Membranes containing immobilized anticoagulants and methods for making same

Abstract

The present disclosure relates to anticoagulant-coated microporous hollow fiber membranes that exhibit reduced thrombogenicity. The disclosure further relates to methods for making the membranes, and to filtration and / or diffusion devices that include the membranes.

Description

[Technical Field]

[0001] The present disclosure relates to anticoagulant-coated microporous hollow fiber membranes with reduced thrombogenicity. The disclosure further relates to methods for making the membranes and filtration and / or diffusion devices containing the membranes. [Background technology]

[0002] In extracorporeal blood circuits, systemic anticoagulation is commonly used to prevent blood clotting, i.e., the formation of microthrombi and blood coagulation. Heparin is the most commonly used anticoagulant. Systemic anticoagulation using heparin is associated with an increased risk of bleeding, and heparin is quite expensive. Therefore, a membrane capable of immobilizing heparin is highly desirable, as it can reduce or even eliminate the need for systemic heparin administration.

[0003] US2003 / 0021826A1 proposes the stable binding of an anticoagulant to the surface of a semipermeable support membrane essentially consisting of a copolymer of acrylonitrile and at least one anionic or anionizable monomer. The anticoagulant can exert its anticoagulant activity without leaching into the blood or plasma during extracorporeal circulation treatment, thereby reducing the amount of anticoagulant used systemically in patients during extracorporeal blood treatment sessions. The surface of the semipermeable support membrane intended to come into contact with blood or plasma is successively coated with a cationic polymer, e.g., polyethyleneimine (PEI), bearing cationic groups capable of forming ionic bonds with the anionic or anionizable groups of polyacrylonitrile, and an anticoagulant (e.g., heparin) bearing anionic groups capable of forming ionic bonds with the cationic groups of the cationic polymer.

[0004] US5840190A discloses a surface-modified biocompatible membrane. The membrane is composed of polysulfone, polyvinylpyrrolidone, and polyethyleneimine (PEI). The membrane surface is modified by reacting with a biologically active compound and a coupling agent. In the examples, heparin partially decomposed with nitrous acid is covalently coupled to the membrane surface using sodium cyanoborohydride as the coupling agent.

[0005] EP1024886A1 discloses a method for producing a composite membrane having a hydrophilic coating layer on a hydrophobic support membrane, in which the hydrophobic support membrane is impregnated with an aqueous solution of a reactant, the reactant being dispersed on the surface and in the pores of the hydrophobic support membrane, and a thin coating of a hydrophilic polymer is formed on the hydrophobic support membrane through a reaction between the hydrophilic polymer and the reactant. The hydrophobic support membrane is made of polysulfone or polyetherimide, and the hydrophilic polymer is made of sodium alginate, chitosan, or polyvinyl alcohol. When chitosan is used as the hydrophilic polymer, the reactant is sulfuric acid, and the concentration of the reactant in the aqueous solution is in the range of 0.5 to 20%.

[0006] US10500549B2 discloses a multilayer composite dialysis membrane comprising a base membrane based on polysulfone and at least one pore-forming hydrophilic additive, which is polyvinylpyrrolidone, a short-chain glycol, triethylene glycol, propylene glycol, or polyethylene glycol / polyethylene oxide; a functional layer disposed on the base membrane and formed from a layer of a polycationic binder, which is polyethyleneimine, chitosan, polylysine, polyarginine, or polyornithine; and another layer of a polymeric polyanion, which is a carboxylated or sulfated polysaccharide selected from dextran sulfate having a molecular weight (Mw) of 15 kDa to 1 MDa, sulfated chitosan having a molecular weight (Mw) of 30 kDa to 750 kDa, cellulose sulfate having a molecular weight (Mw) of 20 kDa to 1 MDa, and mixtures thereof.

[0007] US 2019 / 076787 A1 discloses a method for forming a microporous hollow fiber membrane, which includes extruding a polymer dope solution through an outer ring conduit of a spinneret while simultaneously passing a precipitation liquid through the inner hollow core of the spinneret. The polymer dope solution contains a membrane-forming polymer selected from polysulfone, polyarylsulfone, polyarylethersulfone, polyvinylidene fluoride, polyacrylonitrile, or copolymers thereof, and optionally a hydrophilic polymer such as polyvinylpyrrolidone or polyethylene glycol. The precipitation liquid contains an additive with limited water solubility, such as polyurethane, chitosan, or other N-containing polymers with oxygen-containing moieties. When chitosan is used as the additive, it can be protonated using a weak acid such as acetic acid, acetic anhydride, lactic acid, formic acid, or a combination thereof, before being introduced into the precipitation liquid.

[0008] WO 2020 / 144244 A1 discloses an anticoagulant-coated microporous hollow fiber membrane exhibiting reduced thrombogenicity. The hollow fiber membrane comprises a blend of i) polysulfone, polyethersulfone, or polyarylethersulfone, ii) polyvinylpyrrolidone, and iii) at least one polymer having ammonium groups selected from the group consisting of polyvinylpyridine having ammonium groups and copolymers of vinylpyridine and styrene having ammonium groups. The anticoagulant is grafted onto at least one surface of the membrane.

[0009] WO2007 / 069983A1 describes a method for extracorporeal removal of pathogenic microorganisms, inflammatory cells, or inflammatory proteins from mammalian blood. The method uses a device containing heparin immobilized on a solid substrate. Preparative Example 4 discloses the covalent attachment of heparin to an aminated polymer surface. The polymer surface is etched with an oxidizing agent, subsequently treated with polyamine, polyethyleneimine (PEI), or chitosan, and further stabilized by ionic cross-linking with heparin.

[0010] Linhardt, Robert et al., "Immobilization of Heparin: Approaches and Applications," Current Topics in Medicinal Chemistry, Vol. 8, No. 2, 2008-01-01, pp. 80-100, discusses and compares various techniques used to immobilize heparin and the applications of these immobilized heparins. The use of chitosan as a linker between reactive groups present on the substrate surface and heparin molecules is disclosed. The surface of a polysulfone membrane was first treated with ozone and then with an aqueous solution of acrylic acid (AA) containing FeSO4 to generate carboxyl groups on the surface. Chitosan molecules were coupled to these carboxyl groups using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Heparin was then conjugated to the chitosan molecules using glutaraldehyde (GA).

[0011] It has now been discovered that anticoagulants such as heparin can be immobilized on membranes prepared from spinning solutions containing polyethersulfone, polyvinylpyrrolidone, and chitosan. The resulting heparin-coated microporous hollow fiber membranes exhibit reduced thrombogenicity. Filtration and / or diffusion devices containing heparin-coated membranes can reduce or even eliminate the need for systemic anticoagulation in extracorporeal blood circuits. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] US Patent Application Publication No. 2003 / 0021826 [Patent Document 2] U.S. Patent No. 5,840,190 [Patent Document 3] European Patent Application Publication No. 1024886 [Patent Document 4] U.S. Patent No. 10,500,549 [Patent Document 5] US Patent Application Publication No. 2019 / 076787 [Patent Document 6] International Publication No. 2020 / 144244 [Patent Document 7] International Publication No. 2007 / 069983 [Non-patent literature]

[0013] [Non-Patent Document 1] Linhardt, Robert et al., "Immobilization of Heparin: Approaches and Applications," Current Topics in Medicinal Chemistry, Vol. 8, No. 2, 2008-01-01, pp. 80-100 Summary of the Invention

[0014] The present disclosure provides a porous hollow fiber membrane having an anticoagulant immobilized thereon. The membrane comprises i) polysulfone, polyethersulfone, or polyarylethersulfone, ii) polyvinylpyrrolidone, and iii) chitosan. The present disclosure also provides a method for producing the porous hollow fiber membrane having an anticoagulant immobilized thereon. The present disclosure further provides a filtration and / or diffusion device including the porous hollow fiber membrane having an anticoagulant immobilized thereon. The filtration and / or diffusion device, e.g., a hemodialysis device, can be used in extracorporeal blood treatment, for example, in hemodialysis, hemodiafiltration, and hemofiltration. DETAILED DESCRIPTION OF THE INVENTION

[0015] In one aspect of the present invention, a porous hollow fiber membrane having an anticoagulant immobilized thereon is provided, the membrane comprising a blend of i) polysulfone (PSU), polyethersulfone (PESU), or polyarylethersulfone (PAES), ii) polyvinylpyrrolidone (PVP), and iii) chitosan.

[0016] Examples of suitable polysulfones include those having a weight average molecular weight M of about 40,000 to 80,000 Da. wIn one embodiment, the polysulfones include those having a weight average molecular weight M in the range of 45 to 65 kDa (as determined by GPC). w An example is a polysulfone having a weight average molecular weight M of 52 kDa. w (determined by GPC) and a polydispersity M of 3.5 w / M n Another example is a polysulfone having a weight average molecular weight M of 60 kDa. w (determined by GPC) and a polydispersity M of 3.7 w / M n It is a polysulfone having the formula:

[0017] Examples of suitable polyethersulfones include those having a weight average molecular weight M of about 40,000 to 100,000 Da. w In one embodiment, the polyethersulfone has a weight average molecular weight M in the range of 50 to 60 kDa (as determined by GPC). w An example is a polyethersulfone having a weight average molecular weight M of 58 kDa. w (determined by GPC) and polydispersity M of 3.3 w / M n In another embodiment, the polyethersulfone has a weight average molecular weight M in the range of 65 to 80 kDa. w An example is a polyethersulfone having a weight average molecular weight M of 75 kDa. w (determined by GPC) and polydispersity M of 3.4 w / M n In yet another embodiment, the polyethersulfone has a weight average molecular weight M in the range of 80 to 100 kDa. w An example is a polyethersulfone having a weight average molecular weight M of 92 kDa. w (determined by GPC) and a polydispersity M of 3.0 w / M n It is a polyethersulfone having the formula:

[0018] Suitable polyvinylpyrrolidones have a weight average molecular weight M in the range of 50 kDa to 2,000 kDa. wThese homopolymers generally have a number average molecular weight M in the range of 14 kDa to 375 kDa. n Examples of polyvinylpyrrolidones suitable for preparing the membranes of the present invention are Luvitec® K30, Luvitec® K85, Luvitec® K90, and Luvitec® K90HM, all available from BASF SE.

[0019] One embodiment of the porous hollow fiber membrane of the present disclosure has a weight average molecular weight M of about 1,100 kDa. w , and a number average molecular weight M of approximately 250 kDa n The polyvinylpyrrolidone homopolymer has the formula:

[0020] Further embodiments of the porous hollow fiber membranes of the present disclosure include those having a weight average molecular weight M of about 1,400 kDa. w , and a number average molecular weight M of approximately 325 kDa n The polyvinylpyrrolidone homopolymer has the formula:

[0021] Chitosan is a linear polysaccharide composed of randomly distributed β-(1→4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan is commercially produced by deacetylation of chitin, a structural component of the exoskeleton of crustaceans (such as crabs and shrimp) and the cell walls of fungi. The degree of deacetylation (%DD) can be determined by NMR spectroscopy, and the %DD of commercially available chitosan ranges from 60 to 100%. The weight-average molecular weight M of commercially produced chitosan is w is generally in the range of 3 kDa to 400 kDa.

[0022] One embodiment of the porous hollow fiber membrane of the present disclosure comprises chitosan having a degree of deacetylation of at least 75%, i.e., up to 25% of the hydroxyl groups of the chitosan are acetylated. Another embodiment comprises chitosan having a degree of deacetylation of at least 80%, e.g., at least 90%, or even at least 95%.

[0023] One embodiment of the porous hollow fiber membrane of the present disclosure has a weight average molecular weight M as determined by GPC analysis in the range of 5 kDa to 375 kDa, e.g., 50 to 190 kDa. w The chitosan has the formula:

[0024] In one embodiment, the porous hollow fiber membranes of the present disclosure comprise 0.1 to 5 wt % chitosan based on the total weight of the porous hollow fiber membrane. In a further embodiment, the porous hollow fiber membranes of the present disclosure comprise 0.5 to 3 wt % chitosan based on the total weight of the porous hollow fiber membrane.

[0025] The hollow fiber membranes of the present disclosure have either a symmetric wall structure or an asymmetric wall structure. In one embodiment, the membrane wall has a symmetric spongy structure. In another embodiment, the membrane wall has an asymmetric spongy structure, i.e., the pore size of the hollow fiber wall increases from the inner surface to the outer surface of the membrane. In yet another embodiment of the method, the membrane wall has an asymmetric wall structure, including layers with a finger structure, i.e., layers characterized by macrovoids with a volume-equivalent diameter greater than 5 μm.

[0026] In one embodiment, the hollow fiber membranes of the present disclosure have an inner diameter of 150-250 μm, e.g., 180-250 μm. In another embodiment, the inner diameter is in the range of 185 μm-195 μm. In yet another embodiment, the inner diameter is in the range of 195 μm-205 μm. In yet another embodiment, the inner diameter is in the range of 210 μm-220 μm.

[0027] In one embodiment, the wall thickness of the hollow fiber membrane ranges from 15 μm to 60 μm. In one embodiment, the wall thickness is from 33 μm to 37 μm. In another embodiment, the wall thickness is from 38 to 42 μm. In yet another embodiment, the wall thickness is from 43 μm to 47 μm. In yet another embodiment, the wall thickness is from 48 μm to 52 μm.

[0028] In one embodiment, the membrane contains 1.0% of vitamin B 12has a sieving coefficient, measured at 37°C in accordance with EN1283 in bovine plasma having a protein content of 60 g / l, of 1.0 for inulin, at least 0.7 for β2-microglobulin and less than 0.01 for albumin.

[0029] In one embodiment, the membrane has a molecular weight cutoff (MWCO) in whole blood in the range of 10 kDa to 40 kDa. In a further embodiment, the average pore size in the selective layer of the membrane is in the range of 2 to 5 nm.

[0030] In one embodiment, the membrane is 10 10 -4 cm 3 / (cm 2 ·bar·sec)~280·10 -4 cm 3 / (cm 2 ·bar·sec), e.g., 15·10 -4 cm 3 / (cm 2 ·bar·sec)~130·10 -4 cm 3 / (cm 2 ·bar·sec), or 20·10 -4 cm 3 / (cm 2 ·bar·sec)~80·10 -4 cm 3 / (cm 2 This indicates the permeability (Lp) for water in the range (bar sec).

[0031] The hollow fiber membrane of the present disclosure has an anticoagulant grafted onto its surface. In one embodiment, the anticoagulant is grafted onto the luminal surface of the hollow fiber membrane. In another embodiment, the anticoagulant is grafted onto the outer surface of the hollow fiber membrane. In yet another embodiment, the anticoagulant is grafted onto the luminal surface, outer surface, and surface of the pore channels of the hollow fiber membrane. The anticoagulant forms an ionic bond with the ammonium group of the protonated chitosan. The anticoagulant may include at least one compound from the glycosaminoglycan group having anticoagulant activity, preferably selected from the group consisting of unfractionated heparin, fractionated heparin, danaparoid, heparin derivatives, and mixtures of such products. In one embodiment, unfractionated heparin is used. The surface concentration of the deposited anticoagulant is typically 1,000 to 30,000 IU / m. 2 range, e.g., 1,000-10,000 IU / m 2 or 1,000-5,000 IU / m 2 The range is.

[0032] The present disclosure also provides a filtration and / or diffusion device comprising a plurality of porous hollow fiber membranes of the present disclosure. The filtration and / or diffusion device is a device for extracorporeal purification of blood, such as a hemodialysis device. The surface area of ​​a hemodialysis device comprising hollow fiber membranes of the present disclosure varies, but is typically between 1.0 and 2.3 m. 2 Hemodialysis devices containing membranes of the present disclosure can be assembled as known in the art.

[0033] Sterilization of the device is typically accomplished by steam sterilization, gamma irradiation, or the use of ETO. In one embodiment, the device is sterilized with steam at 121°C for at least 20 minutes. In another embodiment, the gamma radiation dose used is in the range of 25-50 kGy, e.g., 25 kGy. In yet another embodiment, the device is sterilized with ETO.

[0034] The present disclosure also provides for the use of the porous hollow fiber membranes of the present disclosure in hemodialysis, hemodiafiltration, or hemofiltration.

[0035] The present disclosure also provides a method for making a hollow fiber membrane having an anticoagulant grafted onto its surface, the method comprising fabricating a microporous hollow fiber support membrane and then grafting an anticoagulant onto at least one surface of the support membrane.

[0036] In one embodiment, a porous hollow fiber support membrane is prepared by the following steps: a) forming a polymer solution comprising at least one polysulfone, polyethersulfone or polyarylethersulfone, at least one polyvinylpyrrolidone, at least one chitosan, and N-methyl-2-pyrrolidone; b) extruding the polymer solution through an outer ring slit of a nozzle having two concentric openings into a precipitation bath; c) forcing the central fluid through an inner opening of the nozzle; d) washing the resulting membrane, and then e) Drying the membrane The polymer solution is produced by a continuous solvent phase inversion spinning method, and comprises 10 to 15 wt % of polysulfone, polyethersulfone, or polyarylethersulfone based on the total weight of the polymer solution, 1 to 10 wt % of polyvinylpyrrolidone based on the total weight of the polymer solution, and 0.05 to 0.6 wt % of chitosan based on the total weight of the solution.

[0037] The concentration of polyethersulfone in the polymer solution is generally in the range of 10 to 15% by weight, for example, 12 to 14% by weight.

[0038] In one embodiment of the method, the polymer solution has a weight average molecular weight M in the range of 90 to 95 kDa. w An example includes a polyethersulfone having a weight average molecular weight M of 92 kDa. w and polydispersity M of 3 w / M n In another embodiment, the polymer solution is a polyethersulfone having a weight average molecular weight M in the range of 70 to 80 kDa. wAn example includes a polyethersulfone having a weight average molecular weight M of 75 kDa. w and a polydispersity M of 3.4 w / M n It is a polyethersulfone having the formula:

[0039] The concentration of polyvinylpyrrolidone in the polymer solution is generally in the range of 1 to 10% by weight, for example, 2 to 8% by weight.

[0040] In one embodiment of the method, the polymer solution has a weight average molecular weight M of 1,100 kDa. w The composition contains 1 to 5% by weight of polyvinylpyrrolidone having the formula:

[0041] In a further embodiment of the method, the polymer solution has a weight average molecular weight M of 50 kDa. w The composition contains 3 to 6% by weight of polyvinylpyrrolidone having the formula:

[0042] In one embodiment of the method, the polymer solution comprises 0.05-0.6 wt. %, such as 0.1-0.5 wt. %, or 0.2-0.4 wt. % chitosan, based on the total weight of the solution.

[0043] In one embodiment, the chitosan has a degree of deacetylation of 75% to 100% and a weight average molecular weight, as determined by GPC, in the range of 5 kDa to 375 kDa.

[0044] In certain embodiments, the chitosan has a degree of deacetylation of 85% to 95%, for example 90%, and a weight average molecular weight as determined by GPC analysis in the range of 50 kDa to 190 kDa.

[0045] In one embodiment of the method, preparing the polymer solution comprises preparing an acidic aqueous solution of chitosan by dissolving chitosan in an aqueous solution of a weak organic acid. In one embodiment, the organic acid is lactic acid. In a further embodiment, a 90% w / w aqueous solution of lactic acid is used to dissolve the chitosan. In another embodiment, the organic acid is citric acid. In yet another embodiment, the organic acid is acetic acid. In yet another embodiment, the organic acid is tartaric acid.

[0046] In another embodiment of the method, preparing the polymer solution comprises preparing an acidic solution of chitosan by dissolving chitosan in pure lactic acid.

[0047] Lactic acid has been found to be advantageous over other weak organic acids such as acetic acid, citric acid, or tartaric acid. Because lactic acid is liquid at room temperature, adding water is not necessary to prepare chitosan solutions. It has been found that polymer solutions made with citric acid have poor long-term stability, and fibers made with the polymer solutions tend to break during spinning. Chitosan solutions in aqueous acetic acid form gels upon the addition of polyvinylpyrrolidone, and polyvinylpyrrolidone is insoluble in chitosan solutions in aqueous tartaric acid.

[0048] In one embodiment, the concentration of chitosan in the lactic acid solution ranges from 0.2% to 5% by weight, for example, from 1% to 4% by weight, or from 1.5% to 3% by weight.

[0049] Mix the chitosan solution with N-methyl-2-pyrrolidone and add polyvinylpyrrolidone. After the polyvinylpyrrolidone has dissolved, add polyethersulfone and stir the mixture until a clear solution is formed.

[0050] In one embodiment, the polymer solution comprises 1 to 4 wt. % water, for example, 1.5 to 3 wt. % water, based on the total weight of the solution, hi another embodiment, the polymer solution does not contain added water.

[0051] In one embodiment of the method for preparing the membrane, the center fluid comprises 40-60 wt. % water and 40-60 wt. % NMP, e.g., 50-60 wt. % water and 40-50 wt. % NMP, or 52-55 wt. % water and 45-48 wt. % NMP, e.g., 54 wt. % water and 46 wt. % NMP, based on the total weight of the center fluid.

[0052] In one embodiment of the method, the precipitation bath consists of water. In one embodiment of the method, the precipitation bath has a temperature in the range of 10 to 30°C, for example, 15 to 25°C.

[0053] In one embodiment of the method for preparing the membrane, the temperature of the spinneret ranges from 40 to 60°C, for example, from 52 to 56°C.

[0054] In one embodiment of the method, the distance between the nozzle opening and the precipitation bath ranges from 10 to 120 cm, for example, from 90 to 110 cm.

[0055] In one embodiment of the method, the spinning speed is in the range of 20 to 80 m / min, for example, 30 to 50 m / min.

[0056] The membrane is then washed to remove residual solvent and low molecular weight components. In certain embodiments of the continuous method for producing membranes, the membrane is guided through several water baths. In certain embodiments of this method, the individual water baths have different temperatures. For example, each water bath can have a higher temperature than the preceding water bath.

[0057] An anticoagulant is then grafted onto at least one surface of the membrane. In one embodiment, the surface is the luminal surface of the membrane, i.e., its inner wall surface. In another embodiment, the surface is the outer surface of the membrane, i.e., its outer wall surface. In yet another embodiment, the anticoagulant is grafted onto both the inner wall surface and the surface of the pore channels in the membrane. In yet another embodiment, the anticoagulant is grafted onto both the outer wall surface and the surface of the pore channels in the membrane. In yet another embodiment, the anticoagulant is grafted onto the inner wall surface, the pore channels in the membrane, and the outer wall surface of the membrane.

[0058] The anticoagulant forms an ionic bond with the ammonium groups present on the membrane surface. The anticoagulant adheres to the membrane surface by electrostatic interaction between the negatively charged anticoagulant and the positively charged membrane surface. In one embodiment, the anticoagulant comprises at least one member of the glycoaminoglycan family, which has anticoagulant activity. In a further embodiment, the anticoagulant is selected from the group consisting of unfractionated heparin, fractionated heparin, danaparoid, heparin derivatives, and mixtures of said products. In one embodiment, the anticoagulant is unfractionated heparin. In one embodiment, the concentration of the anticoagulant on the membrane surface after the grafting step is between 1,000 and 30,000 IU / m 2 range, e.g., 1,000-10,000 IU / m 2 or 1,000-5,000 IU / m 2 range, e.g., 1,500-3,500 IU / m 2 The range is.

[0059] Grafting can be accomplished by contacting the surface of the support membrane with an aqueous solution of the anticoagulant. In one embodiment, the support membrane is rinsed with the aqueous solution of the anticoagulant, followed by washing with water or saline to remove excess anticoagulant. In another embodiment, the support membrane is rinsed with the aqueous solution of the anticoagulant, followed by drying to evaporate the solvent. In yet another embodiment, the support membrane is immersed in the aqueous solution of the anticoagulant, utilizing capillary forces generated by the hollow fibers.

[0060] Grafting can be performed before or after the membrane is incorporated into a diffusion and / or filtration device. In other words, grafting can also be performed on a diffusion and / or filtration device comprising a positively charged porous hollow fiber membrane of the present disclosure. For example, a diffusion and / or filtration device comprising a bundle of positively charged porous hollow fiber membranes, such as a hemodialysis device, can be flushed with an aqueous solution of an anticoagulant to perform grafting, and then the device is washed with water or saline to remove excess anticoagulant. In another embodiment, the device is flushed with an aqueous solution of an anticoagulant to perform grafting, and then the membranes in the device are dried and the solvent is evaporated.

[0061] After grafting, the membrane can be post-treated, examples of which include extensive rinsing of the membrane to remove loosely bound anticoagulant, irradiating the membrane to cross-link the anticoagulant with other polymers present in the membrane, drying the membrane, and heating the membrane to cross-link the polymer chains of the polymers present in the membrane.

[0062] Thus, the present disclosure provides a simple method for producing membranes coated with an anticoagulant, such as heparin. An advantage of the membranes of the present disclosure is that there is no intermediate or primer layer between the base material and the anticoagulant coating, and the method for making the membrane does not require an additional step to form such a primer layer. The method of the present disclosure also does not involve an additional chemical reaction step to covalently couple the anticoagulant to the membrane surface, and therefore does not involve the use of an additional coupling agent. The present disclosure provides a simple and scalable procedure for the low-cost production of heparin-coated membranes.

[0063] The subject matter of the present disclosure is further described in the following examples.

[0064] method <Preparation of Mini-Module> The mini-modules [=fibers in a housing] are produced by cutting the fibers to a length of 20 cm, drying the fibers at 40 °C and below 100 mbar for 1 hour, and then transferring the fibers into a housing. Both ends of the fibers are embedded with polyurethane. After the polyurethane has hardened, the ends of the embedded membrane bundle are cut to reopen the fibers. The mini-modules ensure the protection of the fibers. In this example, a 360 cm 2 A mini-module with an effective membrane (luminal) surface A of 1000 nm was used.

[0065] <Mini module water permeability (Lp)> The water permeability of a mini-module is determined by forcing a defined amount of water under pressure into a sealed mini-module and measuring the time required. The water permeability is calculated from the determined time t, the effective membrane surface area A, the applied pressure p, and the volume of water passing through the membrane V according to equation (1). Lp=V / [p·A·t] (1)

[0066] The effective membrane surface area A is calculated from the fiber length and the inner diameter of the fiber according to equation (2). A=π d i· l·[cm 2 ] (2) where: d i = inner diameter of fiber [cm] l = effective fiber length [cm]

[0067] The mini-modules are moistened 30 minutes before the Lp test is carried out. For this purpose, they are placed in a box containing 500 mL of ultrapure water. After 30 minutes, the mini-modules are transferred to the test system. The test system consists of a water bath maintained at 37°C and a device into which the mini-modules can be attached. The filling height of the water bath must be such that the mini-modules are located below the water level of the designated device.

[0068] To avoid membrane leaks leading to erroneous test results, an integrity test of the mini-module and the test system is carried out beforehand. The integrity test is performed by forcing air into a mini-module closed on one side. Air bubbles indicate a leak in the mini-module or the test device. It is necessary to verify whether the leak is due to incorrect installation of the mini-module in the test device or whether the membrane is leaking. If a membrane leak is detected, the mini-module must be discarded. The pressure applied in the integrity test must be at least the same as the pressure applied during the hydraulic conductivity determination to ensure that no leaks occur during the hydraulic conductivity determination due to the applied pressure being too high.

[0069] <Heparin coating of hollow fibers in mini-modules> i) A solution of heparin (heparin sodium 194 IU / mg, Celsus Laboratories Inc., Cincinnati, USA) in isotonic saline solution (0.9%) with a concentration of 200 IU / ml was prepared. The dialysate side of the mini-module was closed to prevent filtration from the blood side to the dialysate side. 25 ml of heparin solution was recirculated through the blood side of the mini-module at a flow rate of 1 ml / min for 30 minutes. After coating, the mini-module was then rinsed with 300 ml of isotonic saline and 100 ml of water at a flow rate of 20 ml / min. The module was then dried by passing compressed air through the blood side of the mini-module for 80 minutes.

[0070] ii) A solution of heparin (heparin sodium 194 IU / mg, Celsus Laboratories Inc., Cincinnati, USA) in ultrapure water (Milli-Q® Advantage A10, Merck KGaA, Darmstadt, Germany) with a concentration of 200 IU / ml was prepared. The dialysate side of the mini-module was closed to prevent filtration from the blood side to the dialysate side. 25 ml of heparin solution was recirculated through the blood side of the mini-module at a flow rate of 1 ml / min for 120 minutes. After coating, the mini-module was then rinsed with 300 ml of isotonic saline and 100 ml of water at a flow rate of 20 ml / min. The module was then dried by passing compressed air through the blood side of the mini-module for 80 minutes.

[0071] <Quantitative measurement of immobilized heparin> Glycine buffer was prepared from 3.84 g / L glycine (Merck KGaA, Darmstadt, Germany), 2.8 g / L NaCl (Merck KGaA, Darmstadt, Germany), and water. The pH was adjusted to 11 ± 0.1 with 30% NaOH solution (VWR, Darmstadt, Germany). The mini-module was rinsed with this glycine buffer at a flow rate of 2.3 ml / min. After 10 min, the mini-module was completely emptied. Samples were taken from the extract after 10 min, and the heparin concentration in the sample was determined using the Azure A assay.

[0072] Azure A is a blue metachromatic dye that changes color from blue to purple-red upon binding to sulfated polysaccharides such as heparin. This color change is measured photometrically at a wavelength of 630 nm. Heparin concentrations were determined based on a standard curve covering the heparin concentration range of 0 mg / L to 12 mg / L. Control samples with known heparin concentrations of 1.2 mg / L, 6 mg / L, and 10.8 mg / L were measured simultaneously for each run. 200 μl of an aqueous Azure A solution (dye content approximately 80%, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) with a concentration of 25 mg / L of Azure A was added to 100 μl of the standard, control, and test samples in microplate wells. The microplate was stored in the dark for 15 minutes, and then measurements were performed using a microplate photometer (EL 808 Ultra Microplate Reader, BioTek Instruments Inc., Winorski, USA).

[0073] Determination of Heparin Release First, the minimodule was rinsed with approximately 150 ml of isotonic saline (0.9%) at 37°C. Next, a 30 ml reservoir of solvent / detergent-treated pooled human plasma containing 45–70 g / L human plasma protein, 4.4–7.4 g / L sodium citrate dihydrate, 0.3–1.2 g / L sodium dihydrogen phosphate dihydrate, and 4.0–6.0 g / L glycine, and exhibiting a coagulation factor activity of at least 0.5 IU / mL (Octaplas® LG, Octapharma Pharmazeutika Produktionsgesellschaft mBH, A-1100 Vienna, Austria), was recirculated through the minimodule at a flow rate of 9 ml / min for 120 min. Samples were collected at 0, 60, and 120 min. Heparin concentrations in samples were determined using a heparin anti-Xa chromogenic assay (Stachrom® Heparin, Stago Deutschland GmbH, 40474 Düsseldorf, Germany).

[0074] <Blood compatibility determination> The blood compatibility of the membrane was examined by perfusion of the mini-module with PRP (platelet-rich human plasma containing 100,000 platelets per μl) in vitro.

[0075] First, the blood side of the mini-module was rinsed with approximately 250 ml of isotonic saline solution (0.9%) at 37 °C, then the filtrate side of the mini-module was rinsed with approximately 120 ml of isotonic saline (0.9%) at 37 °C, and subsequently closed with a stopper. A 50 ml reservoir of PRP containing 0.05 IU / ml of heparin was recirculated through the mini-module at a flow rate of 9 ml / min for 120 minutes. Two samples with a volume of 450 μl were taken at 0 minutes, 10 minutes, 20 minutes, 40 minutes, 60 minutes, 90 minutes, and 120 minutes. In each case, one of the two samples was transferred to a microtube (Micro Tube 1,5 ml EASY CAP, Sarstedt AG & Co. KG, Nümbrecht, Germany) containing 50 μl of 10 wt% trisodium citrate solution to inhibit further coagulation of the sample and stored in an ice bath. The other sample was transferred to a microtube (Micro Tube 1,5 ml EASY CAP, Sarstedt AG & Co. KG, Nümbrecht, Germany) containing 50 μl of CTAD (citrate-theophylline-adenosine-dipyridamole) solution to inhibit further coagulation of the sample and stored in an ice bath.

[0076] <Determination of platelet fluid decrease> The platelet content of the plasma samples was analyzed using a cell counting system (XP-300, Sysmex Deutschland GmbH, Norderstedt 22848, Germany). Data are shown relative to an initial concentration of 100,000 THR / μL (corresponding to 100%).

[0077] <Determination of PF-4> The formation of human platelet factor 4 (PF-4) was examined by sandwich enzyme-linked immunosorbent assay (ELISA) (Asserachrom(R) PF4, Stago Deutschland GmbH, Düsseldorf 40474, Germany).

[0078] After thawing, aliquots of plasma samples containing CTAD were diluted with human plasma. The dilution factor was 1:50. Standard samples (2–60 μg / L), control samples, and diluent plasma were analyzed in duplicate, but a single measurement of the samples was sufficient. 50 μl of the dilution solution and 50 μl of the sample were transferred to a microplate well, and PF-4 was allowed to bind to the antibody on the microplate. After a washing process, 100 μl of a second enzyme-conjugated antibody was added and washed away after a 30-minute incubation. The reaction with 3,3',5,5'-tetramethylbenzidine (TMB) was measured photometrically at 450 nm (EL 808 Ultra Microplate Reader, BioTek Instruments Inc., Winorski, USA).

[0079] <Determination of human TAT complex> Activation of coagulation by membrane surfaces was examined by quantitatively determining thrombin-antithrombin III complexes (TAT) in human plasma using a sandwich enzyme-linked immunosorbent assay (ELISA) (Enzygnost® TAT micro, Siemens Healthcare Diagnostics Products GmbH, Germany).

[0080] After thawing, aliquots of plasma samples containing trisodium citrate were diluted with human plasma. The dilution factor was selected depending on the expected TAT concentration and varied between 1:1 and 1:40. Standard samples (ranging from 2 to 60 μg / L), control samples, and dilution plasma were analyzed in duplicate, but a single measurement of the sample was sufficient. 50 μl of the dilution solution and 50 μl of the sample were transferred to a microplate well, allowing the TAT conjugate to bind to the antibody on the microplate. After a washing process, 100 μl of the second enzyme-conjugated antibody was added and, after a 30-minute incubation, was removed by washing. The enzymatic reaction resulting from the addition of the chromogen was measured photometrically at 490 nm within 1 hour (EL 808 Ultra Microplate Reader, BioTek Instruments Inc., Winorski, USA). [Example]

[0081] [Comparative Example 1] Mini-modules were fabricated using fibers taken from a commercial dialysis machine (Revaclear®, Gambro Lundia AB). The fibers had an inner diameter of 190 μm and a wall thickness of 35 μm and were composed of a blend of polyarylethersulfone and polyvinylpyrrolidone.

[0082] The hemocompatibility of the membranes was examined as described above. After 90 minutes, clotting was observed. The experiment was terminated after 110 minutes due to clotting. Platelet reduction, formation of PF-4, and human TAT complexes were determined as described above. The results are summarized in Table 1.

[0083] [Table 1]

[0084] Comparative Example 2 Mini-modules were fabricated using fibers taken from a commercial dialysis machine (Evodial®, Gambro Lundia AB), washed with a mixture of 60 wt% glycerol and 40 wt% water by recirculation at 100 ml / min for 60 min, and dried with compressed air for 2 h. The fibers had an inner diameter of 210 μm and a wall thickness of 42 μm and consisted of a copolymer of acrylonitrile and sodium methallylsulfonate grafted with polyethyleneimine, and had a densitometric value of 3,000 IU / m 2 It is coated with unfractionated heparin.

[0085] The hemocompatibility of the membranes was investigated as described above. Platelet reduction, the formation of PF-4 and human TAT complexes were determined as described above. The results are summarized in Table 2.

[0086] [Table 2]

[0087] <Preparation of polymer solution> For 4,500 g (=100% w / w) of polymer solution, 11.25 g (0.25% w / w) of chitosan (Chitosan 90 / 100 / A1, Kraeber & Co. GmbH, 25474 Ellerbach, Germany) was dissolved in 720 g (16% w / w) of a 90% w / w aqueous solution of lactic acid (GPR Rectapur®, VWR International GmbH, 64295 Darmstadt, Germany). This chitosan solution was mixed with 3,048.75 g (67.75% w / w) of N-methyl-2-pyrrolidone, and 90.00 g (2% w / w) of polyvinylpyrrolidone (Lubitec® K85, BASF SE) with a weight-average molecular weight of approximately 1,100 kDa was added. After the polyvinylpyrrolidone was dissolved, 630.00 g (14% w / w) of polyethersulfone (Ultrason® E 6020, BASF SE) was added and the mixture was stirred until a clear solution was formed. It is also possible to dissolve the PVP and polyethersulfone in NMP and then add the chitosan lactic acid solution in a second step.

[0088] [Example 1] A solution of 14.0% w / w polyethersulfone (Ultrason® E 6020, BASF SE) with a weight-average molecular weight of approximately 75 kDa, 2.0 w / w PVP (Luvitec® K85, BASF SE) with a weight-average molecular weight of approximately 1100 kDa, 0.25% w / w chitosan (Chitosan 90 / 100 / A1, Kraeber & Co GmbH, 25474 Ellerbeck, Germany), 14.4% w / w lactic acid, 1.6% water, and 67.75% NMP was extruded through the outer ring slit of a spinneret with two concentric openings (diameter of outer ring slit: 500 μm, diameter of inner ring slit: 350 μm, diameter of central hole: 180 μm) into a coagulation bath containing water. A solution containing 50% w / w water and 50% w / w NMP was used as the center fluid and extruded through the internal orifice of the spinneret. The spinneret temperature was 48°C, the spinning shaft temperature was 45°C, the air gap was 100 cm, and the coagulation bath was at room temperature. Fibers were spun at a speed of 40 m / min. The fibers were guided through a series of water baths containing demineralized water at temperatures ranging from 50°C to 75°C.

[0089] The resulting fibers had an inner diameter of 190 μm and a wall thickness of 35 μm. The membrane exhibited an asymmetric spongy structure, with the average pore size increasing in the direction from the inner wall surface to the outer wall surface.

[0090] The mini-modules were fabricated as described above, and the fiber hydraulic permeability was tested as described above. The Lp of the mini-modules was (45±5)·10 -4 cm 3 / (cm 2 ·bar·sec) was determined (n=2).

[0091] A number of mini-modules were sterilized by steam at 121°C for 20 minutes. After sterilization, the Lp of the mini-modules was (70±3)·10 -4 cm 3 / (cm 2 The sieving coefficients of the sterilized membrane were determined to be (31±1)% for myoglobin and (0.7±0.1)% for albumin (n=2).

[0092] The membranes of three mini-modules were coated with heparin according to step i) above, and the amount of heparin immobilized on the membrane was determined as described above. After 10 minutes of extraction, (92 ± 4) IU of heparin was extracted from each mini-module (n = 3).

[0093] Heparin release from three mini-modules (n=3) was tested as described above. At 0 min, the plasma heparin concentration was below the detection limit (<0.1 IU / ml). After 60 min of extraction, the heparin concentration in the plasma samples was below the detection limit (<0.1 IU / ml) for the first and second mini-modules and 0.1 IU / ml for the third mini-module. After 120 min of extraction, the heparin concentration in the plasma samples was below the detection limit (<0.1 IU / ml) for the first and third mini-modules and 0.3 IU / ml for the second mini-module.

[0094] The Lp of the heparin-coated mini-module was (65±2)·10 -4 cm 3 / (cm 2 The sieving coefficients of the heparin-coated membrane were determined to be (33±1)% for myoglobin and (0.8±0.1)% for albumin (n=3).

[0095] [Example 2] A solution of 14.0% w / w polyethersulfone (Ultrason® E 6020, BASF SE) having a weight-average molecular weight of approximately 75 kDa, 2.0% w / w PVP (Luvitec® K85, BASF SE) having a weight-average molecular weight of approximately 1,100 kDa, 0.25% w / w chitosan (Chitosan 90 / 100 / A1, Kraeber & Co GmbH, 25474 Ellerbeck, Germany), 14.4% w / w lactic acid, 1.6% water, and 67.75% w / w NMP was extruded through the outer ring slit of a spinneret with two concentric openings (outer boundary diameter of the ring slit: 700 μm, inner boundary diameter of the ring slit: 350 μm, diameter of the central hole: 180 μm) into a coagulation bath containing water. A solution containing 50% w / w water and 50% w / w NMP was used as the center fluid and extruded through the internal orifice of the spinneret. The spinneret temperature was 49°C, the spinning shaft temperature was 45°C, the air gap was 100 cm, and the coagulation bath was at room temperature. Fibers were spun at a speed of 30 m / min. The fibers were guided through a series of water baths containing demineralized water at temperatures ranging from 50°C to 75°C.

[0096] The resulting fibers had an inner diameter of 190 μm and a wall thickness of 35 μm. The membrane exhibited an asymmetric spongy structure, with the average pore size increasing in the direction from the inner wall surface to the outer wall surface.

[0097] The mini-modules were fabricated as described above, and the fiber hydraulic permeability was tested as described above. The Lp of the mini-modules was (44±1)·10 -4 cm 3 / (cm 2 The sieving coefficients of the membrane were determined to be (31±0.5)% for myoglobin and (1.5±0.0)% for albumin (n=3).

[0098] A number of mini-modules were sterilized by steam at 121°C for 20 minutes. After sterilization, the Lp of the mini-modules was (82±4)·10 -4 cm 3 / (cm 2The sieving coefficients of the sterilized membrane were determined to be (39±2)% for myoglobin and (1.7±0.1)% for albumin (n=3).

[0099] The membranes of the minimodules were coated with heparin according to procedure i) above, and the hemocompatibility of the membranes was investigated as described above. Platelet reduction, the formation of PF-4 and human TAT complexes were determined as described above. The results are summarized in Table 3.

[0100] [Table 3]

[0101] [Example 3] 14.0% w / w polyethersulfone (Ultrason® E 6020, BASF SE) with a weight-average molecular weight of approximately 75 kDa, 2.0% w / w PVP (Luvitec® K85, BASF SE) with a weight-average molecular weight of approximately 1,100 kDa, 0.1% w / w chitosan (Chitosan 90 / 100 / A1, Kraeber & Co GmbH, 25474 Ellerbeck, Germany), 14.4% w / w lactic acid, 1.6% water, and 67.9% NMP were extruded through the outer ring slit of a spinneret with two concentric openings (outer ring slit diameter: 500 μm, inner ring slit diameter: 350 μm, central hole diameter: 180 μm) into a coagulation bath containing water. A solution containing 50% w / w water and 50% w / w NMP was used as the center fluid and extruded through the inner orifice of the spinneret. The temperature of the spinneret was 49°C, the temperature of the spinning shaft was 45°C, the air gap was 100 cm, and the coagulation bath was at room temperature. Fibers were spun at a speed of 40 m / min. The fibers were subsequently guided through a series of water baths containing demineralized water at temperatures ranging from 50°C to 75°C.

[0102] The resulting fibers had an inner diameter of 190 μm and a wall thickness of 35 μm. The membrane exhibited an asymmetric spongy structure, with the average pore size increasing in the direction from the inner wall surface to the outer wall surface.

[0103] Mini module Lp is (73±1)·10 -4 cm 3 / (cm 2 After steam sterilization, the Lp of the mini-module was (91±1)·10 -4 cm 3 / (cm 2 The sieving coefficient of the sterilized membrane was (44±1)% for myoglobin and (0.8±0.0)% for albumin (n=2).

[0104] The membranes of three mini-modules were coated with heparin according to step ii) above, and the amount of heparin immobilized on the membrane was determined as described above. After 10 min of extraction, (43 ± 2) IU of heparin was extracted from each mini-module (n = 3).

[0105] The Lp of the heparin-coated mini-module was (82±2)·10 -4 cm 3 / (cm 2 The sieving coefficients of the heparin-coated membrane were determined to be (42±1)% for myoglobin and (0.8±0.0)% for albumin (n=2).

[0106] [Example 4] A solution containing 14.0% w / w polyethersulfone (Ultrason® E 6020, BASF SE) with a weight-average molecular weight of approximately 75 kDa, 2.0% w / w PVP (Luvitec® K85, BASF SE) with a weight-average molecular weight of approximately 1,100 kDa, 0.4% w / w chitosan (Chitosan 90 / 100 / A1, Kraeber & Co. GmbH, 25474 Ellerbeck, Germany), 14.4% w / w lactic acid, 1.6% water, and 67.6% w / w NMP was prepared. To reduce the viscosity of the polymer solution, 30 wt% NMP was added based on the total weight of the polymer solution. The diluted polymer solution was extruded through the outer ring slit of a spinneret with two concentric openings (diameter of outer ring slit boundary: 500 μm, diameter of inner ring slit boundary: 350 μm, diameter of central hole: 180 μm) into a coagulation bath containing water. A solution containing 60% w / w water and 40% w / w NMP was used as the center fluid and extruded through the inner opening of the spinneret. The temperature of the spinneret was 45°C, the temperature of the spinning shaft was 42°C, the air gap was 100 cm, and the coagulation bath was at room temperature. Fibers were spun at a speed of 40 m / min. The fibers were guided through a series of water baths containing demineralized water at temperatures ranging from 50°C to 75°C.

[0107] The resulting fibers had an inner diameter of 190 μm and a wall thickness of 35 μm. The membrane exhibited a finger structure, i.e., macrovoids extending in the direction of the outer wall surface.

[0108] The Lp of the mini module is (91±6)·10 -4 cm 3 / (cm 2 The sieving coefficient of the membrane was (21±2)% for myoglobin and (0.5±0.0)% for albumin (n=3).

[0109] The membranes of three mini-modules were coated with heparin according to step ii) above, and the amount of heparin immobilized on the membrane was determined as described above. After 10 minutes of extraction, (18±1) IU of heparin was extracted from each mini-module (n=3).

Claims

1. A porous hollow fiber membrane having an anticoagulant immobilized thereon, the porous hollow fiber membrane comprising a blend of i) polysulfone, polyethersulfone or polyarylethersulfone, ii) polyvinylpyrrolidone, and iii) chitosan.

2. The membrane of claim 1 , wherein the anticoagulant is a glycosaminoglycan.

3. The membrane of claim 2 , wherein the anticoagulant is unfractionated heparin.

4. The concentration of the anticoagulant on the porous hollow fiber is 1,000 to 5,000 IU / m ２ The membrane according to any one of claims 1 to 3, wherein the thickness is in the range of

5. 5. The membrane according to claim 1, comprising 0.1 to 5% by weight of chitosan relative to the total weight of the membrane.

6. The chitosan has a weight average molecular weight M determined by GPC analysis in the range of 5 to 375 kDa. ｗ The membrane according to any one of claims 1 to 5, having

7. The membrane according to any one of claims 1 to 6, wherein the chitosan has a degree of deacetylation in the range of 85 to 95%.

8. 1. A method for producing a porous hollow fiber membrane having an anticoagulant immobilized thereon, the method comprising producing a microporous hollow fiber support membrane and then grafting an anticoagulant onto at least one surface of the support membrane, the production of the microporous hollow fiber support membrane comprising the steps of: a) forming a polymer solution comprising at least one polysulfone, polyethersulfone or polyarylethersulfone, at least one polyvinylpyrrolidone, at least one chitosan, and N-methyl-2-pyrrolidone; b) extruding the polymer solution through an outer ring slit of a nozzle having two concentric openings into a precipitation bath; c) forcing the central fluid out of the inner opening of the nozzle; d) washing the resulting membrane, and then e) Drying the membrane wherein the polymer solution comprises 10 to 15 wt % of polysulfone, polyethersulfone or polyarylethersulfone, based on the total weight of the polymer solution, and 1 to 10 wt % of polyvinylpyrrolidone, based on the total weight of the polymer solution, and 0.05 to 0.6 wt % of chitosan, based on the total weight of the solution.

9. The chitosan has a degree of deacetylation of 75% to 100% and a weight average molecular weight M, determined by GPC analysis, ranging from 5 kDa to 375 kDa. ｗ 9. The method of claim 8, comprising:

10. 10. The method of claim 8 or 9, wherein the step of forming the polymer solution comprises dissolving chitosan in lactic acid.

11. 11. The method of claim 10, wherein the chitosan is dissolved in a 90% w / w aqueous solution of lactic acid.

12. 12. The method of any one of claims 8 to 11, wherein the anticoagulant is grafted to the at least one surface of the support membrane by contacting an aqueous solution of the anticoagulant with the at least one surface of the support membrane.

13. The method of any one of claims 8 to 12, wherein the anticoagulant is unfractionated heparin.

14. A filtration and / or diffusion device comprising a plurality of porous hollow fiber membranes, wherein the porous hollow fiber membranes are the porous hollow fiber membranes according to any one of claims 1 to 7.

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