Porous particles and adsorbents and chromatography supports containing them

Abstract

This invention provides porous particles that can be used for the adsorption, separation, and purification of substances that can interact with phosphate ester ligands, particularly those with relatively large molecular weights. [Solution] According to one embodiment, porous particles are provided that comprise a base carrier containing a polysaccharide and a phosphate ester ligand immobilized on the base carrier, wherein the cation exchange capacity is 0.1 meq / mL or more per water swelling volume and the degree of swelling is greater than 8 mL / g.

Description

Technical Field

[0001] The present invention relates to porous particles, an adsorbent containing the same, and a chromatographic carrier. The present invention also relates to a method for producing the porous particles.

Background Art

[0002] Regarding the phosphorylation of polysaccharides, many studies have been conducted on cellulose for a long time. Phosphorylated cellulose fibers and cellulose particles are widely used in ion exchange chromatography and affinity chromatography and are easily available (Patent Document 1). It is known that fibrous phosphocellulose has poor column packing efficiency due to its shape and is not suitable for treatment at high flow rates. Therefore, spherical phosphocellulose particles have attracted attention.

[0003] With the expansion of the biopharmaceutical market, attention has also been increasing for chromatographic materials used for the separation and purification of substances with a relatively large molecular size such as antibodies. Examples of such materials include monoliths, which are integrally molded bodies having through-holes composed of a three-dimensional network-like skeleton and voids therein. It is known that by using a monolith, separation and purification can be performed at a higher throughput and a higher processing speed than conventional chromatography (Patent Document 2).

[0004] In recent years, particles having through-holes, which are monoliths in particle form, have also been proposed (Patent Document 3). The advantages of making particles include that they can be used in the same manner as the packing material for conventional chromatographic separation columns and are easy to handle, and they are easy to scale up. Further, by using particles having through-holes, when separating and purifying biopolymers and the like, the voids of the through-holes inside the particles can also be utilized as adsorption sites, so that separation and purification can be performed at a higher throughput and a higher processing speed.

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 Publication No. 2023-037724 [Overview of the project] [Problems that the invention aims to solve]

[0006] Against this backdrop, there is a need for further improvements to porous particles that can be used in the separation and purification of substances. In particular, developing porous particles that can exhibit superior performance depending on the type and characteristics of the substance to be separated and purified would be beneficial. [Means for solving the problem]

[0007] The inventors of this invention have diligently studied porous particles comprising a polysaccharide-containing base carrier and a phosphate ester ligand immobilized on the base carrier, and have succeeded in producing porous particles having a predetermined cation exchange capacity and degree of swelling. Furthermore, they have 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 polysaccharide-containing base carrier and a phosphate ester ligand immobilized on the base carrier, wherein the cation exchange capacity is 0.1 meq / mL or more per water swelling volume and the degree of swelling is greater than 8 mL / g. [2] The porous particle according to [1], wherein the polysaccharide is cellulose. [3] A porous particle according to either [1] or [2], wherein the 10% dynamic binding capacity (DBC) of IgG at a residence time of 2 minutes is 40 mg / mL or more. [4] A porous particle according to [1] or [2], wherein the 10% dynamic binding capacity (DBC) of IgG at a residence time of 2 minutes is 50 mg / mL or more. [5] A porous particle according to any of [1] to [4], wherein the ratio of swelling degree to water content is 0.5 to 2.0. [6] A porous particle according to any of [1] to [5], wherein the shape is spherical, granular, or a mixture thereof. [7] An adsorbent containing porous particles as described in any of [1] to [6]. [8] [7] A chromatography carrier comprising the adsorbent described above. [9] (1) A step of washing the base carrier by adding an organic solvent to the base carrier containing a polysaccharide, (2) A step of adding a compound to the washed base carrier that reacts with the hydroxyl groups in the polysaccharide to form a phosphate ester. A method for producing porous particles according to any one of [1] to [6], including the above.

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

[11] The organic solvent is selected from the group consisting of dimethylformamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, acetone, and acetonitrile, and the method according to [9] or

[10] . [Effects of the Invention]

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

[0009] [Figure 1] This is a diagram showing the particles of Example 1 observed under a microscope. [Figure 2] This is a diagram showing the particles from Example 2 observed under a microscope. [Figure 3] This is a diagram showing the particles of Example 3 observed under a microscope. [Figure 4] This is a diagram showing the particles of Example 4 observed under a microscope. [Modes for carrying out the invention]

[0010] The embodiments of the present invention will be described in detail below. According to one embodiment, the porous particles of the present invention comprise a base carrier containing 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 swelling volume, and a degree of swelling greater than 8 mL / g.

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

[0012] Furthermore, when porous particles are used as chromatography supports, the strength of the support is required when processing at high flow rates. However, in porous particles containing a polysaccharide-containing base support and a phosphate ester ligand immobilized on the base support, the decomposition reaction of the polysaccharide tends to proceed easily when the reaction is carried out at high temperatures during the manufacturing process, resulting in a problem of poor strength in the resulting porous particles. When porous particles with poor strength are used as chromatography supports, high-speed processing becomes difficult. On the other hand, if a reaction is selected under milder temperature conditions, a problem may arise in which the amount of phosphate ester ligand bound to the base support is insufficient. However, the porous particles according to this embodiment have relatively excellent strength and a sufficient amount of phosphate ester ligand bound, so they can withstand high processing rates and high-speed conditions, and also have excellent adsorption capacity.

[0013] As described above, the porous particles according to the embodiment can be used as an adsorbent for a substance that can interact with a phosphate ester ligand, and also as a chromatography carrier for separating and purifying such a substance. The porous particles according to the embodiment exhibit a high adsorption capacity even for substances with a relatively large molecular weight. Therefore, the porous particles according to the embodiment can also be suitably used as an adsorbent for a substance with a relatively large molecular weight that can interact with a phosphate ester ligand, and as a chromatography carrier for separating and purifying such a substance. Examples of substances with a relatively large molecular weight include substances having a molecular weight of 90 kDa or more, and more specifically, enzymes, nucleic acids (e.g., mRNA), antibodies, viruses, etc., but are not limited thereto. According to the porous particles according to the embodiment, such substances can be efficiently separated and purified.

[0014] Hereinafter, each component, manufacturing method, physical properties, uses, etc. of the porous particles according to the embodiment will be described in detail. 1. Porous particles The 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 since they have high mechanical strength, excellent gel sedimentation properties, and can form a uniform packed bed, they are preferably spherical, granular, or a mixture thereof. Here, the term "spherical" means, for example, that the major axis (the longest diameter) is at most twice the minor axis (the shortest diameter). The term "granular" refers to a shape in which a part of the spherical surface of spherical particles is recessed inward. The porous particles are more preferably substantially spherical and the major axis and the minor axis are close to the same length. Since the porous particles are spherical, granular, or a mixture thereof, uniform packing into a separation column for chromatography, etc. becomes possible, and the target substance can be adsorbed and / or separated and purified more efficiently.

[0015] The particle size of the porous particles is preferably 1 to 500 μm, and from the viewpoint of being usable as a packing material for chromatography, the particle size of the porous particles is particularly preferably 10 to 200 μm. Further, the average particle size of the porous particles is preferably 30 to 150 μm, more preferably 40 to 120 μm. Here, the "particle size" means the actually measured value of the particle size of each porous particle, and the "average particle size" is the average value calculated based on the above particle size, and particularly means the volume average particle size.

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

[0017] Alternatively, the particle size can also be measured using an image taken with an optical microscope. Specifically, a particle size on the image is measured using calipers or the like, and the original particle size is obtained from the magnification of the photograph. Then, the average particle size is calculated by the following formula from the values of the particle sizes obtained from the optical microscope image. Volume average particle size (MV) = Σ(nd 4 ) / Σ(nd 3 ) [In the formula, d represents the value of the particle size of each particle obtained from the optical microscope image, and n represents the number of measured particles.]

[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 15 mL / g or less, and more preferably greater than 8 mL / g and 13 mL / g or less. Swelling degree is an index that indicates how much volume dry porous particles swell to when they absorb water. The swelling degree is calculated by placing water-swelled porous particles in a graduated cylinder or the like, measuring the water-swelled volume (the sum of the volume of the swollen porous particles and the volume of the interparticle voids), and then calculating it from the water-swelled volume and the weight of the dry porous particles. Here, the volume of the interparticle voids refers to the volume of the water-filled portion (void) between the porous particles packed in the column. More specifically, the swelling degree can be measured and calculated by the method described in the examples below. Having the swelling degree within the above range has the advantage of efficiently adsorbing relatively large proteins, especially those with a molecular weight of 90 kDa or more, enabling separation and purification at high processing volume and high processing speed.

[0019] The porous particles according to the embodiment have a cation exchange capacity of 0.1 eq / mL or more per water swelling volume (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 this range is advantageous because it allows for effective adsorption of the target substance and enables diffusion of the target substance into the porous particles, thus maintaining high adsorption performance even at high flow rates. The cation exchange capacity can be measured by the method described in the examples below.

[0020] The porous particles according to this embodiment have a moisture content of preferably 5 to 13, more preferably 5 to 11, and particularly preferably 6 to 10. Moisture content refers to the ratio of the weight of suction-dried porous particles to the weight of dry porous particles, and is calculated using the formula: "Moisture content = Weight of suction-dried porous particles / Weight of dry porous particles". The specific measurement method is 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. Having a ratio of swelling degree to water content within the above range allows for more uniform and dense packing of the porous particles into columns, etc., which has the advantage of enabling efficient adsorption, separation, and purification.

[0022] (Base carrier) The base carrier in porous particles contains a polysaccharide. The polysaccharide used is not limited, but it is a polysaccharide that has a hydroxyl group in its molecule that can be phosphate esterified. For example, it is a polysaccharide that contains one or more of the following as components: glucose, agarose, mannose, gross, idose, galactose, talose, ribose, arabinose, xylose, lyxose, allose, altose, allulose, fructose, sorbose, tagarose, or their uronic acid derivatives, amino sugar derivatives, deoxy sugar derivatives, etc.

[0023] Specifically, the polysaccharide is preferably selected from starch, glycogen, pullulan, dextran, nigellan, cellulose, laminaran, curdlan, gellan, mannan, carrageenan, inulin, levan, xylan, arabinan, pectin, chitin, and chitosan. When the porous particles according to this 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, can be used more preferably than synthetic polymers.

[0024] As the polysaccharide, cellulose, particularly spherical cellulose, is especially preferred. Spherical cellulose has the advantages of being inexpensive, highly biocompatible, having high strength, good column pressure resistance, and being autoclavable. The cellulose used here is not particularly limited and may be a cellulose derivative such as cellulose acetate, and may be crystalline cellulose or amorphous cellulose. 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 "phosphate-esterified cellulose particles". The base carrier only needs to have polysaccharides as its main component, and may or may not contain other materials. Here, "main component" 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 particular restriction, but a degree of acetic acidity of 45-57% is preferred.

[0025] The base carrier can be manufactured, for example, by referring to Japanese Patent Publication No. 55-44312. Specifically, it can be manufactured by dissolving a cellulose raw material in an aqueous solution of a calcium salt mainly composed of calcium thiocyanate, dispersing this solution or gel-like substance in granular form in an organic solvent, and then desalting it using a solvent that dissolves calcium salts and is mixed with the dispersion solvent, thereby regenerating the cellulose into a gel-like state.

[0026] The base carrier may be cross-linked cellulose particles. Using cross-linked cellulose particles as the base carrier offers advantages such as superior mechanical strength and flow rate resistance compared to non-cross-linked materials.

[0027] The crosslinking process can be carried out by referring, for example, to Japanese Patent Publication No. 2009-242770, WO2017 / 141910, etc. More specifically, one example is a method that includes adding, in the presence of at least one inorganic salt selected from the group consisting of hydrochloride, sulfate, phosphate, and borate in a mole amount of 6 to 20 times the mole amount of cellulose monomer, 4 to 15 times the mole amount of cellulose monomer, and 0.1 to 1.5 times the mole amount of alkali to the crosslinking agent, to a suspension of uncrosslinked cellulose particles in the presence of 4 to 15 times the mole amount of cellulose monomer, and 0.1 to 1.5 times the mole amount of crosslinking agent, over a period of 3 hours or more. The amount of crosslinking agent to be added (in moles) is more preferably 7 to 15 times the mole amount of cellulose monomer, and particularly preferably 10 to 15 times.

[0028] Crosslinked cellulose particles have high mechanical strength and can be used under chromatographic conditions with higher flow rates. Here, "cellulose monomer" refers to the glucose unit, which is the constituent unit of cellulose. The number of moles of cellulose monomer (i.e., degree of polymerization) is calculated based on the amount obtained by subtracting water from the glucose unit (i.e., the dry weight of cellulose) (1 mole is defined as a molecular weight of 162). The crosslinking agent can be appropriately selected from crosslinking agents commonly used in this field, but examples include epichlorohydrin, epibromohydrin, dichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, glycerol polyglycidyl ether, etc. Preferably, epichlorohydrin, epibromohydrin, or glycerol glycidyl ether is used.

[0029] (Ligang) The porous particles according to this embodiment contain a phosphate ester ligand immobilized on a polysaccharide-containing base carrier. The phosphate ester ligand can be formed by reacting a compound that forms a phosphate ester with the hydroxyl groups of the polysaccharide with the polysaccharide-containing base carrier. Such compounds are not limited as long as they can form a phosphate ester with the hydroxyl groups in the polysaccharide, but phosphorus compounds, particularly phosphorus pentoxide, phosphorus oxychloride, polyphosphate, and phosphoric acid are preferably used. Of these, phosphorus pentoxide and phosphorus oxychloride are preferred, and phosphorus pentoxide is more preferred. When such compounds are used, the introduction of phosphate ester groups is possible under mild conditions, and they are also inexpensive and readily available, making them advantageous for industrial-scale production.

[0030] 2. Method for producing porous particles The porous particles according to this embodiment are, for example, (1) A step of washing the base carrier by adding an organic solvent to the base carrier containing polysaccharides, (2) A step of adding a compound that reacts with the hydroxyl group in the polysaccharide to form a phosphate ester (hereinafter also referred to as the "phosphate ester forming compound") to the washed base carrier. It is manufactured by a method that includes [a specific process].

[0031] In step (1), an organic solvent is added to the polysaccharide-containing base carrier (gel) to wash the base carrier. This washes the base carrier with the organic solvent, that is, the water in the base carrier is replaced by the organic solvent. The organic solvent used is not particularly limited, but an organic solvent with high affinity for water is preferred, such as dimethylformamide, dimethyl sulfoxide, methanol, ethanol, isopropanol, acetone, and acetonitrile. Washing can be performed by repeatedly adding the 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 compounds are as described above, and the amount of compound added can be adjusted as appropriate depending on the desired amount of phosphate ester ligand supported.

[0033] The phosphate ester-forming compound can be added after being dissolved in a suitable solvent. Such solvents include the organic solvents used in step (1) described above, and it is particularly preferable to use a mixture of water and an organic solvent. Here, 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 in such a molar ratio, a large amount of phosphate ester ligands effective for adsorption of the target substance can be introduced. Furthermore, it is possible to suppress the use of the phosphate ester introduced as a ligand as a crosslinking site.

[0034] The immobilization of phosphate ester ligands onto the base support can be carried out by referring to International Publication No. 2013 / 146669, etc. For example, by adding a phosphate ester-forming compound to the base support and reacting it at 40-70°C for 2-18 hours, the phosphate ester-forming compound reacts with the hydroxyl groups of polysaccharides in the base support to form a phosphate ester ligand.

[0035] Furthermore, a drying step may be included after step (1) and before step (2) above. Including such a drying step allows for efficient introduction of the phosphate ester 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 this embodiment can be used in the 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 substance with a relatively large molecular weight is a substance having a molecular weight of 90 kDa or more, and more specifically, is an enzyme, nucleic acid (e.g., mRNA), antibody, protein, virus, etc.

[0037] According to one embodiment, an adsorbent comprising porous particles according to the embodiment is provided. The adsorbent can be used for the adsorption of a target substance. The adsorbent may consist of porous particles according to the embodiment and may contain further materials. Furthermore, a chromatography support containing the above-mentioned adsorbent is also provided. The chromatography support may consist of the adsorbent according to the embodiment, or it may contain further materials. The chromatography support can be suitably used in the separation and purification of target substances.

[0038] The uses of the adsorbent and chromatography support are not limited, but they can be used, for example, as supports for affinity chromatography and cation exchange chromatography. In these chromatographic applications, for example, the adsorbent and chromatography support according to the embodiment are packed into a chromatography separation column. Alternatively, the porous particles according to the embodiment can be subjected to further treatments such as saponification and modification with substituents before use.

[0039] In separation and purification, the chromatography support is first packed into the column, although the method of packing is not particularly limited. Next, the sample solution containing the target substance is brought into contact with the chromatography support to separate the target substance from impurities. Specifically, the target substance can be purified by packing the above-mentioned chromatography support into the column and flowing the sample solution through it, thereby selectively adsorbing the target substance onto the chromatography support. Alternatively, the target substance and impurities can be adsorbed together onto the chromatography support, and the target substance can be purified by taking advantage of the difference in affinity to the chromatography support by gradually or continuously changing the elution conditions (e.g., salt concentration).

[0040] The degree of adsorption of the target substance to the chromatography support according to the embodiment can be evaluated by the 10% dynamic binding capacity (hereinafter also referred to as "10% DBC"). In the porous particles according to the embodiment, the 10% DBC of IgG at a residence time of 2 minutes is 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 chromatography support has excellent adsorption capacity for IgG and substances with similar properties. Furthermore, obtaining excellent 10% DBC at a residence time of 2 minutes means that the chromatography support has the performance to be suitable for separation and purification at high volumes and high processing speeds.

[0041] In setting chromatography conditions, the difference in affinity between the target substance and impurities to the chromatography support is utilized. For example, conditions are set considering differences in the support structure (ligand species, ligand density, ligand orientation, particle size, pore size, base matrix composition, etc.) and the physicochemical properties of the target substance and impurities (isoelectric point, charge, hydrophobicity, molecular structure, stereochemistry, etc.). The conditions can be adjusted to perform chromatography in either bound elution mode or flow-through mode.

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

[0043] According to the method of this 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, recovery rate means the ratio of the amount of the target substance recovered after purification to the amount of the target substance loaded onto the chromatography support (i.e., the amount of the target substance in the sample solution before purification). [Examples]

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

[0045] (Reduction process) (1) 8500 g-wet (water content 11.12) spherical cellulose particles obtained in the above granulation process were dispersed in 12800 g of pure water, then stirring was started and the mixture was heated to 40°C. (2) While continuing to stir at 40°C, 1480g of pure water, 127g of 48% sodium hydroxide aqueous solution, and 76g of sodium borohydride were added. After the addition was complete, the temperature was raised to 60°C and the reaction was carried out for 16 hours. (3) The reaction solution was cooled to a temperature of 40°C or below. (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] <Manufacturing of Phosphate-Esterified Cellulose Particles> [Example 1] 105 g of reduced spherical cellulose particles (water content 10.50) obtained in Production Example 1 were mixed with 158 g of dimethylformamide and stirred, and the mixture was filtered. This process was repeated 10 times. Subsequently, 83 g of dimethylformamide (DMF) was added as a solvent to the washed cellulose particles. While cooling this reaction solution to below 25°C, 11.39 g of phosphorus pentoxide was added, followed by the addition of 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. Next, it was washed with a 0.5 M aqueous sodium hydroxide solution, and then washed with pure water until the washing solution became neutral. Finally, it was washed with a 0.5 M aqueous hydrochloric acid solution, and then washed with pure water until the washing solution became neutral. Finally, the particles were washed with a 0.5M sodium hydroxide aqueous solution, and then washed with pure water until the washing solution became neutral, thereby obtaining phosphate-esterified cellulose particles.

[0047] [Example 2] Phosphate-esterified 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 reduced spherical cellulose particles (water content 10.50) obtained in Production Example 1 were mixed with 450 g of methanol and stirred. This process was repeated 10 times, followed by vacuum drying (40°C to 60°C) to obtain dried cellulose particles. Separately, 206 g of dimethylformamide (DMF) was mixed with 19.28 g of phosphorus pentoxide, 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 were added to this solution, and the temperature was raised to 65°C and the reaction was allowed to proceed for 16 hours. After the reaction, the solvent was removed by filtration, and the resulting gel was washed twice with pure water. Next, it was washed with a 0.5 M sodium hydroxide aqueous solution, and then washed with pure water until the washing solution became neutral. Finally, it was washed with a 0.5 M hydrochloric acid aqueous solution, and then washed with pure water until the washing solution became neutral. Finally, the particles were washed with a 0.5M sodium hydroxide aqueous solution, and then washed with pure water until the washing solution became neutral, thereby obtaining phosphate-esterified cellulose particles.

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

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

[0051] <Evaluation of Phosphate-Esterified Cellulose Particles> The following evaluations were performed on the phosphated cellulose particles of the examples and comparative examples. Unless otherwise specified, the operations described below were carried out at room temperature (25°C).

[0052] [1] Swelling degree The degree of swelling of the phosphated cellulose particles in 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 and allowed to swell. After degassing this 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. The constant volume was measured, and then the entire amount of phosphated cellulose particles in the graduated cylinder was transferred to a beaker and dried at 80°C. The degree of swelling was determined by measuring the dry weight of the gel 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-swelled volume of phosphated cellulose particles refers to the volume measured after repeating the tapping and standing process until the volume of the swollen phosphated cellulose particles becomes constant. More specifically, the water-swelled 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) between the porous particles packed in the column. In other words, the water-swelled volume is measured by reading the value on the graduated cylinder scale when the volume of the swollen phosphated cellulose particles becomes constant. While there are no particular restrictions on the drying method, in this case, the material was dried in a constant temperature bath at 80°C for two days.

[0054] [2] Cation exchange capacity Approximately 10 g of phosphate-esterified cellulose particles obtained in the above examples and comparative examples were mixed with 0.5 M hydrochloric acid and stirred for about 30 minutes. The mixture was then filtered and washed with pure water until the filtrate was neutral. This was transferred to a beaker and dried overnight in a vacuum dryer (60°C). 1 g of these dried particles was accurately weighed, 50 mL of 0.1 M sodium hydroxide was added and gently mixed, and the mixture was allowed to stand for 24 hours. After that, 10 mL of the supernatant was collected and titrated with 0.1 M hydrochloric acid using phenolphthalein as an indicator. Cation exchange capacity (meq / g) = (10 × f1 - V × f2) × 0.1 × 5 / W

[0055] Here, f1 represents the factor of 0.1M sodium hydroxide, f2 represents the factor of 0.1M hydrochloric acid, V represents the titration volume of 0.1M hydrochloric acid (mL), and W represents the particle weight (g). The factor represents how many times (multiple) the actual concentration of the solution used is compared to the target concentration. When expressing the cation exchange capacity in 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%, and the suspension was placed in a graduated cylinder and allowed to stand for 24 hours. The ratio of the volume of naturally settled phosphated cellulose particles to the total volume of the suspension in the graduated cylinder was then confirmed. After that, the graduated cylinder was shaken to make the contents a uniform suspension again, and based on the ratio of the naturally settled volume to the total volume of the suspension confirmed above, a volume of suspension in which the naturally settled volume of cellulose particles was 100 mL was measured. The water was removed from this suspension by suction filtration for 15 minutes on a 90 mm diameter 5A filter paper (Advantec Toyo Co., Ltd., "Quantitative Filter Paper") to obtain suction-dried phosphated cellulose particles. 1 g of the obtained suction-dried phosphated cellulose particles was placed in a beaker and dried overnight at 80°C, and the weight of the obtained dried phosphated cellulose particles was measured. The water content was calculated using the following formula. Moisture content = Weight of suction-dry phosphated cellulose particles / Weight of dry phosphated cellulose particles

[0057] [4] Adsorption evaluation using IgG [Measurement of IgG's 10% dynamic binding capacity] Phosphate-esterified cellulose particles obtained in the above examples and comparative examples were packed into a mini-column (manufactured by JNC Corporation). Separately, a solution of γ-globulin derived from human serum (Wako Pure Chemical Industries, Ltd.) (2 mg / mL) was prepared as an antibody solution. Next, the column was connected to an LC system, and buffer was passed through it to equilibrate until the UV (ultraviolet absorbance, 280 nm), electrical conductivity, and pH of the column effluent were constant. After that, the baseline UV was set to zero, and 10% DBC was measured under the chromatographic conditions shown below.

[0058] The prepared antibody solution was passed through a column packed with phosphated cellulose particles at a flow rate of 0.53 mL / min (residence time 2 minutes). The binding capacity was measured as 10% DBC, using the 280 nm absorbance of the effluent as an indicator. "10% DBC" is a method of estimating adsorption based on the time required for the concentration of the target substance in the effluent to reach 10% of the initial concentration. The UV of the antibody solution passed through the column was measured beforehand, and the UV of the column effluent was monitored to determine the point at which UV equivalent to 10% of the antibody solution concentration was detected.

[0059] Specifically, 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 flowing the antibody solution until it reaches 10% of the pre-measured UV level of the antibody solution × 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) Column: Diameter 6.7 mm, Length 30 mm

[0061] [5] Observation of particle shape using a microscope Microscopic observation was performed using an OLYMPUS CX41 upright microscope. A few drops of the slurry of particles from Examples 1-4 were placed on a glass slide, a coverslip was placed on top, and the image was taken. Figures 1-4 are microscope images at 57.5x magnification. The magnification was calculated using a Kenis Objective Micrometer OM. The images show that the obtained particles are nearly perfectly spherical (Figures 1-4).

[0062] Table 1 shows the evaluation and analysis results for each example and comparative example. [Table 1]

[0063] In Table 1, all of the phosphated cellulose particles in Examples 1 to 4 had an ion exchange capacity of 0.1 meq / mL or higher and a swelling degree greater than 8 mL / g. Furthermore, when the phosphated cellulose particles of Examples 1-4 were used as a chromatography support, the 10% DBC was consistently higher than 50 mg / mL even under high flow rate conditions such as a residence time of 2 minutes, 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 can efficiently adsorb 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 manufacture of biopharmaceuticals and the like.

[0064] While several embodiments of the present invention have been described, these embodiments are presented as examples only and are not intended to limit the scope of the invention. These novel embodiments can be carried out in a variety of other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims of the invention and its equivalents.

Claims

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

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

3. The porous particle according to claim 1, wherein the 10% dynamic binding capacity (DBC) of IgG at a residence time of 2 minutes is 40 mg / mL or more.

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

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

0.

6. The porous particles according to claim 1, wherein the shape is spherical, granular, or a mixture thereof.

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

8. A chromatography support comprising the adsorbent described in claim 7.

9. (1) A step of washing the base carrier by adding an organic solvent to the base carrier containing polysaccharides, (2) A step of adding a compound that reacts with the hydroxyl groups in the polysaccharide to form a phosphate ester to the base carrier after washing. A method for producing porous particles according to claim 1, including the method described in claim 1.

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

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