Porous particles and adsorbents and chromatography carriers containing the same

Abstract

The present invention provides 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. [Solution] According to one embodiment, there is provided a porous particle comprising a base carrier comprising a polysaccharide 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.

Description

[Technical Field]

[0001] The present invention relates to porous particles and adsorbents and chromatography supports containing the same. The present invention also relates to methods for producing the porous particles. [Background technology]

[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 separating and purifying substances with relatively large molecular sizes, such as antibodies. One such material is a monolith, which is an integrally molded body having a three-dimensional network structure and interconnected pores formed by the voids. 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, particles with interconnected pores, which are formed by converting monoliths into particles, 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 and the like, enabling separation and purification at higher throughputs and speeds. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 3,565,886 [Patent Document 2] International Publication No. 2016 / 063702 [Patent Document 2] Japanese Patent Application Publication No. 2023-037724 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[0007] The present inventors have 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 have succeeded in producing porous particles having a predetermined cation exchange capacity and swelling degree. They have also found that such porous particles can be used for the adsorption, separation, and purification of substances with relatively large molecular weights. The present invention is, for example, as follows: [1] Porous particles comprising a base carrier containing a polysaccharide and a phosphate ester ligand immobilized on the base carrier, the particles 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 particles according to either [1] or [2], which have a 10% dynamic binding capacity (DBC) of IgG at a residence time of 2 minutes of 40 mg / mL or more. [4] The porous particles according to [1] or [2], which have a 10% dynamic binding capacity (DBC) of IgG of 50 mg / mL or more at a residence time of 2 minutes. [5] The porous particles according to any one of [1] to [4], wherein the ratio of the swelling degree to the water content is 0.5 to 2.0. [6] The porous particles according to any one of [1] to [5], which have a spherical shape, a granular shape, or a mixture thereof. [7] An adsorbent comprising the porous particles according to any one of [1] to [6]. [8] A chromatography support comprising the adsorbent according to [7]. [9] (1) adding an organic solvent to a base carrier containing a polysaccharide and washing the base carrier; (2) adding a compound to the washed base carrier that reacts with the hydroxyl groups in the polysaccharide to form a phosphate ester; The method for producing porous particles according to any one of [1] to [6], comprising:

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

[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. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 2 is a microscopic view of the particles of Example 1. [Figure 2] FIG. 1 is a microscopic view of particles of Example 2. [Figure 3] FIG. 1 is a microscopic view of particles of Example 3. [Figure 4] FIG. 1 is a microscopic view of the particles of Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present invention will be described in detail. According to one embodiment, the porous particles of the present invention comprise a base carrier comprising a polysaccharide 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 greater 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 90 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] Hereinafter, each component, production method, physical properties, uses, etc. of the porous particle according to the embodiment will be described in detail. 1.Porous particles Porous particles according to the present embodiment comprise 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 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 portion of its spherical surface 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, 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 the 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 size can be measured using an image taken with an optical microscope. Specifically, particle size on the image is measured using a caliper or the like, and the original particle size is determined from the image magnification. Then, the average particle size is calculated from the particle size values ​​determined 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 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 of 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-swollen 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-swollen 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 90 kDa or greater, and enabling separation and purification at a high throughput and high processing speed.

[0019] The porous particles according to the present 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 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 of 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, the polysaccharide may be glucose, agarose, mannose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, allose, altose, allulose, fructose, sorbose, tagarose, or a polysaccharide containing one or more of these 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 that can form water-insoluble gels, such as cellulose, chitin, chitosan, dextran, agarose, and mannan, are more preferably used than synthetic polymers.

[0024] As the polysaccharide, it is particularly preferable to use cellulose, particularly spherical cellulose. Spherical cellulose has the advantages of being inexpensive, highly biocompatible, strong, and having good column pressure resistance, 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 cellulose or amorphous cellulose. Hereinafter, porous particles containing a cellulose-containing base carrier and a phosphate ester ligand immobilized on the base carrier will also be referred to as "phosphated cellulose particles." The base carrier may contain other materials as long as its main component is a polysaccharide. The main component here refers to a component whose content in the porous particles is 50% by mass or more. When cellulose acetate is used, any material that can be generally defined as cellulose acetate can be used without particular limitations, but it is preferable that the acetylation degree is 45 to 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 a higher flow rate. 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, or glycerol glycidyl ether is preferred.

[0029] (ligand) Porous particles according to an 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 preferred. Of these, phosphorus pentoxide and phosphorus oxychloride are preferred, with phosphorus pentoxide being more preferred. The use of such compounds allows for the introduction of phosphate ester groups under mild conditions and is inexpensively available, making them advantageous for industrial-scale production.

[0030] 2. Manufacturing method of porous particles The porous particles according to the embodiment include, for example, (1) adding an organic solvent to a base carrier containing a polysaccharide and washing the base carrier; (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; It is produced by a method comprising:

[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 adjusted appropriately depending on the desired amount of phosphate ester ligand supported.

[0033] The phosphate ester-forming compound can be dissolved in a suitable solvent and then added. Examples of such solvents include the organic solvents used in step (1) above, and it is particularly preferred 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. By using the phosphate ester-forming compound and water at such a molar ratio, a large amount of phosphate ester ligands effective for adsorption of the target substance can be introduced. Furthermore, the phosphate ester introduced as a ligand can be prevented from being used as a crosslinking 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 above step (1) and before the above 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 Supports As described above, the porous particles according to the embodiment can be used for adsorption, separation, and purification of substances that can interact 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 substances are substances with a molecular weight of 90 kDa or more, more specifically, enzymes, nucleic acids (e.g., mRNA), antibodies, proteins, viruses, etc.

[0037] According to one embodiment, there is provided an adsorbent comprising the porous particles according to the embodiment. 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 contain additional materials. Also provided is a chromatography support comprising the adsorbent. The chromatography support may consist of the adsorbent according to the embodiment, or may contain additional materials. The chromatography support can be suitably used in the separation and purification of target substances.

[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 and passing the sample solution through it, thereby 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 varying the elution conditions (e.g., salt concentration) stepwise or continuously 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 have a 10% DBC of IgG at a residence time of 2 minutes of preferably 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 of IgG falls within the above range, it can be said that the adsorption capacity of IgG and substances with similar properties is excellent. Furthermore, the fact that an excellent 10% DBC is obtained at a residence time of 2 minutes 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 of the target substance and impurities (e.g., isoelectric point, charge, hydrophobicity, molecular structure, conformation). 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, and the like are not particularly limited as long as they have buffering capacity, and examples include 1 to 300 mmol / L phosphate, citrate, acetate, succinate, maleate, borate, Tris (base), HEPES, MES, PIPES, MOPS, TES, Tricine, and the like. 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 purified water. The pH of the buffer solution is preferably in the range of 2-9, more preferably in the range of 3-8. The linear velocity of the buffer solution is preferably in the range of 20 to 1000 cm / h.

[0043] 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 on the chromatography carrier (i.e., the amount of the target substance in the sample solution before purification). [Example]

[0044] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples. <Production of spherical cellulose particles> [Manufacturing Example 1] (granulation process) (1) 128 g of crystalline cellulose (manufactured by Asahi Kasei Chemicals Corporation, trade name: Ceolus PH101) was added to 2000 g of a 60 wt % aqueous solution of calcium thiocyanate, and the mixture was heated to 110 to 120° C. to dissolve. (2) 9600 mL of o-dichlorobenzene was prepared, and 120 g of sorbitan monooleate as a surfactant was added thereto while stirring at 200 to 300 rpm, and the mixture was heated to a temperature of 130 to 140° C. to obtain a dispersion liquid. (3) Next, the solution prepared in (1) was added to the dispersion while stirring at 200 to 300 rpm, and the mixture was cooled to 40° C. 3800 mL of methanol was poured therein to obtain a suspension of cellulose particles. (4) The resulting suspension was filtered to recover the cellulose particles, which were then washed with 3800 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 obtained spherical cellulose particles were passed through sieves with openings of 150 μm and 53 μm to obtain spherical cellulose particles with particle diameters of 53 to 150 μm.

[0045] (Reduction process) (1) 8500 g-wet of spherical cellulose particles (moisture content: 11.12) obtained in the above granulation step were dispersed in 12800 g of pure water, and then stirring was started and the mixture was heated to 40°C. (2) While continuing stirring at 40°C, 1,480 g of purified water, 127 g of 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 mixture was cooled to a temperature of 40°C or less. (4) The reaction solution was filtered to recover the gel, which was then filtered and washed with pure water to obtain reduced spherical cellulose particles.

[0046] <Production of phosphated 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 Production 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 pure 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 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.

[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), and then a mixture of 2.22 g of pure water and 10 g of dimethylformamide was added to obtain a solution (molar ratio of water to phosphorus pentoxide: 0.91). 20 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.

[0049] [Example 4] 225 g of methanol 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, 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: The operations described below were carried out at room temperature (25°C) unless otherwise specified.

[0052] [1] Swelling 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 were 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 measuring cylinder and repeatedly tapped and allowed to stand until the volume of the swollen phosphated cellulose particles reached a constant value. After measuring the constant volume, the entire amount of phosphated cellulose particles in the measuring 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 swelling 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 film was dried in a thermostatic chamber at 80° C. for two 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 rinsing 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. 10mL of the supernatant was then recovered and titrated with 0.1M hydrochloric acid using phenolphthalein as an indicator. Cation exchange capacity (meq / g) = (10 × f1 - V × f2) × 0.1 × 5 / W

[0055] Here, f1 is the factor of 0.1M sodium hydroxide, f2 is the factor of 0.1M hydrochloric acid, V is the titration volume of 0.1M hydrochloric acid (mL), and W is the weight of the particles (g). The factor indicates how many times (multiplication factor) the actual concentration of the solution being 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 in 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 through a 90 mm diameter 5A filter paper (Advantec Toyo Co., Ltd., "Quantitative Filter Paper") to remove water 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 dry phosphated cellulose particles

[0057] [4] Evaluation of adsorption 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 (JNC Corporation). A 2 mg / mL solution of γ-globulin derived from human serum (Wako Pure Chemical Industries) was prepared separately as an antibody solution. The column was then connected to an LC system, and buffer was passed through it to equilibrate the column until the UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH of the column effluent remained constant. The baseline UV was then set to zero, and 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 of 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 effluent 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 carried out in a room at 25°C. 10% DBC (mg / mL) = antibody solution concentration (mg / mL) × {time (min) from the start of antibody solution flow until the UV of the antibody solution reaches 10% (preliminarily measured) × flow rate (mL / min) - dead volume} / column volume [Dead volume in the formula = 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, Ltd.) Column: diameter 6.7 mm, length 30 mm

[0061] [5] Observation of particle shape using a microscope Microscopic observation was carried out using an OLYMPUS upright microscope CX41. 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 photographs were taken. Figures 1 to 4 are micrographs 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] Table 1 shows the evaluation and analysis results for each of the examples and comparative examples. [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 degree of swelling higher 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 90 kDa or more, and can therefore be suitably used in the separation and purification of substances in the production of biopharmaceuticals, etc.

[0064] 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 spherical cellulose 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 according to claim 1, having a 10% dynamic binding capacity (DBC) of IgG of 40 mg / mL or more at a residence time of 2 minutes.

3. The porous particle according to claim 1, having a 10% dynamic binding capacity (DBC) of IgG of 50 mg / mL or more at a residence time of 2 minutes.

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

0.

5. An adsorbent comprising the porous particles according to any one of claims 1 to 4.

6. A chromatography support comprising the adsorbent of claim 5.

7. (1) adding an organic solvent selected from the group consisting of dimethylformamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, acetone, and acetonitrile to a base carrier containing spherical cellulose to wash the base carrier; (2) adding dimethylformamide and a compound that reacts with hydroxyl groups in the spherical cellulose to form a phosphate ester to the washed base carrier; The method for producing the porous particles according to claim 1 , comprising:

8. The method of claim 7, further comprising drying after step (1) and before step (2).

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