JP2009095436A - Blood preparation purification material, purification method of the same, and purified blood preparation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a blood preparation purification material, a purification method of the same, and a purified blood preparation, with which a substance to be removed, such as a microorganism, pyrogenic substance, cell activator, cell producing the cell activator, and medical agent, is selectively removed from the blood preparation containing hemocyte and plasma, which are difficult to sterilize or inactivate bacteria by heat treatment or membrane filtration, in a state retaining useful components such as erythrocyte, thrombocyte, albumin, or blood coagulation factor. <P>SOLUTION: This blood preparation purification material includes fixing at least one type of functional group selected from urea bond, thiourea bond, amide bond, primary amino group, secondary amino group, tertiary amino group, pyridyl group, pyrimidyl group, and imidazole group, on a material surface. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to cell components that are difficult to sterilize by heat treatment or membrane filtration, such as erythrocyte preparations, whole blood preparations, synthetic blood, fresh frozen plasma, concentrated platelets, autologous blood, umbilical cord blood, and hematopoietic stem cell liquid for administration to the human body. This medicine selectively removes harmful components such as pyrogens, cells that produce pyrogens, microorganisms, cell activators, or drugs that cause side effects from blood products to reduce side effects during blood transfusion. The present invention relates to a purification material and a purification method using the material, or a cell prepared using the method or a solution containing cells.

  A blood product is a solution containing blood cells and plasma, and its characteristic is that it cannot be sterilized by heat. This is because the useful substances in these body fluid components are deactivated by heating. Sterilization by membrane filtration is also known, but because useful cells are similar in size to components to be removed, such as microorganisms, the cells must be kept sterile by membrane filtration and completely sterilized. Is difficult.

  Various materials that purify blood as extracorporeal circulation materials have been reported so far. For example, a material for removing cytokines (Patent Document 1) and a liquid processing column for removing endotoxins (Patent Document 2) are known. However, unlike extracorporeal circulation, blood products must be stored for a long time after blood collection, and special operations such as returning the product to the body after storage will cause specific side effects due to blood transfusion and cell transplantation. It is known that a material for purifying a blood product is required to have a performance different from that of a blood purification material by extracorporeal circulation. In the case of blood for preparing blood products, an anticoagulant containing citric acid is used, so that platelet activation is suppressed by calcium chelate, such as heparin and mesyl, which are anticoagulants during extracorporeal circulation. Adsorption performance under different conditions from that of blood collection using acid nafamostat is required.

  Bacterial contamination is known as one of the causes of side effects associated with cell transplantation such as blood transfusion, and it is considered that bacteria are mixed into the preservation solution from the puncture site during blood collection or bone marrow collection. For example, Yersinia infection due to long-term storage may be a problem even in erythrocyte preparations stored in a refrigerator, and bacterial contamination is a more important problem in platelet preparations that require room temperature storage. Regarding blood transfusion, 21 deaths of septicemia (1986-1991), which are thought to be caused by platelet preparations contaminated with bacteria in the United States, were reported to the FDA (Food and Drug Administration) (non-food) Patent Document 1). In fact, the frequency of sepsis due to blood transfusion is said to be once in every 50,000 platelet product transfusions and once in every 500,000 red blood cell transfusions in the United States, which is a major factor that threatens the safety of transfusion products (Non-Patent Documents). 2, 3). The expiration date of platelet preparations in Japan is 72 hours after blood collection, but since it is stored under shaking at 20-24 ° C, it may proliferate and cause post-transfusion sepsis if bacterial contamination occurs. As a result of the sterility test conducted by the Japanese Red Cross Society from 1999 to 2004, 0.04% was detected in platelet concentrate and 0.05% in erythrocyte concentrate (Non-patent Document 4), and blood was detected. Bacterial contamination of the preparation cannot be completely prevented. Methods to prevent bacterial contamination of blood products include: 1) disinfection at collection or removal of contaminated blood, 2) minimizing bacterial growth, 3) detecting contaminated products, 4) bacteria It is conceivable to kill them in some way. Currently, in blood donation, thorough disinfection of the skin at the time of blood collection and disposal of bacteria in the blood sample by preventing 30 ml of the initial flow that is thought to contain bacteria at the start of collection is performed. (Non-Patent Document 5) is not perfect.

  In addition to bacterial contamination, non-hemolytic fever has been reported as a side effect when blood products are administered. Non-hemolytic transfusion side effects include urticaria, anaphylactoid reaction, fever reaction, dyspnea, transfusion-related acute lung injury (TRALI), and decreased blood pressure. As a result of analysis of the shelf life and transfusion side effects of platelet preparations, it was reported that there was a relationship between the storage period and the frequency and severity of transfusion side effects. Non-patent document 6). Therefore, it was considered that a side effect of transfusion may be caused by a pyrogenic substance or a cell activating substance that increases during storage of blood products. In 1993, Muylle and colleagues focused on interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α), which are inflammatory cytokines that are cell activators. In the platelet preparation derived from whole blood, it has been reported that these cytokines increase depending on the number of storage days and the number of mixed leukocytes, and that the level can induce clinical symptoms (Non-patent Document 7). IL-1, IL-6, and TNF-α are all mediators that are key to the inflammatory response, and their synergistic effects are also known. IL-1 and TNF-α stimulate central prostaglandin synthesis, which induces fever by raising the temperature setpoint. Stack et al. Also reported that interleukin-8 (IL-8), which has a neutrophil chemotaxis effect, has a high value, although there is no direct pyrogenic effect. It has been strongly suggested by clinical studies by Muylle et al. And Heddle et al. That these cytokines are involved in non-hemolytic transfusion side effects such as fever (Non-patent Document 8). Currently, pretreatment with a leukocyte removal filter is performed to prevent the increase in cytokine concentration during storage by removing leukocytes producing these cytokines before storage (Patent Documents 3 to 5). Has some effects, but is not complete, and in platelet preparations RANTES produced from platelets during storage, transforming growth factor beta 1 (TGF-β1), soluble hemangiodermal growth factor (soluble VEGF) ), It has been reported that non-hemolytic fever develops due to soluble CD40 ligand (Non-Patent Document 6), so that non-hemolytic transfusion side effects still appear regardless of leukocyte removal. The leukocyte removal filter is specialized for leukocyte removal, and does not have the ability to remove produced cytokines and bacteria.

Also, those who are taking therapeutic drugs at the time of blood donation are not allowed to donate blood, but they are self-reported, and drug detection is virtually impossible even if blood containing the drug enters the blood product. Is possible. Therefore, there is a possibility that non-hemolytic transfusion side effects are expressed by the blood product mixed with the drug.
JP 2001-198213 A Japanese Patent Laid-Open No. 10-225515 JP-A-5-194243 Japanese Patent Laid-Open No. 7-24066 Japanese Patent Laid-Open No. 7-25776 Japanese Journal of Transfusion and Cell Therapy, Vol. 52. No. 4 52 (4): 507-511, 2006. Semin Hematol, Vol. 38 (Suppl 11): 20-26, 2001. Dev. Biol, (Basel). 108: 59-67, 2002. Blood transfusion information 0609-101, Japanese Red Cross Blood Division, Pharmaceutical Information Division. Japanese Journal of Transfusion Medicine, Vol. 49. No. 6 49 (6): 761-766, 2003. Japanese Journal of Transfusion Medicine, Vol. 47. No. 6 47 (6): 829-836, 2002 Transfusion, 33 (3): 195-199, 1993. N. Engl. J. Med., 331: 625-628, 1994.

  The present invention solves the above-mentioned problems of the prior art and is useful for erythrocytes, platelets, albumin, blood coagulation factors, etc. from blood products containing blood cells and plasma that are difficult to sterilize or inactivate bacteria by heat treatment or membrane filtration. A material for purifying blood products, a purification method, and a purification method for selectively removing substances to be removed such as microorganisms, pyrogens, cell activators, cells that produce cell activators, and drugs in a state that the components are retained It is an object to provide a purified product.

The present invention has the following configuration in order to solve the above problems.
1. At least one functional group selected from urea bond, thiourea bond, amide bond, primary amino group, secondary amino group, tertiary amino group, pyridyl group, pyrimidyl group and imidazole group is fixed on the material surface A material for purifying blood products.
2. 2. The blood product purification material according to 1 above, which has a chemical structure represented by the following formula on the surface of the material.
-X- (CH ₂ ) nY
X = urea bond, thiourea bond, amide bond, secondary amino group, at least one functional group selected from tertiary amino group
2. an integer of n = 1 to 20, Y = hydrogen, primary amino group, secondary amino group, at least one functional group selected from tertiary amino groups 3. The blood product purification material according to 1 or 2 above, comprising a fibrous carrier having a diameter of 100 μm or less.
4). 4. The material for purifying blood products according to any one of 1 to 3 above, wherein pyrogens, cell activators, cells that produce cell activators and drugs are removed from blood components. 5. The blood product purification material according to 4, wherein the pyrogenic substance is a microorganism or a toxin produced by the microorganism, and the cell activating substance is any one of a cytokine and a platelet activating factor.
6). 6. The blood product purification material according to any one of 1 to 5 above, wherein a removal rate of the pyrogen and cell activating substance is 60% or more and a removal rate of granulocytes is 40% or more.
7). 7. The blood product purification material according to any one of 1 to 6, wherein the removal rate of erythrocytes, albumin and blood coagulation factors is 30% or less.
8). 8. A blood product purification method comprising using the blood product purification material according to any one of 1 to 7 above.
9. 9. A blood product purified using the method according to 8 above.

  Select microorganisms, pyrogens, cell-activating substances, cells that produce cell-activating substances and drugs that retain useful components from solutions containing blood cells and plasma that are difficult to sterilize or inactivate by heat treatment or membrane filtration By providing selective removal materials, purification methods and purified products for effective removal, the incidence of side effects during blood transfusions (allergic reactions such as urticaria, fever, blood pressure reduction, etc.) is low, and safety is high A blood product can be provided.

  The present invention provides a method for selectively removing pyrogens, cell activators, cells that produce cell activators, and drugs from solutions containing blood cells and plasma that are difficult to sterilize by heat treatment or membrane filtration. The present invention relates to a material and a purification method using the material or a blood product prepared using the method.

  In the present invention, blood products are concentrated red blood cells, concentrated irradiated red blood cells, washed red blood cells, leukocyte-removed red blood cells, thawed red blood cell concentrated liquid, synthetic blood, fresh frozen plasma, concentrated platelets, irradiated concentrated platelets, concentrated platelet HLA, human whole blood, It refers to autologous blood, umbilical cord blood, hematopoietic stem cell fluid, and the like. In addition, blood product purification refers to the use of a selective purification material during the period from when the above blood components are donated or collected to when used for blood transfusion or administration, or during administration, from blood products to microorganisms, pyrogens, This refers to selective removal of cell activators, cells that produce cell activators, and drugs.

It has been reported that a material having a chemical structure capable of forming a hydrogen bond and having a microporous structure on the surface of the material is excellent in microbial removal (Japanese Patent Laid-Open No. 2005-119). On the other hand, it has also been reported that a material having a functional group capable of forming a hydrogen bond is excellent in cytokine removal ability (Japanese Patent Laid-Open No. 10-147518). It has also been reported that it is useful for removing polypeptide antibiotics (Japanese Patent Laid-Open No. 10-85329). However, in the present invention, it has been found that a chemical structure capable of activating cells contained in a blood product exists in a chemical structure capable of forming a hydrogen bond. For example, there is a hydroxyl group as a functional group that easily activates cells, and when this is present on the surface of the material, the complement activating effect is strong. Further, depending on the type of quaternary amino group, hemolysis may be exhibited, and materials in which these functional groups are present on the surface at high density alone are excluded from the present invention. Therefore, in the present invention, the functional group fixed on the surface of the material includes urea bond, thiourea bond, amide bond, primary amino group, secondary amino group, tertiary amino group, pyridyl group, pyrimidyl group, and imidazole group. It was limited to at least one kind of functional group selected from among them. These functional groups are not functional groups of the material itself, but substances having functional groups are introduced and fixed to the material.
In addition, useful components in a solution containing cell components may have an affinity for materials having urea bonds, thiourea bonds, amide bonds, and amino groups. In order to minimize the removal of such useful components Reduces the number of urea bonds, thiourea bonds, etc. in the material, introduces functional groups that have affinity with the removed component with other functional groups, and removes useful components while maintaining the removal rate of removed components What is necessary is just to reduce a rate. Substituents with affinity other than functional groups that form hydrogen bonds such as urea bonds, thiourea bonds, amide bonds, amino groups, etc. are hydrophobic groups, especially aliphatic substituents or aromatic substituents having 4 or more carbon atoms It is preferable to introduce and fix a hydrophobic group such as a group. Specific examples of the aliphatic hydrophobic group having 4 or more carbon atoms include a butyl group, a hexyl group, an octyl group, a dodecyl group, a hexadecyl group, a cyclohexyl group, and the like, and examples of the aromatic substituent include a benzyl group, Examples thereof include compounds containing hydrocarbon such as phenyl group and 3,3 diphenylpropyl group. Moreover, when improving the performance of removing exothermic components such as cytokines, the chain length of the hydrophobic group is controlled, and the log P value (water / octanol partition coefficient) of the functional group is set to 2.5 or more. Is mentioned. Moreover, when it has the following structure among the structures having the functional group, superantigens, cytokines and bacteria are easily removed.
-X- (CH ₂ ) nY
X = At least one functional group selected from urea bond, thiourea bond, amide bond and secondary or tertiary amino group
n = 1-20 integer Y = hydrogen, primary amino group, secondary amino group, at least one functional group selected from among tertiary amino groups In the above structure, as n number increases, hydrophobicity increases. n = 2 to 16 is more preferable.

  As a method of changing the ratio of the number of functional groups per unit area or unit weight when two or more kinds of functional groups coexist, a one-step reaction in which a solution in which all compounds are mixed at a predetermined ratio is reacted. Any one of the multi-stage reactions in which one kind of compound is reacted in one reaction and the reaction is performed for the number of compounds to be reacted is used. preferable. There is no particular limitation on the method for introducing these functional groups and substituents onto the surface of the material, and a method of synthesizing a polymer having these functional groups in the side chain or main chain and then molding the polymer on the surface of a fiber or the like. A method of coating a material having these functional groups, or a method of introducing a functional group on the surface of a carrier by using a chemical reaction after molding the carrier can be considered. Here, the material surface of the carrier shown indicates a boundary surface that directly contacts the components in the blood product when used for purification of the blood product.

Moreover, if the amount of the urea bond, thiourea bond, amide bond, primary amino group, secondary amino group, tertiary amino group, pyridyl group, pyrimidyl group and imidazole group is too small, a pyrogen or cell The ability to remove the activator, the cell producing the cell activator and the drug is difficult to obtain. On the other hand, if the amount is too large, the removal rate of red blood cells, albumin and blood coagulation factors is affected. For example, when polyethyleneimine is used and a functional group having both an amino group and a urea bond is used by further reacting an isocyanate, the nitrogen number of the polyethyleneimine is preferably 2 to 10. In addition, when tetraethylenepentamine and chlorophenyl isocyanate are reacted and immobilized on polystyrene having a chloroacetamidomethyl group as a reactive functional group, the charged amount is 0.005 to 100.0 tetraethylenepentamine per polystyrene repeating unit. Mol, 0.005 to 100.0 mol of chlorophenyl isocyanate is preferable, and 0.01 to 20.0 mol of tetraethylenepentamine and 0.01 to 20.0 mol of chlorophenyl isocyanate are more preferable.
Targets to be removed from blood components include pyrogens that cause an increase in body temperature when taken into a living body such as the human or animal body, cells activated or blood transfused during storage of blood products Later, it is taken into the living body such as the human body or animal body to activate cells in the living body, and is used to prevent corruption of cell activation substances or blood products that induce body temperature rise, allergy induction, blood pressure reduction, or treatment of diseases. Drugs.

  Blood components are concentrated red blood cells, concentrated irradiated red blood cells, washed red blood cells, leukocyte-removed red blood cells, thawed red blood cell concentrated liquid, synthetic blood, fresh frozen plasma, concentrated platelets, irradiated concentrated platelets, concentrated platelet HLA, Refers to washed platelets, whole human blood, autologous blood, umbilical cord blood, hematopoietic stem cell fluid, and the like. Pyrogenic substances include Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus, and Acne, Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, and fungi such as Aspergillus and Candida. Toxins produced by these microorganisms, for example, Staphylococcus aureus toxin, S. aureus pyrogenic exotoxin A (hereinafter abbreviated as SEA), S. aureus pyrogenic exotoxin B, S. aureus pyrogenic Exotoxin C, Toxic shock synthromtoxin-1 (hereinafter abbreviated as TSST-1), hemolytic toxin, leucocidin, coagulase and protein A, cholera toxin of Vibrio cholerae, verotoxin produced by hemorrhagic Escherichia coli and Shigella, and Pseudomonas aeruginosa Constructs secretory toxins such as exotoxin A produced by Streptococcus, pyrogenic exotoxin produced by Streptococcus, and bacteria such as lipopolysaccharide Deoxyribonucleic acid and the like is a gene constituent compounds and bacteria that.

  Examples of cell activators include cytokines produced by leukocytes and platelets, such as interleukin-1β, interleukin-6, interleukin-8, tumor necrosis factor-alpha, RANTES, beta-thromboglobulin, platelet factor-4. , Transglomerating factor β1 (TGF-β1), soluble vascular endothelial growth factor (soluble VEGF), soluble CD40 ligand, sphingosine 1-phosphate, platelet activating factor and the like. Furthermore, cell activating substances released from platelets are regarded as important in platelet preparations. A platelet preparation is a product obtained by concentrating only platelets out of blood components. However, transfusion side effects occur due to cell activating substances released from platelets. That is, by removing leukocytes at the time of blood donation, transfusion side effects caused by cell activating substances released from leukocytes can be reduced, but the platelet preparation cannot be reduced. In addition, since platelet preparations are stored at 22 to 24 ° C. with shaking, it is easy to produce cell activating substances and bacteria can easily grow. Therefore, removal of cell activating substances in platelet preparations is useful. high.

  Examples of cells that produce cell activating substances include leukocytes contained in blood products. The granulocytes referred to in the present invention are neutrophils, monocytes, eosinophils and basophils. Among activated leukocytes, granulocytes such as activated neutrophils, monocytes, eosinophils, and basophils release cell-activating substances during storage of blood products, causing transfusion side effects such as fever, hypotension, and allergies. Let The production of these cell activators is abnormally enhanced in blood products with bacterial contamination.

  The drug refers to a drug that is directly administered to blood or a drug that is orally administered and is present in the blood. Antibiotics, antifungal agents, antipyretic analgesics, antihypertensive agents, immunosuppressants, etc. Is mentioned. Examples of antibiotics include cephem antibiotics such as cefaclor and ceftriaxone, penicillin antibiotics such as penicillin G, aminoglycoside antibiotics such as gentamicin, amikacin and arbekacin, polypeptide antibiotics such as vancomycin, clinda Examples include lincomycin antibiotics such as mycin, quinolone antibiotics such as levofloxacin, antifungal agents such as amphotericin B and miconazole, acetaminophen as antipyretic analgesics, and as immunosuppressants Examples include cyclosporin A and tacrolimus. In addition, antihypertensive agents, vasopressors, steroids, diuretics and the like can be mentioned.

  In the present invention, the blood cell component refers to a red blood cell component, a platelet component, or a white blood cell component composed of granulocytes and lymphocytes. Among them, red blood cells having oxygen carrying ability and platelets important for blood coagulation are useful components. In addition, useful plasma components include plasma protein components such as lipoprotein having binding activity with albumin, globulin or endotoxin, lipid components, saccharide components, hormones, vitamins and the like. The electrolyte component has an important effect on the maintenance of osmotic pressure in the body and the function of nerves and proteins. The blood electrolyte component in the present invention includes sodium ion, potassium ion, calcium ion, magnesium ion, iron ion, manganese. It includes any of cations such as ions and crawl ions and anions such as chlorine ions, phosphate ions, sulfate ions and bicarbonate ions.

  The material for purifying blood products according to the present invention removes the above-mentioned “pyrogenic substances, cell activating substances, cells and drugs that produce cell activating substances”, but pyrogenic substances or cell activating substances. Or it is preferable that the removal rate of both of these is 60% or more, and the removal rate of granulocytes is 40% or more.

  Further, the removal rate of red blood cells, albumin and blood coagulation factors is preferably 30% or less.

  There are various methods for measuring the removal rate of these substances. Differences such as the volume ratio between the removal material and the blood product or the solution before preparing the blood product, the concentration of impurities in the solution to be removed, and the contact It is considered that the removal rate varies depending on the time, presence or absence of stirring, and the like.

  The measurement of the removal rate in this invention is shown below.

In the removal test of a solution containing blood cell components or platelet components and plasma components, the initial concentration before removal, the concentration of the initial bacterial solution, or the initial blood cell count in the solution to be removed is set to C ₀ and purified by contacting with a purification material. The removal rate (%) is obtained by the following calculation method, where C is the concentration in the solution after removal, the concentration of the bacterial solution, or the number of blood cells.

(C ₀ -C) / C ₀ × 100 (1) equation Further, the removal rate is affected by the time during which the solution containing the substance to be removed is brought into contact with the removal material. There is often. Therefore, the contact time when measuring this removal rate is 1 to 10 min. It was decided to measure under the conditions. The calculation method of the contact time is as follows.

Contact time (min.) = Volume of purification material (cm ³ ) / flow rate of solution containing substance to be removed (mL / min.)
When a solution containing blood, platelet component or plasma component is used as a sample, the test is performed using a human sample collected from a healthy adult. As the anticoagulant, it is possible to use a CPD (Citrate Phosphate Dextrose) solution or an ACD-A (Acid Citrate Dextrose-A) solution, which is a citrate anticoagulant used when producing a blood product. . In the case of erythrocyte preparations, MAP liquid is used to maintain red blood cell function, but these solutions may be included. Furthermore, heparin, nafamostat mesylate, EDTA, etc. can be used as an anticoagulant.

  Further, the removal rate of drugs such as antibiotics is measured by bovine serum albumin (fraction V) so that the concentration is 5 mg / ml in 0.1 M phosphate buffer (pH 7.4) containing 0.15 M sodium chloride. A solution in which is dissolved is used. Also for these, the removal rate was calculated using the above equation (1).

  In the present invention, the form of the material is not particularly limited, and beads, fibers, hollow fibers, yarn bundles, yarns, felts, sponges, nets, knitted fabrics, woven fabrics, etc. are used. When a column or filter is used as a filter medium, fibers, cloths, beads, felts, sponges, and nets are preferably used. When fibers are used, the removal performance may vary depending on the fiber diameter. The average fiber diameter after preparation of the removal material is preferably 100 μm or less. However, in order to improve the removal performance of the granulocytes that produce the cell activation substance, the fiber diameter is 10 μm or less in which the activated granulocytes easily adhere. It is more preferable. The average fiber diameter is calculated by randomly taking 10 or more fields of view with a scanning electron microscope, and then selecting 5 or more images at random and measuring the fiber diameter using image analysis software. The average fiber diameter was calculated.

  In addition, the present material can be used not only alone but also as a single column by being fixed to a suitable base material and mixed with other materials. Operation such as immobilization or mixing may be performed before processing into the above shape, or may be performed after processing.

  As a blood product purification method, for example, blood is passed through a blood circuit containing a blood product purification material before or at the time of blood donation or blood collection or before or after blood donation or storage after blood collection. I can do it. The target to be purified is whole blood to which anticoagulant such as citrate anticoagulant CPD or ACD solution, EDTA, heparin, concentrated erythrocytes, concentrated irradiated erythrocytes, washed erythrocytes, leukocyte-removed erythrocytes, Thawed erythrocyte concentrated solution, synthetic blood, fresh frozen plasma, concentrated platelets, irradiated concentrated platelets, concentrated platelet HLA, human whole blood, autologous blood, umbilical cord blood, blood before preparing hematopoietic stem cell fluid, and blood product after preparation Can be purified. The blood circuit containing the blood product purification material is not particularly limited, and may have a blood introduction part, a blood purification material storage part, and a blood lead-out part. More preferably, it has a route. In addition to the blood product purification material described in the present invention in the blood circuit, other removal materials such as a leukocyte removal filter, a potassium adsorption material, and a microaggregate removal filter are incorporated, and commercially available medical treatments are also included. Use with equipment products is also possible. In addition, the platelet preparation can be purified during the operation process when preparing washed platelets.

  There is no particular limitation on the form of the blood purification material, such as a surface of the blood product purification material, such as a cotton container or a flat membrane wound around a pipe or a state where the flat membrane is sandwiched between two flat plates. Any blood may be used as long as it passes through and contacts the fibers.

  In addition, the purified product in the present invention, as described above, is concentrated red blood cells, concentrated irradiated red blood cells, washed after being purified by a method of purifying removed components through a container filled with a blood product purification material, etc. Red blood cells, leukocyte-removed red blood cells, thawed red blood cell concentrate, synthetic blood, fresh frozen plasma, concentrated platelets, irradiated concentrated platelets, concentrated platelet HLA, human whole blood, autologous blood, umbilical cord blood, and hematopoietic stem cell fluid. The purified product can be stored in a storage container attached to the blood circuit, or the purified product can be directly transfused after purification. If there is a possibility that the concentration of the substance to be removed increases in the blood product during storage, it may be possible to purify the blood product with the blood product purification material in two stages before storage and before blood transfusion.

  Details of the invention will be described below using examples, but the content of the invention is not limited to the examples.

Example 1
<Introduction of urea bond-containing compound into reactive polystyrene fiber: preparation of fiber A>
A sea island type described in US Pat. No. 4,661,260 comprising a 50 weight ratio sea component (a mixture of 46 weight ratio polystyrene and 4 weight ratio polypropylene) and a 50 weight ratio island component (polypropylene). 50 g of composite fiber (thickness: 2.6 denier, number of islands: 16) (referred to as control fiber A) is 50 g of N-methylol-α-chloroacetamide, 400 g of nitrobenzene, 400 g of 98% by weight sulfuric acid and 0 It was immersed in a mixed solution consisting of .85 g of paraformaldehyde and reacted at 20 ° C. for 1 hour. The fiber was taken out of the reaction solution, poured into 5 L of 0 ° C. ice water to stop the reaction, washed with water, and then the nitrobenzene adhering to the fiber was extracted and removed with methanol. This fiber was vacuum-dried at 50 ° C. to obtain 71 g of chloroacetamidomethylated crosslinked polystyrene fiber (hereinafter abbreviated as AMPSt fiber).

2.90 g of tetraethylenepentamine was dissolved in 500 ml of dimethyl sulfoxide (hereinafter abbreviated as DMSO). To this solution, 20 g of AMPSt fiber (corresponding to a chloro content of 20 mmol) was added with stirring. The reaction was carried out at 25 ° C. for 6 hours. Thereafter, AMPSt fibers were washed by adding 500 ml of DMSO on a glass filter. After washing, 3 g of AMPSt fiber was added to a solution of 150 ml of DMSO in which 0.49 g of parachlorophenyl isocyanate was dissolved. The reaction was carried out at 25 ° C. for 1 hour. Thereafter, it was washed with 60 ml of DMSO and distilled water on a glass filter. Finally, the reacted fiber on the glass filter is washed with 3 L of distilled water and physiological saline, and the functional group represented by -NH-((CH ₂ ) ₂ NH) ₄ -CO-NH-Ph-Cl is obtained. The imparted fiber (fiber A) was obtained. Photographs were randomly taken from scanning electron microscope S-800 made by Hitachi, Ltd. per 20 fields of view of fiber A, and the images were further arbitrarily selected at five locations using image analysis software Win ROOF (Nihonkai Keisoku Toki Co., Ltd.). The fiber diameter was measured. As a result, the average fiber diameter was 56 μm.
<Introduction of urea bond-containing compound into reactive polystyrene fiber: preparation of fiber B>
A sea island composite fiber of 36 islands, in which the island is further composed of a core-sheath composite, was obtained using the following components under spinning conditions of a spinning speed of 800 m / min and a draw ratio of 3 times.

Island core component: Polypropylene Island sheath component: Polystyrene 90wt%, Polypropylene 10wt%
Sea component: Copolymer polyester containing ethylene terephthalate unit as a main repeating unit and 3% by weight of 5-sodiumsulfoisophthalic acid as a copolymer component Composite ratio (weight ratio); Core: sheath: sea = 40: 40: 20
A sheet-like material composed of 70% by weight of this fiber and 30% by weight of polypropylene fiber having a diameter of 25 μm was produced, and then a nonwoven fabric was obtained by needle punching. This non-woven fabric was treated with an aqueous sodium hydroxide solution (3 wt%) at 90 ° C., and a sea component “copolymerized polyester containing ethylene terephthalate unit as a main repeating unit and 3% by weight of 5-sodium sulfoisophthalic acid as a copolymer component” was obtained. By dissolving, a nonwoven fabric having a core-sheath fiber diameter of 4 μm and a bulk density of 0.05 g / cm ³ (total basis weight 200 g / m ² ) was produced (control fiber B). It was immersed in a mixed solution consisting of 50 g of N-methylol-α-chloroacetamide, 400 g of nitrobenzene, 400 g of 98% by weight sulfuric acid and 0.85 g of paraformaldehyde, and reacted at 20 ° C. for 1 hour. The nonwoven fabric was taken out from the reaction solution, poured into 5 L of 0 ° C. ice water to stop the reaction, washed with water, and then the nitrobenzene adhering to the nonwoven fabric was extracted and removed with methanol. This nonwoven fabric was vacuum dried at 50 ° C. to obtain 68 g of a chloroacetamidomethylated crosslinked polystyrene nonwoven fabric (hereinafter abbreviated as AMPSt nonwoven fabric).

Next, 3.9 g of n-cetylamine was dissolved in 500 ml of DMSO. To this solution, 20 g of AMPSt nonwoven fabric (chloro content equivalent to 20 mmol) was added with stirring. The reaction was carried out at 25 ° C. for 6 hours. Thereafter, the AMPSt non-woven fabric was washed by adding 500 ml of DMSO on a glass filter. Thereafter, it was washed with 60 ml of DMSO and distilled water on a glass filter. Finally, the non-woven fabric after the reaction on the glass filter was washed with 3 L of distilled water and physiological saline to give a non-woven fabric to which a compound containing a functional group represented by -NH- (CH ₂ ) ₁₆ -H was added (Fiber B) ) The fiber diameter of fiber B was measured in the same manner as fiber A. As a result, the average fiber diameter was 8 μm.
(Example 2) Removal characteristics for microorganisms in blood 100 mL of blood was collected from 4 normal humans, and 14 mL of CPD solution (composition: sodium citrate 26.3 g / L, citric acid 3.27 g / L, glucose) 23.20 g / L, sodium dihydrogen phosphate dihydrate 2.51 g / L) was added to each blood, and 114 mL of whole blood solution was added to 10 mL of Staphylococcus epidermidis (ATCC 14990). Added to cfu / mL (human a blood). Similarly, Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), and Escherichia coli (ATCC 8739) of Staphylococcus aureus were added to 114 mL of whole blood solution to 10 cfu / mL. . The blood thus prepared is referred to as human b blood, human c blood, and human d blood, respectively. Next, each human blood was dispensed into four 25 mL portions. Next, it processed with the filter with which the fiber A or B produced in Example 1 and the control fiber A or B were filled. The capacity of the filled blood product purification material was 10 cm ³ for both fibers A and B and control fibers A and B. The blood flow rate is 10 mL / min., And the contact time is 1 min. It was. All filters were sterilized with γ rays. Next, 1 mL of blood after treatment was collected, the blood was cultured, the number of viable bacteria was confirmed, and the removal rate was calculated. As the medium, SCD (Soybean Casein Digest) agar medium (Merck Co., Ltd.) was used and cultured at 37 ° C. for 4 days. The removal rate is as shown in Table 1.

  The calculation method of the removal rate (%) is as follows.

(C ₀ -C) / C ₀ × 100 (1) Formula C ₀ : concentration of the initial bacterial solution in the solution before removal, C is the concentration of the bacterial solution in the solution after removal .
In this way, in the microorganism removal test, the control fibers A and B (fibers before chemical modification) grow microorganisms during the test, whereas in the fibers A and B, the microorganisms are adsorbed on the fibers. Decreased the number of bacteria.

(Example 3) Removal characteristics for blood cell components and albumin in blood.

2 mL of blood before and after the treatment with human blood b containing Staphylococcus aureus (ATCC 6538) shown in Example 2 was collected, and the red blood cell count, white blood cell count, and white blood cell fraction (neutrophil count, monocyte count) The platelet count was measured using an automatic blood cell counter (Sysmex XT-1800i). The results are shown in Table 2. Further, similarly, albumin in plasma obtained by collecting and centrifuging 2 mL of blood before and after filtering is measured using a biochemical analyzer (Fuji Dry Chem Fuji Film Co., Ltd.). About 8 factors, it measured by the coagulation time method. The results are shown in Table 3.
The calculation method of the removal rate (%) is as follows.

(C ₀ -C) / C ₀ × 100 (1) Formula C ₀ : Initial blood cell number in solution before removal, initial concentration or initial%, C is the number of blood cells in solution after removal , Concentration or%.

  As described above, the removal characteristics for blood cell components and albumin in blood were such that the removal rate of useful components of red blood cells and albumin was 30% or less, and the removal rate of blood coagulation factor 8 was also 30% or less. A removal rate of 60% or more was observed for granulocytes (neutrophils and monocytes) activated by bacteria. In addition, it was shown that the granulocytes can be removed at a higher rate with the fiber B having a smaller fiber diameter.

(Example 4) Removal characteristics against microorganisms and albumin and blood coagulation factors in a solution containing platelet components and plasma components.

200 mL of blood was collected from each of 4 normal humans, and 28 mL of CPD solution (composition: sodium citrate 26.3 g / L, citric acid 3.27 g / L, glucose 23.20 g / L, sodium dihydrogen phosphate 2 Containing platelet and plasma components obtained by centrifuging 228 mL of whole blood prepared by adding hydrate (2.51 g / L) at room temperature for 750 g × 5 minutes to separate the platelet and plasma components 100 mL of the solution was prepared. 10 solutions of Staphylococcus epidermidis (ATCC14990) in the solution It added so that it might become cfu / mL (human e solution). Similarly, for Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), and Escherichia coli (ATCC 8739) of S. aureus, 10 ml of each bacterium was added to 100 mL of a solution containing platelet components and plasma components. Cfu / mL was added. The solutions thus prepared are designated as human f solution, human g solution, and human h solution, respectively. Next, each 25 mL of human blood was dispensed into four, and treated with a filter filled with fiber A or B prepared in Example 1 and control fiber A or B. The capacity of the filled blood product purification material was 10 cm ³ for both fibers A and B and control fibers A and B. Assuming transfusion, the blood flow rate is 1 mL / min., And the contact time is 10 min. It was. Each filter was steam sterilized at 121 ° C. for 20 minutes. Next, 1 mL of blood after treatment was collected, the blood was cultured, the number of viable bacteria was confirmed, and the removal rate was calculated. The culture method and the removal rate calculation method were the same as in Example 2.
The results are shown in Table 4.

  Thus, even in a solution containing platelet components and plasma components that do not contain blood cell components, microorganisms grow during the test in the control fibers A and B (fibers before chemical modification) in the microorganism removal test. In the fibers A and B, a decrease in the number of bacteria was observed as microorganisms were adsorbed on the fibers.

Example 5 Removal characteristics for a solution containing a platelet component and a plasma component.

2 mL of blood before and after treatment with human blood g containing Pseudomonas aeruginosa (ATCC 9027) shown in Example 4 was collected, and 2 mL of blood before and after filtering was collected in the same manner as in Example 3. Albumin in plasma obtained by centrifugation was measured using a biochemical analyzer (Fuji Dry Chem Fuji Film Co., Ltd.), and blood coagulation factor 8 was measured by the coagulation time method. The results are shown in Table 5.
The calculation method of the removal rate (%) is as follows.

(C ₀ -C) / C ₀ × 100 (1) Formula C ₀ : Initial concentration or initial% in the solution, C is the concentration or% in the solution after removal.

  Thus, even in a solution containing platelets and plasma components that do not contain blood cell components, the removal rate for albumin was 30% or less, and the removal rate of blood coagulation factor 8 was also 30% or less.

(Example 6) Removal characteristics for cell activating substances in a solution containing platelet component and plasma component 50 mL of blood was collected from a normal human and 7 mL of CPD solution (composition: sodium citrate 26.3 g / L, citric acid 3 .57 g / L, glucose 23.20 g / L, sodium dihydrogen phosphate dihydrate 2.51 g / L) and 57 ml of whole blood prepared by centrifugation at 750 g × 5 minutes at room temperature 25 mL of a solution containing platelet components and plasma components obtained by separating the components and plasma components was prepared. Human natural IL-1, IL-6, IL-8 and S. aureus pyrogenic exotoxin A (SEA) and toxic shock synthromtoxin-1 (TSST-1) were added to the solution. It was prepared to be 500 pg / mL.

Next, it processed with the filter with which the fiber A or B produced in Example 1 and the control fiber A or B were filled. The capacity of the filled blood product purification material was 10 cm ³ for both fibers A and B and control fibers A and B. Assuming transfusion, the blood flow rate is 1 mL / min., And the contact time is 10 min. It was. All filters were sterilized with 25 kGy of γ rays. Next, the removal rate was calculated by obtaining the amount of cytokine before and after removal. The EIA method is used for quantification, and a commercially available kit (IL-1: human IL-1β ELISA kit manufactured by R & D System, IL-6, IL-8, SEA, and TSST-1: manufactured by Kamakura Technoscience) is used. It was. The results are shown in Table 6.

  Thus, it was shown that IL-1β, IL-6, IL-8, SEA and TSST-1 which are cell activators are removed at a removal rate of 60% or more.

(Example 7) Removal characteristics of drug in protein solution The removal rate of drug in protein solution was measured at a concentration of 5 mg / ml in 0.1 M phosphate buffer (pH 7.4) containing 0.15 M sodium chloride. Thus, 25 mL of a solution in which bovine serum albumin (fraction V) was dissolved was prepared. The drugs were vancomycin, penicillin G and adrenaline, with initial concentrations of vancomycin and adrenaline being 50 μg / mL and penicillin G being 300 μg / mL. The protein solution to which the drug was added was treated with a filter filled with fiber A or B prepared in Example 1 and control fiber A or B. The capacity of the filled blood product purification material was 10 cm ³ for both fibers A and B and control fibers A and B. The blood flow rate is 10 mL / min., And the contact time is 1 min. It was. Each filter was steam sterilized at 121 ° C. for 20 minutes. Next, the concentration of the drug before and after removal was measured to calculate the removal rate. As for the measurement method, vancomycin was measured by fluorescence polarization immunoassay, and penicillin G and adrenaline were measured by HPLC. The results are shown in Table 7.

  Thus, it was shown that vancomycin, penicillin G and adrenaline contained in the protein solution can be removed at a removal rate of 60% or more.

Claims (9)

At least one functional group selected from urea bond, thiourea bond, amide bond, primary amino group, secondary amino group, tertiary amino group, pyridyl group, pyrimidyl group and imidazole group is fixed on the material surface A material for purifying blood products. The material for purifying blood products according to claim 1, wherein the material surface has a chemical structure represented by the following formula.
-X- (CH ₂ ) nY
X = urea bond, thiourea bond, amide bond, secondary amino group, at least one functional group selected from tertiary amino group
n = 1-20 integer Y = hydrogen, primary amino group, secondary amino group, at least one functional group selected from tertiary amino group The blood product purification material according to claim 1 or 2, comprising a fibrous carrier having a diameter of 100 µm or less. The material for purifying blood products according to any one of claims 1 to 3, wherein pyrogens, cell activators, cells that produce cell activators and drugs are removed from blood components. The blood product purification material according to claim 4, wherein the pyrogenic substance is a microorganism or a toxin produced by the microorganism, and the cell activating substance is one of a cytokine and a platelet activating factor. 6. The blood product purification material according to any one of claims 1 to 5, wherein a removal rate of the pyrogen and the cell activation material is 60% or more, and a removal rate of granulocytes is 40% or more. . The blood product purification material according to any one of claims 1 to 6, wherein the removal rate of erythrocytes, albumin and blood coagulation factors is 30% or less. A blood product purification method comprising using the blood product purification material according to claim 1. A blood product purified using the method according to claim 8.