JP2009165667A - Filter having prion removal function - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a filter having a function removing an abnormal prion from biological fluid such as blood product; and a filter capable of removing leucocyte simultaneously with the abnormal prion. <P>SOLUTION: This filter having a prion removal function has a carrier superposed in a housing having a fluid outlet and inlet, wherein the superposed carrier consists of the at least following formed of A and B. A: The carrier consists of at least one or more porous body layers and at least one or more porous bead layers. B: The porous surface (including the inside of pores) of the porous beads and/or the surface of the porous body are provided with polymer formed of three units of a unit derived from hydrophobic polymerizable monomer of not less than 20 mol% and not more than 40 mol%, a unit derived from polymerizable monomer containing basic nitrogen-containing part of not less than 5 mol% and not more than 13 mol%, and a residual unit derived from polymerizable monomer containing protogenic neutral hydrophilic part. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a filter having a function of removing abnormal prions from biological fluids such as blood products. More specifically, the present invention relates to a filter that selectively removes abnormal prions that may be present in blood products such as whole blood products, concentrated red blood cells, and platelet concentrates.

Abnormal prions are infectious agents that cause spongiform encephalopathy of the central nervous system in humans and animals. Abnormal prions are different from bacteria, viruses and viroids. The current strong hypothesis is that no nucleic acid component is required for the infectivity of an abnormal prion protein. Furthermore, an abnormal prion that infects one species of animal (eg, a human) was thought not to infect another species (eg, a mouse).
An important step in the study of abnormal prions and the diseases caused thereby was the discovery and purification of a protein termed prion protein (“PrP”) (Non-Patent Documents 1 to 3). Subsequently, the complete gene encoding the aberrant prion protein was cloned, sequenced, and expressed in transgenic animals. PrP ^c is encoded by a single copy host gene (Non-Patent Document 4) and is usually found on the outer surface of nerve cells. The hypothesis that prion disease is caused by the conversion of PrP ^c into a variant called PrP ^Sc is promising.

  Later, it was pointed out that mutant Creutzfeldt-Jakob disease (mutant CJD), mainly in the United Kingdom, was the result of beef consumption from cattle infected with BSE (Non-Patent Documents 5 and 6). Furthermore, in addition to transmission of beef from bovine to human beings due to consumption of beef, mutant CJD has a risk of transmission of infection from human to human due to transfusion of blood products and transmission of mutant abnormal prion protein present in tissue grafts. Has been suggested. In recent years, two cases have been reported in which recipients who received blood from donors who developed mutant CJD after donation in 2004 were infected with mutant CJD. In 2006, the third case was reported. Therefore, the possibility of transmission of mutant CJD by blood transfusion is increasing. In addition, there are a considerable number of infected humans who have not yet developed mutant CJD, and it has been pointed out that blood products from these humans may spread the infection. Therefore, a method is needed to remove abnormal prions from blood products.

  On the other hand, in the field of blood transfusion, so-called leukocyte-removed blood transfusion, in which mixed leukocytes contained in blood products are removed and transfused, has come to be performed. This may include side effects such as headache, nausea, chills, and non-hemolytic fever reactions associated with blood transfusions, alloantigen sensitization that has a more serious effect on recipients, post-transfusion graft-versus-host disease (GVHD), viral infections, etc. This is because the serious side effects were caused mainly by leukocytes mixed in blood products used for blood transfusion. In the method of removing leukocytes from a blood product, the filter method is widely used because of its high leukocyte removal ability, simple operation, and low cost. In recent years, the filtration method, that is, the treatment of blood products and the like using a leukocyte removal filter, is becoming increasingly common in blood centers prior to storage in order to thoroughly control the quality of leukocyte-removed blood products. Normally, when filtering blood using a leukocyte removal filter at a blood center, place the blood bag containing the blood product to be filtered in a position 70 to 150 cm higher than the bag for collecting the filtered blood product. It is common practice to filter blood products by the action of gravity.

  Two sets of SCD type and in-line type are widely used as a set for adjusting blood products from which leukocytes have been removed. In the SCD type, a leukocyte removal filter and a blood bag that collects the filtered blood product are connected to aseptically join the bag containing the blood product for which leukocytes are to be removed and the set to aseptically join. . In the in-line type, since blood collection from a blood donor to blood product adjustment is performed in an integrated manner, the blood bag usually contains a preservation solution and an anticoagulant. For these sets of sterilization methods, radiation sterilization is generally employed because the cost for sterilization is low in the SCD type. However, since the preservative and anticoagulant are decomposed by radiation sterilization, high-pressure steam sterilization is generally used for the in-line type.

One of the indicators for measuring the quality of blood products containing red blood cells is the degree of hemolysis, and the degree of hemolysis is less than 0.8% in order to supply leukocyte-removed blood products containing high-quality red blood cells. It is calculated | required (nonpatent literature 7).
As a method for removing abnormal prions from a blood product, a method capable of simultaneously removing leukocytes in consideration of operability in the blood center, cost, and amount of blood loss is preferable.

  Patent Document 1 discloses a leukocyte removal filter comprising a unit derived from a hydrophobic polymerizable monomer, a unit derived from a polymerizable monomer containing a basic nitrogen-containing moiety, and a unit derived from a polymerizable monomer containing a protic neutral hydrophilic moiety. Although the material coating polymer is disclosed as a material having a high leukocyte removal ability, there is no disclosure or suggestion regarding the removal of abnormal prions. As blood filtration for leukocyte removal before storage, room temperature filtration that filters at room temperature within one day after blood collection and refrigeration filtration that filters after storage for about 1 to 3 days in a refrigerator often coexist. However, while the filtration time is short, leukocytes are more likely to leak than refrigerated blood. In refrigerated filtration, the filtration time is long, and the tendency that leukocyte leakage is relatively prevented is recognized. However, in patent document 1, examination of the filtration time in refrigeration filtration is not performed.

  Patent Document 2 discloses a method in which a polymer matrix to which a hydrophilic, hydrophobic or amphiphilic functional group is bonded, or a prion-binding substance composed of alumina or silica and a prion in a biological fluid form a complex. Is disclosed. However, in the examples, the functional group is bonded to the resin, but leukocyte removal cannot be performed, and a leukocyte removal filter must be used separately even if it is practically used in the blood center. Separately removing leukocytes and abnormal prions increases the amount of blood product loss, labor at the blood center, and cost. Alumina and silica are said to cause activation of the coagulation system and have high non-selective adsorption of proteins, and are not suitable for blood products.

  In Patent Document 3, the surface of an apparatus such as a flow-through column or a spherical polymer porous bead is coated with a prion complexing agent such as a metal salt of phosphorus langstenoic acid (for example, sodium), and an arbitrary liquid sample. A method for use in removing prions from the like is disclosed. However, it is necessary to expose the sample to the complexing agent for a time sufficient to form a complex with substantially all the prion complexing agent in the sample. For example, the sample is incubated at about 30 ° C. to 45 ° C. (preferably 37 ° C.) for about 1 hour to 16 hours. However, a temperature of 37 ° C. is not appropriate as a storage temperature for blood products, and refrigeration, room temperature filtration or refrigeration filtration is common in current methods of using leukocyte removal filters, and filtration is performed by the action of gravity. Therefore, this method is not suitable for removing prions from blood products or removing leukocytes.

Patent Document 4 discloses a method for forming a complex of a prion protein and a prion-binding substance comprising a hydrophilic, hydrophobic, or amphiphilic functional group for removing and detecting prion, or aluminum or silica. Is disclosed. However, the prion-binding substances used in the examples cannot remove leukocytes, and if leukocytes and abnormal prions are removed separately, the amount of blood product loss, labor at the blood center, and cost increase. there were. Alumina and silica are said to cause activation of the coagulation system and have high non-selective adsorption of proteins, and are not suitable for blood products.
From the above, for the purpose of removing abnormal prions from blood products, there is a demand for a method that allows efficient and simple removal, and further, it is desired that leukocytes can be removed simultaneously.
Bolton, et. Al., (1982) Science 218: 1309-11 Prusiner, et. Al., (1982) Biochemistry 21: 6942-50 McKinley, et. Al., (1983) Cell 35: 57-62 Basler, et. Al., (1986) Cell 46: 417-28 G. Chazot, et. Al., (1996) Lancet 347: 1181 RGWill, et. Al., (1996) Lancet 347: 921-25 Guide to the preparation, use and quality assurance of blood components 9th edition / Council of Europe Publishing International Publication No. 03/011924 Pamphlet US Patent Application Publication No. 2005/0014196 Japanese translation of PCT publication No. 2002-539081 JP-T-2006-522344

  In view of the above problems, an object of the present invention is to provide a filter having a function of removing abnormal prions from a biological fluid such as a blood product, and further to provide a filter capable of removing white blood cells simultaneously with abnormal prions. It is in.

  As a result of intensive studies to solve the above problems, the present inventors have found that a hydrophobic polymerizable polymer, a polymerizable monomer containing a basic nitrogen-containing moiety, or a polymerizable monomer containing a protic neutral hydrophilic moiety alone When using a filter with porous beads coated with these terpolymers instead of a polymer consisting of these, the abnormal surface prion is surprisingly simple and efficient from the blood product because the specific surface area is much larger than the porous body. Has been found to be able to be removed, and the present invention has been obtained. That is, the present invention relates to the following.

(1) A filter for selectively reducing a target substance in a biological fluid, which has a carrier laminated in a housing having an outlet and an inlet for the fluid, and the laminated carrier is at least the following: A filter having a prion removal function, characterized by comprising the configurations of A and B.
A: It consists of at least one porous body layer and at least one porous bead layer.
B: 20 mol% or more and 40 mol% or less of a hydrophobic polymerizable monomer-derived unit, 5 mol% or more and 13 mol% or less of a polymerizable monomer-containing unit containing a basic nitrogen-containing moiety, and the balance is a protonic neutral hydrophilic A polymer composed of three units, which are units derived from a polymerizable monomer containing a functional moiety, is present on the porous surface (including the pores) of the porous beads and / or the surface of the porous body.
(2) It has a porous body layer on the outlet side of the porous bead layer, and the average pore diameter of at least one porous body of the porous body layer is equal to or less than the average particle diameter of the porous beads ( The filter according to 1).
(3) The filter according to (1) or (2), wherein at least one of the porous bodies has a leukocyte removing ability.
(4) The filter according to (3), wherein the porous body having leukocyte removal ability is a fibrous body.
(5) The filter according to (4), wherein the fibrous body having leukocyte removal ability is a nonwoven fabric.
(6) The filter according to (1), wherein the average particle diameter of the porous beads is 20 μm to 5 mm.
(7) The hydrophobic polymerizable monomer, the polymerizable monomer containing a basic nitrogen-containing moiety, and the polymerizable monomer containing a protic neutral hydrophilic moiety are acrylic acid derivatives and / or methacrylic acid derivatives. The filter described.
(8) The filter according to (1) or (7), wherein the basic nitrogen-containing moiety is a tertiary amino group.
(9) The filter according to (1) or (7), wherein the protonic neutral hydrophilic portion is a hydroxyl group.
(10) The filter according to any one of (1) to (9), wherein the filter is autoclaved after radiation sterilization.

  By using the present invention, abnormal prions can be easily and efficiently removed from blood products, and further, leukocytes can be removed simultaneously with abnormal prions. In addition, when the filter of the present invention is used, it is possible to obtain a high-quality blood product with good hemodynamics and less hemolysis.

Hereinafter, the present invention will be described in more detail.
The polymer referred to in the present invention is composed of a unit derived from a hydrophobic polymerizable monomer, a unit derived from a polymerizable monomer containing a basic nitrogen-containing moiety, and a unit derived from a polymerizable monomer containing a protonic neutral hydrophilic moiety. It is a polymer.

The unit referred to in the present invention means a minimum repeating unit derived from each polymerizable monomer of a polymer molecule. As an example of the unit, when the double bond is simply opened and addition polymerization is performed, the vinyl compound represented by CH ₂ = CXY (X: substituent other than H or H, Y: substituent other than X) the minimum repeating units of the polymerizable monomer _- (CH 2 -CXY) _- is a unit. For example, when polymer is synthesized by polycondensation, A- (R) -B (R: a portion not desorbed by polymerization, A, B: reaction and desorption by polymerization) -(R)-, which is the minimum repeating unit when AB is desorbed and polymerized from the portion), can be exemplified as the unit.

  Specific examples of the hydrophobic polymerizable monomer include styrene, methylstyrene, methyl methacrylate, methyl acrylate, ethyl methacrylate, ethyl methacrylate, butyl acrylate, butyl methacrylate, phenyl acrylate, and phenyl from the viewpoint of easy availability and handling. One acrylate or methacrylate such as methacrylate, ethylhexyl acrylate, ethylhexyl methacrylate, trichloroethyl acrylate, trichloroethyl methacrylate, alkenes such as pentene, hexene, heptene, octene, organosilicon compounds such as silicone and siloxane, one hydrogen atom of ethylene Examples of the organic fluorine-polymerizable monomer substituted with fluorine atoms can be mentioned above. It is not limited to the substance. Among them, a vinyl polymer can be obtained by addition polymerization (vinyl polymerization) for reasons such as easy availability of monomers and easy handling. Monomers in which the polymerizable moiety is a vinyl group are preferred. Among these, acrylic acid derivatives or methacrylic acid derivatives are more preferable as the hydrophobic polymerizable monomer. Most preferred as the hydrophobic polymerizable monomer is acrylate or methacrylate.

  A material having a basic nitrogen-containing functional group becomes positively charged on the surface of the material in a biological fluid, and this also shows an effect of improving the removal performance of abnormal prions. The polymerizable monomer containing a basic nitrogen-containing moiety in the present invention refers to a polymerizable monomer having a basic nitrogen-containing moiety described below. Examples of basic nitrogen-containing moieties include primary amino groups, secondary amino groups, tertiary amino groups, quaternary amino groups, and nitrogen-containing aromatics such as pyridyl groups and imidazolyl groups. it can. As the basic nitrogen-containing moiety, a tertiary amino group is particularly preferable. Specifically, as a polymerizable monomer containing a basic nitrogen-containing moiety, vinylamine, 2-vinylpyridine, 4-vinylpyridine, 2-methyl-5-vinylpyridine, 4 from the viewpoint of easy availability and handling. -Vinyl derivatives of nitrogen-containing aromatic rings such as vinylimidazole, N-vinyl-2-ethylimidazole, N-vinyl-2-methylimidazole, dimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, diethylaminoethyl acrylate, diethylaminoethyl methacrylate, Acrylates and methacrylates such as 3-dimethylamino-2-hydroxypropyl acrylate and 3-dimethylamino-2-hydroxypropyl methacrylate, N-dimethylaminoethyl acrylate, N-dimethylaminoethyl methacrylate Acrylic acid amides and methacrylic acid amide derivatives such as lauric acid amide, N-diethylaminoethylacrylic acid amide, N-diethylaminoethyl methacrylic acid amide, styrene derivatives such as p-dimethylaminomethylstyrene, p-diethylaminoethylstyrene, and Although the derivative | guide_body etc. which made the said polymerizable monomer the quaternary ammonium salt by the halogenated alkyl group are mentioned, the polymerizable monomer containing a basic nitrogen-containing part is not limited to the said substance. As the monomer containing a nitrogen-containing basic functional group, acrylate or methacrylate is more preferable. Of these, dimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, diethylaminoethyl acrylate, and diethylaminoethyl methacrylate containing a tertiary amino group are particularly preferable. Among them, a monomer in which a polymerizable moiety from which a vinyl polymer can be obtained by addition polymerization (vinyl polymerization) is a vinyl group is preferable because of easy availability of the monomer and easy handling. As the polymerizable monomer containing a basic nitrogen-containing moiety, an acrylic acid derivative or a methacrylic acid derivative is preferable. As the polymerizable monomer containing a basic nitrogen-containing moiety, acrylate or methacrylate is more preferable. Of these, dimethylaminoethyl acrylate, dimethylaminoethyl methacrylate, diethylaminoethyl acrylate, and diethylaminoethyl methacrylate are particularly preferable.

The polymerizable monomer containing a protic neutral hydrophilic portion as used in the present invention is one in which a non-polymerizable portion can be dissociated to release a proton (H ⁺ ), and as in a carboxylic acid or a basic amino group. A monomer that does not exhibit extreme acidity or basicity. A monomer having a protic neutral hydrophilic portion exhibits higher hydrophilicity than a monomer having an aprotic neutral hydrophilic portion, and is excellent in blood priming properties and blood channeling prevention properties. Examples of the protic neutral hydrophilic moiety include a hydroxyl group, an aldehyde group having a proton at the α-position, an amide group having a proton at the α-position, and a 1,3-dicarbonyl group. As the non-polymerizable protonic neutral hydrophilic portion, a hydroxyl group is particularly preferable. Examples of the monomer containing a protic neutral hydrophilic moiety include 2-hydroxyxyethyl methacrylate, hydroxypropyl methacrylate, acrylamide, methacrylamide, etc., but the monomer containing a protic neutral hydrophilic moiety is limited to the above substances. It is not something. Among them, a monomer in which a polymerizable moiety from which a vinyl polymer can be obtained by addition polymerization (vinyl polymerization) is a vinyl group is preferable because of easy availability of the monomer and easy handling. Of these, acrylic acid derivatives or methacrylic acid derivatives are preferred as the polymerizable monomer containing a protic neutral hydrophilic portion. As the polymerizable monomer containing a protic neutral hydrophilic portion, acrylate or methacrylate is most preferable. The vinyl polymer in the present invention is a vinyl polymer in a broad sense and means a polymer having a non-cyclic main chain. Specific examples thereof include α-substituted polyacrylic acid and its α-substitution shown in “J Brandrup; EHImmergut. 1989.“ Polymer Hand book Third Edition “A Willey-interscience Publication, pVII-5 to VII-18”. Derivatives, polyvinyl ether, polyvinyl alcohol, polyvinyl ester, polystyrene and derivatives thereof, and copolymers containing these.

  In order to highly remove abnormal prions in blood products containing plasma components, units derived from the above-mentioned hydrophobic polymerizable monomers, units derived from polymerizable monomers containing basic nitrogen-containing moieties, and protonic neutral hydrophilicity Polymers containing three of the units derived from polymerizable monomers containing moieties must be used. Abnormal prions cannot be removed to a high degree by a polymer composed of each single unit, and in particular, a high ability to remove abnormal prions from blood products containing plasma components cannot be obtained.

  An abnormal prion has three different binding regions that bind to a positively charged functional group, a negatively charged functional group, and a hydrophobic functional group. On the other hand, the isoelectric point of the abnormal prion is set at pH 4.6, and the pH of the blood product is between about 5 and 7.5, so that the abnormal prion in the blood product is negatively charged. Therefore, abnormal prions can be highly removed from blood products by using a monomer having basic nitrogen and a hydrophobic polymerizable monomer. In addition, it is said that the contact between a negatively charged material and a blood product causes the production of bradykinin that causes anaphylaxis such as blood pressure drop, flushing of the face, conjunctival redness, smooth muscle contraction, and pain. Is not suitable for coating the surface of a carrier for filtering blood products.

  On the other hand, when a blood product is filtered with a filter, it is required to provide a blood product with the same quality as when filtering with a current leukocyte removal filter. The protonic neutral hydrophilic part of the polymer is an essential part for ensuring wettability for blood products to spread throughout the filter, especially for smooth priming operation that fills the blood product with the filter at the beginning of filtration of the blood product. It is. Further, when the composition of the basic nitrogen-containing monomer and the hydrophobic polysynthetic monomer exceeds a certain level in the polymer, the quality deteriorates such as hemolysis or prolonged filtration time. Therefore, in order to obtain a blood product from which abnormal prions are highly removed, the composition of a hydrophobic polymerizable monomer, a polymerizable monomer containing a basic nitrogen-containing moiety, and a polymerizable monomer containing a protic neutral hydrophilic moiety is required. It is essential that it is set appropriately.

  The hydrophobic polymerizable monomer in the present invention is a polymerizable monomer having an extremely low affinity for water, and is a monomer containing neither a basic nitrogen-containing part nor a protonic neutral hydrophilic part in the molecule, It means a polymerizable monomer having a solubility in water at 20 ° C. of 12 g / 100 g or less of water. If the solubility exceeds 12 g / 100 g of water, such a high abnormal prion removing ability as obtained in the present invention tends to be not obtained, which is not preferable. As a more preferable range, the solubility is 2 g / 100 g or less of water. For the measurement of solubility, when the polymerizable monomer is a solid, a known measurement method such as a dew point method, a thermal analysis method, an electrical method for measuring the electromotive force or conductivity of a solution, a gas chromatography analysis method, a tracer method, etc. Can be measured. When the polymerizable monomer is a liquid, it can be measured by the same measurement method as when it is solid, but can also be measured by a known method such as a volumetric method, a light scattering method, a vapor pressure method or the like. Further, as a simpler method, when the boiling point of the polymerizable monomer is sufficiently higher than that of water, it can be obtained by a method of evaporating water from a saturated aqueous solution of the polymerizable monomer and measuring the weight of the remaining amount.

  In order to realize more advanced abnormal prion removal performance, and further leukocyte removal performance, the composition (molar percentage) of each of the monomers constituting the polymer is 20 mol% or more and 40 mol% or less of the hydrophobic polymerizable monomer, The composition of the polymerizable monomer containing a basic nitrogen-containing moiety is 5 mol% or more and 13 mol% or less, and the composition (mol%) of the polymerizable monomer containing a protic neutral hydrophilic moiety in the polymer is 100 mol. % Is preferably a residue obtained by subtracting the sum of the composition (mol%) of the hydrophobic polymerizable monomer in the polymer and the composition (mol%) of the polymerizable monomer containing a basic nitrogen-containing moiety in the polymer. When the composition of the hydrophobic polymerizable monomer in the polymer is less than 20 mol% or the composition of the polymerizable monomer containing a basic nitrogen-containing moiety is less than 5 mol%, there is a tendency that improvement in abnormal prion removal performance is not observed. It is not preferable. When the composition of the hydrophobic polymerizable monomer in the polymer exceeds 40 mol%, the wettability is deteriorated with respect to the blood product stored in the refrigerator, and as a result, the blood product is obtained with the filter filled with the carrier coated with the polymer of the present invention. This is not preferable because the filtration rate of the blood product tends to decrease. When the composition of the polymerizable monomer containing a basic nitrogen-containing moiety in the polymer exceeds 13 mol%, a tendency such as hemolysis is observed with respect to a blood product stored in a refrigerator, which is not preferable. In order to realize a higher leukocyte removal ability and abnormal prion removal performance, the composition (mole percentage) of each monomer constituting the polymer is 25 mol% or more and 35 mol% or less of the hydrophobic polymerizable monomer, and The composition of the polymerizable monomer containing a basic nitrogen-containing moiety is 7 mol% or more and 12 mol% or less, and the composition (mol%) of the polymerizable monomer containing a protic neutral hydrophilic moiety in the polymer is 100 mol%. It is preferable that the residue is obtained by subtracting the sum of the composition (mol%) of the hydrophobic polymerizable monomer in the polymer and the composition (mol%) of the polymerizable monomer containing a basic nitrogen-containing moiety in the polymer. The copolymerization composition of the hydrophobic polymerizable monomer in the polymer is 27 to 33 mol%, the copolymerization composition of the polymerizable monomer having a basic nitrogen-containing moiety in the polymer is 11% or less, and the remainder is the polymer Most preferred is a polymerizable monomer containing a protonic neutral hydrophilic moiety therein.

  The composition of the monomer in the polymer can be measured by a general physicochemical method. If it illustrates about the physicochemical method which measures a copolymer composition, it can measure using well-known methods, such as a nuclear magnetic resonance spectrum method (NMR, -1H, -13C), and a pyrolysis GC mass spectrum method. It is also possible to confirm that the polymerization has been carried out as prepared and that there are lot-to-lot variations. In addition, the polymer coated on the surface of the carrier is dissolved and extracted using a solvent of the polymer, and the copolymer composition of each monomer in the polymer as the extraction substance is measured by the same method as described above. You can also. Also, the copolymer and the polymer present on the surface of the carrier are dissolved in a deuterated solvent and measured by the nuclear magnetic resonance spectroscopy (NMR, -1H, -13C) method to measure the copolymer composition. Available as a method. The molecular weight of the polymer can be measured by a known gel permeation chromatography method. A preferred range of weight average molecular weight (Mw) is 50,000 to 3,000,000, more preferably 100,000 to 2,000,000, and most preferably 150,000 to 1,500,000. When the weight average molecular weight Mw is less than 50,000, it is not preferable because the polymer tends to be eluted into the blood product when abnormal prions and leukocytes are removed from the blood product. Moreover, when the weight average molecular weight Mw exceeds 3 million, there is a concern that the solubility in a solvent during coating is reduced, and there is a concern that it cannot be stably produced during polymerization. The polymer may be either a random copolymer or a block copolymer. However, the block copolymer has a tendency to decrease the solubility in a solvent during coating, and a uniform coating such as micelle formation in the solution. Random copolymers are preferred because they tend to impair the properties. Either a straight-chain polymer or a branched polymer may be used, but the branched polymer chain tends to decrease the solubility in a solvent during coating, and the coating uniformity is impaired due to micelle formation in the solution. A linear polymer chain is more preferable. A general polymerization method may be used as a polymer synthesis method. Addition polymerization (vinyl polymerization) or the like that is a chain reaction may be used, and isomerization polymerization, elimination reaction that is a sequential reaction, polyaddition, polycondensation, addition polycondensation, or the like may be used. In polymerizing the polymer, radicals, ions, or the like may be used as the chain carrier. Examples of the polymerization method include solution polymerization, bulk polymerization, precipitation polymerization, and emulsion polymerization. Among these, solution polymerization is preferable.

The polymerization method is illustrated below.
Ethanol is used as a polymerization solvent, and an ethanol solution in which each monomer and diazo initiator are dissolved is dropped and reacted in a nitrogen atmosphere while stirring at a constant temperature below the boiling point of ethanol. Moreover, you may add a stabilizer etc. suitably. The reaction yield is measured and confirmed by a known method such as gas chromatography. Purify by using a general chemical purification method to remove impurities such as low molecular weight components and unreacted substances that are likely to be dissolved during the processing of pharmaceuticals existing in the polymer or in the reaction liquid containing the polymer. Can do. Illustrative purification methods are: a method in which impurities are dissolved and a polymer is not dissolved, poured into a solvent and precipitated, and the precipitate is separated by filtration, decantation, etc. If necessary, a solvent having a slightly higher solubility than the precipitation solvent (precipitation) There can be mentioned a method in which the precipitate is washed with a solvent and a solvent mixture, etc., and dried under reduced pressure until the precipitate has a constant weight, and is obtained as a solid polymer.

  The carrier of the present invention is not particularly limited as long as it has a pore capable of filtering blood, and includes any carrier form. Specific examples include natural fibers, glass fibers, and porous beads. Particularly preferred are fibrous media such as knitted fabrics, woven fabrics and nonwoven fabrics, porous membranes, and sponge-like structures having three-dimensional network-like continuous pores. There are no particular limitations as long as they do not easily damage blood cells, and various materials can be used, and examples include organic polymer materials, inorganic polymer materials, and metals. Among them, organic polymer materials are preferable carriers because they are excellent in processability such as cutting. For example, in porous materials, polyester, polyolefin, polyacrylonitrile, polyamide, polystyrene, polymethyl methacrylate, polyvinyl fluoride, polyurethane, polyvinyl alcohol, polyvinyl acetal, polysulfone, polyvinylidene fluoride, polytrifluorochlorovinyl, fluoride Vinylidene-tetrafluoroethylene copolymer, polyethersulfone, polyacrylate, butadiene-acrylonitrile copolymer, polyether-polyamide block copolymer, ethylene-vinyl alcohol copolymer, cellulose, cellulose acetate, and the like. The carrier of the present invention is not limited to the above examples. Polyester and polyolefin are preferable, and polyester is particularly preferable.

The carrier coated with a polymer in the present invention refers to a carrier having a polymer fixed on the surface of the carrier so that the polymer does not easily elute during the preparation process. The method of fixing the polymer to the support surface may be a chemical covalent bond or a physicochemical non-covalent bond. The abundance is preferably 0.6 mg / m ² or more and 83 mg / m ² . When the abundance is less than 0.6 mg / m ² per unit area of the total surface area of the carrier, abnormal prion removal ability and further leukocyte removal ability tend to decrease, and when it exceeds 83 mg / m ² , it depends on coating unevenness. This is not preferable because performance variation tends to increase. More preferably, the abundance is 5 mg / m ² or more and 50 mg / m ² or less per total surface area of the support, and even more preferably the abundance is 10 mg / m ² or more and 40 mg / m ² or less per total area of the support. Furthermore, the amount of the polymer present on the surface of the carrier can be measured by a general physicochemical method. If the method of measuring the amount of the polymer on the surface of the carrier is exemplified, both the coated carrier and the polymer present on the surface are dissolved in a deuterated solvent, and nuclear magnetic resonance (NMR, -1H, -13C) The method of measuring by this method can be illustrated. In the present invention, a carrier coated with a polymer is sometimes referred to as a coated carrier.

  In the present invention, the method of coating the carrier with the polymer is, for example, a method in which each polymerizable monomer or polymer described above is fixed to the carrier by a chemical covalent bond (such as a graft), Examples thereof include known methods such as bonding (fan bonding, der Waals force, etc.) (such as coating) and embedding. More specifically, a method in which each polymerizable monomer or polymer is directly grafted onto the surface of the carrier by a graft polymerization method such as radiation grafting or plasma grafting, or a carrier obtained by impregnating a carrier in a solution in which the polymer is dissolved or coating the polymer on a roll. The method of coating on the surface by transferring the film to the surface is more preferable because it can be produced relatively easily and is stable and excellent in performance. The method of coating the carrier of the polymer of the present invention is not particularly limited as long as the pores of the carrier are not significantly blocked and the carrier surface can be uniformly coated within a certain range, and various methods can be used. it can. For example, a method of impregnating a carrier with a solution in which a polymer is dissolved, a method of spraying a solution in which a polymer is dissolved on a carrier, a method of applying and transferring a solution in which a polymer is dissolved to a carrier using a gravure roll, etc. The method of coating the polymer on the carrier of the present invention is not limited to the above examples. Among them, the method of impregnating the carrier with a solution in which the polymer is dissolved and squeezing the coated carrier after impregnation, and the method of applying and transferring the solution in which the polymer is dissolved to the carrier using a gravure roll or the like are excellent in continuous productivity and low in cost. This is a more preferable method.

  As the solvent for dissolving the polymer, various solvents can be used without any particular limitation as long as the carrier is not significantly dissolved. Aqueous solutions containing water and inorganic salts, alcohols such as methanol, ethanol, propanol and butanol, acetone , Ketones such as methyl ethyl ketone, esters such as methyl acetate and ethyl acetate, hydrocarbons such as benzene and cyclohexane, halogenated hydrocarbons such as chloroform and dichloromethane, sulfur-containing solvents such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide And the like, and a mixture of the above-mentioned plurality of solvents in a soluble range, but the solvent for dissolving the polymer of the present invention is not limited to the above examples.

  In addition, as drying after coating, a method such as mechanical compression, removal of excess solution by blowing a gas such as air or nitrogen, and a method such as normal temperature or heating in a dry gas or under reduced pressure can be used. . Further, before coating, in order to further improve the adhesion between the polymer of the present invention and the carrier, the surface of the carrier can be treated with an appropriate chemical such as acid or alkali, or can be irradiated with plasma. Furthermore, the adhesion between the carrier and the polymer can be further strengthened by applying a heat treatment after the coating of the polymer, or a post-processing by irradiating radiation such as γ rays or electron beams. The coating may be performed when the carrier is manufactured or after the carrier is manufactured.

The physical structure of the coat carrier is known to greatly contribute to the removal of abnormal prions and leukocytes, and the selection of the coat carrier is an important factor for improving the ability to remove abnormal prions and leukocytes.
In order to improve the removal performance of abnormal prions, the carrier comprises at least one porous body layer and at least one porous bead layer. The porous body as used in the present invention refers to a fibrous medium such as natural fiber, glass fiber, knitted fabric, woven fabric, and nonwoven fabric, a porous film, and a sponge-like structure having a three-dimensional network-like continuous hole.

As the physical structure of the coated porous body, the specific surface area is 1.0 m ² / g or more and 5.0 m ² / g or less, preferably 1.1 m ² / g or more and 3.0 m ² / g or less, more preferably 1 .3m is preferably ² / g or more 2.0 m ² / g or less. When the specific surface area of the coated porous body is less than 1.0 m ² / g, it is difficult to remove leukocytes at a high rate. If the specific surface area of the coated porous body is 5.0 m ² / g or more, the coated carrier can no longer be produced stably.
On the other hand, the specific surface area of the porous beads is 5.0 m ² / g or more, preferably 10 m ² / g or more. This value is preferably as high as possible, and can be used up to about 1000 m ² / g. If the specific surface area of the porous beads is not 5.0 m ² / g or more, good prion removal performance tends not to be obtained.

  In addition, any material can be used as the porous beads according to the present invention as long as they are insoluble in water. However, preferable materials in terms of low moldability and cytotoxicity are listed as polyester, nylon, polystyrene, fluororesin, acrylic resin. Synthetic polymer compounds such as polysulfone, polyurethane and polycarbonate, cellulose and its derivatives, natural polymer compounds such as chitin, dextran and agarose, ceramics such as alumina, hydroxyapatite, titania and silica, and inorganic compounds such as glass It is done.

  The particle diameter of the porous beads of the present invention is 20 μm to 5 mm, more preferably 20 μm to 1 mm. If it is less than 20 μm, it is difficult to form pores (5 μm or more) into which the cells enter, and if it exceeds 5 mm, it becomes difficult for the cells to enter the pores. The pore size of the porous beads according to the present invention is 5 to 500 μm, more preferably 5 to 100 μm. If it is less than 5 μm, the cells hardly enter the pores, and if the pore diameter exceeds 500 μm, the matrix portion becomes too thin, and the beads tend to be broken.

Furthermore, the exclusion limit molecular weight of the porous beads of the present invention is in the range of 1 × 10 ^{3 to} 1 × 10 ⁸ . The exclusion limit molecular weight is a value indicating the lower limit of the molecular weight of a molecule that cannot penetrate into the porous beads. In the case of coating a porous bead with a polymer, the exclusion limit molecular weight can be measured by a known method using a standard substance having a known molecular weight after the porous bead is filled in a container such as a column. The exclusion limit molecular weight of the porous beads tends to be unfavorable for the adsorption of abnormal prions if it is less than 1 × 10 ³ , and it tends to be difficult to produce those exceeding 1 × 10 ⁸ .

  As a physical structure of the coated porous body, the porosity is preferably 65% or more and 90% or less, and more preferably 75% or more and 88% or less. If the porosity is less than 65%, the filtration rate of blood or the like is lowered, and it takes a long time to remove abnormal prions and leukocytes. Further, when the porosity is 90% or more, there are few entangled portions of fibers and fibers where abnormal prions and leukocytes are likely to adhere, and high abnormal prion removal performance and leukocyte removal performance cannot be obtained. Further, when a fibrous medium such as a nonwoven fabric is used as a coated porous body, the average fiber diameter is 0.3 μm or more and 3.0 μm or less, preferably 0.5 μm or more and 2.5 μm or less, more preferably 1 μm or more and 2 μm or less. Is preferred. In preparation treatment with an actual blood filter, it is preferable to dispose a plurality of coated carriers having an average fiber diameter so as to become finer toward the outlet side. In the preparation treatment using an actual blood filter, a carrier having an average fiber diameter of 10 μm or more and 40 μm or less, which is mainly used for removing microaggregates, may be disposed on the inlet side of the coated carrier as necessary.

The average pore diameter is a value (mean flow pore size: MFP) measured using a Coulter R porometer manufactured by Coulter Electronics, for example, and a sample of about 50 mg. The average pore diameter is 1 μm or more and 60 μm or less, preferably 1 μm or more and 30 μm or less, more preferably 1 μm or more and 20 μm or less. If the average pore size is less than 1 μm, the whole blood product is difficult to flow, and if it exceeds 60 μm, the leukocyte removal ability tends to decrease, such being undesirable. In preparation treatment with an actual blood filter, it is preferable to dispose a plurality of coated carriers having an average pore size so as to become smaller toward the outlet side. In the preparation treatment with an actual blood filter, a carrier having an average pore diameter of 50 μm or more and 200 μm or less, which is mainly used for the purpose of removing microaggregates, may be arranged on the inlet side of the filter material as necessary.
Further, in the preparation treatment with an actual blood filter, a porous body having an average pore diameter of 50 μm or more and 200 μm or less, which is mainly intended to prevent drifting, may be disposed on the outlet side of the coat carrier as necessary.

The filling density when the fibrous medium is filled in a container for removing abnormal prions and leukocytes is 0.1 g / cm ³ or more and 0.5 g / cm ³ or less, more preferably 0.1 g / cm ^3. It is preferable that it is 0.3 g / cm ³ or more. An example of a method for measuring the packing density will be described. The non-woven fabric to be filled can be cut into a filling cut size (cm ² ), its weight (g) can be measured, and it can be determined from the distance (cm) in the compressed state in the actual container.
When the average fiber diameter is less than 0.3 μm, the average pore diameter is less than 1 μm, or the packing density exceeds 0.5 g / cm ³ , clogging of blood cells and increase in pressure loss are caused, and the average fiber diameter exceeds 3.0 μm. If the average pore diameter exceeds 60 μm, or the packing density is less than 0.1 g / cm ³ , the ability to remove abnormal prions and leukocytes decreases, which is not preferable.
When a porous film or a sponge-like structure having three-dimensional network continuous pores is used as a coating carrier, it preferably has an average pore diameter of 1 μm or more and 60 μm or less. 5 μm or more and 50 μm or less is more preferable. More preferably, it is 10 μm or more and 40 μm or less. If the average pore diameter is less than 1 μm, clogging of blood cells and pressure loss are caused, and if the average pore diameter exceeds 60 μm, the ability to remove leukocytes is not preferable.

The abnormal prion in the present invention means a mutant prion protein known to cause a disease called spongiform encephalopathy in humans and animals. Abnormal prions are proteins having the same amino acid sequence as normal prions but having a changed structure rich in β-sheet, and are resistant to digestion by protease K. Abnormal prions infect animals and are widely known for scrapie, an infectious degenerative disease of the sheep and goat nervous system, BSE in cattle. The forms of human disease include sporadic Creutzfeldt-Jakob disease (sCJD), iatrogenic Creutzfeldt-Jakob disease, Gerstmann-Straeussler-Scheinker (GSS) syndrome, lethal familial isomnia (FFI) ), Kuru and there are mutant CJDs known to be infected by BSE meat. The prions used in the present invention include all or any one of the above diseases, or all types of abnormal prion proteins that cause the disease in any animal, particularly humans and livestock animals. By the way, 2006 was held in March ^{Cambridge Healthtech Institut's10 th Annual Transmissible Spongiform Encephalopathies-} The Definitive American TSE Meeting - of According to the announcement of Samuel Coker, scrapie, variant CJD and sporadic CJD origin of the abnormal prions The filter (LAPRF made by Pall) was removed in the same manner, and there was almost no difference in the removal rate. This indicates that the abnormal prion protein structure related to filter removal performance is almost the same regardless of the type of animal or disease, and the removal rate of human-derived prions can be estimated from the removal rate of abnormal prions derived from animals. Show.

  The filter in the present invention is a container having a blood product inlet and outlet filled with the coated carrier of the present invention, and there may be a carrier for capturing microaggregates upstream of the blood product. There may be a carrier on the downstream side.

  The material of the container may be either a hard resin or a soft resin. In the case of a hard resin, examples of the material include phenol resin, acrylic resin, epoxy resin, formaldehyde resin, urea resin, silicon resin, ABS resin, nylon, hard polyurethane, polycarbonate, vinyl chloride, polyethylene, polypropylene, polyester, and the like. In the case of a soft resin, a blood product inlet and outlet formed on a flexible synthetic resin sheet and welded at the periphery, a cylindrical molded product having a blood product inlet and outlet, or the like is used. . In the case where the material is welded to the container together with the coat carrier, the thermal and electrical properties of the coat carrier are preferably similar. For example, soft polyvinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, Polyolefins such as polyethylene and polypropylene, hydrogenated products of styrene-butadiene-styrene copolymers, thermoplastic elastomers such as styrene-isoprene-styrene copolymers or hydrogenated products thereof, and thermoplastic elastomers and polyolefins, ethylene- A suitable material is a mixture with a softening agent such as ethyl acrylate. Among these, soft vinyl chloride, polyurethane, ethylene-vinyl acetate copolymer, polyolefin, and thermoplastic elastomers containing these as main components are particularly preferred, and soft vinyl chloride and polyolefin are particularly preferable.

  The shape of the container is not particularly limited as long as it has a blood product inlet and an abnormal prion, and further has a blood product outlet from which leukocytes have been removed. However, the shape is preferably in accordance with the shape of the carrier. For example, when the carrier has a flat plate shape, a flat container made of a polygon such as a quadrangle or a hexagon, or a curve such as a circle or an ellipse can be used. More specifically, the container is composed of an inlet side container having a blood product inlet and an outlet side container having a blood product outlet, both of which divide the inside of the carrier into two chambers by sandwiching the carrier directly or via a support, The shape which forms a flat filter can be illustrated. As another example, when the carrier is cylindrical, the container is preferably cylindrical as well. More specifically, the container is composed of a cylindrical body portion for containing a carrier, an inlet header having a blood product inlet, and an outlet header having a blood product outlet, and the inside of the container is introduced from the inlet by potting. A shape that forms a cylindrical filter divided into two chambers so that the liquid flows from the outer peripheral portion of the cylindrical carrier to the inner peripheral portion (or from the inner peripheral portion to the outer peripheral portion) can be exemplified.

  The blood product of the present invention is a whole blood or a liquid composed of one or more kinds of blood components prepared from whole blood, which may contain abnormal prions, or an anticoagulant or storage in these liquids. This is a general term for liquids to which liquids and the like are added. Specific examples include, but are not limited to, whole blood preparations, red blood cell preparations, platelet preparations, plasma preparations, washed red blood cell suspensions, thawed red blood cell concentrates, synthetic blood, platelet rich plasma, buffy coats, etc. No.

  The whole blood preparation of the present invention refers to Citrate Phosphate Dextrose (CPD), Citrate Phosphate Dextrose Adenine-1 (CPDA-1), Citrate Phosphate-2-Dextrose (CP2D), Acid Citrate Dextrose (CP2D). It is a blood product to which preservatives such as Formula-A (ACD-A), Acid Citrate Dextrose Formula-B (ACD-B), heparin, and an anticoagulant are added.

  Next, a method for removing abnormal prions from the blood product of the present invention will be described focusing on the procedure. First, one embodiment of a method for preparing various blood products will be described, but the present invention is not limited thereto.

(Preparation of abnormal prion-removed whole blood product)
Preserved solutions such as CPD, CPDA-1, CP2D, ACD-A, ACD-B, and heparin, and anticoagulants are added to the collected whole blood, and abnormal prions are removed from the whole blood by filtering with the filter of the present invention. Remove the abnormal prion-free whole blood product.
The filtration of the whole blood product can be performed using a system in which at least a bag containing the blood product, a filter, and a bag for collecting the blood product from which the abnormal prion has been removed are connected aseptically by a circuit in this order. A blood collection needle, a blood collection bag, a bag for separating each component after centrifugation, or a circuit for collecting blood products remaining in the filter may be connected. Filtration may be performed by the blood product flowing from the bag containing the blood product placed higher than the filter to the filter through a conduit by gravity drop, or by using a means such as a pump. The abnormal prion may be removed by flowing the blood product under pressure from the inlet side of the filter and / or depressurizing from the outlet side of the filter. This filtration method is not limited to whole blood products, but is the same for other blood products.
In the preparation of abnormal prion whole blood preparations, when removing abnormal prions before storage, whole blood stored preferably at room temperature or refrigerated is preferably within 72 hours after collection, more preferably within 24 hours, particularly preferably. The abnormal prion-removed whole blood product is obtained by removing abnormal prions using the filter of the present invention within 12 hours, most preferably within 8 hours at room temperature or under refrigeration. When removing abnormal prions after storage, an abnormal prion-removed whole blood product is obtained by removing abnormal prions from whole blood stored at room temperature or refrigerated, preferably using a filter within 24 hours before use. . In the present invention, it is also possible to obtain a whole blood preparation from which leukocytes are removed together with abnormal prions.

(Preparation of abnormal prion-removed erythrocyte preparation)
A preservation solution such as CPD, CPDA-1, CP2D, ACD-A, ACD-B, and heparin and an anticoagulant are added to the collected whole blood. The subsequent separation into blood components is carried out by centrifuging after removing abnormal prions from whole blood, or by separating red blood cells or red blood cells and buffy coat (hereinafter referred to as “BC”) after centrifuging whole blood. ) May remove abnormal prions.
When performing centrifugation after removing abnormal prions from whole blood, after obtaining abnormal prion-removed whole blood in the same manner as in the preparation of the abnormal prion-removed whole blood preparation, the abnormal prion-removed whole blood is centrifuged and concentrated in the lower layer. By collecting red blood cells, an abnormal prion-removed red blood cell preparation is obtained.
When centrifuging whole blood before removing abnormal prions, the centrifugation conditions are weak centrifugation conditions that separate into red blood cells and platelet-rich plasma (PRP), and strong centrifugation that separates into red blood cells, BC, and platelet poor plasma (PPP). There are two types of conditions. Red blood cells separated and collected from whole blood as needed, or red blood cells containing BC, Saline Adenine Glucose Mannitol solution (SAGM), Additive solution-1 (AS-1), Additive solution-3 (AS-3), Additive After adding a preservative solution such as solution-5 (AS-5) or Mannitol Adenine Phosphate (MAP), the red blood cell preparation is filtered with the filter of the present invention to obtain an abnormal prion-removed red blood cell preparation.

  In preparation of abnormal prion-removed erythrocyte preparation, whole blood stored preferably at room temperature or under refrigeration is preferably within 72 hours after blood collection, more preferably within 48 hours, particularly preferably within 24 hours, most preferably within 12 hours. Centrifuge. Also, when removing abnormal prions before storage, it is preferably within 120 hours after blood collection, more preferably within 72 hours, particularly preferably within 24 hours, most preferably from a red blood cell preparation stored at room temperature or under refrigeration. Abnormal prion-removed erythrocyte preparation is obtained by removing abnormal prions by filtering with a filter at room temperature or under refrigeration within 12 hours. When removing abnormal prions after storage, preferably remove abnormal prions from the red blood cell preparation stored at room temperature or under refrigeration by filtering with an abnormal prion removal filter within 24 hours before use. obtain.

(Preparation of abnormal prion-removed platelet preparation)
A preservation solution such as CPD, CPDA-1, CP2D, ACD-A, ACD-B, and heparin and an anticoagulant are added to the collected whole blood.
The subsequent separation into blood components may be performed after centrifugation after removing abnormal prions from whole blood, or after removing abnormal prions from PRP or platelets after centrifugation of whole blood.
When centrifugation is performed after removing abnormal prions from whole blood, the abnormal prion-removed whole blood is obtained in the same manner as in the preparation of the abnormal prion-removed whole blood preparation, and then the abnormal prion-removed whole blood is centrifuged to obtain an upper layer PRP. Is collected, and further centrifuged to collect the concentrated platelet (PC) in the lower layer, thereby obtaining an abnormal prion-removed platelet preparation.
When whole blood is centrifuged before removing abnormal prions, there are two types of centrifugation conditions: weak centrifugation conditions for separating red blood cells and PRP, and strong centrifugation conditions for separating red blood cells, BC and PPP. In the case of weak centrifugal conditions, PRP separated from whole blood is filtered through a filter to remove abnormal prions, and then centrifuged again to collect the lower layer PC to obtain an abnormal prion-removed platelet preparation, or PRP After centrifugation to obtain platelets and PPP, the lower layer PC is filtered through a filter to remove abnormal prions, and an abnormal prion-removed platelet preparation is obtained. In the case of strong centrifugation conditions, by adding a preservative solution, plasma, etc. as necessary to a pool of BC separated from whole blood or a unit of several to several tens of units, and performing centrifugation, and collecting the upper layer The obtained concentrated platelets are filtered through a filter to remove abnormal prions, thereby obtaining an abnormal prion-removed platelet preparation.

  In preparing an abnormal prion-removed platelet preparation, preferably whole blood stored at room temperature is centrifuged within 24 hours, more preferably within 12 hours, and particularly preferably within 8 hours after blood collection. When removing abnormal prions, a platelet preparation stored at room temperature is preferably within 120 hours after blood collection, more preferably within 78 hours, particularly preferably within 24 hours, and most preferably within 12 hours at room temperature. The abnormal prion-removed platelet preparation is obtained by removing the abnormal prion by filtering with a filter. When removing abnormal prions after storage, an abnormal prion-removed platelet preparation is preferably obtained by removing abnormal prions from a platelet preparation stored at room temperature or refrigerated with a filter within 24 hours before use.

(Preparation of abnormal prion-removed plasma preparation)
A preservation solution such as CPD, CPDA-1, CP2D, ACD-A, ACD-B, and heparin and an anticoagulant are added to the collected whole blood.
Subsequent methods for separating each blood component include a case where centrifugation is performed after removing abnormal prions from whole blood, and a case where abnormal prions are removed from PPP or PRP after centrifugation of whole blood.
When centrifugation is performed after removing abnormal prions from whole blood, the abnormal prion-removed whole blood is obtained in the same manner as in the preparation of the abnormal prion-removed whole blood preparation. Is collected to obtain a leukocyte-free plasma preparation.
When whole blood is centrifuged before removal of abnormal prions, there are two types of centrifugation conditions: weak centrifugation conditions for separating red blood cells and PRP, and strong centrifugation conditions for separating red blood cells, BC and PPP. In the case of weak centrifugation conditions, PRP is filtered through a filter to remove abnormal prions, and then filtered again, and then centrifuged again to collect the supernatant PPP to obtain an abnormal prion-removed plasma preparation, or by centrifugation. After separating PRP into PPP and platelets, the abnormal prion-removed plasma preparation is obtained by filtering the PPP with a filter to remove the abnormal prion. In the case of strong centrifugal conditions, the abnormal prion-removed plasma preparation is obtained by removing abnormal prions by filtering PPP through a filter.

  In the preparation of abnormal prion-removed plasma preparations, whole blood stored preferably at room temperature or refrigerated is preferably within 72 hours after blood collection, more preferably within 48 hours, particularly preferably within 24 hours, most preferably within 12 hours. Centrifuge. In addition, it is preferably within 120 hours after blood collection from a plasma preparation stored at room temperature or refrigerated, more preferably within 72 hours, particularly preferably within 24 hours, most preferably within 12 hours at room temperature or refrigerated. The abnormal prion-removed plasma preparation is obtained by removing the abnormal prion by filtering through a filter. When removing abnormal prions after storage, preferably the abnormal prions are removed by removing the abnormal prions from the plasma preparation stored at room temperature, refrigerated or frozen within 24 hours before use. Get.

The leukocyte removal filter in the present invention is a filter that can remove 99% or more of the white blood cell count by filtering the blood product. The filter is preferably 99.9% or more, more preferably 99.99% or more. In other words, the leukocyte removal performance refers to a filter that exhibits a removal performance of 2 or more with a value calculated according to the following equation (1). The filter preferably exhibits a removal performance of 3 or more, more preferably 4 or more.
Leukocyte removal performance = -Log ₁₀ {[White blood cell concentration (cells / μL) (blood after filtration)]
÷ [White blood cell concentration (pieces / μL) (blood before filtration)]} (1)

  As a filter sterilization method in the present invention, any of ethylene oxide gas sterilization, radiation sterilization such as γ-ray sterilization or electron beam sterilization, and high-pressure steam sterilization can be used. More preferred is radiation sterilization or autoclaving. In order to obtain a high level of abnormal prion removal performance from a whole blood product, it is preferable to perform both radiation sterilization and autoclaving. The order of performing radiation sterilization and autoclaving is not particularly limited, but it is more preferable to perform autoclaving after radiation sterilization. When a polymer-coated carrier that has been subjected to autoclaving is used, the amount of abnormal prions adsorbed on the polymer-coated carrier may be reduced in whole blood preparations. This is presumed to be because the chemical properties of the polymer on the surface of the carrier do not change during autoclaving and competitive adsorption with proteins occurs in the whole blood product. On the other hand, high abnormal prion removal performance can be obtained even with whole blood preparations by radiation sterilization. The reason for this is not clear, but because the chemical properties of the polymer on the surface of the carrier change due to radiation sterilization of the filter, the charge state and composition balance of the three units that make up the polymer become favorable for adsorption with abnormal prions. It is estimated to be. Surprisingly, however, the present inventors have found that, after radiation sterilization, even if high-pressure steam sterilization is performed, a high level of abnormal prion removal performance can be obtained. When autoclave sterilization is performed after radiation sterilization, the adsorption specificity with the abnormal prion is maintained, and it is estimated that high abnormal prion removal performance can be obtained despite autoclaving. In the case of using an in-line type set, high abnormal prion removal performance can be obtained even if high-pressure steam sterilization is performed by preliminarily sterilizing only the filter.

[Example]
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to this.
Various numerical values used in Examples and Comparative Examples were measured by the following methods.

(Specific surface area of filter material)
Specific surface area (m ² / g) as used in the present invention is a non-woven fabric unit determined by gas adsorption method (BET method) using Shimadzu Corporation “Acousobe 2100” or an apparatus having equivalent specifications. It is the surface area per weight. A carrier weighed in the range of 0.50 g to 0.55 g is filled in a sample tube, and after degassing with the above-mentioned accusorb body at a reduced pressure (room temperature) of 1 × 10 ⁻⁴ mmHg for 20 hours, as an adsorbed gas Using a krypton gas whose adsorption occupation area is known, it is adsorbed on the surface of the nonwoven fabric under the temperature of liquid nitrogen, and the total surface area in the nonwoven fabric weighed is obtained from the amount of adsorption, and divided by the weight of the weighed nonwoven fabric.

(Measurement of average fiber diameter)
Electron micrographs of the non-woven fabric were randomly taken at about five locations per non-woven fabric, and the transparent sheet on which the lattice was drawn was superimposed on the photograph taken above, and the intersection of the lattice overlapped with polystyrene latex with a known diameter as a control, The diameters of 100 fibers were measured, and the average was calculated as the average fiber diameter.

(Amount of polymer retained per unit area of the entire surface of the material)
The material total surface area (m ² ) referred to in the present invention refers to a value obtained from the product of the material weight (g) and the material specific surface area (m ² / g). The polymeric retention amount of unit area (m ²⁾ per Material entire surface in the present invention (mg / m ²⁾ can be prepared by dissolving the materials of certain area (weight) carrier, the coating agent common deuterated solvents NMR measurements Determined by As a specific example, a certain weight of a material obtained by coating a polyester nonwoven fabric with a polymer composed of methyl methacrylate, dimethylaminoethyl methacrylate, and 2-hydroxy methacrylate is added to deuterated 1,1,1,3,3,3-hexa. Intensity ratio between signals dissolved in fluoro-2-propanol and clearly belonging to the nonwoven fabric (for example, protons on the benzene ring) and signals clearly belonging to the coating agent (for example, protons on the methyl group adjacent to the ester of methyl methacrylate) From the copolymer composition of the coating agent measured separately, the amount of polymer retained per unit weight of the nonwoven fabric was determined. Further, the amount of polymer retained per unit weight of the nonwoven fabric can be converted from the specific surface area of the material filled in the filter into the amount of polymer retained per unit area of the entire surface of the material.

(Blood filtration test)
The blood product used for blood evaluation is an erythrocyte product, and 14 mL of a CPD solution as an anticoagulant is added to 100 mL of blood immediately after blood collection, and the mixture is stored in a refrigerator for 72 hours, and then centrifuged to separate the separated BC. SAGM was added to the contained red blood cells and allowed to stand for 1 hour (hereinafter referred to as “pre-filtration blood”).
The container is filled with a polyester nonwoven fabric C having an average fiber diameter of 1.2 μm, a basis weight of 40 g / m ² , a specific surface area of 1.47 m ² / g, and a pore diameter of 5 μm coated with the polymer prepared in each example and comparative example ( Comparative Example 6 has no coating). After filling 8 coated non-woven fabrics into a column with an effective filtration area of 1.3 cm ² and connecting the syringe filled with blood before filtration and the column inlet with a tube made of vinyl chloride with an inner diameter of 3 mm and an outer diameter of 4.2 mm. Then, 3 mL was collected by flowing through the column at a flow rate of 0.175 mL / min with a syringe pump in the refrigerator (hereinafter referred to as “blood after filtration”). The coated nonwoven fabric (Comparative Example 6 has no coating) was subjected to 25 KGy γ-ray sterilization in Examples 1 to 5 and 11 and Comparative Examples 1 to 9, and Examples 8 and 12 were subjected to 30 KGy electron beam sterilization. In Examples 6, 7, 10, and 13, high-pressure steam sterilization was performed at 115 ° C. for 59 minutes, and in Example 9, high-pressure steam sterilization was performed at 115 ° C. for 59 minutes after electron beam sterilization of 30 KGy.

[Leukocyte removal performance]
The leukocyte removal performance was determined from the leukocyte concentration before and after filtration according to the formula (1). The leukocyte concentration was measured by sampling 100 μL of each blood and using a Leucocount kit with porous beads (Nippon Becton, Dickinson) by flow cytometry (device: FACSCalibur, manufactured by BECTON DICKSON).
[Blood processing pressure]
The blood treatment pressure was measured at the end of filtration by connecting a pressure gauge to the tube on the column inlet side.
[Hemolysis degree after filtration]
The degree of hemolysis after 42 days of the blood after filtration was determined from the amount of free hemoglobin in plasma relative to the total amount of hemoglobin in the erythrocyte preparation after filtration.
A specific method for calculating the degree of hemolysis is as follows.
(1) The total hemoglobin concentration (g / dL) and hematocrit value (%) of the erythrocyte preparation after filtration are measured with an automatic blood cell counter.
(2) Collect 2 mL of the erythrocyte preparation sample after filtration, and centrifuge it at 1,750 × g for 10 minutes.
(3) The absorbance in the wavelength range of 630 nm to 500 nm of the supernatant is measured by scanning with a spectrophotometer.
(4) The free hemoglobin concentration in plasma is calculated according to the method of Clin. Biochem. 8, 96-102 (1975).
(5) The degree of hemolysis is calculated from the following equation (2).
Hemolysis (%) = (100−hematocrit value (%))
× (Free hemoglobin concentration (g / dL)) / Total hemoglobin concentration (g / dL) (2)
(Evaluation of prion removal performance: Examples 1 to 13, Comparative Examples 1, 3, 5 to 8, 10)

[Create filter]
As a filter medium, an average fiber diameter of 12 μm, a basis weight of 30 g / m ² , a specific surface area of 0.24 m ² / g, a polyester nonwoven fabric P having a pore diameter of 35 μm, an average fiber diameter of 2.5 μm, a basis weight of 60 g / m ² , and a specific surface area of 0.8 m ² / G, polyester nonwoven fabric A with a pore diameter of 8 μm, average fiber diameter of 1.8 μm, basis weight of 60 g / m ² , specific surface area of 1.1 m ² / g, polyester nonwoven fabric B with a pore diameter of 8 μm, prepared in each example and comparative example. Polyester non-woven fabric C having an average fiber diameter of 1.2 μm coated with a polymer and a basis weight of 40 g / m ^{2 and a} specific surface area of 1.47 m ² / g, and porous beads D coated with the polymer prepared in each example and comparative example are used. (Comparative Example 6 has no coating.)
The porous beads are composed of 100 g of vinyl acetate, 24.1 g of triallyl isocyanate, 124 g of ethyl acetate, 124 g of heptane, 3.1 g of polyvinyl acetate (degree of polymerization 500) and 3.1 g of 2,2′-azobisbutyronitrile. A homogeneous mixture and 400 ml of water in which 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate and 1.5% by weight of disodium hydrogen phosphate dodecahydrate are dissolved are placed in a flask. After sufficiently stirring, suspension polymerization was carried out by heating and stirring at 58 ° C. for 18 hours and further at 80 ° C. for 5 hours to obtain a spherical copolymer. After washing with filtered water and extraction with acetone, the copolymer was transesterified in a solution consisting of 46.5 g of caustic soda and 2000 ml of methanol at 50 ° C. for 18 hours. The average particle diameter of the obtained porous beads was 180 μm, the specific surface area was 22.5 m ² / g, and the exclusion limit molecular weight was about 2 million.

The filter media P, A, B, C, and D are stacked in order of P-A-B-C-D in order from the upstream, and B and the filter media B ′, and A and the filter media A ′ and P are further downstream. And P ′ of the same filter medium were overlapped to produce a filter medium having a structure of P-A-B-C-D-B′-A′-P ′ (Examples 1 to 13 and Comparative Examples 1 to 9) as a whole. The filter medium is sandwiched between flexible vinyl chloride resin sheets having ports that serve as blood inlets or outlets, and the periphery of the filter medium and the flexible sheet is welded and integrated using a high-frequency welding machine, so that the effective filtration area is 56 cm ^2. Created a filter.
In Examples 1 to 5 and 11 and Comparative Examples 1 to 9, 25 KGy of γ-ray sterilization was performed, in Examples 8 and 12, 30 KGy of electron beam sterilization was performed, and in Examples 6, 7, 10 and 13, 115 ° C. In Example 9, high-pressure steam sterilization was carried out for 59 minutes at 115 ° C. after electron beam sterilization of 30 KGy.

[Preparation of homogenate]
The hamster brain was ultrasonically disrupted in 320 mM sucrose to prepare a homogenate of 10 weight (g) / volume (100 ml)% (hereinafter referred to as “W / V%”). The homogenate was further centrifuged at 4 ° C., 80 × g for 1 minute, and used as a homogenate to be added (hereinafter abbreviated as “homogenate”).

[Preparation of microsomal fraction]
The homogenate was centrifuged at 5000 × g for 20 minutes, and the supernatant was further centrifuged at 100,000 × g for 1 hour. The precipitate after centrifugation was resuspended in a solution of 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, and further centrifuged at 100,000 × g for 1 hour. The precipitate after centrifugation was resuspended in a solution of 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, and further centrifuged at 100,000 × g for 1 hour.

[Normal prion removal performance test] A leukocyte-removed erythrocyte preparation prepared according to European standards was purchased and used. After storing in a refrigerator at 4 ° C. for 2 to 4 days, add 9.5 mL of microsomal fraction to 1 unit of leukocyte-removed erythrocyte preparation at room temperature to prepare pre-filtered blood. This was filtered with a natural head having a head of 100 cm using a filter including a polyester-coated non-woven fabric C coated with a polymer, and blood was collected and added and filtered to give blood. The pre-filtered blood and post-filtered blood were centrifuged at 4000 xg for 30 minutes, and the supernatant was dispensed. The supernatants were centrifuged at 100,000 × g, 4 ° C. ± 2 ° C. for 1 hour. After centrifugation, the precipitate was resuspended in 100 μL of sample buffer and kept at 100 ° C. ± 5 ° C. for 5-10 minutes.

[Analysis of normal prion titer]
Normal prion protein was detected by a general Western blotting method using 3F4 that specifically binds to normal prion protein as a primary antibody in blood products before and after addition filtration heated by adding sample buffer. Detection of all pre- and post-filtration blood was performed in a serial 0.5Log dilution series. The maximum dilution ratio at which the signal of normal prion protein can be visually confirmed is defined as the normal prion titer.

[Normal prion removal performance]
Normal prion removal performance is determined by the following equation (3).
Normal prion removal performance = normal prion titer (pre-filtered blood)
-The titer of normal prion (blood after addition filtration) (3)

(Polymer synthesis)
Ethanol was used as a polymerization solvent, and while stirring at 78 ° C. in a nitrogen atmosphere, an ethanol solution in which the polymerizable monomer and diazo initiator were dissolved was dropped to perform polymerization. The charge of each polymerizable monomer was 20 mol% of methyl methacrylate (hereinafter abbreviated as “MMA”) as a hydrophobic polymerizable monomer, and dimethylaminoethyl methacrylate (hereinafter “DM”) as a polymerizable monomer containing a basic nitrogen-containing moiety. 5 mol%, and 75 mol% 2-hydroxyethyl methacrylate (hereinafter abbreviated as “HEMA”) as a monomer containing a protonic neutral hydrophilic portion. The polymerization solution was purified with excess water and dried under reduced pressure. The copolymer composition of the polymer was measured by 1H-NMR. The results were almost the same as the charging ratio. The copolymer composition of MMA in the polymer was 20 mol%, the copolymer composition of DM in the polymer was 5 mol%, and the HEMA in the polymer was 75 mol%. The weight average molecular weight Mw was 210,000.
(Coating on porous material)
Ethanol was used as the coating solvent, and the polymer concentration was 0.8 W / V%. The polyester non-woven fabric C, which is the material, was coated at the above polymer concentration. The amount of porous polymer retained was 21 mg / m ² per unit area of the entire surface of the material.
(Polymer bead on porous beads)
The polymer is coated on the porous surface (including the pores) of the porous beads by placing the porous beads in a 0.1 W / V% ethanol solution of the polymer and stirring at reduced pressure for 1 hour, followed by drying at 30 ° C. did. The amount of the polymer retained in the porous beads was 6.8 mg / m ² per unit area of the entire surface of the material.
(Filtration test)
When the leukocyte removal performance test, the recovery pressure test, the hemolysis test after filtration and the normal prion removal performance test 1 were performed by the above-described methods, the leukocyte removal performance was 4.20, the blood treatment pressure was 10.2 kPa, and the hemolysis was 0. .4%, normal prion removal performance was 2.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 20 mol%, DM 13 mol%, and HEMA 67 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 20 mol%, the copolymer composition of DM in the polymer was 13 mol%, HEMA in the polymer was 67 mol%, and the weight average molecular weight Mw was 250,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 7.2 mg / m ^2.
The leukocyte removal performance was 5.72, the blood treatment pressure was 8.1 kPa, the hemolysis was 0.5%, and the normal prion removal performance was 3.0.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, DM 10 mol%, and HEMA 60 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 60 mol%. The weight average molecular weight Mw was 210,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 20 mg / m ^2, was 8.0 mg / m ^2.
The leukocyte removal performance was 5.64, blood treatment pressure was 11.7 kPa, hemolysis was 0.3%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 40 mol%, DM 5 mol%, and HEMA 55 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 40 mol%, the copolymer composition of DM in the polymer was 5 mol%, and HEMA in the polymer was 55 mol%. The weight average molecular weight Mw was 170,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 6.8 mg / m ^2.
The leukocyte removal performance was 5.22, the blood treatment pressure was 12.0 kPa, hemolysis was 0.4%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 40 mol%, DM 13 mol%, and HEMA 47 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 40 mol%, the copolymer composition of DM in the polymer was 13 mol%, and HEMA in the polymer was 47 mol%. The weight average molecular weight Mw was 180,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 7.3 mg / m ^2.
The leukocyte removal performance was 5.66, blood treatment pressure was 10.6 kPa, hemolysis was 0.6%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, DM 10 mol%, and HEMA 60 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 60 mol%. The weight average molecular weight Mw was 210,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 8.0 mg / m ^2.
The leukocyte removal performance was 5.70, blood treatment pressure was 13.2 kPa, hemolysis was 0.5%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, DM 10 mol%, and HEMA 60 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 60 mol%. The weight average molecular weight Mw was 210,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 7.5 mg / m ^2.
The leukocyte removal performance was 5.10, blood treatment pressure was 10.9 kPa, hemolysis was 0.2%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, DM 10 mol%, and HEMA 60 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 60 mol%. The weight average molecular weight Mw was 210,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 7.8 mg / m ^2.
The leukocyte removal performance was 4.60, blood treatment pressure was 11.5 kPa, hemolysis was 0.3%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, DM 10 mol%, and HEMA 60 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 60 mol%. The weight average molecular weight Mw was 210,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 7.2 mg / m ^2.
The leukocyte removal performance was 5.51, blood treatment pressure was 11.0 kPa, hemolysis was 0.2%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charge ratio of each polymerization monomer was 30 mol% ethyl methacrylate (hereinafter abbreviated as “EMA”), 10 mol% DM, and 60 mol% HEMA. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of EMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 60 mol%. The weight average molecular weight Mw was 220,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 19 mg / m ^2, was 7.0 mg / m ^2.
The leukocyte removal performance was 5.02, the blood treatment pressure was 10.1 kPa, hemolysis was 0.4%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1, except that the charging ratio of each polymerization monomer was MMA 30 mol%, diethylaminoethyl methacrylate (hereinafter abbreviated as “DE”) 10 mol%, and HEMA 60 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of EMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 60 mol%. The weight average molecular weight Mw was 200,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 20 mg / m ^2, was 7.2 mg / m ^2.
The leukocyte removal performance was 4.48, the blood treatment pressure was 11.9 kPa, hemolysis was 0.4%, and normal prion removal performance was 3.5.

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, DM 10 mol%, and hydroxypropyl methacrylate (hereinafter abbreviated as “HPMA”) 60 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HPMA in the polymer was 60 mol%. The weight average molecular weight Mw was 280,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 6.8 mg / m ^2.
The leukocyte removal performance was 4.80, blood treatment pressure was 14.8 kPa, hemolysis was 0.4%, and normal prion removal performance was 3.5.

The charge ratio of each polymerizable monomer is 40 mol% of butyl acrylate (hereinafter abbreviated as “BA”) as a hydrophobic polymerizable monomer, and diethylaminoethyl acrylate (hereinafter “DEA” as a polymerizable monomer containing a basic nitrogen-containing moiety). Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that 10 mol% and 50 mol% of hydroxybutyl acrylate (hereinafter abbreviated as “HBA”) as a monomer containing a protonic neutral hydrophilic portion. The copolymer composition of the polymer was measured by 1H-NMR. The copolymerization composition of BA in the polymer was 40 mol%, the copolymerization composition of DEA in the polymer was 10 mol%, and HPA in the polymer was 50 mol%. The weight average molecular weight Mw was 150,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 21 mg / m ^2, was 8.0 mg / m ^2.
The leukocyte removal performance was 4.68, blood treatment pressure was 11.0 kPa, hemolysis was 0.4%, and normal prion removal performance was 3.5.

Comparative Example 1

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, DM 3 mol%, and HEMA 67 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 3 mol%, and HEMA in the polymer was 67 mol%. The weight average molecular weight Mw was 200,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 19 mg / m ^2, was 6.4 mg / m ^2.
The leukocyte removal performance was 4.90, blood treatment pressure was 10.6 kPa, hemolysis was 0.2%, and normal prion removal performance was 1.0. When the polymerization composition of the polysynthetic monomer containing a basic nitrogen-containing moiety was 5 mol% or less, the adsorption of normal prions decreased, resulting in a decrease in normal prion removal performance.

Comparative Example 2

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, DM 16 mol%, and HEMA 54 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of DM in the polymer was 16 mol%, and HEMA in the polymer was 54 mol%. The weight average molecular weight Mw was 190,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 21 mg / m ^2, was 7.1 mg / m ^2.
The blood treatment pressure was 11.5 kPa and hemolysis was 2.3%. In the refrigerated filtration, hemolysis occurred when the polymerization composition of the polysynthetic monomer containing a basic nitrogen-containing portion increased, and the result of 0.8% was not satisfied.

Comparative Example 3

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 15 mol%, DM 10 mol%, and HEMA 75 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 15 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 75 mol%. The weight average molecular weight Mw was 220,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 20 mg / m ^2, was 7.3 mg / m ^2.
The leukocyte removal performance is 5.20, the blood treatment pressure is 8.2 kPa, the hemolysis is 0.3%, the normal prion removal performance is 1.0, and if the hydrophobic polymerizable monomer is less than 20 mol%, the normal prion removal performance is The result decreased.

Comparative Example 4

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 45 mol%, DM 10 mol%, and HEMA 45 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 45 mol%, the copolymer composition of DM in the polymer was 10 mol%, and HEMA in the polymer was 45 mol%. The weight average molecular weight Mw was 180,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 6.4 mg / m ^2.
The same operation as in Example 1 was performed except that this polymer was used. However, the flowability of the blood product was poor, the blood processing pressure exceeded 60 kPa, and the tube and syringe were damaged, and the test was stopped midway. Therefore, the leukocyte removal performance and hemolysis could not be measured.

Comparative Example 5

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was 3 mol% DM and 97 mol% HEMA. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of DM in the polymer was 3 mol%, and the copolymer composition of HEMA was 97 mol%. The weight average molecular weight Mw was 550,000. The same operation as in Example 1 was performed except that this polymer was used. Retention of the polymer of the porous material and porous beads per unit area of each material total surface 7.4 mg / m ^2, was 2.1 mg / m ^2.
The leukocyte removal performance was 4.90, the blood treatment pressure was 7.2 kPa, hemolysis was 0.4%, and normal prion removal performance was 0.5.

Comparative Example 6

The same operation as in Example 1 was performed except that the porous body and the porous beads were used without polymer coating.
The leukocyte removal performance was 4.60, the blood treatment pressure was 19.9 kPa, the hemolysis was 0.2%, and the normal prion removal performance was 0.

Comparative Example 7

Methacrylic acid having a carboxyl group (hereinafter abbreviated as “MAA”) was used as the acidic monomer.
Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 30 mol%, MAA 10 mol%, and HEMA 60 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 30 mol%, the copolymer composition of MAA in the polymer was 10 mol%, and the HEMA in the polymer was 60 mol%. The weight average molecular weight Mw was 230,000. The same operation as in Example 1 was performed except that this polymer was used. Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 22 mg / m ^2, was 6.6 mg / m ^2.
The leukocyte removal performance was 5.45, the blood treatment pressure was 10.6 kPa, hemolysis was 0.3%, and the normal prion removal performance was 0. The result was that normal prions could not be removed with polymers having acidic monomers.

Comparative Example 8

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was 10 mol% DM and 90 mol% HEMA. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of DM in the polymer was 10 mol%, and the copolymer composition of HEMA was 90 mol%. The weight average molecular weight Mw was 500,000. The same operation as in Example 1 was performed except that this polymer was used.
Retention of the polymer of the porous material and porous beads per unit area of each material the entire surface 18 mg / m ^2, was 6.8 mg / m ^2.
The leukocyte removal performance was 3.60, the blood treatment pressure was 14.4 kPa, hemolysis was 0.2%, and normal prion removal performance was 1.0.

Comparative Example 9

Polymerization, purification, and drying were carried out under the same conditions as in Example 1 except that the charging ratio of each polymerization monomer was MMA 90 mol% and DM 10 mol%. The copolymer composition of the polymer was measured by 1H-NMR. The copolymer composition of MMA in the polymer was 90 mol%, and the copolymer composition of DM in the polymer was 10 mol%. The weight average molecular weight Mw was 240,000. The same operation as in Example 1 was performed except that this polymer was used. The retained amount of the porous body was 21 mg / m ² per unit area of the entire surface of the material. However, the flowability of the blood product was poor, the blood processing pressure exceeded 60 kPa, and the test and the hemolysis were stopped in the middle because of fear of damage to the tube and syringe, so the leukocyte removal performance and hemolysis could not be measured.

The method for removing abnormal prions from the blood product according to the present invention is useful for preventing transfusion infection of transmissible spongiform encephalopathy (TSE) due to abnormal prions in the field of transfusion medicine, and further preventing transfusion side effects due to leukocytes. .

Claims (10)

A filter for selectively reducing a target substance in a biological fluid, comprising a carrier laminated in a housing having an outlet and an inlet for the fluid, wherein the laminated carriers are at least the following A and B: A filter having a prion removal function, characterized by comprising:
A: It consists of at least one porous body layer and at least one porous bead layer.
B: 20 mol% or more and 40 mol% or less of a hydrophobic polymerizable monomer-derived unit, 5 mol% or more and 13 mol% or less of a polymerizable monomer-containing unit containing a basic nitrogen-containing moiety, and the balance is a protonic neutral hydrophilic A polymer composed of three units, which are units derived from a polymerizable monomer containing a functional moiety, is present on the porous surface (including the pores) of the porous beads and / or the surface of the porous body.   The porous bead layer has a porous body layer on the outlet side, and the average pore diameter of at least one porous body of the porous body layer is equal to or less than the average particle diameter of the porous beads. The filter described.   The filter according to claim 1 or 2, wherein at least one of the porous bodies has a leukocyte removing ability.   4. The filter according to claim 3, wherein the porous body having leukocyte removal ability is a fibrous body.   The filter according to claim 4, wherein the fibrous body having leukocyte removing ability is a nonwoven fabric.   2. The filter according to claim 1, wherein the average particle diameter of the porous beads is 20 μm to 5 mm.   The filter according to claim 1, wherein the hydrophobic polymerizable monomer, the polymerizable monomer containing a basic nitrogen-containing moiety, and the polymerizable monomer containing a protic neutral hydrophilic moiety are an acrylic acid derivative and / or a methacrylic acid derivative. .   The filter according to claim 1 or 7, wherein the basic nitrogen-containing moiety is a tertiary amino group.   The filter according to claim 1 or 7, wherein the protonic neutral hydrophilic portion is a hydroxyl group.   The filter according to claim 1, wherein the filter is autoclaved after radiation sterilization.