Porous polymer bead aggregate, method for producing same, and method for producing nucleic acid

Abstract

The purpose of the present invention is to provide porous resin beads for suppressing an increase in back pressure in a reaction vessel during a step for synthesizing a nucleic acid and for stably producing the nucleic acid without a liquid feeding defect. Said problem is solved by a porous polymer bead aggregate including a styrene-based copolymer, the porous polymer bead aggregate being characterized in that: (a) the porous polymer bead aggregate includes particles having a particle diameter of 20-50 μm in a particle frequency range of no more than 10% in accordance with a number standard in an image analysis method; and (b) in a particle mass configured from porous polymer beads having a particle diameter of at least 20 μm in the porous polymer bead aggregate, the median value of the particle diameter is 50-140 μm and the coefficient of variation (CV value) of the particle diameter is no more than 30%.

Description

Porous polymer bead assembly, method for producing the same, and method for producing nucleic acid

[0001] The present invention relates to a collection of porous polymer beads and a method for producing the same, as well as a method for producing nucleic acids.

[0002] Solid-phase synthesis using the phosphoramidite method is widely used for the chemical synthesis of nucleic acids. When porous polymer beads are used as a support for solid-phase synthesis, it is desirable for the porous polymer beads to swell to a certain extent in organic solvents so that nucleic acids can be efficiently synthesized on the support. However, if the degree of swelling of the porous polymer beads varies in various organic solvents, when nucleic acids are sequentially synthesized on the support in different organic solvents, for example, when nucleic acids are chemically synthesized on a support filled in a reaction vessel of a fixed volume, changes in the volume of the porous polymer beads used as the support can cause an increase in back pressure during solution delivery within the reaction vessel, resulting in poor solution delivery. Furthermore, these problems can result in a decrease in the yield of the desired nucleic acid.

[0003] Attempts have been made to improve nucleic acid synthesis performance by suppressing fluctuations in the swelling ratio of porous polymer beads in various organic solvents using (meth)acrylonitrile (Patent Document 1). Furthermore, in order to increase oligonucleotide yield, i.e., oligonucleotide synthesis performance, when oligonucleotides are synthesized, the use of a collection of porous polymer beads having a specific particle size calculated using a laser diffraction / scattering particle size analyzer has been investigated (Patent Document 2). However, while the porous polymer beads in Patent Document 1 suppress fluctuations in the swelling ratio of porous polymer beads in various organic solvents, they are insufficiently effective in suppressing back pressure increase in the reaction vessel, and further improvements are needed. Furthermore, it has been found that the laser diffraction / scattering particle size analyzer used to measure particle size in Patent Document 2 does not accurately measure particle frequency in the small particle size range, and therefore further improvements are needed to suppress back pressure increase and improve nucleic acid synthesis productivity.

[0004] JP 2008-74979 A JP 2009-249478 A

[0005] The present invention aims to provide a porous polymer bead assembly that suppresses an increase in back pressure in a reaction vessel during the nucleic acid synthesis process, and that allows nucleic acid to be produced stably without liquid delivery problems, a method for producing the same, and a method for producing nucleic acid.

[0006] As a result of extensive research to solve the above-mentioned problems, the present inventors discovered that by using an assembly of porous polymer beads having specific parameters calculated on a number basis using an image analysis method, it is possible to suppress an increase in back pressure in a reaction vessel during the nucleic acid synthesis process and to stably produce nucleic acids without poor liquid delivery. Further research led to the completion of the present invention.

[0007] That is, the present invention relates to the following: [1] A collection of porous polymer beads containing a styrene-based copolymer, characterized in that: (a) the collection of porous polymer beads contains particles having a particle diameter of 20 μm or more and 50 μm or less at a particle frequency of 10% or less, based on the number of particles in an image analysis method; and (b) a particle group of the collection of porous polymer beads composed of porous polymer beads having a particle diameter of 20 μm or more has a median particle diameter of 50 μm or more and 140 μm or less, and a coefficient of variation (CV value) of particle diameter of 30% or less. [2] The collection of porous polymer beads according to [1], wherein the collection of porous polymer beads contains particles having a particle diameter of 20 μm or more and 40 μm or less at a particle frequency of 3% or less, based on the number of particles in an image analysis method. [3] A method for producing the porous polymer bead aggregate according to [1] or [2], comprising: (a) a step of suspension polymerizing a monomer mixture containing a styrene-based monomer to obtain porous polymer beads; (b) a step of dispersing the porous polymer beads obtained in the step (a) in an organic solvent, and then leaving the dispersion to stand for a standing time T (seconds) that satisfies the following formula 1 to obtain a dispersion; and (c) a step of collecting the dispersion obtained in the step (b) to a depth D (cm) from the liquid surface to obtain a porous polymer bead aggregate. Formula 1: T = D × α T: standing time (seconds) D: sampling depth (cm) 20≦α (seconds / cm)≦60 [4] A method for producing the porous polymer bead collection according to [1] or [2], comprising: (a) a step of suspension polymerizing a monomer mixture containing a styrene-based monomer to obtain porous polymer beads, and (b) a step of classifying the porous polymer beads obtained by the step (a) using an elbow jet classifier to obtain a porous polymer bead collection. [5] A method for producing an oligonucleotide, comprising sequentially binding nucleosides or nucleotides directly or indirectly to the porous polymer beads in the porous polymer bead collection according to [1] or [2].

[0008] By using the porous polymer bead assembly of the present invention, an increase in back pressure in a reaction vessel can be suppressed in the nucleic acid synthesis step, and nucleic acids can be stably produced without liquid delivery failure.Furthermore, the yield of the target nucleic acid can be improved.

[0009] Fig. 1 shows the particle frequency by number distribution by image analysis for the porous polymer bead aggregates obtained in Examples 1 and 2 and Comparative Examples 1 and 2. Fig. 2 shows the particle frequency by volume distribution by laser diffraction / scattering particle size distribution measurement for the porous polymer bead aggregates obtained in Examples 1 and 2 and Comparative Examples 1 and 2. Fig. 3 shows the results of the liquid transportability test.

[0010] The present invention is described in detail below with reference to preferred embodiments, but is not limited to these. The present invention relates to a collection of porous polymer beads containing a styrene copolymer, characterized in that (a) the collection of porous polymer beads contains particles having a particle diameter of 20 μm or more and 50 μm or less at a particle frequency of 10% or less, as determined by number analysis using an image analysis method, and (b) the particle group composed of porous polymer beads having a particle diameter of 20 μm or more in the collection of porous polymer beads has a median particle diameter of 50 μm or more and 140 μm or less, and a coefficient of variation (CV value) of particle diameter is 30% or less.

[0011] In the present invention, particle analysis of the porous polymer bead collection is performed by image analysis. The image analysis method in the present invention may be dynamic image analysis or static image analysis. The porous polymer bead collection of the present invention preferably contains particles having a particle diameter of 20 μm or more and 50 μm or less at a particle frequency of 10% or less, for example, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% or less, based on the number of particles in the image analysis method, and preferably 4% or less, more preferably 3% or less. The porous polymer bead collection of the present invention preferably contains particles having a particle diameter of 20 μm or more and 40 μm or less at a particle frequency of 10% or less, for example, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, or 3% or less, based on the number of particles in the image analysis method, and preferably 3% or less, more preferably 1% or less.

[0012] Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 50 μm to 120 μm and a particle diameter coefficient of variation (CV value) of 30% or less. Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 50 μm to 100 μm and a particle diameter coefficient of variation (CV value) of 30% or less. Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 60 μm to 85 μm, and preferably has a particle diameter coefficient of variation (CV value) of 30% or less. Among the porous polymer bead aggregates of the present invention, the particle population composed of porous polymer beads having a particle diameter of 20 μm or more has a median particle diameter of 61.5 μm or more and 80.3 μm or less, and preferably has a coefficient of variation (CV value) of particle diameter of 30% or less.

[0013] Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 50 μm or more and 140 μm or less, and a coefficient of variation (CV value) of particle diameter of 25% or less. Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 50 μm or more and 120 μm or less, and a coefficient of variation (CV value) of particle diameter of 25% or less. Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 50 μm or more and 100 μm or less, and a coefficient of variation (CV value) of particle diameter of 25% or less. In the porous polymer bead collection of the present invention, a particle collection composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 60 μm or more and 85 μm or less, and a coefficient of variation (CV value) of particle diameter of 25% or less. In the porous polymer bead collection of the present invention, a particle collection composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 61.5 μm or more and 80.3 μm or less, and a coefficient of variation (CV value) of particle diameter of 25% or less.

[0014] Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 50 μm or more and 140 μm or less, and a coefficient of variation (CV value) of particle diameter of 23.8% or less. Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 50 μm or more and 120 μm or less, and a coefficient of variation (CV value) of particle diameter of 23.8% or less. Among the porous polymer bead aggregates of the present invention, a particle population composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 50 μm or more and 100 μm or less, and a coefficient of variation (CV value) of particle diameter of 23.8% or less. In the porous polymer bead collection of the present invention, a particle collection composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 60 μm or more and 85 μm or less, and a coefficient of variation (CV value) of particle diameter of 23.8% or less. In the porous polymer bead collection of the present invention, a particle collection composed of porous polymer beads having a particle diameter of 20 μm or more preferably has a median particle diameter of 61.5 μm or more and 80.3 μm or less, and a coefficient of variation (CV value) of particle diameter of 23.8% or less.

[0015] In one aspect, the present invention relates to a method for producing a collection of porous polymer beads of the present invention, comprising: (a) a step of suspension polymerizing a monomer mixture containing a styrene-based monomer to obtain porous polymer beads; (b) a step of dispersing the porous polymer beads obtained in the step (a) in an organic solvent and then leaving the dispersion to stand for a standing time T (seconds) that satisfies the following formula 1 to obtain a dispersion; and (c) a step of collecting the dispersion obtained in the step (b) to a depth D (cm) from the liquid surface to obtain a collection of porous polymer beads. Formula 1: T=D×α T: standing time (seconds) D: collection depth (cm) 20≦α (seconds / cm)≦60

[0016] In the above method, the standing time is not particularly limited, but is preferably 40 to 360 seconds, and most preferably 180 seconds. In the above method, the sampling depth (D) is not particularly limited, but is preferably 2 to 6 cm, and most preferably 4 cm. In the above method, α is not particularly limited, but is 20 to 60, preferably 30 to 50, and most preferably 45.

[0017] In one aspect, the present invention relates to a method for producing a collection of porous polymer beads, comprising: (a) a step of suspension polymerizing a monomer mixture containing a styrene-based monomer to obtain porous polymer beads; and (b) a step of classifying the porous polymer beads obtained in step (a) using an elbow jet classifier to obtain a collection of porous polymer beads. In the present invention, classification can be performed, for example, using an elbow jet classifier. The classification edge angle on the fine powder side can be set, for example, to 26 mm to 30 mm, preferably 30 mm. The air pressure can be adjusted so that the yield of particles recovered as fine powder is 70% to 97%, and it is preferable to adjust the air pressure so that the yield is 80% to 97%. The air pressure can be set to 0.04 MPa to 0.07 MPa, preferably 0.05 MPa.

[0018] In the present invention, the styrene copolymer is, for example, a copolymer of styrene, hydroxystyrene, and divinylbenzene, and typical examples of the structural units of styrene, hydroxystyrene, and divinylbenzene are as shown in the following formulae. Here, the structural units (A) to (C) may be substituted as follows.

[0019] (A) One or more hydrogen atoms in the styrene structural unit (including hydrogen atoms in the benzene ring) are substituted with an alkyl group having 1 to 5 carbon atoms, a halogen atom, a carboxyl group, a sulfonic acid group (—SO ３ It may be substituted with a substituent such as a hydroxyl group, a cyano group, an alkoxy group having 1 to 5 carbon atoms, or a nitro group, provided that a hydroxyl group is excluded as a substituent.

[0020] One or more hydrogen atoms in the (B) hydroxystyrene structural unit (including hydrogen atoms in the benzene ring but excluding hydrogen atoms in the hydroxyl group) may be substituted with a substituent such as an alkyl group having 1 to 5 carbon atoms, a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms, or a nitro group.

[0021] One or more hydrogen atoms in the (C) divinylbenzene structural unit (including hydrogen atoms in the benzene ring) may be substituted with a substituent such as an alkyl group having 1 to 5 carbon atoms, a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms, or a nitro group.

[0022] The porous polymer bead aggregate of the present invention may contain (meth)acrylonitrile structural units, and the (meth)acrylonitrile structural units may contain either acrylonitrile structural units or methacrylonitrile structural units alone, or may contain both units.

[0023] In the present invention, the styrene-based monomer refers to styrene or a substituted styrene (excluding acyloxy groups as a substituent), preferably unsubstituted styrene. Examples of the substituted styrene include compounds in which one or more hydrogen atoms of styrene are substituted with an alkyl group having 1 to 5 carbon atoms (e.g., methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-amyl group, isoamyl group, sec-amyl group, tert-amyl group), a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms (e.g., methoxy group, ethoxy group, propoxy group, butoxy group, pentyloxy group), a nitro group, or the like.

[0024] Specific examples of the styrene-based monomer include styrene, alkylstyrenes such as ethylstyrene, methylstyrene, dimethylstyrene, trimethylstyrene, and butylstyrene, halogenated styrenes such as chlorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, and bromostyrene, halogenated alkylstyrenes such as chloromethylstyrene and fluoromethylstyrene, vinyl benzoate, sodium styrenesulfonate, cyanostyrene, methoxystyrene, ethoxystyrene, butoxystyrene, and nitrostyrene. When divinylbenzene is used as the divinylbenzene-based monomer described below, commercially available divinylbenzene contains ethylstyrene as an impurity, and this ethylstyrene may actually function as the styrene-based monomer.

[0025] In the present invention, the monomer mixture containing a styrene-based monomer may contain, in addition to the styrene-based monomer, an acyloxystyrene-based monomer, a divinylbenzene-based monomer, and (meth)acrylonitrile. Note that "(meth)acrylonitrile" refers to "acrylonitrile" or "methacrylonitrile", or "both acrylonitrile and methacrylonitrile".

[0026] The term "acyloxystyrene-based monomer" refers to acyloxystyrene or a substituted acyloxystyrene, preferably p-acetoxystyrene. Examples of substituted acyloxystyrene include compounds in which one or more hydrogen atoms other than the acyloxy group are substituted with an alkyl group having 1 to 5 carbon atoms (e.g., methyl, ethyl, propyl, butyl, or amyl), a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms (e.g., methoxy, ethoxy, propoxy, butoxy, or pentyloxy), or a nitro group. The acyloxy group is a substituent represented by the general formula X-CO-O- (wherein X is an alkyl group or a phenyl group), preferably an acyloxy group in which X is an alkyl group having 1 to 5 carbon atoms (e.g., methyl, ethyl, propyl, butyl, or amyl), and more preferably an acetoxy group. The acyloxy group is preferably positioned para to the vinyl group, but may also be positioned ortho or meta. Specific examples of the acyloxystyrene monomer include p-acetoxystyrene and p-benzoxystyrene.

[0027] The divinylbenzene monomer refers to divinylbenzene or a substituted divinylbenzene, preferably divinylbenzene. Substituted divinylbenzene includes, for example, compounds in which one or more hydrogen atoms of divinylbenzene are substituted with an alkyl group having 1 to 5 carbon atoms (e.g., methyl, ethyl, propyl, butyl, or amyl), a halogen atom, a carboxyl group, a sulfonic acid group, a cyano group, an alkoxy group having 1 to 5 carbon atoms (e.g., methoxy, ethoxy, propoxy, butoxy, or pentyloxy), or a nitro group. The two vinyl groups may be located at the para, meta, or ortho positions. Specific examples of the divinylbenzene monomer include p-divinylbenzene, m-divinylbenzene, o-divinylbenzene, or mixtures thereof.

[0028] In the present invention, the monomer mixture containing a styrene-based monomer preferably contains a styrene-based monomer as well as p-acetoxystyrene, divinylbenzene and methacrylonitrile.

[0029] In the present invention, suspension polymerization is carried out by stirring and emulsifying a mixture of a monomer mixture containing a styrene-based monomer and an organic solvent in water. The organic solvent in the present invention refers to a solvent other than water in the suspension polymerization system, and preferably includes hydrocarbons and alcohols. Examples of the hydrocarbon include saturated or unsaturated aliphatic hydrocarbons and aromatic hydrocarbons. These hydrocarbons are preferably aliphatic hydrocarbons having 5 to 12 carbon atoms, more preferably n-hexane, n-heptane, n-octane, isooctane, undecane, and dodecane. Examples of the alcohol include aliphatic alcohols, preferably aliphatic alcohols having 5 to 12 carbon atoms, more preferably 2-ethylhexyl alcohol, t-amyl alcohol, nonyl alcohol, 2-octanol, decanol, lauryl alcohol, and cyclohexanol.

[0030] The dispersion stabilizer is not particularly limited, and examples thereof include conventionally known hydrophilic protective colloids such as polyvinyl alcohol, polyacrylic acid, gelatin, starch, and carboxymethyl cellulose, and sparingly soluble powders such as calcium carbonate, magnesium carbonate, calcium phosphate, barium sulfate, calcium sulfate, and bentonite.

[0031] The polymerization initiator is not particularly limited, and examples thereof include conventionally known dibenzoyl peroxide, dilauroyl peroxide, distearoyl peroxide, 1,1-di(t-butylperoxy)-2-methylcyclohexane, 1,1-di(t-hexylperoxy)-3,3,5-trimethylcyclohexane, 1,1-di(t-hexylperoxy)cyclohexane, 1,1-di(t-butylperoxy)cyclohexane, di-t-hexyl peroxide, t-butylcumyl peroxide, and the like. Examples of suitable suspension polymerization initiators include peroxides such as di-t-butyl peroxide, 1,1,3,3-tetramethylbutylperoxy-2-ethylhexanoate, t-hexylperoxy-2-ethylhexanoate, t-butylperoxy-2-ethylhexanoate, and t-butylperoxyisopropyl monocarbonate, and azo compounds such as 2,2'-azobisisobutyronitrile, 2,2'-azobis-2-methylbutyronitrile, and 2,2'-azobis-2,4-dimethylvaleronitrile. The reaction conditions for suspension polymerization can be appropriately set, and examples include stirring at 60 to 90°C for 2 to 48 hours.

[0032] In one aspect, the present invention relates to a method for producing oligonucleotides, in which nucleosides or nucleotides are sequentially bound directly or indirectly to the porous polymer beads in the collection of porous polymer beads of the present invention. In the present invention, the method for producing an oligonucleotide can be a conventionally known method, and for example, can be carried out using the so-called phosphoramidite method, which may include, for example, the following steps: (a) removing (deprotecting) a protecting group from a protected nucleoside that is directly or indirectly supported on porous polymer beads and has a protecting group bound to a hydroxyl group, thiol group, or amino group at the 3'- or 5'-position; (b) binding (coupling) a nucleoside phosphoramidite to the hydroxyl group, thiol group, or amino group at the 3'- or 5'-position of the nucleoside that is directly or indirectly supported on the porous polymer beads from which the protecting group has been removed, in the presence of an activating agent; (c) sulfurizing or oxidizing the bond formed in step (b); and (d) capping any unbound hydroxyl group, thiol group, or amino group at the 3'- or 5'-position of the nucleoside that is directly or indirectly supported on the porous polymer beads.

[0033] In the present invention, a nucleoside refers to a compound in which a nucleoside base and a sugar are bonded, and may be a naturally occurring nucleoside such as adenosine, thymidine, guanosine, cytidine, or uridine, or a modified nucleoside. Modified nucleosides include, but are not limited to, nucleosides in which the hydroxyl group at the 3' or 5' position of the nucleoside is substituted with a thiol group or an amino group. The nucleoside base may be a naturally occurring base such as adenine, guanine, cytosine, thymine, or uracil, or a modified nucleoside base. The sugar portion of the nucleoside may be naturally occurring deoxyribose or ribose, and may be in the D-configuration or the L-configuration.

[0034] In the present invention, a nucleotide refers to a compound in which a nucleoside base, a sugar, and a phosphate are bonded, and may be a naturally occurring nucleotide such as adenosine triphosphate, thymidine triphosphate, guanosine triphosphate, cytidine triphosphate, or uridine triphosphate, or a modified nucleotide. The nucleoside base portion of the nucleotide may be a naturally occurring base such as adenine, guanine, cytosine, thymine, or uracil, or a modified nucleoside base. The sugar portion of the nucleoside may be naturally occurring deoxyribose or ribose, and may be in the D- or L-configuration. The phosphate portion may be, for example, phosphorothioate, phosphorodithioate, methylphosphonate, or methylphosphate.

[0035] In the present invention, an oligonucleotide refers to a compound having a structure in which nucleoside bases, sugars, and phosphates are linked together via phosphodiester bonds. This term includes naturally occurring oligonucleotides, such as 2'-deoxyribonucleic acid (hereinafter "DNA") and ribonucleic acid (hereinafter "RNA"), as well as nucleic acids containing modified sugar moieties, modified phosphate moieties, or modified nucleobases. Modifications to the sugar moiety include replacing the ribose ring with a hexose, cyclopentyl, or cyclohexyl ring. Alternatively, the D-ribose ring of naturally occurring nucleic acids may be replaced with an L-ribose ring, or the β-anomer of naturally occurring nucleic acids may be replaced with an α-anomer. An oligonucleotide may also contain one or more abasic moieties. Modified phosphate moieties include phosphorothioates, phosphorodithioates, methylphosphonates, and methylphosphates. Such nucleic acid analogs are known to those skilled in the art. Oligonucleotides comprising mixtures of two or more of the above can be prepared, for example, from oligonucleotides comprising mixtures of deoxyribonucleosides and ribonucleosides, particularly mixtures of deoxyribonucleosides and 2'-O-substituted ribonucleosides such as 2'-O-methyl or 2'-O-methoxyethyl ribonucleosides. Examples of oligonucleotides comprising mixtures of nucleosides include ribozymes.

[0036] In the present invention, a nucleoside phosphoramidite refers to a nucleoside derivatized with an amidite. In the present invention, a nucleoside phosphoramidite has either the 3'- or 5'-hydroxyl group of the nucleoside phosphoramidite converted to a phosphoramidite, and the other hydroxyl group is bound to a protecting group. The amiditization can be carried out, for example, by using 1H-tetrazole as an activating agent and reacting a suitably protected nucleoside with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite.

[0037] In the present invention, an activating agent refers to an agent that activates a nucleoside phosphoramidite, and is used to react with a nucleoside, nucleotide, or oligonucleotide. It is also referred to as an activator or coupling agent. In the present invention, activating agents commonly used in the phosphoramidite method can be used. Examples of activating agents that can be used in the present invention include, but are not limited to, 4,5-dicyanoimidazole, 5-(ethylthio)-1H-tetrazole, 5-(benzylthio)-1H-tetrazole, and saccharin 1-methylimidazole.

[0038] In the method for producing the oligonucleotide of the present invention, nucleosides or nucleotides may be sequentially bound to the porous polymer beads indirectly. For example, a linker may be bound to the hydroxyl group of the porous polymer beads of the present invention, and phosphoramidites may be bound to the end of the linker so as to form a predetermined base sequence. The linker may be cleavable, for example, a linker that is cleaved by hydrolysis. By cleaving the linker, nucleosides or nucleotides may be sequentially bound to the porous polymer beads via the cleavable linker, and then the linker may be cleaved to obtain the desired oligonucleotide.

[0039] 1. Preparation of porous polymer bead aggregates [Preparation of porous polymer beads] (Suspension copolymerization) A 2 L separable flask equipped with a cooler, a stirrer, and a nitrogen inlet tube was placed in a hot water circulator, and 6.5 g of polyvinyl alcohol (manufactured by Kuraray) and 650 g of distilled water were added and dissolved by stirring at 150 rpm. Separately, 120.2 g of styrene (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (66.8% by mass relative to the total monomers), 27 g of methacrylonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (15% by mass relative to the total monomers), 14.8 g of p-acetoxystyrene (manufactured by Tosoh Finechem Co., Ltd.) (8.2% by mass relative to the total monomers), 18 g of divinylbenzene (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 55%) (10% by mass relative to the total monomers), 206.6 g of 2-ethylhexanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 88.6 g of isooctane (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 4 g of benzoyl peroxide (manufactured by Nippon Oil & Fats Co., Ltd., 2% water content) were mixed and dissolved, and the resulting solution was added to the separable flask. Under a nitrogen stream, the mixture was stirred at 325 rpm using a stirring blade at room temperature, and then the temperature was raised to 80 ° C., and suspension copolymerization was carried out for 10 hours.

[0040] (Washing) The obtained polymerization product was filtered and washed using distilled water, acetone (Fujifilm Wako Pure Chemical Industries, Ltd.), and methanol (Fujifilm Wako Pure Chemical Industries, Ltd.). (Hydrolysis) The above styrene-methacrylonitrile-acetoxystyrene-divinylbenzene copolymer powder and 280 g of 2-propanol (Fujifilm Wako Pure Chemical Industries, Ltd.) were placed in a 2 L separable flask and stirred at 280 rpm to disperse. A mixed solution of 5.2 g of sodium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd.) and 150 g of distilled water was added to the mixture, and the temperature was raised to 80°C to carry out a hydrolysis reaction for 1 hour. The mixture was neutralized with hydrochloric acid and then filtered and washed using distilled water, acetone, and methanol. The dispersion was filtered and dried under reduced pressure to obtain a roughly purified porous polymer bead aggregate consisting of a powdered styrene-methacrylonitrile-acetoxystyrene-divinylbenzene copolymer.

[0041] [Purification of Porous Polymer Bead Aggregates] Example 1: 50 g of the crudely purified porous polymer bead aggregate obtained above was placed in a 900 mL reagent bottle, and methanol (viscosity at 25°C: 0.54) was added so that the liquid level was 10 cm above the bottom of the reagent bottle to prepare a test solution. A stirring blade was placed in the test solution at a height of approximately 1 cm above the bottom of the reagent bottle. After stirring and dispersing the mixture with the stirring blade at 200 rpm for 1 minute, the rotation of the stirring blade was stopped and the mixture was allowed to stand for 3 minutes. The test solution (dispersion) was then drawn up for 1 minute at a rate of 250 mL / min using a syringe placed 4 cm below the liquid surface, and an equal amount of methanol was added to the reagent bottle. This procedure was repeated 10 times, and the resulting test solution was dried under reduced pressure to obtain the porous polymer bead aggregate of Example 1.

[0042] Example 2 300 g of the roughly purified porous polymer bead aggregate obtained above was placed in an elbow jet classifier (manufactured by Nittetsu Mining Co., Ltd.) The classification edge angle on the fine powder side was set to 30 mm, and the air pressure was adjusted so that the yield of particles recovered as fine powder would be 85%, thereby obtaining the porous polymer bead aggregate of Example 2.

[0043] Comparative Example 1: The partially purified porous polymer bead aggregate obtained above was sieved and classified using a 70 μm mesh to obtain a porous polymer bead aggregate of Comparative Example 1. Comparative Example 2: 50 g of the partially purified porous polymer bead aggregate obtained above was placed in a 900 mL reagent bottle, and acetone (viscosity at 25°C: 0.30) was added so that the liquid level was 10 cm above the bottom of the reagent bottle to prepare a test solution. A stirring blade was placed in the test solution at a height of approximately 1 cm above the bottom of the reagent bottle. After stirring and dispersing with the stirring blade at 200 rpm for 1 minute, the rotation of the stirring blade was stopped and the solution was allowed to stand for 30 minutes (until the precipitate was no longer disturbed when tilted). The test solution (dispersion) was then drawn up for 1 minute at a rate of 250 mL / min using a syringe placed 4 cm below the liquid surface, and an equal amount of acetone was added to the reagent bottle. This procedure was repeated five times, and the resulting test solution was dried under reduced pressure to obtain a porous polymer bead aggregate of Comparative Example 2.

[0044] 2. Test Methods The following tests were performed on the porous polymer bead aggregates obtained in Examples 1 and 2 and Comparative Examples 1 and 2. [Particle Size Distribution Test] (Image Analysis Method) Porous polymer bead aggregates were dispersed in a 1:1 (volume:volume) mixture of ethanol and water. For this dispersion, the particle size distribution on a number basis was measured using a particle shape image analyzer (PITA-04, manufactured by Seishin Enterprises), and the following were calculated: - Particle frequency in particle size - Median value of particle size in a particle population composed of particle sizes of 20 μm or more - CV value of particle size in a particle population composed of particle sizes of 20 μm or more Here, the coefficient of variation (CV) value is calculated using the following formula: CV value [%] = Standard deviation of particle size [μm] ÷ Arithmetic mean value of particle size [μm] × 100 (Laser Diffraction / Scattering Particle Size Distribution Measurement Method) Porous polymer bead aggregates were dispersed in a 1:1 (volume:volume) mixture of ethanol and water. The particle size distribution of this dispersion was measured on a volume basis using a particle shape image analyzer (Mastersizer 3000, manufactured by Malvern), and the following was calculated: particle frequency at particle diameter

[0045] [Liquid Transportability Test] 3.09 g of porous polymer beads were packed into a synthesis column (diameter 2 cm, height 6 cm) and set in an oligonucleotide synthesizer (AKTA oligopilot plus 100, manufactured by Cytiva). Toluene was pumped into the column at 100 cm / h for 5 minutes, and then acetonitrile was pumped at a flow rate of 100 to 900 cm / h (the flow rate was changed in 100 cm / h increments) for 5 minutes each, and the maximum pressure of the synthesizer was measured. The liquid transportability test was terminated when the maximum pressure reached 20 bar.

[0046] 3. Test Results [Particle Size Distribution Test] (Number Distribution by Image Analysis) Table 1 shows the particle frequency by number distribution by image analysis for the porous polymer bead aggregates obtained in Examples 1 and 2 and Comparative Examples 1 and 2, and FIG. 1 shows a graph thereof.

[0047] Table 2 shows the median value and CV value of particle diameters in a particle group consisting of particles with diameters of 20 μm or more. (Laser diffraction / scattering particle size distribution measurement method) Figure 2 shows the particle frequency by volume distribution by laser diffraction / scattering particle size distribution measurement method for the porous polymer bead aggregates obtained in Examples 1 and 2 and Comparative Examples 1 and 2. Compared with the particle frequency by number distribution by image analysis method, the particle frequency is displayed as very low in the small particle size range, and it is thought that the particle frequency in the small particle size range is not captured in detail by the volume distribution by laser diffraction / scattering particle size distribution measurement method.

[0048] [Liquid Transportability Test] Table 3 and Figure 3 show the results of the maximum pressure at each flow rate (linear velocity). In Comparative Examples 1 and 2, the maximum column pressure reached 20 bar when the flow rate (linear velocity) reached 500 cm / h, and it was not possible to achieve a flow rate (linear velocity) of 500 cm / h or higher. On the other hand, in Examples 1 and 2, even when liquid was transported at a high flow rate (linear velocity) of 600 to 800 cm / h, the column pressure was at a low level, and it was expected that nucleic acids would be produced stably without causing liquid transport problems.

[0049]

Claims

1. A porous polymer bead assembly comprising a styrene copolymer, characterized in that: (a) the porous polymer bead assembly contains particles having a particle diameter of 20 μm or more and 50 μm or less at a particle frequency of 10% or less, as determined by counting using an image analysis method; and (b) the particle group of porous polymer beads having a particle diameter of 20 μm or more has a median particle diameter of 50 μm or more and 140 μm or less, and the coefficient of variation (CV value) of the particle diameter is 30% or less.

2. The porous polymer bead collection described in claim 1, wherein the porous polymer bead collection contains particles having a particle diameter of 20 μm or more and 40 μm or less at a particle frequency of 3% or less, based on the number of particles in the image analysis method.

3. A method for producing a collection of porous polymer beads according to claim 1 or 2, comprising the steps of: (a) suspension polymerizing a monomer mixture containing a styrene-based monomer to obtain porous polymer beads; (b) dispersing the porous polymer beads obtained in step (a) in an organic solvent and then leaving the mixture to stand for a standing time T (seconds) that satisfies the following formula 1 to obtain a dispersion; and (c) sampling the dispersion obtained in step (b) to a depth D (cm) from the liquid surface to obtain a collection of porous polymer beads. Formula 1: T = D x α T: standing time (seconds) D: sampling depth (cm) 20≦α (seconds / cm)≦60 4. A method for producing the porous polymer bead aggregate according to claim 1 or 2, comprising: (a) a step of suspension polymerizing a monomer mixture containing a styrene-based monomer to obtain porous polymer beads; and (b) a step of classifying the porous polymer beads obtained in step (a) using an elbow jet classifier to obtain a porous polymer bead aggregate.

5. A method for producing oligonucleotides, comprising sequentially binding nucleosides or nucleotides directly or indirectly to the porous polymer beads in the porous polymer bead collection according to claim 1 or 2.

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