JP2021101671A - Structure and method for manufacturing the same, adsorbent using the same, and method for purifying biological particles - Google Patents

Abstract

To provide an inexpensive structure that has low adsorption of nonspecific protein and high adsorption of biological particles, a method for manufacturing thereof, adsorbent using the structure, and a method for purifying biological particles.SOLUTION: There is provided a structure having: a spacer; a water-insoluble base material connected to one end of the spacer; and an ion-exchange ligand connected to the other end of the spacer, in which the ion-exchange group density of the structure is less than 50 μmol/g.SELECTED DRAWING: Figure 1

Description

The present invention relates to a structure, a method for producing the same, an adsorbent using the structure, and a method for purifying biological particles.

Biological particles such as viruses, virus-like particles, or viral vectors are very important molecules in the medical field such as gene therapy, vaccines, laboratory diagnosis, and blood purification.
In recent years, these medical fields have attracted particular attention, and there is a strong demand for a method for efficiently recovering biological particles from biological samples such as cell culture medium and blood.
As a conventional virus purification method, a method of purification by various chromatographys after clarification by membrane separation and removal of impurities is known (Patent Document 1, Non-Patent Documents 1 and 2, etc.).
However, in the conventional chromatography using a bead carrier or a membrane carrier, the bead carrier and the membrane carrier are expensive and difficult to prepare, and their pore diameters are small and the amount of protein adsorbed is large. There was a problem in efficient recovery.

Therefore, inexpensive, low adsorption of non-specific proteins, high adsorption of bioparticles, structures and methods for producing them, adsorbents using them, and methods for purifying bioparticles are completely unknown. Is strongly required to be provided.

International Publication No. 2018/011599 Pamphlet

Journal of Virtual Methods 2007 140: 183-192 Molecular Therapy Methods & Clinical Development 2018 9: 33-46

An object of the present invention is to solve the above-mentioned problems in the past and to achieve the following object. That is, the present invention provides a structure and a method for producing the same, an adsorbent using the same, and a method for purifying biological particles, which are inexpensive, have low adsorption of non-specific proteins, and have high adsorption of biological particles. The purpose.

As a result of diligent research to achieve the above object, the present inventors have found a spacer, a water-insoluble base material connected to one end of the spacer, and an ion exchange ligand connected to the other end of the spacer. Provided by a structure having an ion exchange group density of less than 50 μmol / g, which is inexpensive, has low nonspecific protein adsorption, and has high bioparticle adsorption. A method for producing a structure is adopted, which comprises a step of connecting a water-insoluble base material to one end of the spacer and connecting an ion exchange ligand to the other end so that the ion exchange group density of the structure is less than 50 μmol / g. By doing so, it was found that a method for producing a structure can be provided, which is inexpensive, has low adsorption of non-specific proteins, and has high adsorption of biological particles.

The present invention is based on the above-mentioned findings by the present inventors, and the means for solving the above-mentioned problems are as follows. That is,
<1> A structure having a spacer, a water-insoluble base material connected to one end of the spacer, and an ion exchange ligand connected to the other end of the spacer, wherein the structure has an ion exchange group density. Is a structure characterized by being less than 50 μmol / g.
<2> A method for manufacturing a structure, in which a water-insoluble substrate is connected to one end of a spacer and an ion exchange ligand is connected to the other end so that the ion exchange group density of the structure is less than 50 μmol / g. It is a method for manufacturing a structure, which comprises a step.
<3> An adsorbent characterized by having the structure according to the above <1>.
<4> A method for purifying biological particles, which comprises using the structure according to <1> or the adsorbent according to <3>.

According to the present invention, the above-mentioned problems in the prior art can be solved, the above-mentioned object can be achieved, the structure is inexpensive, the adsorption of non-specific proteins is low, the adsorption of biological particles is high, the structure and the method for producing the same, and the like. A method for purifying an adsorbent and a biological particle using the above can be provided.

FIG. 1 is a diagram showing a chromatogram of AAV adsorption / desorption by the carrier 4 of Example 4.

(Structure)
The structure has a spacer, a water-insoluble substrate connected to one end of the spacer, an ion exchange ligand connected to the other end of the spacer, and can further have other elements.

The ion exchange group density of the structure is not particularly limited as long as it is less than 50 μmol / g and can be appropriately selected depending on the intended purpose, but is preferably 45 μmol / g or less, more preferably 40 μmol / g or less. , 35 μmol / g or less is more preferable, and 30 μmol / g or less is particularly preferable.

The ion exchange group density can be determined by measuring the ion exchange capacity.
For the ion exchange capacity, the carrier is transferred to a glass filter, washed with pure water three times, then a 0.1 mol / L sodium hydroxide aqueous solution is added, and the carrier is immersed for 3 minutes to obtain 0.01 mol / L sodium hydroxide. After washing with an aqueous solution, wash with pure water until the washing solution becomes neutral, transfer the carrier to a beaker using pure water, add 1 mol / L sodium hydroxide aqueous solution and stir, and collect only the solution in another beaker. This operation is repeated several times, a phenol phthalein solution is added to the recovered solution, and the measurement is carried out by titrating with a 0.01 mol / L aqueous hydrochloric solution solution.

-Spacer-
The spacer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include those containing a polymer, a monomer, a dimer, a trimer, a trimmer, and a tetramer. Among these, those containing a polymer are preferable. The polymer may be a copolymer.

The polymer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include those containing a hydrophilic polymer and a hydrophobic polymer. Among these, those containing a hydrophilic polymer are preferable from the viewpoints of being able to be handled with an aqueous solvent and suppressing the non-specific hydrophobic action between the protein and the spacer.

The hydrophilic polymer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include those containing polyamines and polysaccharides.

The polyamine is not particularly limited and may be appropriately selected depending on the intended purpose. For example, polyethyleneimine, polyallylamine, polyvinylamine, polylysine, ethylenediamine, 1,2-propanediamine, 1,6-hexamethylenediamine. , Piperazine, 2,5-dimethylpiperazin, isophoronediamine, 4,4'-dicyclohexylmethanediamine, 1,4-cyclohexanediamine and other diamines; polyamines such as diethylenetriamine, dipropylenetriamine, triethylenetetramine; hydrazine, N Hydrazines such as N'-dimethylhydrazine and 1,6-hexamethylenebishydrazine; dihydrazines such as dihydrazid succinate, dihydrazide adipate, dihydrazide glutarate, dihydrazide sebacic acid, and dihydrazide isophthalate can be mentioned. These may be used alone or in combination of two or more. Among these, those containing polyethyleneimine or polyallylamine are preferable because molecules having different molecular weights can be easily obtained.

The polysaccharide is not particularly limited and may be appropriately selected depending on the intended purpose. For example, chitosan, chitin, cellulose, agarose, carrageenan, heparin, hyaluronic acid, pectin, xyloglucan, glucomannan, starch and glycogen. And so on.
These may be used alone or in combination of two or more. Among these, chitosan is preferable because it contains an amino group.

The lower limit of the molar mass of the polymer is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of adsorptivity of biological particles, 500 g / mol or more is preferable, and 5000 g / mol or more is more preferable. Preferably, 10,000 g / mol or more is more preferable, and 60,000 g / mol or more is particularly preferable.
The upper limit of the molar mass of the polymer is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of adsorptivity of biological particles, 2,000,000 g / mol or less is preferable, and 1, It is more preferably ,000,000 g / mol or less, further preferably 500,000 g / mol or less, and particularly preferably 200,000 g / mol or less.

The polymer may be a polymer having a branched chain or a linear polymer, but a polymer having a branched chain is preferable from the viewpoint of adsorptivity of biological particles.

-Water-insoluble substrate-

The lower limit of the thickness of the water-insoluble substrate is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of production stability, 0.08 mm or more is preferable, and 0.10 mm or more is preferable. More preferably, 0.12 mm or more is further preferable.
The upper limit of the thickness of the water-insoluble substrate is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of uniformity of basis weight, 0.50 mm or less is preferable, and 0.40 mm or less is preferable. More preferably, 0.30 mm or less is further preferable.

The lower limit of the basis weight of the water-insoluble substrate is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of production stability, 5 g / m ² or more is preferable and 10 g / m ^{2 is preferable.} The above is more preferable.
The upper limit of the basis weight of the water-insoluble base material is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of the uniformity of the water-insoluble base material, 100 g / m ² or less is preferable, and 90 g. / M ² or less is more preferable, 80 g / m ² or less is further preferable, and 70 g / m ² or less is particularly preferable.

The lower limit of the bulk density of the water-insoluble base material is not particularly limited and may be appropriately selected depending on the intended purpose. However, the structure of the water-insoluble base material does not change much after being charged into the device. 50 kg / m ³ or more is preferable, 60 kg / m ³ or more is more preferable, and 70 kg / m ³ or more is further preferable.
The upper limit of the bulk density of the water-insoluble substrate is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of liquid flow performance, 400 kg / m ³ or less is preferable, and 350 kg / m ^{3 is preferable.} The following is more preferable, and 300 kg / m ³ or less is further preferable.
The bulk density refers to a value obtained by measuring the weight per ^{1 m 3 of the} water-insoluble base material.

The shape of the water-insoluble base material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a circle, a quadrangle, a triangle, and a bale shape.

The surface of the water-insoluble substrate may be modified by graft polymerization, polymer coating, chemical treatment with alkali, acid or the like, plasma treatment or the like.
The graft polymerization is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a graft polymerization method in which electron beam irradiation is performed.

In the graft polymerization method in which the electron beam irradiation is performed, a water-insoluble base material is irradiated with an electron beam in advance to generate radicals, and then a radical-polymerizable compound is added to the water-insoluble base material in which the generated species are generated. Subsequent post-polymerization promotes the polymerization of the graft-polymerizable compound, the so-called pre-irradiation method, and after imparting a radical-polymerizable compound to a water-insoluble substrate, it is irradiated with an electron beam to generate radicals, and then radicals are generated. There is a so-called simultaneous irradiation method in which the polymerization of the graft polymerization compound is promoted by post-polymerization. In the present invention, both the pre-irradiation method and the simultaneous irradiation method can be adopted.

In either of the pre-irradiation method and the simultaneous irradiation method, after the radically polymerizable compound is applied to the water-insoluble base material, a film is laminated on the surface of the water-insoluble base material until the post-polymerization is completed. It is preferable to keep it. As a result, the volatilization of the radically polymerizable compound can be prevented, the graft polymerization is uniformly started, and the deactivation of radicals due to oxygen in the air is suppressed by blocking the air. Further, in the case of the simultaneous irradiation method, the surface of the water-insoluble base material is sealed with a film even during electron beam irradiation, and the water-insoluble base material and the radically polymerizable compound are blocked from oxygen in the air. Oxidation of the water-insoluble base material by oxygen is less likely to occur.

As the film, a polymer film having a thickness of 0.01 to 0.20 mm and having an appropriate thickness is used according to the penetrating power of the electron beam to be used.
The material of the film is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include polyester-based films such as polyethylene terephthalate and polyolefin-based films. Among these, polyethylene terephthalate is preferable. In particular, in the case of the simultaneous irradiation method, a polyethylene terephthalate film having low efficiency of polymer radical generation by electron beam irradiation and low oxygen permeability is preferable.

In the pre-irradiation method, first, a radical (polymer radical or the like) that induces a polymerization reaction is generated by irradiating a water-insoluble substrate with an electron beam. As for the atmospheric temperature at the time of electron beam irradiation, the radical generation efficiency increases when the temperature is low, but it may be a normal room temperature.

In the case of the pre-irradiation method, the irradiation conditions of the electron beam are not particularly limited and may be appropriately selected depending on the intended purpose. However, preferably, the acceleration voltage is set to 300 ppm or less in the irradiation atmosphere. It is appropriately determined in the range of 100 to 2000 kilovolts (hereinafter abbreviated as “kV”), more preferably 120 to 300 kV, and a current of 1 to 100 mA, depending on the thickness of the water-insoluble substrate, the target graft ratio, and the like.

The irradiation dose of the electron beam may be appropriately determined in consideration of the target graft rate and the deterioration of the physical properties of the water-insoluble substrate due to irradiation, and is usually about 10 to 300 kilogray (hereinafter abbreviated as "kGy"). It is suitable, preferably 10 to 200 kGy. If the irradiation dose is less than 10 kGy, the radicals required for a sufficient amount of graft polymerization do not occur, and if it exceeds 300 kGy, the physical properties deteriorate due to the breakage of the main chain even when the water-insoluble substrate is made of a radiation-resistant polymer material. Is not preferable because

In the case of the pre-irradiation method, the irradiation atmosphere is preferably an inert gas atmosphere such as nitrogen gas, but an air atmosphere may also be used. However, in an air atmosphere, oxygen in the air may oxidize the water-insoluble substrate.

Next, a radically polymerizable compound is added to the water-insoluble base material after the electron beam irradiation. For example, a water-insoluble group is immersed in a tank of a radically polymerizable compound solution from which dissolved oxygen has been removed by aerating nitrogen gas, or is allowed to stay for a predetermined time by being immersed and passed through a tank of the radically polymerizable compound solution. Sufficiently impart a radically polymerizable compound to the material.

The term "immersion" in the present invention means that the water-insoluble substrate comes into contact with the radically polymerizable compound solution. Therefore, various coating methods can be used as a method for imparting the radically polymerizable compound to the water-insoluble substrate. Among them, an impregnated coat, a comma direct coat, a comma reverse coat, a kiss coat, a gravure coat and the like can be efficiently coated, and are preferable.

The water-insoluble base material to which the radically polymerizable compound is added is taken out from the solution tank. At that time, it is preferable to laminate the film on the surface of the water-insoluble base material. For example, when the water-insoluble base material is in the form of a sheet or fibrous, the water-insoluble base material to which the radically polymerizable compound solution is applied is sandwiched between the two films and brought into close contact with each other. By adhering the film to the water-insoluble base material to which the radical-polymerizable compound solution is applied, the application rate of the radical-polymerizable compound solution can be controlled to be constant, and the radical-polymerizable compound is uniformly applied to the water-insoluble substrate. it can. Further, the graft reaction is further promoted by reacting the water-insoluble base material with the radically polymerizable compound in a space sealed by laminating the film.
The term "laminate" in the present invention means that the water-insoluble base material is brought into contact with the film.

The water-insoluble substrate taken out from the solution tank is allowed to stay in the polymerization tank after a predetermined temperature for a predetermined time to promote the graft polymerization of the radically polymerizable compound (post-polymerization). The post-polymerization temperature at this time is 0 ° C. to 130 ° C., more preferably 40 ° C. to 70 ° C. This promotes the graft polymerization reaction between the water-insoluble substrate and the radically polymerizable compound. Then, by washing and drying, a grafted base material can be obtained. The post-polymerization atmosphere is preferably an inert gas atmosphere such as nitrogen gas, but an air atmosphere may be used when the post-polymerization is performed with the film laminated.

Further, in the case of the simultaneous irradiation method, water is allowed to stay for a predetermined time by immersing it in a tank of a radically polymerizable compound solution from which dissolved oxygen has been removed by aerating nitrogen gas, or by letting the tank pass through the bath. The radically polymerizable compound is sufficiently imparted to the insoluble substrate. Then, it is taken out from the solution tank and irradiated with an electron beam. When taken out from the solution tank, it is preferable to laminate a film on the surface of the water-insoluble base material and irradiate an electron beam in this state.

In the case of the simultaneous irradiation method, the acceleration voltage may be appropriately determined according to the type of polymer material, the total thickness of the water-insoluble substrate to which the radically polymerizable compound solution is applied, the laminated film, and the target graft ratio. Usually, an acceleration voltage of about 100 to 2000 kV is suitable. Further, the irradiation dose of the electron beam may be the same as in the case of the pretreatment method. The atmosphere during electron beam irradiation is preferably an inert gas atmosphere such as nitrogen or helium, but when a film is laminated on the surface of a water-insoluble substrate, the irradiation atmosphere does not affect graft polymerization, which is economical. In consideration, air irradiation is appropriate.

The water-insoluble base material irradiated with the electron beam undergoes post-polymerization of the radically polymerizable compound in the same manner as in the case of the pre-irradiation method. Then, by washing and drying, a grafted base material can be obtained. Even in the simultaneous irradiation method, the post-polymerization atmosphere is preferably an inert gas atmosphere such as nitrogen gas, but an air atmosphere may be used when the post-polymerization is performed in the state where the film is laminated.

The radically polymerizable compound used in the present invention is a compound that forms a bond with a polymer radical generated on a water-insoluble substrate by electron beam irradiation. Specifically, unsaturated compounds having acidic groups such as acrylic acid, methacrylic acid, itaconic acid, methacrylic sulfonic acid, and styrene sulfonic acid, unsaturated carboxylic acid amides such as these esters, acrylamide, and methacrylic amide, and glycidyl at the terminal. Examples thereof include unsaturated compounds having groups and hydroxyl groups, unsaturated organic phosphoric acid esters such as vinyl phosphonates, basic methacrylic acid esters such as tetraammonium salt and tertiary ammonium salt, fluoroacrylates, and acrylonitrile. It is not limited to these. These can be used alone or in combination of two or more. By using two or more kinds of radically polymerizable compounds, a composite grafted base material having a graft chain made of a copolymer of at least two kinds of radically polymerizable compounds can be obtained.

Among these radically polymerizable compounds, in the present invention, it is preferable to use an acrylic monomer from the viewpoint of graft ratio. Further, from the viewpoint of reactivity with a ligand having an amino group, a hydroxyl group, a thiol group or the like, an acrylic monomer having a carboxy group or an epoxy group at the molecular terminal is preferable, and acrylic acid, methacrylic acid and glycidyl methacrylate are more preferable. It is at least one species selected from the group consisting of (hereinafter abbreviated as "GMA").

The above-mentioned radically polymerizable compound may be an organic solvent such as water or a lower alcohol, or a diluted solution using a mixed solution thereof as a solvent. The concentration of the radically polymerizable compound in this diluted solution varies depending on the desired graft ratio, but can be prepared in an amount of 1 to 70% by volume. When a radically polymerizable compound that easily forms a homopolymer is used, the formation of the homopolymer may be suppressed by adding a metal salt of copper or iron to the diluted solution of the radically polymerizable compound.

The lower limit of the concentration of the radically polymerizable compound in the solution is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1% by weight or more, more preferably 2.5% by weight or more. 5% by weight or more is more preferable, and 10% by weight or more is particularly preferable.
The upper limit of the concentration of the radically polymerizable compound in the solution is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 70% by weight or less, more preferably 60% by weight or less, and 50% by weight or less. It is more preferably 0% by weight or less, and particularly preferably 40% by weight or less.

Further, when the radically polymerizable compound solution contains a solvent, the graft ratio is improved. In this case, the concentration of the emulsifier in the solvent is preferably adjusted to be in the range of 0.1 to 5% by weight.
The emulsifier is not particularly limited and may be appropriately selected depending on the intended purpose. For example, polysorbate is preferable, and polysorbate 20, 60, 65, 80 and the like are mentioned as the polysorbate, and among these, hydrophilic. Polysorbate 20 having a high property is more preferable.

It is desirable to remove dissolved oxygen by blowing an inert gas such as nitrogen gas into the radically polymerizable compound solution in advance.

The graft ratio of the graft polymerization reaction is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50% or more.
In the present invention, the "graft ratio" is a value calculated as follows from the dry weight of the water-insoluble base material (W1) before the graft reaction and the dry weight of the grafted base material (W2) after the graft reaction.
Graft rate = [(W2-W1) / W1] x 100 (%)

The water-insoluble substrate is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include water-insoluble fibers, water-insoluble resins, and water-insoluble films. Among these, water-insoluble fibers are preferable because they have good liquid-passing characteristics in which clogging is unlikely to occur.

The material of the water-insoluble fiber is not particularly limited and may be appropriately selected depending on the intended purpose. For example, polypropylene, polypropylene anhydride, modified polypropylene, cellulose, regenerated cellulose, cellulose acetate, cellulose diacetate, cellulose Triacetate, ethyl cellulose, cellulose acetate, polyethylene terephthalate (PET), polybutylene terephthalate (PBT), acrylic resin, polyethylene, polycarbonate, polyester, polyolefin, polyacrylonitrile, polyamide, polystyrene, brominated polystyrene, polyalkyl (meth) acrylate, Polyvinyl chloride, polychloroprene, polyurethane, polyvinyl alcohol, polyvinyl acetate, polysulfone, polyether sulfone, polybutadiene, butadiene-acrylonitrile copolymer, styrene-butadiene copolymer, ethylene-vinyl alcohol copolymer, aramid, glass, nylon , Polymer, etc. These may be used alone or in combination of two or more. Among these, polypropylene or cellulose is preferable, and polypropylene is more preferable, from the viewpoint of good reactivity of electron beam graft polymerization.

The lower limit of the average fiber diameter of the water-insoluble fiber is not particularly limited and may be appropriately selected depending on the intended purpose, but is 0.3 μm or more from the viewpoint of having good tensile strength or productivity. Is preferable, 0.4 μm or more is more preferable, and 0.5 μm or more is further preferable.
The upper limit of the average fiber diameter of the water-insoluble fiber is not particularly limited and may be appropriately selected depending on the intended purpose. However, from the viewpoint of high purification performance, 15 μm or less is preferable, 10 μm or less is more preferable, and 5 μm is more preferable. The following is more preferable, and 3 μm or less is particularly preferable.
Is. If the average fiber diameter exceeds 15 μm, the purification performance is low, which is not preferable.

The water-insoluble fiber is not particularly limited and may be appropriately selected depending on the intended purpose. It may be a non-woven fabric, a woven fabric or a knitted fabric, but from the viewpoint of simplification of the manufacturing process, the non-woven fabric Is preferable.

The method for producing the non-woven fabric is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a wet method, a dry method, a melt blow method, an electrospinning method, a flash spinning method, a papermaking method, a spunbond method, etc. Examples include a thermal bond method, a chemical bond method, a needle punch method, a spunlace method (water flow entanglement method), a stitch bond method, and a steam jet method. Among these, the melt blow method, the electrospinning method, the flash spinning method, the papermaking method and the like are preferable from the viewpoint of obtaining ultrafine fibers.

The melt blow method is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the thermoplastic resin melted by the extruder is blown from the melt blow die into a thread by a high temperature and high speed air flow to form a fiber. Examples thereof include a method in which fibers are entangled with each other and fused by accumulating the stretched resin on a conveyor to obtain a non-woven fabric of self-adhesive ultrafine fibers without binder. At this time, by adjusting the resin viscosity, melting temperature, discharge amount, hot air temperature, wind pressure, DCD (distance from the spinneret and the surface to the conveyor), etc., the fiber diameter, texture, fiber orientation, and fiber dispersibility of the non-woven fabric are adjusted. Can be controlled. Further, it is possible to control the thickness and average pore size of the non-woven fabric by hot pressing or tentering.

-Ion exchange ligand-

The adsorption target of the ion exchange ligand is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include biomolecules.
The biomolecule is not particularly limited and may be appropriately selected depending on the intended purpose. For example, viruses, virus-like particles, viral vectors, nucleic acids, amino acids, sugars, lipids, vitamins, coenzymes, hormones, and the like. Complexes and metabolites of. Among these, viruses, virus-like particles, and viral vectors are preferable.
The virus-like particles are all or part of the virus shell protein that mainly constitutes the capsid, and since they do not contain nucleic acids, there is no concern about infection, but they induce an immune response and are therefore used as the active ingredient of the vaccine. be able to.

The virus, virus-like particles, and virus vector are not particularly limited and may be appropriately selected depending on the intended purpose. For example, adeno-associated virus (AAV), adenovirus, enterovirus, parvovirus, papovavirus, human papillomavirus. , Rotavirus, coxsackie virus, sapovirus, norovirus, poliovirus, echovirus, hepatitis A virus, hepatitis E virus, rhinovirus, astrovirus, circovirus, simian virus and other non-enveloped viruses, retrovirus, lentivirus, Herpesviruses such as Sendai virus and simple herpesvirus, enveloped viruses such as vaccinia virus, measles virus, baculovirus, influenza virus, leukemia virus, Sindbiswill, and capsids of these viruses, or virus vectors containing these genes, etc. Can be mentioned. Among these, adeno-associated virus, capsid of adeno-associated virus, or adeno-associated virus gene is preferable.

The adeno-associated virus is a virus belonging to the parvoviridae family, which contains a linear single-stranded DNA in a capsid. Examples of the adeno-associated virus include type 1 AAV virus (AAV1), type 2 AAV virus (AAV2), type 3 AAV virus (AAV3), type 4 AAV virus (AAV4), type 5 AAV virus (AAV5), and type 6 AAV. Viruses (AAV6), type 7 AAV viruses (AAV7), type 8 AAV viruses (AAV8), type 9 AAV viruses (AAV9), type 10 AAV viruses (AAV10), and the like are known, and any of them is preferable. It is a type 2 AAV virus (AAV2).

The ion exchanger of the ion exchange ligand is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a cation exchanger and an anion exchanger.
The cation exchanger is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include those containing a sulfo group, those containing a carboxyl group, and those containing phosphoric acid.
The anion exchanger is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include those containing a quaternary ammonium group and those containing an amino group.
Among these, those containing a quaternary ammonium group are preferable, and compounds containing a trimethylammonium group are more preferable.

-Dynamic binding capacity of protein (5% dynamic binding capacity, DBC)-
The dynamic binding capacity (5% dynamic binding capacity, DBC) of the protein of the structure is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 50 mg / mL-volume or less, preferably 40 mg / mL. It is more preferably mL-volume or less, still more preferably 30 mg / mL-volume or less, and particularly preferably 25 mg / mL-volume or less.

The dynamic binding capacity (5% dynamic binding capacity, DBC) of the protein of the structure is measured by the following method.

The prepared ligand-immobilized non-woven fabrics are laminated one by one and filled in a column. As the column, a column made of acrylic resin having an inner diameter of 15 mm and a height of 9 mm, which is manufactured by Ring Co., Ltd. by cutting, is used.
The following solutions A to E are prepared and filtered through a 0.2 μm filter before use.
Solution A: Tris-HCl (50 mmol / L), pH 8.5
Solution B: Tris-HCl (50 mmol / L), NaCl (1 mol / L), pH 8.5
Solution C: NaCl (1 mol / L)
Liquid D: NaOH (0.5 mol / L)
Solution E: A protein solution prepared by dissolving bovine serum albumin (BSA, manufactured by Sigma) in solution A to 1 mg / mL. A column packed with a non-woven fabric is used for column chromatography (GE Healthcare "AKTA Avant 25"). , Wash with solution C, and equilibrate with solution A. After that, the E solution was passed through the carrier for a contact time of 4 minutes, and based on the value of the absorbance monitor at 280 nm, the amount of BSA adsorbed by 5% breakthrough and the volume of the carrier were used to determine the BSA dynamic binding capacity ( DBC) is calculated. After the passage of the E solution is completed, the column is washed with the B solution, the C solution, and the D solution.

-AAV binding capacity-
The AAV binding capacity of the structure is not particularly limited and may be appropriately selected depending on the intended purpose, but 1.0 × 10 ¹² vg / mL-volume or more is preferable, and 5.0 × 10 ¹² vg / More than mL-volume, more preferably 1.0 × 10 ¹³ vg / mL-volume or ^{more, and particularly preferably 1.5 × 10 13} vg / mL-volume or more.

The AAV binding capacity of the structure is measured by the following method.

The crude AAV purified solution is purified by chromatography on a non-woven fabric carrier with reference to a non-patent document (Journal of Virologic Methods 2007 140: 183-192).
The following solutions A to E are prepared and filtered through a 0.2 μm filter before use.
Solution A: Tris (20 mmol / L), ammonium acetate (100 mmol / L), pH 9.0
Solution B: Tris (20 mmol / L), ammonium acetate (300 mmol / L), pH 9.0
Solution C: NaCl (1 mol / L)
Liquid D: NaOH (0.5 mol / L)
Liquid E: A solution prepared by diluting the crude AAV solution with solution A to pH 9.0 ( ^{about 2-3 × 10 12} vg) (so that the amount of AAV in solution E is larger than the AAV binding volume of the carrier). To do)
A column packed with a carrier is connected to AKTA Avant 25, washed with solution C, and equilibrated with solution A. Then, the E solution is passed through, the AAV is retained on the column carrier, washed with the A solution, the B solution is made 100% and eluted in one step, and the AAV binding volume is determined from the amount of AAV contained in the elution peak. Is calculated.
The amount of AAV contained in each fraction is quantified by quantitative PCR (qPCR) using a Quant Studio 3 real-time PCR system (manufactured by Thermo Fisher Scientific).

(Manufacturing method of structure)
The method for producing the structure includes a step of connecting a water-insoluble base material to one end of the spacer and connecting an ion exchange ligand to the other end so that the ion exchange group density of the structure is less than 50 μmol / g. Further other steps can be included.
The spacer, the water-insoluble substrate, and the on-exchange ligand are as described above.

Here, the order of connecting the water-insoluble substrate to one end of the spacer and the connection of the ion exchange ligand to the other end of the spacer does not matter, but the reaction control for connection is easy. After connecting the water-insoluble substrate to one end of the spacer, it is preferable to connect the ion exchange ligand to the other end of the spacer.

The connection of the water-insoluble base material to one end of the spacer is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a method of adding a spacer solution to the water-insoluble base material and inversion mixing is used. Can be mentioned.
The time for the inversion mixing is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 hour or more and 48 hours or less, more preferably 2 hours or more and 48 hours or less, and 3 hours. From the above, 24 hours or less is more preferable, and 8 hours or more and 24 hours or less is particularly preferable.

The connection of the ion exchange ligand to the other end of the spacer is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a method of adding an ion exchange ligand to a water-insoluble substrate and inversion mixing is used. Can be mentioned.
The time for the inversion mixing is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 hour or more and 48 hours or less, more preferably 2 hours or more and 48 hours or less, and 3 hours. From the above, 24 hours or less is more preferable, and 8 hours or more and 24 hours or less is particularly preferable.

(Adsorbent)
The adsorbent may have the above-mentioned structure and may further have other components.
The adsorption target of the adsorbent is the same as the adsorption target of the ion exchange ligand described above.

(Method for purifying biological particles)
The method for purifying biological particles is a method for purifying the above-mentioned structure or the above-mentioned adsorbent.
The purification target of the method for purifying biological particles is the same as the above-mentioned adsorption target of the ion exchange ligand.

The purification is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include purification using the structure or a column packed with the adsorbent.

The material of the column is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include acrylic resin.

-AAV elution rate into flow-through (FT) and AAV recovery rate-
The elution rate of AAV into the flow-through (FT) in the method for purifying biological particles is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably less than 100%, more preferably 50% or less. , 20% or less is more preferable, and 0% is particularly preferable.

The AAV recovery rate in the method for purifying biological particles is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5% or more, more preferably 10% or more, still more preferably 50% or more. 70% or more is particularly preferable.

The elution rate of AAV into the flow-through (FT) and the AAV recovery rate are measured by the following methods.

The crude AAV purified solution is purified by chromatography on a non-woven fabric carrier with reference to a non-patent document (Journal of Virologic Methods 2007 140: 183-192).
The following solutions A to E are prepared and filtered through a 0.2 μm filter before use.
Solution A: Tris (20 mmol / L), ammonium acetate (100 mmol / L), pH 9.0
Solution B: Tris (20 mmol / L), ammonium acetate (300 mmol / L), pH 9.0
Solution C: NaCl (1 mol / L)
Liquid D: NaOH (0.5 mol / L)
Liquid E: A solution prepared by diluting AAV crude purified liquid with liquid A to pH 9.0 ( ^{about 2-3 × 10 12} vg).
A column packed with a carrier is connected to AKTA Avant 25, washed with solution C, and equilibrated with solution A. Then, the solution E is passed through, the AAV is held on the column carrier, and then washed with the solution A to gradually increase the ratio of the solution B to the solution A to elute the AAV. After the elution is completed, the column is washed with solutions C and D.
The amount of AAV contained in each fraction is quantified by quantitative PCR (qPCR) using a Quant Studio 3 real-time PCR system (manufactured by Thermo Fisher Scientific), and the AAV amount of the elution peak with respect to the total amount of AAV of the passed E solution is recovered. Calculate as. Further, the amount of AAV in the flow-through (FT) solution with respect to the total amount of AAV of the passed E solution is calculated as the elution rate.

Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

<Manufacturing Example 1: Fabrication of non-woven fabric A>
Using polypropylene (hereinafter abbreviated as PP) as a material, a non-woven fabric having an average fiber diameter of 1.19 μm, a basis weight of 30 g / m ² , and a thickness of 0.30 mm was produced by a melt blow method. The radically polymerizable compound solution was mixed with GMA / polysorbate 20 / water so as to be 10/2/88 by weight, and dissolved oxygen was removed by aerating nitrogen gas. The prepared non-woven fabric was irradiated with an electron beam at room temperature in a nitrogen gas atmosphere under the conditions of an acceleration voltage of 250 kV and an irradiation dose of 50 kGy. The non-woven fabric after electron beam irradiation was immersed in a radically polymerizable compound solution and heated in a reaction vessel at 50 ° C. for 40 minutes to accelerate the polymerization reaction. Then, the non-woven fabric was washed with water at room temperature and then dried to obtain a non-woven fabric A having a graft ratio of 50%.

The average fiber diameter of the non-woven fabric was determined based on the observation with a scanning electron microscope. From the obtained electron microscope images, the diameters of 100 or more randomly selected fibers were measured. The average number of measured values of 100 or more was defined as the average fiber diameter. The graft ratio is a value calculated as follows by measuring the weight of the non-woven fabric (W1) before the graft reaction and the weight (W2) of the non-woven fabric after the graft reaction.
Graft rate = [(W2-W1) / W1] x 100 (%)

<Manufacturing Example 2: Fabrication of non-woven fabric B>
Using polypropylene as a material, a non-woven fabric having an average fiber diameter of 0.56 μm, a basis weight of 15 g / m ^{2, and a thickness of 0.14 mm was produced by the melt blow method.} The radically polymerizable compound solution was mixed with GMA / ethanol so as to be 10/90 by mass, and dissolved oxygen was removed by aerating nitrogen gas. The prepared non-woven fabric was irradiated with an electron beam at room temperature in a nitrogen gas atmosphere under the conditions of an acceleration voltage of 200 kV and an irradiation dose of 10 kGy. The non-woven fabric after electron beam irradiation was immersed in a radically polymerizable compound solution and heated in a reaction vessel at 50 ° C. for 60 minutes to accelerate the polymerization reaction. Then, the non-woven fabric was washed with ethanol at room temperature and then dried to obtain a non-woven fabric B having a graft ratio of 100%.

<Manufacturing Example 3: Fabrication of Nonwoven Fabric C>
Nonwoven fabric C having a graft ratio of 180% was obtained in the same manner as in Production Example 2 except that the irradiation dose was set to 15 kGy.

<Manufacturing Example 4: Fabrication of Nonwoven Fabric D>
A non-woven fabric D having a graft ratio of 270% was obtained in the same manner as in Production Example 2 except that the irradiation dose was set to 20 kGy.

<Production Example 5: Fabrication of non-woven fabric E>
A non-woven fabric E having a graft ratio of 130% was obtained in the same manner as in Production Example 2 except that a non-woven fabric having an average fiber diameter of 1.13 μm, a basis weight of 40 g / m ^{2, and a thickness of 0.30 mm was used.}

<Manufacturing Example 6: Fabrication of non-woven fabric F>
A non-woven fabric F having a graft ratio of 180% was obtained in the same manner as in Production Example 5 except that the irradiation dose was set to 15 kGy.

Table 1 shows the non-woven fabrics produced in Production Examples 1 to 6 above.
The non-woven fabric shown in Table 1 was used for immobilization of spacers and ion exchange ligands, and various measurements.

<Production Example 7: Preparation of water-insoluble base material disc>
Using a punch, the non-woven fabrics A to F produced in Production Examples 1 to 6 were punched into a circle having a diameter of 15 mm to prepare a water-insoluble base material disk (nonwoven fabric disk) for filling the column.

<Example 1>
Example 1-1: Preparation of carrier 1 (immobilization of polyethyleneimine (PEI) spacer on non-woven fabric A, and subsequent immobilization of trimethylammonium (Q) ligand)
A PEI solution (manufactured by Wako Co., Ltd., Mw: 600, straight chain) was added to the non-woven fabric A of Production Example 7, and the mixture was inverted and mixed at 37 ° C. for 16 hours. After the reaction, the non-woven fabric was transferred to a glass filter and washed with pure water to obtain a non-woven fabric on which the PEI spacer was immobilized. Glycidyltrimethylammonium chloride (manufactured by TCI) was added thereto, and the mixture was again mixed by inversion at 37 ° C. for 16 hours. After the reaction, it was washed with pure water to obtain a non-woven fabric (carrier 1) on which the Q ligand was immobilized.

Example 1-2: Measurement of ion exchange capacity The carrier 1 was transferred to a glass filter and washed with pure water three times. A 0.1 mol / L aqueous sodium hydroxide solution was added and the carrier was immersed for 3 minutes. After washing with a 0.01 mol / L sodium hydroxide aqueous solution, the washing was carried out with pure water until the washing liquid became neutral. The carrier was transferred to a beaker using pure water, a 1 mol / L sodium chloride aqueous solution was added and stirred, and only the solution was recovered in another beaker. This operation was repeated several times, a phenolphthalein solution was added to the recovered solution, and the mixture was titrated with a 0.01 mol / L hydrochloric acid aqueous solution. Table 2 shows the ion exchange capacity per volume and weight.

Example 1-3: Measurement of Dynamic Binding Capacity (DBC) The prepared ligand-immobilized non-woven fabrics were laminated one by one and packed in a column. The column was made of acrylic resin having an inner diameter of 15 mm and a height of 9 mm, and was manufactured by Ring Co., Ltd. by cutting and used.
The following solutions A to E were prepared and filtered through a 0.2 μm filter before use.
Solution A: Tris-HCl (50 mmol / L), pH 8.5
Solution B: Tris-HCl (50 mmol / L), NaCl (1 mol / L), pH 8.5
Solution C: NaCl (1 mol / L)
Liquid D: NaOH (0.5 mol / L)
Solution E: A protein solution prepared by dissolving bovine serum albumin (BSA, manufactured by Sigma) in solution A to 1 mg / mL. A column packed with a non-woven fabric is used for column chromatography (GE Healthcare "AKTA Avant 25"). Was washed with solution C and equilibrated with solution A. After that, the E solution was passed through the carrier for a contact time of 4 minutes, and based on the value of the absorbance monitor at 280 nm, the amount of BSA adsorbed by 5% breakthrough and the volume of the carrier were used to determine the BSA dynamic binding capacity ( DBC) was calculated. After the completion of the passage of the E solution, the column was washed with the B solution, the C solution, and the D solution. The results are shown in Table 3.

<Example 2: Preparation of carrier 2 (immobilization of PEI spacer on non-woven fabric A, and subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 2) on which a Q ligand was immobilized was produced and carried out in the same manner as in Example 1-1 except that a PEI solution (manufactured by Wako Co., Ltd., Mw: 10,000, straight chain) was used as a spacer. The ion exchange capacity per volume and weight was measured by the same method as in Example 1-2, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Example 3: Preparation of carrier 3 (immobilization of PEI spacer on non-woven fabric A, and subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 3) on which a Q ligand was immobilized was produced and carried out in the same manner as in Example 1-1 except that a PEI solution (manufactured by Wako Co., Ltd., Mw: 70,000, straight chain) was used as a spacer. The ion exchange capacity per volume and weight was measured by the same method as in Example 1-2, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Example 4: Preparation of carrier 4 (immobilization of PEI spacer on non-woven fabric A, and subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 4) on which a Q ligand was immobilized was produced in the same manner as in Example 1-1 except that a PEI solution (manufactured by Alfa Aesar, Mw: 70,000, branched chain) was used as a spacer. The ion exchange capacity per volume and per weight was measured by the same method as in Example 1-2, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Example 5: Preparation of carrier 5 (immobilization of polyallylamine (PAA) spacer on non-woven fabric A, and subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 5) on which a Q ligand was immobilized was produced in the same manner as in Example 1-1 except that a PAA solution (manufactured by Polysciences, Mw: 150,000) was used as a spacer, and Example 1- The ion exchange capacity per volume and weight was measured by the same method as in No. 2, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Example 6: Preparation of carrier 6 (immobilization of PEI spacer on non-woven fabric B, and subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 6) on which a Q ligand was immobilized was produced in the same manner as in Example 4-1 except that the non-woven fabric B of Production Example 7 was used, and the volume per volume was the same as in Example 1-2. , The ion exchange capacity per weight was measured, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Example 7: Preparation of carrier 7 (immobilization of PEI spacer on non-woven fabric C, subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 7) on which a Q ligand is immobilized is produced by the same method as in Example 4-1 except that the non-woven fabric C of Production Example 7 is used, and the volume per volume is the same as in Example 1-2. , The ion exchange capacity per weight was measured, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Example 8: Preparation of carrier 8 (immobilization of PEI spacer on non-woven fabric D, subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 8) on which a Q ligand is immobilized is produced by the same method as in Example 4-1 except that the non-woven fabric D of Production Example 7 is used, and the volume per volume is the same as in Example 1-2. , The ion exchange capacity per weight was measured, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Example 9: Preparation of carrier 9 (immobilization of PEI spacer on non-woven fabric E, subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 9) on which a Q ligand was immobilized was produced in the same manner as in Example 4-1 except that the non-woven fabric E in Production Example 7 was used, and the volume per volume was the same as in Example 1-2. , The ion exchange capacity per weight was measured, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Example 10: Preparation of carrier 10 (immobilization of PEI spacer on non-woven fabric F, subsequent immobilization of Q ligand)>
A non-woven fabric (carrier 10) on which a Q ligand was immobilized was produced in the same manner as in Example 4-1 except that the non-woven fabric F of Production Example 7 was used, and the volume per volume was the same as in Example 1-2. , The ion exchange capacity per weight was measured, and the results are shown in Table 2. In addition, the dynamic binding capacity of the protein was measured by the same method as in Example 1-3, and the results are shown in Table 3.

<Comparative Example 1: Bead Carrier>
Comparative Example 1-1: Measurement of ion exchange capacity A carrier of 1 mL-gel of POROS 50HQ (manufactured by Thermo Fisher Scientific), which is a commercially available anion exchange carrier, was transferred to a glass filter and washed with pure water three times. A 0.1 mol / L aqueous sodium hydroxide solution was added and the carrier was immersed for 3 minutes. After washing with a 0.01 mol / L sodium hydroxide aqueous solution, the washing was carried out with pure water until the washing liquid became neutral. After transferring the carrier to a beaker using pure water, a 1 mol / L sodium chloride aqueous solution was added to exchange the counterion of the carrier. A phenolphthalein solution was added thereto, and the mixture was titrated with a 0.01 mol / L hydrochloric acid aqueous solution. The amount of hydroxide ion exchanged with the counterion was calculated from the titration amount, and the ion exchange capacity of the carrier was determined. The ion exchange capacity per volume and weight was measured and shown in Table 2.

Comparative Example 1-2: Measurement of Dynamic Binding Capacity (DBC) POROS 50HQ was measured by 1 mL-gel in a wet volume and packed in a Tricorn 5/50 column (manufactured by GE Healthcare).
The dynamic binding capacity of the protein was measured by the same method as in Example 1-3 except that the non-woven fabric was replaced with beads, and the results are shown in Table 3.

<Comparative Example 2: Membrane Carrier>
Comparative Example 2-1: Measurement of ion exchange capacity Using a commercially available anion exchange carrier, Mustang Q (manufactured by Pall), the ion exchange capacity was determined in the same manner as in Comparative Example 1-1. The ion exchange capacity per volume and weight was measured and shown in Table 2.

Comparative Example 2-2: Measurement of Dynamic Binding Capacity (DBC) Mustang Q (Acrodisc unit with Mustang Q membrane) was used by connecting to AKTA Avant 25.
The dynamic binding capacity of the protein was measured in the same manner as in Example 1-3, and the results are shown in Table 3.

From the results in Table 2, the commercially available carriers POROS 50HQ and Mustang Q both have high ion exchange capacities exceeding 200 μmol / g, whereas the carriers 1-10 produced this time both have an ion exchange capacity of 50 μmol. The value was lower than / g.

From the results in Table 3, the commercially available carriers POROS 50HQ and Mustang Q showed high values of 60 mg / mL-volume and 72 mg / mL-volume, respectively, for 5% DBC. On the other hand, it was found that the 5% DBC of each of the carriers 1-10 produced this time was about 10 mg / mL-volume, and the adsorption amount of the non-specific protein was lower than that of the commercially available product.

<Production Example 8: Production of adeno-associated virus (AAV) by animal cells and preparation of AAV pretreatment solution>
A plasmid for AAV2 preparation expressing VENUS (GenBank: ACQ43955.1), which is a variant of the fluorescent protein GFP, was prepared using an AAV vector preparation kit (“AAVpro (R) Helper Free System” manufactured by Takara Bio Inc.).
Cultured HEK293 cells were transfected with a plasmid prepared using a transfection reagent (“Polyethylenimine MAX” manufactured by Polysciences, MW: 40,000) to produce AAV. After completion of the culture, the cells were detached and the cell culture solution was collected. This was centrifuged and the supernatant was removed to obtain AAV-producing cells.
The AAV-producing cells obtained above were suspended in darbecolinic acid buffered saline (manufactured by Sigma-Aldrich, hereinafter abbreviated as "PBS") containing 0.1% Triton X-100, and 20 in ice. The cells were disrupted with stirring for 1 minute. To the obtained cell disruption solution, 7.5 v / v% 1M magnesium chloride aqueous solution and 0.1 v / v% 250 kU / mL Kaneka Endonucleose (manufactured by Kaneka Corporation) were added, and the mixture was allowed to stand at 37 ° C. for 30 minutes. , Cell-derived nucleic acids were degraded. After the reaction, a 0.5 M EDTA solution of 15 v / v% was added to the reaction solution, and the mixture was centrifuged to collect the supernatant. The obtained supernatant was used as an AAV pretreatment solution.

<Production Example 9: Preparation of crude AAV purified solution>
The AAV pretreatment solution was purified by cation exchange chromatography with reference to a non-patent document (Journal of Virologic Methods 2007 140: 183-192).
POROS 50HS (manufactured by Thermo) was filled in Tricorn 10/150 (manufactured by GE Healthcare) to prepare a column for cation exchange purification.
The following solutions A to F were prepared and passed through a 0.2 μm filter before use.
Solution A: NaPO4 (20 mmol / L), NaCl (100 mmol / L), pH 7.4
Solution B: NaPO4 (20 mmol / L), NaCl (100 mmol / L), Sarkosyl (5 mmol / L), pH 7.4
Solution C: NaPO4 (20 mmol / L), NaCl (370 mmol / L), pH 7.4
Solution D: NaCl (1 mol / L)
Liquid E: NaOH (0.5 mol / L)
Solution F: A solution prepared by diluting the AAV pretreatment solution with solution A to pH 7.4 The column was connected to AKTA Avant 25, washed with solution D, and equilibrated with solution A. Then, the F solution was passed through, the AAV was held on the column carrier, the A solution, the B solution, and the A solution were washed in this order, and the AAV was eluted with the C solution. After the elution was completed, the column was washed with solution D and solution E.
For the crudely purified AAV, the degree of purification was confirmed by SDS-PAGE, and the amount of AAV in the solution was quantified by quantitative PCR (qPCR).

<Example 11: AAV purification using a non-woven fabric carrier>
The AAV crude purified solution was purified by chromatography on a non-woven fabric carrier with reference to a non-patent document (Journal of Virologic Methods 2007 140: 183-192).
The following solutions A to E were prepared and filtered through a 0.2 μm filter before use.
Solution A: Tris (20 mmol / L), ammonium acetate (100 mmol / L), pH 9.0
Solution B: Tris (20 mmol / L), ammonium acetate (300 mmol / L), pH 9.0
Solution C: NaCl (1 mol / L)
Liquid D: NaOH (0.5 mol / L)
Liquid E: A solution prepared by diluting AAV crude purified liquid with liquid A to pH 9.0 (about 2-3 × 1012 vg).
The column packed with carriers 1 to 4 prepared in Examples 1 to 4 was connected to AKTA Avant 25, washed with solution C, and equilibrated with solution A. After that, the solution E was passed, the AAV was retained on the column carrier, washed with the solution A, and the ratio of the solution B to the solution A was gradually increased to elute the AAV. After the elution was completed, the column was washed with solutions C and D. A chromatogram of AAV adsorption / desorption by carrier 4 is shown in FIG.
In addition, the amount of AAV contained in each fraction was quantified by quantitative PCR (qPCR) using a Quant Studio 3 real-time PCR system (manufactured by Thermo Fisher Scientific), and the amount of AAV at the elution peak with respect to the total amount of AAV in the passed E solution was determined. Calculated as recovery rate. Further, the amount of AAV in the flow-through (FT) solution with respect to the total amount of AAV of the passed E solution was calculated as the elution rate. The results are shown in Table 4.

From the results in Table 4, it was found that the AAV recovery rate was improved by increasing the molecular weight of the spacer. Moreover, when considered together with the results in Table 2, it was found that the carrier can efficiently purify the virus through the spacer even if the ion exchange group density is low.

<Example 12: Calculation of AAV binding capacity of various carriers>
The amount of AAV in the solution E is set to be larger than the AAV binding volume of the carrier, and at the time of elution, the solution B is made 100% and eluted in one step, and the AAV binding volume is calculated from the amount of AAV contained in the elution peak. The AAV binding capacity was calculated in the same manner as in Example 1116 except that it was calculated. The results are shown in Table 5.

From the results shown in Table 5, the commercially available POROS 50HQ and the carrier 7 had an AAV binding capacity ^{exceeding 1 × 10 13} vg / mL-volume and showed a high AAV adsorption amount.
On the other hand, the BSA binding capacity of the carrier 7 is about 1/6 to 1/7 that of POROS 50HQ and Mustang Q, which is remarkably low.
Summarizing the above results, it can be said that the carrier of the present application is a structure in which the ion exchange capacity and the adsorption of non-specific proteins are low, and the adsorption of biological particles is remarkably high as compared with the ion exchange capacity.

Examples of aspects of the present invention include the following.
<1> A structure having a spacer, a water-insoluble base material connected to one end of the spacer, and an ion exchange ligand connected to the other end of the spacer, wherein the structure has an ion exchange group density. Is a structure characterized by being less than 50 μmol / g.
<2> The structure according to <1>, wherein the spacer contains a polymer.
<3> The structure according to <2>, wherein the polymer contains a hydrophilic polymer.
<4> The structure according to <3>, wherein the hydrophilic polymer contains a polyamine.
<5> The structure according to <4>, wherein the polyamine contains polyethyleneimine or polyallylamine.
<6> The structure according to any one of <1> to <5>, wherein the polymer has a molar mass of 10,000 g / mol or more and 200,000 g / mol or less.
<7> The structure according to any one of <1> to <5>, wherein the polymer has a molar mass of 60,000 g / mol or more and 200,000 g / mol or less.
<8> The structure according to any one of <1> to <7>, wherein the polymer has a branched chain.
<9> The structure according to any one of <1> to <8>, wherein the water-insoluble base material is a water-insoluble fiber.
<10> The structure according to <9>, wherein the water-insoluble fiber contains polypropylene.
<11> The structure according to any one of <9> to <10>, wherein the water-insoluble fiber is a non-woven fabric.
<12> The structure according to any one of <1> to <11>, wherein the ion exchange ligand can adsorb a biomolecule.
<13> The structure according to <12>, wherein the biomolecule is a virus, a virus-like particle, or a viral vector.
<14> The structure according to <13>, wherein the virus, virus-like particles, or viral vector is an adeno-associated virus, or a capsid of an adeno-associated virus, or a viral vector containing an adeno-associated virus gene.
<15> The structure according to any one of <1> to <14>, wherein the ion exchange ligand contains a trimethylammonium group.
<16> A method for manufacturing a structure, in which a water-insoluble substrate is connected to one end of a spacer and an ion exchange ligand is connected to the other end so that the ion exchange group density of the structure is less than 50 μmol / g. It is a method for manufacturing a structure, which comprises a step.
<17> An adsorbent having the structure according to any one of <1> to <15>.
<18> A method for purifying biological particles, which comprises using the structure according to any one of <1> to <15> or the adsorbent according to <17>.

Claims (18)

With spacers
A water-insoluble substrate connected to one end of the spacer,
A structure having an ion exchange ligand connected to the other end of the spacer.
A structure characterized in that the ion exchange group density of the structure is less than 50 μmol / g. The structure according to claim 1, wherein the spacer contains a polymer. The structure according to claim 2, wherein the polymer comprises a hydrophilic polymer. The structure according to claim 3, wherein the hydrophilic polymer contains a polyamine. The structure according to claim 4, wherein the polyamine contains polyethyleneimine or polyallylamine. The structure according to any one of claims 1 to 5, wherein the polymer has a molar mass of 10,000 g / mol or more and 200,000 g / mol or less. The structure according to any one of claims 1 to 5, wherein the polymer has a molar mass of 60,000 g / mol or more and 200,000 g / mol or less. The structure according to any one of claims 1 to 7, wherein the polymer has a branched chain. The structure according to any one of claims 1 to 8, wherein the water-insoluble base material is a water-insoluble fiber. The structure according to claim 9, wherein the water-insoluble fiber contains polypropylene. The structure according to any one of claims 9 to 10, wherein the water-insoluble fiber is a non-woven fabric. The structure according to any one of claims 1 to 11, wherein the ion exchange ligand is capable of adsorbing a biomolecule. The structure according to claim 12, wherein the biomolecule is a virus, a virus-like particle, or a viral vector. The structure according to claim 13, wherein the virus, virus-like particles, or viral vector is an adeno-associated virus, or a capsid of an adeno-associated virus, or a viral vector containing an adeno-associated virus gene. The structure according to any one of claims 1 to 14, wherein the ion exchange ligand contains a trimethylammonium group. It is a method of manufacturing structures
Manufacture of a structure comprising a step of connecting a water-insoluble substrate to one end of the spacer and connecting an ion exchange ligand to the other end so that the ion exchange group density of the structure is less than 50 μmol / g. Method. An adsorbent having the structure according to any one of claims 1 to 15. A method for purifying biological particles, which comprises using the structure according to any one of claims 1 to 15 or the adsorbent according to claim 17.

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