Porous particles, their manufacturing method, and chromatography packing material using the same

Abstract

To provide a porous particle having a controlled three-dimensional network-shaped skeleton and a communication hole consisting of cavities of the skeleton, further in which the communication hole penetrates from a surface of the particle to the inside thereof, a production method thereof, and a filler for chromatography with the porous particle.SOLUTION: A porous particle, of which the main component is cellulose acetate or cellulose, is spherical, and has a three-dimensional network-shaped skeleton and a communication hole structure consisting of cavities of the skeleton, further in which the communication hole penetrates from a surface of the particle to the inside thereof. When the porous particles are filled in a column with an inside diameter of 0.78 cm and a length of 30 cm and the flow rate is set to be 0.4 ml / minute, retention time of dextran with molecular weight of 2,000,000 is 20 minutes or more.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to porous particles, a method for producing the same, and a chromatography packing material using the same. [Background technology]

[0002] Monoliths, which are integrally molded bodies with a three-dimensional network structure and interconnected pores, have been attracting attention as a material for chromatography used in the separation and purification of biopharmaceuticals. It is known that the use of monoliths enables separation and purification with higher throughput and speed than conventional chromatography (Patent Document 1).

[0003] In recent years, particles with interconnected pores, which are formed by converting monoliths into particles, have been proposed. The advantages of forming particles include ease of handling, as they can be used in the same way as packing materials for conventional chromatography separation columns, and ease of scale-up. Furthermore, particles with interconnected pores can utilize the voids of the interconnected pores within the particles as adsorption sites during the separation and purification of biopolymers and other substances, making them a material that enables high-throughput, high-speed separation and purification. Methods for producing interconnected pore particles include, for example, a method of pulverizing monoliths into particles (Patent Document 2) and a method of directly granulating particles with interconnected pores (Patent Documents 3, 4, and 5). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2016 / 063702 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-148456 [Patent Document 3] International Publication No. WO2016 / 013568 [Patent Document 4] International Publication No. WO2017 / 026424 [Patent Document 5] Japanese Patent Publication No. 2020-026511 [Patent Document 6] International Publication No. WO2015 / 029790 [Non-patent literature]

[0005] [Non-Patent Document 1] P DePhillips, AM Lenhoff, J Chromatogr A. 2000, 883, 39 DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0006] In the method of crushing monoliths to form particles, the particles obtained by crushing have irregular shapes, which makes it impossible to uniformly pack them into a chromatography separation column, resulting in a failure to achieve the expected performance. Furthermore, in the method of directly granulating spherical particles, there are problems such as difficulty in forming interconnected pores on the particle surface, which can lead to blockage, uneven pore size for each particle, and difficulty in controlling the pore size.

[0007] Therefore, an object of the present invention is to provide porous particles having a controlled three-dimensional network skeleton and interconnected pores formed by voids therein, the interconnected pores penetrating from the particle surface to the interior, a method for producing the same, and a chromatography packing material using the same. [Means for solving the problem]

[0008] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have discovered porous particles having a controlled three-dimensional network skeleton and interconnected pores formed by voids therein, with the interconnected pores penetrating from the particle surface to the interior, a method for producing the same, and a chromatography packing material using the same, and have completed the present invention.

[0009] That is, the present invention is as follows. [1] Porous particles whose main component is cellulose acetate or cellulose, characterized in that they are spherical, have a three-dimensional network-like skeleton and an interconnected pore structure consisting of voids therebetween, and the interconnected pores penetrate from the particle surface to the interior, and when packed into a column with an inner diameter of 0.78 cm and a length of 30 cm, the retention time of dextran with a molecular weight of 2 million is 20 minutes or more when the column is filled at a flow rate of 0.4 ml / min. [2] The porous particles according to [1], characterized in that when packed in a column having an inner diameter of 0.78 cm and a length of 30 cm and at a flow rate of 0.4 ml / min, the retention time of silica nanoparticles having a particle size of 30 nm is 16 minutes or more. [3] The porous particles according to [1] or [2], which have a mode diameter in the range of 1 to 1000 μm. [4] The specific surface area measured by the BET multipoint method is 1 to 200 m 2 The porous particles according to any one of [1] to [3], wherein the range is 1 / g. [5] A method for producing porous particles, comprising: (a) preparing a cellulose acetate solution by heating and dissolving cellulose acetate in a mixed solvent of a solvent in which cellulose acetate is soluble and a solvent in which cellulose acetate is insoluble; (b) dispersing the cellulose acetate solution in water containing an emulsion stabilizer to obtain a dispersion; and (c) cooling the dispersion to precipitate cellulose acetate particles, wherein the mixed solvent is an organic solvent that is immiscible with water. [6] The method for producing porous particles according to [5], wherein in (a), the solvent in which cellulose acetate is soluble is benzyl alcohol, ethyl acetate, cyclohexanone, or isophorone. [7] The method for producing porous particles according to [5] or [6], wherein in (a), the solvent in which cellulose acetate is insoluble is an alcohol, a glycol, an ether, or an ester. [8] The method for producing porous particles according to any one of [5] to [7], wherein in (a), the volume ratio of the solvent in which cellulose acetate is soluble to the solvent in which cellulose acetate is insoluble in the mixed solvent is 5:95 to 95:5. [9] The method for producing porous particles according to any one of [5] to [8], wherein in (a), the content of cellulose acetate in the mixed solvent is 0.1 to 30% by weight based on the cellulose acetate solution.

[10] The method for producing porous particles according to any one of [5] to [9], wherein in (a), the mixed solvent contains a third component which is a polymer, and the third component is polyethylene glycol or polypropylene glycol.

[11] (b) The method for producing porous particles according to any one of [5] to

[10] , wherein the emulsion stabilizer is sodium dodecylbenzenesulfonate, polyvinyl alcohol, carboxymethyl cellulose, hydroxyethyl cellulose, or a mixture thereof.

[12] The method for producing porous particles according to any one of [5] to

[11] , wherein in (c), the cooling rate of the dispersion system is 0.1°C / min to 10°C / min.

[13] A chromatography packing material comprising the porous particle according to any one of [1] to [4], or the porous particle which has been saponified, crosslinked, or modified.

[14] The chromatography packing material according to

[13] , which is used for separating and purifying virus particles. [Effects of the Invention]

[0010] The porous particles of the present invention have a controlled three-dimensional network structure and interconnected pores formed by the voids therein, and these interconnected pores penetrate from the particle surface to the interior. Therefore, when separating and purifying biopolymers and other substances, the voids in the interconnected pores inside the particles can also be used as adsorption sites, making them a useful material that can achieve high throughput and high processing speed. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the characteristics when the particles produced in Production Examples 1 to 4 are packed into a column, and is a graph showing the relationship between the molecular weight of a sample and the retention time. [Figure 2] FIG. 2 is a photograph (magnification: 1200 times) showing the appearance of the particles produced in Production Example 1. [Figure 3] FIG. 3 is a photograph (magnification: 700 times) showing the appearance of the particles produced in Production Example 2. [Figure 4]FIG. 4 is a photograph (magnification: 10,000 times) showing the appearance of the particles produced in Production Example 3. [Figure 5] FIG. 5 is a photograph (magnification: 800 times) showing the appearance of the particles produced in Production Example 4. [Figure 6] FIG. 6 is a photograph (magnification: 700 times) of the internal cross section of the particle produced in Production Example 1. [Figure 7] FIG. 7 is a photograph (magnification: 900 times) of the internal cross section of the particle produced in Production Example 2. [Figure 8] FIG. 8 is a graph showing the relationship between pressure loss and linear velocity for the particles produced in Production Examples 5 and 6. DETAILED DESCRIPTION OF THE INVENTION

[0012] The porous particles of the present invention have a spherical shape. Here, the spherical shape means, for example, that the major axis (longest axis) is no more than twice the minor axis (shortest axis). It is preferable that the porous particles of the present invention have a true spherical shape, and that the major axis and minor axis are nearly the same length.

[0013] The porous particles of the present invention have an interconnected pore structure consisting of a three-dimensional network skeleton and voids therein, and the interconnected pores penetrate from the particle surface to the interior. Whether or not the particles have a three-dimensional network skeleton and interconnected pores can be determined, for example, from the results of confocal laser microscope observation or SEM observation of the cross section of the particles. Furthermore, whether or not the interconnected pores penetrate from the particle surface to the interior can be confirmed, for example, from a photograph of the surface of the porous particles of the present invention. In the porous particles of the present invention, the interconnected pores are not blocked at the particle surface but are open at the particle surface.

[0014] The porous particles of the present invention satisfy the following characteristics: When packed into a column with an inner diameter of 0.78 cm and a length of 30 cm and at a flow rate of 0.4 ml / min, the retention time of dextran with a molecular weight of 2 million is 20 minutes or longer. From the perspective of utilizing the voids inside the particles as adsorption sites, the retention time of dextran with a molecular weight of 2 million is preferably 25 minutes or longer, more preferably 27 minutes or longer. The column is packed by dispersing the particles in pure water to form a slurry, then packing it into the column, and then flowing pure water at a flow rate of 0.4 ml / min for 1 hour or longer to compact it. The retention time here refers to the time it takes from the injection of a sample into the column until the elution peak appears. The elution peak can be determined, for example, from the value at which the RI detection intensity is maximized using an HPLC device (Agilent Technologies, 1260 Infinity). The retention time measurement in the present invention is performed using pure water as the mobile phase and at a temperature of 25°C.

[0015] In the field of chromatography, when measuring the pore size of porous particles, it is common to measure dextran with a molecular weight of 2 million as the exclusion limit molecular weight (Patent Document 6). This is based on the premise that dextran with a molecular weight of 2 million cannot enter the interior of porous particles. The porous particles of the present invention have a longer retention time for dextran with a molecular weight of 2 million compared to conventional particles. This indicates that the porous particles of the present invention have interconnected pores that allow dextran with a molecular weight of 2 million to enter the interior of the particles.

[0016] It is generally known that when spherical porous particles are packed into a column, the porosity is 30 to 40% (Non-Patent Document 1). The porosity here is calculated by dividing the total volume of the gaps between the particles inside the column by the total volume of the column. For example, assuming that a sample passes only through the gaps between the packed particles inside a column with an inner diameter of 0.78 cm and a length of 30 cm, the theoretical retention time at a flow rate of 0.4 ml / min is 10.7 to 14.3 minutes. The fact that the retention time of dextran with a molecular weight of 2 million in the porous particles of the present invention is 20 minutes or more indicates that the dextran with a molecular weight of 2 million penetrates not only into the gaps between the particles but also into the interior of the particles.

[0017] The porous particles of the present invention more preferably satisfy the following characteristics: When packed into a column having an inner diameter of 0.78 cm and a length of 30 cm and at a flow rate of 0.4 ml / min, the retention time of 30 nm silica nanoparticles is 16 minutes or longer. From the viewpoint of utilizing the voids inside the particles as adsorption sites, the retention time of 30 nm silica nanoparticles is more preferably 18 minutes or longer. The column packing method, retention time, and measurement temperature are as described above. The particles of the present invention have a longer retention time of 30 nm silica nanoparticles compared to conventional particles. This indicates that the particles of the present invention have interconnected pores that allow 30 nm silica nanoparticles to enter the interior of the particles.

[0018] The main component of the porous particles of the present invention is cellulose acetate or cellulose. The term "main component" as used herein means a component whose content in the porous particles is 50% by mass or more. The cellulose acetate used in the present invention is not particularly limited as long as it can be generally defined as cellulose acetate, but it is preferable that the acetylation degree is 45 to 57%.

[0019] The porous particles of the present invention have a mode diameter of 1 to 1000 μm. From the viewpoint of usability as a packing material for chromatography, the mode diameter of the porous particles is particularly preferably 35 μm to 200 μm. However, it is not limited to these values. The mode diameter referred to here was calculated by measuring the mode diameter using a particle size distribution analyzer: Laser Scattering Particle Size Distribution Analyzer Partica LA-950 manufactured by HORIBA.

[0020] The specific surface area of the porous particles of the present invention measured by the BET multipoint method is 1 to 200 m 2 / g. The specific surface area by the BET multipoint method can be measured, for example, using a high-speed specific surface area / pore distribution analyzer (BELSORP MAXII manufactured by MicrotrackBell). For example, the particles of the present invention can be freeze-dried overnight, and then pretreated by vacuum degassing at 100°C for 2 hours, before measurement. The specific surface area can be determined from an adsorption isotherm (straight line) obtained by measuring the amount of nitrogen gas adsorbed on the solid surface at a measurement temperature of 77.3K and a relative pressure range of 0.05 to 0.30 at three or more points.

[0021] Hereinafter, the method for producing porous particles of the present invention includes the steps of: (a) preparing a cellulose acetate solution by heating and dissolving cellulose acetate in a mixed solvent of a solvent in which cellulose acetate is soluble and a solvent in which cellulose acetate is insoluble; (b) dispersing the cellulose acetate solution in water containing an emulsion stabilizer to obtain a dispersion; and (c) cooling the dispersion to precipitate cellulose acetate particles, wherein the mixed solvent is an organic solvent that is immiscible with water.

[0022] According to the production method of the present invention, it is possible to preferably produce porous particles whose main component is cellulose acetate, which have a spherical shape, a three-dimensional network-like skeleton, and an interconnected pore structure consisting of voids therebetween, and the interconnected pores extend from the particle surface to the interior. The production method of the present invention makes it possible to easily control the particle size and interconnected pore size of the resulting porous particles. For example, this can be controlled by the amount of cellulose acetate raw material used, the type and ratio of the good and poor solvents used, the molecular weight and concentration of the polymer (third component), the type and amount of surfactant added, the cooling rate, etc. The porous particles of the present invention described above can be preferably produced by carrying out the above-mentioned control in the production method of the present invention. Each of the above steps will be explained below in order.

[0023] [Step (a)] In (a), cellulose acetate as a raw material is dissolved in a mixed solvent to prepare a cellulose acetate solution. Cellulose acetate is a semi-synthetic polymer obtained by acetic acid esterification of cellulose, a natural polymer. The cellulose acetate used in the present invention is not particularly limited as long as it can be generally defined as cellulose acetate, but it preferably has a degree of polymerization of 50 to 300 and an acetylation degree of 45 to 57%. By using cellulose acetate with a degree of polymerization of 50 to 300 and an acetylation degree of 45 to 57%, it can be dissolved in a wider variety of solvents.

[0024] In (a), the solvent in which the cellulose acetate solution is soluble, i.e., the good solvent, is not particularly limited as long as it can dissolve cellulose acetate, but an organic solvent with low solubility in water is preferred. The term "good solvent" as used herein refers to a solvent in which the solute dissolves in the solvent alone, yielding a clear solution containing no solids. In particular, the term "good solvent" refers to a solvent that yields a solution of preferably 1% by weight or more at a temperature below the boiling point of the solvent. The solvent may be used alone or in combination of two or more solvents. Specific examples include benzyl alcohol, ethyl acetate, cyclohexanone, isophorone, and mixtures thereof. Of these, benzyl alcohol is preferred in terms of forming interconnected pores.

[0025] In (a), examples of solvents insoluble in the cellulose acetate solution, i.e., poor solvents, include lower alcohols, glycols, ethers, esters, and mixtures thereof. A poor solvent is a solvent that has no or low solubility for a solute compared to a good solvent. Specifically, it is a solvent that cannot dissolve the solute by itself at temperatures below its boiling point and does not yield a transparent solution. In particular, the poor solvent is a solvent that cannot dissolve 1% by weight or more of the solute at temperatures below its boiling point. Solvents with low solubility in water are preferred. Lower alcohols are particularly preferred, and 1-hexanol is particularly preferred in terms of forming interconnected pores.

[0026] In (a), the mixed solvent preferably contains a solvent in which cellulose acetate is soluble (i.e., a good solvent) and a solvent in which cellulose acetate is insoluble (i.e., a poor solvent) in a proportion of 5 to 95 v / v % good solvent and 95 to 5 v / v % poor solvent. The higher the proportion of poor solvent in the mixed solvent, the larger the pore size of the continuous pores tends to be.

[0027] In (a), the proportion of cellulose acetate in the mixed solvent is preferably 0.1 to 30% by weight, more preferably 5 to 20% by weight. Within this range, a homogeneous solution is obtained, and porous particles with clear interconnected pores are easily obtained. Furthermore, precipitation due to phase separation is likely to occur upon cooling, resulting in sufficient strength and easy maintenance of particle shape. Furthermore, the lower the cellulose acetate concentration in the cellulose acetate solution, the smaller the particle size of the resulting porous particles tends to be and the larger the pore size of the interconnected pores tends to be.

[0028] In step (a), the mixed solvent is an organic solvent that is not miscible with water. If it becomes miscible with water, a dispersion cannot be obtained in step (b).

[0029] In (a), a third component, which is a polymer, can be contained in the mixed solvent. The third component polymer is preferably polyethylene glycol or polypropylene glycol, and more preferably polypropylene glycol.

[0030] In (a), when the third component is polypropylene glycol, its molecular weight is not particularly limited, but from the viewpoints of ease of handling during the production process and the pore size of the interconnected pores of the porous particles, it is preferable that the average molecular weight (Mw) is 100 to 3000. Furthermore, the higher the molecular weight of the polymer of the third component, the larger the pore size of the interconnected pores tends to be.

[0031] In (a), from the viewpoint of the pore size of the interconnected pores of the porous particles, the concentration of the third component in the mixed solvent is preferably 0.1 to 10% by weight, and the higher the concentration of the third component, the larger the pore size of the interconnected pores tends to be.

[0032] In (a), the heating temperature for dissolving by heating is preferably, for example, in the range of 50 to 130°C. Within this temperature range, cellulose acetate is preferably dissolved in the mixed solvent. The heating and dissolving time is not particularly limited, and may be, for example, 3 to 24 hours.

[0033] [Step (b)] In step (b), the cellulose acetate solution is dispersed in high-temperature water containing an emulsion stabilizer. The volume ratio of the cellulose acetate solution to the high-temperature water in this step is not particularly limited, as long as it is within a range that allows for a dispersion system in which the cellulose acetate solution is the dispersed phase and the high-temperature water is the continuous phase. From the viewpoint of obtaining a stable dispersion system, the volume ratio of the cellulose acetate solution to the high-temperature water (dispersed phase / continuous phase) is preferably 1.0 or less. The temperature of the high-temperature water is preferably 50 to 100°C, more preferably 60 to 95°C.

[0034] Regarding the dispersion method in (b), any known method can be applied. Examples include a method using a mixer such as a stirrer, a method using a homogenizer, and a method using ultrasound. Other examples include a method using what is generally called a microreactor to extrude a cellulose acetate solution through a thin nozzle to obtain droplets, and a method in which a cellulose acetate solution or a mixture of a cellulose acetate solution, water, and an emulsion stabilizer is extruded through a porous membrane with uniform pore size and subjected to shear. In the present invention, the simplest dispersion method using a stirrer was investigated.

[0035] In (b), the emulsion stabilizer is not particularly limited as long as it enhances the stability of the dispersion and prevents particle aggregation. Examples include natural polymers such as starch, pectin, alginic acid, alginates, and gelatin; processed natural polymers such as methylcellulose, carboxymethylcellulose, and hydroxyethylcellulose; alcoholic OH-containing synthetic polymers such as polyvinyl alcohol and partially saponified polyvinyl acetate; SOH group-containing polymers such as sulfonated styrene; COOH group-containing polymers such as acrylic esters; nitrogen-containing synthetic polymers such as polyvinylpyrrolidone; inorganic powders such as barium sulfate, talc, bentonite, and titanium oxide; anionic surfactants such as linear sodium alkylbenzenesulfonate; cationic surfactants such as alkyltrimethylammonium salts; amphoteric surfactants such as alkyldimethylamine oxide; nonionic surfactants such as fatty acid sorbitan esters; and mixtures thereof. In particular, sodium dodecylbenzenesulfonate, polyvinyl alcohol, carboxymethylcellulose, hydroxyethylcellulose, and mixtures thereof are preferred from the perspective of stabilizing the dispersion.

[0036] The amount of emulsion stabilizer used in (b) is preferably 0.01 wt % or more in high-temperature water to stabilize the dispersion system. In this concentration range, increasing the amount of emulsion stabilizer tends to result in smaller particle sizes of the resulting porous particles. Conversely, decreasing the amount of emulsion stabilizer tends to result in larger particle sizes of the resulting porous particles.

[0037] [Step (c)] In step (c), the dispersion obtained in step (b) is cooled, which causes thermally induced phase separation, resulting in porous particles having interconnected pores.

[0038] In (c), the cooling rate of the dispersion is preferably 0.1°C / min to 10°C / min. The slower the cooling rate in (c), the larger the pore size of the continuous pores tends to be. Conversely, the faster the cooling rate, the smaller the pore size of the continuous pores tends to be. When producing the porous particles of the present invention described above, the cooling rate is preferably 0.1°C / min to 5°C / min, and more preferably 0.1°C / min to 2°C / min.

[0039] In (c), the temperature to which the dispersion is cooled is not particularly limited as long as it is a temperature at which cellulose acetate precipitates, but the cooling temperature is preferably 0°C to 50°C in order to completely precipitate the porous particles.

[0040] [Porous particles or cross-linked, modified porous particles] According to one embodiment of the present invention, porous particles that can be produced by the above-mentioned production method are provided. The porous particles can be used in the separation and purification of various substances. Furthermore, the porous particles may be used in a form that has been subjected to a saponification reaction, a crosslinking reaction, or modified with a substituent.

[0041] By saponifying and crosslinking porous particles, it is possible to impart mechanical strength and flow rate resistance sufficient for use as a chromatography packing material. The crosslinking treatment of porous particles can be carried out with reference to JP 2009-242770 A. By appropriately modifying the experimental conditions, it is possible to impart the desired mechanical strength and flow rate resistance.

[0042] Furthermore, by adding a ligand to at least a portion of the reactive functional groups of the porous particles of the present invention, adsorbents capable of adsorbing various substances can be easily obtained. For example, they can be used as virus adsorbents for influenza virus, hepatitis virus, rabies virus, human papillomavirus, adeno-associated virus, etc., adsorbents for purifying antibody drugs, exosome adsorbents, nucleic acid adsorbents, etc. Specifically, by saponifying and crosslinking the porous particles of the present invention and then introducing a sulfated polysaccharide, a chromatography packing suitable for separating or purifying virus particles can be provided. The introduction of sulfated polysaccharides can be carried out with reference to, for example, JP 2011-220992 A. Other ligands include, but are not limited to, ion exchange groups such as 2-diethylaminoethyl (DEAE), carboxymethyl (CM), sulfone, and quaternary ammonium (Q); hydrophobic groups such as phenyl and butyl; ligands that have both ion exchange and hydrophobic groups and can be used for so-called mixed-mode separation; ligands for protein adsorption such as protein A and antibodies; polycations such as polylysine; and functional polymers such as polyanions such as heparin and polyglutamic acid. These can be selected as desired depending on the intended use of the porous particles, and their introduction can be carried out as desired according to known methods.

[0043] The adsorption performance of the chromatography packing material of the present invention for influenza viruses can be evaluated, for example, by the method described in JP 2011-220992 A. The influenza viruses used for evaluation can be type A, type B, influenza viruses derived from embryonated chicken eggs, or influenza viruses derived from cultured cells such as MDCK cells. [Example]

[0044] The present invention will be described in more detail below with reference to examples. It is not limited to:

[0045] [Example 1] 14 g of cellulose acetate (L-20, Daicel) was added to a mixed solvent of 52.5 ml of benzyl alcohol, 47.5 ml of 1-hexanol, and 2 ml of polypropylene glycol (Wako Pure Chemical Industries, diol type, average molecular weight 1,000) and stirred. The mixture was then heated to 120°C and stirred for 4 hours to dissolve the cellulose acetate, yielding a clear cellulose acetate solution. 60 mg of PVA (JP-18E, Nippon Vinyl Acetate & Poval Co., Ltd.) and 4.5 g of sodium carboxymethylcellulose (Wako Pure Chemical Industries, Ltd.) were added to 300 ml of pure water saturated with the mixed solvent, heated to 80°C, and stirred for over 1 hour to obtain a dispersion medium. 100 g of the cellulose acetate solution was quickly poured into 300 ml of the dispersion medium and stirred at 80°C for 15 minutes at 250 rpm to obtain a dispersion. This dispersion was then left at room temperature (20°C) and then cooled to 40°C. The cooling rate was 0.5°C / min on average. When the temperature reached 40°C, cellulose acetate precipitated, yielding spherical cellulose acetate particles. The resulting cellulose acetate particles were then thoroughly washed with a large amount of water and then with methanol. After washing again with water, the resulting spherical cellulose acetate particles were passed through sieves with 250µm and 45µm openings to yield porous particles with particle sizes of 45-250µm.

[0046] [Example 2] 14 g of cellulose acetate (L-20, Daicel) was added to a mixed solvent of 52.5 ml of benzyl alcohol, 47.5 ml of 1-hexanol, and 2 ml of polypropylene glycol (Wako Pure Chemical Industries, Ltd., triol type, average molecular weight 700) and stirred. The mixture was then heated to 120°C and stirred for 4 hours to dissolve the cellulose acetate, yielding a clear solution. This was then granulated, washed, and classified in the same manner as in Example 1 to yield porous particles.

[0047] [Example 3] 16 g of cellulose acetate (L-20, Daicel) was added to a mixed solvent of 55 ml of benzyl alcohol and 45 ml of 1-hexanol and stirred. The mixture was then heated to 120°C and stirred for 4 hours to dissolve the cellulose acetate, yielding a clear cellulose acetate solution. 6 g of hydroxyethyl cellulose (Wako Pure Chemical Industries, Ltd.) and 6 g of sodium carboxymethyl cellulose (Wako Pure Chemical Industries, Ltd.) were added to 600 ml of pure water saturated with the mixed solvent, heated to 80°C, and stirred and dissolved for over 1 hour to obtain a dispersion medium. 100 g of the cellulose acetate solution was quickly poured into 600 ml of the dispersion medium and stirred at 80°C and 300 rpm for 15 minutes to obtain a dispersion. This dispersion was then rapidly cooled in a 20°C water bath. The cooling rate was 4°C / min on average. Spherical particles were obtained. The mixture was then washed and classified in the same manner as in Example 1 to obtain porous particles.

[0048] [Example 4] 16 g of cellulose acetate (L-20, Daicel) was added to a mixed solvent of 55 ml of benzyl alcohol, 45 ml of 1-hexanol, and 1.5 ml of polypropylene glycol (Wako Pure Chemical Industries, Ltd., triol type, average molecular weight 1,500) and stirred. Thereafter, the mixture was granulated, washed, and classified in the same manner as in Example 3 to obtain porous particles.

[0049] An example of the production of crosslinked porous particles, which is one of the preferred uses of the porous particles of the present invention, will be described below. [Manufacturing Example 1] 55 g of the porous particles obtained in Example 1 (water content: 6.31 g) were added to a solution prepared by dissolving 32 g of NaSO in 124 g of pure water, and the mixture was stirred at 40°C for 30 minutes. Next, 2.93 mol equivalents of NaOH were added, and the mixture was stirred at 40°C for 2 hours to react and saponify the particles. After the reaction, the temperature was raised to 50°C, and while continuing to stir, 0.9 g of 48% NaOH solution and 0.5 g of NaBH were added, and the mixture was dissolved and reacted for 30 minutes. After dissolution, 22 g of 48% NaOH solution and 30 g of epichlorohydrin were added in four equal portions every 60 minutes over approximately 3 hours. After the addition was completed, the mixture was allowed to react for 16 hours at 50°C. The mixture was cooled to below 40°C, and 5.8 g of acetic acid was added for neutralization. The reaction mixture was filtered to recover the gel, which was then filtered and washed with pure water to obtain the desired crosslinked porous particles.

[0050] [Manufacturing Examples 2 to 4] The resulting particles of Examples 2 to 4 were subjected to a crosslinking step in the same manner as in Production Example 1, to obtain crosslinked porous particles of Production Examples 2 to 4.

[0051] [Particle evaluation] Retention time measurement The particles obtained in Production Examples 1 to 4 above were packed into a stainless steel column (manufactured by Tosoh) with an inner diameter of 0.78 cm and a length of 30 cm, and the retention time of each sample was measured. A commercially available GCL-2000HF (manufactured by JNC) was used for particle comparison. The particles were packed into a column by dispersing them in pure water to form a slurry, and then pure water was passed through the column at a flow rate of 0.4 ml / min for at least 1 hour to compact the particles. The samples used were those listed in Table 1 below. Pure water was used as the mobile phase during measurement. The value at which the RI detection intensity reached its maximum was taken as the elution peak, and the time at this time was taken as the retention time. The equipment used for the measurements was as follows: Equipment: 1260 Infinity HPLC equipment (Agilent Technologies) Retention times were measured using the above equipment. The results are shown in Figure 1 and Table 2.

[0052] [Table 1] 10 μl of each sample was used for the measurement.

[0053] [Table 2]

[0054] As a result of the measurements, the particles from Production Examples 1 and 2 had a retention time of 2 million molecular weight dextran of 20 minutes or more. On the other hand, the particles from Production Examples 3 and 4 and GCL-2000HF had a retention time of less than 20 minutes, indicating that the pore size of the interconnected pores was small (Table 2). Even when other polymer samples were used, the particles from Production Examples 1 and 2 had a longer sample retention time than the particles from Production Examples 3 and 4 and GCL-2000HF (Figure 1).

[0055] The retention time of the 30 nm silica nanoparticles of Production Example 1 was 16 minutes or more. On the other hand, the retention times of the 30 nm silica nanoparticles of Production Examples 2 to 4 and GCL-2000HF were less than 16 minutes (Table 2).

[0056] 2. Mode diameter measurement The particle size distribution was measured and the mode diameter was determined for the particles obtained in the above Production Examples 1 to 4. The following apparatus was used for the measurement. Equipment: Laser Scattering Particle Size distribution Analyzer Partica LA-960 (manufactured by HORIBA) The mode diameter was measured using the above device.

[0057] 3. Measurement of BET specific surface area The BET specific surface area was measured for the particles obtained in Production Examples 1 to 4 above. The BET specific surface area was measured using a high-speed specific surface area / pore distribution analyzer (BELSORP MAXII manufactured by MicrotrackBell). The freeze-dried particles of Production Examples 1 to 4 were further pretreated by degassing at 100°C for 2 hours (vacuum degassing) to prepare samples. The BET specific surface area was determined from an adsorption isotherm (straight line) obtained by measuring the amount of nitrogen gas adsorbed on the solid surface at a measurement temperature of 77.3 K and at five or more pressure points in the relative pressure range from 0.05 to 0.30. The evaluation results are shown in Table 3.

[0058] [Table 3]

[0059] 4. SEM Observation SEM observations were carried out using an ultra-high resolution field emission scanning electron microscope "SU8020" manufactured by Hitachi High-Technologies. The particles of Production Examples 1 to 4, which had been freeze-dried as a pretreatment, were coated with Au for photography purposes. Electron microscope photographs show that the obtained particles are nearly spherical, with interconnected pores on the surface and cross section (Figures 2 to 7).

[0060] The following describes a production example of a chromatography packing material, which is one of the preferred uses of the porous particles of the present invention.

[0061] [Use as a chromatography packing] [Production Example 5] (Binding of porous particles to sulfated polysaccharides) 1) Epoxidation of crosslinked porous particles 20 g of the crosslinked porous particles (water content 6.25%) obtained in Preparation Example 1 were placed in a reactor equipped with a stirrer along with 24 ml of purified water. The internal temperature was adjusted to 30°C while stirring vigorously, and 15.6 g of epichlorohydrin was added. After 15 minutes of stirring, 14.6 g of 48% aqueous NaOH solution was added and the reaction was allowed to proceed for 2 hours. After completion of the reaction, the reaction solution was filtered and thoroughly washed with water until the filtrate became neutral. The mixture was suction-dried for 30 minutes to obtain 20 g of epoxy-activated wet gel. 1.0 g of the epoxy-activated wet gel was shaken with 3.0 ml of 1.3 M sodium thiosulfate solution in a shaker set at 30°C for 1 hour, and the epoxy content was determined by titration with 0.1 mol L hydrochloric acid. The epoxy group content was 230 μmol / g-dry.

[0062] 2) Sulfated polysaccharide binding 1.63 g of dextran sulfate sodium DS-500 (Meito Sangyo Co., Ltd.) was placed in a reactor equipped with a stirrer and stirred until the dextran sulfate sodium was completely dissolved. Then, 15 g of the epoxy-activated wet gel (water content: 5.78, epoxy group content: 230 μmol / g-dry) prepared in step 1) was added to the reactor. The internal temperature was adjusted to 30°C with vigorous stirring. After 15 minutes of stirring, 8.4 g of Na2SO4, 13.5 g of Na2HPO4, and 1.3 g of 48% NaOH aqueous solution were added. The temperature was raised to 40°C, and the reaction was continued for 6 hours. After completion of the reaction, a solution of 0.2 g of NaBH4 dissolved in 0.9 ml of 48% NaOH aqueous solution in 9.7 ml of pure water was added, and the reaction was continued for an additional 16 hours. After the reaction, the reaction mixture was filtered and thoroughly washed with water until the filtrate was neutral. After suction drying for 30 minutes, 15 g-wet of dextran sulfated porous particles was obtained.

[0063] [Manufacturing Example 6] The crosslinked porous particles obtained in Production Example 4 were bound with a sulfated polysaccharide in the same manner as in Production Example 5 to give Production Example 6.

[0064] 3) Virus evaluation test (1) Comparison of commercially available Cellufine Sulfate with the chromatography packing material of Preparation Example 5 The packing materials used were commercially available Cellufine Sulfate (manufactured by JNC) and the chromatography packing materials of Production Examples 5 and 6. The commercially available Cellufine Sulfate used for comparison is a dense cellulose particle having sulfated polysaccharides as ligands on its surface. Each filler was dispersed in water and degassed while stirring under reduced pressure. Each column was packed with each chromatography packing material, attached to an AKTA avant (Cytiva), and equilibrated with 0.01 M phosphate buffer, 0.12 M NaCl, pH 7.4, at least 10 times the column volume. Then, 30 ml of H1N1 strain test virus solution (total HA titer at time of use: 76,800) filtered through a 0.45 μm cellulose acetate membrane filter (product name: GD / X Syringe Filter) was passed through the column, and 1.5 ml of effluent was collected. After the test virus solution was passed through, 0.01 M phosphate buffer, 0.12 M NaCl, pH 7.4 was passed through to wash out the non-adsorbed fraction. After washing out the non-adsorbed fraction, 0.01 M phosphate buffer, 2 M NaCl, pH 7.4 was passed through until the virus adsorbed to the column was completely eluted. After elution, 50 μL of the 1.5 mL fraction of the collected effluent was used to measure the HA titer. Furthermore, the total volume (μL) up to the fraction immediately before the HA titer reached 1 / 10 of the HA titer of the 50 μL test virus solution was calculated. The 10% DBC in this measurement system was calculated as follows: 10% DBC (HA value / ml-gel) = (total volume up to the fraction just before reaching 1 / 10 of the HA titer of 50 μL of test virus solution × 1 / 50 × HA titer of 50 μL of test virus solution) / column volume (ml-gel)

[0065] (2) Measurement of viral activity HA titer Add 50 μL of saline to each well of a round-bottom 96-well plate, and place the evaluation sample in the first vertical row. Purification (i.e., the elution fraction obtained above and the inactivated virus-containing solution as a reference) 50 μL of this 2-fold diluted sample was added to the adjacent well. The same procedure was repeated until the 12th well, with the concentration increased from 2 to 4096 times. Dilution samples were prepared. 50 μL of 0.5% chicken red blood cell suspension was added to each sample and mixed. After 1 hour, the blood cells that had not aggregated and settled to the bottom were counted. The virus was considered to be active, and the dilution factor just before the point where activity was no longer observed was 50μ. The HA titer per 50 μL (HAU / 50 μL) was calculated. Saline was used.

[0066] As a result, the virus adsorption amount was 64,000 HA / ml-gel when commercially available Cellufine Sulfate was used, 217,600 HA / ml-gel when the chromatography packing material of Production Example 5 was used, and 112,000 HA / ml-gel when the chromatography packing material of Production Example 6 was used. Compared to commercially available Cellufine Sulfate, a popular particle, the chromatography packing materials of Preparation Examples 5 and 6 exhibited 3.4-fold and 1.75-fold higher 10% DBC values for influenza virus, respectively. Preparation Example 5, which utilized porous particles with a retention time of 29.6 minutes for dextran with a molecular weight of 2 million, exhibited a high 10% DBC. On the other hand, Preparation Example 6, which utilized porous particles with a retention time of 15.0 minutes for dextran with a molecular weight of 2 million, exhibited a lower 10% DBC value than Preparation Example 5.

[0067] [Relationship between pressure loss and linear velocity] A chromatography column (manufactured by JNC) with an inner diameter of 10 mm was packed with the particles obtained in Production Example 5 and Production Example 6 to a depth of 10 cm (±1 cm). Pure water at 20°C was passed through this column, and the pressure loss, which is the pressure difference between the column inlet and outlet, was measured. The flow rate was initially started at 0.5 ml / min, and then the flow rate was increased stepwise. After the flow had continued for more than 1 minute, the pressure loss was measured. As a blank, the pressure loss on the column alone without any particles was measured, and the pressure on the particles was calculated using the following formula. Pressure loss due to particles (MPa) = Pressure at column inlet (MPa) - Pressure at column outlet (MPa) - Pressure loss due to column only (MPa) The linear velocity was calculated using the following formula: Linear velocity (cm / h) = flow rate at measurement (ml / h) / column cross-sectional area (cm 2 )

[0068] The results are shown in Figure 8. As is clear from Figure 8, Production Examples 5 and 6 exhibited less pressure loss and maintained high flow rate characteristics compared to commercially available Cellufine Sulfate at the same linear velocity.

[0069] The results show that when the porous particles of the present invention were used as a chromatography packing, the 10% DBC was 3.4 times higher than that of the commercially available Cellufine Sulfate control, and the pressure loss characteristics were also good. [Industrial Applicability]

[0070] This invention demonstrates porous particles that have a controlled three-dimensional network structure and interconnected pores formed by the voids between the pores, with the interconnected pores penetrating from the particle surface to the interior, a method for producing the particles, and the use of the particles as a chromatography packing. The porous particles obtained by the production method of the invention can be used as chromatography packings in which the interconnected pores within the particles can also be used as adsorption sites. This allows for high throughput and high processing speed to be achieved in separation and purification processes for pharmaceuticals and other substances, making them extremely useful industrially.

Claims

1. Porous particles whose main component is cellulose acetate or cellulose, characterized in that they are spherical, have a three-dimensional network-like skeleton and an interconnected pore structure consisting of voids therebetween, and the interconnected pores penetrate from the particle surface to the interior, and when packed in a column having an inner diameter of 0.78 cm and a length of 30 cm, the retention time of dextran having a molecular weight of 2,000,000 is 20 minutes or more when the column is used at a flow rate of 0.4 ml / min.

2. 2. The porous particles according to claim 1, wherein the retention time of silica nanoparticles having a particle size of 30 nm when packed in a column having an inner diameter of 0.78 cm and a length of 30 cm and at a flow rate of 0.4 ml / min is 16 minutes or more.

3. 3. The porous particles according to claim 1, wherein the mode diameter is in the range of 1 to 1000 μm.

4. Specific surface area measured by BET multipoint method is 1 to 200 m ２ The porous particles according to any one of claims 1 to 3, wherein the molecular weight is in the range of / g.

5. A method for producing porous particles, comprising the steps of: (a) preparing a cellulose acetate solution by heating and dissolving cellulose acetate in a mixed solvent of a solvent in which cellulose acetate is soluble and a solvent in which cellulose acetate is insoluble; (b) dispersing the cellulose acetate solution in water containing an emulsion stabilizer to obtain a dispersion; and (c) cooling the dispersion at a rate of 0.1°C / min to 2°C / min to precipitate cellulose acetate particles, wherein the mixed solvent is a water-immiscible organic solvent.

6. 6. The method for producing porous particles according to claim 5, wherein in (a), the solvent in which cellulose acetate is soluble is benzyl alcohol, ethyl acetate, cyclohexanone, or isophorone.

7. 7. The method for producing porous particles according to claim 5, wherein in (a), the solvent insoluble in cellulose acetate is an alcohol, a glycol, an ether, or an ester.

8. 8. The method for producing porous particles according to claim 5, wherein in (a), the volume ratio of the solvent in which cellulose acetate is soluble to the solvent in which cellulose acetate is insoluble in the mixed solvent is 5:95 to 95:

5.

9. 9. The method for producing porous particles according to claim 5, wherein in (a), the content of cellulose acetate in the mixed solvent is 0.1 to 30% by weight based on the weight of the cellulose acetate solution.

10. The method for producing porous particles according to any one of claims 5 to 9, wherein in (a), the mixed solvent contains a third component that is a polymer, and the third component is polyethylene glycol or polypropylene glycol.

11. The method for producing porous particles according to any one of claims 5 to 10, wherein in (b), the emulsion stabilizer is sodium dodecylbenzenesulfonate, polyvinyl alcohol, carboxymethyl cellulose, hydroxyethyl cellulose, or a mixture thereof.

12. A chromatographic packing material comprising the porous particles according to any one of claims 1 to 4, or the porous particles which have been saponified, crosslinked or modified.

13. The chromatography packing material according to claim 12, which is used for separating and purifying virus particles.

Images