Coated membranes for blood purification applications

Abstract

The present disclosure relates to semipermeable membranes comprising a base membrane and a coating with a vinyl polymer derived from vinyl monomers carrying an acid group and grafted onto the surface of the base membrane by radiation induced graft copolymerization (RIGC), thereby introducing a coating carrying negative charges. The coated membranes are especially suitable for blood purification applications as they are characterized by an increased selectivity and excellent blood compatibility and an excellent hemocompatibility due to the careful selection of the vinyl monomers used for the coating process and the introduction of negative charges. The invention also relates to a production process for said coated membranes and to their use in hemodialysis devices and in medical applications such as extracorporeal blood purification.

Description

Coated membranes for blood purification applicationsTechnical Field

[0001] The present disclosure relates to semipermeable membranes comprising a base membrane and a coating with a vinyl polymer derived from vinyl monomers carrying an acid group and grafted onto the surface of the base membrane by radiation induced graft copolymerization ( RIGC ) , thereby introducing a coating carrying negative charges . The coated membranes are especially suitable for blood purification applications as they are characterized by an increased selectivity and excellent blood compatibility and an excellent hemocompatibility due to the careful selection of the vinyl monomers used for the coating process and the introduction of negative charges . The invention also relates to a production process for said coated membranes and to their use in hemodialysis devices and in medical applications such as extracorporeal blood purification .Description of the Related Art

[0002] Patients with end stage renal disease in hemodialysis have an increased mortality ris k when compared to the general population . This indicates that the currently available replacement therapies , even though relying on advanced membrane and dialyzer technologies as well as highly effective and sophisticated hemodialysis machines , should be further improved in a way that the clinical outcome for patients and their quality of life during therapy is getting better ( Bowry and Chazot , The scientific principles and technological determinants of haemodialysis membranes . Clin Kidney J 14 ( Suppl 4 ) : i5-il 6 ( 2021 ) ) . One of the still manifold unmet needs that has been accompanying the development of new membranes and dialyzers over decades is the enhanced removal of uremic toxins over a broad range , including higher molecular weight middle molecules ("large middle molecules" ) , whileessentially retaining proteins such as albumin and other higher molecular weight compounds that are deemed to be essential and must essentially be preserved in the treated blood of the patient . In other words , synthetic membranes which are state of the art today are still less selective than the glomerular membrane of the kidney . In consequence , uremic toxins having a higher molecular weight are often not cleared appropriately from the patients ' blood . In addition, hemodialysis is performed generally thrice weekly for approximately 4 hours , while the healthy kidney operates continuously ( Boschetti-de-Fierro et al . , Scientific Reports ( 2015 ) 5 : 18448 ) , which adds to the issues around the accumulation of uremic toxins in the body . Another persistent need in hemodialysis is the hemocompatibility of the materials used in extracorporeal blood treatments , specifically the hemocompatibility of membranes that are in direct blood contact with a significant surface area , making it necessary to use systemic heparin administration in most therapies .

[0003] In response to the above challenges , membrane innovation has recently been focusing on enhancing the effective removal of larger molecular weight uremic toxins or "large middle molecules" and increased membrane permeability . In addition, the industry also focused on the improvement of hemocompatibility and antithrombogenicity of the membranes . The progressive shift towards higher molecular weight toxins and increased membrane permeability also necessitates the provision of membranes with an increasingly improved selectivity . In other words , it remains a maj or target in the industry to close the gap between synthetic membranes and the natural kidney by combining high permeability and high selectivity .

[0004] Typical hallmarks of selectivity of a membrane specifically for use in hemodialysis are the sieving coefficients for albumin ( 66 . 4 kDa ) on the one hand and the sieving coefficients for large middle molecules ( >25-58 kDa ) on the other hand ( Rosner et al . , Classification of Uremic Toxins and Their Role in KidneyFailure: CJASN 16, 1-8 (2021) ) . For albumin, the sieving coefficients should be very low, whereas those for large middle molecules should be high. Combining these two characteristics in one and the same membrane is, of course, challenging.

[0005] The challenge resides in providing membranes that combine both requirements, which is nothing else than high selectivity. Large middle molecules include, for example, YKL-40 (40kDa) , AGEs, lambda-FLC, CX3CL1, CXCL12, or IL-2. However, selectivity does expressly encompass the highly efficient removal of also small middle molecules (0.5-15kDa) such as L2-microglubulin, and medium middle molecules (>15-25kDa) , such as TNF, IL-10, IL-6, kappa-FLC, myoglobin, FGF-2, complement factor D and others. The sieving coefficients for said molecules are reflected, for example, in the sieving curve and including the MWCO and MWRO values of a membrane. These parameters are a means to provide information on the onset of retention of molecules, i.e., where the sieving coefficient is 0.9, and the cut-off of a membrane, i.e. , where the sieving coefficient is 0.1.

[0006] Together, MWRO and MWCO define the steepness of the sieving curve and thus provide for an excellent measure of the selectivity of a membrane. For example, shifting the retention onset to higher molecular weights while keeping the cut-off stable or reducing it, results in increased selectivity, which is apparent from an increasing steepness of the sieving curve. In other words, an increase in selectivity is defined by a decrease of the delta between MWRO and MWCO of a membrane, or by an increase of the absolute value of the slope at the turning point of the sieving curve. Keeping the MWRO of a membrane stable while reducing the MWCO thereof could be another way to improve the selectivity of a membrane. However, it is a technical challenge to achieve that desired shift of either MWRO or MWCO while keeping the respective other parameter stable.

[0007] Hemodialyzers comprising very selective membranes should have a low albumin loss over a typical dialysis treatmentof about 4 hours (or simulated use correlated with in vivo use) , i.e. , the albumin loss should remain below about 7 g / 4h, and preferably should remain below about 4g / 4h. At the same time, the hemodialyzers should provide a good clearance for large middle molecules, including, for example, YKL-40, i.e., they should have a YKL-40 clearance of at least 20 mL / min and higher. They should, in addition, efficiently remove L-2 micro-globulin (L2m) with a clearance of more than 80 mL / min.

[0008] L-2 microglobulin is a well-known marker molecule in hemodialysis. It is a component of MHC class I molecules with a molecular weight of 11 kDa that in patients on long-term hemodialysis can aggregate into amyloid fibers that deposit in joint spaces, a disease, known as dialysis-related amyloidosis. YKL-40 (or chitinase-3-like protein) has recently emerged as a relevant and functional marker protein in hemodialysis (Lorenz et al . , Kidney International (2018) 93: 221-230) , where it has been linked to chronic inflammatory response in chronic kidney disease and HD patients. Its presence in the serum of the patients makes it accessible and traceable.

[0009] Recently, a significant step towards membranes and hemodialyzers with such improved performance was made by the introduction of so-called medium cut-off membranes and dialyzers, which are characterized by membranes having a high molecular weight retention onset (MWRO) , thereby allowing also larger molecular weight toxins to pass the membrane to a significant extent, combined with a low molecular weight cut off (MWCO) , meaning that proteins and other compounds that have a molecular weight of albumin and higher are nevertheless essentially retained (WO 2015 / 118045 Al and WO 2015 / 118046 Al) . In other words, the design of said medium cut-off membranes and dialyzers allows for a significantly improved selectivity, which is a distinguishing feature over other prior art membranes such as the so-called "high cutoff" membranes, or membranes that have been referred to, in the past, as "protein-leaking" (PL) membranes.

[0010] While so-called high cut-off membranes and dialyzers are also able to efficiently remove higher molecular weight toxins , their increased albumin loss recommends their use only in acute situations that are limited in time and strictly controlled as regards albumin loss and replacement thereof . The expression "high cut-off" membrane refers to a type of membrane which has first been disclosed in WO 2004 / 056460 Al . Dialyzers making use of such high cut-off type membrane are , for example , the HCOll OO , SEPTEX and THERALITE dialyzers . Known uses of , for example , the THERALITE dialyzer include treatment of sepsis ( EP 2 281 625 Al ) , chronic inflammation ( EP 2 161 072 Al ) , amyloidosis and rhabdomyolysis , and the treatment of anemia (US 2012 / 0305487 Al ) , the most explored therapy to date being the treatment of myeloma kidney patients (US 7 , 875 , 183 B2 ) . Due to the relatively high loss of albumin of up to 40 g per treatment session, high cut-off membranes so far have been used for acute applications only, although some physicians have contemplated benefits of using them in chronic applications , possibly in conj unction with albumin substitution and / or in addition to or in alternate order with standard high- flux dialyzers . The expression "high cut-off membrane" or "high cut-off" as used herein refers to membranes having a MWRO of between 15 and 20 kDa and a MWCO of between 170-320 kDa ( see Table I ) . The membranes can also be characterized by a pore radius , on the selective layer surface of the membrane , of between 8 -12 nm, typically around 10 nm.

[0011] Protein-leaking membranes , on the other hand, generally have a cut-off that provides for a certain limited loss of albumin and other higher molecular weight compounds beyond albumin, but the retention onset is generally low, resulting in a flat sieving curve that signifies a low selectivity and undesirable weakness in the clearance of smaller and of large uremic tox-ins . In contrast , medium cut-off membranes when combined with a careful physical design of the hollow fiber , membrane bundle and overall dialyzer geometry, lead to a performance which provides for an unprecedented clearance of relevant higher molecular weight toxins in hemodialysis , while albumin loss can be kept within narrowlimits ( Kirsch et al . , Nephrology Dialysis Transplantation ( 2017 ) 32 : 165 ) . A commercially available example for such a medium cutoff membrane and dialyzer is the MCO THERANOVA dialyzer which has made a significant impact on clinical practice over the last few years ( Zhang et al . ( 2022 ) , Effects of Expanded Hemodialysis with Medium Cut-Off Membranes on Maintenance Hemodialysis Patients : A Review . Membranes 12 : 253 ) .

[0012] The significant contribution of medium cut-off membranes to the superior dialyzer performance is achieved by their unique membrane structure which is generated by a careful control of all relevant parameters of the manufacturing process as described in WO 2015 / 118045 Al and WO 2015 / 118046 Al . Such a highly controlled manufacturing process is not easily reproducible throughout the industry, and further improving the selectivity of medium cut-off membranes by further improving such manufacturing process is difficult and / or expensive . However, as mentioned above , it would be desirable to further improve dialysis membranes towards even higher selectivity by easily accessible methods that allow the production of highly selective hemodialysis membranes without further enhancing production costs that add to the price of the final product and make it less accessible for all patient groups .

[0013] Other membranes , especially including high cut-off membranes could equally benefit from methods to improve their selectivity in a simple , cost-effective , and safe way to provide access to improved dialyzers to more patients that may otherwise not be able to benefit from potentially more expensive high-end dialyzers as discussed above or potentially even more expensive and complex therapies such as HDF . Of course , any such methods for improving the selectivity of available membranes and dialyzers must guarantee or further improve the biocompatibility of the membrane and the dialyzer and its general safety for the patient ( Bowry and Chazot ( 2021 ) : The scientific principles and technological determinants of haemodialysis membranes . Clin Kidney J 14 ( Suppl 4 ) : i5 -il 6 ) .

[0014] Conventional dialysis membranes are generally classified as "high-flux". High-flux membranes used in devices, such as, for example, POLYFLUX 170H, REVACLEAR, ULTRAFLUX EMIC2, or OPTIFLUX F180NR, have been on the market for many years. The high- flux membranes used therein are mainly polysulfone or polyethersulfone based membranes and methods for their production have been described, for example, in US 5,891,338 and EP 2 113 298 Al. The expression "high-flux membrane (s)" as used herein refers to membranes having a MWRO between 5 kDa and 10 kDa and a MWCO between 25 kDa and 65 kDa (Table I) , as determined by dextran sieving measurements according to Boschetti et al. (2013) . The average pore radius is in the range of from 3.5 to 5.5 nm, wherein the pore size is determined from the MWCO based on dextran sieving coefficients according to Boschetti-de-Fierro et al. (2013) and Granath et al. (1967) . Molecular weight distribution analysis by gel chromatography on sephadex. J Chromatogr A. 1967;28(C) : 69-81.

[0015] In known polysulfone or polyethersulfone based membranes such as the ones mentioned above, the polymer solution generally comprises between 10 and 20 weight-% of polyethersulfone or polysulfone as hydrophobic polymer and 2 to 11 weight-% of a hydrophilic polymer, in most cases PVP, wherein said PVP generally consists of a low and a high molecular PVP component. The resulting high-flux type membranes generally consist of 80-99% by weight of said hydrophobic polymer and 1-20% by weight of said hydrophilic polymer. During production of the membrane the temperature of the spinneret generally is in the range of from 25-55 °C. Polymer combinations, process parameters and performance data can otherwise be retrieved from the references mentioned or can be taken from the respective data sheets.

[0016] As can be seen from Table I, medium cut-off membranes are characterized by a larger pore size compared to standard high- flux membranes, wherein the pore size distribution is, however, very narrow and highly controlled which contributes to theincreased selectivity of these membranes as well as to their superior clinical performance . Medium cut-off membranes are further characterized by a MWRO of between 9 and 14 kDa and a MWCO of between 55 and 130 kDa .

[0017] In Japan, dialyzers are classified based on p2-micro- globulin clearance . Type I dialyzers are classified as low-flux dialyzers (<10 mL / min clearance ) , type II and I II as high-flux dialyzers ( bi o to <30 mL / min and h30 to <50 mL / min clearance , respectively) , and type IV and V as super high-flux dialyzers ( h50 to <70 mL / min and h70 mL / min clearance , respectively) . Type IV and V dialyzers are also classified as high-performance membrane ( HPM) dialyzers (Abe et al . ( 2022 ) , Super high-flux membrane dialyzers improve mortality in patients on hemodialysis : a 3-year nationwide cohort study . Clin Kidney J 15 ( 3 ) : 473-483 ) , or Super high-flux sharp cut-off dialyzers . Said categories are encompassed herein when discussing low and high flux dialyzers .

[0018] The sieving properties of a membrane , including its permeability to solutes , are generally determined by the pore size which sets the maximum size for the solutes that can be dragged through the membrane with the fluid flow, and by pore size distribution and density across the surface of the membrane ( Reis et al . , Disruptive technologies for hemodialysis : medium and high cutoff membranes . Is the future now? J Bras Nef rol . 2021 43 ( 3 ) : 410-416 ) . The glomerular filtration barrier in the kidney is thought to include a charge-selective component , in addition to a size-selective one , which has been a cornerstone of renal physiology for decades . It is now generally accepted that the glomerular basement membrane represents the maj or permselective barrier for both molecular size and charge , excluding molecules with the size and negative charge of , for example , albumin from glomerular filtration under normal conditions . Therefore , further improvements to existing, already highly selective hemodialysis membranes that are mainly based on size exclusion properties might be available through the introduction of suitable charges . However , it is known that negatively charged membrane surfaces canpromote contact activation ( Ji et al . ( 2023 ) , Advances in Enhancing Hemocompatibility of Hemodialysis Hollow-Fiber Membranes . Advanced Fiber Materials 5 : 1198-1240 ) . Therefore , it is a maj or task to introduce charges in a way that has no negative effects on the hemocompatibility of the resulting membranes , and to avoid any thrombotic ris k in patients . Not much is currently known about how said negative charges can be presented on a membrane surface to reap the benefits without compromising on hemocompatibility .

[0019] To determine and compare the selectivity of given membranes requires suitable tools that , for example , allow to reliably measure the sieving coefficients of a membrane for certain selected substances . These sieving coefficients can serve as a measure for the ability of a membrane to let selected molecules pass to a greater extent than comparable existing hemodialysis membranes . As mentioned above , the shape of the sieving curve of a membrane depends , to a significant extent , on the pore size distribution and on the physical form of appearance of the membrane and its pore structure , which can otherwise only be inadequately described . Sieving coefficients and curves are thus a good description not only of the performance of a membrane but are also descriptive of its specific submacroscopic structure .

[0020] In vi tro characterization of blood purification membranes often includes the determination of the sieving coefficients for small molecules , such as urea, large middle molecules , such as YKL-40 , and for albumin, because these molecules represent the spectrum of molecules which, in the case of small and large middle molecules , should be removed from blood, and in the case of albumin, which should essentially be retained .

[0021] MWRO and MWCO values as used herein for describing membrane characteristics are common terms and are understood by a person skilled in the art according to established technology and may be determined by dextran sieving before blood contact according to the methods of Boschetti-de-Fierro et al . ( 2013 ) . Accordingly, the expressions "as determined by dextran sieving" and"based on dextran sieving" refer to the dextran sieving method as described in Boschetti-de-Fierro et al . ( 2013 ) . Briefly, by way of example , dextrane sieving measurements are performed as follows using a standard setting comprising a pool of dextrane solution, a feed pump , a filtrate pump and a heating / stirring plate : Filtration experiments are carried out under a constant shear rate y (preferably y=750 s-1) , with the ultrafiltration rate preferably set at 20% of the blood side entrance flux Qsin , calculated as :Where QBin is the flux at the blood side entrance in ml / min; n is the number of fibers in the minimodule ;is the inner diameter of the fibers in cm and y is the constant shear rate mentioned above . The filtration conditions are prfereably without backf iltration and the dextran solution is preferably recirculated at 37 ° C ± 1 ° C . Feed (blood side entrance ) , retentate (blood side exit ) , and filtrate ( dialysate exit ) samples are preferably taken after 15 min . Relative concentration and molecular weight of the samples are analyzed via gel permeation chromatography . Samples are prior to analysis filtered and calibration is done against dextran standards . The sieving coefficient SC is calculated as follows :Where cFis the concentration of the solute in the filtrate , cPits concentration in the permeate and cRits concentration in the retentate .

[0022] The MWCO and MWRO values used for describing prior art membranes and the base membranes according to the invention are measured before blood or plasma contact because the sieving properties of synthetic membranes may change upon blood contact . This fact can be attributed to the adhesion of proteins to the membrane surface , which means that the MWRO and MWCO value may be influenced and therefore vary depending on the individual characteristics ofthe medium (blood ) . When proteins adhere to the membrane surface , a protein layer is created on top of the membrane . This secondary layer acts also as a barrier for the transport of substances to the membrane , and the phenomenon is commonly referred to as fouling .TABLE I : General classification of current hemodialysis membranes based on dextran sieving

[0023] Technologies for the modification of membranes , including hemodialysis membranes , by incorporating or immobilizing biologically active or inactive compounds in / on the membrane have generally been described in the prior art . This includes , for example , modifications for improving their biocompatibility, an- tithrombogenicity, or performance . They further encompass adding compounds to the membrane matrix for the unspecific capturing of , for example , hydrophobic or hydrophilic uremic toxins . Modifications also include methods for specifically functionalizing membrane surfaces for the binding of , for example , antibodies that can specifically target molecules that are causative for various diseases , and which are thus removed from the blood of the patient .

[0024] The introduction of charges , specifically negative charges , for example through blending, physical surface treatment , or chemical surface functionalization is a known approach in the industry ( Radu and Voicu ( 2022 ) , Functionalized HemodialysisPolysulfone Membranes with Improved Hemocompatibility. Polymers 14:1130) , and it has been shown that the functionalization approach, the chemical nature of the compounds used, and the reaction conditions by which said compounds are introduced have a massive impact on performances of separation, anticoagulant properties, and hemocompatibility.

[0025] It is therefore hard to predict the performance and hemocompatibility of a membrane created by a given functionalization. It is equally difficult to determine which membranes, both regarding chemical composition and physical properties, in combination with which functionalization in terms of compounds used and processes applied provides for the intended results.

[0026] The most common polymer used in membrane formation is polysulfone (PS) , as well as polyethersulfone (PES) and poly- arlyethersulf on (PAES) . Functionalization attempts for introducing charges include adding by grafting onto the aromatic rings, such as the introduction of a sulfonate group into the polymer structure through electrophilic substitution via sulfonation (Liu et al . (2021) , Vorapaxar-modif ied polysulfone membrane with high hemocompatibility inhibits thrombosis. Mater. Sci. Eng. C 118: 111508) . Aydemir Sezer et al. (2018) , A design achieved by coaxial electrospinning of polysulfone and sulfonated polysulfone as a core-shell structure to optimize mechanical strength and hemocompatibility. Surf. Interfaces 10:176-187) , described the sulfonation treatment of PSF using a chlorosulfonic agent as a sulfonating agent. In addition to sulfonation, another modification method for PS-based membranes is chloromethylation, which will induce the appearance of numerous functional groups on the PS, thereby increasing hydrophilicity, such as hydroxyl group (-OH) , azide group (-N3) , amino (-NH2) , carboxyl (-COOH) and sulfo (-SO3H) groups (Radu and Voicu (2022) , Polymers 14:1130) .

[0027] Wang et al. (2011) , Preparation and characterization of negatively charged hollow fiber nanofiltration membrane by plasma-induced graft polymerization. Desalination 286:138-144,describe the grafting of 2-acrylamido-2-methylpropanesulf onic acid (AMPS) on a polysulfone (PS) hollow fiber ultrafiltration membrane for water treatment (desalination) using a two-step plasma method comprising a pre-treatment of the PS membrane with plasma, soaking the pre-treated membrane in AMPS solution (3-20%, preferably 5% AMPS solution) for about 80s, and grafting AMPS by plasma treatment.

[0028] Xu et al . ( 2012 ) , Nanofiltration hollow fiber membranes with high charge density prepared by simultaneous electron beam radiation-induced graft polymerization for removal of Cr(VI) . Desalination 346:122-130, describe polysulfone hollow fiber membranes for water treatment (removal of chromium) with a MWCO of 20 kDa which are immersed in a 3%-20% AMPS solution (w / v) in a bag under N2 atmosphere followed by irradiation at RT with preferably up to 80 kGy.

[0029] Singh et al. (2017) , Role of poly (2-acrylamido-2-me- thyl-l-propanesulf onic acid) in the modification of polysulfone membranes for ultrafiltration. J Appl Polym Sci 134 (37) :45290, describes polysulf one / PEG based membranes for ultrafiltrationhaving a MWCO of 30 kDa modified with poly AMPS of an average molecular weight of 2000 kDa which is added to the polymer solution in an amount of from 0 to 4 wt.-%. Singh et al. report a decrease in BSA rejection.

[0030] US6183640B1 discloses an asymmetric membrane having permanent internal anionic charges, cast from a solution or suspension comprising polyethersulfone, 2-acrylamido-2-methylpropane sulfonic acid or 1-propanesulf onic acid 2-methyl-2- ( l-oxy-2-pro- penyl amino) in an amount of from 0.4-4 wt.-%, a nonsolvent (H2O) , and a solvent (NMP) . The ultrafiltration membrane has a MWCO of between 10 kDa and up to 100 kDa. The AMPS component is thermally crosslinked with itself and the other membrane components.

[0031] Abidin et al. (2022) , Fouling Prevention in Polymeric Membranes by Radiation Induced Graft Copolymerization. Polymers 14:197, review the use of adding chemical functionalities tomembranes by radiation induced graft copolymerization (RIGC) , wherein polymeric membranes can be modified by grafting polar monomers onto membranes using US, plasma, gamma-rays, or electron beam (e-beam) for fouling prevention. The method allows modifying membranes, such as, for example, PS or PES based membranes, by covalent immobilization of selected moieties such as hydroxyl and amine groups with controllable levels of grafting and without using hazardous chemicals. It is also mentioned that negative surface charges of a membrane are commonly formed by imparting sulfonic and carboxylic acid groups, which dissociate in the feed solution .

[0032] US2022 / 0023805 Al discloses membranes for nano / ultra- filtration comprising an active layer and a porous support layer, which may be used for hemodialysis. The active layer is a nanocellulose or other membrane such as a porous polyethersulf one (PES) membrane. A C02-respons ive polymer is grafted to the active layer, which upon exposure to CO2 changes its charge and either extends or collapses, thereby decreasing or increasing the overall pore size but not specifically increasing selectivity for specific molecules such as large middle molecules or albumin. A MWCO of preferably 20 to 100 kDa and an apparent pore size distribution of preferably 1 to 50 nm or 3 to 10 nm is disclosed. There is no MWRO disclosed.

[0033] US2020 / 0147287 Al discloses uncoated hollow fiber membrane dialyzer for blood purification applications with specific MWCO and M RO. No further modification of these membranes to increase selectivity is disclosed.

[0034] Shao et al. (Macromol. Mater. Eng. 2018, 303, 1700378) discloses PES membranes coated with different polymers like Catechol-conjugated poly (2-acrylamido-methylpropane sulfonic acid) PAMPS, for use in hemodialysis, which show increased blood compatibility, in particular anticoagulation properties . There is no disclosure of increasing selectivity of the membrane by the coating .

[0035] KR 102 428 696 Bl discloses hemodialysis membranes with improved phosphate adsorption . The surface of a membrane such as PES is modified by grafting acrylic acid and subsequently binding gelatin by reaction with the carboxylic acid groups of acrylic acid, the final coating thus does not comprise carboxylic acid groups . There is no mention of increasing selectivity for other molecules than phosphate .

[0036] US 11 185 827 B2 discloses surface modified filters for blood filtering, in particular for separating plasma and serum. The membranes aim at reducing hemolysis and avoiding reduction of the levels of metabolic products such as peptide hormones ( e . g . , ACTH, insulin and leptin ) in plasma , which may influence analysis results of these products . There is no mention of increasing selectivity for retention of albumin and removal of other molecules such as large middle molecules by such coating .

[0037] US 2010 / 135852 Al discloses an oxygenator in the form of a hollow fiber membrane , whereby the part in contact with blood is coated with a methacrylate copolymer as antithrombotic material preventing complement activation, blood coagulation, platelet activation and plasma leakage . The polymers used do not comprise an acid group .

[0038] It was now found that the modification or coating of semipermeable membranes having a defined MWRO and MWCO range with certain vinyl monomers that carry a sulfonic acid or sulfonate group or a carboxylic acid or carboxylate group results in highly selective and hemocompatible membranes which are excellent hemodialysis membranes .Summary of the Invention

[0039] It is an obj ect of the present invention to provide highly selective and hemocompatible semipermeable membranes for extracorporeal blood treatment applications which are characterized by a base membrane which is coated with non-animal derived,synthetic molecules that introduce a negative charge. Said synthetic molecules preferably are based on vinyl monomers that comprise a sulfonic acid or sulfonate group, such as AMPS, or that comprise a carboxylic acid or carboxylate group. The resulting coating accordingly comprises or essentially consists of a vinyl polymer derived from vinyl monomers of formula (A)H2C=CH-R-A (A) , wherein A represents an acidic functional group, and R represents an organic combining group, and wherein A, according to one embodiment, represents a sulfonic acid (-SO3H) or a sulfonate (-SOs-) group; or represents a carboxylic acid (-COOH) or a carboxylate (-C00-) group.

[0040] According to one aspect, the base membrane is a medium cut-off membrane and has a MWRO of from 8.6 to 14.0 kDa and a MWCO of from 50.0 to 130.0 kDa as measured by dextran sieving before blood contact, preferably a MWRO of from 8.6 to 12.5 kDa and a MWCO of from 50.0 to 110.0 kDa as measured by dextran sieving before blood contact. According to yet another aspect, the base membranes have a MWRO of from 9.0 to 14.0 kDa and a MWCO of from 60.0 to 130.0 kDa as measured by dextran sieving before blood contact .

[0041] According to another aspect, the base membrane is a high cut-off membrane having a MWRO of from 15kDa to 20kDa and a MWCO of from 170kDa to 320kDa as measured by dextran sieving before blood contact.

[0042] According to another aspect, the base membrane is a high-flux (HF) membrane having an MWRO of from 5.0 kDa to 8.5 kDa and a MWCO of from 25 kDa to 50 kDa as measured by dextran sieving before blood contact.

[0043] According to one embodiment, the base membrane is characterized by a molecular weight retention onset (MWRO) of between 5.0 kDa and 20.0 kDa and a molecular weight cut-off (MWCO) ofbetween 50 kDa and 320 kDa as determined by dextran sieving before blood contact. According to yet another embodiment, the base membrane is characterized by a molecular weight retention onset of between 9.5 kDa and 20.0 kDa and a molecular weight cut-off (MWCO) of between 60 kDa and 320 kDa as determined by dextran sieving before blood contact.

[0044] According to another embodiment, the base membrane is characterized by a molecular retention onset (MWRO) of between 10.0 kDa and 17.0 kDa and a molecular weight cut-off (MWCO) of between 70 kDa and 300 kDa as determined by dextran sieving before blood contact .

[0045] According to yet another embodiment, the base membrane is characterized by a molecular retention onset (MWRO) of between 10.0 kDa and 17.0 kDa and a molecular weight cut-off (MWCO) of between 70 kDa and 120 kDa as determined by dextran sieving before blood contact .

[0046] According to yet another embodiment, the base membrane is characterized by a molecular retention onset (MWRO) of between 10.5 kDa and 12.5 kDa and a molecular weight cut-off (MWCO) of between 60 kDa and 90 kDa as determined by dextran sieving before blood contact .

[0047] According to another embodiment, the base membrane comprises or essentially consists of a hydrophobic component chosen from the group consisting of polysulfone (PS) , polyethersulfone (PES) , polyarylethersulfone (PAES) , or combinations thereof, and a hydrophilic component chosen from the group consisting of polyvinylpyrrolidone (PVP) , polyethyleneglycol (PEG) , polyvinylalcohol (PVA) , and a copolymer of polypropyleneoxide and polyethyleneoxide (PPO-PEO) . According to one embodiment, the hydrophobic component is PES. According to another embodiment, the hydrophilic component is PVP.

[0048] According to another embodiment of the invention, the membrane is prepared from a polymer solution comprising 10 to 20 wt.-% of at least one hydrophobic polymer component, 5 to 10 wt . - % of at least one hydrophilic polymer component and at least one solvent .

[0049] According to another embodiment the base membrane is characterized by a mean pore size of between 5.0 nm and 12.0 nm. According to yet another embodiment, the base membrane is characterized by a mean pore size of between 5.5 nm and 11.0 nm.

[0050] According to yet another embodiment, the base membrane is a hollow fiber membrane. According to yet another embodiment, the hollow fiber base membrane has an inner diameter of the membrane is below 200 pm and the wall thickness is below 40 pm.

[0051] According to another embodiment, the base membranes in the context of the present invention are polysulfone-based, polyethersulf one-based or poly (aryl) ethersulfone-based synthetic membranes, comprising, in addition, a hydrophilic component such as, for example, PVP and optionally low amounts of further polymers, such as, for example, polyamide or polyurethane.

[0052] According to another embodiment of the invention, the base membrane is prepared from a polymer solution which comprises from 10 to 15 wt.%, relative to the total weight of the polymer solution, of polysulfone, polyethersulfone or polyarylethersulfone, and from 1 to 10 wt.%, relative to the total weight of the polymer solution, of polyvinylpyrrolidone.

[0053] According to another embodiment of the invention, the base membrane before coating comprises from 0.5 wt.-% to 5.0 wt . - % PVP and from 95.0 wt.-% to 99.5 wt . -% PES, PS, or PAES.

[0054] According to another embodiment of the invention, membranes that are preferably used as base membranes can havedifferent physical structures , including varying degrees of asymmetric configuration, ranging from minimum asymmetry in spongelike structures to maximum asymmetry in finger-like structures , such as described, for example , in Ronco and Clark, Nature Revi ews . Nephrology 14 ( 6 ) ( 2018 ) : 394 -410 . In some applications the lumen or inner side of the hollow fibers membranes is coated as it generally provides for the selective layer of the hollow fiber membranes in blood purification applications , wherein the lumen is in contact with blood, or , optionally, with blood plasma . The "lumen" surface of the membrane has pores which may extend from said lumen side of the hollow fiber membrane towards the other side of the membrane . However , devices wherein the outer surface of hollow fiber membranes is in contact with blood whereas the lumen is transfused with a solution ( e . g . , a dialysis solution ) or gas are also known . In devices where the outer side of a hollow fiber membrane is in contact with blood and provides for the selective layer of the membrane , a modification or coating according to the invention would preferably be applied to the outer side of the hollow fiber membranes . If so desired, the complete membrane can be treated with a coating solution according to the invention and will then be coated on the outer surface as well as on the lumen side of the hollow fiber membrane , including the accessible surfaces of the pores of the membrane .

[0055] According to another embodiment , the base membrane is a hollow fiber membrane having a finger-like structure and is modified or coated with a material that provides for biocompatibility, antithrombogenicity, and is stable during extracorporeal blood treatment . Said material further results in a coated membrane surface which is negatively charged in a way that the selectivity of the base membrane is modified towards a higher removal of large middle molecules and concomitant sharp rej ection of albumin in extracorporeal blood treatment applications without negatively impacting hemocompatibility and / or antithrombogenicity .

[0056] According to another embodiment, the compound used for coating the base membrane is a vinyl monomer with an acidic group, wherein the acidic group is selected from sulfonic acid and carboxylic acid and its salts, respectively.

[0057] According to another embodiment, the compound for coating the base membrane is a vinyl monomer of formula (I)H2C=CH-R-SO3H (I) or its salts, wherein R represents an organic combining group.

[0058] According to another embodiment, the compound for coating the base membrane is a vinyl monomer of formula (II)H2C=CH-R-COOH (II) or its salts, wherein R represents an organic combining group.

[0059] R in formula (I) and formula (II) represents an organic combining group. According to a further embodiment, R represents a moiety selected from the moieties 1, 2, 3, 4, or 5, according to Table II:TABLE IIwherein n represents 0 or an integer between 1 and 12, preferably between 1 and 8, especially between 1 and 4; and wherein Ri represents -(CH2)m-, wherein m represents 0 or an integer between 1 and 12, preferably between 1 and 8, especially between 1 and 4, and wherein one or more of the carbon atoms of Ri may be substituted with one or more of methyl, ethyl, propyl, phenyl, or benzyl.

[0060] According to one embodiment of the invention, the compound used for coating the membrane is selected from the group of compounds comprising or consisting of 2-acrylamido-2-methylpro- pane sulfonic acid (AMPS) , sodium 4-vinylbenzensulf onate (VBS) , sodium vinylsulf onate (VS) , potassium 3- ( acryloyloxy ) propane-1- sulf onate (AOPS) , or combinations thereof.

[0061] According to another embodiment of the invention, the compound used for coating the membrane is acrylic acid, methacrylic acid, acryloyl-L-phenylalanine, acryloyl-L leucine.

[0062] According to another embodiment, the present invention is directed at a process for preparing the coated membranes according to the invention (Figure 3) , comprising the steps of(i) providing a base membrane as defined herein,(ii) contacting the base membrane with a coating solution that comprises a compound of formula (A) ,(iii) irradiating the membrane in the presence of said coating solution,( iv) removing the coating solution and rinsing the coated membrane , and(v) optionally drying the membrane , and / or(vi ) optionally sterilizing the membrane .

[0063] According to one embodiment , the process is applied to a hollow fiber base membrane , wherein step ( ii ) comprises immersing the hollow fiber membrane in the coating solution, and wherein all accessible surfaces of the hollow fiber membrane are brought into contact with the compound of formula (A) , or of formula ( I ) or ( II ) during step ( iii ) . The expression "all accessible surfaces" refers to the outer side of the membrane , its inner side or lumen side , and any pore surface that can be brought into contact with a solution, such as pores spanning the membrane wall and through which a solution can flow or be pumped form the lumen side to the outer side of the membrane or vice versa , but also including pores that do not span the membrane wall and represent dead-end pores , but which can nevertheless be contacted or filled with a solution . According to another embodiment , only the lumen side of the membrane and any adj acent pore surface is brought into contact with the coating solution . According to yet another embodiment , only the outside of the membrane and any adj acent pore surface is contacted with the coating solution .

[0064] According to another embodiment , the hollow fiber is provided, in step ( i ) , as part of a fiber bundle within a filter housing , wherein the fiber bundle is encapsulated at each end of the dialyzer in a potting material thereby providing for a first flow space surrounding the hollow fiber membranes on the outside , which is accessible by means of a dialysate inlet and / or a dialysate outlet , and a second flow space formed by the lumen space of the hollow fibers and the flow space above and below said potting material which is in flow communication with said lumen space and which is closed by end caps having a blood inlet and blood outlet , respectively; and wherein the coating solution is provided to the outside of the hollow fiber membranes through said dialyzer inletand / or outlet, and to the lumen of the hollow fibers through said blood inlet / or outlet.

[0065] According to another embodiment of the invention, the membrane of the invention is coated on its accessible surface, including the lumen surface, the outer surface, and the accessible pore surface of the membrane.

[0066] According to yet another embodiment, the membrane is brought into contact with the coating solution for 5 seconds to 60 minutes at room temperatures, for example at a temperature of from 15°C to 28°C, such as from 18°C to 25°C before discarding the coating solution. According to yet another embodiment, the membrane is brought in contact or kept in contact with the coating solution for 20 seconds to 20 minutes. According to yet another embodiment, the membrane is brought into contact with the coating solution for up to 24 hours.

[0067] According to yet another embodiment, the coating solution comprises from 2 to 1000 mg / mL of the compound of formula (I) or formula (II) , such as from 5 to 600 mg / mL, from 5 to 100 mg / mL, from 5 to 75 mg / mL, or from 5 to 50 mg / mL, including, for example, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, 23 mg / mL, 25mg / mL, 28 mg / mL, 30 mg / mL, 33 mg / mL, 35 mg / mL, 38 mg / mL, 40 mg / mL, 43 mg / mL, 45 mg / mL, 48 mg / mL, 50 mg / mL, 53 mg / mL, 55 mg / mL, or 60 mg / mL .

[0068] According to yet another embodiment, the coating solution comprises AMPS, VBS, VS, or AOPS. According to yet another embodiment, the vinyl monomer which is used for coating the base membrane is AMPS.

[0069] According to yet another embodiment, step (iii) comprises e-beam irradiation, gamma ray irradiation, or UV irradiation, wherein the dose is between 20 and 110 kGy, such as from 25to 110 kGy, from 25 to 90 kGy, from 40 to 90 kGy, from 50 to 90 kGy, from 60 to 80 kGy, including, for example, 25 kGy, 30 kGy, 35 kGy, 40 kGy, 45 kGy, 50 kGy, 60 kGy, 65, kGy, 70 kGy, 75 kGy, 80 kGy, 85 kGy, and 90 kGy, and any values in between. According to yet another embodiment, step (iii) comprises e-beam irradiation .

[0070] According to one embodiment, irradiation in step (iii) is done with filter devices comprising the hollow fiber bundle to be coated, wherein the device is completely filled with coating solution, including the lumen side of the hollow fibers, and wherein the coating solution is in contact with all accessible surfaces of the hollow fibers. According to another embodiment, irradiation in step (iii) is done after having emptied the filter devices, wherein the membranes as still wet, i.e., drenched with the coating solution.

[0071] According to another embodiment, the coated membrane after rinsing and optionally drying is sterilized with steam, gamma irradiation, or e-beam. According to yet another embodiment, the coated membrane is sterilized with steam without previously drying the membrane, i.e., in the presence of residual water. According to yet another embodiment, the coated membrane is dried and subsequently sterilized by e-beam sterilization. According to yet another embodiment, the coated membrane is sterilized by e- beam sterilization without previously drying the membrane and in the presence of residual water.

[0072] According to yet another embodiment, rinsing is done with water, such as RO water, or a buffer solution, such PBS or saline, at a temperature of preferably 12°C to 30°C, such as, for example, from 15°C to 25°C, or at room temperature.

[0073] According to another embodiment of the present invention, the amount of coating present on the base membrane as based on the compounds of formula (A) , (I) , or (II) is from 10 mg / m2to 180 mg / m2, such as from 10 mg / m2to 140 mg / m2, from 15 to 80 mg / m2.

[0074] According to another embodiment, the membrane of the invention comprises (i) a base membrane comprising or essentially consisting of a hydrophobic polymer selected from PS, PES, or PAES, and the hydrophilic polymer PVP, has a MWRO of between 10 kDa and 14 kDa and a MWCO of between 68 kDa and 320 kDa as determined by dextran sieving before blood contact, and (ii) a coating deposited on said base membrane comprising or essentially consisting of poly-2-acrylamido-2-methypropane sulfonic acid (pAMPS) which has been grafted onto the base membrane. According to yet another embodiment, said pAMPS has been grafted onto the base membrane by radiation-induced graft polymerization (RIGP) .

[0075] According to a further embodiment, the membrane of the invention and blood purification devices comprising same are designed to increase the clearance of large middle molecules (Figure 1) and extend the removable range of uremic toxins (Figure 2) while maintaining or significantly reducing the level of retention of albumin in comparison with prior art membranes such as high- flux membranes, medium cut-off membranes, or high cut-off membranes .

[0076] According to another embodiment, the coated membrane of the present invention has a sieving coefficient for albumin of between 0.001 and 4.50 and for YKL-40 of between 40 and 98. According to yet another embodiment of the invention, the coated membrane of the present invention has a sieving coefficient for albumin of between 0.001 and 2.50 and for YKL- 40 of between 55 and 98.

[0077] According to another embodiment, the present invention is directed at hemodialysis devices or filters comprising the coated membrane of the invention.

[0078] According to yet another embodiment, the coated membranes of the present invention are designed for safe use inhemodialysis and hemodiaf iltration applications for patients suffering from chronic and / or end stage renal failure.

[0079] The present invention is also directed to methods of using the coated membranes of the invention in blood purification applications, in particular in hemodialysis methods, including CRRT, for treating acute, advanced, and permanent kidney failure.Brief Description of the Drawings

[0080] Figure 1 shows a comparison of the overall clearance of YKL-40, a large middle molecule, during 1 h simulated hemodialysis (HD) with human plasma from healthy donors according to ISO8637 : 2004. QB, QD, and Uf, are indicated for each dialyzer tested. Effective surface areas (m2) are as provided by the manufacturer. The dialyzer comprising a membrane according to the invention had an effective surface area of 1.7m2for the dialyzers comprising a membrane according to Example 1 (MCO 4 base membrane with an average MWRO of 11.7 kDa and an average MWCO of 75 kDa, coating with a solution with 20 mg / mL AMPS, 75 kGy e-beam, rinsing, and steam sterilization) .

[0081] Figure 2 is a general, schematic representation of small, middle and large molecular solutes which are removed by various membrane classes. Membranes according to the invention are designed to extend the spectrum of large middle molecules which can efficiently be removed to higher molecular weights compared to low-flux, high-flux, and even medium cut-off membranes. Retention capabilities for relevant molecules, such as albumin and larger molecules, is not directly shown. However, the said relevant molecules are retained by membranes according to the invention as good as or better than with high-flux or medium cutoff membranes. They are therefore also superior to high cut-off membranes that show some albumin loss.

[0082] Figure 3 is a schematic representation of the process used for coating the base membrane. In said process, the coatingsolution, shown as a droplet, is pumped into the filter device via the lumen side of the hollow fibers which are arranged as a bundle in the dialyzer housing (i) , the filter is closed so the solution remains inside the hollow fibers (ii) . The filter, including the coating solution, is then submitted to irradiation such as by e-beam irradiation (iii) . After irradiation the filter ports are opened, and the coating solution is removed (iv) . The hollow fibers can then be rinsed, and the dialyzer can be tested for leaks and eventually be submitted to steam sterilization or, after drying, to e-beam sterilization.

[0083] Figure 4 is a schematic representation of the radiation induced graft copolymerization (RIGC) reaction that takes place during irradiation. As a result of radiation induced graft copolymerization, the polymeric side chains grow from the surface by polymerizing the vinyl monomers of e.g. , formula (I) , ultimately resulting in a graft copolymer which forms the "coated membrane". Radiation induced graft immobilization (RIGI) of homopolymers generated from monomers according to the invention will potentially occur alongside RIGC which is not shown here.

[0084] Figure 5 depicts the amount of toluidine which is adsorbed on the surface of membranes which have been coated according to the invention, such as with AMPS shown here, as a measure for the amount of AMPS coated on the membrane . The amount of toluidine corresponds to the amount of AMPS in the coating solution, as expected.

[0085] Figure 6 depicts the clearance for YKL-40 in mL / min for a set of dialyzers which are compared with each other regarding their clearance efficiency. Said dialyzers are REVACLEAR 500, comprising a High-Flux membrane; THERANOVA 400 and THERANOVA 500, both comprising a medium cut-off membrane, a device comprising the MCO-4 membrane (also a medium cut-off membrane) without coating and with a 2% (or 20 mg / mL) coating with AMPS followed by irradiation according to the invention.

[0086] Figure 7 compares the albumin loss in mg / L when using MCO-4 (a medium cut-off membrane) without coating (upper curve) and MCO-4 with a coating according to the invention (lower curve) over 60 minutes .

[0087] Figure 8 depicts a combined overview over YKL-40 clearance of various coated membranes according to the invention in comparison with each other and with THERANOVA 400 as a dialyzer of the state of the art, and of the respective albumin loss of the dialyzers shown in mg / h.

[0088] Figure 9 is a Boxplot depiction of the influence of an exemplary membrane according to the invention (MCO4 coated) on coagulation in terms of TAT generation in pg / L in comparison with an uncoated medium cut-off membrane (Example 1.3 from W02015118045A1) and relative to a negative and positive control and a baseline (see Example 4) .

[0089] Figure 10A is a Boxplot depiction of the influence of an exemplary membrane according to the invention (MCO4 coated) on red blood cell count (RBC) in comparison with an uncoated medium cut-off membrane (Example 1.3 from W02015118045A1) and relative to a negative and positive control and a baseline (see Example 4) .

[0090] Figure 10B is a Boxplot depiction of the influence of an exemplary membrane according to the invention (MCO4 coated) on white blood cell count (WBC) in comparison with an uncoated medium cut-off membrane (Example 1.3 from W02015118045A1) and relative to a negative and positive control and a baseline (see Example 4) .

[0091] Figure IOC is a Boxplot depiction of the influence of an exemplary membrane according to the invention (MCO4 coated) on platelets (PLT) in comparison with an uncoated medium cut-offmembrane (Example 1.3 from W02015118045A1 ) and relative to a neg- ative and positive control and a baseline (see Example 4) .

[0092] Figure 11 is a Boxplot depiction of the influence of an exemplary membrane according to the invention (MCO4 coated) on the formation of human platelet factor 4 (PF-4) as a measure for hemocompatibility in comparison with an uncoated medium cut-off membrane (Example 1.3 from W02015118045A1 ) and relative to a negative and positive control and a baseline (see Example 4) .

[0093] Figure 12 is a Boxplot depiction of the influence of an exemplary membrane according to the invention (MCO4 coated) on complement factor C3a activation as a measure for hemocompatibility in comparison with an uncoated medium cut-off membrane (Example 1.3 from W02015118045A1) and relative to a negative and positive control and a baseline (see Example 4) .

[0094] Figure 13 is a Boxplot depiction of the influence of an exemplary membrane according to the invention (MCO4 coated) on formation free hemoglobin (fHB) as a measure for hemolysis and hemocompatibility in general in comparison with an uncoated medium cut-off membrane (Example 1.3 from W02015118045A1) and relative to a negative and positive control and a baseline (see Example 4) .

[0095] Figure 14 depicts the endotoxin retention capabilities as LRV for two membranes according to the invention (MCO-4, both coated and submitted to final sterilization with either steam or e-beam irradiation) , the same MCO-4 membrane without any coating, and for the MCO THERANOVA membrane as a state-of-the-art membrane which is known for its good endotoxin retention capabilities .Detailed Description

[0096] The present invention is directed at membranes comprising a base membrane having a defined MWRO and MWCO, including high-flux , medium cut-off , and high cut-off type membranes , which are coated with a solution comprising vinyl monomers carrying a sulfonic acid or sulfonate group and / or carboxylic acid or carboxylate group by radiation induced graft copolymerization ( RIGC ) and / or radiation induced graft immobilization ( RIGI ) , resulting in a membrane surface which essentially consists of a graft copolymer comprising the base membrane surface and the polymer side chains generated from the vinyl monomers used and which are covalently bound to the base membrane material . The combination of the base membranes and vinyl monomers as disclosed herein results in an unexpectedly pronounced effect on the performance of the coated membranes , which can be summarized as a significant increase in the removal capabilities of large middle molecules , evidenced by increased sieving coefficients and clearance for selected marker molecules , whereas the corresponding values for albumin remain the same or are significantly decreased in comparison to the uncoated membrane . Without wanting to be bound by theory, the membranes provided herein are a synthesis of the sieving capabilities of the base membrane , which largely relies on size exclusion, and effects relating to the careful introduction of charges , that result in surprising changes and improvements of membrane performance , especially regarding selectivity .

[0097] It is also highly surprising that the modifications described herein do not lead to a negative effect on hemocompatibility, thrombogenicity, and / or cytotoxicity, as could be expected when introducing negative charges ( ) . In contrast , the modification results in highly hemocompatible and antithrombo- genic membranes even when compared to their respective base membranes , which are already known for their good performance in this regard .

[0098] As explained in further detail below, it is especially surprising that the selectivity increase takes effect on different membrane classes in different but consistent ways . High-flux membranes , for example , have a very low sieving coefficient for albumin, but their capability to remove larger middle molecules is limited, which is evident from their MWCO ( see Table I ) . The modification as described herein leads to an increase of the removal capabilities regarding larger middle molecules , as can be shown, for example , for an appropriate marked molecule such as YKL-40 ( sieving coefficient and clearance of the membrane and dialyzer, respectively) . The excellent albumin retention capabilities , however, are not impacted .

[0099] In the case of high cut-off membranes ( see Table I ) , the starting situation is essentially the reverse . High cut-off membranes , such as , for example , THERALITE , have excellent removal capabilities for large middle molecules such as YKL-40 , but also show an increased loss of albumin which prohibits their use in maintenance hemodialysis . The modification of a high cut-off base membrane according to the invention results in coated membranes which show a significantly reduced albumin coefficient ( lowered sieving coefficient for albumin) , whereas the excellent removal capabilities for large middle molecules are not negatively impacted and can even turn out to be improved .

[0100] The effect of the coating as disclosed herein is especially interesting in relation to base membranes which are classified as medium cut-off membranes ( see again Table I , or Zhang et al . ( 2022 ) , Membranes 12 , 253 ) . Medium cut-off membranes are advanced membranes that have significantly improved the removal of higher molecular weight toxins while essentially retaining albumin, especially in combination with a tailored design of the fibers , fiber bundles and hemodialyzers in which they are provided . As a result , they can match the results , when used in hemodialysis modality ( HD ) , that can otherwise be achieved only with more complex hemodiaf iltration modalities . It is difficult to further improve said high selectivity of medium cut-offmembranes with further, even though this would be desirable to push membrane and dialyzer performance ever closer to what a kidney can do. However, when subjected to the modifications disclosed herein, medium cut-off base membranes turned out to be even more selective than the untreated base membrane, evidenced by increased sieving coefficients for large middle molecules (such as, for example, YKL-40) as well as improved clearance of such molecules by dialyzers containing said membranes . Importantly, the very low albumin loss of medium cut-off membranes can, at the same time, at least be maintained but generally, albumin loss can be lowered significantly up to almost complete rejection of albumin.

[0101] The expression "coating" or "coated" as used herein refers to the modification of a base matrix, herein disclosed as a base membrane, by depositing selected materials onto said base matrix. In the context of the present invention, said deposition occurs by radiation induced graft copolymerization (RIGC) which leads to the generation of active sites that can lead to the initiation of a graft polymerization and the formation of graft copolymers, i.e. , to the covalent modification of the base membrane with the selected material, which in the present case comprises compounds of formula (I) and / or formula (II) . RIGC, in the context of the present invention encompasses the so-called "graft- ing-from" approach (Figure 3 and Figure 4) , in which polymeric side chains grow directly from the surface by polymerizing the monomers according to the invention (Schmidt et al. (2021) , Radiation-Induced Graft Immobilization (RIGI) : Covalent Binding of Non-Vinyl Polymers 13:1849) , as well as homopolymerisation of the monomers of formula (I) or (II) following initial radical formation in one or more monomers, wherein the resulting homopolymer may get attached to the base membrane in a second step ("grafting- to" approach) . In some cases, the homopolymer may be deposited as such on the base membrane without being covalently bound thereto. When using the expressions "coating" or "coated", it is assumed that the above processes can occur simultaneously, even though it is assumed that in the context of the present invention the"graf ting-f rom" process and covalent coupling of the material prevails .

[0102] The expression "selectivity" as used herein for a hemodialysis membrane refers to the membrane's ability to efficiently remove small (0.5-15kDa) , medium (>15-25kDa) and large (>25-58kDa) middle-molecular weight compounds (see also Rosner et al . : Classification of Uremic Toxins and Their Role in Kidney Failure. Clin J Am Soo Nephrol. 2021) while essentially retaining essential, larger proteins such as, specifically, albumin (66.4 kDa) . Such selectivity is described, for example, by the steepness of the sieving curve (e.g. , dextran sieving curve) of a membrane. The increased steepness of the sieving curve correlates to increased selectivity, indicating that the membrane has an increased ability to remove large middle while efficiently retaining albumin. For example, selectivity of a membrane can be described by its MWRO (molecular weight retention onset) , i.e. , the molecular weight at which the sieving coefficient of the membrane is 0.9, and its MWCO (molecular weight cut-off) , i.e. , the molecular weight at which the sieving coefficient of the membrane is 0.1, as these parameters provide for the necessary information on the shape and steepness of a membrane's sieving curve. By way of example, defining two points, i.e. MWCO and MWRO, on the sieving curve of a membrane allows a better characterization of the sigmoid sieving curve (see Ronco and Clark (2018) , Nature Reviews. Nephrology 14 (6) : 394-410) . It widens the methodology to the second dimension, giving an indication of the pore sizes and also of the pore size distribution. Thereby, MWRO and MWCO are independent parameters . While the slope (steepness) of the curve depends on both, membranes with similar MWCO may have a different MWRO and thus sieving curves with different steepness and different permeability and selectivity, as for instance disclosed in Figure 2 of Ronco et al . , Expanded Hemodialysis - Innovative Clinical Approach In Dialysis. Contrib Nephrol. Basel, Karger, 2017, vol 191, pp 115-126) Together the parameters MWCO and MWRO in combination are an indication to the pore size distribution of a membrane. In general, one can interpret the MWRO value as some reference of where the pore size distribution starts, while the MWCO indicates where it ends (see Boschetti et al . ,Extended characterization of a new class of membranes for blood purification: The high cut-off membranes; Int J Artif Organs 2012; 36 (7) : 455-463) . However, these parameters only provide an indication on the pore size distribution of a membrane, but do not directly correlate thereto or may not be directly derivable from each other or from the pore size therefrom, as besides the pore size distribution also the pore morphology and structure has to be taken into account. In summary, the introduction of the second characteristic point for the dextran sieving curve, the MWRO, allows dialysis membranes to be differentiated using only one simple, in vitro method namely dextran sieving, which is performed as a routine characterization for dialysis membranes. The MWRO as an additional parameter brings out the second dimension of the structural properties in the membranes, so that both the pore size and the pore size distribution are fully considered and unwanted misperceptions are avoided. Alternatively, selectivity can be described by determining the sieving coefficients for a set of relevant marker molecules with different and increasing molecular weights, and which also serve for defining a sieving curve. Similarly, selectivity can be determined by selecting two relevant marker molecules such as albumin and YKL-40. An increasing selectivity can be shown by a high sieving coefficient (SC) for YKL-40 and a low sieving coefficient for albumin, meaning that the higher the SC for YKL-40 gets, while the SC for albumin remains stable or is reduced, the more selective a membrane becomes .

[0103] The expression "medium cut-off membrane (s)" or "medium cut-off dialyzer (s)" as used herein, which is sometimes interchangeably used with the expression "HRO membrane (s)" or "HRO dialyzer (s)" for "High Retention Onset" membrane (s) or dialyzer^) , refers to membranes and dialyzers such as described, for example, in WO 2015 / 118046 Al and WO 2015 / 118046 Al, respectively, and wherein the membrane (s) preferably has (have) a MWRO of from 9 to 12.5 kDa and a MWCO of from 55 to 110 kDa as measured by dextran sieving before blood contact and as otherwise described in WO 2015 / 118046 Al. Preferably, medium cut-off membranes are further defined by a sieving coefficient for albumin (ScAib (%) ) of below 1 (<1) as measured in human plasma at QB=300ml / min andQUF=60ml / min; a sieving coefficient for L-2-microglobulin (ScB2m(%) ) of equal to or more than 95 (>95) as measured in human plasma at QB=300ml / min and QUF=60ml / min; and a sieving coefficient for YKL-40 ( SCYKL-4O (%) ) of equal to or more than 30 (>30) as measured in human plasma at QB=300ml / min and QUF=60ml / min . A medium cutoff dialyzer as mentioned herein is preferably further characterized by a KUF (ml / h / mmHg) of equal to or higher than 20 (>20) as measured according to ISO 8637; a L2m clearance K^m (ml / min)of higher than 80 (>80) as measured according to ISO 8637 with QB=300- 400mL / min; QD=7 OOmL / min; QUF=0-10mL / min; a YKL-40 clearance KYKL-4O (ml / min) of equal to or higher than 20 (>20) as determined in human plasma with QB=300ml / min; QD=500ml / min; QUF=10ml / min and preferably further has an albumin loss (g / 4h in vivo treatment or simulated use correlated with in vivo use) of equal to or below 7 (<7.0) , more preferably of equal to or below 4 (<4.0) .

[0104] The expression "vinyl monomer" as used herein refers to a vinyl monomer of formula (A)H2C=CH-R-A (A) or its salts, wherein A represents an acidic group such as sulfonic acid in formula (I)H2C=CH-R-SO3H (I) or such as carboxylic acid in formula (II)H2C=CH-R-COOH (II) or their salts, wherein R represents an organic combining group. R in formula (I) and formula (II) may represent a moiety selected from the moieties shown in Table II above, wherein R2represents -(CH2)m-, wherein m represents 0 or an integer between 1 and 12, between 1 and 8, between 1 and 4, between 1 and 3, or represents 1 or 2; and wherein one or more of the carbon atoms of R2may besubstituted with one or more of methyl, ethyl, propyl, phenyl, or benzyl.

[0105] The expression "base membrane" as used herein refers to a membrane before being coated with a vinyl monomer according to the invention. When used in the context of describing the final, coated membrane, the expression "base membrane" accordingly refers to the portion of the coated membrane which is derived from the base membrane before coating.

[0106] The expression "blood treatment" as used herein refers to the treatment of whole blood or any blood product, such as, for example, blood plasma, of a human or animal patient. Such treatment comprises, but is not limited to, hemodialysis, hemofiltration or hemodiaf iltration, and further includes extracorporeal blood treatment applications for the support of various organs, such as kidneys, liver, lung, or heart.

[0107] The material of membranes used according to the invention may vary but will generally be uncharged membranes. Suitable base membranes in the context of the present invention comprise, for example, a blend of i) a polysulfone, a polyethersulfone or a polyarylethersulfone and ii) polyvinylpyrrolidone, polyethyleneglycol (PEG) , polyvinylalcohol (PVA) , or a copolymer of polypropyleneoxide and polyethyleneoxide (PPO-PEO) . It I sone aspect of the present invention to use membranes comprising or essentially consisting of a blend of i) a polysulfone, a polyethersulfone or a polyarylethersulfone and ii) polyvinylpyrrolidone, wherein a blend of polyethersulfone and PVP would be used most often. The blend can comprise certain additives, such a vitamins like vitamin E, or other polymers such as polyacrylonitrile, polyurethane, or polyamide. Base membranes according to the invention can also be made from other known materials as the concept of selectivity increase as described herein is connected to the modification of the surface and pores of a given membrane, and the coating is based on RIGP and / or RIGI . Therefore, various materials are accessible for coating. Other membrane materials that canaccordingly be used are, for example, sulfonated polyacrylonitrile (PA or AN69) , poly(methyl methacrylate) (PMMA) , CTA (cellulose triacetate) , or EVAL (ethylene vinyl alcohol copolymer) . For an overview of membrane materials see, for example, Said et al . (2021) , A Review of Commercial Developments and Recent Laboratory Research of Dialyzers and Membranes for Hemodialysis Application. Membranes 11:767.

[0108] Base membranes according to the invention are prepared, for example, from a blend of i) a polysulfone, a polyethersulfone or a polyarylethersulfone and ii) polyvinylpyrrolidone and optionally any additives such as mentioned above. For example, a suitable base membrane can be prepared from a polymer solution comprising 10 to 20 wt.-% or 10 to 15 wt.-% of at least one hydrophobic polymer component, such as PS, PES, or PAES, and from 1 to 10 wt.-% or 5 to 10 wt.-% of at least one hydrophilic polymer component, such as PVP, and at least one solvent. Such base membrane, before coating, accordingly comprises or essentially consists of from 0.5 wt.-% to 5.0 wt.-% PVP and from 95.0 wt.-% to 99.5 wt . — % PES, PS, or PAES.

[0109] Base membranes according to the invention, according to another aspect, can be characterized by a mean pore size of between 5.0 nm and 12.0 nm, such as between 5.5 nm and 11.0 nm or between 5.0 and 8.0 nm.

[0110] According to another aspect of the invention the base membrane is a hollow fiber membrane, wherein according to one specific aspect the inner diameter of the membrane is below 200 pm and the wall thickness is below 40 pm.

[0111] According to one aspect, base membranes according to the invention can have different physical structures, including varying degrees of asymmetric configuration, ranging from minimum asymmetry in sponge-like structures to maximum asymmetry in fin- ger-like structures, such as described, for example, in Ronco andClark ( 2018 ) , Nature Reviews. Nephrology 14 (6) : 394-410) . In caseof hollow fiber membranes for use in hemodialysis , at least the lumen or inner side of the hollow fibers membranes is coated as it generally provides for the selective layer of the hollow fiber membranes that is in contact with blood, or , optionally, with blood plasma . In devices where the outer side of a hollow fiber membrane is in contact with blood and provides for the selective layer of the membrane , a modification or coating according to the invention would preferably be applied to the outer side of the hollow fiber membranes . According to a preferred embodiment , the complete accessible surface of a membrane is coated . As will be understood by the s killed person, the accessible surface includes the surfaces of pores present and accessible from the lumen side and / or the outside of a hollow fiber , which will also be coated according to the invention . The membranes of the present invention can also be produced as flat sheet membranes according to known methods and can be coated accordingly, e . g . , by immersing the flat sheet membranes in a coating solution like what is described for hollow fiber membranes .

[0112] Accordingly, the base membrane of the invention may have an asymmetric foam- or sponge-like structure , or an asymmetric finger-like structure with at least three layers , wherein the separation layer has a thickness of less than 0 . 5 pm . The next layer in the hollow fiber membrane is the second layer , having the form of a sponge structure and serving as a support for said first layer . In a preferred embodiment , the second layer has a thickness of about 1 to 15 pm . The third layer has the form of a finger structure . Like a framework, it provides mechanical stability on the one hand; on the other hand a very low resistance to the transport of molecules through the membrane , due to the high volume of voids . The third layer , in one embodiment of the invention, has a thickness of 20 to 30 pm. In another embodiment of the invention, the membranes also include a fourth layer, which is the outer surface of the hollow fiber membrane . This fourth layer has a thickness of about 1 to 10 pm .

[0113] According to one aspect, the base membrane is a medium cut-off membrane. A representative of such medium cut-off membranes which is available in the market today is the THERANOVA dialyzer. Other medium cut-off type membranes and dialyzers and how they can be prepared are disclosed in WQ2015118045A1 and WQ2015118046A1, including, for example, Examples 1.1 through 1.7. (membranes A to G) , which have the indicated MWRO and MWCO and finger-like structures (A to E and G) , whereas Membrane F has a sponge-like structure. Membrane A, for example, has been used as an experimental membrane in certain studies, where it is generally referred to as "MCO-4" or "MCO-Ci". Said MCO-4 membrane has a mean MWRO of 11.7 kDa and a mean MWCO of 75.0 kDa (see Table III of WQ2015118045A1) and is used herein for demonstrating the effect of the coating according to the invention on selectivity, sieving coefficients, and clearance. Membrane B of WQ2015118045A1, in certain scientific publications is referred to as "MCO3", and Membrane C is referred to as "MCO-2". As mentioned above, Membrane F of WQ2015118045A1 is an example for a membrane having an asymmetric sponge- or foam structure and is used herein as an example for demonstrating the effect of the coating according to the invention .

[0114] Accordingly, the base membrane for coating according to the invention has a MWRO of from 8.6 to 14.0 kDa and a MWCO of from 50.0 to 130.0 kDa as measured by dextran sieving before blood contact, preferably a MWRO of from 8.6 to 12.5 kDa and a MWCO of from 50.0 to 110.0 kDa as measured by dextran sieving before blood contact. According to yet another aspect, the base membrane has a MWRO of from 9.0 to 14.0 kDa and a MWCO of from 60.0 to 130.0 kDa as measured by dextran sieving before blood contact.

[0115] According to another aspect, the base membrane is a high cut-off type membrane which has an MWRO of from 15 kDa to 20kDa and a MWCO of from 170kDa to 320kDa as measured by dextran sieving before blood contact. Such membranes are described, for example, in WO 2004 / 056460 Al or in Gondouin and Hutchison (2011) , High Cut-Off Dialysis Membranes: Current Uses and FuturePotential. Advances in Chronic Kidney Disease 18:180-187. A representative of such high cut-off membranes available in the market today is the THERALITE dialyzer which comprises such membrane. The THERALITE dialyzer is described in some further detail, for example, in US20120305487A1, where also another high cut-off type membrane is described (HCOllOO dialyzer) . The membrane used in the THERALITE dialyzer has, for example, a MWRO of 15 kDa and a MWCO of 300 kDa. The membrane used in the HCOllOO dialyzer has an MWRO of 19.3 kDa and a MWCO of 200 kDa.

[0116] According to another aspect, the base membrane according to the invention is a high-flux (HF) membrane having an MWRO of from 5.0 kDa to 8.5 kDa and a MWCO of from 25 kDa to 50 kDa as measured by dextran sieving before blood contact. A representative of such high-flux membrane and dialyzer which is commercially available today is the REVACLEAR dialyzer.

[0117] In summary, a relatively broad range of membranes can be used as base membranes in the context of the present invention. Taking the above-described membrane classes together, the base membrane which can be successfully coated and modified according to the invention to achieve an enlarged range of uremic toxins to be removed combined with an improved albumin retention is characterized by a molecular weight retention onset (MWRO) of between 5.0 kDa and 20.0 kDa and a molecular weight cut-off (MWCO) of between 25 kDa and 320 kDa as determined by dextran sieving before blood contact. According to yet another embodiment, the base membrane is characterized by a molecular weight retention onset of between 7.0 kDa and 20.0 kDa and a molecular weight cut-off (MWCO) of between 30 kDa and 320 kDa as determined by dextran sieving before blood contact. An especially interesting subgroup in this context is the group of membranes characterized by a molecular retention onset (MWRO) of between 10.0 kDa and 17.0 kDa and a molecular weight cut-off (MWCO) of between 70 kDa and 120 kDa as determined by dextran sieving before blood contact, as demonstrated with Examples 1.1 to 1.7 (Membranes A to G) as disclosed in WQ2015118045A1 (Table III) . Another subgroup which mayespecially benefit from a coating according to the invention are the high cut-off membranes, including those disclosed as Example 1.8 to Example 1.11 in WQ2015118045A1 (THERALITE, HCOllOO, SEPTEX, PHYLTER HF 22 SD (Bellco) ) , see also Table III.TABLE III

[0118] A further subgroup which could benefit from a coating according to the invention are High Flux membranes such as used, for example, in the REVACLEAR dialyzers, FILTRYZER BK-F dialyzers (Toray) , VIE series dialyzers (Asahi) , ELISIO-HX and ELISIO H- dialyzers (both from Toray) , and FX CORDIAX or FX CORAL dialyzers (all from Fresenius Medical Care) .

[0119] The base membrane according to the invention is coated with a vinyl polymer that is generated or derived from vinyl monomers of formula (A)H2C=CH-R-A (A)or its salts, wherein A represents and acidic group such as sulfonic acid (or sulfonate) in formula (I)H2C=CH-R-SO3H (I) or of carboxylic acid (or carboxylate) in formula (II)H2C=CH-R-COOH (II) wherein "R" represents an organic combining group. The expression "organic combining group" refers to a linear or branched, substituted or unsubstituted alkyl or aryl group.

[0120] According to one embodiment, R represents a moiety selected from the moieties shown in Table II above, wherein n represents 0 or an integer between 1 and 12, and Ri represents -(CH2)m-, wherein m represents 0 or an integer between 1 and 12, and wherein one or more of the carbon atoms of Ri may be substituted with one or more of methyl, ethyl, propyl, phenyl, or benzyl. Examples of residues R according to the invention and according to Formula (A) are provided in Table IV, which is not intended to be a conclusive list but only serves to illustrate possible residues R.TABLE IV

[0121] According to one aspect, the compound for coating the base membrane is a vinyl monomer carrying a sulfonic acid group or a salt thereof, such as a sodium or potassium salt.

[0122] According to one aspect of the invention, the compound used for coating the membrane is selected from the group of compounds comprising or consisting of 2-acrylamido-2-methylpropane sulfonic acid (AMPS) , sodium 4-vinylbenzensulf onate (VBS) , sodium vinylsulf onate (VS) , potassium 3- ( acryloyloxy) propane-l-sulf onate (AOPS) , or combinations thereof.

[0123] According to another aspect of the invention, the compound used for coating the membrane is selected from at least one of acrylic acid, methacrylic acid, acryloyl-L-phenylalanine, ac- ryloyl-L leucine.

[0124] The process for preparing the coated membranes according to the invention (Figure 3) comprises several steps which are accessible to industrially producing the membrane of the present invention. The process comprises the steps of(i) providing a base membrane as defined herein,(ii) contacting the base membrane with a coating solution that comprises a compound of formula (I) or formula (ID ,(iii) irradiating the membrane in the presence of coating solution,(iv) removing the coating solution and rinsing the coated membrane, and(v) optionally drying the membrane, and / or(vi) optionally sterilizing the membrane.

[0125] According to one embodiment, the process is applied to a hollow fiber base membrane, wherein step (ii) comprises contacting or immersing the hollow fiber membrane in / with the coating solution, thereby bringing all accessible surfaces of the hollow fiber membrane in contact with the compound of formula (I) or (II) including during step (iii) .[ 0012 6 ] In one aspect of the invention, the hollow fiber is provided, in step ( i ) , as part of a fiber bundle placed longitudinally within a filter housing, wherein the fiber bundle is encapsulated at each end of the dialyzer in a potting material thereby providing for a first flow space surrounding the hollow fiber membranes on the outside ("filtrate side" or "filtrate space" ) , wherein said flow space is in connection with and accessible through a dialysate inlet and a dialysate outlet . A second flow space is formed by the lumen space ("lumen side" or "lumen space" ) of the hollow fibers and the flow space above and below said potting material which is in fluid communication with said lumen space and which is closed by end caps having a blood inlet and blood outlet , respectively . In one aspect of the invention, the coating solution is provided to the filtrate side of the hollow fiber membranes through said dialyzer inlet and / or outlet , and to the lumen side of the hollow fibers through said blood inlet / or outlet , wherein the complete accessible surface of the membrane , including all accessible pores , are brought into contact with the coating solution .

[0127] In one aspect , the irradiation of step ( iii ) is done in the presence of the coating solution on both the filtrate and lumen side of the membranes , including the pore surfaces , e . g . , by submitting the complete housing and fiber bundle in its filled state to irradiation ("filled" state ) . In another aspect , the coating solution is drained from the filtrate space but not from the lumen space of the housing holding the hollow fiber membrane bundle ( the "semi-filled" state ) . According to yet another aspect , the coating solution is drained from both the filtrate space and the lumen space of the housing holding the hollow fiber membrane bundle before irradiation, wherein the membrane is in a "wet" state .

[0128] According to one aspect , the coating process is applied to a hollow fiber base membrane , wherein step ( ii ) comprises immersing the hollow fiber membrane in the coating solution, thereby contacting all accessible surfaces of the hollow fiber membranewith the coating solution and the compound of formula (I) or (II) during step (iii) .

[0129] According to one aspect, the membrane is brought into contact with the coating solution during coating at room temperature, such as, for example, at a temperature of from 12 °C to 32 °C, generally at a temperature of from 18 °C to 25 °C. The time during which the membrane is contacted with the coating solution can vary over a brought range, such as from few minutes (including the time required for filling the filtrate and / or lumen space of a filter comprising the base hollow fiber membrane bundle) to several days before being irradiated, which allows for different processes comprising online irradiation during production wherein irradiation immediately follows the said contacting of the membrane with the coating solution, or, alternatively, a delocalized irradiation of the filled, semi-filled, or wet filter device in / on a different production line, building, or facility. Preferably, the filled, semi-filled or wet devices before and after irradiation, especially where a delocalized production step is involved, should not be submitted to extreme temperatures (below 4 °C or above 50°C) for prolonged periods of time.

[0130] According to one aspect, the solution for coating the base membrane is prepared, for example, by dissolving the vinyl monomer according to the invention in water, such as RO water, or a suitable buffer. The solution can then be applied to the lumen of a hollow fiber membrane for the coating of the lumen of the membrane and any adjacent pore surfaces. For example, the coating solution can be pumped into the filtrate and / or lumen space of the fibers via the dialysate inlet / outlet or the blood inlet / out- let, respectively. Alternatively, the coating solution can be sucked into the lumen space of the fibers. The flow rate for filling and / or rinsing of a hollow fiber membrane module can be varied over a relatively broad range, e.g., from about 10 mL / min to 250 ml / min. The rinsing time can also be varied over a broad range but is mostly done from about 2 to 40 minutes, such as, forexample, for about 5 to 20 minutes. The flow rate during rinsing can be varied as well. Generally, flow rates of about 200 to 800 ml / min will be chosen, depending on the targeted rinsing time and production preferences.

[0131] According to yet another embodiment, the coating solution comprises from 2 to 1000 mg / mL of a vinyl compound carrying an acidic group, such as from 5 to 600 mg / mL, from 5 to 100 mg / mL, from 5 to 75 mg / mL, or from 5 to 50 mg / mL, including, for example, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, 23 mg / mL, 25mg / mL, 28 mg / mL, 30 mg / mL, 33 mg / mL, 35 mg / mL, 38 mg / mL, 40 mg / mL, 43 mg / mL, 45 mg / mL, 48 mg / mL, 50 mg / mL, 53 mg / mL, 55 mg / mL, or 60 mg / mL.

[0132] According to yet another embodiment, the coating solution comprises a compound of formula (I) or formula (II) , such as, for example, AMPS, VBS, VS, or AOPS . According to yet another embodiment, the coating solution comprises AMPS.

[0133] According to one aspect of the invention, step (iii) comprises e-beam irradiation, gamma ray irradiation, or UV irradiation, wherein the irradiation dose is between 20 and 110 kGy, such as from 25 to 110 kGy, from 25 to 90 kGy, from 40 to 90 kGy, from 50 to 90 kGy, from 60 to 80 kGy, including, for example, 25 kGy, 30 kGy, 35 kGy, 40 kGy, 45 kGy, 50 kGy, 60 kGy, 65, kGy, 70 kGy, 75 kGy, 80 kGy, 85 kGy, and 90 kGy, and any single values in between. According to yet another embodiment, step (iii) comprises e-beam irradiation.

[0134] After irradiation, the filter devices are drained as required and optionally blown out to remove excess coating solution. In the next step, the membrane will generally be rinsed to remove any residual coating material that is not covalently bound to the membrane surface. According to another aspect of the invention, the coated membrane after rinsing can be dried with standard processes, such as with online drying as described inUS11154820B2. Dry membranes can then be submitted to final sterilization with, for example, e-beam. In another aspect, the coated membrane will be kept moist (residual water) , for example when submitted to final sterilization by steam.

[0135] The coated membranes and devices comprising them can be sterilized with steam, gamma irradiation, ethylene oxide, or e-beam. According to one aspect of the invention, the coated membrane is sterilized with steam without previously drying the membrane, i.e. , in the presence of residual water. According to yet another embodiment, the coated membrane is dried and subsequently sterilized by e-beam sterilization. According to yet another embodiment, the coated membrane sterilized by e-beam sterilization without previously drying the membrane and in the presence of residual water.

[0136] According to yet another embodiment, rinsing is done with water, such as RO water. However, it is also possible to use buffers, such as PBS or saline. Rinsing can be done over a broad range of temperatures and will usually be done at room temperature, such as at a temperature of from 12°C to 30°C, or from 15°C to 25°C.

[0137] According to another embodiment of the present invention, the amount of a compound of formula (I) or (II) that is deposited on the base membrane according to the invention (coating) is from 10 mg / m2to 180 mg / m2, such as from 10 mg / m2to 140 mg / m2, from 15 to 80 mg / m2.

[0138] In one aspect, the coated membrane of the invention after irradiation and sterilization as described above comprises (i) a base membrane according to the invention, and (ii) a coating deposited on said base membrane essentially comprising or consisting of the polymer of a compound of formula (I) or formula (II) which has been grafted onto the base membrane by irradiation (RIGP, RIGI) . For example, after irradiation, such as e-beam radiation, in the presence of AMPS, the coating will comprise oressentially consist of poly-2 -acrylamido-2-methypropane sulfonic acid (herein referred to as "pAMPS" ) which has been grafted onto the base membrane by radiation-induced grafting polymerization ( RIGP ) and / or immobilization .

[0139] According to another embodiment , the membrane of the invention comprises ( i ) a base membrane comprising or essentially consisting of a hydrophobic polymer selected from PS , PES , or PAES , and PVP , and having a MWRO of between 10 kDa and 14 kDa and a MWCO of between 65 kDa and 130 kDa , and ( ii ) a coating deposited on said base membrane essentially comprising or consisting of poly-2-acrylamido-2 -methypropane sulfonic acid (pAMPS ) which has been grafted onto the base membrane as mentioned above . According to yet another embodiment , pAMPS has been grafted onto the base membrane by radiation-induced grafting polymerization ( RIGP ) , potentially in combination with RIGI .

[0140] The coated membranes of the invention have been designed to further increase the clearance of large middle molecules ( Figure 1 ) , thereby extending the removable range of uremic toxins while maintaining or further increasing the level of retention of albumin in comparison with prior art membranes such as high-flux membranes , medium cut-off membranes , or high cut-off membranes . Surprisingly, the combination of a coating with vinyl monomers carrying an acidic group ( see formula ( I ) and ( II ) on base membranes as defined herein via irradiation shows excellent effects on the selectivity of a membrane thereby rendering said membranes highly suitable for blood purification applications . Accordingly, the present invention provides for an improved selectivity of the modified base membranes while at the same time the post-spinning modification of the membrane and the resulting medical filter device comprising such coated membrane is , as such, not very complex and cost effective , and will therefore provide excess to advanced blood purification therapies to more patients in need . For example , compared to earlier approaches using (poly) dopamine or chondroitin for the coating of membranes , the current approach has the benefit of a simplified modification procedure , noleaching, excellent endotoxin retention, and no need to use animal-derived compounds for the modification. In consequence, the coated membranes described herein provide for a highly hemocompatible and antithrombogenic, affordable blood purification membrane which stands out with a so far unprecedented removal capacity for large middle molecules at an extremely low albumin loss.

[0141] The sieving coefficients for human serum albumin (HSA) and for YKL-40 were used herein, among other parameters, to determine the performance of the coated membranes of the invention and to compare them with membranes of the prior art, preferably with membranes that already show an excellent selectivity, i.e., a low albumin sieving coefficient and a high YKL-40 sieving coefficient. According to one aspect, the coated membranes of the present invention have a sieving coefficient ("SC") for HSA of from 0.00 (including values such as, for example, 0.001 or 0.002) and from 0.1 (including values, such as, for example, 0.005, 0.006, and up to 0.009) to 4.50 and any values in between. According to another aspect, the coated membranes of the present invention have a sieving coefficient for HSA of from 0.00 to 3.50. According to yet another aspect, the coated membranes of the present invention have a sieving coefficient for HSA of from 0.00 to 2.50, or from 0.00 to 2.00. According to another aspect, the coated in combination with said SC for HSA, have a sieving coefficient for YKL-40 of from 40 to 98, such as from 45 to 98, from 45 to 95, from 50 to 95, from 50 to 80, or from 55 to 85. The aforementioned ranges can be combined with each other, meaning that a coated membrane of the invention can have a SC for HSA of from 0.00 to 2.00 and a SC for YKL-40 of from 50 to 90. For example, the coated membrane according to the invention has a SC for HSA of from 0.01 to 1.2 and a SC for YKL-40 of from 45.0 to92.0.

[0142] The marker compounds that can be used for assessing said influence on selectivity by determining their respective sieving coefficients are advantageously selected from medium tolarge middle molecules , such as , for example , the above mentioned YKL-40 . In addition, albumin is advantageously used as another or second marker molecule . For determining the effect of the coating according to the invention on the sieving performance and selectivity of a given base membrane , sieving coefficients for the selected marker molecules can be determined before and after the modification with vinyl monomers according to the invention .

[0143] According to another aspect of the invention, the modified or coated membranes according to the invention retain their hemocompatibility after coating and final sterilization . According to one aspect of the invention, the membranes coated according to the invention can be used as membranes in medical applications , including for applications that require a direct contact of the coated surface with the blood or blood components of a patient . For example , coated membranes according to the invention can be used for preparing dialyzers for use in hemodialysis , including chronic and acute ( CRRT ) applications , and encompassing hemodia- filtration and hemofiltration .

[0144] According to yet another aspect of the invention, filters or hemodialyzers are provided that comprise hollow fiber membranes that have been coated according to the invention on the lumen side , the outer surface , or all accessible surfaces of the hollow fiber membranes . According to one aspect , said filters or hemodialyzers comprise hollow fiber membranes comprising or essentially consisting of polysulfone or polyethersulfone and PVP, and which are coated with AMPS according to the invention, or, in other words , which have a coating layer that comprises or essentially consists of covalently bound pAMPS .

[0145] According to another aspect of the invention, the filters and dialyzers of the invention that comprise hollow fiber membranes which are modified and / or coated according to the invention can be used in extracorporeal blood purification applications or extracorporeal blood treatment applications , including,for example, hemodialysis, hemodiaf iltration and hemofiltration, including CRRT .

[0146] It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0147] The present invention will now be illustrated by way of non-limiting examples to further facilitate the understanding of the invention.ExamplesExample 1 : Basic Materials and MethodsExample 1.1: Preparation of Minimodules

[0148] Fibers for Minimodules which are used for the determination of sieving coefficients are prepared by cutting fibers out of or corresponding to a standardized filter to a length of 20 cm and drying the fibers for 1 h at 40°C at <100 mbar . The fibers are then transferred into a Minimodule housing. The ends of the fibers are potted with polyurethane. After the polyurethane has hardened, the ends of the potted membrane bundle are cut to reopen the fibers . The Minimodules ensure protection and an adequate as well as reproducible presentation of the fibers. In the present examples, minimodules having an effective membrane (lumen) surface A of 360 cm2were used. The number of fibers required for an effective surface A of 360 cm2is calculated according to Equation (1)A=nxdi><lxn [cm2] (Equation 1) , wherein di is the inner diameter of fiber [cm] , n represents the number of fibers, and 1 represents the effective fiber length [cm] . Each Minimodule has an effective length of approx. 170 mm(without PU potting) and an inner diameter of 10 mm. The internaldiameter of fibers and the wall thickness depends on the specific membranes used. Hence, the packing density generally varies between 23% and 31%.Example 1.2: Hydraulic Permeability (Lp) of Minimodules

[0149] The hydraulic permeability of a Minimodule is determined by pressing a defined volume of water under pressure through the Minimodule, which has been sealed on one side, and measuring the required time. The hydraulic permeability is calculated from the determined time t, the effective membrane surface area A, the applied pressure p and the volume of water pressed through the membrane V, according to Equation (2) :Lp = V / [p - A • t] (Equation 2)

[0150] The effective membrane surface area A is calculated from the fiber length and the inner diameter of the fiber according to Equation (3)A = 7t • di - 1 • [cm2] (Equation 3) wherein di represents the inner diameter of fiber [cm] and 1 represents the effective fiber length [cm] .

[0151] The Minimodule is wetted thirty minutes before the Lp- test is performed. For this purpose, the Minimodule is put in a box containing 500 mL of ultrapure water. After 30 minutes, the Minimodule is transferred into the testing system. The testing system consists of a water bath that is maintained at 37 °C and a device where the Minimodule can be mounted. The filling height of the water bath must ensure that the Minimodule is located underneath the water surface in the designated device. In order to avoid that a leakage of the membrane leads to a wrong test result, an integrity test of the Minimodule and the test system is carried out in advance. The integrity test is performed by pressing air through the Minimodule that is closed on one side. Air bubblesindicate a leakage of the Minimodule or the test device . It must be checked if the leakage is due to an incorrect mounting of the Minimodule in the test device or if the membrane leaks . The minimodule must be discarded if a leakage of the membrane is detected . The pressure applied in the integrity test must be at least the same value as the pressure applied during the determination of the hydraulic permeability in order to ensure that no leakage can occur during the measurement of the hydraulic permeability because the pressure applied is too high .Example 1 . 3 : Measurement of sieving coefficients in human plasma

[0152] Large-Middle molecules , consisting mostly of peptides and small proteins with molecular weights in the range of 500- 60 , 000 Da, accumulate in renal failure and contribute to the uremic toxic state . Beta2-microglobulin (beta2 -MG or P2 -M) with a molecular weight of 11 kDa is considered one of the smaller representatives of these molecules . Myoglobin has a molecular weight (MW) of about 17 kDa is already larger . It will not be completely cleared from blood by known high-flux dialyzers , whereas it is readily removed by medium cut-off or high cut-off dialyzers . YKL- 40 with a molecular weight of 40 kDa is considered a representative of higher molecular weight middle molecules that should still be removed as efficiently as possible . Albumin with a MW of about 67 kDa is a key element in describing the sieving characteristics of membranes , as albumin should preferably not be allowed to pass a membrane for chronic hemodialysis to a significant extent , whereas molecules having a lower molecular weight , such as p2-M and YKL- 40 should be removed as efficiently as possible , i . e . , the sieving curve of a given membrane should be steep and not flat , as is typically found with protein-leaking membranes .

[0153] For the experiments , human plasma ( such as Octaplas LG from Octapharma ) is thawed and brought to 37 ° C under careful stirring . Other marker substances can be added to the plasma as required . For each minimodule 150g of human plasma is used . Thefinal protein concentration of the test solution is 4515 g / 1. The minimodules are rinsed with 40 mL 0.9% NaCl solution on the blood and dialysate side. Then the blood and dialysate sides are blown out at 0.3 ± 0.1 bar for 10 ± 5 s. QB was 8,7 mL / min (= 7,71 SKT) and QUF was 1,7 mL / min (=1,58 SKT) . The minimodules are connected to a new tubing set with the open filtrate side on top. The recirculating tubes are returned to the solution reservoir. The blood-in pump is started. When the minimodules are free of air bubbles, the filtrate pump is started. As soon as filtrate flows back from the tube into the pool time is started. After 30 minutes of testing, a sample of the test solution (A) (minimum 1,1 mL for HSA and 550 pL for YKL-40) can be taken. The sampling time is 1 min for QUF and 1 min for QBout- From these measurements, the re- tentate sample (R) and filtrate sample (F) are used for further analysis of the marker molecules. The sieving coefficient SC is calculated according to Equation (4) .2*F / (A+R) (Equation 4)Example 1.4: Coating of membranes with AMPS

[0154] A hose pump is set to a flow of 100 mL / min. The filter or minimodule (the "device") is attached to a stand with a clamp and the hose set is connected to both the blood side and the filtrate side. The device is then filled with a pump flow of 100 mL / min of AMPS solution with desired concentration, first on the blood side, whereas the filtrate side is clamped off. The filtrate side is also filled to about half in the course of the process, even though the filtrate side is clamped off. Filling the blood side takes 3 minutes. To then fill the filtrate side, the blood outlet is clamped off after 3 minutes and the filtrate side is opened. The device is then filled with the remaining coating solution. Care is taken to avoid air bubbles in the device. If necessary, the device is tapped and turned. After the device has been filled, the inlet and outlet tubes on the blood side are clamped off using an artery clamp. The tubes on the filtrate sideare removed and the device is held vertically. The filtrate side is then opened and the liquid on the filtrate side is drained.

[0155] The devices are then closed with connectors / caps on both the filtrate side and the blood side, weighed, and packed for e-beam irradiation at the desired dosage. After the devices have been irradiated with e-beam, the caps are removed on the filtrate and blood side and the device is drained, followed by blowing out the device for approx. 30 seconds at 0.5 bar. The filtrate and blood side are then rinsed with RO water for 20 minutes at a flow rate of 400 mL / min. The devices are then dried overnight at 2 bar. After drying, the filtrate side of the devices can be filled with 40 g RO water and then subjected, for example, to steam sterilization. After the steam sterilization step, the filters are ready for testing albumin loss and clearance of YKL- 40 values as described herein.Example 1.5 : Clearance Performance

[0156] The clearance "C" (mL / min) refers to the volume of a solution from which a solute is completely removed per unit time. In contrast to the sieving coefficient which is the best way to describe a membrane as the essential component of a hemodialyzer, clearance is a measure of the overall dialyzer function and hence dialysis effectiveness. Accordingly, the coating as described before was also applied to complete hemodialyzers, such as commercially available dialyzers like THERANOVA 400 (Gambro Dialysatoren GmbH, Hechingen, Germany) , or hemodialyzers were prepared from other MCO-type membranes, such as the MCO-CI 400 dialyzer (Gambro Dialysatoren GmbH, Hechingen, Germany) ; see, for example, Boschetti-de-Fierro et al. (2015) , Scientific Reports 5: 18448, DOI: 10.1038 / srepl8448 ) and were coated according to the invention. If not indicated otherwise, the clearance performance of a dialyzer was determined according to ISO 8637 : 2004 (E ) . The set-up of the test circuit was as shown in ISO 8637:2004 (E) . Flows are operated in single path.

[0157] To measure clearance, human plasma (protein content 55 ± 10 g / L) is spiked with myoglobin and p2-M (Lee Biosolutions) , to a concentration of 0.50 mg / L and 5.0 mg / L, respectively. Albumin and YKL-40, for example, are measured directly from plasma (e.g., OctaplasLG 45-70 mg / mL from Octapharma) . The test is performed by recirculating human plasma for 60 ± 5 minutes at QB = 300mL / min, QD = 500mL / min, and UF = 10 mL / min at a temperature of 37 °C. Samples are in each case taken from the blood outlet, dialysate outlet and blood inlet, respectively. Samples are taken at t [min]= 0, 4, 10, 20, 30, 40, 50, 60. Clearance (Cl) is calculated according to Equation (5)wherein c(t) is the concentration of the marker (e.g., YKL-40) protein at time (t) , V is the plasma volume and co is the concentration of the marker at t=0.Example 1.6: Quantitative determination of YKL-40 in human plasma

[0158] For determining the concentration of YKL-40, the Quan- tikine Human Chitinase 3-like (CHI3L1) was used according to the supplier's instructions. The assay is a solid phase ELISA assay designed to measure CHI3L1 in cell culture supernates, serum, plasma, and urine. The assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for the analyte has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any analyte present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for the analyte is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of CHI3L1 in the initial step. Once the color development stops, the intensity of the color is measured.

[0159] For determining the concentration of p2-M, myoglobin and albumin a nephelometer (BN ProSpec, Siemens Medical Solutions) is used in combination with the supplier's quantification kits. p2-M, myoglobin and albumin are quantified using the N Latex p2 Microglobulin kit, N Latex Myoglobin kit and N Antiserum to Human albumin kit (Siemens Health solutions) , respectively, all according to the supplier's instructions. Each determinant is measured separately by formation of an insoluble antigen-antibody complex upon addition of the provided antibody. The complex formation proceeds in excess of antibody, is measured using light scattering by the nephelometer and compared to standards of known concentration .Example 1.7: Determination of albumin loss

[0160] Albumin loss is determined in the same setup as described for the determination of clearance (Example 1.5) . The test setup comprises using an AK 200 Ultra S monitor for plasma and dialysate cycle. For the test setup with UF= 10 ml / min, substitution solution (dialysate acetate) is needed to maintain constant pool volumes, controlled by a separate substitution pump besides the monitor and dosing of required volumes into the pool. Correct fluid volumes are controlled via pool volume on scales. Samples are taken at the dialysate outlet at t [min] = 4, 10, 20, 30, 40, 50, 60. Albumin concentration at different time points samples can be determined by spectrophotometry according to known protocols. The quantification of HSA is done with "N Antiserum to Human albumin" Kit of "Siemens Medical Solutions" with nephelometer BN ProSpec (Siemens Healthcare Diagnostics) , see Example 1.6. The HSA (=antigen) reacts with added antibodies and forms an antigen- antibody-complex. For details, references is made to the protocol and operation manual from the manufacturer.Example 1.8: Quantification of poly-AMPS deposited on the membrane

[0161] For determining how much AMPS is immobilized on the membrane during coating (see Example 1.4) , various mini modules with different AMPS concentrations were tested. The AMPS coating introduces a negative charge due to the SOs- group. To determine the number of negative charges, a positively charged dye called toluidine blue is used, which binds to the negatively charged membrane, thereby turning blue. For a quantitative statement, a reference membrane, MCO-4 without coating, is tested in direct comparison .

[0162] Before starting the test, 200 pL of 0.1 pmol / mL toluidine blue solution are measured in a photometer. For this purpose, a wavelength scan from 400 nm to 800 nm is carried out. In addition, the pump flow on the blood side is calibrated to 8.7 mL / min (SKT=8.00) and for the filtrate pump to 1.7 mL / min (SKT=1.50) . For each mini module, 30 mL of 0.1 pmol / mL toluidine blue solution is placed in a 50 mL beaker and a stir bar is added.

[0163] The blood pump is started first, so that the blood side can be filled without air bubbles . As soon as no air bubbles can be detected any more on the blood side, the filtrate pump is started. Time is measured as soon as the first drop drips from the filtrate tube back into the receiver. The mini modules are rinsed in recirculation mode for 60 minutes. After that, 200 pL of the toluidine blue solution are taken from each receiver and measured in the photometer. A wavelength scan from 400 nm to 800 nm is also carried out here. After the end of the test, the mini modules are first rinsed on the blood side (the filtrate side is clamped off) with RO water for 10 minutes at a pump flow of 20 mL / min. The clamps on the filtrate side are then opened and the blood side at the top is clamped off. The mini modules are now also rinsed with filtering for 10 minutes and then dried (approx. 0.4 bar compressed air for 80 minutes) .

[0164] The number of toluidine molecules immobilized on the membrane surface corresponds to the number of the AMPS moleculesdue to the negatively charged SO3 groups . The AMPS amounts coated on the surface can be calculated accordingly .[ 001 65 ] As can be seen in Figure 5 , the amount of toluidine in pmol shows a good alignment with the amount of AMPS in the coating solution after coating the membrane in accordance with Example 1 . 4 . The reference (MCO-4 without coating ) shows now binding of toluidine due to the absence of the negatively charged SOs- groups of immobilized AMPS . Table V provides for the AMPS amounts in mg / m2on membranes coated according to the invention with an e- beam dose of 75 kGy and after steam sterilization . Apart from the AMPS amounts , Table V also shows that steam sterilization does not negatively influence the stability of the coating . For the avoidance of doubt , AMPS appears as pAMPS on the membrane surface tested, as mentioned above . However , reference is made to AMPS for reasons of clarity regarding the amount or number of molecules deposited on the membrane surface .TABLE V: Amount of AMPS* coated on a MCO-4 membrane according to the invention after e-beam irradiation and final sterilization with steam. 0 . 5 wt . -% AMPS corresponds to 5 mg / mL AMPS in the coating solution, 1 wt . -% corresponds to 10 mg / mL, 2% corresponds to 20 mg / mL .^present as pAMPS after e-beam treatmentExample 2 : Influence of Coating on sieving coefficients of YKL-40 and Human Serum Albumin (HSA)The sieving coefficients ( SC ) for YKL-40 and HSA were determined as described above with Minimodules (MM) . Table VI shows theeffect of coating a hollow fiber membrane extracted from a THERANOVA 400 dialyzer (TN400) with 2 wt.-% (20 mg / mL) AMPS on RO (Example 1.4) after e-beam irradiation and final steam sterilization. In the comparative example, the same process was applied to the membrane, however no AMPS was added to the coating solution. It is notable that the SC (in %) for HSA is significantly reduced from 1.18 to 0.13, thereby arriving at very low values, whereas the SC (in %) is significantly increased from 31.2 to 47.3. In essence, the combination of a lower SC for HSA and an increased SC for YKL-40 means a steeper sieving curve and thus a significantly increased selectivity of the membrane. In other words, the coated membrane will be able to remove higher molecular weight molecules, such as YKL-40, with increased efficiency, whereas the albumin loss will be further decreased compared to state-of-the- art membranes .TABLE VI : Influence of coating with AMPS on sieving coefficients for YKL-40 and HSA of TN400 with resulting selectivity increaseThe data shown in Table VII were generated in analogy to what is provided in Table VI. Table VII shows the effect of the coating on another base membrane that corresponds to Membrane F (Example 1.6, Figure 6) of W02015118045A1. This membrane (MCO-SF) has an asymmetric sponge structure (TN400 in Table VI has an asymmetric finger structure) . It is obvious that the effect of the coatingas disclosed herein is the same for MCO-SF and is , therefore , independent from membrane structure .TABLE VII : Influence of coating with AMPS on sieving coefficients for YKL-40 and HSA of MCO-SF with resulting selectivity increaseTable VIII summarizes the results of coating a High Cut-Off type membrane , prepared from a THERALITE 2100 dialyzer . The data show was again prepared in accordance to those shown in Table VI . The THERALITE 2100 dialyzer membrane was coated with 5 mg / mL AMPS in RO water and 50 mg / mL AMPS in RO water , respectively, and submitted to e-beam irradiation at 75 kGy to show that the amount of AMPS can be varied according to the invention to achieve the desired effect on selectivity in a simple manner . For the THERALITE 1200 membrane , somewhat higher amounts of AMPS lead to a significant selectivity increase . As can be seen in Table VIII , coating with a solution comprising 50 mg / mL AMPS results in a significant reduction of the sieving coefficient for human serum albumin ( HSA) in combination with a notable increase of the sieving coefficient for YKL-40 . The resulting membrane thus has a very sharp sieving curve . It lets more than 90% of the YKL-40 molecules ( having a MWof 40 kDa) pass, whereas it virtually completely rejects HSA(having a MW of 66 kDa) .TABLE VIII: Influence of coating a High Cut-Off type membrane (THERALITE 2100) with AMPS on sieving coefficients for YKL-40 and HSA. "SC" represents "Sieving Coefficient", "HSA" represents "Human Serum Albumin" .Example 3: Influence of Coating on YKL-40 Clearance and Albumin Loss

[0166] Figure 6 shows the effect of the coating according to the invention on the clearance for middle molecules in mL / min, exemplified with YKL-40. Comparative commercial membranes were tested (REVACLEAR 500 dialyzer, a high flux dialyzer, as well as MCO THERANOVA 400 and MCO THERANOVA 500, both medium cut-off dialyzers) along with an uncoated medium cut-off membrane (MCO-4, see above) and the same medium cut-off membrane MCO-4 coated with 2% AMPS according to Examples 1.4 and 1.8. Clearance was measured according to Example 1.5. The clearance for the middle molecule YKL-40 of THERANOVA 400 and 500 dialyzers is higher compared to the REVACLEAR 500 dialyzers, as is known. Accordingly, the clearance is again higher for the MCO-4 dialyzer (uncoated) . The clearance efficacy is again significantly increases by a 2% AMPS coating according to the invention. Importantly, the increase in clearance should be compared with the development of albumin loss . In contrast to what would generally be expected, the increase in clearance seen upon coating of the MCO-4 membrane does not comewith an increase in albumin loss . Figure 7 provides for the respective values. By comparing the albumin loss of the coated MCO- 4 membrane (lower curve) that achieves a very high clearance for YKL-40 (Figure 6) , with the uncoated MCO-4 membrane (upper curve) , it is obvious that in contrast to expectations the albumin loss is significantly decreased.100167] Figure 8 brings together how the coating of the inven- tion influences clearance (YKL-40) and albumin loss in comparison with the MCO THERANOVA 400 dialyzer. The membranes tested were MCO-4 membranes (previously steam sterilized as indicated) and coated with a solution of 20 mg / mL AMPS followed by e-beam treatment (75 kGy) and steam sterilization as described above. VI through V4 indicates individual tests with MCO-4 membranes, wherein VI through V4 experiments were identical apart from using different membrane samples. V4 is different in as far as the respective MCO-4 membrane was not pre-treated (sterilized) with steam before coating. YKL-40 clearance and albumin loss were determined as described above. Figure 8 underlines the significant effect the coating has on selectivity, specifically in comparison with an already very selective membrane (prepared from an MCO THERANOVA 400 dialyzer) . The MCO-4 membranes have a significantly increased clearance for YKL-40 (columns) after coating according to the invention in comparison with the THERANOVA 400 membrane. Strikingly, albumin loss (filled circles) is significantly reduced in comparison with said THERANOVA 400 membrane.Example 4 : Influence of Coating on Hemocompatibility

[0168] Testing the hemocompatibility of the membranes of the invention was done according to ISO10993-4 with the following endpoints: (1) Coagulation (TAT) , (2) Platelet Activation (PF4) , (3) Complement (C3a) , and (4) Complete Blood count. As an additional endpoint, free plasma hemoglobin ("fHb") was determined, which is not covered by the above ISO norm but was also carried out here. The membrane used in the hemocompatibility experiments below is MCO-4, coated with an AMPS solution of 20 mg / mL in ROwater as described above, and irradiated with e-beam (75kGy) followed by steam sterilization. An uncoated medium cut-off membrane according to Example 1.3 from W02015118045A1 was included for evaluating the effect of the coating.

[0169] Priming on the blood side was done with 500 mL NaCl, QB = 100 mL / min and on the dialysate side with 500 mL NaCl, QD= 150 mL / min. After that, the test solution (fresh, donated human whole blood (pool of 2 donations) , 1.5 lU / mL Heparin, hematocrit set to 32%) was pumped into the filters' blood side (fill to capacity) . The filters were kept at static incubation conditions at 37°C for 60 min in a warming chamber under agitation. After 60 minutes of incubation, the following samples were tested: i. Baseline control: Blood for 60min at RT ii . Negative control: Blood for 60min at 37°C iii . Positive control coagulation: Blood + Glasbeads, 60min at 37°C iv. Positive control complement: Blood + Yeast, 60min at 37 °C v. Positive control RBC : Blood frozen at -20°C for 60min

[0170] The activation of coagulation by the coated membrane was examined by the quantitative determination of thrombin-antithrombin ITT complex (TAT) by means of a sandwich enzyme-linked immunosorbent assay (ELISA) , Enzygnost TAT Micro (Siemens Healthi- neers 10446632) . Aliquoted samples comprising trisodium citrate were diluted with human plasma. The dilution factor was chosen depending on the expected TAT concentration and varied between 1:1 and 1:40. 50 pL of diluent and 50 pL of the samples were transferred into a well of a microplate and the TAT complex bonded to the antibodies on the microplate. After washing, 100 pL of the second enzyme-conjugated antibody were added and removed by washing after 30 minutes of incubation. An enzymatic reaction which occurs by addition of chromogen was measured photometrically (EL 808 Ultra Microplate Reader, BioTek Instruments Inc. , Winoo- ski / USA) at 490 nm within one hour. Figure 9 shows the resultsfor baseline, negative and positive controls as defined above. The TAT activation for the MCO-4 membrane coated according to the invention is as low as for an uncoated medium cut-off membrane (Example 1.3 from W02015118045A1) , the Baseline and Negative Control. No significant TAT activation could be observed.

[0171] The formation of human platelet factor 4 (PF-4) was examined by means of a sandwich enzyme-linked immunosorbent assay (ELISA) , Asserachrom PF4, Stago Deutschland GmbH, Dusseldorf, Germany according to the supplier's protocols. Figure 11 depicts the results for the coated MCO-4 membrane in comparison with negative and positive control as well as the baseline and an uncoated medium cut-off membrane (Example 1.3 from W02015118045A1 ) . The PF-4 activation of "MCO-4 coated" is comparable to the baseline and the negative control with about 6000 lU / mL, and is slightly below the value for the uncoated medium cut-off membrane. No significant PF-4 activation could be observed, and both the uncoated medium cut-off membrane and the "MCO4 coated" of the invention are clearly below the positive control.

[0172] Complement factor C3a was determined with the MicroVue C3a Plus Enzyme Immunoassay from QuidelOrtho Corporation, USA. Figure 12 depicts the results of C3A (complement) activation for "MCO4 coated" according to the invention in comparison with the Theranova 400 membrane, negative and positive control, and the baseline. C3a results for "MCO4 coated" with 220 ng / mL show an excellent hemocompatibility which is comparable with the baseline as well as with the uncoated medium cut-off membrane.

[0173] Complete Blood Count (CBC) (platelet drop, white blood cells, red blood cells) was measured by a cell counting system (XP-300, Sysmex Deutschland GmbH, Germany) based on flow cytometry and light scattering and a starting concentration of 100.000 THR / pL which corresponds to 100%. Figure 10 shows the results for said platelet drop (PLT) , Figure IOC, and white and red blood cell count (WBC, RBC) , Figures 10B and 10A, respectively. Compared to the baseline, "MCO4 coated" according to the invention shows aslight drop in platelet count, which is comparable to the negative control. Figure 10A shows no influence on the RBC for "MCO4 coated" according to the invention. For WBC (Figure 11B) the drop for "MC04 coated" according to the invention is limited to acceptable values (75%) in the chosen setup.

[0174] Free hemoglobin (fHB) was measured directly by photometry at 540 nm and 680 nm. Figure 13 shows the results for "MCO4 coated" according to the invention in comparison with the an uncoated medium cut-off membrane, baseline, and negative and positive control. fHb for "MCO4 coated" according to the invention is comparable to the baseline. No fHB could be observed, i.e., there is no hemolysis.Example 5 : Influence of Coating on Endotoxin Retention

[0175] Tests on the ability of the membranes according to the invention and devices comprising same to retain (reject) endotoxins was tested with minimodules comprising the membrane used also in Example 4 (MCO-4 coated with a 20% AMPS solution followed by e-beam irradiation with 25kGy or 75kGy as indicated and subsequently sterilized with e-beam irradiation (25kGy) or with steam, respectively. Uncoated MCO-4 membranes were also tested for comparison. In addition, the THERANOVA 400 membrane was included in the tests as well (also in minimodules) as its good endotoxin retention capabilities are known (Hulko et al . , Pyrogen retention: Comparison of the novel medium cut-off (MCO) membrane with other dialyser membranes. Sci Rep. 2019, 9(1) :6791) .

[0176] Figure 14 shows the results for the respective log retention values (LRV) of the tested membranes in minimodules, THERANOVA 400, MCO-4 (uncoated) , MCO-4 coated and irradiated with e-beam (75 kGy) followed by steam sterilization, MCO-5 coated and irradiated with e-beam (25 kGy) and sterilized with e-beam (25 kGy) . The LRPs of all tested membranes are comparable and seem to be fully suitable for hemodialysis under ISO standard dialysis fluid quality in terms of endotoxin retention.

Claims

Claims1. A membrane for use in blood purification applications, characterized in that it comprises a base membrane having a molecular weight retention onset (MWRO) of between 5.0 kDa and 20.0 kDa and a molecular weight cut-off (MWCO) of between 50 kDa and 320 kDa as determined by dextran sieving before blood contact, and in that it is coated with a vinyl polymer derived from vinyl monomers of formula (A)H2C=CH-R-A (A) , wherein A represents an acidic functional group, and R represents an organic combining group.

2. The membrane of claim 1, characterized in that the vinyl polymer is derived from a vinyl monomer of formula (I) or (II)H2C=CH-R-SO3H (I)H2C=CH-R-COOH (II) or combinations thereof, wherein R represents a group selected from - (CH2)n-, - (CO) -NH-Ri-, -(CO)-O-Ri-,wherein n represents 0 or an integer between 1 and 12, and Ri represents -(CH2)m-, wherein m represents 0 or an integer between 1 and 12, and wherein one or more of the carbon atoms of Ri may be substituted with one or more of methyl, ethyl, propyl, phenyl, or benzyl.

3. The membrane of claim 1 or claim 2, characterized in that the amount of vinyl polymer deposited on the base membrane is from 10 mg / m2to 180 mg / m2.

4. The membrane of any of claims 1 to 3, characterized in that the vinyl monomer used for coating the base membrane is selected from the group of vinyl monomers consisting of 2-acrylamido-2- methypropane sulfonic acid (AMPS) , sodium 4-vinylbenzensulfonate (VBS) , sodium vinylsulf onate (VS) , potassium 3- (acryloyloxy) pro- pane-l-sulf onate (AOPS) , acrylic acid, methacrylic acid, acry- loyl-L-phenylalanine, acryloyl-L leucine, or combinations thereof .

5. The membrane of any of claims 1 to 4, characterized in that the base membrane comprises a hydrophobic component chosen from polysulfone (PS) , polyethersulfone (PES) , polyarylethersulfone (PAES) , or combinations thereof, and a hydrophilic component chosen from polyvinylpyrrolidone (PVP) , polyethyleneglycol (PEG) , polyvinylalcohol (PVA) , or a copolymer of polypropyleneoxide and polyethyleneoxide (PPO-PEO) .

6. The membrane of claim 5, characterized in that the base membrane is prepared from a polymer solution comprising 10 to 20 wt.-% of at least one hydrophobic polymer component, 5 to 10 wt . - % of at least one hydrophilic polymer component and at least one solvent .

7. The membrane of any of claims 1 to 6, characterized in that the membrane is a hollow fiber membrane.

8. The membrane of any of claims 1 to 7, characterized in that it comprises (i) a base membrane comprising a hydrophobic polymer selected from PS, PES, or PAES, and (ii) a coating comprising poly-2-acrylamido-2-methypropane sulfonic acid (pAMPS) .

9. The membrane of any of claims 1 to 8, characterized in that it has a sieving coefficient for albumin of between 0.001 and 4.50 and a sieving coefficient for YKL-40 of between 40 and 98.

10. A process for preparing the membrane of any of claims 1 to9, comprising the steps of(vii) providing a base membrane having a molecular weight retention onset (MWRO) of between 5.0 kDa and 20.0 kDa and a molecular weight cut-off (MWCO) of between 50 kDa and 320 kDa as determined by dextran sieving before blood contact,(viii) contacting the base membrane with a coating solution that comprises a vinyl monomer of formula (A) H2C=CH-R-A (A) , wherein A represents an acidic functional group, and R represents an organic combining group,(ix) irradiating the membrane in the presence of coating solution,(x) removing the coating solution and rinsing the coated membrane, and(xi) optionally drying the membrane, and / or(xii) optionally sterilizing the membrane.

11. The process of claim 10, wherein all accessible surfaces of the base membrane are brought into contact with the coating solution of step.

12. The process of claim 10 and 11, wherein the coating solution comprises from 2 to 1000 mg / mL of the vinyl monomer of formula (A) .

13. The process of any of claims 10 to 12, wherein step (iii) comprises submitting the membrane in the presence of coating solution to e-beam irradiation, gamma ray irradiation, or UV irradiation .

14. A method for increasing the selectivity of a membrane for use in blood purification applications, comprising coating a base membrane having a molecular weight retention onset (MWRO) of between 8.6 kDa and 20.0 kDa and a molecular weight cut-off (MWCO) of between 50 kDa and 320 kDa as determined by dextran sievingbefore blood contact with a vinyl polymer derived from vinyl monomers of formula (A)H2C=CH-R-A (A) , wherein A represents an acidic functional group, and R represents an organic combining group .15 . A hemodialyzer comprising a membrane according to any of claims 1 to 9 .16 . The membrane according to any of claims 1 to 9 or prepared according to any of claims 9 to 13 for use in the manufacture of a blood purification device .

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