Porous particles, adhesive containing same, and chromatographic support

Abstract

One embodiment of the present invention provides porous particles that each include a base support containing a polysaccharide, and a phosphate ligand immobilized on the base support, wherein the cation ion exchange capacity per water-swelled particle volume is 0.1 meq / mL or more, and the degree of swelling is more than 8 mL / g.

Description

Porous particles and adsorbents and chromatography carriers containing the same

[0001] The present invention relates to porous particles and adsorbents and chromatography carriers containing the same. The present invention also relates to a method for producing the porous particles and a purification method using the porous particles.

[0002] Regarding the phosphorylation of polysaccharides, much research has been conducted on cellulose for a long time. Phosphated cellulose fibers and cellulose particles are widely used in ion exchange chromatography and affinity chromatography and are readily available (Patent Document 1). Fibrous cellulose phosphate is known to have poor column packing efficiency due to its shape and is not suitable for high flow rate processing. Therefore, spherical cellulose phosphate particles have attracted more attention.

[0003] As the biopharmaceutical market expands, attention is also growing on chromatographic materials used for the separation and purification of relatively large molecular substances such as antibodies. One such material is a monolith, which is an integrally molded body having a three-dimensional network structure and interconnected pores. It is known that the use of monoliths enables separation and purification at higher throughputs and speeds than conventional chromatography (Patent Document 2).

[0004] In recent years, monolith-based particles with interconnected pores have also been proposed (Patent Document 3). The advantages of using particles include ease of handling, as they can be used in the same way as packing materials in conventional chromatography separation columns, and ease of scaling up. Furthermore, by using particles with interconnected pores, the voids within the interconnected pores can also be used as adsorption sites during the separation and purification of biopolymers, etc., enabling separation and purification at higher throughputs and speeds.

[0005] US Patent No. 3,565,886 International Publication No. 2016 / 063702 Japanese Patent Application Laid-Open No. 2023-037724

[0006] In light of the above, there is a need for further improvements in porous particles that can be used in the separation and purification of substances. In particular, it would be beneficial to develop porous particles that can demonstrate excellent performance depending on the type and properties of the substances to be separated and purified.

[0007] The present inventors conducted extensive research into porous particles comprising a polysaccharide-containing base carrier and a phosphate ester ligand immobilized on the base carrier, and as a result, succeeded in producing porous particles having a predetermined cation exchange capacity and swelling degree. They also found that such porous particles can be used for adsorption and separation / purification of substances with relatively large molecular weights. The present invention includes, for example, the following: [1] A porous particle comprising a polysaccharide-containing base carrier and a phosphate ester ligand immobilized on the base carrier, the porous particle having a cation exchange capacity of 0.1 meq / mL or more per water-swollen volume and a swelling degree of greater than 8 mL / g. [2] The porous particle according to [1], wherein the polysaccharide is cellulose. [3] The porous particle according to either [1] or [2], wherein the 10% dynamic binding capacity (DBC) of IgG at a 2-minute residence time is 40 mg / mL or more. [4] The porous particle according to [1] or [2], wherein the 10% dynamic binding capacity (DBC) of IgG at a 2-minute residence time is 50 mg / mL or more. [5] The porous particle according to any one of [1] to [4], having a ratio of swelling degree to water content of 0.5 to 2.0. [6] The porous particle according to any one of [1] to [5], having a spherical shape, a granular shape, or a mixture thereof. [7] An adsorbent comprising the porous particle according to any one of [1] to [6]. [8] A chromatography carrier comprising the adsorbent according to [7]. [9] A method for producing the porous particle according to any one of [1] to [6], comprising: (1) a step of adding an organic solvent to a base carrier containing a polysaccharide to wash the base carrier; and (2) a step of adding, to the washed base carrier, a compound that reacts with hydroxyl groups in the polysaccharide to form a phosphate ester.

[10] The method according to [9], further comprising drying after step (1) and before step (2).

[11] The method according to [9] or

[10] , wherein the organic solvent is selected from the group consisting of dimethylformamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, acetone, and acetonitrile.

[12] A purification method comprising: contacting the porous particles according to any one of [1] to [6] with a solution containing a substance to be purified to adsorb the substance to the porous particles; and eluting and recovering the substance to be purified.

[13] The purification method according to claim

[12] , wherein the substance to be purified comprises one or more selected from the group consisting of an antibody, an enzyme, a nucleic acid, an antibody, a protein, and a virus.

[14] The porous particle according to any one of [1] to [6], for use in purifying a substance.

[0008] According to the present invention, it is possible to provide porous particles that can be used for adsorption, separation and purification of substances that can interact with phosphate ester ligands, particularly substances with relatively large molecular weights.

[0009] Fig. 1 is a microscopic view of particles in Example 1. Fig. 2 is a microscopic view of particles in Example 2. Fig. 3 is a microscopic view of particles in Example 3. Fig. 4 is a microscopic view of particles in Example 4. Fig. 5 is a diagram showing a flow of crude purification of a T7 RNA polymerase solution.

[0010] The porous particles of the present invention comprise a polysaccharide-containing base carrier and a phosphate ester ligand immobilized on the base carrier, and have a cation exchange capacity of 0.1 meq / mL or more per water-swollen volume and a swelling degree of more than 8 mL / g.

[0011] As a result of extensive research into porous particles comprising a polysaccharide-containing base carrier and a phosphate ester ligand immobilized on the base carrier, the present inventors have succeeded in producing porous particles having a cation exchange capacity of 0.1 meq / mL or more per water-swollen volume and a swelling degree of more than 8 mL / g. They have also found that such porous particles can be used for the adsorption, separation, and purification of substances that can interact with the phosphate ester ligand, particularly substances with relatively large molecular weights.

[0012] Furthermore, when porous particles are used as a chromatography carrier, the carrier's strength is required when processing at a high flow rate. However, in the case of porous particles containing a base carrier containing a polysaccharide and a phosphate ester ligand immobilized on the base carrier, the polysaccharide decomposition reaction is likely to proceed when a reaction is carried out at high temperatures during the production process, resulting in a problem of poor strength of the resulting porous particles. When porous particles with poor strength are used as a chromatography carrier, high-speed processing becomes difficult. On the other hand, when a reaction is carried out under milder temperature conditions, a problem may arise in which the amount of phosphate ester ligand bound to the base carrier is insufficient. However, the porous particles according to the embodiment have relatively excellent strength and a sufficient amount of phosphate ester ligand bound, and therefore can withstand high processing volumes and high-speed conditions, and also have excellent adsorption capacity.

[0013] As described above, the porous particles according to the embodiments can be used as adsorbents for substances capable of interacting with phosphate ester ligands, and as chromatography carriers for separating and purifying such substances. The porous particles according to the embodiments exhibit high adsorption capacity even for substances with relatively large molecular weights. Therefore, the porous particles according to the embodiments can be suitably used as adsorbents for substances with relatively large molecular weights that can interact with phosphate ester ligands, and as chromatography carriers for separating and purifying such substances. Examples of substances with relatively large molecular weights include, but are not limited to, substances having a molecular weight of 50 kDa or more, more specifically, enzymes, nucleic acids (e.g., mRNA), antibodies, viruses, etc. The porous particles according to the embodiments can efficiently separate and purify such substances.

[0014] The components, manufacturing method, physical properties, applications, etc. of porous particles according to the embodiment are described in detail below. 1. Porous Particles Porous particles according to the embodiment include a base carrier containing a polysaccharide and a phosphate ester ligand immobilized on the base carrier. The shape of the porous particles is not particularly limited, but spherical, granular, or a mixture thereof is preferred because of their high mechanical strength, excellent gel sedimentation, and the ability to produce a uniform packed bed. Here, spherical means, for example, that the major axis (longest diameter) is no more than twice the minor axis (shortest diameter). Granular refers to a spherical particle with a spherical surface partially recessed inward. More preferably, the porous particles are substantially spherical, with the major axis and minor axis being nearly equal in length. Porous particles that are spherical, granular, or a mixture thereof enable uniform packing into a chromatographic separation column or the like, thereby enabling more efficient adsorption and / or separation and purification of target substances.

[0015] The particle diameter of the porous particles is preferably 1 to 500 μm, and from the viewpoint of usability as a chromatography packing, the particle diameter of the porous particles is particularly preferably 10 to 200 μm. The average particle diameter of the porous particles is preferably 30 to 150 μm, more preferably 40 to 120 μm. Here, "particle diameter" refers to the measured particle diameter of each porous particle, and "average particle diameter" refers to an average value calculated based on the above particle diameters, and particularly refers to the volume-average particle diameter.

[0016] In this specification, the particle size and average particle size of porous particles can be measured, for example, using a laser diffraction / scattering particle size distribution analyzer. With this analyzer, a particle group is irradiated with laser light, and the particle size distribution is determined from the intensity distribution pattern of the diffracted / scattered light emitted from the particle group, and the particle size and average particle size are calculated based on this. A specific example of a measuring device that can be used is the laser diffraction / scattering particle size distribution analyzer LA-960 (Horiba, Ltd.).

[0017] Alternatively, particle diameters can be measured using images taken with an optical microscope. Specifically, particle diameters on the image are measured using a caliper or the like, and the original particle diameters are determined from the magnification of the image. Then, the average particle diameter is calculated from the values ​​of the particle diameters determined from the optical microscope image using the following formula: Volume average particle diameter (MV) = Σ(nd ４ ) / Σ(nd ３ ) (wherein d represents the particle diameter value of each particle determined from an optical microscope image, and n represents the number of particles measured.)

[0018] The porous particles according to the embodiment have a swelling degree greater than 8 mL / g. For example, the swelling degree is preferably greater than 8 mL / g and not greater than 15 mL / g, and more preferably greater than 8 mL / g and not greater than 13 mL / g. The swelling degree is an index indicating the volume to which a dry porous particle swells when it absorbs water. The swelling degree is calculated by placing porous particles swollen with water in a measuring cylinder or the like, measuring the water-swelled volume (the total volume of the swollen porous particles and the volume of the interparticle voids), and then calculating the swelling degree from the water-swelled volume and the weight of the dry porous particle. Here, the volume of the interparticle voids refers to the volume of the water-filled portions (voids) present between the porous particles packed in a column. More specifically, the swelling degree can be measured and calculated by the method described in the Examples below. A swelling degree within the above range has the advantage of efficiently adsorbing relatively large proteins, particularly those with a molecular weight of 50 kDa or more, and enabling separation and purification at a high throughput and high processing speed.

[0019] The porous particles according to the embodiment have a cation exchange capacity per water-swollen volume of 0.1 eq / mL or more (e.g., 0.1 to 1.0 meq / mL), more preferably 0.15 meq / mL or more (e.g., 0.15 to 0.7 meq / mL), and particularly preferably 0.2 meq / mL or more (e.g., 0.2 to 0.5 meq / mL). Having a cation exchange capacity within the above range provides the advantage of more effective adsorption of target substances and allows diffusion of the target substances into the interior of the porous particles, thereby maintaining high adsorption performance even under high flow rates. The cation exchange capacity can be measured by the method described in the Examples below.

[0020] The porous particles according to the embodiment preferably have a moisture content of 5 to 13, more preferably 5 to 11, and particularly preferably 6 to 10. The moisture content refers to the ratio of the weight of the suction-dried porous particles to the weight of the porous particles in a dry state, and is calculated by the formula "moisture content = weight of suction-dried porous particles / weight of dry porous particles." Specific measurement methods are as described in the Examples below.

[0021] The porous particles according to the embodiment have a ratio of swelling degree to water content (swelling degree / water content) of preferably 0.5 to 2.0, more preferably 0.8 to 1.7, and particularly preferably 1.0 to 1.5. When the ratio of swelling degree to water content is within the above range, the porous particles can be packed more uniformly and densely into a column or the like, which has the advantage of enabling efficient adsorption, separation, and purification.

[0022] (Base Carrier) The base carrier in the porous particles includes a polysaccharide. The polysaccharide to be used is not limited, but is a polysaccharide having a hydroxyl group in the molecule that can be phosphated. For example, it is a polysaccharide containing one or more of glucose, agarose, mannose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, allose, altose, allulose, fructose, sorbose, tagarose, or uronic acid derivatives, amino sugar derivatives, deoxy sugar derivatives, etc. as constituent elements.

[0023] Specifically, the polysaccharide is preferably selected from starch, glycogen, pullulan, dextran, nigeran, cellulose, laminaran, curdlan, gellan, mannan, carrageenan, inulin, levan, xylan, arabinan, pectin, chitin, and chitosan. When the porous particles according to the embodiment are used in so-called affinity chromatography, which utilizes biochemical affinity as the driving force for adsorption, polysaccharides capable of forming water-insoluble gels, such as cellulose, chitin, chitosan, dextran, agarose, and mannan, are more preferably used than synthetic polymers. The polysaccharides may be used alone or in combination of two or more.

[0024] As the polysaccharide, cellulose, particularly spherical cellulose, is preferably used. This is intended to indicate that an embodiment containing cellulose (particularly spherical cellulose) as the primary polysaccharide is preferred, i.e., other polysaccharides may be used in addition to cellulose. Spherical cellulose has the advantages of being inexpensive, highly biocompatible, strong, and resistant to column pressure, and is also autoclavable. The cellulose used here is not particularly limited and may be a cellulose derivative such as cellulose acetate, or may be crystalline or amorphous cellulose. Hereinafter, porous particles containing a cellulose-containing base carrier and a phosphate ester ligand immobilized on the base carrier are also referred to as "phosphated cellulose particles." The base carrier may contain a polysaccharide as its main component, and may or may not contain other materials. The term "main component" as used here refers to a component whose content in the porous particles is 50% by mass or more. When using cellulose acetate, any material that can be generally defined as cellulose acetate can be used without any particular limitation, but it is preferable for the acetylation degree to be 45-57%.

[0025] The base carrier can be produced by, for example, referring to Japanese Patent Application Laid-Open No. 55-44312. Specifically, the base carrier can be produced by dissolving a cellulose raw material in an aqueous calcium salt solution containing calcium thiocyanate as the main component, dispersing this solution or gel-like substance in a granular form in an organic solvent, and then desalting it with a solvent that dissolves calcium salts and is mixed with the dispersion solvent, thereby regenerating the cellulose into a gel-like form.

[0026] The base carrier may be crosslinked cellulose particles, which have the advantage of being superior in mechanical strength, flow rate resistance, etc. compared to non-crosslinked cellulose particles.

[0027] The crosslinking step can be carried out with reference to, for example, JP 2009-242770 A and WO 2017 / 141910. More specifically, for example, a method can be used that includes a step of continuously dropping or adding in portions to a suspension of uncrosslinked cellulose particles over a period of 3 hours or more in the presence of at least one inorganic salt selected from the group consisting of hydrochlorides, sulfates, phosphates, and borates in an amount 6 to 20 times the number of moles of cellulose monomer. A crosslinking agent in an amount 4 to 15 times the number of moles of cellulose monomer and an alkali in an amount 0.1 to 1.5 times the number of moles of crosslinking agent are added. The amount (in moles) of crosslinking agent added is preferably 7 to 15 times the number of moles of cellulose monomer, and particularly preferably 10 to 15 times the number of moles of cellulose monomer.

[0028] Crosslinked cellulose particles have high mechanical strength and can be used under chromatographic conditions with faster flow rates. Here, "cellulose monomer" refers to a glucose unit, which is a structural unit of cellulose. The number of moles of cellulose monomer (i.e., degree of polymerization) is calculated based on the amount of glucose unit minus water (i.e., the dry weight of cellulose) (a molecular weight of 162 is defined as 1 mole). The crosslinking agent can be appropriately selected from those commonly used in the field. Examples include epichlorohydrin, epibromohydrin, dichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, and glycerol polyglycidyl ether. Epichlorohydrin, epibromohydrin, and glycerol glycidyl ether are preferred.

[0029] (Ligand) Porous particles according to the embodiment include a phosphate ester ligand immobilized on a base carrier containing a polysaccharide. The phosphate ester ligand can be formed by reacting a base carrier containing a polysaccharide with a compound that forms a phosphate ester with a hydroxyl group of the polysaccharide. Such compounds are not limited as long as they can form a phosphate ester with a hydroxyl group in the polysaccharide. Phosphorus compounds, particularly phosphorus pentoxide, phosphorus oxychloride, polyphosphoric acid, phosphoric acid, and the like, are preferably used. Of these, phosphorus pentoxide and phosphorus oxychloride are preferred, with phosphorus pentoxide being more preferred. The use of such compounds allows the introduction of phosphate ester groups under mild conditions and is inexpensively available, making them advantageous for industrial-scale production.

[0030] 2. Method for Producing Porous Particles Porous particles according to the embodiment are produced by a method including, for example, the steps of: (1) adding an organic solvent to a base carrier containing a polysaccharide to wash the base carrier; and (2) adding a compound that reacts with hydroxyl groups in the polysaccharide to form a phosphate ester (hereinafter also referred to as a "phosphate ester-forming compound") to the washed base carrier.

[0031] In step (1), an organic solvent is added to a base carrier (gel) containing polysaccharides to wash the base carrier. This washes the base carrier with the organic solvent, i.e., the water in the base carrier is replaced with the organic solvent. The organic solvent used is not particularly limited, but organic solvents with high affinity for water are preferred, such as dimethylformamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, acetone, and acetonitrile. Washing can be performed by repeatedly adding an organic solvent to the base carrier, stirring, and filtering.

[0032] In step (2), a phosphate ester-forming compound is added to the washed base carrier. Specific examples of the compound are as described above, and the amount of the compound added can be appropriately adjusted depending on the desired amount of phosphate ester ligand supported.

[0033] An appropriate solvent may be added when adding the phosphate ester-forming compound, or the phosphate ester-forming compound may be dissolved in an appropriate solvent before addition. Examples of such solvents include the organic solvents used in step (1) above. It is particularly preferable to use a mixture of water and an organic solvent. The molar ratio of water to the phosphate ester-forming compound is preferably 0.5 to 2.0, more preferably 0.7 to 1.9, and particularly preferably 1.0 to 1.8. Using the phosphate ester-forming compound and water at such a molar ratio allows for the introduction of a large amount of phosphate ester ligands that are effective for adsorbing the target substance. Furthermore, the phosphate ester introduced as a ligand can be prevented from being utilized as a cross-linking site.

[0034] The immobilization of a phosphate ester ligand to a base carrier can be carried out with reference to WO 2013 / 146669, etc. For example, a phosphate ester-forming compound is added to the base carrier and reacted at 40 to 70°C for 2 to 18 hours, whereby the phosphate ester-forming compound reacts with the hydroxyl groups of the polysaccharide in the base carrier to form a phosphate ester ligand.

[0035] Furthermore, a drying step may be further included after the step (1) and before the step (2). By including such a drying step, the phosphate ester can be efficiently introduced as a ligand. The drying method is not particularly limited, but examples include heat drying, vacuum drying, and freeze drying. Heat drying is preferably carried out at 40 to 70°C for 6 to 30 hours, more preferably at 50 to 60°C for 16 to 24 hours. Vacuum drying is preferably carried out at 25 to 70°C for 4 to 120 hours, more preferably at 40 to 60°C for 10 to 96 hours.

[0036] 3. Adsorbents and Chromatography Carriers As described above, the porous particles according to the embodiments can be used for adsorption, separation, and purification of substances capable of interacting with phosphate ester ligands (hereinafter also referred to as "target substances"), particularly substances with relatively large molecular weights. In one embodiment, the relatively large molecular weight substance is a substance having a molecular weight of 50 kDa or more (or 60 kDa or more, 70 kDa or more, 80 kDa or more, or 90 kDa or more), and more specifically, an enzyme, a nucleic acid (e.g., mRNA), an antibody, a protein, a virus, or the like.

[0037] According to one embodiment, an adsorbent including the porous particles according to the embodiment is provided. The adsorbent can be used for adsorption of a target substance. The adsorbent may consist of the porous particles according to the embodiment, or may include an additional material. Also provided is a chromatography support including the adsorbent. The chromatography support may consist of the adsorbent according to the embodiment, or may include an additional material. The chromatography support can be suitably used in the separation and purification of a target substance.

[0038] The use of the adsorbent and chromatography carrier is not limited, and they can be used, for example, as carriers for affinity chromatography or cation exchange chromatography. In these chromatography applications, for example, the adsorbent or chromatography carrier according to the embodiment is packed into a chromatographic separation column. In addition, the porous particles according to the embodiment can also be used after further treatment such as saponification or modification with a substituent.

[0039] In the separation and purification process, a column is first packed with a chromatography carrier, although the manner of packing is not particularly limited. A sample solution containing the target substance is then brought into contact with the chromatography carrier, thereby separating the target substance from impurities. Specifically, the target substance can be purified by packing the above-described chromatography carrier into a column, passing the sample solution through the column, and selectively adsorbing the target substance onto the chromatography carrier. Alternatively, the target substance can be purified by adsorbing both the target substance and impurities onto the chromatography carrier and gradually or continuously changing the elution conditions (e.g., salt concentration) to utilize the difference in affinity for the chromatography carrier.

[0040] The degree of adsorption of a target substance to a chromatography support according to an embodiment can be evaluated by its 10% dynamic binding capacity (hereinafter also referred to as "10% DBC"). The porous particles according to an embodiment preferably have a 10% DBC for IgG at a 2-minute residence time of 40 mg / mL or more (e.g., 40 to 200 mg / mL), more preferably 45 mg / mL or more (e.g., 45 to 200 mg / mL), and particularly preferably 50 mg / mL or more (e.g., 50 to 200 mg / mL). If the 10% DBC for IgG is within the above range, it can be said that the support has excellent adsorption capacity for IgG and substances with similar properties. Furthermore, the fact that an excellent 10% DBC is obtained at a 2-minute residence time means that the chromatography support has performance suitable for separation and purification at high throughput and high processing speed.

[0041] Chromatography conditions are designed to take advantage of the differences in affinity between the target substance and impurities for the chromatography support. For example, the conditions are designed taking into account differences in support structure (e.g., ligand type, ligand density, ligand orientation, particle size, pore size, base matrix composition) and the physicochemical properties (e.g., isoelectric point, charge, hydrophobicity, molecular structure, conformation) of the target substance and impurities. Conditions can be adjusted to perform chromatography in either bind-elute or flow-through mode.

[0042] Components contained in buffer solutions that can be used for sample solution, column washing, elution, etc. are not particularly limited as long as they have buffering capacity, but examples include 1 to 300 mmol / L phosphate, citrate, acetate, succinate, maleate, borate, Tris (base), HEPES, MES, PIPES, MOPS, TES, Tricine, etc. The above salts can also be used in combination with other salts, such as sodium chloride, potassium chloride, calcium chloride, sodium citrate, sodium sulfate, and ammonium sulfate. Furthermore, the buffer may contain amino acids such as glycine, alanine, arginine, serine, threonine, glutamic acid, aspartic acid, and histidine, sugars such as glucose, sucrose, lactose, and sialic acid, or derivatives thereof. Elution is preferably performed with pure water. The pH of the buffer is preferably in the range of 2 to 9, more preferably 3 to 8. The linear velocity of the buffer solution is preferably in the range of 20 to 1000 cm / h.

[0043] According to one embodiment, a purification method is provided, including the steps of contacting the porous particles (or adsorbent or chromatography support) with a solution containing a target substance (a target substance to be purified that is adsorbed to the adsorbent) to adsorb the target substance onto the porous particles, and eluting and recovering the target substance adsorbed onto the porous particles. Examples of target substances include antibodies, enzymes, nucleic acids (e.g., mRNA), antibodies, proteins, viruses, etc. As described above, the porous particles according to the embodiment can be used to adsorb and separate and purify substances with relatively large molecular weights. For example, the target substance has a molecular weight of 50 kDa or more. The elution step after the adsorption step can be carried out by a conventional method using an eluent commonly used in the field. According to the method according to 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 refers to the ratio of the amount of target substance recovered after purification to the amount of target substance loaded onto the chromatography support (i.e., the amount of target substance in the sample solution before purification).

[0044] The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited thereto. <Production of Spherical Cellulose Particles> [Production Example 1] (Granulation Step) (1) 128 g of crystalline cellulose (manufactured by Asahi Kasei Chemicals Corporation, product name: Ceolus PH101) was added to 2,000 g of a 60 wt % aqueous solution of calcium thiocyanate, and the mixture was heated to 110-120°C to dissolve. (2) 9,600 mL of o-dichlorobenzene was prepared, and 120 g of sorbitan monooleate as a surfactant was added thereto while stirring at 200-300 rpm. The mixture was then heated to 130-140°C to obtain a dispersion. (3) Next, while continuing to stir at 200-300 rpm, the solution prepared in (1) was added to the dispersion, and the mixture was cooled to 40°C. 3,800 mL of methanol was poured into the mixture to obtain a suspension of cellulose particles. (4) The resulting suspension was filtered to recover cellulose particles, which were then washed with 3,800 mL of methanol. This washing procedure was repeated several times. (5) The particles were further washed with a large amount of pure water to obtain spherical cellulose particles. (6) The resulting spherical cellulose particles were passed through sieves with 150 μm and 53 μm openings to obtain spherical cellulose particles with particle diameters of 53 to 150 μm.

[0045] (Reduction step) (1) 8,500 g-wet (water content 11.12) of the spherical cellulose particles obtained in the granulation step was dispersed in 12,800 g of pure water, and then stirring was initiated and the mixture was heated to 40°C. (2) While continuing stirring at 40°C, 1,480 g of pure water, 127 g of a 48% aqueous sodium hydroxide solution, and 76 g of sodium borohydride were added, and after the addition was completed, the temperature was raised to 60°C and the reaction was carried out for 16 hours. (3) The reaction liquid was cooled to a temperature of 40°C or below. (4) The reaction liquid was filtered to recover the gel, which was then filtered and washed with pure water to obtain reduced spherical cellulose particles.

[0046] <Preparation of Phosphate-Esterified Cellulose Particles> [Example 1] 158 g of dimethylformamide was added to 105 g of the reduced spherical cellulose particles (water content: 10.50) obtained in Preparation Example 1, followed by stirring and filtration. This procedure was repeated 10 times. Subsequently, 83 g of dimethylformamide (DMF) was added as a solvent to the resulting washed cellulose particles. While cooling the reaction solution to below 25°C, 11.39 g of phosphorus pentoxide was added, followed by a mixture of 1.11 g of purified water and 5 g of dimethylformamide to obtain a solution (molar ratio of water to phosphorus pentoxide: 0.77). The resulting solution was heated to 65°C and reacted for 16 hours. After the reaction, the solvent was removed by filtration, and the resulting gel was washed twice with purified water. It was then washed with 0.5 M aqueous sodium hydroxide solution, followed by pure water until the washing solution became neutral. It was further washed with 0.5 M aqueous hydrochloric acid solution, followed by pure water until the washing solution became neutral. Finally, the particles were washed with a 0.5 M aqueous solution of sodium hydroxide, and then with pure water until the washing liquid became neutral, thereby obtaining phosphated cellulose particles.

[0047] Example 2 Phosphated cellulose particles were produced in the same manner as in Example 1, except that the amount of phosphorus pentoxide added was 16.4 g (molar ratio of water to phosphorus pentoxide: 0.53).

[0048] Example 3: 300 g of the reduced spherical cellulose particles (water content: 10.50) obtained in Production Example 1 were added with 450 g of methanol, stirred, and filtered. This procedure was repeated 10 times, followed by vacuum drying (40°C to 60°C) to obtain dried cellulose particles. Separately, 19.28 g of phosphorus pentoxide was added to 206 g of dimethylformamide (DMF), followed by a mixture of 2.22 g of pure water and 10 g of dimethylformamide to obtain a solution (molar ratio of water to phosphorus pentoxide: 0.91). 20 g of the dried cellulose particles obtained above were added to this solution, heated to 65°C, and reacted for 16 hours. After the reaction, the solvent was removed by filtration, and the resulting gel was washed twice with pure water. It was then washed with 0.5 M aqueous sodium hydroxide solution, followed by pure water until the washing solution became neutral. It was further washed with 0.5 M aqueous hydrochloric acid solution, followed by pure water until the washing solution became neutral. Finally, the particles were washed with a 0.5 M aqueous solution of sodium hydroxide, and then with pure water until the washing liquid became neutral, thereby obtaining phosphated cellulose particles.

[0049] Example 4: 150 g of the reduced spherical cellulose particles (water content: 10.50) obtained in Production Example 1 were added with 225 g of methanol, stirred, and filtered. This procedure was repeated nine times, followed by vacuum drying (40°C to 60°C) to obtain dried cellulose particles. Separately, 8.76 g of phosphorus pentoxide was added to 120.2 g of dimethylformamide (DMF), followed by a mixture of 1.11 g of pure water and 5 g of dimethylformamide to obtain a solution (molar ratio of water to phosphorus pentoxide: 1.00). 10 g of the dried cellulose particles obtained above was added to this solution, heated to 65°C, and reacted for 16 hours. After the reaction, the solvent was removed by filtration, and the resulting gel was washed twice with pure water. It was then washed with 0.5 M aqueous sodium hydroxide solution, followed by pure water until the washing solution became neutral. It was further washed with 0.5 M aqueous hydrochloric acid solution, followed by pure water until the washing solution became neutral. Finally, the particles were washed with a 0.5 M aqueous solution of sodium hydroxide, and then with pure water until the washing liquid became neutral, thereby obtaining phosphated cellulose particles.

[0050] Comparative Example 1 Cellufine Phosphate (manufactured by JNC Corporation) was used.

[0051] <Evaluation of Phosphated Cellulose Particles> The phosphated cellulose particles of the Examples and Comparative Examples were evaluated as follows. Unless otherwise specified, the operations described below were carried out at room temperature (25°C).

[0052] [1] Swelling degree The swelling degree of the phosphated cellulose particles of the Examples and Comparative Examples was measured as follows. First, 10 g of the phosphated cellulose particles obtained in the above Examples and Comparative Examples was suspended in approximately 60 g of water to allow swelling. After degassing the suspension for 1 hour, the swollen phosphated cellulose particles were placed in a 100 mL graduated cylinder, and tapping and standing were repeated until the volume of the swollen phosphated cellulose particles became constant. After measuring the constant volume, the entire amount of phosphated cellulose particles in the graduated cylinder was transferred to a beaker and dried at 80°C. The dry weight of the gel was measured to calculate the swelling degree using the following formula: Swelling degree (mL / g) = Water-swollen volume of phosphated cellulose particles (mL) / Dry weight of phosphated cellulose particles (g)

[0053] Here, the water-swollen volume of phosphated cellulose particles refers to the volume measured after repeatedly tapping and leaving the swollen phosphated cellulose particles until the volume becomes constant. More specifically, the water-swollen volume refers to the sum of the volume of the swollen porous particles and the volume of the interparticle voids. Here, the volume of the interparticle voids refers to the volume of the water-filled portions (voids) present between the porous particles packed in the column; that is, the water-swollen volume is measured by reading the value on the graduated cylinder when the volume of the swollen phosphated cellulose particles becomes constant. The drying method is not particularly limited, but here, the particles were dried in a thermostatic chamber at 80°C for 2 days.

[0054] [2] Cation Exchange Capacity: 0.5M hydrochloric acid was added to the phosphated cellulose particles (approximately 10g) obtained in the above Examples and Comparative Examples, and the mixture was stirred for approximately 30 minutes before filtering and washing with pure water until the filtrate became neutral. The mixture was transferred to a beaker and dried overnight in a vacuum dryer (60°C). 1g of the dried particles was precisely weighed, 50mL of 0.1M sodium hydroxide was added, the mixture was gently mixed, and the mixture was allowed to stand for 24 hours. Then, 10mL of the supernatant was recovered and titrated with 0.1M hydrochloric acid using phenolphthalein as an indicator. Cation Exchange Capacity (meq / g) = (10xf1-Vxf2)x0.1x5 / W

[0055] Here, f1 is the factor of 0.1 M sodium hydroxide, f2 is the factor of 0.1 M hydrochloric acid, V is the titration amount (mL) of 0.1 M hydrochloric acid, and W is the weight of the particles (g). The factor indicates how many times (multiplication factor) the actual concentration of the solution used is compared to the target concentration. When expressing the cation exchange capacity in terms of volume, it was calculated using the following formula: Cation exchange capacity (meq / mL) = Cation exchange capacity (meq / g) / Swelling degree (mL / g)

[0056] [3] Moisture Content The phosphated cellulose particles obtained in the above Examples and Comparative Examples were suspended in water to a concentration of approximately 50% V / V. The suspension was then placed in a measuring cylinder and allowed to stand for 24 hours. The ratio of the spontaneous sedimentation volume of the phosphated cellulose particles to the total volume of the suspension in the measuring cylinder was then determined. The measuring cylinder was then shaken to re-suspend the contents into a uniform suspension. Based on the ratio of the spontaneous sedimentation volume to the total volume of the suspension determined above, an amount of suspension was measured to obtain a spontaneous sedimentation volume of 100 mL of cellulose particles. The suspension was subjected to suction filtration for 15 minutes on a 90 mm diameter 5A filter paper (Advantec Toyo Co., Ltd., "Quantitative Filter Paper") to remove moisture and obtain suction-dried phosphated cellulose particles. 1 g of the resulting suction-dried phosphated cellulose particles was placed in a beaker and dried overnight at 80°C. The weight of the resulting dried phosphated cellulose particles was measured. The moisture content was calculated using the following formula: moisture content = weight of suction-dried phosphated cellulose particles / weight of dried phosphated cellulose particles.

[0057] [4] Adsorption Evaluation Using IgG [Measurement of 10% Dynamic Binding Capacity of IgG] The phosphated cellulose particles obtained in the above Examples and Comparative Examples were packed into a minicolumn (manufactured by JNC Corporation). Separately, a solution (2 mg / mL) of γ-globulin derived from human serum (Wako Pure Chemical Industries) was prepared as an antibody solution. Next, the column was connected to an LC system, and a buffer was passed through it to equilibrate the column effluent until the UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH were constant. After that, the baseline UV was set to zero, and the 10% DBC was measured under the following chromatographic conditions.

[0058] The prepared antibody solution was passed through a column packed with phosphated cellulose particles at a flow rate of 0.53 mL / min (retention time: 2 minutes). The binding capacity was measured as 10% DBC, using the absorbance at 280 nm of the solution eluted from the column as an index. "10% DBC" is an estimate of the amount of adsorption from the time required for the concentration of the target substance in the eluate from the column to reach 10% of the initial concentration. The UV of the antibody solution passed through the column was measured in advance, and the UV of the column effluent was monitored to determine the time point at which UV corresponding to a concentration of 10% of the antibody solution was detected.

[0059] Specifically, the 10% DBC of IgG was calculated using the following formula. This analysis was performed in a room at 25°C: 10% DBC (mg / mL) = antibody solution concentration (mg / mL) × {time (minutes) from the start of antibody solution flow until the UV of the antibody solution reaches 10% of its pre-measured level × flow rate (mL / min) - dead volume} / column volume [where dead volume = system piping volume + column void volume (mL)]

[0060] (Chromatography conditions for 10% DBC measurement) (1) Equipment and reagents used LC system: AKTA avant 25 (registered trademark) Buffer: acetate buffer pH 5.0 (containing 0.05 mol / L NaCl) Polyclonal antibody: γ-globulin, derived from human serum (Wako Pure Chemical Industries) Column: diameter 6.7 mm, length 30 mm

[0061] [5] Observation of particle shape using a microscope Microscopic observation was performed using an upright microscope CX41 manufactured by Olympus Corporation. Several drops of a slurry of the particles of Examples 1 to 4 were placed on a glass slide, and a cover glass was placed on top of them and photographed. Figures 1 to 4 are micrographs magnified at 57.5x magnification. The magnification was calculated using an Objective Micrometer OM manufactured by Kenis. The photographs show that the obtained particles are almost spherical (Figures 1 to 4).

[0062] The evaluation and analysis results for each of the Examples and Comparative Examples are shown in Table 1.

[0063] In Table 1, all of the phosphated cellulose particles of Examples 1 to 4 had an ion exchange capacity of 0.1 meq / mL or more and a swelling degree greater than 8 mL / g. Furthermore, when the phosphated cellulose particles of Examples 1 to 4 were used as chromatography carriers, the 10% DBC was higher than 50 mg / mL in all cases, even under high flow rate conditions such as a 2-minute residence time, demonstrating excellent adsorption performance. On the other hand, the 10% DBC of Comparative Example 1 was low. In other words, the porous particles according to the embodiment are capable of efficiently adsorbing relatively large proteins, particularly those with a molecular weight of 50 kDa or more, and can therefore be suitably used in the separation and purification of substances in the production of biopharmaceuticals, etc.

[0064] [6] Purification of T7 RNA Polymerase <Production of Phosphate-Esterified Cellulose Particles> [Example 5] 225 g of acetone was added to 150 g of the reduced spherical cellulose particles (water content: 10.50) obtained in Production Example 1, followed by stirring and filtration. This procedure was repeated nine times and then vacuum dried (40°C to 60°C) to obtain dried cellulose particles. Separately, 11.39 g of phosphorus pentoxide was added to 103 g of dimethylformamide (DMF), followed by a mixture of 1.78 g of purified water and 5 g of dimethylformamide to obtain a solution (molar ratio of water to phosphorus pentoxide: 0.91). 10 g of the dried cellulose particles obtained above was added to this solution, heated to 65°C, and reacted for 16 hours. After the reaction, the solvent was removed by filtration, and the resulting gel was washed twice with purified water. It was then washed with 0.5 M aqueous sodium hydroxide solution, followed by pure water until the washings became neutral. The particles were further washed with a 0.5 M aqueous hydrochloric acid solution, followed by pure water until the washings became neutral. Finally, the particles were washed with a 0.5 M aqueous sodium hydroxide solution, followed by pure water until the washings became neutral, to obtain phosphated cellulose particles. The physical properties of the obtained cellulose particles are shown in Table 2 below. The measurement methods and definitions for each item in Table 2 are the same as those described above.

[0065] T7 RNA polymerase was purified as follows using the phosphated cellulose particles of Example 5 produced above. As a comparative example, the same procedure was carried out using the phosphated cellulose particles of Comparative Example 1 described above.

[0066] <Rough purification of T7 RNA polymerase solution> Escherichia coli strain pAR1219 expressing T7 RNA polymerase was cultured to produce bacterial cells. Protein was extracted from the resulting bacterial cells and roughly purified. The flow chart is shown in Figure 5. Details are as follows: (1) Preparation of carbenicillin sodium aqueous solution Carbenicillin sodium (isomer mixture) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to prepare a 100 mg / mL carbenicillin aqueous solution. This solution was filtered through a syringe filter (manufactured by Merck) with a pore size of 0.22 μm.

[0067] (2) Preparation of Medium 63.2 g of 2xYT Medium (Sigma-Aldrich) was weighed into a 2 L beaker and approximately 1.5 L of purified water was added. The mixture was stirred to dissolve, and then diluted to 2 L with purified water using a measuring cylinder and placed in a stoppered glass bottle. 72 L of this solution was prepared. 12.0 g of agar was added to a portion of the prepared medium (1200 mL), the lid was covered with aluminum foil, and the mixture was autoclaved. After cooling to below 60°C, 1200 μL of the 100 mg / mL carbenicillin sodium aqueous solution prepared above was added and mixed. The resulting mixture was dispensed in 20 mL aliquots into culture dishes (AS ONE Corporation) and allowed to solidify. After solidification was confirmed, the dish was capped, inverted, and dried for 12 hours in a cool incubator (Mitsubishi Electric Engineering Co., Ltd.) set at 37°C to produce Agar / Crb agar medium. The remaining 2xYT Medium aqueous solution was also autoclaved, and immediately before use, the above-prepared 100 mg / mL carbenicillin sodium aqueous solution was added to make the concentration 0.1 mg / mL to prepare 2xYT(+) liquid medium.

[0068] (3) Cultivation of E. coli expressing T7 RNA polymerase. A frozen glycerol stock of E. coli strain pAR1219 expressing T7 RNA polymerase was streaked onto the Agar / Crb agar medium prepared above. After overnight incubation, colonies were confirmed the following morning. The resulting colonies were picked with a toothpick and inoculated into 50 mL of 2xYT(+) liquid medium in a 125 mL fluted Erlenmeyer flask. Twenty-four such flasks were prepared, and shaken at 37°C for approximately 24 hours. The OD600 of the preculture was then confirmed to be 1.7-2.0 using a spectrophotometer (Thermo Fisher SCIENTIFIC, product name: GENESYS 10S UV-VIS).

[0069] Six 2-L Erlenmeyer flasks were filled with 1.5 L of 2xYT(+) liquid medium, and 30 3-L Erlenmeyer flasks were filled with 2 L of 2xYT(+) liquid medium, and the preculture solution was inoculated into them. Shaking culture was carried out at 37°C for approximately 18 to 22 hours, and the OD600 of the culture solution was confirmed to be 0.5 to 1.0 using a spectrophotometer (Thermo Fisher SCIENTIFIC, product name: GENESYS 10S UV-VIS).

[0070] 16.8 g of IPTG (Fujifilm Wako Pure Chemical Industries, Ltd., product name: isopropyl-β-D(-)-thiogalactopyranoside) was weighed out and dissolved in 720 mL of purified water to prepare a 100 mM IPTG aqueous solution. This 100 mM IPTG aqueous solution was filtered through a 0.22 μm syringe filter (Merck). This was added to the above culture solution so that the final IPTG concentration was 1 mM, and shaking culture was carried out at 37°C for 3 hours or more. After completion of the culture, the culture solution was dispensed into 250 mL Nalgene bottles and centrifuged (4°C, 5,000 x g, 5 minutes). The supernatant was removed to obtain wet bacterial cells. 20 mL of PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each bottle, and the cells were collected by centrifugation (4°C, 10,000 x g, 10 minutes). The supernatant was removed to obtain wet bacterial cells A.

[0071] (4) Protein Extraction Crude protein was extracted from the wet bacterial cells A obtained above by the following procedure. A 20 mM Tris-HCl (Fujifilm Wako Pure Chemical Industries, Ltd., product name: 2-amino-2-hydroxymethyl-1,3-propanediol) solution containing protease inhibitors (10 μL / mL PMSF (manufactured by Abcam, product name: protease inhibitor), 100 μM benzamidine (manufactured by Tokyo Chemical Industry Co., Ltd., product name: Benzamidine Hydrochloride), 10 μM bacitracin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product name: BACITRACIN)), 10% sucrose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 1 mM EDTA (manufactured by Dojindo Laboratories, Ltd., product name: 2NA (EDTA·2Na), pH 8.0) was prepared and used as a lysis buffer.

[0072] The wet bacterial mass A obtained above was suspended in 720 mL of the lysis buffer prepared above and then flash-frozen at -80°C. It was then thawed at room temperature (25°C), and lysozyme (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product name: lysozyme, derived from egg white) was added to a concentration of 0.2 mg / mL. This was then incubated at 4°C for 45 minutes. It was then flash-frozen again at -80°C and thawed twice at room temperature (25°C). The solution thawed at 4°C was then added with MgCl . ２ was added to a final concentration of 6 mM, and then benzonase nuclease (Merck, product name: Benzonase Nuclease, purity >99%) was added to a final concentration of 12.9 units / mL. This solution was incubated at 4°C for 0.5 to 1 hour, and a decrease in viscosity was visually confirmed. This solution was centrifuged (4°C, 10,000 x g, 1 hour), and the supernatant was collected. This was designated as Supernatant A and stored at 4°C.

[0073] (5) Crude Purification by Ammonium Sulfate Precipitation 1: 0.35 g of ammonium sulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed out per 1 mL of the supernatant A obtained above and crushed to a powder. While slowly stirring the supernatant A at 4°C using a stirrer, the crushed ammonium sulfate was gradually added over 10 minutes. After the addition of ammonium sulfate, stirring was continued at 4°C for 1 hour. The resulting solution was centrifuged (4°C, 15,000 x g, 1 hour). The supernatant was removed to recover precipitate A, which was then frozen and stored at -80°C if not immediately subjected to further processing.

[0074] (6) Crude purification 2 using Cellufine MAX DEAE (manufactured by JNC Corporation) (Equipment and reagents used) LC system: AKTA avant 25 (registered trademark) Column: diameter 4.4 cm, length 16.5 cm, 250 mL Sample: crude purified solution A Residence time: 10 minutes Buffer A: 10 mM tris-HCl pH 7.5, 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol, protease inhibitor Equilibration buffer A: 10 mM tris-HCl pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, 10% glycerol, protease inhibitor Elution buffer A: 10 mM tris-HCl pH 7.5, 1M NaCl, 0.1mM EDTA, 0.5mM DTT, 10% glycerol, protease inhibitors

[0075] The precipitate A obtained above was suspended in 1800 mL of buffer A so that the conductivity was 10.0 mS / cm or less, and the conductivity was reduced to 8.68 mS / cm. The suspension was then filtered through a syringe filter (manufactured by Merck) with a pore size of 0.22 μm, and this was used as a crudely purified solution A.

[0076] A column was packed with Cellufine MAX DEAE (manufactured by JNC Corporation) and connected to an LC system. Equilibration buffer A was passed through the column at 25 mL / min until the UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH of the column effluent became constant, thereby achieving equilibration. This process was continued at a flow rate of 25 mL / min. 1600 mL of the crude purified solution A prepared above was passed through the column. Five column volumes of equilibration buffer A were then passed through to wash the column. The mobile phase mixture ratio was then continuously changed over 20 column volumes, so that the mixture ratio of equilibration buffer A to elution buffer A changed from 0% (V / V) to 50% (V / V), and the column was purified by gradient elution. This was collected as fractions of 0.2 column volumes each.

[0077] The column was then washed by passing 5 column volumes of elution buffer A through the column. Furthermore, the column was washed by passing 2 column volumes of 0.5 M aqueous sodium hydroxide through the column. Finally, 5 column volumes of buffer A were passed through the column to re-equilibrate the column. SDS-PAGE was performed using each of the 0.2 column volume fractions collected above and commercially available T7 RNA polymerase (manufactured by Takara Bio Inc.) as a positive control. Based on the results, only fractions (fractions 25 to 42) showing a strong band migrating at the same approximately 98 kDa position as the T7 RNA polymerase positive control and few other impurity bands were combined, and 900 mL of crude solution B was collected.

[0078] (7) Affinity Purification: Preparation of Highly Purified T7 RNA Polymerase Solution The crudely purified solution B obtained above was purified to a high purity using Cellufine Phosphate (manufactured by JNC Corporation) according to the following procedure. (Equipment and reagents used) LC system: AKTA avant 25 (registered trademark) Sample: Pre-purified enzyme solution A Column: Diameter 14.6 mm, length 30 mm, 5 mL Residence time: 2 minutes Buffer B: 10 mM potassium phosphate pH 7.5, 0.1 mM EDTA, 0.1 mM DTT, protease inhibitor Equilibration buffer B: 10 mM potassium phosphate pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, protease inhibitor Elution buffer B: 10 mM potassium phosphate pH 7.5, 1 M NaCl, 0.1 mM EDTA, 0.1mM DTT, protease inhibitors

[0079] (Purification step) The crudely purified solution B obtained above was diluted with equilibration buffer B until the conductivity reached 7.60 mS / cm. The resulting solution was filtered through a syringe filter (manufactured by Merck) with a pore size of 0.22 μm, and this was used as pre-purification enzyme solution A. A column was filled with Cellufine Phosphate (manufactured by JNC Corporation) and connected to an LC system. Equilibration buffer B was passed through the column, and the column was equilibrated by passing it through at 2.5 mL / min until the UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH of the column effluent became constant.

[0080] 240 mL of the pre-purification enzyme solution A prepared above was passed through the column at 2.5 mL / min. The column was then washed by passing 10 column volumes of equilibration buffer B and elution buffer B at a mixing ratio such that the conductivity of the mobile phase was approximately 18 mS / cm. Then, 20 column volumes of elution buffer B were passed through the column, recovering fractions of 3 column volumes each, and the first fraction, 15 mL, was recovered as a high-purity T7RNAP solution. The column was then washed by passing 3 column volumes of 0.5 M aqueous sodium hydroxide solution through the column. Finally, 15 column volumes of buffer B were passed through the column to re-equilibrate the column.

[0081] (8) Preparation of T7 RNA Polymerase Solution (Preparation of T7 RNA Polymerase Solution (Enzyme Solution)) The high-purity T7 RNA polymerase solution prepared above was diluted 2-fold with buffer B and dialyzed against a 10-fold volume of equilibration buffer B using a Spectrapore 3 dialysis membrane (Repligen) with a molecular weight cutoff of 3,500. The solution was further diluted with equilibration buffer B and buffer B to achieve a conductivity of approximately 7.5 mS / cm. This diluted high-purity T7 RNA polymerase solution was used as the enzyme solution. The DNA and protein concentrations of the enzyme solution were measured and calculated using the following procedures. The DNA concentration was measured using a Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). The protein concentration was calculated by preparing a dilution series using Albumin Standard (Thermo Fisher Scientific) to prepare a calibration curve, staining with Protein Assay Dye Reagent Concentrate (Bio-Rad), and measuring the absorbance at 595 nm (Bradford method).

[0082] (Enzyme Activity Measurement) The enzyme activity of the enzyme solution obtained above was measured and calculated according to the following procedure. First, a separately prepared high-purity T7RNAP solution was subjected to enzyme activity measurement using a T7 RNA Polymerase Assay Kit (ProFoldin) to confirm in advance that it exhibited an enzyme activity concentration of 1.25 U / μL per mAU of absorbance at 280 nm when using a UV flow cell with a path length of 2 mm. As shown in Table 3, the enzyme solution obtained above had low amounts of residual DNA and protein per enzyme activity value, indicating high purity of T7RNA polymerase. Therefore, the enzyme activity concentration of the enzyme solution was calculated as 1.25 U / μL per mAU of absorbance at 280 nm, and the enzyme activity concentration (U / μL) was calculated. That is, the enzyme activity concentration of the present invention was calculated by measuring absorbance at 280 nm according to the following procedure.

[0083] First, equilibration buffer B was run through the LC system and equilibrated until UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH became constant, and the baseline UV was set to zero. The enzyme solution was then run through the LC system until UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH became constant, and the absorbance (mAU) at 280 nm was calculated by multiplying the absorbance (mAU) value by 1.25 U / (μL mAU). Enzyme activity concentration (U / μL) = 1.25 {U / (μL mAU)} × absorbance (mAU) at 280 nm when a constant value was shown.

[0084] The results of the analysis of the DNA concentration, protein concentration and enzyme activity concentration of the enzyme solution are shown in Table 3 below.

[0085] (9) Adsorption Evaluation Using T7 RNA Polymerase (Measurement of 5% Dynamic Binding Capacity) The phosphated cellulose particles of Example 5 and Comparative Example 1 were packed into Tricorn™ Columns (Cytiva) to a height of 1.5 cm so that the volume was 0.3 mL. The column was then connected to an LC system, and a buffer was passed through it to equilibrate the column effluent until the UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH were constant. The baseline UV was then reset to zero, and the 5% DBC was measured using the following procedure and conditions. The results are shown in Table 4 below.

[0086] (Procedure) The enzyme solution prepared above was passed through a column packed with phosphated cellulose particles at a flow rate of 0.15 mL / min (retention time 2 minutes). The binding capacity was measured as 5% DBC using the absorbance at 280 nm of the solution eluted from the column as an index. "5% DBC" is an estimation of the amount of adsorption from the time required for the concentration of the target substance in the eluate from the column to reach 5% of the initial concentration. The UV of the enzyme solution passed through the column was measured in advance, and the UV of the column effluent was monitored to determine the time point at which UV corresponding to a concentration of 5% of the enzyme solution was detected.

[0087] Specifically, the 5% DBC was calculated using the following formula. The enzyme solution concentration was calculated by converting 400,000 U into 1 mg. This analysis was performed in a room at 25°C. 5% DBC (mg / mL) = enzyme solution concentration (mg / mL) x {time (minutes) from the start of enzyme solution flow until the 5% UV absorption value, which was measured and calculated in advance, was reached x flow rate (mL / min) - dead volume} / column volume [where dead volume = system piping volume + column void volume (mL)]

[0088] (Chromatography conditions) Equipment and reagents used LC system: AKTA avant 25 (registered trademark) Buffer: acetate buffer pH 5.0 (containing 0.05 mol / L NaCl) Sample: enzyme solution Column: Tricorn ＴＭ Columns: diameter 5.0 mm, length 3.0 cm

[0089] [7] Purification of BSA (Albumin Derived from Bovine Serum) The phosphated cellulose particles of Example 5 and Comparative Example 1 were packed into a minicolumn (manufactured by JNC Corporation). Separately, a solution (2 mg / mL) of albumin derived from bovine serum (Wako Pure Chemical Industries, Ltd.) was prepared as a protein solution. The column was then connected to an LC system, and a buffer was run through it to equilibrate the column effluent until the UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH were constant. The baseline UV was then reset to zero, and the 10% DBC was measured using the procedure and conditions described below. The results are shown in Table 4 below.

[0090] (Procedure) The protein solution prepared above was passed through a column packed with phosphated cellulose particles at a flow rate of 0.265 mL / min (retention time: 4 minutes). The binding capacity was measured as 10% DBC, using the absorbance at 280 nm of the solution eluted from the column as an index. "10% DBC" is an estimation of the amount of adsorption from the time required for the concentration of the target substance in the eluate from the column to reach 10% of the initial concentration. The UV of the antibody solution passed through the column was measured in advance, and the UV of the column effluent was monitored to determine the time point at which UV corresponding to a concentration of 10% of the antibody solution was detected.

[0091] Specifically, the 10% DBC of BSA was calculated using the following formula. This analysis was performed in a room at 25°C. 10% DBC (mg / mL) = protein solution concentration (mg / mL) x {time (minutes) from the start of protein solution flow until the protein solution reaches 10% of its UV value (preliminarily measured) x flow rate (mL / min) - dead volume} / column volume [where dead volume = system piping volume + column void volume (mL)]

[0092] (Chromatography conditions) Equipment and reagents used LC system: AKTA avant 25 (registered trademark) Buffer: acetate buffer pH 5.0 (containing 0.05 mol / L NaCl) Protein: albumin, derived from bovine serum (Wako Pure Chemical Industries) Column: diameter 6.7 mm, length 30 mm

[0093] The results of measuring the 5% dynamic binding capacity of T7 RNA polymerase and the 10% dynamic binding capacity of BSA are shown in Table 4 below.

[0094] The T7 RNA polymerase adsorbed in the adsorption assay (5% DBC) was recovered and its DNA concentration, protein concentration, and enzyme activity concentration were measured. The measurement methods were the same as those used for the analysis of the enzyme solution described above. The results are shown in Table 5 below.

[0095] As can be seen from Table 4, the chromatography carrier according to the embodiment exhibited excellent adsorption performance. This effect was more pronounced for larger substances, and excellent adsorption performance was observed for T7 RNA polymerase even under high flow rate conditions, such as a 2-minute residence time. Furthermore, as can be seen from Table 5, the residual amount of impurities (DNA and protein) per enzyme activity value in the recovered fractions of the examples is lower than that of the comparative examples. Therefore, it can be said that the chromatography carrier according to the embodiment also has excellent impurity separation ability and can successfully separate enzymes and impurities produced in the manufacturing process of biopharmaceuticals. In other words, the porous particles according to the embodiment enable efficient purification of relatively large molecules such as T7 RNA polymerase while maintaining high adsorption capacity and excellent impurity separation ability, and therefore can be suitably used in separation and purification processes for biopharmaceuticals, etc.

[0096] Although several embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, and are also included in the scope of the invention and its equivalents as defined in the claims.

Claims

1. Porous particles comprising a base carrier containing a polysaccharide and a phosphate ester ligand immobilized on the base carrier, having a cation exchange capacity of 0.1 meq / mL or more per water-swollen volume and a swelling degree of greater than 8 mL / g.

2. The porous particle of claim 1, wherein the polysaccharide is cellulose.

3. Porous particles described in claim 1 or 2, having a 10% dynamic binding capacity (DBC) of IgG at a residence time of 2 minutes of 40 mg / mL or more.

4. Porous particles described in claim 1 or 2, having a 10% dynamic binding capacity (DBC) of IgG at a residence time of 2 minutes of 50 mg / mL or more.

5. The porous particle according to any one of claims 1 to 4, wherein the ratio of swelling degree to water content is 0.5 to 2.

0.

6. The porous particles according to any one of claims 1 to 5, which have a spherical, granular or mixture thereof shape.

7. An adsorbent comprising the porous particles according to any one of claims 1 to 6.

8. A chromatography support comprising the adsorbent of claim 7.

9. A method for producing porous particles according to any one of claims 1 to 6, comprising: (1) adding an organic solvent to a base carrier containing a polysaccharide to wash the base carrier; and (2) adding a compound to the washed base carrier that reacts with hydroxyl groups in the polysaccharide to form a phosphate ester.

10. The method of claim 9, further comprising drying after step (1) and before step (2).

11. A purification method comprising: bringing the porous particles according to any one of claims 1 to 6 into contact with a solution containing a substance to be purified, thereby adsorbing the substance to the porous particles; and eluting and recovering the substance to be purified.

12. The purification method according to claim 11, wherein the substance to be purified comprises one or more selected from the group consisting of antibodies, enzymes, nucleic acids, antibodies, proteins, and viruses.

13. Porous particles according to any one of claims 1 to 6 for use in purifying a substance.

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