Porous particles, liquid chromatography carrier, liquid chromatography device, and method for separating and purifying biopolymer

Abstract

Provided is a technique for purifying biopharmaceuticals and the like at a high processing speed using a conventional device for filling a tubular column with a filler. The porous particles are substantially spherical, have a three-dimensional network skeleton and a communicating pore structure including voids thereof, and are characterized in that the communicating pores penetrate from the surface of the particles to the inside of the particles, and the porous particles are filled in a tubular column having an inner diameter of 0.78 cm and a length of 30 cm, with pure water as a mobile phase. When a slurry containing 5 mg / mL of silica microparticles having a particle size of 100 nm is introduced at a tubular column temperature of 25 DEG C and a flow rate of 0.4 mL / min, the retention time of the silica microparticles is 15.0 minutes or more.

Description

Technical Field

[0001] The present invention relates to a porous particle having a large pore diameter and a chromatography technique at a high processing speed using the same. Background Art

[0002] In chromatography for separation and purification of biopharmaceuticals and the like, since porous particles have a large surface area, they are used as materials with a high binding capacity. In recent years, efforts have been made to utilize the voids of the interconnected pores inside the particles as adsorption sites by making the pores of the porous particles continuous pores (Patent Document 1, Patent Document 2, Patent Document 3). In addition, conventional porous particles have been optimized for the separation of biological molecules having a size in the range of usually 5 nm to 10 nm, such as antibodies, proteins, and nucleic acids. On the other hand, in order to increase the binding capacity of larger particles such as viruses, efforts have been made to adsorb large particles in the pores by increasing the pore diameter of the porous particles (Patent Document 4).

[0003] In recent years, in order to improve the productivity of biopharmaceuticals and the like, the ability to bind a target substance per unit time has attracted attention. The ability is expressed as the amount of the target substance bound per unit volume and per unit time (mg / mL / min), which helps to reduce the chromatography adsorption material for separation and purification and / or shorten the time required for separation and purification. In the conventional binding to porous particles, the diffusion of the target substance into the pores of the particles becomes the rate-limiting step. Therefore, especially at a high processing speed with a residence time of less than 1 minute, if the flow rate is increased in order to shorten the residence time in the column filled with the particles, the binding capacity of the target substance to the particles is significantly reduced. Therefore, there is a problem that an increase in the flow rate does not contribute to an increase in productivity. As a method for solving the problem of the decrease in the binding capacity at such a high processing speed, a chromatography purification method using a membrane, a monolith, or a fiber instead of porous particles is known. In such materials, since the liquid passes through the widely opened spaces between the fibers, it is not easy to reach the diffusion rate-limiting step, and they have the characteristic of reducing the flow rate dependence of the binding capacity. However, a membrane, a monolith, or a fiber cannot be filled into the conventionally used column, and there is a problem that a dedicated container is required. In addition, when using a membrane, a monolith, or a fiber, there is a problem that the pressure rises sharply when clogged.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: International Publication No. 2016 / 013568

[0007] Patent Document 2: International Publication No. 2017 / 026424

[0008] Patent Document 3: Japanese Patent Laid-Open No. 2020-026511

[0009] Patent Document 4: Japanese Patent Translation of International Publication No. 2022-515769

[0010] Patent Document 5: WO 2015 / 029790

[0011] Non-Patent Document

[0012] Non-Patent Document 1: W.M. Deen, F.G. Smith III, J. Membr. Sci., 1982, 12, 217-237

[0013] Non-Patent Document 2: P. DePhillips, A.M. Lenhoff, J. Chromatogr. A., 2000, 883, 39-54 Summary of the Invention

[0014] Problems to be Solved by the Invention

[0015] As described above, in order to improve the separation and purification efficiency of biopharmaceuticals and the like, while increasing the amount of target substance bound per unit volume and per unit time (productivity), it is required to effectively utilize the packed column as a conventional device and to have a purification method with a small risk of blockage.

[0016] Therefore, an object of the present invention is to provide a technique for purifying biopharmaceuticals and the like at a high processing speed using a conventional device in which a filler is packed into a column.

[0017] Means for Solving the Problems

[0018] The present inventors have made intensive studies to solve the above problems, and as a result, have found porous particles having a three-dimensional network-like skeleton and a specific communication pore structure including the voids thereof and having relatively large communication pores, and have found that by using the porous particles as a chromatographic carrier, a high adsorption amount can be maintained even under high flow rate conditions, thereby completing the present invention.

[0019] That is, the present invention is as follows.

[0020] [1] A porous particle, characterized in that

[0021] it is substantially spherical, has a three-dimensional network-like skeleton and a communication pore structure including the voids thereof, and the communication pores penetrate from the particle surface to the inside,

[0022] When packing the porous particles into a column with an inner diameter of 0.78 cm and a length of 30 cm, using pure water as the mobile phase, and introducing a slurry containing 5 mg / mL of silica microparticles with a particle size of 100 nm at a column temperature of 25°C and a flow rate of 0.4 mL / minute, the retention time of the silica microparticles is 15.0 minutes or more.

[0023] [2] The porous particles according to [1], characterized in that the pore diameter is 650 nm or more.

[0024] [3] A kind of porous particles, modified by ligands, the porous particles are characterized in that

[0025] it is substantially spherical, has a three-dimensional network skeleton and a connected pore structure including its voids, and the connected pores penetrate from the particle surface to the inside,

[0026] the pore diameter is 400 nm or more.

[0027] [4] The porous particles according to any one of [1] to [3], wherein the main component is cellulose acetate or cellulose.

[0028] [5] The porous particles according to any one of [1] to [4], wherein the median particle size is 40 μm to 200 μm.

[0029] [6] The porous particles according to any one of [1] to [5], wherein the specific surface area obtained by the Brunauer-Emmett-Teller (BET) multipoint method is 1 m 2 / g to 200 m 2 / g.

[0030] [7] The porous particles according to [1], [2] and any one of [4] to [6], wherein the porous particles according to [1] or [2] are modified by ligands.

[0031] [8] The porous particles according to [3] or [7], wherein the ligand is one or more selected from the group consisting of ligands containing ion exchange groups, affinity ligands and ligands containing hydrophobic groups.

[0032] [9] A liquid chromatography support, comprising the porous particles according to any one of [1] to [8].

[0033]

[10] A liquid chromatography device, comprising a separation column filled with the liquid chromatography support according to [9].

[0034]

[11] A separation and purification method, which is a separation and purification method for biological macromolecules,

[0035] It includes a step of separating and purifying a biopolymer using the liquid chromatography apparatus described in

[10] .

[0036]

[12] The separation and purification method according to

[11] , wherein the size of the biopolymer is 1 nm to 100 nm.

[0037]

[13] The separation and purification method according to

[11] or

[12] , wherein the biopolymer is an antibody.

[0038] Effects of the Invention

[0039] According to the present invention, when separating and purifying a target substance such as a biopolymer, a large adsorption capacity can be achieved even under high flow rate conditions. Description of the Drawings

[0040] Figure 1 Figure 1 It is a photograph of the appearance of the particles produced in Production Example 1 (magnification: 1000 times).

[0041] Figure 2 Figure 2 It is a photograph of the appearance of the particles produced in Production Example 2 (magnification: 1500 times).

[0042] Figure 3 Figure 3 It is a photograph of the appearance of the particles produced in Production Example 3 (magnification: 1300 times).

[0043] Figure 4 Figure 4 It is a photograph of the internal cross-section of the particles produced in Production Example 1 (magnification: 800 times).

[0044] Figure 5 Figure 5 It is a photograph of the internal cross-section of the particles produced in Production Example 2 (magnification: 700 times).

[0045] Figure 6 Figure 6 It is a graph showing the characteristics when the particles produced in Examples 1 to 4 and Comparative Examples 1 to 6 are packed into a column, and is a graph showing the 10% dynamic adsorption capacity of γ-globulin per unit time and per unit volume at a column residence time of 7.5 seconds when using the particles produced in Examples 1 to 4 and Comparative Examples 1 to 6. Detailed Description of the Invention

[0046] ​​​​​​​​​​​​The shape of the porous particles of the present invention is substantially spherical. The so-called substantially spherical mentioned herein means that, for example, with respect to the minor axis (the shortest diameter), the major axis (the longest diameter) is 2 times or less. The porous particles of the present invention preferably have the major axis and the minor axis being close to the same length, and more preferably are spherical.

[0047] The porous particles of the present invention have a three-dimensional network-like skeleton and a connected pore structure including the voids thereof, and the connected pores penetrate from the particle surface to the inside. Regarding whether there is a three-dimensional network-like skeleton and connected pores, for example, it can be judged according to the measurement results of confocal laser microscope observation and scanning electron microscope (SEM) observation of the cross-section of the particles. In addition, for example, the state where the connected pores penetrate from the particle surface to the inside can be confirmed by the internal cross-section photograph of the porous particles of the present invention. In the porous particles of the present invention, the connected pores are not blocked on the particle surface but open on the particle surface. By the porous particles having a connected pore structure as described above, not only the particle surface of the porous particles but also the voids of the connected pores inside the particles can be used as adsorption points for the target substance, so the separation and purification efficiency of the target substance is improved.

[0048] In addition, the so-called "the connected pores penetrate from the particle surface to the inside" includes not only the state where the connected pores pass through the particle interior from the opening on the particle surface and lead to the surface outside the particle (that is, the state where both ends of the connected pores lead to the particle surface), but also the state where it leads from the particle surface to the inside but does not lead to the surface outside the particle from the inside (that is, the state where only one end of the connected pore leads to the particle surface). In addition, the connected pores leading from the opening on the particle surface to the inside can also continuously form the voids inside the particles with one or more other connected pores.

[0049] The porous particles of the present invention satisfy any one or more of the following (1) to (2) characteristics.

[0050] (1) When the porous particles are packed into a column with an inner diameter of 0.78 cm and a length of 30 cm, and pure water is used as the mobile phase, and a slurry containing 5 mg / mL of silica microparticles with a particle size of 100 nm is passed through at a column temperature of 25 °C and a flow rate of 0.4 mL / minute, the retention time of the silica microparticles is 15.0 minutes or more.

[0051] (2) The pore diameter of the porous particles in the state of being modified by ligands is 400 nm or more.

[0052] (1) When packing porous particles into a column with an inner diameter of 0.78 cm and a length of 30 cm, using pure water as the mobile phase, and passing a slurry containing 5 mg / mL of silica microparticles with a particle size of 100 nm through the column at a column temperature of 25 °C and a flow rate of 0.4 mL / minute, the retention time of the silica microparticles is 15.0 minutes or more, preferably 15.5 minutes or more, more preferably 15.8 minutes or more. In addition, the upper limit value is preferably 30.0 minutes or less, more preferably 25.0 minutes or less, and still more preferably 20.0 minutes or less. Here, the porous particles packed into the column are in a state where they are not modified with ligands.

[0053] Specifically, as a method for packing porous particles into a column, after dispersing the porous particles with pure water to prepare a slurry, it is packed into a column (for example, a stainless-steel column (manufactured by Tosoh)), and then pure water is flowed into the column at a flow rate of 0.4 mL / minute for 1 hour or more to compact it. Then, a slurry containing 5 mg / mL of silica microparticles with a particle size of 100 nm is passed through the column at a flow rate of 0.4 mL / minute. The dispersion medium of the slurry containing 5 mg / mL of silica microparticles and the mobile phase during retention time measurement use pure water. In addition, the column temperature is adjusted to 25 °C. As the silica microparticles with a particle size of 100 nm, commercially available silica microparticles such as Sicaster (manufacturing number: 43-00-102, manufactured by Micromod) can be used, for example.

[0054] In addition, the so-called "retention time" is the time required from when the sample is injected into the column until the elution peak. Regarding the elution peak, for example, it can be obtained by using a high-pressure liquid chromatography (HPLC) device (manufactured by Agilent Technologies, 1260 Infinity) and detecting the value with the maximum intensity based on the refractive index (RI).

[0055] When the retention time of the silica microparticles is 15.0 minutes or more, even under high flow rate conditions, the dynamic adsorption capacity of target substances such as biopolymers can be maintained at a high value.

[0056] In the field of chromatography, when evaluating the pore diameter of porous particles, generally, dextran with a molecular weight of 2 million is used as the exclusion limit molecular weight for measurement (Patent Document 5). This is based on the premise that dextran with a molecular weight of 2 million cannot enter the interior of the porous particles.

[0057] Dextran or polyethylene oxide is a soft material that can move in a liquid while deforming and enter the pores of particles. In contrast, inorganic microparticles such as silica are rigid spheres and cannot deform, so they have the property of being less likely to enter the pores of particles (Non-Patent Document 1). Therefore, generally speaking, compared with dextran with a molecular weight of 2 million or polyethylene oxide with a molecular weight of 780,000 and a Stokes diameter exceeding 40 nm, the retention time of silica microparticles with a smaller particle size of 30 nm is shorter. Here, when the porous particles of the present invention are filled into a column, the retention time of silica microparticles with a particle size of 100 nm, which are rigid spheres and have a larger particle size, is 15.0 minutes or more, which is longer than that of conventional porous particles. This indicates that the porous particles of the present invention have interconnected pores with a pore diameter large enough for silica microparticles with a particle size of 100 nm as rigid spheres to enter the particle interior.

[0058] In addition, generally speaking, it is known that when spherical porous particles are filled into a column, the interparticle porosity is 30% - 40% (Non-Patent Document 2). The interparticle porosity mentioned here is the porosity obtained by dividing the total volume of the gaps between particles in the column interior by the volume of the entire column. For example, assuming that the sample only passes through the gaps between the filled 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 / minute is 10.7 minutes - 14.3 minutes, and this time is the retention time of excluded particles such as silica microparticles. In the porous particles of the present invention, the retention time of silica microparticles with a particle size of 100 nm is 15.0 minutes or more. Thus, the comparison with the retention time of excluded particles also shows that the porous particles of the present invention not only have gaps between particles but also have interconnected pores with a pore diameter large enough for silica microparticles with a particle size of 100 nm to enter the particle interior.

[0059] The pore diameter of the porous particles of the present invention is preferably 650 nm or more. In addition, here, the porous particles refer to particles in a state not modified with ligands.

[0060] When evaluating the pore diameter of particles with a large pore diameter such as the porous particles of the present invention, since it is difficult to prepare excluded particles that do not enter the pores, assuming the retention time of excluded particles according to the above theory and using it for the estimation of the pore diameter is also effective in comparing the size of the pore diameter between particles.

[0061] That is, when calculating based on the assumed retention time of the particles according to the above theory, the pore diameter of the porous particles in the unligand-modified state is preferably 650 nm or more, more preferably 800 nm or more, and still more preferably 1000 nm or more. The upper limit value is preferably 2000 nm or less, more preferably 1800 nm or less, and still more preferably 1700 nm or less.

[0062] The estimated value of the pore diameter can be calculated, for example, in the following order based on the retention time of polyethylene oxide.

[0063] The porous particles in the unligand-modified state of the present invention are filled into a column with an inner diameter of 0.78 cm and a length of 30 cm. Using pure water as the mobile phase, at a column temperature of 25 °C and a flow rate of 0.4 mL / min, a solution containing 5 mg / mL of polyethylene oxide is used to measure the retention time of polyethylene oxide.

[0064] Specifically, as a method for packing the porous particles into the column, after dispersing the porous particles with pure water to form a slurry, it is filled into a column (for example, a stainless-steel column (manufactured by Tosoh)). Then, pure water is flowed in at a flow rate of 0.4 mL / min for 1 hour or more to compact it. Then, each solution or slurry containing 5 mg / mL of polyethylene glycol with a weight average molecular weight of 106, a measurement sample (standard polyethylene oxide), and exclusion particles (for example, silica microparticles with a particle size of 100 nm) is passed through the column at a flow rate of 0.4 mL / min. The solvent or dispersion medium of each solution or slurry and the mobile phase during retention time measurement use pure water. In addition, the column temperature is adjusted to 25 °C. The time when the RI detection intensity becomes the maximum value is taken as the elution peak, and this time is taken as the retention time. As a measurement device, for example, a 1260 Infinity HPLC device (manufactured by Agilent Technologies) can be used.

[0065] (i) Calculate the total volume V (mL) of the mobile phase by the formula "retention time (minutes) of polyethylene glycol with a weight average molecular weight of 106 × flow rate (mL / min) × column volume (mL)". T As the polyethylene glycol with a weight average molecular weight of 106, for example, polyethylene glycol (PEG) 106 manufactured by Agilent Technologies can be used.

[0066] (ii) Calculate the interstitial volume V0 (mL) between the particles by the formula "retention time (minutes) of the exclusion particles × flow rate (mL / min) × column volume (mL)".

[0067] Determine the retention time of the exclusion particles. As the exclusion particles, commercially available silica microparticles with a particle size of 100 nm such as Sicaster (manufacturing number: 43-00-102, manufactured by Micromod) can be used, for example.

[0068] In the case of porous particles with a small pore diameter, high-molecular-weight polymers or silica microparticles can be treated as exclusion particles that do not enter the pores. Therefore, the retention time of the exclusion particles can be determined based on the retention time of high-molecular-weight polymers or silica microparticles. However, in the case of porous particles with a large pore diameter, high-molecular-weight polymers or silica microparticles also enter the pores, so it is difficult to calculate the retention time of the exclusion particles.

[0069] Here, generally, it is known that when spherical porous particles are packed into a column, the interstitial porosity between particles is 30% to 40% (Non-Patent Document 1). When it is assumed that the sample only passes through the interstitial spaces between the particles packed inside a column with an inner diameter of 0.78 cm and a length of 30 cm, the retention time theoretically becomes 10.7 minutes to 14.3 minutes when the flow rate is 0.4 mL / minute. As described later, since the pore diameters of the particles in Production Examples 4 to 6 are relatively small, it is speculated that silica microparticles with a particle diameter of 100 nm as rigid spheres hardly enter the pores. Moreover, the retention times of the silica microparticles with a particle diameter of 100 nm in Production Examples 4 to 6 are all in the range of 13 minutes to 14 minutes. Therefore, based on the average value of the retention times of the silica microparticles in Production Examples 4 to 6, the value of the retention time of the exclusion particles is fixed at 13.6 minutes, and V0 is calculated.

[0070] (iii) Calculate the elution volume V (mL) of the measurement sample by the formula "retention time (minutes) of the measurement sample × flow rate (mL / minute) × column volume (mL)". As the measurement sample, for example, those manufactured by Tosoh with weight average molecular weights of 2.36×10 R Da (SE-2), 3.76×10 4 Da (SE-5), 1.07×10 4 Da (SE-8), 1.49×10 5 Da (SE-15), 2.8×10 5 Da (SE-30), 5.8×10 5 Da (SE-70), and 7.86×10 5 Da (SE-70) and 7.86×10 5Polyethylene oxide of Da(SE-150). In addition, for example, polyethylene glycols with weight average molecular weights of 20440 Da (PEG21300), 15260 Da (PEG16100), 7460 Da (PEG7830), 4110 Da (PEG4040), 1020 Da (PEG1010), 620 Da (PEG610), and 400 Da (PEG400) manufactured by Agilent Technologies can be used.

[0071] (iv) Calculate the partition coefficient K by the following formula d .

[0072] K d = (V R - V0) / (V T - V0)

[0073] (v) Refer to "The Hydrodynamic Radii of Macromolecules and Their Effect on Red Blood Cell Aggregation" (J.K. Armstrong, et al., Biophysical Journal, 2004, 87, 4259 - 4270). Based on the weight average molecular weight M w (Da) and the intrinsic viscosity [η] (mL / g) of each measurement sample, calculate the viscosity radius r s (nm) of the measurement sample by the following formula.

[0074] r s = (3[η]M w / 10πN) 1 / 3 × 10 7

[0075] [where N represents Avogadro's constant]

[0076] (vi) If it is assumed to be a single pore diameter, the partition coefficient K d and the particle depth (hydraulic radius) r hyd are expressed by the following formula.

[0077] K d = (1 - r s / r hyd ) 2

[0078] Take the K when measuring each measurement sampled The 0.5 power is taken as the vertical axis, and the viscosity radius r of each of the measured samples is taken s as the horizontal axis. According to the formula K d 0.5 = 1 - r s / r hyd The slope when performing linear approximation is used to calculate -1 / r hyd , and as a result, in actual porous particles, there is a pore size distribution. When the viscosity radius of the measured sample is 5 nm or less (for example, when the measured sample is PEG21300 to PEG400), there is a large difference in the slope compared to when it is 5 nm or more (for example, when the measured sample is SE-150 to SE-2). Therefore, in order to calculate the pore size of the large pores that affect the adsorption of biopolymers, the 0.5 power of K d is taken as the vertical axis, and the viscosity radius r of each of the samples is taken s as the horizontal axis. The slope when performing linear approximation based on the following formula is used to calculate -1 / r hyd .

[0079] K d 0.5 = C - r s / r hyd

[0080] [In the formula, C represents the intercept when performing linear approximation]

[0081] (vii) If it is assumed that the pores of the particles are cylindrical through-holes, then the pore size r pore and the pore depth r hyd are represented by the following formulas.

[0082] r pore = r hyd ×2

[0083] According to the above formula, the pore size r pore of the particles is calculated as an estimated value of the pore size of the particles.

[0084] The intra-particle porosity (the porosity inside the particles) ε of the porous particles in the unligand-modified state of the present invention p is not particularly limited, and is preferably 50% to 97%, more preferably 60% to 95%, and still more preferably 70% to 90%. If ε p is within the above range, while maintaining the strength of the particles, it is easy to increase the dynamic adsorption capacity even under high flow rate conditions. ε p can be obtained by the following formula based on the void volume V0 between the particles and the total volume V of the mobile phase obtained in the calculation of the estimated value of the pore size. T , and is obtained by the following formula.

[0085] ε p =(V T -V0) / (V c -V0)

[0086] [wherein, V c represents the volume of the column (mL)]

[0087] (2) The pore diameter of the porous particles in the state of being modified with a ligand is 400 nm or more, preferably 700 nm or more, more preferably 750 nm or more. The upper limit value is preferably 1800 nm or less, more preferably 1500 nm or less, and still more preferably 1200 nm or less.

[0088] Regarding the pore diameter of the porous particles modified with a ligand, as the column packing liquid when compacting the porous particles, an aqueous sodium chloride solution with a concentration of 0.1 mol / L is used instead of pure water, and as the solvent or dispersion medium of each solution or slurry containing polyethylene glycol with a weight average molecular weight of 106, the measurement sample (standard polyethylene oxide), and the excluded particles, and as the mobile phase during the holding time measurement, an aqueous sodium chloride solution with a concentration of 1 mol / L is used instead of pure water. Except for this, it can be calculated in the same order as the method for calculating the pore diameter of the unmodified porous particles.

[0089] The intra-particle porosity ε of the porous particles in the state of being modified with a ligand p is preferably 40% to 95%, more preferably 50% to 90%, and still more preferably 60% to 85%. If ε p is within the above range, while maintaining the strength of the particles, it is easy to increase the dynamic adsorption capacity even under high flow rate conditions. ε p can be obtained by the following formula based on the void volume V0 between particles and the total volume V of the mobile phase obtained in the calculation of the estimated value of the pore diameter. T , and is obtained by the following formula.

[0090] ε p =(V T -V0) / (V c -V0)

[0091] [wherein, V c represents the volume of the column (mL)]

[0092] The porous particles of the present invention are not particularly limited as long as they are porous particles satisfying any one or more of the characteristics (1) to (2) described above. For example, the following porous particles can be mentioned.

[0093] Examples include organic porous particles such as synthetic polymer-based porous particles and natural polymer-based porous particles; inorganic porous particles; organic-organic composite porous particles or organic-inorganic composite porous particles formed by combining these. Examples of natural polymer-based porous particles include polysaccharide-based porous particles such as cellulose, agarose, and dextran (preferably cross-linked polysaccharide-based porous particles). Examples of inorganic porous particles include particles containing glass, silica gel, metal, metal oxide, etc.

[0094] Among these, organic porous particles are preferred, polymer-based porous particles are more preferred, and polysaccharide-based porous particles are even more preferred. In addition, among the polysaccharide-based porous particles, porous cellulose particles are preferred. The main component of the porous cellulose particles is preferably cellulose acetate or cellulose, and in terms of having no non-specific adsorption of proteins and having sufficient strength such that the particle shape does not collapse during liquid passage at high flow rates, a component obtained by saponifying and cross-linking cellulose acetate or cellulose is more preferred.

[0095] In addition, the so-called main component mentioned here refers to a component with a content of 50% by mass or more in the porous cellulose particles. When the main component of the porous cellulose particles is cellulose acetate or cellulose, the content of cellulose and / or cellulose acetate in the porous cellulose particles is preferably 60% to 100% by mass, more preferably 70% to 100% by mass. In addition, the degree of acetylation of cellulose acetate is preferably 45% to 57%.

[0096] The median particle size of the porous particles of the present invention is not particularly limited, preferably 40 μm to 200 μm, more preferably 50 μm to 120 μm, and even more preferably 60 μm to 100 μm. If it is 40 μm or more, the pressure loss can be reduced, and if it is 200 μm or less, the surface area of the particles can be increased, enabling a large amount of biological macromolecules to bind. In addition, the median particle size mentioned here can be calculated, for example, by measuring the median particle size using a particle size distribution measuring device: Laser Scattering Particle Size distribution Analyzer Partica LA-960 manufactured by HORIBA.

[0097] The specific surface area of the porous particles of the present invention obtained by the BET multi-point method is preferably 1 m 2 / g to 200 m 2 / g. The specific surface area obtained by the BET multipoint method can be measured, for example, by performing pretreatment by vacuum degassing at 100°C for 2 hours after freeze-drying overnight, and then using a high-speed specific surface area / pore size distribution measuring device (BELSORP MAXII manufactured by MICROTRAC-BEL). In the pressure range of 3 points or more where the measurement temperature is 77.3K and the relative pressure is 0.05 to 0.30, it is obtained from the adsorption isotherm (straight line) obtained based on the measurement of the amount of nitrogen adsorbed on the solid surface.

[0098] The method for manufacturing the porous particles of the present invention is not particularly limited. For example, in the case where the porous particles are porous cellulose particles, they are manufactured by the following method, which includes: (a) a step of 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) a step of dispersing the cellulose acetate solution in water containing an emulsifying stabilizer to obtain a dispersion system; and (c) a step of cooling the dispersion system to precipitate cellulose acetate particles, and the mixed solvent is an organic solvent that is immiscible with water.

[0099] Regarding (a) of the porous cellulose particle manufacturing process, in step (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 acetylation of cellulose, which is a natural polymer. The cellulose acetate used in the present invention is not particularly limited as long as it is a substance generally defined as cellulose acetate, but it is preferably a cellulose acetate having a degree of polymerization of 50 to 300 and an acetylation degree of 45% to 57%. By using cellulose acetate having a degree of polymerization of 50 to 300 and an acetylation degree of 45% to 57%, it can be dissolved in more types of solvents.

[0100] In step (a), the proportion of cellulose acetate in the mixed solvent is preferably 0.1% by mass to 50% by mass, and more preferably 5% by mass to 20% by mass. If it is within the above range, a uniform solution can be obtained, and it is easy to obtain porous particles having openings with a uniform diameter. In addition, during cooling, precipitation due to phase separation is likely to occur, the strength becomes sufficient, and it is easy to maintain a substantially spherical particle shape. In addition, the lower the concentration of cellulose acetate in the cellulose acetate solution, the smaller the particle size of the obtained porous particles and the greater the tendency for the pore diameter to become larger. In addition, the higher the concentration of cellulose acetate in the cellulose acetate solution, the smaller the tendency for the internal void ratio ε p to become smaller.

[0101] In step (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 good solvent mentioned here refers to a good solvent in which the solute dissolves in a single solvent to obtain a transparent solution without solid substances. In particular, here it refers to a solvent that preferably obtains a solution of 1% by mass or more at a temperature below the boiling point of the solvent. The solvent can be used alone or in combination of two or more solvents. Specifically, benzyl alcohol, ethyl acetate, cyclohexanone, isophorone, and mixtures thereof can be cited. Among them, benzyl alcohol is preferred in terms of forming communication pores.

[0102] In step (a), solvents in which the cellulose acetate solution is insoluble, i.e., poor solvents, include alcohols, diols, ethers, esters, and mixtures thereof. The poor solvent refers to a solvent that has no solubility or low solubility in the solute compared to the good solvent. Specifically, it refers to a solvent that cannot dissolve the solute with a single solvent and cannot obtain a transparent solution at a temperature below the boiling point of the solvent. In particular, here it refers to a solvent that preferably cannot dissolve 1% by mass or more of the solute at a temperature below the boiling point of the solvent. A solvent with low solubility in water is preferred. In particular, alcohols are preferred. The alcohols are preferably lower alcohols, and 1-hexanol is more preferred in terms of further forming communication pores.

[0103] In step (a), the volume ratio of the good solvent, i.e., the solvent in which the cellulose acetate is soluble, to the poor solvent, i.e., the solvent in which the cellulose acetate is insoluble, in the mixed solvent is preferably 5:95 to 95:5. The higher the ratio of the poor solvent contained in the mixed solvent, the greater the tendency for the average diameter of the communication pores to increase.

[0104] In step (a), the mixed solvent is an organic solvent that does not mix with water. If it mixes with water, a dispersion system cannot be obtained in step (b).

[0105] In step (a), a third component as a polymer can be contained in the mixed solvent. The polymer as the third component is preferably polyethylene glycol or polypropylene glycol, and more preferably polypropylene glycol.

[0106] In step (a), when the third component is polypropylene glycol, its molecular weight is not particularly limited, but from the viewpoints of operability in the manufacturing process and the pore diameter of the porous particles, an average molecular weight (Mw) of 100 to 3000 is preferred. In addition, the higher the molecular weight of the polymer of the third component, the greater the tendency for the pore diameter to increase.

[0107] In step (a), from the viewpoint of the pore diameter of the porous particles, the concentration of the third component in the mixed solvent is preferably 0.1% by weight to 10% by weight. The higher the concentration of the third component, the more likely the pore diameter is to become larger.

[0108] In step (a), during the heating and dissolution, the heating temperature is preferably in the range of 50°C to 130°C, for example. In this temperature range, it is preferred that cellulose acetate is dissolved in the mixed solvent. The time for the heating and dissolution is not particularly limited. For example, the heating time can be set to 3 hours to 24 hours.

[0109] Regarding step (b) of the porous cellulose particle manufacturing process, the cellulose acetate solution is dispersed in high-temperature water in which an emulsification stabilizer is dissolved. 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 the range that can obtain 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 (dispersed phase / continuous phase) of the cellulose acetate solution to the high-temperature water is preferably 1.0 or less. In addition, the temperature of the high-temperature water is preferably 50°C to 100°C, more preferably 60°C to 95°C.

[0110] In step (b), regarding the method of dispersing it, known methods can be arbitrarily applied. For example, there are methods using a mixer such as a stirrer, methods using a homogenizer, methods using ultrasonic waves, etc. In addition, there are methods such as a method of using a device generally called a microreactor and extruding the cellulose acetate solution from a fine nozzle to obtain droplets, or extruding the cellulose acetate solution, or a mixture of the cellulose acetate solution, water, and an emulsification stabilizer from a uniform fine-diameter porous membrane and performing shearing. Among them, since it is a simple method, the method of dispersing it by a stirrer is preferred.

[0111] In step (b), the emulsifying stabilizer is not particularly limited as long as it has the effect of improving the stability of the dispersion system and preventing particle aggregation. For example, it includes: natural polymers such as starch, pectin, alginic acid, alginate, gelatin; processed natural polymers such as methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose; synthetic polymers containing alcoholic OH such as polyvinyl alcohol and partially saponified polyvinyl acetate; polymers containing SO3H groups such as sulfonated styrene; polymers containing COOH groups such as acrylate; synthetic polymers containing nitrogen such as polyvinylpyrrolidone; inorganic substance powders such as barium sulfate, talc, bentonite, titanium oxide; anionic surfactants such as linear alkylbenzene sulfonate; cationic surfactants such as alkyltrimethylammonium salt; amphoteric surfactants such as alkyldimethylamine oxide; nonionic surfactants such as sorbitan fatty acid ester; or mixtures thereof. Particularly from the viewpoint of stabilizing the dispersion system, sodium dodecylbenzenesulfonate, polyvinyl alcohol, carboxymethyl cellulose, hydroxyethyl cellulose, or mixtures thereof are preferred.

[0112] From the viewpoint of stabilizing the dispersion system, the addition amount of the emulsifying stabilizer used in step (b) is preferably 0.01% by mass or more in terms of the concentration in high-temperature water. If the addition amount of the emulsifying stabilizer is increased within the above concentration range, the particle size of the obtained porous cellulose particles tends to become smaller. On the contrary, if the addition amount of the emulsifying stabilizer is small, the particle size of the obtained porous cellulose particles tends to become larger.

[0113] In step (c) of the porous cellulose particle manufacturing process, the dispersion liquid obtained in (b) is cooled. By cooling, thermally induced phase separation occurs, and thus porous cellulose particles having a connected pore structure can be obtained.

[0114] In step (c), the temperature for cooling the dispersion system is not particularly limited as long as it is a temperature at which cellulose acetate precipitates. However, from the aspect of sufficiently precipitating the porous cellulose particles, the cooling temperature is preferably 0°C to 50°C.

[0115] In step (c), the cooling rate of the dispersion system is preferably 0.1°C / min to 10°C / min, more preferably 0.1°C / min to 5°C / min, and still more preferably 0.1°C / min to 2°C / min. By adjusting the cooling rate, particles having a pore diameter corresponding to the target can be obtained.

[0116] Through the above manufacturing method, porous cellulose particles using cellulose acetate as a raw material can be obtained. When making the porous cellulose particles using cellulose acetate as a raw material hydrophilic, saponification can be carried out in a solution such as an alkali metal hydroxide such as sodium hydroxide to convert cellulose acetate into cellulose.

[0117] The porous particles of the present invention can be further saponified and crosslinked. The saponification and crosslinking treatments can be carried out by known methods. For example, the crosslinking treatment of porous cellulose particles can be carried out with reference to Japanese Patent Laid-Open No. 2009-242770 and the like.

[0118] As the crosslinking agent, if it has two or more functional groups capable of reacting and bonding with the hydroxyl groups of cellulose, it can be used as a crosslinking agent. For example, halogenated alcohols such as epichlorohydrin, epibromohydrin, and dichlorohydrin; difunctional diepoxides (diepoxyethane); polyfunctional polyepoxides (polyethylene oxide) such as glycerol polyglycidyl ether can be cited.

[0119] The porous particles of the present invention can be modified with ligands. That is, at least a part of the reactive functional groups of the porous particles of the present invention can also be modified with ligands. A ligand is a compound that binds directly to the surface of the porous particles or binds via an epoxy group, a formyl group, a vinyl group, etc. introduced to the particle surface, and there is no particular limitation as long as it has an affinity for the adsorption target. For example, ligands containing known ion exchange groups, affinity ligands, ligands containing hydrophobic groups, ligands containing charged groups, etc. can be cited, and these can be introduced individually or a plurality of them can be appropriately combined and introduced.

[0120] As the ion exchange group, for example, 2-diethylaminoethyl (DEAE) group, carboxymethyl (CM) group, sulfonic acid group, quaternary ammonium (Q) group, etc. can be cited.

[0121] As the hydrophobic group, for example, phenyl group, n-butyl group, n-hexyl group, n-octyl group, n-octadecyl group, etc. can be cited.

[0122] In addition, known ligands that can be used in so-called mixed mode separation, which have both an ion exchange group and a hydrophobic group, can be used.

[0123] As the affinity ligand, for example, ligands containing a sulfate group (such as sulfated polysaccharides), protein A, protein adsorption ligands such as antibodies, polycations such as polylysine, polyanions such as heparin or polyglutamic acid, functional polymers containing a phosphate group, etc. can be cited.

[0124] According to the purification purpose, porous particles modified with any ligand can be used. As the ligand, one or more selected from the group consisting of ligands containing an ion exchange group, affinity ligands, and ligands containing a hydrophobic group are preferred.

[0125] When the porous particles of the present invention are modified with a sulfur-containing ligand such as a ligand containing a sulfonic acid group or a ligand containing a sulfate group, the sulfur content of the porous particles is preferably 0.1% by weight to 10.0% by weight, more preferably 1.0% by weight to 5.0% by weight.

[0126] The sulfur content is an index of the modification amount of the sulfur-containing ligand. The more the sulfur content, the more the modification amount of the sulfur-containing ligand. The sulfur content can be calculated, for example, as follows: drying the porous particles modified with the ligand, and determining the sulfur content (% by weight) per unit dry weight by carbon, hydrogen, nitrogen, sulfur (CHNS) elemental analysis using a fully automatic elemental analyzer vario EL cube (manufactured by Elementar).

[0127] The sulfur content can be adjusted according to the addition amount of the compound having a ligand (such as 2-acrylamido-2-methylpropanesulfonic acid, dextran sulfate, etc.), as long as it is appropriately determined according to the purification purpose, etc.

[0128] The ligand can be introduced by a known method. The porous particles modified with the ligand can be suitably used as a carrier for various chromatographies such as affinity chromatography, ion exchange chromatography, chelation chromatography, and hydrophobic interaction chromatography. Regarding the porous particles of the present invention, in terms of the size of their pore diameters, they are suitable for the separation and purification of biological macromolecules as the target substance. Chromatographic carriers into which an affinity ligand, a charged group, a hydrophobic group, etc. are introduced can be preferably used for the purification of biological macromolecules.

[0129] The size of the purified biological macromolecule is preferably 1 nm to 100 nm, more preferably 1 nm to 25 nm, and further preferably 5 nm to 10 nm. If it is within the above range, since the biological macromolecule easily enters the pores of the porous particles of the present invention, it is easy to maintain a high adsorption amount even under high flow rate conditions.

[0130] Examples of biological macromolecules include: polypeptides, such as polypeptides obtained by expression in Escherichia coli, yeast, animal cells and separated by conventional methods, proteins, synthetic polypeptides or synthetic proteins and other synthetic substances, and nucleic acids (such as deoxyribonucleic acid (DNA), etc.), and derivatives thereof (such as glycoproteins, DNA conjugates), viruses (such as adeno-associated viruses, influenza viruses). Among these, as biological macromolecules, polypeptides, proteins and their derivatives are preferred, and proteins and their derivatives, especially antibodies, are more preferred.

[0131] The liquid chromatography carrier of the present invention contains the porous particles of the present invention.

[0132] In addition, the liquid chromatography apparatus of the present invention includes a separation column packed with the liquid chromatography support of the present invention.

[0133] The separation column, except for being packed with the liquid chromatography support of the present invention, is the same as a normal liquid chromatography separation column. Specifically, a separation column including a column container and the liquid chromatography support of the present invention packed into the column container can be exemplified.

[0134] There is no particular limitation on the method for packing the liquid chromatography support of the present invention into the separation column. For example, it can be carried out by flow packing, dynamic axial compression, or pack-in-place.

[0135] The liquid chromatography apparatus of the present invention, in addition to including a separation column packed with the liquid chromatography support of the present invention, can also use a conventional liquid chromatography apparatus. The liquid chromatography apparatus of the present invention can form a part of various analytical apparatuses such as HPLC, Liquid Chromatography / Mass Spectrometry (LC / MS), and LC / MS / MS. The liquid chromatography apparatus, for example, has an autosampler as an introduction part for a sample solution, has a liquid feeding device for feeding the sample solution to the separation column, and has a detection part for optically detecting the sample separated by the separation column. The detection result in the detection part is subjected to necessary analysis processing in a data processing device in the liquid chromatograph in the form of digital information and output to an output device such as a printing device or a display device. Alternatively, it can also be output to other data processing devices or data storage media.

[0136] The method for separating and purifying a biological polymer of the present invention includes a step of separating and purifying a biological polymer using the liquid chromatography apparatus of the present invention. According to the method for separating and purifying a biological polymer of the present invention, even under high flow rate conditions, for example, conditions where the column residence time of the mobile phase containing the biological polymer is set to 20 seconds or less, 10 seconds or less, 7.5 seconds or less, etc., the productivity of the target biological polymer bound per unit volume and per unit time can be improved. In addition, the so-called "column residence time" refers to the value obtained by dividing the "column volume" by the "flow rate when injecting the mobile phase containing the biological polymer into the separation column".

[0137] The description related to the biological polymer refers to the above description.

[0138] Examples

[0139] Hereinafter, the present invention will be described more specifically by way of examples, but the present invention is not limited to the forms of these examples.

[0140] Particles of Production Examples 1 to 7 were produced in the following order. In addition, Production Examples 1 to 3 and Production Example 7 correspond to Examples, and Production Examples 4 to 6 correspond to Comparative Examples.

[0141] [Production Example 1]

[0142] ·Granulation step

[0143] (1) 100 g of cellulose acetate (L-20, manufactured by Daicel Corporation) was added to a mixed solvent of 391 g of benzyl alcohol, 276 g of 1-hexanol, and 14 g of polypropylene glycol (Wako Pure Chemical Industries, Ltd., diol type, average molecular weight of 1,000), and stirred.

[0144] (2) The temperature was raised and stirred at 120 °C for 4 hours to dissolve cellulose acetate and obtain a transparent cellulose acetate solution.

[0145] (3) 2 g of polyvinyl alcohol (PVA) (JP-18E, manufactured by Nippon Vinylon K.K.) and 30 g of sodium carboxymethyl cellulose (CMC1140, manufactured by Daicel Miraizu Co., Ltd.) were added to 2300 g of pure water saturated with the mixed solvent, the temperature was raised and stirred and dissolved at 80 °C for 1 hour or more to obtain a dispersion medium.

[0146] (4) 770 g of the cellulose acetate solution was quickly injected into 2300 g of the dispersion medium, stirred at 80 °C to obtain a dispersion system. Next, the dispersion system was cooled to 35 °C to obtain spherical cellulose acetate particles.

[0147] (5) The obtained cellulose acetate particles were thoroughly washed with a large amount of water, then with methanol, and then washed again with pure water.

[0148] ·Gelation step

[0149] (1) 630 g - wet (water content 6.86) of the obtained cellulose acetate particles were added to a mixed solution of 1072 g of pure water and 242 g of methanol, the temperature was set to 35 °C and stirred for 30 minutes.

[0150] (2) 201 g of 20% NaOH was added, stirred at 35 °C for 2 hours and allowed to react to carry out saponification.

[0151] (3) Cool to below 30°C, neutralize with acetic acid, thoroughly wash with pure water, and then pass the obtained saponified cellulose spherical particles through sieves with mesh sizes of 106 μm and 32 μm to obtain saponified cellulose particles with a particle size of 32 μm to 106 μm.

[0152] · Crosslinking step

[0153] (1) Disperse 96.4 g of the obtained saponified cellulose particles - wet (water content 7.93%) - in 194 g of pure water, and then dissolve 78.6 g of Na2SO4.

[0154] (2) Set the temperature to 50°C and stir for 30 minutes, then add 4.0 g of 48% NaOH and 0.69 g of NaBH4, and allow them to dissolve and react over 30 minutes.

[0155] (3) After dissolution, add the amounts obtained by dividing 36.4 mL of 48% NaOH solution and 62 g of epichlorohydrin into eight equal parts every 30 minutes for approximately 4 hours.

[0156] (4) After the addition is complete, react at 50°C for 16 hours. Cool the temperature to below 30°C, add acetic acid and neutralize.

[0157] (5) Filter the reaction mixture and recover the gel, and filter and wash with pure water to obtain crosslinked porous particles.

[0158] [Production Example 2]

[0159] (1) Add 681 g of cellulose acetate (L-20, manufactured by Daicel Corporation) to a mixed solvent of 2671 g of benzyl alcohol, 1883 g of 1-hexanol, and 97 g of polypropylene glycol (Wako Pure Chemical Reagent, diol type, average molecular weight of 1,000), and stir.

[0160] (2) Raise the temperature and stir at 120°C for 4 hours to dissolve the cellulose acetate and obtain a transparent cellulose acetate solution.

[0161] (3) Add 14 g of PVA (JP-18E, manufactured by Japan VAM & POVAL Co., Ltd.) and 252 g of sodium carboxymethyl cellulose (CMC1140, manufactured by Daicel Miraizu Co., Ltd.) to 18 kg of pure water saturated with the mixed solvent, raise the temperature, and stir and dissolve at 80°C for more than 1 hour to obtain a dispersion medium.

[0162] (4) Inject 5300 g of the cellulose acetate solution into 16 kg of the dispersion medium, and stir at 80 °C to obtain a dispersion system. Next, cool the dispersion system from 80 °C to 35 °C in about 1 hour to obtain spherical cellulose acetate particles.

[0163] (5) Thoroughly wash the obtained cellulose acetate particles with a large amount of water, then with methanol, and then wash again with pure water. Saponify the obtained cellulose acetate particles in the same order as in Production Example 1, and then pass the obtained saponified cellulose spherical particles through sieves with a mesh size of 150 μm and 45 μm to obtain saponified cellulose particles with a particle size of 45 μm to 150 μm. Then, crosslink in the same order as in Production Example 1 to obtain crosslinked porous particles.

[0164] [Production Example 3]

[0165] Cool the dispersion system obtained by mixing and stirring cellulose acetate prepared in the same manner as in Production Example 2 with the dispersion medium from 80 °C to 35 °C over about 50 minutes to obtain spherical cellulose acetate particles.

[0166] Saponify, classify, and crosslink the obtained cellulose acetate particles in the same order as in Production Example 2 to obtain crosslinked porous particles.

[0167] [Production Example 4]

[0168] Cool the dispersion system obtained by mixing and stirring cellulose acetate prepared in the same manner as in Production Example 2 with the dispersion medium from 80 °C to 35 °C over about 90 minutes to obtain spherical cellulose acetate particles.

[0169] Saponify, classify, and crosslink the obtained cellulose acetate particles in the same order as in Production Example 2 to obtain crosslinked porous particles.

[0170] [Production Example 5]

[0171] Add 100 g of cellulose acetate (L-20, manufactured by Daicel Corporation) to a mixed solvent of 391 g of benzyl alcohol, 276 g of 1-hexanol, and 7 g of polypropylene glycol (Wako Pure Chemical Industries, diol type, average molecular weight of 1,000) and stir. Then, obtain spherical cellulose acetate particles in the same order as in Production Example 1.

[0172] Saponify, classify, and crosslink the obtained cellulose acetate particles in the same order as in Production Example 1 to obtain crosslinked porous particles.

[0173] [Production Example 6]

[0174] · Granulation process

[0175] (1) 6.4 g of crystalline cellulose (manufactured by Asahi Kasei Chemicals Corporation, trade name: Ceolus PH101) was added to 100 g of a 60 wt% aqueous solution of calcium thiocyanate and dissolved by heating to 110°C to 120°C.

[0176] (2) 6 g of sorbitan monooleate was added as a surfactant to the solution, and the mixture was dropped into 480 mL of o-dichlorobenzene preheated to 130°C to 140°C and stirred for dispersion.

[0177] (3) The dispersion was cooled to 40°C or lower and poured into 190 mL of methanol to obtain a suspension of particles.

[0178] (4) The suspension was filtered and separated, and the particles were washed with 190 mL of methanol and then filtered and separated. This washing operation was repeated several times.

[0179] (5) After washing with a large amount of water, spherical cellulose particles were obtained.

[0180] (6) The spherical cellulose particles were passed through sieves with a mesh size of 125 μm and 53 μm to obtain cellulose particles with a particle size of 53 μm to 125 μm.

[0181] · Crosslinking step

[0182] (1) 121 g of pure water was added to 100 g of the obtained cellulose particles, and the mixture was heated with stirring. When the temperature reached 30°C, 3.3 g of a 45 wt% aqueous NaOH solution and 0.5 g of NaBH4 were added and stirred. The initial alkali concentration was 0.69% (w / w).

[0183] (2) After 30 minutes, 60 g of Na2SO4 was added to the reaction solution and dissolved. When the temperature of the mixture reached 50°C, stirring was continued for 2 hours.

[0184] (3) While continuing to stir the mixture at 50°C, an amount obtained by dividing 48 g of a 45 wt% aqueous NaOH solution and 50 g of epichlorohydrin into 25 equal portions was added every 15 minutes over a period of about 6 hours.

[0185] (4) After the addition was completed, the mixture was reacted at 50°C for 16 hours.

[0186] (5) After the mixture was cooled to a temperature of 40°C or lower, 2.6 g of acetic acid was added for neutralization.

[0187] (6) The reaction mixture was filtered to recover the gel, and the gel was washed by filtration with pure water to obtain crosslinked cellulose particles.

[0188] [Production Example 7]

[0189] (1) 28 g of cellulose acetate (L-20, manufactured by Daicel Corporation) was added to a mixed solvent of 110 g of benzyl alcohol, 77 g of 1-hexanol, and 4 g of polypropylene glycol (Wako Pure Chemical Industries, Ltd., diol type, average molecular weight: 1,000), and the mixture was stirred.

[0190] (2) The temperature was raised and the mixture was stirred at 120°C for 4 hours to dissolve the cellulose acetate and obtain a transparent cellulose acetate solution.

[0191] (3) 0.46 g of PVA (JP-18E, manufactured by Japan VAM & Poval Co., Ltd.) and 6.8 g of sodium carboxymethyl cellulose (CMC1140, manufactured by Daicel Miraizu Co., Ltd.) were added to 570 g of pure water saturated with the mixed solvent. The temperature was raised and the mixture was stirred and dissolved at 80°C for 1 hour or more to obtain a dispersion medium.

[0192] (4) 219 g of the cellulose acetate solution was poured into 577 g of the dispersion medium, and the mixture was stirred at 80°C to obtain a dispersion system. Next, the dispersion system was cooled from 80°C to 35°C in about 1 hour to obtain spherical cellulose acetate particles.

[0193] (5) The obtained cellulose acetate particles were thoroughly washed with a large amount of water, then with methanol, and then with pure water again. The obtained cellulose acetate particles were saponified in the same order as in Production Example 1, and the obtained saponified cellulose spherical particles were passed through sieves with mesh sizes of 150 μm and 45 μm to obtain saponified cellulose particles with a particle size of 45 μm to 150 μm. Then, crosslinking was carried out in the same order as in Production Example 1 to obtain crosslinked porous particles.

[0194] [Example 1]

[0195] ·Ligand modification step

[0196] After dissolving 21 g of 2-acrylamido-2-methylpropanesulfonic acid in 40 g of pure water, 8.4 g of a 48.7% (w / w) aqueous sodium hydroxide solution was added for neutralization. Further, 20 g of the porous particles obtained in Production Example 1 - wet was added to prepare a slurry. After performing decompression of the reaction vessel and nitrogen filling three times, while stirring under a nitrogen atmosphere, a liquid obtained by dissolving 0.9 g of cerium ammonium nitrate in 9.5 mL of a 0.17 mol / mL nitric acid aqueous solution was slowly added to the reaction vessel. After the addition, the reaction vessel was heated to 40°C under a nitrogen atmosphere and reacted for 16 hours. The reaction mixture was filtered to recover the gel, and washed three times with pure water. Then, after washing 10 times with 80 mL of 1 mol / L sulfuric acid, it was washed with pure water until the washing liquid became neutral. Then, it was washed with 80 mL of 0.5 mol / L sodium hydroxide, and finally washed with pure water by filtration until the washing liquid became neutral to obtain ligand-modified porous particles.

[0197] [Example 2]

[0198] Using the crosslinked porous particles obtained in Production Example 2, except for this, the ligand modification reaction was carried out in the same order as in Example 1 to obtain ligand-modified porous particles.

[0199] [Example 3]

[0200] Using the crosslinked porous particles obtained in Production Example 3, except for this, the ligand modification reaction was carried out in the same order as in Example 1 to obtain ligand-modified porous particles.

[0201] [Example 4]

[0202] ·Ligand modification process

[0203] (Epoxidation process)

[0204] (1) 60 g - wet (water content 5.01) of the crosslinked porous particles obtained in Production Example 7 was added to 103 mL of pure water and stirred.

[0205] (2) The internal temperature was adjusted to 30°C, and 58.5 g of epichlorohydrin was added.

[0206] (3) After stirring for 15 minutes, 54.7 g of a 48% NaOH aqueous solution was added and reacted for 2 hours.

[0207] (4) After the reaction was completed, it was neutralized with acetic acid and then washed thoroughly with water until the filtrate became neutral to obtain epoxy-activated porous particles.

[0208] (5) 1.0 g of epoxy-activated porous particles and 3.0 mL of 1.3 M sodium thiosulfate solution were shaken on a shaker set at 30 °C for 1 hour, and titrated with 0.1 mol / L hydrochloric acid to quantify the epoxy amount. The amount of epoxy groups was 229 μmol / g - dry.

[0209] (Binding step of sulfated polysaccharide)

[0210] (1) 4.0 g of dextran sulfate sodium DS-500 (manufactured by Meito Sangyo Co., Ltd.) was added to 51 mL of pure water and stirred until the dextran sulfate sodium was completely dissolved.

[0211] (2) 30 g - wet of epoxy-activated porous particles were added to the reactor.

[0212] (3) While stirring thoroughly, the internal temperature was adjusted to 30 °C. After stirring for 15 minutes, 20.7 g of sodium sulfate, 22.1 g of disodium hydrogen phosphate, and 3.2 g of 48% NaOH aqueous solution were added.

[0213] (4) The temperature was raised to 40 °C and the reaction was carried out for 6 hours.

[0214] (5) After the reaction was completed, a solution obtained by dissolving 3.3 mL of 48% NaOH aqueous solution and 0.5 g of sodium borohydride in 23.8 mL of pure water was added, and the reaction was further carried out for 16 hours.

[0215] (6) After the reaction, the reaction solution was filtered and washed thoroughly with water until the filtrate was neutral to obtain dextran-sulfated porous particles.

[0216] [Comparative Example 1]

[0217] Using the cross-linked porous particles obtained in Production Example 4, ligand modification reaction was carried out in the same order as in Example 1 to obtain ligand-modified porous particles.

[0218] [Comparative Example 2]

[0219] Using the cross-linked porous particles obtained in Production Example 5, ligand modification reaction was carried out in the same order as in Example 1 to obtain ligand-modified porous particles.

[0220] [Comparative Example 3]

[0221] ·Ligand modification step

[0222] After dissolving 8.7g of 2-acrylamide-2-methylpropane sulfonic acid in 64.4g of pure water, 3.4g of 48.7% (w / w) sodium hydroxide aqueous solution was added for neutralization, and then 80g of porous particles obtained by manufacturing example 6 were added to make a slurry. After the reaction container was decompressed and filled with nitrogen three times, 5.19g of cerium ammonium nitrate dissolved in 18.1mL of 0.17mol / mL nitric acid aqueous solution was slowly added to the reaction container while stirring under a nitrogen environment. After the addition, the reaction container was heated to 40°C under a nitrogen environment and reacted for 16 hours. The reaction mixture was filtered and the gel was recovered, and washed with pure water three times. Then, after washing 10 times with 80mL of 1mol / L sulfuric acid, it was washed with pure water until the washing liquid was neutral. Then, the mixture was washed with 80 mL of 0.5 mol / L sodium hydroxide, and finally, filtered and washed with pure water until the washing liquid became neutral, thereby obtaining ligand-modified porous particles.

[0223] [Comparative Example 4]

[0224] As the ligand-modified porous particles, a strong cation exchange resin POROS XS purchased from Thermo Fisher Scientific Inc. was used.

[0225] [Comparative Example 5]

[0226] As the ligand-modified porous particles, a strong cation exchange resin POROS 50HS purchased from Thermo Fisher Scientific Inc. was used.

[0227] [Comparative Example 6]

[0228] Ligand modification process

[0229] (Epoxidation process)

[0230] (1) 40 g of the crosslinked porous particles obtained in Production Example 6 were added wet to 30 mL of pure water and stirred.

[0231] (2) The internal temperature was adjusted to 30°C, and 24.7 g of epichlorohydrin was added.

[0232] (3) After stirring for 15 minutes, 23.1 g of a 48% NaOH aqueous solution was added and the mixture was reacted for 2 hours.

[0233] (4) After the reaction is completed, the mixture is neutralized with acetic acid and then washed thoroughly with water until the filtrate becomes neutral, thereby obtaining epoxy-activated porous particles.

[0234] (5) 1.0 g of the epoxy-activated porous particles and 3.0 mL of a 1.3 M sodium thiosulfate solution were shaken on a shaker set at a temperature of 30 °C for 1 hour, and titrated with 0.1 mol / L hydrochloric acid to quantify the epoxy amount. The amount of epoxy groups was 230 μmol / g - dry.

[0235] (Binding step of sulfated polysaccharide)

[0236] (1) 3.3 g of dextran sulfate sodium DS-500 (manufactured by Meito Sangyo Co., Ltd.) was added to 25.6 mL of pure water and stirred until the dextran sulfate sodium was completely dissolved.

[0237] (2) 40 g - wet of the epoxy-activated porous particles were added to the reactor.

[0238] (3) While stirring thoroughly, the internal temperature was adjusted to 30 °C. After stirring for 15 minutes, 16.7 g of sodium sulfate, 26.8 g of disodium hydrogen phosphate, and 2.6 g of a 48% NaOH aqueous solution were added.

[0239] (4) The temperature was raised to 40 °C and the reaction was carried out for 6 hours.

[0240] (5) After the reaction was completed, a solution obtained by dissolving 3.1 mL of a 48% NaOH aqueous solution and 0.2 g of sodium borohydride in 28.3 mL of pure water was added, and the reaction was further carried out for 16 hours.

[0241] (6) After the reaction, the reaction solution was filtered and washed thoroughly with water until the filtrate was neutral to obtain the dextran-sulfated porous particles.

[0242] [Measurement method 1: Measurement of median particle size]

[0243] For the particles obtained in Production Examples 1 to 7, Examples 1 to 4, and Comparative Examples 1 to 6, the particle size distribution was measured to determine the median particle size. The apparatus used in the measurement is as described below.

[0244] Apparatus: Laser Scattering Particle Size distribution Analyzer Partica LA-960 (manufactured by HORIBA)

[0245] The median particle size was measured using the above apparatus.

[0246] [Measurement method 2: Measurement of retention time of crosslinked particles, estimation of pore diameter, and estimation of void fraction inside particles]

[0247] For the particles obtained in Production Examples 1 to 7, after packing them into a stainless steel column (manufactured by Tosoh) with an inner diameter of 0.78 cm and a length of 30 cm, the retention times of the respective samples shown in Table 1 were measured. Regarding the packing method, after dispersing the particles in pure water to prepare a slurry, the slurry was packed into the column, and then pure water was flowed at a flow rate of 0.4 mL / minute for 1 hour or more to compact it. Pure water was used as the dispersion medium for each sample and as the mobile phase when measuring the retention time of each sample. In addition, the column temperature during measurement was adjusted to 25°C. The value at which the RI detection intensity became maximum was set as the elution peak, and its time was set as the retention time. As the measuring device, a 1260 Infinity HPLC device (manufactured by Agilent Technologies) was used.

[0248] [Table 1]

[0249] [Table 1]

[0250]

[0251] The sample concentration was set to 5 mg / mL, and 10 μL of each sample was used in the measurement.

[0252] The pore diameter of the particles was calculated in the following order.

[0253] (i) The total volume V of the mobile phase was calculated by the formula "retention time (minutes) of polyethylene glycol (PEG106) with a weight average molecular weight of 106 × flow rate (mL / minute) × column volume (mL)". T (mL). In addition, with a flow rate of 0.4 mL / minute and a column volume of 14.34 mL, V was calculated. T .

[0254] (ii) The interstitial volume V0 (mL) between the particles was calculated by the formula "retention time (minutes) of the exclusion particles × flow rate (mL / minute) × column volume (mL)". In addition, with a flow rate of 0.4 mL / minute and a column volume of 14.34 mL, V0 was calculated. Also, in any of the production examples, the value of the retention time of the exclusion particles was fixed at 13.6 minutes (i.e., the average value of the retention times of the silica microparticles in Production Examples 4 to 6), and V0 was calculated.

[0255] (iii) The elution volume V of the measurement sample (standard polyethylene oxide) was calculated by the formula "retention time (minutes) of the measurement sample × flow rate (mL / minute) × column volume (mL)". R (mL). In addition, with a flow rate of 0.4 mL / minute and a column volume of 14.34 mL, V was calculated. R .

[0256] (iv) Calculate the distribution coefficient K by the following formula d .

[0257] K d =(V R -V0) / (V T -V0)

[0258] (v) Based on the weight-average molecular weight M w (Da) and the intrinsic viscosity [η] (mL / g) of each measurement sample, calculate the viscosity radius r s (nm) of the measurement sample by the following formula.”

[0259] r s =(3[η]M w / 10πN) 1 / 3 ×10 7

[0260] [In the formula, N represents Avogadro's constant]

[0261] (vi) If it is assumed to be a single pore diameter, the distribution coefficient K d and the particle depth (hydraulic radius) r hyd are expressed by the following formula.

[0262] K d =(1 - r s / r hyd ) 2

[0263] In addition, similar to each sample shown in Table 1, the retention times were also measured for the 7 samples from PEG-21300 to PEG-400 shown in Table 2. However, due to the following reasons, the measurement results of the 7 samples shown in Table 2 are not used for the estimation of the pore diameter. That is, taking the 0.5 power of K d of each measurement sample as the vertical axis and taking the viscosity radius r s of each measurement sample as the horizontal axis, according to the formula K d 0.5 =1 - r s / r hyd when performing linear approximation, the slope is used to calculate -1 / r hyd, as a result, in actual porous particles, there is a pore size distribution. There is a large difference in slope between the region where the viscosity radius of the measurement sample is below 5 nm (when the measurement sample is PEG21300 to PEG400) and the case where it is above 5 nm (when the measurement sample is SE-150 to SE-2). Therefore, in order to calculate the pore diameter of the large pores that affect the adsorption of biopolymers, take the K when taking polyethylene oxide of 7 samples from SE-150 to SE-2 with a measured viscosity radius above 5 nm d to the 0.5th power as the vertical axis, and take the viscosity radius r s of each of the above samples as the horizontal axis, and find -1 / r from the slope when performing linear approximation based on the following formula hyd .

[0264] K d 0.5 = C - r s / r hyd

[0265] [In the formula, C represents the intercept when performing linear approximation]

[0266] (vii) If it is assumed that the pores of the particles are cylindrical through-holes, then the pore diameter r pore and the pore depth r hyd are represented by the following formulas.

[0267] r pore = r hyd ×2

[0268] According to the above formula, calculate the pore diameter r pore of the particles as the estimated value of the pore diameter of the particles.

[0269] The internal porosity ε p of the particles is calculated based on the void volume V0 between the particles and the total volume V T of the mobile phase obtained in the calculation of the estimated value of the pore diameter, and is calculated by the following formula.

[0270] ε p = (V T - V0) / (V c - V0)

[0271] In addition, assume that the column volume V c = 14.34 mL for calculation.

[0272] [Measurement method 3: Measurement of the retention time of ligand-modified particles, estimation of pore diameter, and estimation of internal porosity of particles]

[0273] For the particles of Examples 1 to 4 and Comparative Examples 1 to 6, after filling them into a stainless steel column (manufactured by Tosoh) with an inner diameter of 0.78 cm and a length of 30 cm, the retention time of each sample was measured. The column packing liquid during compaction was set as an aqueous sodium chloride solution with a concentration of 0.1 mol / L, and the dispersion medium or solvent of each sample and the mobile phase during the retention time measurement were set as an aqueous sodium chloride solution with a concentration of 1 mol / L. Except for this, the measurement was carried out in the same order as the measurement of the retention time of the particles of Production Examples 1 to 7. In the said aqueous sodium chloride solution, since the silica nanoparticles aggregate and precipitate, the silica nanoparticles were not used in the measurement. Except for this, the same samples as in Table 1 were used to measure the retention time. In addition, the pore diameter of the particles was also calculated in the same order as the estimation of the pore diameter of the said crosslinked particles. However, since the pore diameter of the particles of Comparative Example 3 was small, the molecular weight dependence of the retention time of polyethylene oxide with a molecular weight of 580,000 to 786,000 was small, and there was a concern of overestimating the pore diameter. Therefore, the K of 5 samples from SE-30 to SE-2 was used d to calculate the pore diameter. Furthermore, in the particles of Comparative Example 6, the retention times of SE-150 to SE-5 were all about 12.4 minutes. Excluding the particles with a retention time lower than the assumed value of 13.6 minutes, only in Comparative Example 6, the retention time of the excluded particles was set as 12.4 minutes, and the pore diameter was estimated. In addition, as the samples for calculating the pore diameter of Comparative Example 6, the K when measuring 7 samples of PEG-21300 to PEG-400 shown in Table 2 was used d to estimate the pore diameter. In addition, the intra-particle porosity was also calculated in the same order as the estimation of the intra-particle porosity of the said crosslinked particles.

[0274] [Table 2]

[0275] [Table 2]

[0276]

[0277] The sample concentration was set as 5 mg / mL, and 10 μL of each sample was used in the measurement.

[0278] [Measurement Method 4: Determination of Sulfur Content of Ligand-Appended Particles]

[0279] Dry the particles of Examples 1 to 4 and Comparative Examples 1 to 6, and determine the sulfur content (wt%) per unit dry weight by CHNS (carbon, hydrogen, nitrogen, sulfur) elemental analysis. The device used for the measurement is as described below.

[0280] Device: Fully automatic elemental analyzer vario EL cube (manufactured by Elementar)

[0281] Determine the sulfur content using the said device.

[0282] [Assay Method 5: Determination of 10% dynamic binding capacity (DBC) using polyclonal antibody and calculation of 10% dynamic binding capacity (DBC) per unit time and per unit volume]

[0283] (1) Equipment and reagents used

[0284] LC system: AKTA avant 25 (registered trademark)

[0285] Buffer: Acetate buffer pH 5.0 (containing 0.05 mol / L NaCl)

[0286] Polyclonal antibody: γ-globulin, human serum-derived (Wako Pure Chemical Industries, Ltd.)

[0287] Column: diameter 6.7 mm, length 30 mm

[0288] (2) Assay method

[0289] First, dissolve the polyclonal antibody in the buffer to prepare an antibody solution of 2 mg / mL. Then, fill the ligand-modified particles (particles of Examples 1 to 4 and Comparative Examples 1 to 6) into each column without gaps. Next, connect the column to the LC system and flow in the buffer to equilibrate until the ultraviolet (UV) (ultraviolet absorbance, 280 nm), conductivity, and pH of the column effluent are constant. Then, set the baseline UV to zero. Next, flow the antibody solution into the column at a flow rate of 8.48 mL / min (column residence time 7.5 seconds). Monitor the UV of the column effluent and read the time for flowing in the antibody solution until the UV of the column effluent reaches 10% of the pre-determined UV of the antibody solution. Calculate the 10% dynamic binding capacity of the antibody using the following formula. In addition, the analysis is carried out in a room at 25°C.

[0290] {Antibody solution concentration (mg / mL) × Time (min) from the start of flowing in the antibody solution until the UV of the pre-determined antibody solution reaches 10% × Flow rate (mL / min) - Dead volume} / Column volume = 10% dynamic binding capacity (mg / mL)

[0291] [In the formula, the dead volume is the volume (mL) obtained by adding the system piping volume and the column void volume]

[0292] In addition, the dead volume was determined by measuring the flow rate of the following sodium chloride solution. The flow rate of the sodium chloride solution was obtained by flowing a 1 mol / L sodium chloride solution into the column for equilibration until the UV (ultraviolet absorbance, 280 nm), conductivity, and pH of the column effluent became constant. Then, an antibody solution with a concentration of 2 mg / mL and an amount of about 1% of the column volume was injected. Subsequently, the flow rate of the sodium chloride solution before detecting the UV peak when flowing the 1 mol / L sodium chloride solution again was measured.

[0293] In addition, using the value of 10% dynamic binding capacity (mg / mL) calculated according to the above formula, the 10% dynamic binding capacity (mg / mL / min) of the antibody per unit time and per unit volume was calculated under the condition of a column residence time of 7.5 seconds by the following formula.

[0294] 10% dynamic binding capacity (mg / mL) / column volume (mL) / 0.125 (min) = 10% dynamic binding capacity per unit time and per unit volume (mg / mL / min)

[0295] [Measurement method 6: SEM observation]

[0296] SEM observation was carried out using a super-high-resolution field emission scanning electron microscope "SU8020" manufactured by Hitachi High-technologies. As a pretreatment, the particles of Production Examples 1 to 3 after freeze-drying were coated with Au for photography. In the electron micrographs, it was confirmed that the obtained particles were approximately spherical, and had through-holes on the surface and in the cross-section ( Figures 1 to 5 ).

[0297] The evaluation results of each production example, example, and comparative example are shown in Tables 3 to 5, Figure 6 .

[0298] Table 3 shows the median particle size, pore diameter, intra-particle porosity of the particles of Production Examples 1 to 7, and the retention time of silica microparticles with a particle size of 100 nm.

[0299] [Table 3]

[0300] [Table 3]

[0301]

[0302] Table 4 shows the median particle size, pore diameter, intra-particle porosity of the particles of Examples 1 to 4 and Comparative Examples 1 to 6, the types of functional groups of the modified ligands, sulfur content, 10% dynamic adsorption capacity of γ-globulin at a column residence time of 7.5 seconds, and its dynamic adsorption capacity per unit time and per unit volume.

[0303] [Table 4]

[0304] [Table 4]

[0305]

[0306] In Figure 6 is shown the 10% dynamic adsorption capacity per unit time and per unit volume of γ-globulin when the column residence time is 7.5 seconds using the particles of Examples 1 to 4 and Comparative Examples 1 to 6.

[0307] According to Figure 6 it is clear that in the Examples, the dynamic adsorption capacity per unit time and per unit volume is higher than that in the Comparative Examples.

[0308] Industrial Applicability

[0309] In the present invention, there is shown a chromatographic purification method at a high processing speed using porous particles having a three-dimensional network-like skeleton and large communicating pores including the voids thereof, the communicating pores penetrating from the particle surface to the inside, and filling the porous particles into a column. In order to improve the separation / purification efficiency of biopharmaceuticals and the like, the present invention can increase the amount of the target substance bound per unit volume and per unit time (productivity), effectively utilize a packed column as a conventional device, and reduce the risk of clogging. Therefore, in the separation / purification process of biopharmaceuticals and the like, high throughput and high processing speed can be achieved, which is very useful industrially.

Claims

1. A porous particle, characterized in that, it is substantially spherical, has a three-dimensional network-like skeleton and a connected pore structure including its voids, and the connected pores penetrate from the particle surface to the interior, when the porous particles are filled into a column with an inner diameter of 0.78 cm and a length of 30 cm, and pure water is used as the mobile phase, and a slurry containing 5 mg / mL of silica microparticles with a particle size of 100 nm is passed through at a column temperature of 25 °C and a flow rate of 0.4 mL / minute, the retention time of the silica microparticles is 15.0 minutes or more.

2. The porous particles according to claim 1, wherein The pore diameter is 650 nm or more.

3. A porous particle modified with a ligand, the porous particle is characterized in that, it is substantially spherical, has a three-dimensional network-like skeleton and a connected pore structure including its voids, and the connected pores penetrate from the particle surface to the interior, The pore diameter is 400 nm or more.

4. The porous particles according to any one of claims 1 to 3, wherein The main component is cellulose acetate or cellulose.

5. The porous particles according to any one of claims 1 to 3, wherein, The median particle size is 40 μm to 200 μm.

6. The porous particles according to any one of claims 1 to 3, wherein, The specific surface area obtained by the BET multipoint method is 1 m 2 / g to 200 m 2 / g.

7. The porous particles according to claim 1 or 2, wherein, The porous particle according to claim 1 or 2 is modified with a ligand.

8. The porous particles according to claim 7, wherein, The ligand is one or more selected from the group consisting of ligands containing an ion exchange group, affinity ligands, and ligands containing a hydrophobic group.

9. A liquid chromatography support comprising the porous particle according to any one of claims 1 to 3.

10. A liquid chromatography device comprising a separation column filled with the liquid chromatography support according to claim 9.

11. A separation and purification method, which is a separation and purification method for biological macromolecules, comprising a step of separating and purifying biological macromolecules using the liquid chromatography device according to claim 10.

12. The separation and purification method according to claim 11, wherein, The size of the biological macromolecule is 1 nm to 100 nm.

13. The separation and purification method according to claim 11, wherein, The biological macromolecule is an antibody.

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