JP2012213635A - Hollow fiber membrane for removing protein and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a hollow fiber membrane for highly efficiently removing a harmful protein such as a cytokine or the like from blood or the like.SOLUTION: The hollow fiber membrane for removing a protein has a uniform membrane structure, and contains a modified polyalkyleneimine obtained by introducing a functional group containing an amide bond to a polyalkyleneimine to partially replace an amine group.

Description

  The present invention relates to an adsorbent material, and in particular, in a purification module that removes harmful proteins such as cytokines and β2-microglobulin by binding to proteins present in high concentration protein solutions such as in human blood, Or it is related with the hollow fiber membrane used suitably as a coating | covering material, and its manufacturing method.

  Cytokines and β2-microglobulin (hereinafter referred to as β2-MG) are proteins harmful to the human body. Cytokines are described as follows. The cytokines are produced from various cells including immunocompetent cells by stimulation such as infection and trauma. A group of proteins that are released and act on, such as interferon alpha, interferon beta, interferon gamma, interleukin-1 to interleukin-15, tumor necrosis factor alpha, tumor necrosis factor beta, erythropoietin, and monocyte chemotactic factor Many are known. Cytokines are originally considered to be substances produced by the living body for defense of the body, but are classified as inflammatory cytokines such as tumor necrosis factor, interleukin-6, interleukin-8, and monocyte chemotactic activator ( Hereinafter, it has been clarified that excessive production of a group of proteins such as MCAF / MCP-1 is involved in tissue damage and pathology in various inflammatory diseases. For example, it has been reported that administration of tumor necrosis factor to animals induces septic shock, and inhibiting the action of this cytokine is useful for improving pathological conditions. In hypercytokinemia in which these cytokines are released in the blood at high concentrations, such as human sepsis, the blood concentration of interleukin-6 shows a marked increase, and these concentrations are related to the pathophysiology and prognosis. Has been found to correlate with In addition, an autoimmune disease such as rheumatoid arthritis or an allergic disease has been pointed out to be associated with overproduction and disease state. In addition, β2-MG is said to be a causative substance such as dialyzable amyloidosis, and its molecular weight is relatively close to that of albumin, which is a protein useful for the human body, and is easy to remove by dialysis using a conventional membrane. I can not say.

  In addition, in order to treat the above inflammatory diseases by suppressing the action of inflammatory cytokines so far, it specifically binds to target cytokines such as antibodies and soluble receptors and acts on them. It has been attempted to administer in vivo a protein that inhibits or a protein that binds to a receptor competitively with a cytokine, such as a receptor antagonist. However, preparation of a large amount of protein for biological administration is very expensive, and if the administered protein is foreign to the living body, it may induce an immune reaction that is inconvenient for the patient. In many cases, the amount and frequency of administration were limited.

  In addition, attempts have been made so far to remove inflammatory cytokines using urea, thiourea, or a composite fiber (Patent Document 1) that binds polyurea or polythiourea having a plurality of these in the molecular structure. However, since the material combined with urea or thiourea in this invention is insoluble in water, it must be dissolved in an organic solvent and introduced into a substrate, and cannot be introduced into a substrate soluble in an organic solvent. There is a concern that by immersing the base material in the organic solvent, shrinkage and destruction of the fine structure contained in the base material may be caused when the solvent evaporates. Therefore, attempts have been made to remove inflammatory substances using a composite fiber (Patent Document 2) in which a water-soluble polyalkyleneimine is bonded to a base material soluble in an organic solvent. However, the composite fiber bonded with polyalkyleneimine in this invention has a small amount of removal of inflammatory cytokines, and the conventional invention uses fibers or beads, so that it is less than that of porous hollow fiber membranes. There is a problem that the image characteristics are small and the removal efficiency is low because removal of harmful proteins such as cytokines is performed only by the force of diffusion because the force of filtration does not work. Further, although an invention for introducing a urea group into polyethyleneimine in a dry composite semipermeable membrane has been disclosed (Patent Document 3), the urea group is introduced as a functional group for a crosslinking reaction and adsorbs a cytokine. It did not have a function, and the protein could not be removed effectively as a membrane design.

JP 2007-202635 A JP 2002-35117 A JP-A-57-94308

  The subject of this invention is providing the hollow fiber membrane which removes protein from blood etc. efficiently.

The present invention has the following configuration in order to solve the above problems.
1. A hollow fiber membrane for protein removal comprising a modified polyalkyleneimine having a uniform membrane structure, wherein a functional group containing an amide bond is introduced into the polyalkyleneimine and a part of the amine group is substituted.

Here, since the hollow fiber membrane according to the present invention is for protein removal, it is necessary that the functional group containing an amide bond can adsorb and remove the protein and not the functional group for the crosslinking reaction. is there.
2. 2. The protein-removing hollow fiber membrane according to 1, wherein 0.01 mol% or more and 43 mol% or less of the amine group in the polyalkyleneimine is substituted with the functional group containing the amide bond.
3. 3. The hollow fiber membrane for protein removal according to 1 or 2, wherein the functional group containing an amide bond is at least one selected from a urea group, a thiourea group, and an amide group.
4). The hollow fiber membrane for protein removal according to any one of 1 to 3, wherein the albumin sieving coefficient is 0.5% or more and less than 5%.
5. The hollow fiber membrane for protein removal according to any one of 1 to 4 containing a negative charge.
6). The hollow fiber membrane for protein removal according to any one of 1 to 5, wherein the functional group having a negative charge is at least one selected from a sulfo group, a carboxyl group, and an ester group.
7). The hollow fiber membrane for protein removal according to any one of 1 to 6, wherein the material of the hollow fiber membrane includes polymethyl methacrylate or a derivative thereof.
8). The hollow fiber membrane for protein removal according to any one of 1 to 7, wherein the protein is a cytokine.
9. The hollow fiber membrane for protein removal according to 8, wherein the cytokine is an inflammatory cytokine.
10. 10. The hollow fiber membrane for protein removal according to 9, wherein the inflammatory cytokine is interleukin-6.
11. The hollow fiber membrane module in which the hollow fiber membrane in any one of said 1-10 was incorporated.
12 Irradiating a hollow fiber membrane for blood purification having a uniform structure while immersed in a modified polyalkyleneimine solution in which a functional group containing an amide bond is introduced into polyalkyleneimine and a part of the amine group is substituted The manufacturing method of the hollow fiber membrane for protein removal obtained by this.
13. 13. The method for producing a hollow fiber membrane for protein removal according to 12, wherein 0.01 mol% or more and 43 mol% or less of the amine group in the polyalkyleneimine is substituted with a functional group containing the amide bond.
14 14. The method for producing a hollow fiber membrane for protein removal according to 12 or 13, wherein the functional group containing an amide bond is at least one selected from a urea group, a thiourea group, and an amide group.
15. The method for producing a hollow fiber membrane for protein removal according to any one of 12 to 14, wherein the albumin sieving coefficient is 0.5% or more and less than 5%.
16. The method for producing a hollow fiber membrane for protein removal according to any one of 12 to 15, which comprises negative charge.
17. The method for producing a hollow fiber membrane for protein removal according to any one of 12 to 16, wherein the protein is an inflammatory cytokine.
18. The method for producing a hollow fiber membrane for protein removal according to any one of 12 to 17, wherein the inflammatory cytokine is interleukin-6.

  ADVANTAGE OF THE INVENTION According to this invention, the hollow fiber membrane for protein removal which can remove or inactivate excess protein rapidly with high efficiency also in the solution containing many coexisting proteins like blood is realizable.

1 shows an embodiment of an artificial kidney used in the present invention.

  The protein-removing hollow fiber membrane used in the present invention refers to a hollow fiber membrane that is generally used for hemodialyzers, blood filters, slow blood filters for emergency lifesaving, hemodialysis filters, and the like, which are called artificial kidneys. The advantage of using a hollow fiber membrane for the removal of harmful proteins such as cytokines and β2-MG is that the body fluid flows continuously inside or outside the hollow fiber and dialysate (contained in normal serum) on the other side. By flowing an electrolyte of the same concentration as that of the body (for example, a solution containing sodium, potassium, calcium, magnesium, etc. and sodium bicarbonate with ions), inflammatory cytokines contained in the body fluid are diffused or pressured due to the concentration difference. By using filtration based on the difference, removal can be performed with higher efficiency than the conventional method.

  The structure of the protein-removing hollow fiber membrane preferably has a uniform membrane structure in the thickness direction. As the material, polymethyl methacrylate, polyacrylonitrile, polysulfone, polyethersulfone, cellulose acetate, ethylene vinyl alcohol copolymer, polyethersulfone polyarylate polymer alloy, etc. are used. Derivatives are preferably used. The reason for this is that a polymethyl methacrylate-based film, which is a typical example of a film having a uniform film structure in the thickness direction, has many adsorption sites, and removes cytokines and the like by adding a modified polyalkyleneimine to the entire film. This is because the protein to be removed can be removed not only by permeation but also by adsorption, and a large amount of amino acids can be prevented from being lost.

  Moreover, it is desirable that the protein-removing hollow fiber membrane has a negative charge. By having a negative charge, the graft amount of the modified polyalkyleneimine having a positive charge can be improved. It has also been reported that the hydrophilicity increases by including a negatively charged functional group in at least a part of the material, and tends to be finely dispersed (that is, many fine pores are formed). Materials having a substituent such as sulfo group, carboxyl group, phosphoric acid group, phosphorous acid group, ester group, sulfite group, hyposulfite group, sulfide group, phenol group, hydroxysilyl group as the functional group having negative charge Is mentioned. Among these, at least one selected from a sulfo group, a carboxyl group, and an ester group is preferable. As those having a sulfo group, vinyl sulfonic acid, acrylic sulfonic acid, methacryl sulfonic acid parastyrene sulfonic acid, 3-methacryloxypropane sulfonic acid, 3-acryloxypropane sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid and These sodium salts, potassium salts, ammonium salts, pyridine salts, quinoline salts, tetramethylammonium salts and the like can be mentioned. The negative charge amount is preferably 5 μeq or more and 30 μeq or less per 1 g of the dried hollow fiber membrane. The negative charge amount can be measured using, for example, a titration method.

In the hollow fiber membrane according to the present invention, it is important to adjust the sieving coefficient of albumin. Usually, when used as an artificial kidney, for example, albumin is an essential protein for biological activities. For example, when continuously used as an artificial kidney, it is necessary to minimize leakage of albumin. Therefore, the upper limit of the albumin sieving coefficient of the hollow fiber membrane according to the present invention is less than 5%, and preferably less than 3%. However, in the present invention, in order to remove inflammatory cytokines such as interleukin-6 (IL-6) with high efficiency, membrane design may be performed so that the sieving coefficient of albumin is 0.5% or more. is important. The albumin sieving coefficient is preferably 0.8% or more. The albumin sieving coefficient here is a protein anticoagulated using an ACD-A solution in accordance with a method defined by the Japanese Society for Dialysis Medicine (Performance Evaluation Method of Blood Purifier, Journal of Dialysis Society 29 (8) 1231-1245, 1996). Using blood plasma at a concentration of 6-7 g / dL as a blood side solution, blood inlet side fluid, blood from the test solution after 60 minutes when circulating at a blood side flow rate of 200 mL / min and a filtration flow rate of 10 mL / min / m ² It is a value obtained by collecting the outlet side liquid and the filtrate and measuring the albumin concentration in the liquid. A density | concentration is measured using the change of the color tone by the coupling | bonding with pigment | dyes, such as albumin, bromcresol green (henceforth BCG), and bromcresol purple. The filtrate obtained during circulation is returned to the blood side. The calculation formula of the sieve coefficient is in accordance with JIST3250 (2005).

In addition, adjustment of water permeability (water permeability) is also important for the protein removal ability of the hollow fiber membrane. In order to increase the removal amount of proteins such as cytokines, it is effective to increase the water permeability of the membrane. On the other hand, if the water permeability is too high, the surface area inside the membrane decreases, and removal by cytokine adsorption. The amount will decrease. Therefore, the water permeability (UFR) of the protein-removing hollow fiber membrane is 1 mL / hr / m ² / mmHg or more, 100 mL / hr / m ² / mmHg or less, and further 30 mL / hr / m ² / mmHg or more, 80 mL. / Hr / m ² / mmHg or less is preferable. The upper limit is more preferably 70 mL / hr / m ² / mmHg or less. The range may be any one of the above lower limit and the upper limit or a combination thereof. As a method for measuring water permeability, a water pressure of 100 mmHg is applied to the inside of the hollow fiber of a glass tube mini-module (36 pieces: effective length 10 cm) sealed at both ends of the hollow fiber, and the amount of filtration per unit time flowing out to the outside Can be measured. The water permeability (UFR) is calculated by the following formula.
UFR (mL / hr / m ² / mmHg) = Qw / (P × T × A)
Here, Qw: filtration amount (mL), T: outflow time (hr), P: pressure (mmHg), and A: hollow fiber membrane surface area (m ² ).

  Further, in order to efficiently remove inflammatory cytokines such as IL-6, it is important to control the pore radius of the hollow fiber membrane. The average pore radius is preferably 1 nm to 5 μm, more preferably 5 nm to 60 nm. The average pore radius was calculated according to the method using DSC of Ishikiriyama et al. (Ishikiriyama K et al., Thermochimica Acta 267, 169-180 (1995)).

  In the present invention, a functional group containing an amide bond is introduced into a polyalkyleneimine so that a part of the amine group in the polyalkyleneimine is substituted. Here, since the hollow fiber membrane according to the present invention is for protein removal, a functional group containing an amide bond adsorbs any one of cytokines, β2-MG, or a plurality of proteins and removes them from body fluids. It must be possible. Examples of the functional group containing an amide bond include a urea group, a thiourea group, an amide group, and the like, which have good affinity for a living body. A plurality of urea groups, thiourea groups, and amide groups may exist in one compound.

As the compound for introducing a urea group or a thiourea group into the polyalkyleneimine, an isocyanate compound or an isothiocyanate compound can be used. The mixing ratio of the polyalkyleneamine and the isocyanate compound or isothiocyanate compound can be arbitrarily selected. Examples of isocyanate compounds or isothiocyanate compounds include ethyl isocyanate, stearyl isocyanate, n-butyl isocyanate, iso-butyl isocyanate, n-propyl isocyanate, methyl isocyanate, ethyl isocyanate, n-butyl isothiocyanate, benzyl isothiocyanate, hexamethylene diisocyanate. Any of aliphatic isocyanate compounds such as cyclohexyl isocyanate, cyclohexyl isothiocyanate, cyclohexyl diisocyanate or isothiocyanate compounds can be used, but more preferred are phenyl isocyanate, chlorophenyl isocyanate, fluorophenyl isocyanate, bromophenyl isocyanate, nitrophenyl. Socyanate, tolyl isocyanate, methoxyphenyl isocyanate, 1-naphthyl isocyanate, 4,4′-diphenylmethane diisocyanate, 3,3 ′, 5,5′-tetraethyl-4,4′-diisocyanatodiphenylmethane, phenylisothiocyanate, chlorophenyliso An aromatic isocyanate compound such as thiocyanate, fluorophenyl isothiocyanate, nitrophenyl isothiocyanate, tolyl isothiocyanate, methoxyphenyl isothiocyanate, 1-naphthyl isothiocyanate, or isothiocyanate compound is used.
On the other hand, the adsorption performance and hydrophilicity of a polyalkyleneimine having a basic nitrogen atom are determined by the ratio of the number of carbon atoms contained in the main chain alkyleneimine to the number of basic nitrogen atoms, the degree of polymerization of the alkyleneimine, and N-substitution. It varies depending on the ratio of the groups, that is, the N-substitution rate of at least one functional group selected from the group consisting of a urea group, a thiourea group and an amide group. Here, the N-substitution rate is the ratio of the nitrogen in the polyalkyleneimine replaced by nitrogen in the functional group.

If the N-substitution rate of the polyalkyleneimine is too small, the adsorptive capacity decreases, and if it is too large, the solubility in water becomes poor. Therefore, it is 0.05% or more and 60% or less, more preferably 1% or more and 25% or less. The range may be any one of the above lower limit and the upper limit or a combination thereof.
Further, the N-substitution rate can be improved by reducing the steric hindrance of the amine group in the alkyleneimine. For example, by arranging an aliphatic hydrocarbon as a spacer arm between the main chain and the amine group, the efficiency of introducing a urea group, a thiourea group, an amide group, or the like is increased. In addition, protein adsorption can be performed more effectively by reducing steric hindrance using this technique.

  The ratio of the number of carbon atoms to the number of basic nitrogen atoms is difficult to process if the ratio of basic nitrogen atoms is too large, and conversely, if it is too small, the adsorptive capacity decreases, so preferably 1 to 8: 1, more preferably It may be in the range of 1-5: 1.

  In addition, the larger the weight average molecular weight (Mw) of the polyalkyleneimine, the more the inflammatory protein adsorption performance increases. However, when the weight average molecular weight (Mw) is too large, it becomes difficult to fix the grafted chain in an extended form. On the other hand, if the weight average molecular weight is too small, the solubility in water becomes poor when an N-substituent is introduced. Therefore, it is good to be 600-90000, more preferably 1000-750,000. As an upper limit, 100,000 or less is more preferable. The range may be any one of the above lower limit and the upper limit or a combination thereof.

  Based on the above, the introduction ratio to the at least one alkyleneimine selected from urea group, thiourea group and amide group on the surface can range from 0.01 mol% to 43 mol% in terms of the substance amount ratio It is. More preferably, in the case of an amide group alone, the amount ratio is preferably 0.01 mol% or more, more preferably 0.25 mol% or more, preferably 36 mol% or less, more preferably 15 mol% or less, and thiourea group alone. In this case, 0.02 mol% or more is preferable, 0.37 mol% or more is more preferable, 43 mol% or less is preferable, 18 mol% or less is more preferable, and in the case of a urea group alone, 0.02 mol% or more is preferable, and 0.31 mol% % Is more preferable, more preferably 3 mol% or more, and a preferable upper limit is 40 mol% or less, and 18 mol% or less is preferable for ensuring hydrophilicity, and 17 mol% or less is more preferable. The range may be any one of the above lower limit and the upper limit or a combination thereof.

  The synthesis reaction of the above polyalkyleneimine and a compound having a urea group, a thiourea group or an amide bond is typically carried out at a reaction temperature of 0 to 150 ° C. and a reaction time of 0.1 to 24 hours. In addition, a reaction solvent is not necessarily required, but it is generally performed in the presence of a solvent. Solvents that can be used include aliphatic hydrocarbons such as methanol, ethanol, isopropanol, n-butanol, hexane, acetone, N, N-dimethylformamide, and dimethyl sulfoxide, and aromatic hydrocarbons such as benzene, toluene, and xylene. And halogenated hydrocarbons such as dichloromethane, chloroform and chlorobenzene, and ethers such as diethyl ether, tetrahydrofuran and dioxane. The reaction solution after completion of the reaction can be purified by operations such as column chromatography, recrystallization and the like after usual treatments such as filtration and concentration, if necessary. In the case of a water-insoluble material, it is also a preferable method to wash using a glass filter or the like.

  The weight average molecular weight of the modified polyalkyleneimine can be measured using gel permeation chromatography (GPC). Solvents that can be used in GPC analysis include aliphatic hydrocarbons such as methanol, ethanol, isopropanol, n-butanol, hexane, acetone, N, N-dimethylformamide, and dimethyl sulfoxide, and aromatics such as benzene, toluene, and xylene. Group hydrocarbons, halogenated hydrocarbons such as dichloromethane, chloroform and chlorobenzene, ethers such as diethyl ether, tetrahydrofuran and dioxane, etc. are used, and the molecular weight is preferably a value in terms of polystyrene.

  For example, in a N, N-dimethylformamide solvent, polyethyleneimine (weight average molecular weight: 10,000) is used as a polyalkyleneimine, and chlorophenylisocyanate added in the reaction process when chlorophenylisocyanate is used as an amino compound is 1 mol of polyethyleneimine. Preferably, it is in the range of 1 mmol to 100,000 mmol, more preferably 10 mmol to 2000 mmol, but the addition amount when other solvents are used is not limited to this. In addition, the range which combined any of the said upper limit and a minimum may be sufficient.

  As a method for introducing the modified polyalkyleneimine into the hollow fiber membrane containing polymethyl methacrylate or a derivative thereof, there is a method using a radiation graft method. In particular, when a graft reaction is used, it is a preferable method because the graft reaction and sterilization can be performed simultaneously. That is, after the separation membrane is incorporated in the module, the inside of the module is filled with an aqueous solution of a modified polyalkyleneimine, and irradiation is performed by irradiating a sterilizing dose. If irradiation is performed after modularization, the entire module can be sterilized at the same time, which is preferable. For example, examples of radiation to be used include α rays, β rays, γ rays, X rays, ultraviolet rays, and electron beams. Particularly, γ rays and electron beams are preferably used. In order to sterilize the blood purification module with γ-rays, a dose of 15 kGy or more is preferable.

  As a method for producing a protein removal module using a hollow fiber membrane in the present invention, there are various methods depending on its use. As a rough process, a process for producing a separation membrane for blood purification and the separation membrane as a module are provided. It can be divided into the process of incorporating into the process.

  When the modified base material of the present invention is a separation membrane, a polymer containing an ester group in the main chain or side chain and having a modified polyalkyleneimine may be synthesized and molded into the separation membrane. Further, after the separation membrane is formed, a graft reaction of the modified polyalkyleneimine onto the separation membrane by irradiation with radiation may be used.

  Next, an example about the manufacturing method of the protein removal module using a hollow fiber membrane is shown. As a manufacturing method of the hollow fiber membrane incorporated in the protein removal module, there are the following methods. That is, 5 parts by weight of iso-polymethyl methacrylate and 20 parts by weight of syn-polymethyl methacrylate are added to 75 parts by weight of dimethyl sulfoxide and dissolved by heating to obtain a film forming stock solution. This membrane-forming stock solution is discharged from an orifice-type double-cylinder die, allowed to pass through 300 mm in air, and then guided into a 100% water coagulation bath to obtain a hollow fiber separation membrane. At this time, dry nitrogen is used as the internal injection gas.

  The method of incorporating the hollow fiber membrane in the module is not particularly limited, but an example is as follows. First, the hollow fiber membrane is cut to a required length, bundled in a necessary number, and then put into a cylindrical case. Then, a temporary cap is put on both ends, and a potting agent is put on both ends of the hollow fiber membrane. At this time, the method of adding the potting agent while rotating the module with a centrifuge is a preferable method because the potting agent is uniformly filled. After the potting agent is solidified, both ends are cut so that both ends of the hollow fiber membrane are open, and a hollow fiber membrane module is obtained.

  Further, the filling rate of the protein-removing hollow fiber membrane into the module case can be obtained from the following equation using the outer diameter of the hollow fiber membrane.

Filling factor = (((hollow fiber ^{Makugai径) 2} × hollow fiber membrane number) / (the module case body inner ^{diameter) 2)} × 100 [%]
Thus, when the outer diameter of the hollow fiber membrane is reduced, the filling rate is lowered, and when the outer diameter of the hollow fiber membrane is increased, the filling rate is also increased. However, when the outer diameter of the hollow fiber membrane is too large, the amount of blood introduced into the module decreases, so that the protein removal efficiency decreases. Therefore, the outer diameter of the hollow fiber membrane is preferably 220 μm or more and 300 μm or less. Here, the outer diameter of the hollow fiber membrane is the average value obtained by measuring the diameter of each of the 16 hollow fiber membranes randomly extracted from the bundle of hollow fiber membranes using a laser displacement meter or a microwatcher. It is taken. The filling rate is preferably 50% or more and 62% or less, more preferably 55% or more and 60% or less, and may be a range in which any of the above upper limit and lower limit is combined. By designing the module within this range, a short path is unlikely to occur, so that the diffusion performance is improved and the module can be easily inserted into the module case. Further, in order to further improve the diffusion performance, a fiber may be wound around the hollow fiber membrane for protein removal as a spacer.

  An example of the basic structure of a blood purification module using the hollow fiber membrane obtained as described above is shown in FIG. A bundle of hollow fiber membranes 5 is inserted into a cylindrical module case 7, and both ends of the hollow fiber are sealed with a potting part 10. The case 7 is provided with an inlet 8 and an outlet 9 for dialysate, and dialysate, physiological saline, filtered water and the like flow outside the hollow fiber membrane 5. An arterial header 1 and a venous header 2 are provided at the ends of the case 7, respectively. The blood 6 is introduced from the blood introduction port 3 provided in the arterial header 1 and is guided into the hollow fiber membrane 5 by the funnel-shaped arterial header 1. The blood 6 filtered by the hollow fiber membrane 5 is collected in the venous header 2 and discharged from the blood outlet 4. A blood circuit 11 is connected to the blood inlet 3 and the blood outlet 4.

  The protein-removing hollow fiber membrane in the present invention can be used not only as a single unit but also as a single module by further immobilizing on a suitable base material or mixing with other materials. When a column using the material produced in the present invention is used as an extracorporeal circulation column, blood derived outside the body may be passed directly through the column, or may be used in combination with a plasma separation membrane or the like.

  The materials produced in the present invention can be removed or inactivated with high efficiency when materials capable of adsorbing inflammatory cytokines are selected, but such inflammatory cytokines are particularly limited. Instead, it generally removes or inactivates a group of proteins such as generally classified tumor necrosis factor (TNF), IL-6, IL-8, and monocyte chemotaxis activator (MCAF / MCP-1). . Especially, the material in this invention can be used suitably for hypercytokinemia treatment.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

-Preparation of substrate (1) 31.7 parts by weight of syn-PMMA having a weight average molecular weight of 400,000, 31.7 parts by weight of syn-PMMA having a weight average molecular weight of 1.4 million, and an iso having a weight average molecular weight of 500,000 -Mixing 20 parts by weight of PMMA copolymer containing 16.7 parts by weight of PMMA and 1.5 mol% of parastyrene sulfonic acid soda with 376 parts by weight of dimethyl sulfoxide, and stirring at 110 ° C for 8 hours. Was prepared. The obtained spinning dope had a viscosity at 110 ° C. of 1240 poise. The resulting spinning dope is heated in air at a rate of 2.5 g / min from a double tube hollow fiber membrane die having an outer diameter / inner diameter of 2.1 / 1.95 mmφ of an annular slit portion kept at 99 ° C. Was discharged. At the same time, nitrogen gas was injected into the inner tube portion of the double tube, and the air portion was allowed to travel 50 cm before being led to a coagulation bath. The water temperature used in the coagulation bath (coagulation bath temperature) was changed between 38 to 48 ° C. as shown in Table 1, and different hollow fiber membranes were obtained. After washing each hollow fiber membrane with water, glycerin was added as a 63% by weight aqueous solution as a humectant, the heat treatment bath temperature was changed from 78 ° C. to 85 ° C. as shown in Table 1, and excess glycerin was removed. Was wound up at 60 m / min. The resulting hollow fiber separation membrane had an inner diameter of 0.2 mm and a film thickness of 0.03 mm.

(2) 12,000 hollow fibers obtained were inserted into a cylindrical plastic case having a dialysate inlet and a dialysate outlet as shown in FIG. A hollow fiber membrane module for protein removal of 6 m ² was prepared. The membrane area is a value calculated by multiplying the hollow fiber inner surface area (excluding the portion sealed with urethane resin) calculated from the hollow fiber inner diameter by the number of yarns inserted into the module and the end face length.
2. Method for producing modified hollow fiber membrane (1) An aqueous solution was prepared by dissolving the polymer used for modifying the hollow fiber membrane produced in Example 1 described later in degassed pure water. Here, degassed water means water stirred at room temperature for 30 minutes to 1 hour under a reduced pressure of −500 to −760 mmHg. Dissolved oxygen in water becomes a radical initiator during γ-ray irradiation. Therefore, care must be taken because non-degassed water contributes to variations in subsequent experiments.

(2) The above aqueous solution is used for 1. The hollow fiber membrane module prepared in (2) is inserted from the blood inlet 3, guided from the blood outlet 4 to the dialysate outlet 9, and discharged from the dialysate inlet 8, and the aqueous solution is used for the hollow fiber membrane module Filled. At this time, the flow rate of the aqueous solution was 450 mL / min, and the passing time was 2 minutes. The hollow fiber membrane module was irradiated with 25 kGy of γ-rays to simultaneously modify the hollow fiber membrane and sterilize the hollow fiber membrane module.
Example 1
A mixed solution consisting of 2.3 g of polyethyleneimine (weight average molecular weight: 10,000), 3.8 g of 4-chlorophenyl isocyanate, and 60 ml of N, N-dimethylformamide was reacted at 30 ° C. for 1 hour. After the reaction, 80 ml of pure water was added to form a white precipitate. The supernatant from which the white precipitate had been removed was applied to an evaporator and dried under reduced pressure to obtain a modified polyalkyleneimine.

1. 1. Using the aqueous solution containing 0.1% by weight of modified polyalkyleneimine (hereinafter referred to as 0.1% by weight ethanol aqueous solution) to the hollow fiber membrane module obtained in (1) and (2). (1) The hollow fiber membrane was simultaneously modified and sterilized according to the procedure of (2). The introduction rate of 4-chlorophenyl isocyanate in the modified polyalkyleneimine was measured using nuclear magnetic resonance (NMR). In nuclear magnetic resonance, 1H-NMR measurement was performed using a 270 MHz Super-conducting magnet device manufactured by JEOL Ltd. In the obtained spectrum, the integrated value of the hydrogen peak contained in the polyalkyleneimine main chain (2 to 3 ppm based on tetramethylsilane) is B, and the hydrogen peak contained in the amine in the urea group (8 based on tetramethylsilane). When the integral value of 0.5 to 9.5 ppm) is A, the introduction rate of urea groups into the polyalkyleneimine was determined by the following formula.
Introduction rate (mol%) = A / (A + B) × 100
Using the prepared modified hollow fiber membrane module, a removal test for IL-6 (hereinafter, h-IL-6) (manufactured by KTS), which is a human-derived cytokine, was performed. h-IL-6 was dissolved in fetal bovine serum (manufactured by Sigma) so as to have a concentration of 10 ng / ml to obtain a solution to be removed. 10 ml of this solution to be removed was circulated through the mini-module to carry out a removal reaction, and the h-IL-6 concentration in the reaction solution before and after the removal was measured by ELISA (enzyme-linked immunosorbent method). For the ELISA method, a human IL-6 ELISA kit (manufactured by KTS) was used. As a specific technique, a 96-well antibody plate coated with anti-human IL-6 polyclonal antibody was first washed with 400 μl of phosphate buffer. Discard the solution by a general method using ELISA, add 50 μl of Tris buffer to each well, add 100 μl of standard h-IL-6 prepared to the concentration for the sample and the calibration curve, and then add room temperature (20 ° C. to 30 ° C.). ) For 60 minutes, the solution was discarded and washing was performed 3 times. After adding 100 μl of HRP-labeled anti-human IL-6 mouse monoclonal antibody to each well, the mixture was shaken at room temperature (20 ° C. to 30 ° C.) for 30 minutes, then the solution was discarded and washing was performed 3 times. Each well was charged with 100 μl of a TMB (3,3 ′, 5,5′-tetramethylbenzidine) solution as a color developing solution, and then subjected to a shaking reaction at room temperature (20 ° C. to 30 ° C.) for 60 minutes. 100 μl of 0.5 mol / l sulfuric acid as a stop solution was added to the well. Immediately after completion of the reaction, absorbance at a wavelength of 450 nm was measured using a microplate reader (MPR-A4iII manufactured by TOSOH) (600 nm was used as a control wavelength).
Example 2
A mixed solution consisting of 2.3 g of polyethyleneimine (weight average molecular weight: 10,000), 5.2 g of 4-chlorophenyl isocyanate, and 60 ml of N, N-dimethylformamide was reacted at 30 ° C. for 1 hour. After the reaction, 80 ml of pure water was added to form a white precipitate. The supernatant from which the white precipitate had been removed was applied to an evaporator and dried under reduced pressure to obtain a modified polyalkyleneimine.
Subsequent steps and evaluation were in accordance with Example 1.
Example 3
A mixed solution consisting of 2.3 g of polyethyleneimine (weight average molecular weight: 1800), 3.8 g of 4-chlorophenyl isocyanate, and 60 ml of N, N-dimethylformamide was reacted at 30 ° C. for 1 hour. After the reaction, 80 ml of pure water was added to form a white precipitate. The supernatant from which the white precipitate had been removed was applied to an evaporator and dried under reduced pressure to obtain a modified polyalkyleneimine.
Subsequent steps and evaluation were in accordance with Example 1.
Example 4
A mixed solution consisting of 2.3 g of polyethyleneimine (weight average molecular weight: 750000), 3.8 g of 4-chlorophenyl isocyanate, and 60 ml of N, N-dimethylformamide was reacted at 30 ° C. for 1 hour. After the reaction, 80 ml of pure water was added to form a white precipitate. The supernatant from which the white precipitate had been removed was applied to an evaporator and dried under reduced pressure to obtain a modified polyalkyleneimine. Subsequent steps and evaluation were in accordance with Example 1.
Example 5
A mixed solution consisting of 2.3 g of polyallylamine (weight average molecular weight: 5000), 3.8 g of 4-chlorophenyl isocyanate, and 60 ml of N, N-dimethylformamide was reacted at 30 ° C. for 1 hour. After the reaction, 80 ml of pure water was added to form a white precipitate. The supernatant from which the white precipitate had been removed was applied to an evaporator and dried under reduced pressure to obtain a modified polyalkyleneimine.
Subsequent steps and evaluation were in accordance with Example 1.
Example 6
A mixed solution consisting of 2.3 g of polyethyleneimine (weight average molecular weight: 10,000), 5.2 g of 4-chlorophenyl isocyanate, and 60 ml of N, N-dimethylformamide was reacted at 30 ° C. for 1 hour. After the reaction, 80 ml of pure water was added to form a white precipitate. The supernatant from which the white precipitate had been removed was applied to an evaporator and dried under reduced pressure to obtain a modified polyalkyleneimine.
Subsequent steps were in accordance with Example 1. About the obtained modification | reformation hollow fiber membrane module, the clearance measurement of (beta) 2-MG was performed by the method shown below. The results are shown in Table 2.
a. Using the prepared modified hollow fiber membrane module, a removal test for β2-MG, which is a human-derived protein, was performed. β2-MG was dissolved in fetal bovine serum (manufactured by Sigma) to a concentration of 1 mg / l to obtain a solution to be removed. 10 ml of this solution to be removed was circulated through the mini-module to carry out the removal reaction, and the β2-MG concentration in the sample in the reaction solution before and after the removal was measured using a latex agglutination immunoassay (for reference, medical and test equipment). -Reagent 26 (2) 127-134, 2003).
Comparative Example 1
1. (1) For the hollow fiber membrane module obtained in (2), 2. (1) Instead of the modified polyalkyleneimine aqueous solution used for modification in the procedure shown in (2), pure water is filled; (1) Sterilization was performed according to the procedure shown in (2). Thereafter, the same removal test as in Example 1 was performed, and h-IL-6 concentrations before and after removal were measured in the same manner as in Example 1 by ELISA.
Comparative Example 2
1. (1) For the hollow fiber membrane module obtained in (2), 2. (1) Instead of the aqueous solution of the modified polyalkylenimine used for the modification in the procedure shown in (2), a 0.1% by weight polyethyleneimine (weight average molecular weight: 10,000) aqueous solution is filled; (1) Sterilization was performed according to the procedure shown in (2). Thereafter, the same removal test as in Example 1 was performed, and h-IL-6 concentrations before and after removal were measured in the same manner as in Example 1 by ELISA.
Comparative Example 3
A mixed solution consisting of 2.3 g of tetraethylenepentamine, 3.8 g of 4-chlorophenyl isocyanate and 60 ml of N, N-dimethylformamide was reacted at 30 ° C. for 1 hour. After the reaction, 80 ml of pure water was added to form a white precipitate. The supernatant from which the white precipitate was removed was applied to an evaporator and dried under reduced pressure to obtain a low molecular weight modified polyalkyleneimine.
1. The low molecular weight modified polyalkyleneimine In order to obtain a 0.1 wt% aqueous solution by dissolving in water according to the procedure shown in (1), it was not possible to obtain a uniform aqueous solution due to poor solubility in water.
Comparative Example 4
1. (1) For the hollow fiber membrane module obtained in (2), 2. (1) Instead of the modified polyalkyleneimine aqueous solution used for modification in the procedure shown in (2), pure water is filled; (1) Sterilization was performed according to the procedure shown in (2). Thereafter, the obtained modified hollow fiber membrane module was subjected to a. The clearance measurement of β2-MG was performed by the method shown in FIG. The results are shown in Table 2.

Thus, in the material used in Example 1, the IL-6 removal rate was improved by 5.7% compared to Comparative Example 1 and 26.2% compared with Comparative Example 2. Further, as shown in Example 2, the IL-6 removal rate tended to further increase by increasing the introduction rate of the modified polyalkyleneimine. Thus, it was shown that IL-6 can be efficiently removed by using a modified polyalkyleneimine.
As the weight average molecular weight of polyethyleneimine used as polyalkyleneimine, it was shown that it can be used from 1800 to 750,000 as shown in Examples 1, 3, and 4. However, as can be seen from Example 3, when the molecular weight was small, the length of the graft chain was not sufficient, and the IL-6 removal rate tended to decrease. In addition, when the weight average molecular weight of Example 4 was 750,000, the IL-6 removal rate was lower than that of Example 1, and thus it was considered difficult to immobilize the graft chain in an extended form because the molecular weight was large. .
As can be seen from Comparative Example 3, when tetraethylenepentamine having a molecular weight of 189 was used, a uniform aqueous solution containing the modified polyalkyleneimine could not be obtained. This is considered due to an increase in the proportion of hydrophobic groups in the modified polyalkyleneimine.
Also when polyallylamine was used as the polyalkyleneimine, the IL-6 removal rate was improved by 4.7% as compared with Comparative Example 1, as shown in Example 5. This is considered to be because urea groups were introduced more efficiently by using an alkyleneimine containing a primary amine having a small steric hindrance such as allylamine.

Thus, in the material used in Example 6, the β2-MG removal rate was improved by 24.0% compared to Comparative Example 3. Further, as shown in Example 2, the β2-MG removal rate tended to further increase by increasing the introduction rate of the modified polyalkyleneimine. Thus, it was shown that β2-MG can be efficiently removed by using a modified polyalkyleneimine.

1. 1. Arterial header 2. Venous header 3. Blood introduction port 4. Blood outlet Hollow fiber membrane6. Blood 7. Module case 8. Dialysate inlet 9. Dialysate outlet 10. Potting unit 11. Blood circuit

Claims (18)

A hollow fiber membrane for protein removal comprising a modified polyalkyleneimine having a uniform membrane structure, wherein a functional group containing an amide bond is introduced into the polyalkyleneimine and a part of the amine group is substituted. The hollow fiber membrane for protein removal according to claim 1, wherein 0.01 mol% or more and 43 mol% or less of the amine group in the polyalkyleneimine is substituted with a functional group containing the amide bond. The hollow fiber membrane for protein removal according to claim 1 or 2, wherein the functional group containing an amide bond is at least one selected from a urea group, a thiourea group, and an amide group. The hollow fiber membrane for protein removal according to any one of claims 1 to 3, wherein the albumin sieving coefficient is 0.5% or more and less than 5%. The hollow fiber membrane for protein removal according to any one of claims 1 to 4, comprising a negative charge. The hollow fiber membrane for protein removal according to any one of claims 1 to 5, wherein the functional group having a negative charge is at least one selected from a sulfo group, a carboxyl group, and an ester group. The hollow fiber membrane for protein removal according to any one of claims 1 to 6, wherein the material of the hollow fiber membrane contains polymethyl methacrylate or a derivative thereof. The protein-removing hollow fiber membrane according to any one of claims 1 to 7, wherein the protein is a cytokine. The hollow fiber membrane for protein removal according to claim 8, wherein the cytokine is an inflammatory cytokine. The hollow fiber membrane for protein removal according to claim 9, wherein the inflammatory cytokine is interleukin-6. The hollow fiber membrane module in which the hollow fiber membrane in any one of said 1-10 was incorporated. Irradiating a hollow fiber membrane for blood purification having a uniform structure while immersed in a modified polyalkyleneimine solution in which a functional group containing an amide bond is introduced into polyalkyleneimine and a part of the amine group is substituted The manufacturing method of the hollow fiber membrane for protein removal obtained by this. The method for producing a hollow fiber membrane for protein removal according to claim 12, wherein 0.01 mol% or more and 43 mol% or less of the amine group in the polyalkyleneimine is substituted with a functional group containing the amide bond. The method for producing a hollow fiber membrane for protein removal according to claim 12 or 13, wherein the functional group containing an amide bond is at least one selected from a urea group, a thiourea group, and an amide group. The method for producing a hollow fiber membrane for protein removal according to any one of claims 12 to 14, wherein the albumin sieving coefficient is 0.5% or more and less than 5%. The method for producing a hollow fiber membrane for protein removal according to any one of claims 12 to 15, wherein the method comprises a negative charge. The method for producing a hollow fiber membrane for protein removal according to any one of claims 12 to 16, wherein the protein is an inflammatory cytokine. The method for producing a hollow fiber membrane for protein removal according to any one of claims 12 to 17, wherein the inflammatory cytokine is interleukin-6.

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