Porous particles, and adsorbent and chromatography carrier containing same

Abstract

One embodiment of the present invention provides porous particles that contain a base carrier containing cellulose, and an oligo (dT) ligand that is immobilized on the base carrier, and that have a peak at a pore diameter of 200 nm or more in a pore distribution.

Description

Porous particles and adsorbents and chromatography supports containing them

[0001] This invention relates to porous particles, as well as adsorbents and chromatography carriers containing them. The invention also relates to a method for producing porous particles and a method for purifying nucleic acids using porous particles.

[0002] Monoliths, which are integrally molded bodies with a three-dimensional network-like framework and interconnected pores formed by its voids, are attracting attention as materials for chromatography used in the separation and purification of biopharmaceuticals. It is known that using monoliths enables separation and purification at higher volumes and processing speeds than conventional chromatography (Patent Document 1).

[0003] In recent years, particles with interconnected pores, formed from monoliths, have been proposed (Patent Document 2). Advantages of using particles include ease of handling, similar to packing materials for conventional chromatography separation columns, and ease of scale-up. Furthermore, by using particles with interconnected pores, the voids within the pores can be utilized as adsorption sites during the separation and purification of biomacromolecules, enabling higher processing volumes and faster processing speeds.

[0004] International Publication No. 2016 / 063702, Japanese Patent Publication No. 2023-037724

[0005] Against the background described above, there is a need for further improvement of porous particles that can be used in the separation and purification of substances. In particular, it is beneficial to develop porous particles that can exhibit excellent performance depending on the type and characteristics of the substance to be separated and purified. The present invention aims to provide porous particles that are particularly suitable for the adsorption and separation and purification of substances that can bind to oligo(dT) ligands.

[0006] As a result of diligent research, the present inventors have found that by immobilizing an oligo(dT) ligand onto cellulose particles having relatively large pore sizes, porous particles suitable for the adsorption, separation, and purification of relatively large molecular weight substances can be obtained, having a structure (e.g., poly(A) sequence) capable of binding to the ligand. The present invention is as follows, for example: [1] Porous particles comprising a cellulose-containing base carrier and an oligo(dT) ligand immobilized on the base carrier, wherein the pore distribution has a peak at a pore size of 200 nm or more. [2] The porous particles according to [1], wherein the ratio of the total volume of pores with a pore size of 200 nm or more to the total volume of pores with a pore size of 5 nm or more and less than 200 nm is 5.0 or more. [2-1] The porous particles according to [1] or [2], wherein the ratio of the total volume of pores with a pore size of 500 nm or more to the total volume of pores with a pore size of 500 nm or more and less than 500 nm is 4.0 or more. [2-2] The porous particle is spherical, as described in [1], [2], or [2-1]. [3] The porous particle is crosslinked cellulose, as described in [1], [2], [2-1], or [2-2]. [4] The BET specific surface area of ​​the porous particle is 1.0 to 50 m². ２[3] A porous particle according to [3], wherein the amount of oligo(dT) is 0.01 mg / g-dry or more. [5] A porous particle according to [3] or [4], wherein the crosslinked cellulose has a dry weight increase rate of 20% or more with respect to the dry weight of the cellulose before the crosslinking process. [6] A porous particle according to any one of [1] to [5], [2-1] and [2-2], wherein the oligo(dT) ligand comprises 5 or more thymine(T) repeating units. [7] A porous particle according to any one of [1] to [6], [2-1] and [2-2], wherein the amount of oligo(dT) supported is 0.01 mg / g-dry or more, based on the dry weight of the porous particle. [8] A porous particle according to any one of [1] to [7], [2-1] and [2-2], wherein the oligo(dT) ligand is immobilized on the base carrier via one or more active groups that are reactive with the ligand, and the active group is selected from the group consisting of a formyl group, a divinylsulfone group and an NHS group. [9] A porous particle according to any one of [1] to [8], [2-1], and [2-2], having a particle size of 1 to 300 μm.

[10] A porous particle according to any one of [1] to [9], [2-1], and [2-2], used in the separation and purification of nucleic acids having a poly(A) sequence.

[11] The porous particle according to

[10] , wherein the nucleic acid is mRNA.

[12] An adsorbent containing the porous particle according to any one of [1] to

[11] , [2-1], and [2-2].

[13] A chromatography carrier containing the adsorbent according to

[12] . [13-1] The chromatography carrier according to

[13] , having a 10% dynamic binding capacity (DBC) of 0.01 mg / mL-gel or more.

[14] A method for producing porous particles according to any one of [5] to

[11] , comprising: preparing crosslinked cellulose by adding a crosslinking agent to cellulose; and immobilizing an oligo(dT) ligand on a base carrier containing the crosslinked cellulose, wherein the crosslinking agent is added such that the crosslinked cellulose after the crosslinking step has a dry weight increase rate of 20% or more compared to the dry weight of the cellulose before the crosslinking step.

[15] The method according to

[14] , wherein the crosslinking agent is added in a number of moles 4 to 15 times the number of moles of monomers constituting the cellulose.

[16] A method for purifying a nucleic acid having a poly(A) sequence, comprising contacting a solution containing the nucleic acid having a poly(A) sequence with porous particles according to any of [1] to

[11] , [2-1], and [2-2].

[0007] According to the present invention, porous particles suitable for the adsorption, separation, and purification of substances that can bind to oligo(dT) ligands can be provided.

[0008] This figure shows the pore size distribution curve measured by the mercury porosimeter method (Example 3). This figure shows the absorbance measurement results at 260 nm of the poly(A) eluate (Example 3). This figure shows the absorbance measurement results at 260 nm of the poly(A) eluate (Example 4). This figure shows the absorbance measurement results at 260 nm of the poly(A) eluate (Example 5). This figure shows the absorbance measurement results at 260 nm of the mRNA (approximately 2,000 bases) eluate (Example 3). This figure shows the absorbance measurement results at 260 nm of the mRNA (approximately 2,000 bases) eluate (Example 8). This figure shows the absorbance measurement results at 260 nm of the mRNA (approximately 4,000 bases) eluate (Example 3).

[0009] The embodiments of the present invention will be described in detail below. According to one embodiment, porous particles are provided that comprise a cellulose-containing base carrier and an oligo(dT) ligand immobilized on the base carrier, and have a peak in the pore distribution at a pore size of 200 nm or more.

[0010] The inventors have found that by using porous particles with relatively large pore sizes to which oligo(dT) ligands are immobilized, substances that can bind to oligo(dT) ligands, particularly substances with relatively large molecular weights, can be successfully adsorbed and / or separated and purified. The porous particles according to the embodiment have interconnected pores consisting of a controlled three-dimensional network-like framework and voids, and these interconnected pores penetrate from the surface of the porous particle to the interior. Therefore, during the adsorption, separation, and purification of substances, the voids of the interconnected pores inside the particles can also be used as adsorption sites, enabling high processing rates and high-speed processing. Furthermore, because the pores have relatively large pore sizes, substances with relatively large molecular weights can be successfully adsorbed and / or separated and purified.

[0011] Substances that can bind to oligo(dT) ligands include those having a poly(A) sequence. The poly(A) sequence is located at the 3' end of mRNA, for example, and is a structure that plays an important role in mRNA stability, translation efficiency, and nuclear export. Oligo(dT) has the effect of recognizing and specifically adsorbing the poly(A) sequence. Therefore, the porous particles according to the embodiment can be used as an adsorbent for substances having a poly(A) sequence, and as a chromatography support for separating and purifying substances having a poly(A) sequence. In particular, the porous particles according to the embodiment can be suitably used as an adsorbent for substances with relatively large molecular weights, such as nucleic acids (e.g., mRNA), and as a chromatography support for separating and purifying nucleic acids (e.g., mRNA). Since mRNA is a very large molecule of about 30 to 50 nm, it has been difficult to use with conventional chromatography supports, and only surface adsorption has been achieved. In contrast, the porous particles according to the embodiment have a relatively large pore size as described above, and the voids of the interconnected pores inside the particles can also be used as adsorption sites. Therefore, the adsorption and separation / purification of mRNA, which was previously difficult, can be performed more efficiently.

[0012] The following describes in detail the constituent elements, manufacturing method, physical properties, and applications of the porous particles according to the embodiment. In this specification, "pore size" refers to the diameter of the pore. 1. Porous Particles The porous particles according to the embodiment include a cellulose-containing base carrier and an oligo(dT) ligand immobilized on the base carrier. The shape of the porous particles is not particularly limited, but a spherical shape is preferred because it has high mechanical strength, excellent gel sedimentation properties, and allows for the creation of a uniform packed bed. Here, "spherical" means, for example, that the major axis (longest diameter) is 2 times or less than the minor axis (shortest diameter). It is more preferable that the porous particles are approximately spherical, with the major axis and minor axis being close to the same length. The spherical shape of the porous particles allows for uniform packing into chromatography separation columns, etc., enabling more efficient adsorption and / or separation and purification of the target substance.

[0013] The porous particles according to this embodiment have a three-dimensional network-like framework and a communication pore structure consisting of its voids, and these communication pores penetrate from the particle surface to the interior. Whether or not a three-dimensional network-like framework and communication pores are present can be determined, for example, from measurements obtained by confocal laser microscopy or SEM observation of the cross-section of the porous particle. Furthermore, whether or not the communication pores penetrate from the particle surface to the interior can be confirmed, for example, by photographs of the surface and cross-section of the porous particle. In the porous particles according to this embodiment, the communication pores are not blocked at the particle surface but are open at the particle surface.

[0014] The porous particles according to the embodiment have a peak in the pore size distribution at a pore size of 200 nm or more (preferably 500 nm or more, more preferably 1 μm or more, and even more preferably 2 μm or more). The pore size distribution can be measured by the method described in the examples below, and "having a peak in the pore size distribution at a pore size of 200 nm or more" means that when a pore size distribution curve is created according to the method described in the examples, the peak of the curve is located at a pore size of 200 nm or more. This means that the particles contain many pores with relatively large diameters, which allows for more favorable adsorption and separation / purification of substances with relatively large molecular weights.

[0015] Furthermore, in the porous particles according to the embodiment, it is preferable that the ratio of the total volume of pores with a pore diameter of 200 nm to 10 μm to the total volume of pores with a pore diameter of 5 nm to less than 200 nm is 5.0 or more. The above ratio is more preferably 10 or more, and even more preferably 10 to 800. In particular, from the viewpoint of suitably adsorbing and then eluting and recovering substances having a poly(A) arrangement, it is preferable that the above ratio is 10 to 500, 10 to 100, or 10 to 50. Alternatively, in the porous particles according to the embodiment, it is preferable that the ratio of the total volume of pores with a pore diameter of 500 nm to 10 μm to the total volume of pores with a pore diameter of 5 nm to less than 500 nm is 4.0 or more. The above ratio is more preferably 5.0 or more, and even more preferably 5.0 to 100, or 5.0 to 50. In particular, from the viewpoint of suitably adsorbing and then eluting and recovering substances having a poly(A) arrangement, it is preferable that the above ratio is 5.0 to 40 or 5.0 to 15. By having relatively small pores (pore size 5 nm to less than 200 nm or 5 nm to less than 500 nm) and relatively large pores (pore size 200 nm to 10 μm or 500 nm to 10 μm) in the above-mentioned ratio, target substances, especially those with relatively large molecular weights, can be more effectively adsorbed and separated / purified.

[0016] The particle size of the porous particles is preferably 1 to 300 μm, and from the viewpoint of being usable as a packing material for chromatography, the particle size of the porous particles is particularly preferably 30 μm to 200 μm. Here, "particle size" refers to the measured value of the particle size of each porous particle, and "average particle size" refers to the average value calculated based on the above particle sizes, and in particular refers to the volume average particle size.

[0017] In this specification, the particle diameter and average particle diameter of porous particles can be measured, for example, using a laser diffraction / scattering particle size distribution analyzer. This analyzer irradiates a group of particles with laser light, determines the particle size distribution from the intensity distribution pattern of the diffracted / scattered light emitted, and calculates the particle diameter and average particle diameter based on this distribution. Specific measurement devices that can be used include the LA-960 laser diffraction / scattering particle size distribution analyzer (Horiba, Ltd.).

[0018] Alternatively, particle size can be measured using images taken with an optical microscope. Specifically, the particle size on the image is measured using a caliper or similar tool, and the original particle size is determined from the magnification. Then, the average particle size is calculated from the individual particle size values ​​obtained from the optical microscope image using the following formula: Volume average particle size (MV) = Σ(nd ４ ) / Σ(nd ３ [In the formula, d represents the particle diameter value of each particle obtained from the optical microscope image, and n represents the number of particles measured.]

[0019] The specific surface area of ​​the porous particles according to this embodiment, determined by the BET multipoint method (hereinafter also referred to as "BET specific surface area"), is preferably 1.0 to 50 m². ２ The value is / g. From the viewpoint of suitably adsorbing and then eluting and recovering the adsorbed poly(A) sequence, the BET specific surface area is more preferably 2.0 to 30 m². ２ / g, particularly preferably 2.0 to 20m ２ The value is / g. The BET specific surface area can be measured using, for example, a high-speed specific surface area / pore distribution analyzer (BELSORP MAXII manufactured by Microtrac Bell), as described in the examples below. With a BET specific surface area of ​​the above value, substances with relatively large molecular weights, such as those having a poly(A) arrangement, can be efficiently adsorbed and / or separated and purified.

[0020] (Base carrier) The base carrier in porous particles contains cellulose. The cellulose used here is not particularly limited and may be a cellulose derivative such as cellulose acetate, and may be crystalline cellulose or amorphous cellulose. The base carrier only needs to have cellulose as its main component and may or may not contain other materials. The main component here means a component whose content in the porous particles is 50% by mass or more. When using cellulose acetate, it can be used without particular restriction as long as it can be generally defined as cellulose acetate, but it is preferable that the degree of acetic acid is 45 to 57%. The base carrier can be manufactured, for example, by referring to Japanese Patent Application Publication No. 2023-037724.

[0021] Specifically, the base carrier can be manufactured by a method comprising: (a) heating and dissolving cellulose in a mixed solvent of a solvent in which cellulose is soluble (good solvent) and a solvent in which cellulose is insoluble (poor solvent) to prepare a cellulose solution; (b) dispersing the obtained cellulose solution in water containing an emulsifying stabilizer to obtain a dispersion system; and (c) cooling the obtained dispersion system to precipitate cellulose particles. The mixed solvent used is preferably an organic solvent that is immiscible with water.

[0022] While not limited to these, the above manufacturing method preferably produces a base carrier whose main component is cellulose, which is spherical, has a three-dimensional network-like framework and a pore structure consisting of interconnected holes, and whose interconnected holes penetrate from the particle surface to the interior. Furthermore, the particle size of the base carrier and the pore size of the interconnected holes can be easily controlled by changing the manufacturing conditions. For example, this can be controlled by the amount of cellulose used, the type and ratio of good and poor solvents used, the molecular weight and concentration of the polymer as the third component, the type and amount of surfactant added, the cooling rate, etc.

[0023] The base carrier is preferably, in particular, cross-linked cellulose particles. By using cross-linked cellulose particles as the base carrier, the porous particles according to the embodiment can adsorb poly(A) more efficiently, and excellent properties can be obtained when used in separation and purification. Furthermore, cross-linked cellulose particles have the advantage of being superior in mechanical strength, flow rate resistance, etc., compared to non-cross-linked particles.

[0024] The degree of crosslinking is not particularly limited, but it is preferable that the crosslinking agent be added such that the crosslinked cellulose has a dry weight increase rate of 20% or more compared to the dry weight of the cellulose before the crosslinking process. Here, the dry weight increase rate (%) is the ratio of the increase in the dry weight (g) of the cellulose after the crosslinking process to the dry weight (g) of the cellulose before the crosslinking process, and is a value calculated by [(dry weight of crosslinked cellulose after crosslinking process - dry weight (g) of cellulose before crosslinking process) / dry weight (g) of cellulose before crosslinking process × 100]. The above dry weight increase rate is more preferably 40% or more (e.g., 40 to 200%), even more preferably 50% or more (e.g., 50 to 200%), and particularly preferably 80% or more or 90% or more (e.g., 80 to 200%, 80 to 150%, 80 to 120%, 90 to 200%, 90 to 150%, or 90 to 120%). By appropriately designing and changing the experimental conditions, the crosslinking process can be carried out to achieve the desired dry weight increase rate.

[0025] The crosslinking process can be carried out by referring, for example, to Japanese Patent Publication No. 2009-242770, WO2017 / 141910, etc. More specifically, one example is a method that includes adding, in the presence of at least one inorganic salt selected from the group consisting of hydrochloride, sulfate, phosphate, and borate in a mole amount of 6 to 20 times the mole amount of cellulose monomer, 4 to 15 times the mole amount of cellulose monomer, and 0.1 to 1.5 times the mole amount of alkali to the crosslinking agent, to a suspension of uncrosslinked cellulose particles in the presence of 4 to 15 times the mole amount of cellulose monomer, and 0.1 to 1.5 times the mole amount of crosslinking agent, over a period of 3 hours or more. The amount of crosslinking agent to be added (in moles) is more preferably 7 to 15 times the mole amount of cellulose monomer, and particularly preferably 10 to 15 times.

[0026] Crosslinked cellulose particles have high mechanical strength and can be used under high flow rate chromatographic conditions. Here, "cellulose monomer" refers to the monomer that makes up cellulose, that is, the glucose unit which is the constituent unit of cellulose. The number of moles of cellulose monomer (i.e., degree of polymerization) is calculated based on the amount obtained by subtracting water from the glucose unit (i.e., the dry weight of cellulose) (1 mole is defined as a molecular weight of 162). The crosslinking agent can be appropriately selected from crosslinking agents commonly used in this field, but examples include epichlorohydrin, epibromohydrin, dichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, glycerol polyglycidyl ether, etc. Preferably, epichlorohydrin, epibromohydrin, or glycerol glycidyl ether is used.

[0027] (Ligand) The porous particles according to the embodiment include an oligo(dT) ligand immobilized on a cellulose-containing base carrier. The oligo(dT) ligand preferably contains 5 or more (e.g., 5 to 30), more preferably 10 or more (e.g., 10 to 30), and particularly preferably 15 or more (e.g., 15 to 30) thymine(T) repeating units. By using an oligo(dT) ligand containing thymine repeating units in the above range, substances having a poly(A) sequence can be specifically adsorbed and / or separated and purified.

[0028] The amount of oligo(dT) loaded onto the base carrier is not particularly limited, but is preferably 0.01 mg / g-dry or more (e.g., 0.01 to 20 mg / g-dry), more preferably 0.1 mg / g-dry or more (e.g., 0.1 to 20 mg / g-dry), and particularly preferably 1.0 mg / g-dry or more (e.g., 1.0 to 20 mg / g-dry), based on the dry weight of the porous particles. By using such a loading amount, a chromatography carrier can be obtained that maintains high adsorption capacity to the target substance, has excellent washability, and can be used repeatedly.

[0029] 2. Method for Manufacturing Porous Particles Porous particles according to the embodiment are manufactured by immobilizing oligo(dT) ligands on a base carrier. For example, when the base carrier is crosslinked cellulose, the particles are manufactured by a method that includes the steps of preparing crosslinked cellulose by adding a crosslinking agent to cellulose, and immobilizing oligo(dT) ligands on a base carrier containing crosslinked cellulose.

[0030] The immobilization of oligo(dT) ligands onto a base support can be carried out by referring to U.S. Patent Application Publication No. 2016 / 0024140, etc. For example, the base support can be pre-modified with one or more active groups that are reactive with the ligand, and then the ligand can be attached thereto. Specifically, one method is to add a modified oligo(dT) having an amino group at its terminus by reacting it with a base support into which active groups have been introduced. Examples of active groups include formyl groups, epoxy groups, divinyl sulfone groups, and N-hydroxysuccinimide (NHS) groups, with formyl groups or divinyl sulfone groups being preferred, and formyl groups being more preferred. Two or more of the active groups listed above may be used in combination, and other active groups may also be used in combination.

[0031] Alternatively, a method can be used in which a modified oligo(dT) is reacted with a base support using a condensing agent such as carbodiimide. The modified oligo(dT) here refers to an oligo(dT) whose 5'-terminus or 3'-terminus is modified with a reactive functional group (e.g., a carboxyl group, amino group, thiol group, etc.). Furthermore, an alkyl group or the like may be present as a linker between the oligo(dT) and the reactive functional group.

[0032] In order to obtain the desired properties, the base carrier can be appropriately modified before immobilizing the oligo(dT) ligand on the base carrier. For example, by adding a substance such as dextran to the base carrier, the pore diameter of the base carrier can be reduced. It is also expected to act as a spacer between the oligo(dT) ligand. The substance for modification is not particularly limited, and substances commonly used in the art can be appropriately selected and used according to the purpose.

[0033] 3. Adsorbent and chromatography carrier As described above, the porous particles according to the embodiment are preferably used in the adsorption and separation purification of substances having a poly(A) sequence. In one embodiment, the substance having a poly(A) sequence is a nucleic acid, and more specifically, mRNA.

[0034] According to one embodiment, an adsorbent containing the porous particles according to the embodiment is provided. The adsorbent can be used for the adsorption of substances having a poly(A) sequence (nucleic acids, particularly mRNA). The adsorbent may consist of the porous particles according to the embodiment or may contain additional materials. Also provided is a chromatography carrier containing the above adsorbent. The chromatography carrier may consist of the adsorbent according to the embodiment or may contain additional materials. The chromatography carrier can be preferably used in the separation and purification of substances having a poly(A) sequence (nucleic acids, particularly mRNA; hereinafter also referred to as "target substance"). Therefore, according to one embodiment, a method for purifying a substance having a poly(A) sequence (nucleic acids, particularly mRNA), which includes contacting a solution containing the substance having a poly(A) sequence (nucleic acids, particularly mRNA) with the porous particles according to the embodiment, is provided.

[0035] The usage modes of the adsorbent and the chromatography carrier are not limited. For example, the adsorbent or the chromatography carrier according to the embodiment can be filled in a separation column for chromatography and used. Also, the porous particles according to the embodiment can be further subjected to treatments such as saponification and modification with substituents before use.

[0036] In purification, first, a chromatographic carrier is packed into a column, and the packing mode is not particularly limited. Next, a sample solution containing the target substance is brought into contact with the chromatographic carrier to separate the target substance from the impurities. Specifically, the column is packed with the above-described chromatographic carrier, and the sample solution is passed therethrough to selectively adsorb the target substance onto the chromatographic carrier, whereby the target substance can be purified. Alternatively, both the target substance and the impurities are adsorbed onto the chromatographic carrier, and the elution conditions (e.g., salt concentration) are changed stepwise or continuously to utilize the difference in affinity for the chromatographic carrier to purify the target substance.

[0037] The degree of adsorption of the poly(A) sequence to the chromatographic carrier according to the embodiment can be evaluated by 10% dynamic binding capacity (DBC). When the chromatographic carrier according to the embodiment is used, for example, a 10% dynamic binding capacity (DBC) of 0.01 mg / mL-gel or more, 0.05 mg / mL-gel or more, 0.1 mg / mL-gel or more, 0.2 mg / mL-gel or more, or 0.3 mg / mL-gel or more can be achieved. In particular, a 10% dynamic binding capacity (DBC) of 0.05 to 0.17 mg / mL-gel, 0.08 to 0.15 mg / mL-gel, 0.09 to 0.13 mg / mL-gel, 0.05 to 1.0 mg / mL-gel, 0.05 to 0.60 mg / mL-gel, 0.20 to 0.60 mg / mL-gel, 0.30 to 0.60 mg / mL-gel, etc. can be achieved.

[0038] In setting the chromatographic conditions, the difference in affinity between the target substance and the impurities for the chromatographic carrier is utilized. For example, conditions are set in consideration of differences in carrier structure (ligand species, ligand density, ligand orientation, particle diameter, pore diameter, base matrix composition, etc.) and physicochemical properties (isoelectric point, charge, hydrophobicity, molecular structure, three-dimensional structure, etc.) of the target substance and the impurities. The conditions can be adjusted to perform chromatography in a binding-elution mode or a flow-through mode.

[0039] The components of the buffer solution, which can be used for washing sample solutions and columns, elution, etc., are not particularly limited as long as they have buffering capacity, but examples include phosphates, citrates, acetates, succinates, maleates, borates, Tris(base), HEPES, MES, PIPES, MOPS, TES, Tricinene, etc., in concentrations of 1 to 300 mmol / L. The above salts can also be used in combination with other salts such as sodium chloride, potassium chloride, calcium chloride, sodium citrate, sodium sulfate, ammonium sulfate, etc. Furthermore, the buffer solution may also contain amino acids such as glycine, alanine, arginine, serine, threonine, glutamic acid, aspartic acid, histidine, sugars such as glucose, sucrose, lactose, sialic acid, or derivatives thereof. Elution is preferably performed with pure water. The pH of the buffer solution is preferably in the range of 2 to 9, and more preferably in the range of 3 to 8. The linear velocity of the buffer solution is preferably in the range of 20 to 1000 cm / h.

[0040] According to the method of the embodiment, the target substance can be purified with a recovery rate of preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more. Here, the recovery rate means the ratio of the amount of the target substance recovered after purification to the amount of the target substance loaded onto the chromatography support (i.e., the amount of the target substance in the sample solution before purification).

[0041] Hereinafter, the present invention will be described in detail with reference to examples, but the content of the present invention is not limited thereto. <Example 1> (1) Production of Cellulose Particles 78 g of cellulose acetate (L-20, manufactured by Daicel) was added to a mixed solvent of 292 mL of benzyl alcohol, 264 mL of 1-hexanol, and 11 mL of polypropylene glycol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., diol type, average molecular weight 1,000), and stirred. Further, the temperature was raised and stirred at 120 °C for 4 hours to dissolve the cellulose acetate, and a transparent cellulose acetate solution was obtained. Also, 1.44 g of PVA (JP-18E, manufactured by Nippon Vinyl Poval Co., Ltd.) and 25.2 g of sodium carboxymethyl cellulose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to 1800 mL of saturated water saturated with the above mixed solvent, the temperature was raised and stirred at 80 °C for 1 hour or more to dissolve, thereby obtaining a dispersion medium. 600 g of the above cellulose acetate solution was quickly poured into 1800 mL of the dispersion medium, and stirred at 80 °C and a rotation speed of 200 rpm for 15 minutes to obtain a dispersion system. Subsequently, this dispersion system was cooled to 40 °C. When the temperature reached 40 °C, cellulose acetate precipitated, and spherical cellulose acetate particles were obtained. Thereafter, the obtained cellulose acetate particles were thoroughly washed with a large amount of water and then with methanol. After washing again with water, 600 g-wet of the obtained cellulose acetate particles were added to a mixed solution of 540 g of pure water and 160 g of methanol, the temperature was set to 35 °C and stirred for 15 minutes. Next, 140 g of 20% NaOH was added and stirred for 2 hours to react, thereby saponifying. The reaction mixture was filtered to recover the gel, and after filtering and washing with pure water, it was passed through sieves with a mesh size of 150 μm and a mesh size of 45 μm to obtain cellulose particles with a particle size of 45 to 150 μm. When the particle size of the obtained cellulose particles was measured with a particle size distribution measuring device LA-960 (manufactured by Horiba, Ltd.), the volume average particle size was 95 μm.

[0042] (2) Crosslinking Reaction 80 g-wet (dry weight 8.44 g-dry) of the cellulose particles obtained above was charged into a reactor equipped with a stirrer together with 122 mL of pure water. The temperature was raised to 40 °C, and while stirring was continued, 55 g of Na ２ SO ４ , 2.8 g of a 48% NaOH solution, 0.5 g of NaBH ４The mixture was dissolved and reacted over 30 minutes. After dissolution, 6.8 g of Denacol EX-313 (manufactured by Nagase ChemteX) was added and the mixture was reacted for more than 16 hours. Then, the temperature of the reaction solution was raised to 50°C, and 38 g of 48% NaOH solution and 43 g of epichlorohydrin, each divided into eight equal parts, were added at 30-minute intervals over approximately 4 hours. After the addition was complete, the mixture was reacted at 50°C for more than 16 hours. The temperature was lowered to below 30°C, and acetic acid was added to neutralize the mixture. The reaction mixture was filtered to recover the gel, which was then filtered and washed with pure water to obtain cross-linked cellulose particles. The dry weight increase rate of the obtained cross-linked cellulose particles was 56%. The dry weight increase rate (%) was calculated using the formula: [(Dry weight of cross-linked cellulose after the cross-linking process - Dry weight of cellulose before the cross-linking process (g)) / Dry weight of cellulose before the cross-linking process (g) × 100]. Furthermore, for every mole of cellulose monomer, 0.5 moles of Denacol EX-313 and 9.0 moles of epichlorohydrin were added.

[0043] (3) Formylation of cross-linked cellulose particles 20 g-wet of the cross-linked cellulose particles obtained above was charged into a reactor with a stirrer along with 66 mL of pure water. The internal temperature was adjusted to 30°C while stirring well. After stirring for 15 minutes, 20 mL of 1 wt% sodium periodate aqueous solution was added and the reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was filtered and thoroughly washed with water. Suction drying was performed to obtain 20 g-wet of formyl-activated cellulose particles.

[0044] (4) Modified oligo(dT) 24mer (manufactured by Bio-synthesis), which has a structure in which a linear alkyl group with 12 carbon atoms and a primary amino group at the binding end of the oligo(dT) is bonded to the 5' end of the oligo(dT), was dissolved in pure water to prepare a 10 mg / mL modified oligo(dT) aqueous solution. 189 μL of the modified oligo(dT) aqueous solution and 2.0 mL of 0.1 M sodium phosphate + 1.5 M sodium sulfate solution (pH = 12) were placed in a 15 mL tube. After mixing at 30°C for 15 minutes using a tube rotator, 1 g-wet of formyl-activated cellulose particles prepared above was added to the reaction mixture. After reacting at 30°C for 2 hours while mixing well using a rotator, 6 mg of NaBH ４The mixture was then added and the reaction was allowed to continue for at least one hour. After the reaction, the supernatant of the reaction solution was collected, and the reaction rate of oligo(dT) was calculated from the absorbance at 260 nm. The reaction rate was 54%, and the amount of oligo(dT) loaded was 5.0 mg / g-dry. The reacted gel was thoroughly washed with water and then collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles (porous particles). The absorbance used to calculate the reaction rate of oligo(dT) was measured using BioSpec-nano (Shimadzu Corporation).

[0045] <Example 2> Steps (1) to (3) were carried out in the same manner as in Example 1. (4) Modified oligo(dT) 15mer (manufactured by Eurofins Genomics), which has a structure in which a linear alkyl group with six carbon atoms and a primary amino group at the binding end of the oligo(dT) is bonded to the 5' end, was dissolved in pure water to prepare a 10 mg / mL aqueous solution of modified oligo(dT). 189 μL of the 10 mg / mL aqueous solution of modified oligo(dT) 15mer, 2.0 mL of 0.1 M sodium phosphate solution (pH = 10.0), and 0.9 g of sodium sulfate were placed in a 15 mL tube and mixed using a tube rotator at 30°C for 15 minutes. Then, 1 g-wet of formyl-activated cellulose particles obtained in step (3) was added to the reaction mixture and reacted for 2 hours. After the reaction, 7 mg of NaBH ４ The mixture was then added and reacted for more than one hour. The supernatant of the reaction solution was collected, and the reaction rate of oligo(dT) was calculated from the absorbance at 260 nm. The reaction rate was 88%, and the amount of oligo(dT) loaded was 7.6 mg / g-dry. The reacted gel was thoroughly washed with water and then collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles.

[0046] <Example 3> Step (1) was carried out in the same manner as in Example 1. (2) A crosslinking reaction was performed on the cellulose particles obtained in step (1). The reaction was carried out under the same conditions as in Example 1, except that Denacol EX-313 (manufactured by Nagase ChemteX) was changed to 13.6 g, and crosslinked cellulose particles were obtained. The dry weight increase rate of the obtained crosslinked cellulose particles was 91%. For every mole of cellulose monomer, 1.0 mole of Denacol EX-313 and 9.0 moles of epichlorohydrin were added.

[0047] (3) Formylation of cross-linked cellulose particles 15 g-wet of cross-linked cellulose particles obtained above was charged into a reactor with a stirrer along with 55 mL of pure water. The internal temperature was adjusted to 30°C while stirring well. After stirring for 15 minutes, 16 mL of 1 wt% sodium periodate aqueous solution was added and the reaction was carried out for 2 hours. After the reaction was completed, the reaction solution was filtered and thoroughly washed with water. Suction drying was performed to obtain 15 g-wet formyl-activated cellulose particles.

[0048] (4) Binding of Oligo(dT) 197 μL of a 10 mg / mL aqueous solution of modified oligo(dT) 15 mer (manufactured by Eurofins Genomics) used in Example 2, 2.0 mL of 0.1 M sodium phosphate solution (pH = 10.0), and 0.9 g of sodium sulfate were placed in a 15 mL tube and mixed using a tube rotator at 30°C for 15 minutes. Then, 1 g-wet of formyl-activated cellulose particles obtained above was added to the reaction mixture and reacted for 2 hours. After the reaction, 7 mg of NaBH ４ The mixture was then added and reacted for more than one hour. The supernatant of the reaction solution was collected, and the reaction rate of oligo(dT) was calculated from the absorbance. The reaction rate was 77%, and the amount of oligo(dT) loaded was 6.7 mg / g-dry. The reacted gel was thoroughly washed with water and then collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles.

[0049] <Example 4> Step (1) was carried out in the same manner as in Example 1. (2) A crosslinking reaction was performed on the cellulose particles obtained in step (1). The reaction was carried out under the same conditions as in Example 1, except that Denacol EX-313 (manufactured by Nagase ChemteX) was changed to 20.3 g, and crosslinked cellulose particles were obtained. The dry weight increase rate of the obtained crosslinked cellulose particles was 114%. For every mole of cellulose monomer, 1.5 moles of Denacol EX-313 and 9.0 moles of epichlorohydrin were added.

[0050] (3) Formylation of cross-linked cellulose particles 15 g-wet of cross-linked cellulose particles obtained above was charged into a reactor with a stirrer along with 66 mL of pure water. The internal temperature was adjusted to 30°C while stirring well. After stirring for 15 minutes, 20 mL of 1 wt% sodium periodate aqueous solution was added and the reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was filtered and thoroughly washed with water. The mixture was dried by suction to obtain 15 g-wet formyl-activated cellulose particles.

[0051] (4) Binding of Oligo(dT) 255 μL of 10 mg / mL aqueous solution of modified oligo(dT) 15 mer (manufactured by Eurofins Genomics) used in Example 2, 2.0 mL of 0.1 M sodium phosphate solution (pH = 10.0), and 0.9 g of sodium sulfate were placed in a 15 mL tube and mixed using a tube rotator at 30°C for 15 minutes. Then, 1 g-wet of formyl-activated cellulose particles obtained above was added to the reaction mixture and reacted for 2 hours. After the reaction, 7 mg of NaBH ４ The mixture was then added and reacted for more than one hour. The supernatant of the reaction solution was collected, and the reaction rate of oligo(dT) was calculated from the absorbance. The reaction rate was 77%, and the amount of oligo(dT) loaded was 6.7 mg / g-dry. The reacted gel was thoroughly washed with water and then collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles.

[0052] <Example 5> Step (1) was carried out in the same manner as in Example 1. (2) A crosslinking reaction was performed on the cellulose particles obtained in step (1). The reaction was carried out under the same conditions as in Example 1, except that the amount of Denacol EX-313 (manufactured by Nagase ChemteX) was changed to 27.1 g, and crosslinked cellulose particles were obtained. The dry weight increase rate of the obtained crosslinked cellulose particles was 196%. In addition, 2.0 moles of Denacol EX-313 and 9.0 moles of epichlorohydrin were added for every 1 mole of cellulose monomer.

[0053] (3) Formylation of cross-linked cellulose particles 15 g-wet of the cross-linked cellulose particles obtained above was charged into a reactor with a stirrer along with 86 mL of pure water. The internal temperature was adjusted to 30°C while stirring well. After stirring for 15 minutes, 26 mL of 1 wt% sodium periodate aqueous solution was added and the reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was filtered and thoroughly washed with water. The mixture was dried by suction to obtain 15 g-wet formyl-activated cellulose particles.

[0054] (4) Binding of Oligo(dT) 331 μL of 10 mg / mL aqueous solution of modified oligo(dT) 15 mer (manufactured by Eurofins Genomics) used in Example 2, 2.0 mL of 0.1 M sodium phosphate solution (pH = 10.0), and 0.9 g of sodium sulfate were placed in a 15 mL tube and mixed using a tube rotator at 30°C for 15 minutes. Then, 1 g-wet of formyl-activated cellulose particles obtained above was added to the reaction mixture and reacted for 2 hours. After the reaction, 12 mg of NaBH ４ The mixture was then added and reacted for more than one hour. The supernatant of the reaction solution was collected, and the reaction rate of oligo(dT) was calculated from the absorbance. The reaction rate was 26%, and the amount of oligo(dT) loaded was 2.3 mg / g-dry. The reacted gel was thoroughly washed with water and then collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles.

[0055] <Example 6> Step (1) was carried out in the same manner as in Example 1. (2) 80 g-wet cellulose particles obtained in the crosslinking reaction step (1) were charged into a reactor with a stirrer together with 122 mL of pure water. The temperature was raised to 50°C, and while stirring was continued, 55 g of Na ２ SO ４2.8 g of 48% NaOH solution and 0.5 g of NaBH4 were added and dissolved and reacted over 30 minutes. Then, 38 g of 48% NaOH solution and 43 g of epichlorohydrin, each divided into eight equal parts, were added at 30-minute intervals over approximately 4 hours. After the addition was complete, the reaction was carried out at 50°C for more than 16 hours. After the reaction, the temperature was lowered to below 30°C and neutralized with acetic acid. The reaction mixture was filtered to recover the gel, which was then filtered and washed with pure water to obtain cross-linked cellulose particles. The dry weight increase rate of the obtained cross-linked cellulose particles was 40%. Note that 9.0 moles of epichlorohydrin were added for every 1 mole of cellulose monomer.

[0056] (3) Dextran addition and formylation of cross-linked cellulose particles 42 g wet cross-linked cellulose particles obtained above were charged into a stirrer-equipped reactor with 71 mL of pure water and 34 g of epichlorohydrin, and the temperature was adjusted to 30°C. 38 g of 48% NaOH solution was added over 1 hour, and the reaction was carried out for a further 3 hours. After neutralizing the reaction solution with acetic acid, the gel was thoroughly washed with water to obtain epoxy-activated cellulose particles. Next, 24 g of dextran EH (manufactured by Meito Sangyo Co., Ltd., average molecular weight approximately 200,000) and 60 g of pure water were charged into a stirrer-equipped reactor, and after adjusting the temperature to 30°C, the mixture was stirred and dissolved. 40 g wet epoxy-activated cellulose particles obtained and 6 g of 48% NaOH solution were added, and the reaction was carried out at 30°C for 18 hours or more. After the reaction, 0.6 g of NaBH ４ The mixture was added and the reaction was allowed to continue for more than 16 hours. The reaction mixture was filtered to collect the gel, which was then filtered and washed with pure water to obtain dextran-added particles. Next, 12 g of wet dextran-added particles were placed in a reactor with a stirrer along with 40 mL of pure water. The internal temperature was adjusted to 30°C while stirring well. After stirring for 15 minutes, 12 mL of 1 wt% sodium periodate aqueous solution was added and the reaction was carried out for 2 hours. After the reaction was complete, the reaction solution was filtered, thoroughly washed with pure water, and dried by suction to obtain formyl-activated dextran-added particles.

[0057] (4) Binding of Oligo(dT) 169 μL of a 10 mg / mL aqueous solution of modified oligo(dT) 15 mer (manufactured by Eurofins Genomics) used in Example 2, 2.0 mL of 0.1 M sodium phosphate solution (pH = 10.0), and 0.9 g of sodium sulfate were placed in a 15 mL tube and mixed using a tube rotator at 30°C for 15 minutes. Then, 1 g-wet of the formyl-activated dextran-added particles obtained above was added to the reaction mixture and reacted for 2 hours. After the reaction, 6 mg of NaBH ４ The mixture was then added and reacted for more than one hour. The supernatant of the reaction solution was collected, and the reaction rate of oligo(dT) was calculated from the absorbance. The reaction rate was 83%, and the amount of oligo(dT) loaded was 7.2 mg / g-dry. The reacted gel was thoroughly washed with water and then collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles.

[0058] <Example 7> Steps (1) and (2) were carried out in the same manner as in Example 6. (3) Divinyl sulfonation step of cross-linked cellulose particles 8 g-wet obtained in step (2) was charged into a reactor with a stirrer together with 34 mL of 0.25 M sodium carbonate solution, and 106 μL of divinyl sulfonate was added and the reaction was carried out for 1 hour. The reacted gel was thoroughly washed with water and then recovered by suction filtration to obtain divinyl sulfonate-activated cellulose particles.

[0059] (4) Binding of Oligo(dT) 155 μL of a 10 mg / mL aqueous solution of modified oligo(dT) 15 mer (Eurofin Genomics) used in Example 2, 2.0 mL of 0.1 M sodium phosphate solution (pH = 10.0), and 0.9 g of sodium sulfate were placed in a 15 mL tube and mixed using a tube rotator at 30°C for 15 minutes. Then, the 1 g-wet divinylsulfone activated cellulose particles obtained above were added to the reaction mixture and reacted for 16 hours or more. The supernatant of the reaction mixture was collected and the reaction rate of oligo(dT) was calculated from the absorbance. The reaction rate was 99%, and the amount of oligo(dT) loaded was 8.6 mg / g-dry. Subsequently, 120 μL of 10% thioglycerin solution was added and the reaction was continued for another 16 hours or more to inactivate the unreacted divinylsulfone groups. The reacted gel was thoroughly washed with water and then collected by suction filtration to obtain 1 g-wet of oligo(dT)-modified cellulose particles.

[0060] <Example 8> Steps (1) and (2) were carried out in the same manner as in Example 6. (3) Epoxidation step of cross-linked cellulose particles 40 g-wet obtained in step (2) was charged into a reactor with a stirrer together with 54 mL of pure water. After adding 33.7 g of epichlorohydrin while stirring well, the internal temperature was adjusted to 30°C. Then, while controlling the rate of addition so that the liquid temperature was 30 ± 2°C, 31.6 g of 48% NaOH aqueous solution was added. The reaction was carried out at 30°C for 3 hours, after which 2.77 g of acetic acid was added and stirred for 10 minutes. The reaction solution was filtered and thoroughly washed with pure water. Suction filtration was performed to obtain 38 g-wet epoxy-activated cellulose particles.

[0061] (4) Carboxification of epoxy-activated cellulose particles 34 g-wet epoxy-activated cellulose particles obtained above were charged into a reactor with a stirrer along with 24 mL of pure water. After adding 13.4 g of aminohexanoic acid while stirring well, the internal temperature was adjusted to 45°C and the reaction was carried out with stirring for 2 hours. After that, the temperature was lowered to 30°C, and the reaction solution was filtered and thoroughly washed with pure water. Suction filtration was performed to obtain 35 g-wet carboxy-activated cellulose particles.

[0062] (5) NHS conversion of carboxy-activated cellulose particles 34 g-wet carboxy-activated cellulose particles obtained above were washed three times with dimethylformamide (DMF), and then charged with 71 g of DMF into a reactor with a stirrer. 2.5 g of N,N-hydroxysuccinimid was added while stirring well, and the internal temperature was adjusted to 30°C and stirred for 10 minutes. Then, 4.9 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added, and the mixture was stirred overnight at 30°C. After that, it was washed with methanol and isopropanol to obtain NHS-activated cellulose particles as an isopropanol slurry.

[0063] (6) 15 mer of modified oligo(dT) (manufactured by Integrated DNA Technologies), which has a structure in which a linear alkyl group with six carbon atoms and a primary amino group at the binding end of the oligo(dT) is bonded to the 5' end, was dissolved in a 0.1 M sodium borate + 3.0 M sodium chloride solution (pH = 8.5) to prepare a 10.8 mg / mL aqueous solution of modified oligo(dT). The isopropanol slurry obtained in step (5) was filtered through a glass filter and washed three times with a 3.0 M sodium chloride solution pre-cooled at 4°C. Then, 1.1 g-wet NHS activated cellulose particles filtered through a glass filter and 0.95 mL of the modified oligo(dT) solution were placed in a 5 mL tube and reacted by mixing at 25°C for 3 hours using a tube rotator. After the reaction, the supernatant of the reaction solution was collected, and the salt and desorbed NHS were removed from the supernatant using a centrifugal ultrafiltration device with an ultramolecular weight of 3,000 MWt. The reaction rate of oligo(dT) was calculated from the absorbance of the supernatant at 260 nm. The reaction rate was 22%, and the amount of oligo(dT) loaded was 11.1 mg / g-dry. After binding of oligo(dT), a 1.0 M monoethanolamine solution (pH = 9.0) was added, and the mixture was mixed using a tube rotator at 25°C for more than 1 hour to inactivate the unreacted NHS ester. The reacted gel was thoroughly washed with water and collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles.

[0064] <Example 9> Steps (1) and (2) were carried out in the same manner as in Example 6. (3) After replacing 129 g-wet of cross-linked cellulose particles obtained in step (2) of the NHS conversion of cross-linked cellulose particles with acetone, it was charged into a reactor with a stirrer together with 200 mL of anhydrous acetone and 16 g of N,N-disuccinimidyl carbonate. After stirring for 15 minutes, a mixed solvent of 15 mL of anhydrous triethylamine and 200 mL of dehydrated pyridine was added to the reaction solution over 45 minutes. After the addition was complete, the reaction was allowed to continue for another hour. The reacted gel was thoroughly washed with anhydrous acetone and anhydrous isopropanol, and then recovered as a slurry dispersed in anhydrous isopropanol.

[0065] (4) Modified oligo(dT) 23mer (manufactured by Integrated DNA Technologies), which has a structure in which a linear alkyl group with six carbon atoms and a primary amino group at the binding end of the oligo(dT) is bonded to the 5' end, was dissolved in a 0.1 M sodium borate + 0.5 M sodium chloride solution (pH = 8.5) to prepare a 10.0 mg / mL aqueous solution of modified oligo(dT). The anhydrous isopropanol slurry obtained in step (3) was filtered through a glass filter and washed three times with MilliQ water pre-cooled at 4°C. Then, 1.0 g-wet of the filtered particles and 1.00 mL of the modified oligo(dT) solution were placed in a 5 mL tube and reacted by mixing at 25°C for 3 hours using a tube rotator. After the reaction, the supernatant of the reaction solution was collected, and the salt and desorbed NHS were removed from the supernatant using a centrifugal ultrafiltration device with an ultramolecular weight of 10,000 MWt. The reaction rate of oligo(dT) was calculated from the absorbance of the supernatant at 260 nm. The reaction rate was 11%, and the amount of oligo(dT) loaded was 5.8 mg / g-dry. After the binding of oligo(dT), a 1.0 M monoethanolamine solution (pH = 9.0) was added, and the mixture was mixed using a tube rotator at 25°C for more than 1 hour to inactivate the unreacted NHS ester. The reacted gel was thoroughly washed with water and collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles.

[0066] <Comparative Example 1> (1) 10 g-wet of divinyl sulfonated cellulose particles Cellfine GH-25 (manufactured by JNC Corporation) was charged with 85 mL of 0.25 M sodium carbonate solution in a reactor with a stirrer, and 131 μL of divinyl sulfone was added and the reaction was carried out for 2 hours. After thoroughly washing the reacted gel with water, it was recovered by suction filtration to obtain divinyl sulfone-activated cellulose particles.

[0067] (2) Binding of Oligo(dT) 15 mer (Eurofin Genomics) modified oligo(dT) used in Example 2 was combined in a 15 mL tube with 182 μL of a 10 mg / mL aqueous solution, 2.0 mL of 0.1 M sodium phosphate solution (pH = 10.0), and 0.9 g of sodium sulfate. The mixture was mixed at 30°C for 15 minutes using a tube rotator. Then, 1 g-wet divinylsulfone activated GH-25 obtained above was added to the reaction mixture and the mixture was reacted for 16 hours or more. The supernatant of the reaction mixture was collected, and the reaction rate of oligo(dT) was calculated from the absorbance. The reaction rate was 99%, and the amount of oligo(dT) loaded was 4.3 mg / g-dry. Subsequently, 280 μL of 10% thioglycerin solution was added, and the mixture was reacted for another 16 hours or more to inactivate the unreacted divinylsulfone groups. The reacted gel was thoroughly washed with water and then recovered by suction filtration to obtain 1 g-wet oligo(dT)-modified GH-25.

[0068] <Comparative Example 2> (1) 25 g-wet of formylated cellulose particles Cellfine GH-25 (manufactured by JNC Corporation) was charged into a reactor with a stirrer along with 66 mL of pure water. The internal temperature was adjusted to 30°C while stirring well. After stirring for 15 minutes, 10 mL of 1 wt% sodium periodate aqueous solution was added and the reaction was carried out for 2 hours. After the reaction was completed, the reaction solution was filtered and thoroughly washed with water. Suction drying was performed to obtain 30 g-wet formyl-activated cellulose particles.

[0069] (2) Modified oligo(dT) 23mer (manufactured by Integrated DNA Technologies), which has a structure in which a linear alkyl group with six carbon atoms and a primary amino group at the binding end of the oligo(dT) is bonded to the 5' end, was dissolved in 0.1 M sodium borate solution (pH = 10.0) to prepare a 9.7 mg / mL aqueous solution of modified oligo(dT). 517 μL of the 9.7 mg / mL aqueous solution of modified oligo(dT) 23mer, 1.90 mL of 0.1 M sodium phosphate solution (pH = 10.0), and 0.896 g of sodium sulfate were placed in a 15 mL tube and mixed using a tube rotator at 30°C for 15 minutes. Then, 0.95 g-wet of formyl-activated cellulose particles obtained in step (1) was added to the reaction mixture and reacted for 2 hours. After the reaction, 4 mg of NaBH ４ The mixture was then added and reacted for more than one hour. The supernatant of the reaction solution was collected, and the reaction rate of oligo(dT) was calculated from the absorbance at 260 nm. The reaction rate was 98%, and the amount of oligo(dT) loaded was 13.0 mg / g-dry. The reacted gel was thoroughly washed with water and then collected by suction filtration to obtain 1 g-wet oligo(dT)-modified cellulose particles.

[0070] <Evaluation> The particles obtained in the above examples and comparative examples were evaluated as follows. The evaluation results are shown in Tables 1 and 2. (1) Pore distribution and pore volume The pore distribution and pore volume of the particles obtained in the examples and comparative examples were measured. The measurements were performed using a mercury porosimeter ("Autopore V9620" manufactured by micromeritics). From the obtained pore distribution curve (differential pore volume), the peaks of 10 μm or more, which are measured as interparticle gaps according to the measurement principle of the instrument, were excluded, and the pore diameter (pore size) located at the peak of the pore distribution curve (corresponding to) was read. For the particles obtained in Examples 1 to 9, the pore sizes corresponding to the peaks of the pore distribution curve were 2.4 μm (Example 1), 2.4 μm (Example 2), 2.5 μm (Example 3), 2.4 μm (Example 4), 2.4 μm (Example 5), 1.8 μm (Example 6), 2.0 μm (Example 7), 3.2 μm (Example 8), and 3.3 μm (Example 9), respectively. In other words, the peaks of the pore distribution curve exist at the positions of the above pore size values. For example, as shown in Figure 1, when the particles obtained in Example 3 were measured, the peak of the pore distribution curve was found at 2.5 μm.

[0071] Furthermore, the total volume of pores with a diameter of 5 nm or more and less than 200 nm (A) and the total volume of pores with a diameter of 200 nm or more and less than 10 μm (B) were calculated from the cumulative pore volume at each pore size. The ratio of these two values ​​[(B) / (A)] was also calculated. That is, for example, "total volume of pores with a diameter of 5 nm or more and less than 200 nm" means the sum of the volumes of each pore with a diameter of 5 nm or more and less than 200 nm present in the porous particles of each example. Similarly, the total volume of pores with a diameter of 5 nm or more and less than 500 nm (C) and the total volume of pores with a diameter of 500 nm or more and less than 10 μm (D) were calculated. The ratio of these two values ​​[(D) / (C)] was also calculated. Note that no pores were observed in the particles obtained in Comparative Example 1 and Comparative Example 2.

[0072] (2) BET specific surface area The BET specific surface area was measured for the particles obtained in the examples and comparative examples. The BET specific surface area was measured using a high-speed specific surface area / pore distribution analyzer (BELSORP MAX II, Microtrac-BEL). Freeze-dried porous particles were pre-treated by degassing at 100°C for 2 hours (vacuum degassing) to prepare the measurement samples. The BET specific surface area was calculated from the adsorption isotherm (straight line) obtained from the measurement of the amount of nitrogen gas adsorbed on the solid surface at a measurement temperature of 77.3 K and a relative pressure range of 0.05 to 0.30 at five or more points.

[0073] (3) Adsorption evaluation using poly(A) The particles obtained in the examples and comparative examples were used as a packing material for chromatography to evaluate the adsorption properties of poly(A). Each particle was dispersed in a buffer (0.25 M NaCl dissolved in 50 mM phosphate buffer; pH 7.0) and degassed under reduced pressure while stirring. After degassing, 0.29 mL-gel porous particles (5 mm inner diameter x 150 mm height) were packed into a Cytiva Tricorn 5 / 20 column. The column was mounted on an AKTA avant 25 (Cytiva) and equilibrated by flowing 10 times the column volume of the above buffer (pH 7.0).

[0074] Subsequently, 15 mL of a 0.02 mg / mL poly(A) solution, prepared by dissolving poly(A) 20 mer (Eurofin Genomics) in a buffer (0.25 M NaCl dissolved in 50 mM phosphate buffer; pH 7.0), was passed through the column. After the passage of the poly(A) solution was complete, the column was washed with 6 mL of the buffer (pH 7.0). After washing, linear gradient elution was performed using the buffer (pH 7.0) and pure water to elute the poly(A) adsorbed on the porous particles. After elution, washing was performed with 0.1 M NaOH. While the poly(A) solution was passing through the column, the absorbance of the eluate from the column outlet at 260 nm was measured. The total volume (mL) passed through until the absorbance of the eluate reached 1 / 10 of the absorbance of the 0.02 mg / mL poly(A) solution passed through was measured, and the 10% dynamic binding capacity (DBC) in this measurement system was calculated as follows. The absorbance was measured using the variable UV-Vis monitor of the AKTA Avant 25.

[0075] 10% DBC (mg / mL-gel) = {Total volume of eluate (mL) until the absorbance of the eluate at 260 nm reaches 1 / 10 of the absorbance of the POLIA solution passed through at 260 nm - Column volume (mL-gel)} × Concentration of POLIA solution (mg / mL) / Column volume (mL-gel)

[0076] (4) Adsorption evaluation using mRNA The particles obtained in the examples and comparative examples were used as chromatographic packing material to evaluate the adsorption of mRNA. The mRNA used for adsorption was produced by the following procedure. <Production of mRNA with approximately 4,000 base pairs> (Preparation of template DNA) A plasmid containing the target gene (SARS-Cov-2 S antigen) was incorporated into a psaRVac vector containing a transcription unit (T7 promoter, polyA sequence). This plasmid was transformed into E. coli (HST08) and cultured on an LB agar plate (with 100 μg / mL kanamycin added) at 37°C for 16 hours. Subsequently, single colonies were isolated from the colonies that appeared on the agar plate to obtain kanamycin-resistant transformants. The obtained transformants were cultured in Yeast extract medium (40 g / L, NaH) containing 50 μg / mL kanamycin and phosphate. ２ PO ４ ・2H ２ E. coli was inoculated into 50 mL of solution (14.5 g / L, pH 7) and incubated overnight at 37°C. After incubation, E. coli was recovered by centrifugation (6,000 g, 10 min, 4°C), and plasmid DNA was extracted using NucleoSnap Plasmid Midi (Takara Bio). The obtained plasmid DNA was further treated with restriction enzyme SapI, and then purified by phenol-chloroform extraction and ethanol precipitation to obtain a precipitate of linear DNA containing the target gene (SARS-Cov-2 S antigen). The obtained precipitate was dissolved in Nuclear-Free Water and analyzed by NanoDrop, yielding 160 μL of linear DNA solution with a concentration of 1.1 μg / μL and an OD260 / 280 ratio of 1.8 or higher.

[0077] (IVT reaction) Using linear DNA, the following IVT reaction solution was prepared and reacted at 32°C for 4 hours. Nuclease-Free Water 130 μL 10×T7 RNA Polymerase Buffer 40 μL ATP, UTP, GTP, CTP 10 mM each RNase Inhibitor Ver2 20 U Pyrophosphatase 0.1 U T7 RNA Polymerase Ver2.0 200 U Linear DNA 20 μg Total 400 μL

[0078] Lithium chloride solution (7.5 M) was added to the IVT reaction mixture to a final concentration of 3.0 M or less, and the mixture was thoroughly mixed. The mixture was allowed to stand at -20°C for 2 hours to precipitate the mRNA, and then centrifuged (15,000 g, 10 minutes, 4°C), after which the supernatant was removed. The precipitate was washed with 1 mL of 70% ethanol and centrifuged again under the same conditions. After removing the ethanol and lightly drying the precipitate at room temperature, it was dissolved in 100 μL of RNase Storage Sol. The obtained RNA solution was analyzed using an Agilent Fragment Analyzer. The main peak of mRNA was observed around the target base number (approximately 4,000 bases), confirming that full-length mRNA was the main component.

[0079] <Production of mRNA with approximately 2,000 base pairs> Linear DNA precipitate was obtained using the same procedure as for mRNA with approximately 4,000 base pairs, except that Positive Control Template (Fluc) (manufactured by Takara Bio) and restriction enzyme Hind III were used. Analysis with NanoDrop yielded 170 μL of linear DNA solution with a concentration of 2.0 μg / μL and an OD260 / 280 ratio of 1.8 or higher. (IVT reaction) Using template DNA, an RNA solution was obtained using the same procedure as for mRNA with approximately 4,000 base pairs, except that the reaction temperature was changed to 37°C for 2 hours. Analysis of the obtained RNA solution with a fragment analyzer revealed that the main peak of mRNA was around the target base pair (approximately 2,000 base pairs), confirming that full-length mRNA was the main component.

[0080] <Procedure for 10% DBC Measurement> Each particle was dispersed in a buffer (0.5 M NaCl dissolved in 50 mM phosphate buffer; pH 7.0) that had been pre-treated with diethyl pyrocarbonate (DEPC) to inactivate RNase, and degassed under reduced pressure while stirring. After degassing, 0.29 mL gel (5 mm inner diameter x 150 mm height) of the particles obtained in the examples and comparative examples was packed into a Cytiva Tricorn 5 / 20 column. The column was mounted on an AKTA avant 25 (Cytiva), and washing and equilibration were performed by running 0.1 M NaOH + 0.002 MEDTA and 10 times the amount of the above buffer (pH 7.0) through the column.

[0081] Subsequently, 20 mL (Example 3) and 18 mL (Example 8, Comparative Example 2) of a 0.03 mg / mL mRNA solution, prepared by mixing the mRNA solution prepared above with the buffer, were passed through the respective columns. After the mRNA solution had passed through, the columns were washed with 6 mL of the buffer (pH 7.0). After washing, linear gradient elution was performed using the buffer (pH 7.0) and DEPC-treated pure water to elute the mRNA adsorbed on the porous particles. After elution, washing was performed with 0.1 M NaOH. While the mRNA solution was passing through the column, the absorbance of the eluate at 260 nm from the column outlet was measured using the variable UV-Vis monitor of AKTA avant 25. The total volume (mL) passed through until the absorbance of the eluate reached 1 / 10 of the absorbance of the 0.02 mg / mL mRNA solution was measured, and the 10% dynamic binding capacity (DBC) in this measurement system was calculated as follows. 10% DBC (mg / mL-gel) = {Total volume of eluate (mL) until the absorbance of the eluate at 260 nm reaches 1 / 10 of the absorbance of the mRNA solution at 260 nm - Column volume (mL-gel)} × Concentration of mRNA solution (mg / mL) / Column volume (mL-gel)

[0082] Table 1 shows the 10% DBC values ​​for poly(A) and mRNA when using the particles obtained in the examples and comparative examples. Note that the mRNA (approximately 2,000 bases) used in Example 3 showed an absorbance of approximately 35 mAU (260 nm) immediately after loading, possibly due to impurities without the poly(A) moiety being present in the sample during production. Therefore, the 10% DBC was calculated by subtracting the absorbance immediately after loading from the absorbance of the sample (approximately 140 mAU) and using this value as the absorbance of the mRNA (approximately 2,000 bases). Similarly, for the mRNA (approximately 2,000 bases) used in Example 8 and Comparative Example 2, the 10% DBC was calculated by subtracting the absorbance of approximately 15 mAU (260 nm) detected immediately after loading and using this value as the absorbance of the mRNA (approximately 2,000 bases). Furthermore, for the mRNA (approximately 4,000 bases) used in Example 3, the absorbance of approximately 67 mAU (at 260 nm) detected immediately after loading was subtracted from the absorbance of the mRNA (approximately 4,000 bases) to calculate the 10% DBC. In addition, considering the effect of components eluted through flow-through immediately after loading, the concentration of the mRNA solution was corrected as follows: mRNA concentration (corrected) = mRNA concentration analyzed by NanoDrop × (absorbance at 260 nm in the chromatograph when the sample solution was passed through - absorbance at 260 nm detected immediately after loading) / absorbance at 260 nm in the chromatograph when the sample solution was passed through

[0083] When the porous particles of Examples 1 to 9 were used, poly(A) adsorption was confirmed, whereas when the particles of Comparative Examples 1 to 2 were used, poly(A) adsorption was not observed. From these results, it can be seen that the porous particles of the examples are useful as adsorbents for substances having poly(A) sequences. This is also evident from the fact that mRNA adsorption was confirmed when the porous particles of Examples 3 and 8 were used, while mRNA adsorption was not observed when the porous particles of Comparative Example 2 were used.

[0084] Furthermore, when poly(A) adsorbed on the porous particles was eluted with pure water, elution of poly(A) was confirmed in all examples. In particular, in Examples 3 to 5, the adsorbed poly(A) could be almost completely recovered by elution with pure water. This was confirmed by measuring the absorbance peak at 260 nm during the subsequent washing step with 0.1 M NaOH, and observing almost no peaks originating from poly(A) (Figure 2: Example 3, Figure 3: Example 4, Figure 4: Example 5). In Figures 2 to 4, the peaks appearing between 30 and 35 on the horizontal axis for the total liquid volume (mL) are peaks originating from poly(A) eluted in the washing step with 1 M NaOH. From these findings, it can be seen that by using the porous particles of the examples, substances containing poly(A) arrangements can be efficiently separated and purified.

[0085] Furthermore, when mRNA (approximately 2,000 bases) adsorbed on the porous particles of Example 3 and Example 8 was eluted with pure water, the elution of mRNA (approximately 2,000 bases) was confirmed (Figure 5: Example 3, Figure 6: Example 8). In addition, it was confirmed that almost no absorbance peak at 260 nm was observed during the subsequent washing step with 0.1 M NaOH. In Figures 5 and 6, the peaks appearing between 35 and 40 on the horizontal axis for the total volume of liquid passed through (mL) are peaks originating from mRNA eluted in the washing step with 1 M NaOH, and it was possible to recover almost the entire amount of mRNA (approximately 2,000 bases) by elution with pure water. Similarly, as shown in Figure 7, it was confirmed that mRNA (approximately 4,000 bases) adsorbed on the porous particles of Example 3 was also eluted with pure water, and that almost no absorbance peak at 260 nm was observed during the washing step with 0.1 M NaOH (between 38 and 43 on the horizontal axis for the total volume of liquid passed through (mL)). These findings indicate that mRNA, a substance having a poly(A) sequence, can be efficiently separated and purified by using the porous particles described in the examples.

[0086] While several embodiments of the present invention have been described, these embodiments are presented as examples only and are not intended to limit the scope of the invention. These novel embodiments can be carried out in a variety of other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims of the invention and its equivalents.

Claims

1. A porous particle comprising a cellulose-containing base carrier and an oligo(dT) ligand immobilized on the base carrier, wherein the pore distribution has a peak at a pore size of 200 nm or larger.

2. The porous particle according to claim 1, wherein the ratio of the total volume of pores with a pore size of 200 nm to 10 μm to the total volume of pores with a pore size of 5 nm to less than 200 nm is 5.0 or more.

3. The porous particle according to claim 1 or 2, wherein the cellulose is cross-linked cellulose.

4. The BET specific surface area of ​​the porous particles is 1.0 to 50 m². ２ The porous particles according to claim 3, wherein the particle size is / g.

5. The porous particles according to claim 3 or 4, wherein the crosslinked cellulose has a dry weight increase rate of 20% or more with respect to the dry weight of the cellulose before the crosslinking process.

6. The porous particle according to any one of claims 1 to 5, wherein the oligo(dT) ligand comprises five or more thymine(T) repeating units.

7. The porous particle according to any one of claims 1 to 6, wherein the amount of oligo(dT) loaded is 0.01 mg / g-dry or more, based on the dry weight of the porous particle.

8. The porous particle according to any one of claims 1 to 7, wherein the oligo(dT) ligand is immobilized on the base carrier via one or more active groups that are reactive with the ligand, and the active groups are selected from the group consisting of a formyl group, a divinylsulfone group, and an NHS group.

9. A porous particle according to any one of claims 1 to 8, wherein the particle size is 1 to 300 μm.

10. A porous particle according to any one of claims 1 to 9, used in the separation and purification of nucleic acids having a poly(A) sequence.

11. The porous particle according to claim 10, wherein the nucleic acid is mRNA.

12. An adsorbent comprising porous particles according to any one of claims 1 to 11.

13. A chromatography carrier comprising the adsorbent described in claim 12.

14. A method for producing porous particles according to any one of claims 5 to 11, comprising: preparing crosslinked cellulose by adding a crosslinking agent to cellulose; and immobilizing an oligo(dT) ligand on a base carrier containing the crosslinked cellulose, wherein the crosslinking agent is added such that the crosslinked cellulose after the crosslinking step has a dry weight increase rate of 20% or more compared to the dry weight of the cellulose before the crosslinking step.

15. The method according to claim 14, wherein the crosslinking agent is added in a number of moles 4 to 15 times the number of moles of monomers constituting the cellulose.

16. A method for purifying a nucleic acid having a poly(A) sequence, comprising contacting a solution containing a nucleic acid having a poly(A) sequence with porous particles according to any one of claims 1 to 11.

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