JP5875779B2 - Porous particles, method for producing porous particles, and carrier - Google Patents

Description

  The present invention relates to porous particles, a method for producing porous particles, and a carrier.

  In recent years, advances in medicine, in particular internal medicine, hematology, immunology, and clinical laboratory methods, are revealing malignant substances in blood that are considered to be closely related to the cause or progression of disease. For example, autoantibodies and immune complexes against autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis.

  Conventionally known techniques for removing malignant substances from body fluids include (1) removing malignant substances from blood with activated carbon or activated carbon whose surface is coated with a hydrophilic polymer material, and (2) blood. Plasma exchange therapy in which patient plasma containing malignant substances such as antigens, antibodies, immune complexes, etc. in autoimmune diseases is replaced with other fresh plasma, as in (3) and (2) The adsorbent removes the malignant substances contained in the plasma from the separated plasma, and removes the high molecular weight malignant substances through the filter as in (4) and (2). The thing which is going to be mentioned.

  However, although the method (1) can directly treat blood, activated carbon has poor adsorption selectivity and can hardly adsorb high molecular weight malignant substances because of its small pores. In many types of diseases, malignant substances that are closely related to the progression or cause of the disease have become known, and there is an increasing demand for selectively removing these malignant substances from body fluids. Adsorbents that do not meet this requirement.

  In addition, the method (2) requires (i) a large amount of fresh frozen plasma and is not generally spread due to supply limitations. (Ii) Since other people's plasma is used, a virus or unknown There are drawbacks such as high risk of infection with pathogens or Hb antigens.

  Furthermore, although the method (3) overcomes the drawbacks of (2), the presence of an adsorbent used for the removal of malignant substances (iii) priming volume is inevitably increased. Requires a device and highly trained practitioner to simultaneously control the steps of (iv) separating plasma from blood and removing malignant substances from plasma, which increases the patient's physiological burden at times Such problems are inherent.

  Although the method (4) is one of the important therapeutic methods for the purpose of effectively removing high molecular weight malignant substances, the adsorption removal process of (3) is replaced with a filtration process as a configuration of technical elements. Only. In principle, substances are to be separated by the molecular sieve effect utilizing the pore size of the filtration membrane, and only a removal effect limited to a specific molecular weight range can be expected, and selective removal cannot be performed. Moreover, it does not essentially solve the problem of (3).

  In solving these problems, a blood purifier or a blood purification device that can directly remove malignant substances from blood by direct blood perfusion is significant. For example, in an attempt to adsorb and remove malignant substances such as corresponding antigens and antibodies by immobilizing antibodies, antigens, enzymes, etc. on the inner and outer surfaces of the hollow fiber as seen in Patent Document 1, a plasma separation step And removal of malignant substances are performed simultaneously in one device, so it is expected that problems such as an increase in priming volume and an increase in the degree of difficulty in treatment technology can be solved. There are few that use the whole body surface, and there is nothing that has been put to practical use because of its low adsorption capacity.

  Elements required for a carrier for providing a practical and highly versatile adsorbent capable of removing malignant substances in body fluids are (I) capable of direct blood perfusion, (II) malignant to be removed It has a pore that can effectively exert a molecular sieving effect in the molecular weight region of the substance and allow the malignant substance to permeate the inside of the carrier, and (III) has a total surface area of the carrier enough to sufficiently adsorb the malignant substance to be removed. , And so on.

  Originally, affinity chromatography carriers are well known as those having the characteristics (II) and (III) described above, and natural polymer carriers, polyacrylamide carriers, glass carriers, and the like. This is a representative example. As a technique for separating molecules having a molecular weight of less than 200,000 Da, such as IgG (molecular weight of 150,000 Da), which is considered to be deeply involved in the autoimmune disease, the above-described affinity is used in the separation / purification step in the bioprocess. Examples using chromatographic carriers are widely known. However, in the separation / purification process in the bioprocess, a sample containing molecules having a molecular weight of less than 200,000 such as IgG used for the separation material composed of the carrier for affinity chromatography as described above is roughly purified through several pretreatment steps. In general, insoluble matters such as fine particles and unnecessary molecules are removed. By such a pretreatment process, problems such as pressure loss when a sample containing molecules having a molecular weight of less than 200,000, such as IgG, flows through a separation material composed of an affinity chromatography carrier as described above are drastically reduced. Selectivity is improved.

  On the other hand, when the affinity chromatography carrier as described above is used for extracorporeal circulation treatment for adsorbing and removing malignant substances in body fluids, there are various problems as follows. In other words, natural polymer carriers represented by cellulose, dextran, or agarose are soft gels in addition to chemical and physiological problems that lead to leucopenia and thrombocytopenia. However, there is a physical problem that body fluid cannot flow at a high flow rate. Although the polyacrylamide carrier has the advantage of being relatively physically and chemically stable, it has the disadvantage of non-specifically adsorbing plasma proteins and the residual toxicity of acrylamide cannot be ignored. Although the glass-based carrier also has physical and chemical stability, it is not preferable because nonspecific adsorption of plasma protein is remarkable and thrombus formation occurs when used for blood treatment. Due to the above problems, it has been difficult to use a known affinity chromatography carrier for extracorporeal circulation treatment, particularly for direct blood perfusion.

  On the other hand, the ethylene-vinyl alcohol copolymer is known to be a material having a slight effect on blood coagulation / fibrinolytic factors and platelets and having excellent blood anticoagulation properties. In addition, the effect on the complement / immune system is mild, and it has been put to practical use as an artificial dialysis membrane having high biocompatibility.

Japanese Patent No. 2814399

  However, to date, an adsorbent carrier comprising an ethylene-vinyl alcohol copolymer, which can be directly used for body fluid perfusion, can remove malignant substances having a molecular weight of less than 200,000 Da with high efficiency and high selectivity, and its The manufacturing method has not been put to practical use.

  Therefore, the present invention provides an adsorbent carrier that can be directly used for body fluid perfusion and can remove malignant substances having a molecular weight of less than 200,000 Da from body fluids such as blood and plasma with high efficiency and high selectivity, and a method for producing the same. The purpose is to provide.

  The present invention provides the following [1] to [10].

[1] A method for producing porous particles comprising an ethylene-vinyl alcohol copolymer and polyvinylpyrrolidone,
A stock solution preparation step of preparing a stock solution having a viscosity at 50 ° C. of 1000 mPa · s or more and 6500 mPa · s or less from an ethylene-vinyl alcohol copolymer, polyvinyl pyrrolidone and these good solvents;
A molding step of solidifying particles in a poor solvent of the ethylene-vinyl alcohol copolymer at 5 ° C. or more and 55 ° C. or less after discharging the stock solution from a nozzle and passing through an idle running portion;
To produce a porous particle comprising an ethylene-vinyl alcohol copolymer and polyvinylpyrrolidone and having an exclusion limit molecular weight of 200,000 Da or more and 600,000 Da or less.
[2] The production method according to [1], further comprising a surface modification step of surface-modifying particles obtained in the molding step with hypochlorous acid.
[3] The production method according to [1] or [2], wherein the ethylene-vinyl alcohol copolymer has an ethylene content of 20 mol% to 50 mol%.
[4] The production method according to any one of [1] to [3], wherein the good solvent is dimethyl sulfoxide and / or dimethylacetamide.
[5] The production method according to any one of [1] to [4], wherein the poor solvent is water or water containing the good solvent. The good solvent that can be added to the poor solvent is the same as the good solvent used in the stock solution, but there are many good solvents that can be used. Need not be the same solvent.
[6] A porous particle obtainable by the production method according to any one of [1] to [5].
[7] A porous particle comprising an ethylene-vinyl alcohol copolymer and polyvinylpyrrolidone having an exclusion limit molecular weight of 200,000 Da or more and 600,000 Da or less and a specific surface area of 10 m ² / g or more and 65 m ² / g or less.
[8] A carrier for a direct body fluid perfusion adsorbent comprising the porous particles according to [6] or [7]. In the case where the carrier has a ligand, the invention can also be expressed as the use of the porous particles according to [6] or [7] for producing a carrier for a direct body fluid perfusion adsorbent having a ligand.
[9] The carrier according to [8], wherein the body fluid is blood or plasma.
[10] The carrier according to [8] or [9], wherein the adsorbent is an IgG adsorbent.

  According to the present invention, porous particles for producing an adsorbent that can be directly used for body fluid perfusion and can remove malignant substances having a molecular weight of less than 200,000 Da from body fluids such as blood and plasma with high efficiency and high selectivity, and A manufacturing method thereof can be provided. Moreover, the porous particles of the present invention are used as a carrier to produce an adsorbent that can be directly used for body fluid perfusion and can remove malignant substances having a molecular weight of less than 200,000 Da from body fluids such as blood and plasma with high efficiency and high selectivity. It is possible.

2 is a cut-off molecular weight curve of porous particles produced in Example 2. FIG. 2 is a cut-off molecular weight curve of porous particles produced in Example 12. 2 is a cut-off molecular weight curve of porous particles produced in Comparative Example 1.

  Hereinafter, embodiments of the present invention will be specifically described. Note that the embodiments described below do not limit the present invention.

  Porous particles that can be used as an adsorbent capable of removing a pathogenic substance having a molecular weight of less than 200,000 Da from body fluids such as blood and plasma according to an embodiment of the present invention with high efficiency and high selectivity are ethylene-vinyl alcohol. Manufactured with a composition comprising a copolymer and polyvinylpyrrolidone. The ethylene content of the ethylene-vinyl alcohol copolymer is preferably 20 mol% or more and 50 mol% or less. Further, the porous particles according to the embodiment are preferably porous particles having an average particle diameter of 90 μm or more and 600 μm or less and a porosity of 70% or more. If the adsorbent is produced using the porous particles according to the embodiment as a carrier, malignant substances (pathogenic substances) having a molecular weight of less than 200,000 Da can be removed from body fluids such as blood and plasma with high efficiency and high selectivity. obtain. At this time, the particles may be processed into a monolith and used as a monolith.

  The “ethylene-vinyl alcohol copolymer” here is a copolymer of ethylene and vinyl alcohol. Generally, an ethylene-vinyl acetate copolymer obtained by copolymerizing a vinyl acetate monomer and ethylene. Manufactured by saponifying the coalescence. The ethylene-vinyl alcohol copolymer may be any type of copolymer such as random, block, or graft.

  In addition, in the ethylene-vinyl alcohol copolymer, if the ethylene content is less than 20%, the hydrophilicity is increased, so that the solubility in a poor solvent that should be used for production to form a coagulum is increased, It becomes difficult to form porous particles. In addition, when the ethylene content exceeds 50 mol%, the tendency to make it difficult to granulate spherically becomes strong, and at the same time, dissolution in a good solvent for dissolving ethylene-vinyl alcohol copolymer and polyvinylpyrrolidone used during production Sexuality decreases. The ethylene content of the ethylene-vinyl alcohol copolymer is more preferably 24 mol% or more and 48 mol% or less.

  The range of the weight average molecular weight (Mw) of the ethylene-vinyl alcohol copolymer is preferably from 10,000 Da to 3 million Da, and more preferably from 15,000 Da to 1.5 million Da. When Mw is less than 10,000 Da, the molecular weight of the polymer decreases when sterilization treatment, particularly radiation sterilization treatment is performed, and the elution amount with respect to a poor solvent such as water tends to increase. Moreover, when Mw exceeds 3 million Da, there exists a concern that the solubility in a solvent tends to be lowered, and that there is a tendency that it cannot be stably produced during polymerization. The Mw of the ethylene-vinyl alcohol copolymer is determined by various known methods, but in the embodiment of the present invention, measurement by gel permeation chromatography using polyethylene oxide as a standard is employed.

  The weight average molecular weight (Mw) of polyvinylpyrrolidone constituting the porous particles together with the ethylene-vinyl alcohol copolymer is preferably in the range of 2,000 Da to 2,000,000 Da, more preferably in the range of 2,000 Da to 1,000,000 Da. Preferably, the range from 2,000 Da to 100,000 Da is more preferable, and from the viewpoint of obtaining a larger exclusion limit molecular weight, the range from 20,000 Da to 2,000,000 Da is more preferable, and from 200,000 Da to 2,000,000 Da. A range is further preferred. When the weight average molecular weight is smaller than 2,000 Da, when the porous particles have fibrils, the effect of developing a structure having voids inside the fibrils tends to be low. Viscosity increases and molding tends to be difficult. In addition, Mw of polyvinylpyrrolidone can be measured by the same method as Mw of ethylene-vinyl alcohol copolymer. In addition, the ratio (mass ratio) of the ethylene-vinyl alcohol copolymer and polyvinyl pyrrolidone in the stock solution is 0.01 to 100 parts by mass of polyvinyl pyrrolidone with respect to 100 parts by mass of the ethylene-vinyl alcohol copolymer, preferably 0.00. 1-100 mass parts, More preferably, it is 0.5-100 mass parts, Most preferably, it is 7.5-100 mass parts.

  According to the embodiment of the present invention, porous particles having an exclusion limit molecular weight of 200,000 Da or more and 600,000 Da or less are prepared by using a stock solution composed of a mixture of an ethylene-vinyl alcohol copolymer, polyvinyl pyrrolidone and a good solvent. After discharging from the nozzle and passing through the free running part (that is, after passing through the free running part), it is immersed in a poor solvent, and the polymer is gelled (particulate solidification) by solvent exchange to make it porous The principle is a wet phase separation method for forming a body. During these processes, the proportion of good solvent decreases, and as a result, microphase separation occurs, polymer globules are formed, grow, entangle, fibrils are formed, fibril gaps become communication holes, and porous particles are formed. It is formed.

  As described above, the porous particles are formed of a plurality of fibrils, and it is preferable that the fibrils have voids therein and have an open surface. The average diameter of the fibrils is usually 0.01 μm to 50 μm, and the opening diameter of the fibril surface is usually 0.001 μm to 5 μm. Moreover, the clearance gap between fibrils is 0.01 micrometer-5 micrometers normally.

  The good solvent only needs to dissolve both the ethylene-vinyl alcohol copolymer and polyvinylpyrrolidone. Examples thereof include dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), dimethylacetamide (DMAC), dimethylformamide (DMF), and the like, and preferably dimethyl sulfoxide and / or dimethylacetamide. These good solvents may be used alone or as a mixed solvent. The content of the good solvent in the stock solution is preferably 50% by mass or more and 98% by mass or less, and more preferably 70% by mass or more and 95% by mass or less, based on the total mass of the stock solution.

  The poor solvent may be any one that does not dissolve the ethylene-vinyl alcohol copolymer but dissolves polyvinylpyrrolidone. For example, water, water containing the good solvent, and the like. By containing a good solvent in the poor solvent, the rate of coagulation can be changed variously, and the degree of elution of polyvinylpyrrolidone from the ethylene-vinyl alcohol copolymer and the polyvinylpyrrolidone mixture can be controlled.

  Polyvinylpyrrolidone, which is a water-soluble polymer, extends part of its molecular chain from the surface of the fibril in the above phase separation process, as if it were a mustache, so that the surface of the fibril is kept hydrophilic and has the effect of suppressing hydrophobic adsorption. Can be expected.

  In the method for producing a porous particle, “pass through the free-running portion” means once in the air (or a gas such as an inert gas) so that the stock solution discharged from the nozzle does not immediately contact the poor solvent. Middle).

  The mixture of ethylene-vinyl alcohol copolymer, polyvinylpyrrolidone and good solvent used as a stock solution in the production of porous particles must have a viscosity at 50 ° C. of 1000 mPa · s to 6500 mPa · s. The viscosity at 50 ° C. is more preferably 1000 mPa · s or more and 5300 mPa · s or less. If it is less than 1000 mPa · s, the polymer concentration in the discharged stock solution is low, so the progress of solidification in the poor solvent is slow, and the particles are deformed by the impact received at the gas-liquid interface when immersed in the poor solvent. In particular, the solidification of the droplets becomes extremely difficult at 600 mPa · s or less. On the other hand, when it exceeds 6500 mPa · s, it becomes difficult to discharge from the nozzle. The viscosity of the stock solution is a measured value when the rotational speed is 20 rpm using a rotational viscometer.

In the method for producing porous particles, the temperature of the poor solvent for performing phase separation by immersing the stock solution must be 5 ° C. or higher and 55 ° C. or lower. The temperature of the poor solvent is more preferably 10 ° C. or higher and 40 ° C. or lower, and further preferably 10 ° C. or higher and 30 ° C. or lower. If it is lower than 5 ° C., it tends to be difficult to produce because it approaches the freezing point of the poor solvent. In addition, when the temperature is higher than 55 ° C., the specific surface area of the obtained porous particles is less than 10 m ² / g, and the molecular weight is less than 200,000 Da from the body fluid including blood and plasma according to the embodiment of the present invention. It is difficult to obtain porous particles that can be used as an adsorbent that can remove malignant substances (pathogenic substances) with high efficiency and high selectivity.

  In the production of the porous particles according to the embodiment of the present invention, the particles may be modified by a process such as etching or annealing after the above-described forming step. Examples of the modification method include hypochlorous acid treatment and incubation under a temperature condition that is higher than the glass transition temperature of the polymer and lower than the crystallization temperature. About the density | concentration at the time of using hypochlorous acid, 100 ppm or more and 8000 ppm or less are preferable. By modifying the surface of the porous particles by hypochlorous acid treatment, the pore diameter, porosity, and specific surface area can be increased.

  The “average particle diameter” of the porous particles indicates the average value of the diameters of 10 arbitrarily selected porous particles in the case of a true sphere, and 10 arbitrarily selected pores in the case of a non-spherical sphere. The average value when the particle in a given direction is measured twice per particle with a microscope. The average particle size of the porous particles is preferably 90 μm or more and 600 μm or less. When the average particle size is smaller than 90 μm, the voids between the particles are narrowed, making it difficult for blood to pass, and the pressure loss tends to increase. When the average particle size is larger than 600 μm, the particle surface area per unit volume tends to be small, and the adsorption efficiency tends to decrease.

The “specific surface area” of the porous particles is preferably 10 m ² / g or more and 65 m ² / g or less, more preferably 25 m ² / g or more and 65 m ² / g or less, and further preferably 45 m ² / g or more. 65 m ² / g or less. When the specific surface area is less than 10 m ² / g, it is not possible to secure a surface area for sufficiently adsorbing malignant substances (pathogenic substances) having a molecular weight of less than 200,000 Da from body fluids such as blood and plasma. On the other hand, if it is larger than 65 m ² / g, the porosity of the porous particles will increase, and it will not be possible to secure a strength sufficient to withstand pressure loss when perfused with body fluids such as blood and plasma, Alternatively, the exclusion limit molecular weight tends to be less than 200,000 Da.

The specific surface area is represented by the surface area occupied by the nitrogen gas adsorbed per unit weight of the dry particles. In other words, the specific surface area indicates how effectively the substances constituting the unit weight particles form a surface in a dry state. The specific surface area of the porous particles is determined by the most common BET method using nitrogen gas when determining the specific surface area of the polymer gel. In general, a polymer gel swells in a medium having an affinity for the gel and shrinks when dried. In the case of a soft gel in which the pores filled with the medium at the time of swelling are maintained only by the crosslinked network, the network is crushed when dried, and the pores are almost lost. In this case, the specific surface area is a value almost only outside the particle, and thus generally shows a low value of 1 m ² / g or less. Since agarose conventionally known for affinity chromatography is a soft gel, the pores disappear upon drying. Furthermore, since it has a soft mesh that is easily crushed, body fluid cannot flow at a high flow rate for a long time even when packed in a column and used for extracorporeal circulation. On the other hand, if the pore has a firm structure and is a hard gel that can withstand freeze-drying, the pore will shrink somewhat when dried, but it will remain almost in a swollen state. That is, the hard gel has permanent pores, and the specific surface area generally shows a higher value than the soft gel.

  Preparation of the stock solution as described above (stock solution preparation step), polymer gelation (particulate coagulation) step (molding step), and modification of the particles, if necessary, particularly surface modification of the particles (surface) The porous particle which concerns on embodiment of this invention is obtained by the method which passes through a modification process. In this method, by setting the viscosity of the stock solution in the stock solution in the stock solution preparation step, the temperature of the poor solvent in the molding step in the specific range, and performing the surface modification step under appropriate conditions as necessary, It becomes possible to obtain porous particles having an exclusion limit molecular weight in a specific range of 200,000 Da or more and 600,000 Da or less.

  The body fluid perfusion adsorbent can be produced directly by binding various ligands to the porous particles using a known method. As the ligand, an antibody, an antibody fragment, a protein, a peptide, or a low molecular compound can be used, and may be appropriately selected according to the purpose. By binding a ligand to the porous particles, an adsorbent capable of removing malignant substances such as antigens and antibodies having a molecular weight of less than 200,000 Da, particularly IgG, with high efficiency and high selectivity can be obtained.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples of the present invention. However, the present invention is not limited to these examples, and various modifications can be made without departing from the technical idea of the present invention. Can be changed. In the examples, various physical properties of the porous particles were measured by the following methods.

(Average particle size)
The particle diameter of the porous particles was measured using a microscope (manufactured by Keyence Corporation, trade name: VH7000). When the porous particles are true spheres, the diameters of 10 arbitrarily selected porous particles are measured, and the average value is taken as the average particle diameter. For the porous particles, the diameter in an arbitrary direction was measured twice with a microscope, and the average value was defined as the average particle diameter.

(Specific surface area)
Using TriStar 3000 (manufactured by Shimadzu Corporation), the specific surface area of the porous particles was determined by the BET method by nitrogen gas adsorption. Since the sample used for the measurement needs to be dried, in the present invention, the porous particles swollen with water were freeze-dried and used as a sample for the measurement.

(Exclusion limit molecular weight)
The porous particles obtained in the examples and comparative examples were swollen with pure water, and after degassing by sonication, an empty column for high performance liquid chromatography having an inner diameter of 7.6 mmφ and a column length of 5 cm (manufactured by GL Sciences Inc.) ) And closed with a spanner to obtain a column for exclusion limit molecular weight evaluation. After the column was connected to a high performance liquid chromatography apparatus (manufactured by Shimadzu Corporation, trade name: LC20A), phosphate buffered saline (PBS) was perfused at a flow rate of 0.1 mL / min, and an RI detector was used. The flow continued until the baseline became constant on the monitor screen. After the baseline is stabilized, pullulan P800 (molecular weight 788 KDa), P400 (molecular weight 404 KDa), P200 (molecular weight 212 KDa), P100 (molecular weight 112 KDa), P50 (molecular weight 47.3 KDa), P20 (molecular weight 22. 8 KDa), P10 (molecular weight 11.8 KDa), P5 (molecular weight 5.9 KDa) (manufactured by Showa Denko KK), ethylene glycol (manufactured by Wako Pure Chemical Industries, Ltd.), and human IgG (manufactured by Sigma Aldrich KK). Each dissolved PBS solution was prepared, and these standard molecular weight substance solutions were sequentially injected to measure the retention time (minutes). Furthermore, the fractionation molecular weight curve was obtained by plotting the elution volume (mL) calculated by the following formula with respect to the molecular weight.
Elution volume (mL) = flow rate (mL / min) x retention time (min)

In the fractional molecular weight curve, the intersection of the straight line A parallel to the vertical axis of the graph and passing through the point of pullulan P800 and the tangent line B at the point with the smallest elution capacity among the points located on the right side of the graph from the straight line A is C The molecular weight at the intersection C was taken as the exclusion limit molecular weight.
The exclusion limit molecular weight refers to the molecular weight of the smallest molecular weight substance that cannot generally enter the fine pores of the packing material packed in the column. A substance having a molecular weight higher than the exclusion limit molecular weight cannot enter the fine pores of the filler and flows in the same volume space outside the fine pores regardless of the molecular weight. give. On the other hand, with respect to substances having a molecular weight less than the exclusion limit molecular weight, the elution capacity becomes larger as the substance has a lower molecular weight because there is a difference in the ease of penetration into the micropores depending on the molecular weight. However, if the substance has a molecular weight lower than a certain molecular weight, it completely penetrates into the micropores, so that the same elution volume is given.

Example 1
Ethylene-vinyl alcohol copolymer (EVOH) (manufactured by Kuraray Co., Ltd., Eval (registered trademark) F101 (trade name), ethylene content 32 mol%) 200 g, and polyvinylpyrrolidone (PVP) (manufactured by BASF Japan Ltd., Luvitec K85 ( (Product name)) 90 g and 1710 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved by heating at 50 ° C. in a Separa flask to obtain a uniform polymer stock solution (mixed solution). .

  The obtained polymer stock solution is discharged from a nozzle (inner diameter 0.55 mm), and the resulting polymer droplet is allowed to pass through a free running portion of about 50 cm, and then is placed in a coagulation bath made of water at 55 ° C. and solidified. It was. Further, washing was performed to obtain porous particles having an average particle size of 410 μm. The physical properties are shown in Table 1.

  The obtained porous particles were packed into a column, and priming was performed by flowing 10 mL of heparinized physiological saline (heparin concentration 5000 IU / L) at room temperature. Then, 10.3 mL of human fresh blood (heparin concentration 5000 IU / L) with heparin added as an anticoagulant was flowed by a syringe pump at room temperature at a flow rate of 12.3 mL / h, and confirmed to flow without hemolysis did. At this time, the internal pressure of the circuit connected to the column was maintained at 2.1 KPa or less.

(Example 2)
Except for coagulation in a coagulation bath made of water at 50 ° C., the polymer stock solution was coagulated by the same method as in Example 1 and washed to obtain porous particles having an average particle size of 433 μm. The physical properties are shown in Table 1, and the molecular weight cut-off curve is shown in FIG. Further, it was confirmed that blood flowed in the same manner as in Example 1.

  10 mL of the obtained porous particles were added to a reaction solution consisting of 2.6 mL of an aqueous sodium hydroxide solution (concentration 16.7 M), 14 mL of DMSO and 12 mL of epichlorohydrin, and stirred at 30 ° C. for 2 hours. The porous particles after the reaction are collected by filtration with a glass filter 3G, washed thoroughly with pure water, and then buffered with sodium bicarbonate at pH 9.6 containing 2- (2-aminoethyl) thiophene at a concentration of 5.9 g / L. The mixture was stirred in 20 mL of the liquid at 50 ° C. for 17 hours to be reacted. The porous particles after the reaction were collected by filtration with a glass filter 3G and washed with hot water at 50 ° C. From the ultraviolet absorbance change of the reaction solution before and after the reaction, it was found that 87 μmol of 2- (2-aminoethyl) thiophene was immobilized per 1 mL of porous particles.

After washing 1 mL of the obtained 2- (2-aminoethyl) thiophene-immobilized porous particles with 10 mL of heparinized physiological saline (heparin concentration 5000 IU / L) at room temperature, heparin was used as an anticoagulant in a polypropylene tube. The mixture was mixed with 2 mL of added human fresh blood (heparin concentration: 5000 IU / L) and stirred at room temperature for 2 hours using a mix rotor. After the stirring, the porous particles were removed by filtration with a top filter, and the plasma was separated from the collected blood by centrifugation at 3500 rpm for 10 minutes. Concentration measurement of albumin, IgG, and fibrinogen was performed on the obtained plasma. The removal rates defined by the following formulas were 34%, 82% and 5%, respectively.
Removal rate (%) = {1− (density after treatment / density before treatment)} × 100

(Example 3)
Except for coagulation in a coagulation bath made of water at 40 ° C., the polymer stock solution was coagulated by the same method as in Example 1 and washed to obtain porous particles having an average particle size of 587 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

Example 4
Except for coagulation in a coagulation bath made of water at 30 ° C., the polymer stock solution was coagulated by the same method as in Example 1 and washed to obtain porous particles having an average particle size of 596 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

(Example 5)
Except for coagulation in a coagulation bath made of 20 ° C. water, the polymer stock solution was coagulated by the same method as in Example 1 and washed to obtain porous particles having an average particle size of 549 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

(Example 6)
Except for coagulation in a coagulation bath made of water at 10 ° C., the polymer stock solution was coagulated by the same method as in Example 1 and washed to obtain porous particles having an average particle size of 550 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

(Example 7)
Except for coagulation in a coagulation bath made of water at 5 ° C., the polymer stock solution was coagulated by the same method as in Example 1 and washed to obtain porous particles having an average particle diameter of 562 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

(Example 8)
80 g of ethylene-vinyl alcohol copolymer (EVOH) (manufactured by Kuraray Co., Ltd., EVAL (registered trademark) F101 (trade name), ethylene content 32 mol%), and polyvinylpyrrolidone (PVP) (manufactured by BASF Japan Ltd., Luvitec K85 ( (Product Name)) 6 g and 414 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved by heating to 50 ° C. in a Separ flask to obtain a uniform polymer stock solution (mixed solution). .
The obtained polymer stock solution was discharged from a nozzle, and the resulting polymer droplets were allowed to pass through an idle running portion of about 50 cm, and then were placed in a coagulation bath made of water at 40 ° C. and coagulated. Furthermore, washing was performed to obtain porous particles having an average particle size of 531 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

Example 9
Ethylene-vinyl alcohol copolymer (EVOH) (Kuraray Co., Ltd., EVAL (registered trademark) F101 (trade name), ethylene content 32 mol%) 40 g, and polyvinylpyrrolidone (PVP) (BASF Japan Co., Ltd., Luvitec K85 ( (Product Name)) 18 g and 442 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved by heating to 50 ° C. in a Separa flask to obtain a uniform polymer stock solution (mixed solution). .
The obtained polymer stock solution was discharged from a nozzle, and the resulting polymer droplets were allowed to pass through an idle running portion of about 50 cm, and then were placed in a coagulation bath made of water at 40 ° C. and coagulated. Further, washing was performed to obtain porous particles having an average particle diameter of 532 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

(Example 10)
38.5 g of ethylene-vinyl alcohol copolymer (EVOH) (manufactured by Kuraray Co., Ltd., EVAL (registered trademark) M100B (trade name), ethylene content 24 mol%), and polyvinylpyrrolidone (PVP) (Luvitec, manufactured by BASF Japan Ltd.) 38.5 g of K85 (trade name) and 423 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved by heating to 50 ° C. in a Separa flask, and a uniform polymer stock solution (mixed solution) )
The obtained polymer stock solution was discharged from a nozzle, and the resulting polymer droplets were allowed to pass through an idle running portion of about 50 cm, and then were placed in a coagulation bath made of water at 40 ° C. and coagulated. Furthermore, washing was performed to obtain porous particles having an average particle diameter of 460 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

(Example 11)
Ethylene-vinyl alcohol copolymer (EVOH) (manufactured by Kuraray Co., Ltd., Eval (registered trademark) G156B (trade name), ethylene content 48 mol%) 28.5 g, and polyvinylpyrrolidone (PVP) (manufactured by BASF Japan Ltd., Luvitec) 28.5 g of K85 (trade name) and 443 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved by heating to 50 ° C. in a Separ flask, and a uniform polymer stock solution (mixed solution) Got.
The obtained polymer stock solution was discharged from a nozzle, and the resulting polymer droplets were allowed to pass through an idle running portion of about 50 cm, and then were placed in a coagulation bath made of water at 40 ° C. and coagulated. Further, washing was performed to obtain porous particles having an average particle diameter of 495 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

(Example 12)
10 mL of porous particles obtained through the same steps as in Example 2 were transferred to a falcon tube, 5 mL of a 5000 ppm sodium hypochlorite aqueous solution was added, and the mixture was shaken at 50 ° C. for 2 hours. The shaken porous particles were collected and washed with pure water to obtain porous particles having an average particle size of 433 μm. The physical properties are shown in Table 1, and the molecular weight cut-off curve is shown in FIG. Further, it was confirmed that blood flowed in the same manner as in Example 1.
Using the porous particles obtained above, 2- (2-aminoethyl) thiophene-immobilized porous particles were prepared in the same manner as in Example 2, and the removal rate of albumin, IgG, and fibrinogen in blood Were 40%, 90%, and 10%, respectively.

(Example 13)
Except for discharging using a nozzle having an inner diameter of 0.15 mm, the polymer stock solution was coagulated by the same method as in Example 5 and washed to obtain porous particles having an average particle diameter of 101 μm. The physical properties are shown in Table 1. Further, it was confirmed that blood flowed in the same manner as in Example 1.

(Comparative Example 1)
Except for coagulation in a coagulation bath made of water at 60 ° C., the polymer stock solution was coagulated by the same method as in Example 1 and washed to obtain porous particles having an average particle diameter of 400 μm. The physical properties are shown in Table 1. Further, a fractional molecular weight curve is shown in FIG.
Using the porous particles obtained above, 2- (2-aminoethyl) thiophene-immobilized porous particles were obtained in the same manner as in Example 2, and the same washing operation was performed. 80 mL of 2- (2-aminoethyl) thiophene was immobilized per mL of particles. Using these particles, the removal rates of albumin, IgG, and fibrinogen in blood were determined in the same manner as in Example 2, and were 10%, 7%, and 5%, respectively.

(Comparative Example 2)
Ethylene-vinyl alcohol copolymer (EVOH) (manufactured by Kuraray Co., Ltd., EVAL (registered trademark) F101 (trade name), ethylene content 32 mol%) 50 g, and polyvinylpyrrolidone (PVP) (manufactured by BASF Japan Ltd., Luvitec K85 ( (Trade name)) 13.5 g and 436.5 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved by heating to 50 ° C. in a Separa flask, and a uniform polymer stock solution (mixed solution) )
The obtained polymer stock solution was discharged from a nozzle, and the resulting polymer droplets were allowed to pass through an idle running portion of about 50 cm, and then were placed in a coagulation bath made of water at 60 ° C. and coagulated. Furthermore, washing was performed to obtain porous particles having an average particle diameter of 399 μm. The physical properties are shown in Table 1.

(Comparative Example 3)
Ethylene-vinyl alcohol copolymer (EVOH) (manufactured by Kuraray Co., Ltd., EVAL (registered trademark) F101 (trade name), ethylene content 32 mol%) 50 g, and polyvinylpyrrolidone (PVP) (manufactured by BASF Japan Ltd., Luvitec K85 ( (Product Name)) 6 g and 444 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved by heating to 50 ° C. in a Separ flask to obtain a uniform polymer stock solution (mixed solution). .
The obtained polymer stock solution was discharged from a nozzle, and the resulting polymer droplets were allowed to pass through an idle running portion of about 50 cm, and then were placed in a coagulation bath made of water at 60 ° C. and coagulated. Furthermore, washing was performed to obtain porous particles having an average particle diameter of 398 μm. The physical properties are shown in Table 1.

(Comparative Example 4)
Ethylene-vinyl alcohol copolymer (EVOH) (manufactured by Kuraray Co., Ltd., EVAL (registered trademark) F101 (trade name), ethylene content 32 mol%) 60 g, and polyvinylpyrrolidone (PVP) (manufactured by BASF Japan Ltd., Luvitec K85 ( (Product name)) 27 g and 413 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were heated to 50 ° C. and dissolved in a Separ flask to obtain a uniform polymer stock solution (mixed solution). .
The obtained polymer stock solution was tried to be discharged from the nozzle, but the viscosity of the polymer stock solution was high and could not be discharged. The physical properties are shown in Table 1.

(Comparative Example 5)
Ethylene-vinyl alcohol copolymer (EVOH) (manufactured by Kuraray Co., Ltd., EVAL (registered trademark) F101 (trade name), ethylene content 32 mol%) 25 g, and polyvinylpyrrolidone (PVP) (manufactured by BASF Japan Ltd., Luvitec K85 ( Product name)) 11.5 g and 463 g of dimethyl sulfoxide (DMSO) (manufactured by Wako Pure Chemical Industries, Ltd.) were dissolved by heating to 50 ° C. in a Separa flask, and a uniform polymer stock solution (mixed solution) was obtained. Obtained.
The obtained polymer stock solution is ejected from a nozzle, and the resulting polymer droplets are allowed to pass through a free running portion of about 50 cm, and then placed in a coagulation bath made of 50 ° C. water. As a result, the polymer stock solution is solidified. There wasn't. The physical properties are shown in Table 1.

  The method for producing porous particles of the present invention is useful as a method for producing a carrier for direct perfusion adsorbent that removes malignant substances having a molecular weight of less than 200,000 Da from body fluids such as blood and plasma with high efficiency and high selectivity. .

Claims (9)

A method for producing porous particles comprising an ethylene-vinyl alcohol copolymer and polyvinylpyrrolidone,
A stock solution preparation step of preparing a stock solution having a viscosity at 50 ° C. of 1000 mPa · s or more and 6500 mPa · s or less from an ethylene-vinyl alcohol copolymer, polyvinyl pyrrolidone and these good solvents;
A molding step of solidifying particles in a poor solvent of the ethylene-vinyl alcohol copolymer at 5 ° C. or more and 55 ° C. or less after discharging the stock solution from a nozzle and passing through an idle running portion;
To produce a porous particle comprising an ethylene-vinyl alcohol copolymer and polyvinylpyrrolidone and having an exclusion limit molecular weight of 200,000 Da or more and 600,000 Da or less.   The manufacturing method of Claim 1 further equipped with the surface modification process of surface-modifying the particle | grains obtained at the said formation process with hypochlorous acid.   The manufacturing method according to claim 1 or 2, wherein the ethylene-vinyl alcohol copolymer has an ethylene content of 20 mol% or more and 50 mol% or less.   The manufacturing method according to claim 1, wherein the good solvent is dimethyl sulfoxide and / or dimethylacetamide.   The manufacturing method according to any one of claims 1 to 4, wherein the poor solvent is water or water containing the good solvent.   A porous particle comprising an ethylene-vinyl alcohol copolymer and polyvinyl pyrrolidone having an exclusion limit molecular weight of 200,000 Da or more and 600,000 Da or less and a specific surface area of 10 m 2 / g or more and 65 m 2 / g or less. A porous particulate according to claim 6 Symbol mounting, direct fluid perfusion adsorbent carrier. The carrier according to claim 7 , wherein the body fluid is blood or plasma. The carrier according to claim 7 or 8 , wherein the adsorbent is an IgG adsorbent.

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