬであることがさらに好ましい。エンベロープ型ウイルス様粒子の濃度を上記の範囲内とすることにより、エンベロープ型ウイルス様粒子および宿主培養細胞等由来の夾雑物をカラムに吸着させることができる。 The concentration of the enveloped virus-like particles in the liquid containing the enveloped virus-like particles is preferably 0.01 mg / mL to 10 mg / mL, more preferably 0.1 mg / mL to 5 mg / mL, More preferably, it is 0.2 mg / mL to 3 mg / mL. By setting the concentration of the envelope-type virus-like particles in the above range, the envelope-type virus-like particles and contaminants derived from host culture cells and the like can be adsorbed on the column.
　カラム内におけるエンベロープ型ウイルス様粒子を含む液の流速は、カラムにタンパク質を吸着させることができれば特に制限されないが、２ＣＶ／分～１２．５ＣＶ／分であることが好ましく、２．５ＣＶ／分～５ＣＶ／分であることがより好ましい。エンベロープ型ウイルス様粒子を含む液をカラムに通液することにより、この液に含まれるエンベロープ型ウイルス様粒子及び宿主培養細胞等由来の夾雑物を、カラムに吸着させることができる。 The flow rate of the liquid containing the envelope-type virus-like particles in the column is not particularly limited as long as the protein can be adsorbed to the column, but is preferably 2 CV / min to 12.5 CV / min, and more preferably 2.5 CV / min. More preferably, it is 5 CV / min. By passing a liquid containing the envelope-type virus-like particles through the column, the envelope-type virus-like particles and impurities derived from host culture cells and the like contained in this liquid can be adsorbed to the column.
　カラムにエンベロープ型ウイルス様粒子を吸着させる際、カラムおよびエンベロープ型ウイルス様粒子を含む液の温度は、特に制限されないが、２℃～５０℃であることが好ましく、４℃～４０℃であることがより好ましく、８℃～３０℃であることがさらに好ましい。カラムおよび液の温度を、前記の範囲にすることにより、エンベロープ型ウイルス様粒子溶液の凍結やエンベロープ型ウイルス様粒子の破壊を抑制することができる。 When the envelope-type virus-like particles are adsorbed to the column, the temperature of the column and the liquid containing the envelope-type virus-like particles is not particularly limited, but is preferably 2 ° C to 50 ° C, and preferably 4 ° C to 40 ° C. Is more preferable, and the temperature is more preferably 8 ° C to 30 ° C. By setting the temperature of the column and the liquid within the above ranges, freezing of the envelope-type virus-like particle solution and destruction of the envelope-type virus-like particles can be suppressed.
＜エンベロープ型ウイルス様粒子の溶出工程＞
　溶出工程では、エンベロープ型ウイルス様粒子が吸着したモノリス型カラムから、エンベロープ型ウイルス様粒子を選択的に溶出させる。溶出工程では、カラムの平衡化に用いた緩衝液と、無機塩を溶解させた緩衝液である無機塩緩衝液とを適当な比率で混合して混合液を用いる。この混合液を移動相として、エンベロープ型ウイルス様粒子を吸着させたモノリス型カラムに通すことにより、エンベロープ型ウイルス様粒子を選択的に溶出させる。選択的な溶出は、エンベロープに挿入されているタンパク質のエンベロープの外側領域の等電点より高いｐＨの緩衝液と無機塩緩衝液との混合液からなる移動相を通液することにより、行うことができる。 <Elution process of enveloped virus-like particles>
In the elution step, the envelope-type virus-like particles are selectively eluted from the monolith-type column to which the envelope-type virus-like particles are adsorbed. In the elution step, a buffer solution used for equilibrating the column and an inorganic salt buffer solution in which an inorganic salt is dissolved are mixed at an appropriate ratio, and a mixed solution is used. By passing this mixed solution as a mobile phase through a monolithic column to which the envelope-type virus-like particles are adsorbed, the envelope-type virus-like particles are selectively eluted. Selective elution is performed by passing a mobile phase consisting of a mixture of a buffer solution having a pH higher than the isoelectric point in the outer region of the envelope of the protein inserted in the envelope and an inorganic salt buffer solution. Can be.
　無機塩緩衝液に用いる無機塩は、エンベロープ型ウイルス様粒子を選択的に溶出させることができるものであれば制限されず、硫酸アンモニウム、塩化ナトリウムが好ましい。前記緩衝液と無機塩緩衝液との混合液における無機塩の濃度は、好ましくは５０ｍＭ～５００ｍＭ、より好ましくは７５～４００ｍＭ、さらに好ましくは１００～３００ｍＭである。無機塩を溶解する緩衝液の種類、濃度およびｐＨは、エンベロープ型ウイルス様粒子を含む液に含まれる緩衝液と同様であってもよいし、異なっていてもよい。移動相の流速は、カラムに吸着させたエンベロープ型ウイルス様粒子を選択的に溶出させることができれば、特に制限されないが、２ＣＶ／分以上であることが好ましく、２ＣＶ／分～１２．５ＣＶ／分であることがより好ましく、２．５ＣＶ／分～５ＣＶ／分であることがさらに好ましい。 無機 The inorganic salt used for the inorganic salt buffer is not limited as long as it can selectively elute envelope-type virus-like particles, and ammonium sulfate and sodium chloride are preferable. The concentration of the inorganic salt in the mixture of the buffer and the inorganic salt buffer is preferably 50 mM to 500 mM, more preferably 75 to 400 mM, and still more preferably 100 to 300 mM. The type, concentration and pH of the buffer for dissolving the inorganic salt may be the same as or different from the buffer contained in the solution containing the enveloped virus-like particles. The flow rate of the mobile phase is not particularly limited as long as the envelope-type virus-like particles adsorbed on the column can be selectively eluted, but is preferably 2 CV / min or more, preferably 2 CV / min to 12.5 CV / min. Is more preferable, and more preferably 2.5 CV / min to 5 CV / min.
　カラムに移動相を通す際、カラムおよび移動相の温度は、特に制限されないが、２℃～５０℃であることが好ましく、４℃～４０℃であることがより好ましく、８℃～３０℃であることがさらに好ましい。カラムおよび移動相の温度を、前記の範囲にすることにより、エンベロープ型ウイルス様粒子溶液の凍結、エンベロープ型ウイルス様粒子の破壊を抑制することができる。 When passing the mobile phase through the column, the temperature of the column and the mobile phase is not particularly limited, but is preferably 2 ° C to 50 ° C, more preferably 4 ° C to 40 ° C, and 8 ° C to 30 ° C. It is more preferred that there be. By setting the temperature of the column and the mobile phase within the above ranges, freezing of the envelope-type virus-like particle solution and destruction of the envelope-type virus-like particles can be suppressed.
　なお、エンベロープ型ウイルス様粒子を溶出した後、上記の無機塩緩衝液のみを移動相としてカラムに通すことにより、カラムに吸着されたままになっている宿主培養細胞等由来の夾雑物を溶出させることができる。それらを溶出させた後、カラムに平衡化に用いた緩衝液を再び通すことにより、カラムを再生することができる。 After the envelope-type virus-like particles are eluted, the above-mentioned inorganic salt buffer alone is passed through the column as a mobile phase to elute contaminants derived from host culture cells and the like which are still adsorbed to the column. be able to. After elution of them, the column can be regenerated by passing the buffer used for equilibration through the column again.
　溶出工程において、塩濃度によるグラジエントを用いれば、エンベロープ型ウイルス様粒子や宿主培養細胞等由来の夾雑物を別々に溶出させることができる。エンベロープ型ウイルス様粒子、宿主培養細胞等由来の夾雑物が吸着したカラムからの塩濃度によるグラジエントにおける、画分を分取することにより、エンベロープ型ウイルス様粒子を含有する画分を採取すれば、純度が高いエンベロープ型ウイルス様粒子を高収率かつ高濃度で、高速に得ることができる。
　このようにして得られたエンベロープ型ウイルス様粒子は、医薬品等の原料として有用である。 In the elution step, if a gradient based on the salt concentration is used, it is possible to separately elute envelope-type virus-like particles, host culture cells, and other contaminants. Envelope type virus-like particles, by collecting fractions in the gradient according to the salt concentration from the column to which contaminants derived from host culture cells are adsorbed, if the fraction containing the envelope type virus-like particles is collected, Envelope type virus-like particles with high purity can be obtained at high yield and high concentration at high speed.
The envelope-type virus-like particles thus obtained are useful as raw materials for pharmaceuticals and the like.
　本発明による精製方法は、カラムを容易に再生することができるため、工業的かつ経済的に優れている。さらに、本発明による方法は、カチオン交換基が固定されたモノリス担体を用いるため、液中のアニオン性の夾雑物の吸着を防ぐことができる。カチオン交換基が固定されたモノリス型カラムは従来の粒子状の多孔質高分子担体よりも高いｐＨにおいても、エンベロープ型ウイルス様粒子を高収率、高純度で精製できる。また、モノリス形状の特性から、エンベロープ型ウイルス様粒子のような物質においても、十分な固定化容量を有するため、精製時に必要な担体容量を減らすことができる。あるいは同担体容量の場合には、一度の処理量を増やすことができる。 精製 The purification method of the present invention is industrially and economically excellent because the column can be easily regenerated. Furthermore, the method according to the present invention uses a monolithic carrier to which a cation exchange group is immobilized, so that adsorption of anionic contaminants in the liquid can be prevented. The monolithic column to which the cation exchange group is fixed can purify enveloped virus-like particles with high yield and high purity even at a pH higher than that of a conventional particulate porous polymer carrier. Also, due to the monolithic shape characteristics, even a substance such as an enveloped virus-like particle has a sufficient immobilization capacity, so that the carrier capacity required for purification can be reduced. Alternatively, in the case of the same carrier capacity, the amount of one treatment can be increased.
　以下、実施例および比較例により本発明をさらに具体的に説明するが、本発明は以下の実施例に限定されるものではない。
　実施例および比較例において、精製の評価は以下の方法により算出した。 Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples.
In Examples and Comparative Examples, the evaluation of purification was calculated by the following method.
［総タンパク質収率］
　カラムにロードさせた総タンパク質の全量に対する、精製された総タンパク質の量の比率を総タンパク質収率として評価した。総タンパク質の量はバイオ・ラッド社製のＱｕｉｃｋ　Ｓｔａｒｔプロテインアッセイにより定量した。 [Total protein yield]
The ratio of the amount of purified total protein to the total amount of total protein loaded on the column was evaluated as total protein yield. The amount of total protein was quantified by a Bio-Rad Quick Start protein assay.
［ＨＢｓＡｇ－ＶＬＰ収率］
　カラムにロードさせたＢ型肝炎ウイルス抗原ウイルス様粒子（以下、「ＨＢｓＡｇ－ＶＬＰ」と略す。）の全量に対する、精製されたＨＢｓＡｇ－ＶＬＰの量の比率をＨＢｓＡｇ－ＶＬＰ収率として評価した。ＨＢｓＡｇ－ＶＬＰの量はビークル社製のＨＢ　Ｓ抗原定量キットにより定量した。 [HBsAg-VLP yield]
The ratio of the amount of purified HBsAg-VLP to the total amount of hepatitis B virus antigen virus-like particles (hereinafter abbreviated as “HBsAg-VLP”) loaded on the column was evaluated as the HBsAg-VLP yield. The amount of HBsAg-VLP was determined using an HBS antigen quantification kit manufactured by Vehicle.
［Ｐｕｒｉｆｉｃａｔｉｏｎ　ｆａｃｔｏｒ（ＰＦ）］
　カラムで精製された後のＨＢｓＡｇ－ＶＬＰの比活性に対する、精製前のＨＢｓＡｇ－ＶＬＰの比活性の比をＰｕｒｉｆｉｃａｔｉｏｎ　ｆａｃｔｏｒ（ＰＦ）として評価した。比活性は、上記の総タンパク質量に対するＨＢｓＡｇ－ＶＬＰ量の比である。 [Purification factor (PF)]
The ratio of the specific activity of HBsAg-VLP before purification to the specific activity of HBsAg-VLP after purification on the column was evaluated as Purification factor (PF). The specific activity is a ratio of the amount of HBsAg-VLP to the amount of the total protein.
［実施例１］
　非特許文献２に従って、酵母細胞にエンベロープ型ウイルス様粒子を発現させ、培養上清を調製した。具体的には、組換え酵母Ｓ．ｃｅｒｅｖｉｓｉａｅを７２時間培養した。得られた培養液を遠心し、沈殿した酵母を７．５Ｍ尿素、０．１ＭＴｒｉｓ、５０ｍＭリン酸水素二ナトリウム、１５ｍＭ　ＥＤＴＡ、０．１％（ｖ／ｖ）Ｔｗｅｅｎ８０、４ｍＭ　ＰＭＳＦ混合液（ｐＨ７．４）で懸濁し、０．５ｍｍのガラスビーズで破砕した。破砕抽出液を沈殿し、１ｍＭ　ＥＤＴＡを含むＰＢＳで透析した。透析液を７０℃で２０分インキュベートし、すぐに氷上で静置した。不溶分を遠心で除去し、得られた上清を０．４５μｍのフィルターで濾過した。
　挿入されているタンパク質のエンベロープの外側領域の等電点の理論値は５．６だった。なお等電点の理論値の計算はゼネティックス社の解析ソフトウェアＧＥＮＥＴＹＸ　Ｖｅｒ．１０で計算を行った。 [Example 1]
According to Non-Patent Document 2, envelope-type virus-like particles were expressed in yeast cells, and a culture supernatant was prepared. Specifically, recombinant yeast S. cerevisiae was cultured for 72 hours. The obtained culture solution was centrifuged, and the precipitated yeast was mixed with 7.5 M urea, 0.1 M Tris, 50 mM disodium hydrogen phosphate, 15 mM EDTA, 0.1% (v / v) Tween 80, and 4 mM PMSF mixed solution (pH 7.0). The suspension was suspended in 4) and crushed with 0.5 mm glass beads. The crushed extract was precipitated and dialyzed against PBS containing 1 mM EDTA. The dialysate was incubated at 70 ° C. for 20 minutes and immediately placed on ice. Insoluble matter was removed by centrifugation, and the obtained supernatant was filtered with a 0.45 μm filter.
The theoretical value of the isoelectric point of the region outside the envelope of the inserted protein was 5.6. The calculation of the theoretical value of the isoelectric point is performed by the analysis software GENETYX Ver. The calculation was performed at 10.
（１）カラムの平衡化
　装置はＧＥヘルスケア社製のＡＫＴＡｐｕｒｉｆｉｅｒ１００を用いた。カラムとして、ＢＩＡ　ｓｅｐａｒａｔｉｏｎｓ社製のＣＩＭｍｕｌｔｕｓ（登録商標）　ＳＯ３－１　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎ（厚さ：６ｍｍ、外径：１８．６ｍｍ、内径６．７ｍｍ、長さ：４．２ｍｍ、空隙率：約６０ｖ／ｖ％以上、ＣＶ：　１　ｍＬ）を用い、このカラムに、２０ｍＭ、ｐＨ８．５のトリス緩衝液を５ＣＶ以上通して、このカラムを平衡化した。なお、ＣＩＭｍｕｌｔｕｓ（登録商標）　ＳＯ３－１　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎの担体はグリシジルメタクリレートとエチレングリコールジメタクリレートの共重合体の変性物であって、一部のグリシジルメタクリレートにスルホン酸基が保持されているモノリス型カラムである。 (1) Equilibration of column As an apparatus, AKTApurifier100 manufactured by GE Healthcare was used. As a column, CIMmultus (registered trademark) SO3-1 Advanced Composite column (thickness: 6 mm, outer diameter: 18.6 mm, inner diameter 6.7 mm, length: 4.2 mm, porosity: about 60 v /) manufactured by BIA separations v% or more, CV: 1 mL), and the column was equilibrated by passing at least 5 CV of 20 mM, pH 8.5 Tris buffer through the column. The carrier of CIMmultus (registered trademark) SO3-1 Advanced Composite column is a modified product of a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, and is a monolithic type in which a sulfonic acid group is retained in some glycidyl methacrylates. It is a column.
（２）エンベロープ型ウイルス様粒子の吸着
　培養上清のｐＨが８．５になるように、水酸化ナトリウム水溶液で調整し、試料溶液とした。試料溶液の電気伝導度は１０ｍＳ／ｃｍ程度であった。この試料溶液２０ｍＬを、（１）で平衡化したカラムに注入し、試料溶液中に含まれるエンベロープ型ウイルス様粒子をカラムに吸着させた。試料溶液の流速は５ＣＶ／分とした。 (2) Adsorption of enveloped virus-like particles A sample solution was prepared by adjusting the pH of the culture supernatant to 8.5 with an aqueous sodium hydroxide solution. The electric conductivity of the sample solution was about 10 mS / cm. 20 mL of this sample solution was injected into the column equilibrated in (1), and the envelope-type virus-like particles contained in the sample solution were adsorbed on the column. The flow rate of the sample solution was 5 CV / min.
（３）エンベロープ型ウイルス様粒子の溶出
　２０ｍＭ、ｐＨ８．５のトリス緩衝液と、２ＭのＮａＣｌ溶液とを、ＮａＣｌ濃度が１Ｍになるように混合して、混合液を調製した。なお、２ＭのＮａＣｌ溶液は、２０ｍＭ、ｐＨ８．５のトリス緩衝液にＮａＣｌを溶解させたものである。
　エンベロープ型ウイルス様粒子を吸着させたカラムに、この混合液を５ｍｌ通すことにより、エンベロープ型ウイルス様粒子を溶出させた画分を回収した。混合液の流速は５ＣＶ／分とした。 (3) Elution of envelope-type virus-like particles 20 mM, pH 8.5 Tris buffer and a 2 M NaCl solution were mixed so that the NaCl concentration became 1 M to prepare a mixed solution. The 2M NaCl solution is obtained by dissolving NaCl in a 20 mM, pH 8.5 Tris buffer.
By passing 5 ml of this mixture through a column to which the envelope-type virus-like particles were adsorbed, a fraction eluted from the envelope-type virus-like particles was collected. The flow rate of the mixture was 5 CV / min.
（４）宿主培養細胞等由来不純物の溶出
　その後、２ＭのＮａＣｌ溶液を、５ｍｌ通液することにより、宿主培養細胞等由来不純物を溶出させ、カラムを洗浄した。溶液の流速は５ＣＶ／分とした。
　なお、実施例１において、カラム、タンパク質溶液および移動相の温度は２５℃とし、カラムの圧力は最大許容圧１．８ＭＰａを超えない範囲とした。 (4) Elution of impurities derived from host culture cells After that, 5 ml of a 2M NaCl solution was passed through to elute impurities derived from host culture cells and the like, and the column was washed. The solution flow rate was 5 CV / min.
In Example 1, the temperature of the column, the protein solution and the mobile phase was 25 ° C., and the pressure of the column was within a range not exceeding the maximum allowable pressure of 1.8 MPa.
［実施例２］
　培養上清のｐＨを７．５に調整し、緩衝液を２０ｍＭ、ｐＨ７．５のリン酸緩衝液としたこと以外は実施例１と同様に実施した。回収画分の電子顕微鏡観察した結果を図１に示す。 [Example 2]
The procedure was performed in the same manner as in Example 1 except that the pH of the culture supernatant was adjusted to 7.5 and the buffer was changed to a phosphate buffer of 20 mM and pH 7.5. FIG. 1 shows the results of electron microscopic observation of the collected fraction.
［比較例１］
　カラムとして、ＧＥヘルスケア社製のＨｉＴｒａｐ（登録商標）　ＳＰ　ＦＦを使用し、試料溶液および緩衝液の通液速度を１ＣＶ／ｍｉｎとしたこと以外は実施例１と同様に実施した。ＨｉＴｒａｐ　ＳＰ　ＦＦは、架橋されたアガロースで構成される、粒子サイズが４５－１６５μｍ粒子状で、スルホン酸基が修飾されたゲルを充填したカラムである。単体容量は１ｍＬである。 [Comparative Example 1]
The procedure was performed in the same manner as in Example 1 except that HiTrap (registered trademark) SP FF manufactured by GE Healthcare was used as the column, and the flow rate of the sample solution and the buffer solution was 1 CV / min. HiTrap SP FF is a column composed of cross-linked agarose and packed with a gel having a particle size of 45-165 μm and modified with sulfonic acid groups. The unit volume is 1 mL.
［比較例２］
　カラムとして、ＧＥヘルスケア社製のＨｉＴｒａｐ　（登録商標）　ＳＰ　ＦＦを使用し、試料溶液および緩衝液の通液速度を１ＣＶ／ｍｉｎとしたこと以外は実施例２と同様に実施した。 [Comparative Example 2]
The operation was performed in the same manner as in Example 2 except that HiTrap (registered trademark) SP FF manufactured by GE Healthcare was used as the column, and the flow rate of the sample solution and the buffer solution was 1 CV / min.
［比較例３］
　カラムとして、ＢＩＡ　ｓｅｐａｒａｔｉｏｎｓ社製のＣＩＭｍｕｌｔｕｓ（登録商標）　ＱＡ－１　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎを使用したこと以外は実施例１と同様に実施した。ＣＩＭｍｕｌｔｕｓ　ＱＡ－１　Ａｄｖａｎｃｅｄ　Ｃｏｍｐｏｓｉｔｅ　ｃｏｌｕｍｎは、ＣＩＭｍｕｌｔｕｓ　ＳＯ３－１　Ａｄｖａｎｃｅｄ　ｃｏｌｕｍｎと同様の基材に、４級アンモニウム基が修飾された、モノリス型カラムである。単体容量は１ｍＬである。 [Comparative Example 3]
The procedure was performed in the same manner as in Example 1 except that CIMmultus (registered trademark) QA-1 Advanced Composite column manufactured by BIA separations was used as a column. The CIMmultus QA-1 Advanced Composite column is a monolithic column in which a quaternary ammonium group is modified on the same base material as that of the CIMmultus SO3-1 Advanced column. The unit volume is 1 mL.
［比較例４］
　カラムとしてＪＮＣ社製のＣｅｌｌｕｆｉｎｅ（登録商標）　ｓｕｌｆａｔｅ（５ｍＬ）を使用し、培養上清のｐＨを７．５に調整し、緩衝液を１０ｍＭ、ｐＨ７．５のリン酸緩衝液とし、さらに試料溶液および緩衝液の通液速度を０．５ＣＶ／ｍｉｎとしたこと以外は実施例１と同様に実施した。Ｃｅｌｌｕｆｉｎｅ　ｓｕｌｆａｔｅは、セルロースで構成される、粒子サイズは４０－１３０μｍである、硫酸基が修飾された、粒子状のゲルが充填されたカラムである。単体容量は５ｍＬである。 [Comparative Example 4]
JNC's Cellufine (registered trademark) sulfate (5 mL) was used as the column, the pH of the culture supernatant was adjusted to 7.5, the buffer was changed to 10 mM, pH 7.5 phosphate buffer, and the sample solution was further adjusted. The procedure was performed in the same manner as in Example 1 except that the flow rate of the buffer was 0.5 CV / min. Cellufine sulfate is a column composed of cellulose, having a particle size of 40-130 μm, and modified with a sulfate group and packed with a particulate gel. The unit volume is 5 mL.
　実施例１～３および比較例１～５について、総タンパク質収率、ＨＢｓＡｇ－ＶＬＰ収率及びＰＦを算出し、結果を表１に示す。表１の結果から明らかなように、本発明による方法により、粒子状の充填剤を用いた方法や、従来のカチオン交換基を有するモノリス型カラムを用いた方法よりも培養液中に含まれるＨＢｓＡｇ－ＶＬＰを高収率かつ高活性で精製することができた。 に つ い て For Examples 1 to 3 and Comparative Examples 1 to 5, the total protein yield, HBsAg-VLP yield, and PF were calculated, and the results are shown in Table 1. As is clear from the results in Table 1, the HBsAg contained in the culture solution by the method according to the present invention is larger than the method using a particulate filler or the conventional method using a monolithic column having a cation exchange group. -VLP could be purified with high yield and high activity.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001

Claims (6)

  1. 　カチオン交換基が固定されたモノリス型カラムに、エンベロープ型ウイルス様粒子を含む液を通液してエンベロープ型ウイルス様粒子を前記カラムに吸着させる工程、および前記カラムに吸着したエンベロープ型ウイルス様粒子を選択的に溶出させる工程を含み、
    　前記のエンベロープ型ウイルス様粒子を含む液のｐＨが、エンベロープに挿入されているタンパク質のエンベロープの外側領域の等電点より高く、ｐＨ１０．０以下である、エンベロープ型ウイルス様粒子の精製方法。     A step of allowing the liquid containing the envelope-type virus-like particles to pass through the monolith-type column to which the cation exchange group is fixed to adsorb the envelope-type virus-like particles to the column, and the envelope-type virus-like particles adsorbed to the column. Including a step of selectively eluting,
    A method for purifying enveloped virus-like particles, wherein the pH of the solution containing the enveloped virus-like particles is higher than the isoelectric point of the outer region of the envelope of the protein inserted into the envelope and is pH 10.0 or less.
  2. 　前記モノリス型カラムが、グリシジルメタクリレート由来のモノマー単位とエチレングリコールジメタクリレート由来のモノマー単位を含む共重合体にカチオン交換基が固定されたモノリス担体から構成されるモノリス型カラムである、請求項１に記載の精製方法。 The monolithic column is a monolithic column composed of a monolithic carrier having a cation exchange group fixed to a copolymer containing a monomer unit derived from glycidyl methacrylate and a monomer unit derived from ethylene glycol dimethacrylate. Purification method as described.
  3. 　前記カチオン交換基がスルホン酸基である、請求項１あるいは２のいずれかに記載の精製方法。 精製 The purification method according to claim 1, wherein the cation exchange group is a sulfonic acid group.
  4. 　前記エンベロープ型ウイルス様粒子に挿入されているタンパク質がＢ型肝炎ウイルス抗原（ＨＢｓＡｇ）を含む、請求項１～３のいずれか１項に記載の精製方法。 (4) The purification method according to any one of (1) to (3), wherein the protein inserted into the envelope-type virus-like particle includes a hepatitis B virus antigen (HBsAg).
  5. 　前記エンベロープ型ウイルス様粒子が酵母細胞により調製されたものである、請求項１～４のいずれか１項に記載の精製方法。 (5) The purification method according to any one of (1) to (4), wherein the envelope-type virus-like particles are prepared by yeast cells.
  6. 　前記モノリス型カラムの通液方向の厚さが１～１００ｍｍである、請求項１～５のいずれか１項に記載の精製方法。 (6) The purification method according to any one of (1) to (5), wherein the monolith-type column has a thickness in a flow direction of 1 to 100 mm.
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