Highly selective dialysis membranes and methods for preparing same

Abstract

The present disclosure relates to a membrane with significantly increased selectivity for use in hemodialysis applications, wherein the membrane is coated with chondroitin. The membrane can be obtained by contacting a base membrane, which for example comprises a blend of i) a polysulfone, polyethersulfone or polyarylethersulfone and ii) polyvinylpyrrolidone, to a solution of chondroitin followed by e-beam radiation. The disclosure further relates to a process for producing the selective hemodialysis membrane, and to a method of increasing the selectivity of a membrane. The disclosure further relates to a filtration / diffusion device comprising such membrane.

Description

P51141PC00 Highly selective dialysis membranes and methods for preparing same Technical Field

[0001] The disclosure relates to a highly selective membranefor use in hemodialysis applications which is coated with chon-droitin. The membrane can be obtained by contacting a base mem-brane, which for example comprises a blend of i) a polysulfone,polyethersulfone or polyarylethersulfone and ii) polyvinylpyrrol-idone, to a solution of chondroitin followed by e-beam radiation.The disclosure further relates to a process for producing the selective hemodialysis membrane, and to a method of increasingthe selectivity of a membrane. The disclosure further relates toa filtration / diffusion device comprising such membrane.Description of the Related Art

[0002] Patients with end stage renal disease in hemodialysishave an increased mortality risk when compared to the general population. This indicates that the currently available replace- ment therapies, even though relying on advanced membrane and di- alyzer technologies as well as highly effective and sophisticatedhemodialysis machines, should be further improved in a way thatthe clinical outcome for patients and their quality of life duringtherapy is getting better (Bowry and Chazot, The scientific prin-ciples and technological determinants of haemodialysis membranes.Clin Kidney J 14 (Suppl 4): i5-i16 (2021)). One of the stillmanifold unmet needs that has been accompanying the developmentof new membranes and dialyzers over decades is the enhanced re-moval of uremic toxins over a broad range, including highermolecular weight middle molecules, while essentially retaining proteins such as albumin and other higher molecular weight com- pounds that are deemed to be critical. In other words, synthetic membranes which are state of the art today are still less selectivethan the glomerular membrane of the kidney. In consequence, uremictoxins having a higher molecular weight are still 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-Fi-erro et al., Scientific Reports (2015) 5: 18448), which adds tothe 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, specif- ically the hemocompatibility of membranes that are in direct bloodcontact with a significant surface area, making it necessary touse systemic heparin administration in most therapies.

[0003] In response to the above challenges, membrane innova-tion has recently been focussing on enhancing the removal oflarger molecular weight uremic toxins and increased membrane per-meability. In addition, the industry also focused on the improve-ment of hemocompatibility and antithrombogenicity of the mem- branes. The progressive shift towards higher molecular weighttoxins and increased membrane permeability also necessitates theprovision of membranes with an increasingly improved selectivity.In other words, it remains a major target in the industry to closethe gap between synthetic membranes and the natural kidney bycombining high permeability and high selectivity.

[0004] Typical hallmarks of selectivity of a membrane specif-ically for use in hemodialysis are the sieving coefficients for albumin (^66.4kDa) on the one hand and the sieving coefficients for large middle molecules (>25-58kDa) on the other hand (Rosner et al., Classification of Uremic Toxins and Their Role in Kidney Failure: CJASN 16, 1-8 (2021)). For albumin, the sieving coeffi- cients should be very low, whereas those for large middlemolecules should be high. The challenges resides in providing membranes that are able to combine both requirements, which is nothing else than high selectivity. Large middle molecules in- clude, 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 ß2-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 saidmolecules are reflected, for example, in the sieving curve repre-sented by the MWCO and MWRO values of a membrane. These parametersare a means to provide information on the onset of retention ofmolecules, 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.

[0005] Together, MWRO and MWCO define the steepness of thesieving curve and thus provide for an excellent measure of theselectivity of a membrane. For example, shifting the retentiononset to higher molecular weights while keeping the cut-off stableor reducing it, results in increased selectivity, which is appar-ent from an increasing steepness of the sieving curve which isdetermined by the molecular weight retention onset (MWRO) and themolecular weight cut-off (MWCO) of a membrane (FIG. 1). In otherwords, an increase in selectivity is defined by a decrease of thedelta ^ between MWRO and MWCO of a membrane, or by an increase ofthe absolute value of the slope at the turning point of the sievingcurve. 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 challenge to achieve that desired shiftof either MWRO or MWCO while keeping the respective other param-eter stable.

[0006] Hemodialyzers comprising such selective membranesshould 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, andpreferably should remain below about 4g / 4h. At the same time, thehemodialyzers should provide for a good clearance for large middlemolecules, including, for example, YKL-40, i.e., they should havea YKL-40 clearance of at least 20 mL / min and higher. They should,in addition, efficiently remove ß-2 microglobulin (ß2m) with aclearance of more than 80 mL / min.

[0007] ß-2 microglobulin is a well-known marker molecule inhemodialysis. It is a component of MHC class I molecules with amolecular weight of 11 kDa that in patients on long-term hemodi-alysis can aggregate into amyloid fibers that deposit in jointspaces, 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 linkedto chronic inflammatory response in chronic kidney disease and HDpatients. Its presence in the serum of the patients makes it accessible and traceable.

[0008] Recently, a significant step towards membranes and he-modialyzers with such improved performance was made by the intro-duction of so-called medium cut-off membranes and dialyzers, whichare 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, com-bined with a low molecular weight cut off (MWCO), meaning thatproteins and other compounds that have a molecular weight of al-bumin and higher are essentially retained (WO 2015 / 118046 A1 andWO 2015 / 118046 A1). In other words, the design of said medium cut-off membranes allows for a significantly improved selectivity,which is a distinguishing feature over other prior art membranessuch as the so-called “high cut-off” membranes, or membranes thathave been referred to, in the past, as “protein-leaking” (PL)membranes. While high cut-off membranes are also able to effi-ciently remove higher molecular weight toxins, their increasedalbumin loss recommends their use only in acute situations thatare limited in time and strictly controlled as regards albuminloss and replacement thereof. Protein-leaking membranes, on theother hand, generally have a cut-off that provides for a limitedloss of albumin and other higher molecular weight compounds beyondalbumin, but the retention onset is generally low, resulting in aflat sieving curve that signifies a low selectivity and undesir-able weakness in the clearance of smaller and of large uremictoxins. In contrast, medium cut-off membranes when combined witha careful physical design of the hollow fibers, membrane bundleand overall dialyzer, lead to a performance which provides for anunprecedented clearance of relevant higher molecular weight toxins in hemodialysis, while albumin loss can be kept within narrow limits (Kirsch et al., Nephrology Dialysis Transplantation (2017)32: 165). A commercially available example for such a medium cut-off membrane and dialyzer is the MCO Theranova dialyzer (Baxter).

[0009] The significant contribution of medium cut-off mem-branes to the final superior dialyzer performance is achieved bytheir unique membrane structure which is generated by a carefulcontrol of all relevant parameters of the manufacturing processas described in WO 2015 / 118045 A1 and WO 2015 / 118046 A1. Suchhighly controlled manufacturing process is not easily reproduciblethroughout the industry, and further improving the selectivity ofmedium cut-off membranes by further improving the process is dif-ficult and / or expensive. However, as mentioned above, it would bedesirable to further improve dialysis membranes towards evenhigher selectivity by easily accessible methods that allow the production of highly selective hemodialysis membranes without ex- cessive production costs that add to the price of the final prod- uct.

[0010] Other membranes, such as standard hemodialysis mem-branes and dialyzers, including, for example, high cut-off as well as high-flux membranes that are today widely used in hemodialysis(HD) could especially benefit from methods to improve their se-lectivity in a simple, cost-effective, and safe way to provideaccess to improved dialyzers to more patients that may otherwise not be able to benefit from potentially more expensive high-enddialyzers 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 the biocompatibility of the membrane and the dia-lyzer and its general safety for the patient (Bowry and Chazot(2021): The scientific principles and technological determinantsof haemodialysis membranes. Clin Kidney J 14 (Suppl 4): i5-i16).

[0011] Technologies for the modification of membranes, in-cluding hemodialysis membranes, by incorporating or immobilizingbiologically active or inactive compounds in / on the membrane havegenerally been described in the prior art. This includes, for example, modifications for improving their biocompatibility, an-tithrombogenicity, or performance. They further encompass addingcompounds to the membrane matrix for the unspecific capturing of,for example, hydrophobic or hydrophilic uremic toxins. Modifica-tions also include methods for specifically functionalizing mem-brane 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.

[0012] Chondroitin is a compound which has already been usedin the past for modifying structures or materials of medical de-vices as it can safely be used as a component for medical appli-cations (Miraglia et al. (2016) Food and Chemical Toxicology 93:89-101). Chondroitin, or chondroitin sulfate (herein generallyreferred to as “CS”), is an anionic linear polysaccharide, whichis synthesized as part of proteoglycan (PG) molecules in verte-brates and invertebrates. CS, with different disaccharides andoverall carbohydrate backbones, may be biosynthesized possessingvarious numbers and positions of sulfate groups. Consequently, CSis a relatively heterogeneous molecule having different molecularweights and being formed of alternate and variously sulfated di- saccharides of GlcA and GalNAc linked by β(1→3) bonds. Moreover,the different disaccharides can be linked to each other by β(1→4)bonds. CS-A and CS-C are the predominant CS variants and are constituted by disaccharides sulfated in position C4 or C6 of theGalNAc residue, respectively. Minor percentages of the monosul-fated disaccharide on C2 of GlcA exist as well. CS-A or CS productscontaining predominantly CS-A (as used herein, this expressionmeans that more than 50%, 60%, 70%, 80%, 90% or 95% of the CS areof the CS-A type) may be preferable in connection with the presentinvention.

[0013] Besides the main presence of these two kinds of disac-charide units monosulfated in position C4 or C6 of GalNAc, disac- charides with a different number and position of sulfate groups can be present, in various percentages, within the carbohydratechains (Volpi, molecules (2019), 24: 1447; Awofiranye et al.,Fermentation (2022), 8: 323). CS is mostly produced from the car-tilage of animals such as cows, pigs, chicken, or marine organisms and therefore subject to certain variations depending on the spe-cies on the process used for extraction. However, it can also beproduced synthetically or by fermentation which may have a benefitin terms of purity and homogeneity. An example of fermentation-derived CS is CS from Mythocondro.

[0014] In Ning et al., Progress in Polymer Science (2018),81: 144-162, polypyrrole (PPy) nanostructures for use as regener-ative biomaterials in tissue regeneration were doped with, for example, chondroitin sulfate (CS).

[0015] WO 2017 / 030502 A1 describes biomimetic membranes foruse in nanofiltration. The membranes have deposited thereon afirst mixture comprising a first polyelectrolyte and a transmem- brane protein such as aquaporin, and a second mixture comprising a second polyelectrolyte and a cross-linking agent, wherein the second polyelectrolyte has a charge opposite to the charge on thefirst polyelectrolyte. Chondroitin sulfate is mentioned as an an-ionic polyelectrolyte which can be used for producing such biomi- metic membranes.

[0016] Ren et al., Chemical Engineering Journal (2019), 370:1027-1038, describe aligned porous poly (l-lactic acid) (PLLA)electrospun fibrous membrane with a biomimetic surface for therapid regeneration of cartilage tissue. The PLLA electrospun fi-bers aligned in a single direction and generated ellipse-shaped nanopores in situ onto the surface of the fibers. Subsequently, chondroitin sulfate (CS) was coated on the surface of the fibers using polydopamine (PDA) as an adhesive polymeric bridge.

[0017] WO 2023117812 A1 discloses hemodialysis membranes pre-pared from a blend comprising polysulfone, polyethersulfone orpolyarylethersulfone, and polyvinylpyrrolidone, wherein the mem-brane is coated with polydopamine and chondroitin, and optionallywith heparin as a third component. WO 2023117812 A1 for the firsttime discloses that a coating with polydopamine and chondroitin,optionally in combination with heparin, can improve the selectiv-ity of a membrane. It is also mentioned that e-beam irradiationis a way to sterilize the membrane as the method does not nega-tively impact the desired effects achieved with the proposed coat-ing. However, polydopamine and chondroitin as well as, where pre-sent, heparin, may be eluted from the membrane surface, therebyentering the blood stream of the patient. Even though only small amounts of polydopamine and chondroitin are “leaking” from the membrane, and even though both are non-toxic, it would be prefer- able to avoid such membrane behavior to avoid any substances en-tering the patient´s blood stream.

[0018] Finally, Schulze et al., Macromolecular Rapid Communi-cations (2010) 31:467-472, showed that polyethersulfone and poly-acrylonitrile membranes were surface modified in a one-step pro-cedure, leading to the stable immobilization of certain surface modifying substances. For this purpose, the membranes were soaked with aqueous solutions of different low-molecular weight molecules bearing diverse hydrophilic functionalities and subject to elec- tron beam treatment. No catalysts, photoinitiators, organic sol- vents or other toxic reagents were used, and no additional syn-thetic or purification steps were required. The concentrations ofvarious molecules varied in the range of from 0.1-2.0% and irra-diation doses in the range of 30-300kGy were applied. Irradiationwas performed in an N-atmosphere with O quantities. Chondroitin,however, is not mentioned in Schulze et al. (2010). Also, thereference is silent on the effects of e-beam radiation in combi-nation with any of the substances used on the selectivity ofmembranes.

[0019] It was now found that the selectivity of a base mem-brane as used, for example, in hemodialysis applications, includ-ing membranes based on polysulfone, polyethersulfone or poly-arylethersulfone, and polyvinylpyrrolidone, can surprisingly beenhanced with a simple coating with chondroitin (CS), if the mem-brane after coating with a solution having a CS concentration offrom 0.1 wt.% to 16 wt.-% (1 mg / mL – 160 mg / mL), from 0.1 wt.% to14 wt.-% (1 mg / mL – 140 mg / mL), or from 0.1 wt.% to 10 wt.-% (1mg / mL – 100 mg / mL) is e-beam treated at doses of from 12.5 kGy to115 kGy. Coating solutions with 3 wt.% to 8 wt.% are especiallysuitable for preparing membranes according to the invention. No elution of chondroitin is observed after e-beam treatment, whilethe selectivity of a given membrane is significantly increased bythe combination of chondroitin alone and e-beam irradiation.

[0020] Other characteristics relevant for hemodialysis appli-cations such as, for example, biocompatibility, or technical pa- rameters such as, for example, Lp, are not negatively affected, making the combination of chondroitin coating in combination with e-beam irradiation a surprisingly effective and simple way to increase the pre-existing selectivity of a given membrane. Summary

[0021] It is an object of the present invention to providehollow fiber and flat sheet membranes for use in separation and / orblood treatment applications such as, for example, hemodialysis,hemofiltration or hemodiafiltration. Said membranes can be ob-tained by contacting a base membranes with a solution of chon-droitin followed by submission to e-beam irradiation. As usedherein, the expression “membrane” or “membranes” encompasses bothhollow fiber membranes and flat sheet membranes. In the context of the present invention, hollow fiber membranes are preferred as they constitute the state-of-the-art membrane configuration,e.g., for use in hemodialysis devices.

[0022] According to one aspect of the present invention, amembrane for use in extracorporeal blood treatment applicationscomprises a base membrane, such as, for example, a membrane com-prising a blend of a polysulfone (PS), polyethersulfone (PES), orpolyarylethersulfone (PAES), and polyvinylpyrrolidone (PVP), anda coating which comprises chondroitin. The membrane is preparedby treating the base membrane with an aqueous solution comprising from 0.1 wt.% to 16 wt.-%, from 0.1 wt.% to 14 wt.-%, or from 0.1wt.-% to 10.0 wt.-% of chondroitin, followed by submitting thecoated membrane to e-beam radiation at a dose of from 12.5 kGy to115 kGy. The membrane is characterized in that the coated membranehas a higher selectivity in comparison with the base membrane. Ahigher selectivity can be expressed in a smaller difference be-tween MWRO and MWCO of a coated membrane in comparison with anuncoated membrane (see also Fig. 1)

[0023] According to one aspect of the invention, the basemembrane 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.

[0024] According to another aspect of the invention, the basemembrane is treated with an aqueous solution comprising from 0.1 wt.-% to 8.0 wt.-% chondroitin sulfate.

[0025] According to yet another aspect of the invention, thebase membrane is coated with chondroitin sulfate in an amount offrom 0.30 µg / cm to 2.00 µg / cm of the membrane area.

[0026] According to another aspect of the invention, the basemembrane is coated with chondroitin sulfate which consists of orpredominantly comprises chondroitin sulfate of the CS-A type (chondroitin-4-sulfate), wherein “predominantly comprises” refersto CS mixtures (e.g., mixtures with CS-C) that comprise more than50% CS-A, preferably more than 60% CS-A, more than 70% CS-A, morethan 80% CS-A, more than 90% CS-A, or more than 95% CA-A.

[0027] According to another aspect of the invention, the basemembrane is coated with chondroitin sulfate which consists of or predominantly comprises chondroitin sulfate of the CS-C type (chondroitin-6-sulfate), wherein “predominantly comprises” refers to CS mixtures (e.g., mixtures with CS-A) that comprise more than 50% CS-C, preferably more than 60% CS-C, more than 70% CS-C, more than 80% CS-C, more than 90% CS-C, or more than 95% CS-C.

[0028] According to another aspect of the invention, the basemembrane is coated with chondroitin sulfate which has an averagemolecular weight (MW) of from 10 kDa to 60 kDa, preferably from10 kDa to 50 kDa, and especially preferably from 10 kDa to 30 kDaor from 20 kDa to 50 kDa. According to another aspect of theinvention, the chondroitin sulfate used is chondroitin sulfate Asodium salt with an average MW of from 10 kDa to 50 kDa or from20 kDa to 30 kDa, or chondroitin sulfate C sodium salt with anaverage molecular weight of from 1 to 5 kDa. According to yetanother aspect of the invention, higher molecular weight chon-droitin sulfate C sodium salt is used, which has an average MW ofup to 70 kDa.

[0029] According to another aspect of the invention, the mem-brane after being contacted with an aqueous solution comprisingchondroitin sulfate is submitted to e-beam radiation at a dose offrom 12.5 kGy to 110 kGy, preferably at a dose of from 20 kGy to 100 kGy, such as from 25 kGy to 100 kGy or from 50 to 90 kGy, such as, for example, 75 kGy.

[0030] According to a further aspect of the invention, e-beamradiation can be done either in the presence of the coating solu-tion or after incubation of the base membrane with a chondroitinsolution according to the invention, followed by removal of thesolution and optionally rinsing the membrane with an aqueoussolution. Preferably, e-beam radiation is done in the presence ofthe coating solution (referred to herein as the “filled” state,as the membrane will be provided together with the coating solu- tion inside of a container that is, accordingly, “filled” with the coating solution surrounding the membrane). The coating solu- tion will generally be removed from the membrane / filter device after e-beam radiation. The membrane and / or filter device can berinsed with water one or several times and then be submitted tofinal sterilization, e.g., by renewed e-beam irradiation or bygamma irradiation according to known practices.

[0031] According to another embodiment of the invention, mem-branes that are preferably used as base membranes can have dif-ferent physical structures, including varying degrees of asymmet- ric configuration, ranging from minimum asymmetry in sponge-like structures to maximum asymmetry in finger-like structures, such as described, for example, in Ronco and Clark, Nature Reviews.Nephrology 14 (6) (2018): 394-410. In some applications the lumenor inner side of the hollow fibers membranes is coated as it generally provides for the selective layer of the hollow fiber membranes in hemodialysis 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 lumenside of the hollow fiber membrane towards the other side of themembrane. 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 thehollow fiber membranes. If so desired, the complete membrane canbe treated with a CS solution and will then be coated on the outersurface as well as on the lumen side of the hollow fiber membrane, including the accessible surfaces of the pores of the membrane.

[0032] Accordingly, it is one aspect of the present inventionthat the base membrane, which is a hollow fiber membrane, iscoated with chondroitin on the lumen side of the membrane includ-ing any adjacent / accessible pores. According to another aspect,the base membrane, which is a hollow fiber membrane, is coatedwith chondroitin on the outer surface of the hollow fiber membraneincluding adjacent / accessible pores. According to yet another as-pect of the invention, the complete surface of a hollow fiber membrane is coated with chondroitin, including the surfaces of accessible pores.

[0033] According to one aspect, the base membrane is a mediumcut-off membrane and has a MWRO of from 8.5 to 14.0 kDa and a MWCOof from 50.0 to 130.0 kDa as measured by dextran sieving before blood contact, preferably a MWRO of from 8.5 to 12.5 kDa and a MWCO of from 55.0 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 12.5 kDa and a MWCO of from 55.0 to 90.0 kDa as measured by dextran sieving before blood contact. Such membranes are generally referred to as medium cut-off membranes, sometimes also as high retention onset or HRO mem-branes. They are described in detail in WO 2015 / 118045 A1 and WO2015 / 118046 A1, respectively. A product which is a representativeof such medium cut-off membranes and dialyzers and which is al-ready available on the market today is the Theranova dialyzer (Baxter).

[0034] According to another aspect, the base membrane is ahigh cut-off membrane having a MWRO of from 15kDa to 20kDa and aMWCO of from 170kDa to 320kDa as measured by dextran sieving before blood contact. Such membranes are described, for example,in WO 2004 / 056460 A1 or in Gondouin and Hutchison: High Cut-OffDialysis Membranes: Current Uses and Future Potential. Advances in Chronic Kidney Disease 18 (2011):180-187. A product which is awell-established representative of such high cut-off membranesand dialyzers, and which is available on the market today, is theTheralite dialyzer (Baxter). High cut-off membranes arecharacterized by a larger average pore size compared to the above-described medium cut-off membranes, whose mean pore size is lowerand whose pore size distribution is narrower compared to standardhigh cut-off membranes.

[0035] According to another aspect, the base membrane is ahigh-flux (HF) membrane having an MWRO of from 5.0 kDa to 8.5 kDaand a MWCO of from 25 kDa to 50 kDa as measured by dextran sieving before blood contact.

[0036] According to yet another aspect of the invention, themembranes can be coated in two consecutive steps comprising (i)contacting a base membrane with a solution comprising chondroitin,and (ii) submitting the base membrane which is or has been incontact with a chondroitin solution to e-beam irradiation, thereby providing for a coated membrane comprising or essentially con-sisting of a base membrane which is coated with an essentiallyhomogenous coating layer comprising or essentially consisting ofchondroitin.

[0037] According to another embodiment of the invention, thebase 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 polyarylethersul- fone, and from 1 to 10 wt.%, relative to the total weight of the polymer solution, of polyvinylpyrrolidone.

[0038] According to another embodiment of the invention, amethod or process for increasing the selectivity of a membrane isprovided, wherein a base membrane is brought into contact with asolution comprising chondroitin and is subsequently submitted toe-beam irradiation at a dose of from 12.5 to 115 kGy, preferablya dose of from 20 to 100 kGy, either in the presence of thechondroitin solution or after discarding the chondroitin solution while the membrane is still wet.

[0039] According to another embodiment of the invention, themethod is carried out with a base membrane comprising or essen- tially consisting of polysulfone (PS), polyethersulfone (PES), or polyarylethersulfone (PAES), and polyvinylpyrrolidone (PVP).

[0040] According to another embodiment of the invention, themethod comprises e-beam irradiating the membrane in the presence of the chondroitin solution.

[0041] According to yet another embodiment of the invention,a process for the preparation a membrane is provided that com-prises the steps of comprising the steps of(a) Providing a base membrane, preferably a base membrane com-prising, for example, a blend of a polysulfone (PS), polyeth-ersulfone (PES) or polyarylethersulfone (PAES), and polyvi-nylpyrrolidone (PVP);(b) Providing a chondroitin solution comprising chondroitin in anamount of from 5 mg / mL and 180 mg / mL;, preferably in an amount of from 10 mg / mL to 140 mg / mL;(c) Contacting and optionally incubating the membrane of step (a)with the solution of step (b);(d) Submitting the membrane of step (c) to e-beam irradiation ata dose of from 12.5 kGy to 115 kGy, preferably at a dose offrom 20 kGy to 100 kGy, either in the presence of the chon-droitin solution of step (b) or after having removed the chondroitin solution and while the membrane is still wet.

[0042] According to one embodiment of the invention, the lumenof a hollow fiber membrane (base membrane) is contacted with thesolution of step (b) before and / or during e-beam irradiation.According to another embodiment, the outer surface of a hollow fiber base membrane is contacted with the solution of step (b) before and / or during e-beam irradiation. According to yet anotherembodiment of the invention, the complete accessible surface of ahollow fiber membrane is contacted with the chondroitin solution before or during e-beam irradiation. As will be understood by theskilled person, the solution may also enter the pores present on the lumen and / or outer surface of the membrane, at least to someextent. The surface of said pores will then be in contact andcoated with chondroitin as well. It is also possible to submitthe complete membrane to coating, wherein the lumen and outersurface as well the accessible surfaces of the pores on both surfaces of the membrane will be contacted with the coating solu- tion.

[0043] According to another embodiment of the invention themembrane or the device comprising said membrane will be submittedto e-beam irradiation in the presence of the coating solution.

[0044] According to another aspect of the invention, the coat-ing solution is removed after e-beam treatment, e.g., by emptying the device, optionally followed by one or more rinsing steps which can be carried out, for example, with water such as RO water. Themembrane and device can then be processes as usual, i.e., it canbe submitted to final sterilization, generally in a wet state (i.e., in the presence of residual water). Sterilization methods encompass e-beam or gamma sterilization according to methods known in the art. Alternatively, steam sterilization can be used.

[0045] According to one embodiment of the invention, a deviceis being provided which is a hemodialyzer or filter device which comprises a membrane coated with chondroitin according to the invention, wherein the membrane has an increased selectivity in comparison to the uncoated membrane. Such device is preferably for use in blood treatment applications, such as hemodialysis,hemofiltration, or hemodiafiltration applications.Brief Description of the Drawings

[0046] Figure 1 is a schematic representation of sievingcurves (e.g., dextran sieving curves) of hypothetic membranes having different selectivities as illustrated by their respectiveMWRO and MWCO values and the delta ^ between said MWRO and MWCOvalues. When the difference or delta (^) between the molecularweight retention onset (MWRO) and molecular weight cut-off (MWCO) of a membrane decreases, the profile of the curve becomes steeper, i.e., the slope at the turning point increases, which correspondsto an increased selectivity of the membrane. Starting from sievingcurve 1, this means that, for example, that the MWCO can remainthe same or become lower (see MWCO2 and MWCO3, respectively),while the MWRO is shifted to higher molecular weights, resulting in MWRO2 or MWCO3. In consequence, the sieving curves 2 and 3 become steeper, i.e., the membrane becomes more selective.

[0047] Figure 2 is a schematic representation of the processfor coating the lumen of a base membrane with chondroitin. (A) shows a hollow fiber membrane (“base membrane”) without coating. A coating solution comprising chondroitin is brought into contactwith the lumen of the hollow fiber membrane in (B) for a givenperiod of time which ranges from several minutes to several hours, wherein chondroitin gets into contact with the surface of themembrane. Preferably, the hollow fiber membrane is then submittedto e-beam irradiation in the presence of the coating solution,resulting in the immobilization of the chondroitin to the membrane surface.

[0048] Figure 3 is a schematic representation of how the coat-ing of the invention is hypothesized to improve the selectivity of a membrane. FIG. 3(A) shows an unmodified membrane, which isnot charged. Negatively charged molecules such as, for example,albumin can pass through a membrane pore of appropriate size. FIG.3(B) shows a membrane which is coated with negatively chargedchondroitin whereby the membrane and pore receive a negativelycharged layer and therefore reject passage of the same molecule which would have otherwise passed the pore. Another or additional effect that may contribute to an increase of rejection after mod- ification is shown in FIG. 3(C). By reducing the pore size of larger pores more than smaller ones the selectivity of the overall membrane can also be increased.

[0049] Figure 4 is a schematic representation of a processfor coating hollow fiber membranes in a filter module, such as aminimodule, in recirculation mode, whereby the coating solutionis pumped through the filter or minimodule. After a certain time,the pump can be stopped, and the filled filter (e.g., a hemodi-alyzer) or minimodule can be submitted to e-beam irradiation.

[0050] Figure 5 shows the effect of chondroitin + e-beamcoating on sieving coefficients of human serum albumin (HSA) in %and YKL-40 in %. The Figure provides for a comparison with theuncoated membrane (MCO Ci-400), a commercially available medium cut-off membrane (Theranova 400, Baxter), and a standard high-flux membrane (#5-WKB-001-01). Said reference membranes are allshown with open circles. Filled circles depict membranes that have been treated with chondroitin and e-beam irradiation under various conditions. Hatched circles depict MCO Ci-400 base membranes withchondroitin but irradiated with gamma rays instead of e-beam (^1and ^2). All membranes have been contacted with a 20 mg / mL chon- droitin solution. Membranes (in minimodules) A and B have been irradiated with 25 kGy and 75 kGy, respectively, both in thepresence of the chondroitin solution (“filled”). Membrane (inminimodule) C was irradiated with 25 kGy in a “dry” state. Mem-branes D and E were irradiated with 75 kGy and 25 kGy, respec-tively, in the “wet” state, i.e., the chondroitin solution was discarded before submitting the membrane / module to e-beam irradi-ation. See Table 6 and Table 7. Theranova 400 was measured “un-sterile” to be able to compare it with the coated membranes whichhave also been measured before final sterilization.

[0051] Figure 6 depicts the effects of the chondroitin / e-beam coating on albumin loss in g / 60 min and YKL-40 clearance inml / min. Albumin loss and clearance have been measured with stand-ard filter modules (hemodialyzers) instead of minimodules. Ther-anova 400 was added as a reference membrane as a measure forselectivity of the membranes of the invention. MCO C1-400 whichwas treated according to the protocols of the invention but inthe absence of chondroitin in the solution was added for illus-trating the impact of the coating. See Table 9. Figure 6 demon-strates that the coating of the invention significantly reducesalbumin loss in comparison with the uncoated membrane but main-tains the excellent clearance capabilities of YKL-40.

[0052] Figure 7 shows the amount of chondroitin which iseluted from a filter device (1.7 m) with ultrapure water insimulated treatment for 4h at 40°C (see Example 6). The filterdevice contained MCO Ci-400 hollow fiber membraned coated with asolution of 40 mg / mL chondroitin and e-beam irradiation with adose of 75 kGy. Standard deviation: 2.6.Detailed Description

[0053] The expression “selectivity” as used herein for a he-modialysis membrane refers to the membrane´s ability to effi-ciently remove small (0.5-15kDa), medium (>15-25kDa) and large(>25-58kDa) middle-molecular weight compounds (see also Rosner etal.: Classification of Uremic Toxins and Their Role in KidneyFailure. Clin J Am Soc Nephrol. 2021) while essentially retainingessential, larger proteins such as, specifically, albumin (66.4kDa). Such selectivity is described, for example, by the steep- ness of the sieving curve (e.g., dextran sieving curve) of amembrane. The increased steepness of the sieving curve correlatesto increased selectivity, indicating that the membrane has anincreased ability to remove large middle while efficiently re-taining albumin. Accordingly, 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 themembrane is 0.9, and its MWCO (molecular weight cut-off), i.e., the molecular weight at which the sieving coefficient of the mem- brane is 0.1, as these parameters provide for the necessary in-formation on the shape and steepness of a membrane´s sievingcurve. For example, two membranes may have the same MWCO whilediffering in their MWRO. The membrane having the lower MWRO, ac- cordingly, is less effective in removing molecules of a molecularweight below the cut-off. Its sieving curve is less steep, andthe membrane is less selective than the one having a higher MWROand the same MWCO. In other words, the selectivity of a membranecan be described by the delta ^ between its MWRO and MWCO. Thesmaller the delta ^ becomes, the higher the selectivity is. Thesteepness of the sieving curve is thus determined largely by theproximity of the values of the parameters MWRO and MWCO as visu-alized in Figure 1.

[0054] The expression “medium cut-off membrane(s)” or “mediumcut-off dialyzer(s)” as used herein, which is sometimes inter-changeably used with the expression “HRO membrane(s)” or “HRO dialyzer(s)” for “High Retention Onset” membrane(s) or dia-lyzer(s), refers to membranes and dialyzers such as described,for example, in WO 2015 / 118046 A1 and WO 2015 / 118046 A1, respec-tively, and wherein the membrane(s) preferably has (have) a MWROof from 9 to 12.5 kDa and a MWCO of from 55 to 110 kDa as measuredby dextran sieving before blood contact and as otherwise describedin WO 2015 / 118046 A1. Preferably, medium cut-off membranes arefurther defined by a sieving coefficient for albumin (Sc (%)) ofbelow 1 (<1) as measured in human plasma at QB=300ml / min andQUF=60ml / min; a sieving coefficient for ß-2-microglobulin (Sc(%)) of equal to or more than 95 (^95) as measured in human plasmaat and QUF=60ml / min; and a sieving coefficient forYKL-40 (Sc (%)) of equal to or more than 30 (^30) as measuredin human plasma at QB=300ml / min and QUF=60ml / min. A medium cut-off dialyzer as mentioned herein is preferably further character- ized by a KUF (ml / h / mmHg) of equal to or higher than 20 (^20) asmeasured according to ISO 8637; a ß2m clearance K (ml / min)ofhigher than 80 (>80) as measured according to ISO 8637 with QB=300-400mL / min; QD=700mL / min; QUF=0-10mL / min; a YKL-40 clearance K(ml / min) of equal to or higher than 20 (^20) asinhuman 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).

[0055] The expression “chondroitin” as used herein refers tochondroitin sulfate (CS), a polymer with a wide molecular weight range composed of an alternating sequence of sulfated and / orunsulfated d-glucuronic acid (GlcA) and N-acetyl- d-galactosamine(GalNAc) residues linked through alternating β-(1 → 3) and β-(1 → 4) bonds and derived from various sources, wherein the pol-ymer can be sulfonated at various positions. The expression en-compasses chondroitin sulfates which are classified as CS- O,A,B,C,D,E,F,K (CS-A, chondroitin sulfate A,[GlcAβ1-3Gal-NAc(4 S)]; CS-C, chondroitin sulfate C, [GlcAβ1-3GalNAc(6 S)];CS-D, chondroitin sulfate D,[GlcA(2 S)β1-3GalNAc(6 S)]; CS-E,chondroitin sulfate E, ,[GlcAβ1-3GalNAc(4 S,6 S)]; CS-F, fucosyl-ated chondroitin sulphate, [GlcA(α1-3Fuc)β1-3GalNAc(4 S)]; CS-O,chondroitin,[GlcAβ1-3GalNAc]) according to their sulfation pat-tern, as well as CS extracted from different sources. The expres-sion further pertains to CS which may consist of combinations ofdifferent types or classes of CS. If not expressly indicated oth-erwise, it preferably refers to the two major chondroitin sulfatesCS-A and CS-C which differ from one another in the position ofthe sulfates. Chondroitin sulfate A is sulfated at position 4, and chondroitin sulfate C is sulfated at position 6.

[0056] The expression “base membrane” as used herein refersto a membrane before being coated with chondroitin according to the invention. Optionally, further compounds such as, for example,heparin may be present in the coating layer that is providedaccording to the present invention. In other words, the base mem-brane and the coated membrane differ regarding the presence of acoating layer comprising or essentially consisting of chondroitin. The base membrane may be altered during the coating process bythe presence of a coating comprising or essentially consisting ofchondroitin and changes to the base membrane due to the e-beam irradiation which is part of the coating process.

[0057] The expression “blood treatment” as used herein refersto 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, hemo- filtration or hemodiafiltration.

[0058] The material of membranes used according to the inven-tion may vary but will generally be uncharged membranes. Suitablebase membranes in the context of the present invention comprise,for example, a blend of i) a polysulfone, a polyethersulfone or apolyarylethersulfone and ii) polyvinylpyrrolidone. Base membranesaccording to the invention can also be made from other knownmaterials as the concept of selectivity increase as described herein is connected to the modification of the surface and pores of a given membrane. Other membrane materials that can 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 mem- brane materials see, for example, Said et al., A Review of Com- mercial Developments and Recent Laboratory Research of Dialyzers and Membranes for Hemodialysis Application. Membranes 11, 767 (2021).

[0059] According to one aspect, base membranes according tothe 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, Nature Reviews. Nephrology 14 (6) (2018): 394-410). In caseof hollow fiber membranes for use in hemodialysis, at least thelumen or inner side of the hollow fibers membranes is coated asit 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 thehollow fiber membranes. It may be practical to coat the completeaccessible surface of a membrane, including hollow fiber mem- branes, during the coating process. As will be understood by the skilled person, the accessible surface of pores on the lumenand / or outer surface of a hollow fiber may also be coated accordingto the invention.

[0060] According to one aspect, the base membrane is a mediumcut-off membrane. A representative of such medium cut-off mem-branes which is available in the market today is the Theranovadialyzer (Baxter).

[0061] According to another aspect, the base membrane has aMWRO of from 8.5 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 8.5 to 12.5 kDa and a MWCO of from 55.0 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 to12.5 kDa and a MWCO of from 55.0 or 60.0 to 90.0 kDa as measuredby dextran sieving before blood contact.

[0062] According to another aspect, the base membrane has anMWRO of from 15kDa to 20kDa and a MWCO of from 170kDa to 320kDa as measured by dextran sieving before blood contact. Such mem-branes are described, for example, in WO 2004 / 056460 A1 or inGondouin and Hutchison: High Cut-Off Dialysis Membranes: Current Uses and Future Potential. Advances in Chronic Kidney Disease 18(2011):180-187. A representative of such high cut-off membranesavailable in the market today is the Theralite dialyzer (Baxter) which comprises such membrane.

[0063] According to another aspect, the base membrane is ahigh-flux (HF) membrane having an MWRO of from 5.0 kDa to 8.5 kDaand a MWCO of from 25 kDa to 50 kDa as measured by dextran sievingbefore blood contact. A representative of such high-flux membraneand dialyzer which is commercially available today is the Re- vaclear dialyzer (Baxter).

[0064] According to one aspect of the invention, the membranescan be coated in a two-step procedure, wherein the membrane ormembrane surface is (i) contacted with a solution comprising oressentially consisting of chondroitin and (ii) subsequently sub-mitted to e-beam irradiation. Generally, the coating step will beperformed with the flat sheet membrane(s) or hollow fibers beingprovided or encased in a filter module according to the state ofthe art. E-beam irradiation will preferably be carried out whilethe coating solution is still present in the filter module. Al-ternatively, e-beam irradiation can be carried out after havingemptied the filter module or container without drying of the mem-brane. Thus, e-beam irradiation can be carried out with “wet”membranes or membranes or modules containing at least residualsolution. Another option includes the irradiation of membraneswhich have been rinsed once or several times, or the irradiationof modules which are filled with rinsing solution (e.g., water)after the chondroitin solution has been removed.

[0065] According to another embodiment of the invention, thecoating step can be repeated once or several times. In this case,the coating solution is removed from the filter module and a freshcoating solution is added to the filter module and the hollow fibers. This process can be repeated several times. Generally,one, two, three, or four rounds of filling, incubating, and re-moving the coating solution can be carried out. Alternatively,the membrane can be contacted with the coating solution by recir-culating the solution through the filter module such as shown,for example, in Fig. 4. Preferably, the coating solution remainsin the filter module after the last incubation step or after acertain pumping time in case of recirculation, and the “filled”module is submitted to e-beam irradiation according to the inven- tion.

[0066] According to one aspect, the solution for coating thebase membrane is prepared, for example, by dissolving chondroitinin water (e.g., deionized water) or a buffer such as, for example,tris buffer. The solution can then be applied to the lumen of ahollow fiber membrane for the coating of the lumen of the membrane and any adjacent pore surfaces. For example, the coating solution can be filled into the lumen of the fibers by aspiration. Alter- natively, the filter modules comprising a bundle of hollow fiber membranes can be filled with a syringe. The hollow fiber membrane can be incubated with the coating solution for a certain timewhich can be varied from several minutes to several hours or evendays. Generally, five minutes to five hours of incubation or re-circulation will be sufficient depending on the base membrane andthe concentration of the chondroitin in the solution. The lumenof the hollow fiber can optionally be rinsed after incubation,e.g., with 0.9% NaCl or distilled water. The flow rate for fillingand / or rinsing of a hollow fiber membrane module can be variedover a relatively broad range, e.g., from about 10 mL / min to 50 ml / min, and the rinsing time can also be varied from about 1 to 20 minutes, such as, for example, from about 2 to 10 minutes.

[0067] As mentioned above, hollow fiber membranes can be sub-mitted to the initial step of coating in recirculation mode,wherein the coating solution is provided in a pool from which it is pumped, for example, through the hollow fibers of a filter device (Fig. 4). The pool volume can vary, but about 100 to 500 mL of a coating solution will be sufficient for an effective initial coating of a standard dialyzer having a surface area of about 1.5 -2.2 m². The pump flow can also be varied over a rela-tively broad range, such as, for example, from 1 mL / min to about20 mL / min. After coating the filters can be rinsed as describedbefore or directly be submitted to e-beam irradiation in a filledstate.

[0068] According to yet another aspect of the invention theconcentration of chondroitin in the coating solution is between 5mg / mL and 160 mg / mL, preferably between 10 mg / mL and 120 mg / mL,more preferably between 10 mg / mL and 100 mg / mL, such as between10 mg / mL and 80 mg / mL, including concentrations of 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL or 80 mg / mL.

[0069] According to one aspect of the invention, chondroitinsulfate A (CS-A) is used for coating the membranes. Chondroitin sulfate A is available in different molecular weights ranging from, for example, 4 kDa to 50 kDa. According to the invention,all chondroitin molecular weight variants can be used for thecoating of a membrane. According to one embodiment of the inven-tion, chondroitin with a higher molecular weight, such as fromabout 10 kDa to 50 kDa, from 20 kDa to 50 kDa, or from 20 kDa to30 kDa is used for coating a membrane. According to yet another aspect of the invention, chondroitin with a lower molecularweight, such from about 1 kDa to 15 kDa, such as from 4 kDa to 10kDa, is used for coating a membrane. According to yet anotheraspect of the invention chondroitin with a higher molecular weight of up to 70 kDa is used for coating a membrane. As mentioned before, also mixtures of different CS variants can be used, e.g., mixtures of CS-A and CS-C. Mixtures that predominantly containCS-A are preferably used according to the inventions as it wasfound that they are specifically well able to increase the selec-tivity of a membrane. However, also CS containing predominantlyCS-C can be used, as well as various combination of CS-A and CS- C.

[0070] According to another aspect of the invention, the mem-branes are coated with chondroitin in an amount of from 0.30 µg / cmto 2.00 µg / cm, such as 0.30 µg / cm, 0.40 µg / cm, 0.50 µg / cm, 0.60µg / cm, 0.70 µg / cm, 0.80 µg / cm, 0.90 µg / cm, 1.00 µg / cm, 1.10 µg / cm, 1.20 µg / cm, 1.30 µg / cm, 1.40 µg / cm, 1.50 µg / cm, 1.60µg / cm, or 1.70 µg / cm,. According to yet another aspect of theinvention, the membranes are coated with chondroitin in an amount of from 0.20 µg / cm to 1.20 µg / cm.

[0071] The presence and amount of chondroitin on the finishedfibers can be tested with known assays such as the dimethyl-methylene blue (DMMB) assay (Farndale et al., Biochimica et Bio-physica Acta 883: 173-177 (1986)).

[0072] The membranes and filters, respectively, are submittedto e-beam irradiation according to the invention. E-beam irradi-ation is carried out according to know procedures, preferably ata dose of from 12.5 kGy to 110 kGy, such as from 12.5 to 90 kGy,from 12.5 to 80 kGy, from 20 to 90 kGy, from 30 to 90 kGy, or from50 to 100 kGy, for example, at 12.5 kGy, 15 kGy, 20 kGy, 25 kGy,30 kGy, 35 kGy, 40 kGy, 45 kGy, 50 kGy, 55 kGy, 60 kGy, 65 kGy, 70 kGy, 75 kGy, 80 kGy, 85 kGy, 90 kGy, 95 kGy, or 100 kGy.

[0073] As mentioned before, it may be preferable to submit amembrane, hollow fiber bundle, or filter device comprising suchmembrane or hollow fiber bundle to e-beam irradiation in thepresence of the coating solution which comprises or essentiallyconsist of chondroitin according to the invention. This is re-ferred to herein as the “filled” state, as in the case of using a filter device or minimodule which comprises, for example, hollowfiber membranes, the device is “filled” with the coating solutionand submitted to irradiation according to the invention. Afterthe irradiation process, the coating solution is removed, e.g., by emptying the filter device, optionally followed by blowing out the device and / or rinsing the device and hollow fibers with water, such as RO water. Optionally, the membranes are dried before sterilization. The membrane and device have to undergo final ster-ilization after the coating procedure. Final sterilization is doneby known methods, such as, for example, e-beam sterilization at25 kGy or gamma irradiation. Steam sterilization is also possiblebut may have a more pronounced impact on the coating of the mem- brane.

[0074] According to one aspect of the invention, the above-described modification and / or coating of a given membrane, suchas a polysulfone, polyarylethersulfone or polyethersulfone basedmembrane, with chondroitin in combination with e-beam irradiation,has an unexpected influence on the sieving performance of and, inconsequence, on the selectivity of the membrane. Surprisingly,the combination of chondroitin coating and e-beam irradiationshows excellent effects on the selectivity of a membrane. Accord-ingly, the present invention provides for an improved selectivity of the treated membranes while at the same time the post-spinningmodification of the membrane and medical device comprising suchmembrane is not very complex. For example, compared to earlierapproaches using (poly)dopamine as an intermediate in the coatingof membranes, the current approach has the benefit of a simplifiedmodification procedure, no leaching of (poly)dopamine, and an im-proved YKL-40 clearance combined with a reduced albumin loss.

[0075] Marker compounds that can be used for assessing saidinfluence on selectivity by determining their respective sievingcoefficients are advantageously selected from medium to largemiddle molecules, such as, for example, YKL-40. In addition, al- bumin is advantageously used as another or second marker molecule. For determining the effect of the coating according to the inven-tion on the sieving performance and selectivity of a given basemembrane, sieving coefficients for the selected marker moleculescan be determined before and after the modification with chon-droitin / e-beam.

[0076] As explained with a view on MWRO and MWCO and the delta^ between same (see Fig. 1), a similar assessment can be madebased on dextran sieving.

[0077] According to another aspect of the invention, the mod-ified or coated membranes according to the invention retain theirhemocompatibility also after coating and final sterilization. Ac-cording to one aspect of the invention, the membranes coated ac-cording to the invention can be used as membranes in medicalapplications, including for applications that require a directcontact of the coated surface with the blood or blood components of a patient. For example, coated membranes according to theinvention can be used for preparing dialyzers for use in hemodi- alysis, including chronic and acute (CRRT) applications and en- compassing hemodiafiltration and hemofiltration.

[0078] According to yet another aspect of the invention, fil-ters or hemodialyzers are provided that comprise hollow fibermembranes that have been coated according to the invention on thelumen side, the outer surface, or all accessible surfaces of thehollow fiber membranes. According to one aspect, said filters or hemodialyzers comprise hollow fiber membranes which are coated with chondroitin, or, in other words, which have a coating layer that comprises or essentially consists of chondroitin.

[0079] According to another aspect of the invention, the fil-ters and dialyzers of the invention that comprise hollow fiber membranes which are modified and / or coated with chondroitin ac- cording to the invention can be used in extracorporeal blood pu-rification applications or extracorporeal blood treatment appli-cations, including, for example, hemodialysis, hemodiafiltration and hemofiltration, including CRRT.

[0080] It will be readily apparent to one skilled in the artthat various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0081] The present invention will now be illustrated by wayof non-limiting examples to further facilitate the understanding of the invention.Examples Example 1: Basic Materials and Methods Example 1.1: Preparation of minimodules

[0082] Minimodules (fibers in a housing used as standardizedfilters for analysis) are prepared by cutting fibers to a length of 20 cm, drying the fibers for 1 h at 40°C and <100 mbar and subsequently transferring the fibers into the 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 minimodule ensures protection and adequate presentation of the fibers. In the present examples, minimodules having an effective membrane (lumen) surface A of 360 cm wereused. The number of fibers required for an effective surface A of360 cm is calculated according to equation (1) A=π×d×l×n[cm2] (1), wherein d is the inner diameter of fiber [cm], n represents the number of fibers, and l 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 internal diameter of fibers and the wall thickness depends on the specific membranes used. Hence, the packing density generally varies be- tween 23% and 31%. Example 1.2: Hydraulic Permeability (Lp) of minimodules

[0083] The hydraulic permeability of a minimodule is deter-mined 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] (2)

[0084] The effective membrane surface area A is calculatedfrom the fiber length and the inner diameter of the fiber according to equation (3) A= ^ · d · l · [cm] (3)wherein d represents the inner diameter of fiber [cm] and lrepresents the effective fiber length [cm].

[0085] The mini-module is wetted thirty minutes before theLp-test is performed. For this purpose, the minimodule is put ina box containing 500 mL of ultrapure water. After 30 minutes, theminimodule is transferred into the testing system. The testingsystem consists of a water bath that is maintained at 37°C and adevice where the minimodule can be mounted. The filling height ofthe water bath must ensure that the minimodule is located under-neath the water surface in the designated device. In order toavoid that a leakage of the membrane leads to a wrong test result,an integrity test of the minimodule and the test system is carriedout in advance. The integrity test is performed by pressing airthrough the minimodule that is closed on one side. Air bubblesindicate a leakage of the minimodule or the test device. It mustbe checked if the leakage is due to an incorrect mounting of theminimodule in the test device or if the membrane leaks. The min-imodule must be discarded if a leakage of the membrane is detected.The pressure applied in the integrity test must be at least thesame value as the pressure applied during the determination ofthe hydraulic permeability in order to ensure that no leakage canoccur during the measurement of the hydraulic permeability becausethe pressure applied is too high. Example 1.3: Dextran sieving measurementsExample 1.3.1: Dextran solutions

[0086] Fractions of dextran supplied by Fluka (Mw 6, 15-20,40, 70, 100, 200, 500 kDa) and Sigma-Aldrich (Mw 9-11 kDa) (bothfrom Sigma-Aldrich Co. LLC, St. Louis, USA) are used without fur-ther purification. Solutions of dextrans with the different mo- lecular weight fractions are combined in Millipore water (i.e., Type 1 ultrapure water, as defined by ISO 3696) at a concentration of 1 g / l for each fraction, which results in an overall concen- tration of 8 g / l.Example 1.3.2: Dextran sieving coefficient tests

[0087] Filtration experiments are carried out under a constantshear rate (γ=750s ) and with the ultrafiltration rate set at 20% of the blood side entrance flux Q , calculated according to equa- tion (4):where Q is the flux at the blood side entrance in ml / min; n is the number of fibers in the minimodule; d is the inner diameter of the fibers in cm and γ is the constant shear rate mentioned above. The filtration conditions are without backfiltration, con- trary to the conditions typical of hemodialysis. The dextran so- lution is recirculated at 37°C ± 1°C. Feed (blood side entrance), retentate (blood side exit), and filtrate (dialysate exit) samplesare taken after 15 min. Relative concentrations and molecularweights of the samples are analyzed via gel permeation chromatog-raphy. The analysis is carried out in a High Performance Liquid Chromatography (HPLC) device (HP 1090A or Agilent 1200; Agilent, Santa Clara, CA, USA) equipped with an RI detector (G1362 from Agilent) and TSKgel columns (PWXL-Guard Column, G 3000 PWXL, G 4000 PWXL; Tosoh, Tessenderlo, Belgium). Samples are filtered through a 0.45 μm filter type OE67 from Schleicher and Schnell, Einbeck, Germany. Calibration is done against dextran standards(Fluka). The sieving coefficient SC is calculated according to equation (5) ^^ =^∙^^(5), ^^^^^wherein c is the concentration of the solute in the filtrate, cits concentration in the permeate and c its concentration in the retentate. Example 1.4: Measurement of sieving coefficients in human plasma

[0088] Middle molecules, consisting mostly of peptides andsmall 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 ^2-M) with a molecularweight of 11 kDa is considered one of the smaller representativesof these middle molecules. Myoglobin has a molecular weight (MW)of about 17 kDa is already larger. It will not be completelycleared 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 representativeof higher molecular weight middle molecules that should still beremoved as efficiently as possible. Albumin with a MW of about 67kDa is a key element in describing the sieving characteristics of membranes, as albumin should preferably not be allowed to pass amembrane for chronic hemodialysis to a significant extent, whereasmolecules having a lower molecular weight, such as β2-M and YKL-40 should be removed as efficiently as possible, i.e., the sievingcurve of a given membrane should be steep and not flat, as istypically found with protein-leaking membranes.

[0089] The sieving coefficients for albumin and YKL-40 whichare naturally contained in human plasma can be determined foruncoated membranes and membranes that were coated according tothe invention or of comparative examples. Determination of sievingcoefficients for these marker molecules can be done, for example,with Octaplas LG from Octapharma.

[0090] For the experiments, human plasma is thawed and broughtto 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. The final protein concentration of the test solu- tion is 45±5 g / l. 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. Q was 8,7 mL / min (= 7,71 SKT) and Q 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 mini- modules are free of air bubbles, the filtrate pump is started. As soon as filtrate flows back from the tube into the pool time isstarted. After some time a sample of the test solution (A) (minimum1,1 mL for HSA and 550 μL for YKL-40) can be taken. The samplingtime is 1 min for Q and 1 min for Q . From these measurements, the retentate sample (R) and filtrate sample (F) are used for further analysis of the marker molecules. The sieving coefficient SC is calculated according to equation (5).Example 1.5: Clearance Performance

[0091] The clearance “C” (mL / min) refers to the volume of asolution 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 be- fore was also applied to complete hemodialyzers, such as commer- cially available dialyzers like Theranova 400 (Baxter Int., Inc.) or hemodialyzers were prepared from other MCO type membranes, suchas the MCO-C1 400 dialyzer (Gambro Dialysatoren GmbH, Hechingen,Germany); see, for example, Boschetti-de-Fierro et al. (2015),Scientific Reports 5: 18448, DOI: 10.1038 / srep18448) and werecoated 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.

[0092] To measure clearance, human plasma (protein content 55± 10 g / L) is spiked with myoglobin and β2-M (Lee Biosolutions),to a concentration of 0.50 mg / L and 5.0 mg / L, respectively. Albu-min and YKL-40, for example, are measured directly from plasma(e.g., OctaplasLG 45-70 mg / mL from Octapharma). The test is per-formed by recirculating human plasma for 60 ± 5 minutes at QB =300mL / min, QD = 500mL / min, and UF = 10 mL / min at a temperature of37°C. Samples are in each case taken from the blood outlet, dia- lysate outlet and blood inlet, respectively. Samples are taken att [min]= 0, 4, 10, 20, 30, 40, 50, 60. Clearance (Cl) is calcu-lated according to equation (6)

[0093] wherein c(t) is the concentration of the marker (e.g.,YKL-40) protein at time (t), V is the plasma volume and c is theconcentration of the marker at t=0.Example 1.6: Quantitative determination of YKL-40 in human plasma

[0094] For determining the concentration of YKL-40 the Quan-tikine Human Chitinase 3-like (CHI3L1) is used according to thesupplier´s instructions. The assay is a solid phase ELISA assaydesigned to measure CHI3L1 in cell culture supernates, serum,plasma, and urine. The assay employs the quantitative sandwichenzyme immunoassay technique. A monoclonal antibody specific forthe analyte has been pre-coated onto a microplate. Standards andsamples are pipetted into the wells and any analyte present isbound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for theanalyte is added to the wells. Following a wash to remove anyunbound 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. The color development is stopped, and the intensity of the color is measured.

[0095] For determining the concentration of β2-M, myoglobinand albumin a nephelometer (BN ProSpec, Siemens Medical Solutions) is used in combination with the supplier’s quantification kits.β2-M, myoglobin and albumin are quantified using the N Latex β2Microglobulin kit, N Latex Myoglobin kit and N Antiserum to Humanalbumin kit (Siemens Health solutions), respectively, all accord-ing to the supplier´s instructions. Each determinant is measuredseparately 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 concentra- tion. Example 1.7: Determination of albumin loss

[0096] Albumin loss is determined in the same setup as de-scribed for the determination of clearance (Example 1.5). Samplesare taken at the dialysate outlet at t [min] = 4, 10, 20, 30, 40,50, 60. Albumin concentration can be determined by spectrophotom-etry according to known protocols, see also Example 1.6.Example 2: Coating of hollow fiber membranes with chondroitin

[0097] A solution of chondroitin A sulfate sodium salt with ahigh molecular weight and an average MW of 10,000 to 50,000 Da from Biosynth was used for the experiments if not indicated oth- erwise. Solutions of other chondroitin sulfate types were prepared the same way. 300 mL of coating solution were used per minimodule(see Example 1.1). The solutions used for coating had final con-centrations of from 20 mg / mL to 160 mg / mL as indicated.

[0098] The chondroitin solutions were prepared by stirringuntil the chondroitin was dissolved completely, which takes longerat higher chondroitin concentrations. The resulting solutions wereimmediately used for the coating process. For example, the lumenand pores of the fibers of a minimodule can be filled with thechondroitin solution by suction, wherein the upper connector thatis fluidly connected to the lumen (blood side) of the hollowfibers is closed, and the bottom connector that is fluidly con-nected to the blood side of the fibers or module is connected to the coating solution and then closed as well. The connector at the bottom of the module that is fluidly connected to the filtrateor dialysate side of the module is also closed, whereas the re-spective (dialysate side) connector at the upper side of the mod-ule is connected to a vacuum pump. About 40 mL of the coatingsolution are pumped into the filter module with the help of a vacuum pump, wherein the blood side connector at the bottom ofthe module is opened after a vacuum has been achieved. The coatingsolution is thus sucked through the lumen side of the membrane to the outside of the membrane, resulting in a complete coating of the membrane surfaces, including the pores. After the coatingsolution is completely sucked in, the module is left standing fora short while, then all connectors are closed, the module isdisconnected and sent to irradiation. During the filling processthe minimodule can optionally be shaken to remove air bubbles.

[0099] Alternatively, the hollow fibers are coated by con-necting the module to a peristaltic pump (“filling”). Tubes areconnected to the blood side of the module whereas the connectorsbeing in fluid communication with the dialysate side of the module are closed. The coating solution is pumped into the module withabout 100 mL / min until the complete solution has been transferredinto the module. Again, the formation of air bubbles is avoided. All connectors are then closed, the module is disconnected andprepared for irradiation.

[0100] The chondroitin coating solution can also be filledinto the minimodules by using a 50 mL syringe filled with about 5mL of the chondroitin solution. Again, minimodules were closed and left for 15 minutes, after which 50 mL syringes filled withair were used to empty them. The emptied “wet” minimodules wereagain closed and sent to e-beam irradiation. Alternatively, the “filled” minimodules still containing the chondroitin solution were closed without emptying them and sent to e-beam irradiation.

[0101] The initial coating with chondroitin can equally beperformed with standard size hemodialyzers, wherein, for example, about 100 mL of the coating solution are used instead of 5 mL as for the minimodules.

[0102] Known hollow fiber membranes such as otherwise usedin commercial hemodialyzers (Theranova 400, Baxter) or in exper-imental membranes which have been described before, such as MCO- Ci 400 (see, for example, Boschetti-de-Fierro et al., ScientificReports (2015) 5: 18448, wherein “MCO 4” corresponds to MCO-C1400, or WO 2015 / 118045 A1), all from Baxter, were used for pre-paring minimodules according to Example 1.1 or filter devices andcoated as described above in this Example 2. An overview overcertain minimodules prepared according to the invention and usedin subsequent tests is provided in Table 1.Table 1: Minimodules prepared according to the invention. #Membrane ChondroitinFilter E-beam dose Designa- 123456789Example 3: Quantification of chondroitin

[0103] The amount of chondroitin on polyethersulfone (PES)fibers after coating can be determined by placing fibers in 2 mLcryovials and adding the respective detection reagents for eachsubstance. For quantifying chondroitin, a dimethylmethylene blue(DMMB) assay was used (Farndale et al., Biochimica et BiophysicaActa (1986) 883: 173-177).

[0104] The tests were carried out by using a 200 µg / mL stocksolution, of which 200 µl were combined with 2mL of reagent. Sincesolid membranes were used, the dilution of the stock solution inthe reagent was utilized to calculate the final standardconcentration and this number was then used to determine the re-spective quantities present on the membranes.Example 4: E-beam irradiation and dose

[0105] E-beam irradiation was done according to known methods.The coated membranes in minimodules or filter modules were sub-mitted to e-beam irradiation with focused electrons (beta parti-cles) at various doses. E-beam irradiation was typically performedat 25 or 75 kGy. The process was performed with “dry”, “wet” and“filled” devices (see also Example 2) when being submitted to e-beam irradiation.Example 5: Influence of process parameters on sieving coeffi-cients, clearance and albumin loss Example 5.1: Influence of e-beam dose and chondroitin concentra- tion

[0106] Experiments based on MCO C1-400 minimodules as de-scribed before were performed to determine the influence of e-beam dose on the selectivity of the membrane. As a measure forselectivity, the sieving coefficients of YKL-40 and HSA were de-termined. The minimodules were irradiated in the “filled” stateto make them comparable and several minimodules were tested foreach value as indicated. Table 2 summarizes the outcome of thecoating experiments with different doses.Table 2: MCO Ci-400 membranes in minimodules were submitted todifferent chondroitin concentrations and doses to test the influ-ence of both parameters on the increase of selectivity of the basemembrane after coating as determined by sieving coefficients forHSA (Human Serum Albumin) and YKL-40. Tests were carried out asdescribed herein with human plasma. Lp is provided in [*10 cm / (bar*s)]. “MV” refers to “Mean Value”, “SD” to “Standard Devia-tion”. “n” designates the number of minimodules / test performed.Chondroitin concentrations having an asterisk (*) mean that chon-droitin from Mythocondro was used (CS-C, MW of 1-5 kDa), whereasall other coatings were done with chondroitin from Biosynth (CS-A, MW of 10-30 kDa).# CoatingIrradia- LPn Sieving Coefficient HSA12345678911111unsterile

[0107] As can be seen in Table 2, higher e-beam doses (75 kGyversus 25 kGy) result in an increased improvement of selectivityversus the uncoated base membrane MCO C1-400 (see Table 5 forcomparison with the uncoated base membrane), even though bothdoses are effective in improving membrane selectivity. Comparing the chondroitin concentrations used, the data further show that higher chondroitin concentrations, such as, for example 40 mg / mLin comparison to 20 mg / mL (e.g., #4 / 5 or #2 / 6), enhance the se-lectivity increase. Example 5.2: Hydraulic permeability and the influence of the coat-ing process

[0108] Table 3 provides for the hydraulic permeability ofmembranes according to the invention. Tests were performed asdescribed in Example 1.2 on minimodules shown in Table 1 at samechondroitin concentrations for better comparability. The results confirm that the Lp values are within expected ranges, indicating that the coating according to the invention does not negatively affect hydraulic permeability of the membranes, which is an im- portant prerequisite for the applicability of the membranes and methods of the invention in the field of dialysis. Lp values for the minimodules submitted to e-beam irradiation in a filled state (shown in bold, 36, 43, 22) showed a lower Lp compared to those that were irradiated in a wet state, indicating that membranecoating is different depending on the process used and indicatingthat irradiation in the filled state might be preferable. Table 3: Lp measurements for membranes / minimodules coated and ir- radiated according to the invention with chondroitin. M m ( 3423344337,7 180 370 12,9 27031 291,4 180 374 13,0 27533Example 5.2: Sieving coefficients and the influence of the coatingprocess

[0109] Sieving coefficients (SC) for albumin and (HAS) andYKL-40 were determined for membranes according to the inventionas described in Example 1.4. The focus in this experiment was onthe influence of the irradiation process. Accordingly, minimodules in a filled state (bold) were tested in comparison with those in a wet state. The results are provided in Table 4. Table 4: Sieving coefficients for human serum albumin (HAS) and YKL-40. Tested membranes (in minimodules) were as described inTable 1. Membranes and minimodules shown in bold are those whichwere irradiated in the “filled” state.

[0110] The results provided in Table 4 show that the coatingof the base membrane with chondroitin followed by e-beam irradi- ation leads to change of the sieving coefficients for HSA and YKL-40 of the coated membrane compared to the uncoated membrane. Thedata further show that the results are different for the differentmethods used for producing the coated membranes. Those modules that were subjected to e-beam irradiation in a filled state (coat- ing solution is left in the modules during irradiation) show asomewhat higher selectivity, i.e., the sieving coefficients forHSA are lower in comparison to those membranes and modules thatwere irradiated in the “wet” state. As a result, the sieving curvefor those membranes that were irradiated in a filled module are steeper than those irradiated in a wet state. It is therefore preferable to submit the membrane or filter modules to e-beamradiation in the “filled” state.

[0111] The impact of coating on the sieving coefficients forhuman serum albumin (HSA) and YKL-40 and on the selectivity of the membrane can be determined by comparing uncoated membranes with the same membranes after having been treated according tothe invention. Table 5 provides for the hydraulic permeability aswell as sieving coefficients for HSA and YKL-40 for reference membrane MCO-Ci 400 (uncoated), which is the same membrane thatwas used in the coating experiments shown before. In addition,Table 5 also shows Lp and SC values measured with a high-flux typemembrane (#5-WBK-001-01, unsterile) which is included for compar-ison of Lp and SC values, and the Theranova 400 membrane (also unsterile for better comparability). Table 5: Lp and SC for the uncoated MCO-Ci-400 membrane. Also shown are the Theranova membrane (unsterile), and #5-WKB-001-01, which is a high-flux type membrane. The values shown are averageval 4 t ti lMT 4 sM#01 (9)

[0112] As can be seen in comparison with the values providedfor SCs in Table 2 and 4, coating a base membrane with a processaccording to the invention results in an improvement of theselectivity of the base membrane, which is especially prominent with regard to the increase in the SC for YKL-40. Such improvementis achieved by e-beam irradiating the membrane in the presence ofthe coating solution (“filled” state) or in the “wet” state. How-ever, irradiation in the “filled” state is preferable.Example 5.3: Influence of irradiation type and dose: gamma versuse-beam radiation

[0113] The influence of the e-beam dose (25 kGy versus 75 kGy)was again tested with minimodules in different states (dry, wet,filled) that are coated with solutions having the same chondroitinconcentration (20 mg / mL). Table 6 shows the results of these testsin terms of the sieving coefficients for HSA and YKL-40 achievedin each case. The results confirm that a higher irradiation dosein combination with a module that is filled with the coatingsolution during irradiation (bold) surprisingly provides for thebest results, even though lower doses (see e.g., #5) are alsoeffective, and also wet modules still show an effect.Table 6: Coating of MCO-C1 400 membranes according to the inven-tion with different e-beam doses (25 kGy or 75 kGy) and in dif- ferent states. “MM” refers to “Minimodule”. # 0 12345(filled)

[0114] Table 7 shows the different impact of the irradiationtype. Using e-beam radiation in the coating process is more ef-fective in comparison to gamma irradiation. Coating was performedwith a chondroitin solution of 20 mg / mL with Biosynth CS-A. Table 7: Minimodules with MCO Ci-400 membranes were irradiatedwith either gamma or e-beam (bold) under otherwise same condi-tions. The coating solution contained chondroitin in a concentra-tion of 20 mg / mL and 40 mg / mL as indicated. The Lp is shown in[10 cm / s^bar]. “SC” refers to “Sieving Coefficient”, “HSA” refersto “Human Serum Albumin”. The values are average values based onthree measurements each. #01234567

[0115] Selected membranes of Table 6 and Table 7 are depictedin Figure 5 with their SC values for HSA and YKL-40. In Figure 5,the position of the data point is a measure of the impact of thecoating and selectivity. As can be seen, the best effect on se-lectivity increase is obtained with filled devices during e-beamtreatment (data points A and B corresponding to minimodules #5and #2 of Table 6, respectively). Data points C, D, and E corre-spond to minimodules #1, #4, and #3, respectively, of Table 6. These minimodules were submitted to e-beam treatment in dry andwet states. Data points ^1 and ^2 correspond to minimodules #1 and#2 of Table 7, respectively, showing that gamma irradiation doesnot have the same beneficial effect as e-beam treatment. MCO C1-400 is the base membrane without coating. Example 5.4: Chondroitin Type CS-C

[0116] For comparison with chondroitin CS-A (Biosynth, MW 10-30 kDa), membranes were coated with chondroitin of the CS-C type(Chondroitin sulfate C sodium salt from Biosynth) on MCO C1-400base membranes at chondroitin concentrations of 40 mg / mL or 80mg / mL, as indicated, and e-beam treated with a dose of 75 kGyeach. Table 8 shows the effects of the coating also in comparisonwith water-filled devices without chondroitin. As can be seen from the HSA values, both CS-A and CS-C can be used to modify thesieving coefficients and selectivity of a base membrane. Astronger concentration effect is visible for CS-C, wherein an increase of the chondroitin concentration in the coating solutionresults in a clearly improved (lower) SC for HSA.Table 8: Membranes were coated with chondroitin of the CS-A andCS-C type according to the invention as indicated. Lp [^10cm / (bar^s)] and sieving coefficients [%] have been measured asdescribed above. The first two modules were only filled with waterto show the effect of chondroitin. All modules were irradiated inthe filled state as indicated. “n” indicates the number of modulestested, “SD” means “standard deviation”. The values for Lp and SCare average values (“AV”).M C M C M C M C(filled)Mem-Coating E-Beam Lp SC HSA [%] SC YKL-40M C M CExample 5.5: Effects on YKL-40 clearance and albumin loss

[0117] To demonstrate the influence of the coating and shiftin sieving coefficients on albumin loss and clearance for YKL-40in filter modules, tests were carried out according to Examples1.5 and 1.7. For the tests, filter devices were used instead ofminimodules. Table 9 shows the results on albumin loss and clear-ance for YKL-40, which confirm the effects already demonstratedwith minimodules for sieving coefficients. The coating with chon-droitin (CS-A from Biosynth, MW 10-30 kDa) results in a signifi-cant reduction of albumin loss of the MCO CI-400 membrane. At thesame time, the clearance of YKL-40 remains very high, resultingin a very efficient filter device for the selective removal of larger middle molecules while being able to excellently retain albumin. Table 9: Albumin loss and YKL-40 clearance of filter devices con-taining membranes of the invention and comparative devices / mem-branes. “n” indicates the number of modules tested, “SD” means“st M400 droitinMem-Coating E-BeamAlbumin Loss Clearance M 4 M 4 T n 4

[0118] The results shown in Table 9 are visualized in Figure6. It is clearly visible that a coating with chondroitin (e.g.,40 mg / mL chondroitin in the coating solution) followed by e-beamtreatment at e.g., 75 kGy in the filled state leads to a signif-icant reduction in albumin loss, whereas the clearance of YKL-40remains stable.Example 6: Stability of chondroitin on the membrane

[0119] Further tests were carried out to determine the sta-bility of chondroitin on the coated membrane and the amount ofchondroitin which can be extracted from the coated membrane. Someextraction or elution of chondroitin from the coating is fully acceptable as chondroitin sulfate is a compound which is present naturally in the body as an essential part of hyaline cartilage.Chondroitin is used as a chondroprotective drug for the treatmentof osteoarthritis. It is suggested in the literature that chon-droitin is partially absorbed by the human body and reaches thejoints, thus leading to a reduction in pain and slowing down ofthe rate of joint destruction and cartilage loss (Zhu et al.: Effectiveness and safety of glucosamine and chondroitin for thetreatment of osteoarthritis: a meta-analysis of randomized con-trolled trials. Journal of Orthopedic Surgery and Research (2018),13:170). Chondroitin was further found to have an excellent safetyprofile (Monfort et al.: Chondroitin sulphate for symptomatic os-teoarthritis: critical appraisal of meta-analyses. Curr Med ResOpin. (2008); 24(5):1303). The recommended oral dose per day is800-1200 mg per day. Therefore, it is important to understand howmuch chondroitin is eluted from a coating according of the inven-tion.

[0120] Filter modules with MCO C1-400 hollow fiber membranebundles (1.7 m) were coated (lumen) with a chondroitin solutioncontaining 40 mg / mL chondroitin (CS-A, average MM 10-30 kDa) andan e-beam irradiation dose of 75 kGy. The filter module was emptiedafter irradiation and rinsed with ultrapure water at 50°C in re-circulation mode (QB=300 mL / min) for 10 minutes in an in vitrotreatment model, i.e., without undergoing final sterilization. After rinsing, elution during dialysis treatment was simulated inthe same setup (recirculation mode, QB = 300 ml / min) at 40°C with500 ml of ultrapure water for 4h. As a reference, a Theranova 400filter (1.7 m) was submitted to a rinsing and elution process asdescribed for the test filter. After 4h of simulated treatmentthe amount of chondroitin that was eluted from the test filterand the Theranova 400 filter was determined with a DMMB assay asmentioned above. Figure 7 shows the results for four measurements.6.0 mg chondroitin are eluted with water per device in 4h of simulated treatment (standard deviation 2.6). As to be expected, no chondroitin is detected in the assay when using Theranova 400.

Claims

P51141PC00 Claims1. A membrane coated with chondroitin for use in extracorpo-real blood treatment applications, wherein the membrane is pre-pared by treating a base membrane with an aqueous solution com-prising from 0.1 wt.-% to 16.0 wt.-% of chondroitin sulfate(CS), followed by submitting the coated membrane to e-beam radi-ation at a dose of from 12.5 kGy to 115 kGy, and wherein thecoated membrane is characterized by a higher selectivity in com- parison to the base membrane.

2. The membrane according to claim 1, wherein the base mem-brane comprises (i) a blend of a polysulfone (PS), polyethersul-fone (PES), or polyarylethersulfone (PAES), and polyvinylpyrrol-idone (PVP), preferably in an amount of from 0.5 wt.-% to 5.0wt.-% PVP and from 95.0 wt.-% to 99.5 wt.-% PES, PS, or PAES.

3. The membrane according to claim 1 or claim 2, wherein thebase membrane is treated with an aqueous solution comprising from 0.1 wt.-% to 8.0 wt.-% chondroitin sulfate.

4. The membrane according to any of claims 1 to 3, whereinthe base membrane is coated with chondroitin sulfate in anamount of from 0.30 µg / cm to 1.20 µg / cm of the membrane area.

5. The membrane according to any of claims 1 to 4, whereinthe base membrane after treatment with an aqueous solution com-prising chondroitin sulfate is e-beam treated with a dose offrom 12.5 kGy to 110 kGy.

6. The membrane according to any of claims 1 to 5, whereinthe base membrane has a MWRO of from 9.0 to 14.0 kDa and a MWCOof from 60.0 to 130.0 kDa as measured by dextran sieving before blood contact.- 2 -7. The membrane according to any of claims 1 to 5, whereinthe base membrane has a MWRO of from 5.0 kDa to 8.5 kDa and aMWCO of from 25 kDa to 50 kDa as measured by dextran sieving be- fore blood contact.

8. The membrane according to any of claims 1 to 5, whereinthe base membrane has a MWRO of from 15kDa to 20kDa and a MWCOof from 170kDa to 320kDa as measured by dextran sieving before blood contact.

9. The membrane according to any of claims 1 to 8 wherein thebase membrane is prepared from a polymer solution which compris- es from 10 to 15 wt.%, relative to the total weight of the poly- mer solution, of polysulfone, polyethersulfone or polyarylether-sulfone, and from 1 to 10 wt.%, relative to the total weight ofthe polymer solution, of polyvinylpyrrolidone.

10. The membrane according to any of claims 1 to 9, whereinthe e-beam radiation is carried out at a dose of from 20 kGy to100 kGy.

11. The membrane according to any of claims 1 to 10, whereinthe membrane is a hollow fiber membrane or a flat sheet mem- brane.

12. The membrane according to any of claims 1 to 11, whereinthe chondroitin sulfate is chondroitin-4-sulfate (CS-A).

13. The membrane according to any of claims 1 to 12, whereinthe chondroitin sulfate and has an average molecular weight (MW) of between 10 and 50 kDa.

14. A process for preparing a membrane according to claim 1,comprising the steps of (a) Providing a base membrane;<sub>- 3 -(b) Providing an aqueous chondroitin solution comprisingchondroitin in an amount of from 5 mg / mL and 160 mg / mL; (c) Contacting the base membrane with the solution ofstep (b); and(d) Submitting the membrane of step (c) to e-beam irradi-ation at a dose of from 12.5 kGy to 110 kGy.

15. The process of claim 14, wherein the base membrane com-prises a blend of a polysulfone (PS), polyethersulfone (PES), orpolyarylethersulfone (PAES), and polyvinylpyrrolidone (PVP).

16. The process of claim 14 or claim 15, wherein the membraneis submitted to the e-beam radiation in the presence of the so- lution of step (b).

17. A medical device for use in blood treatment applications,wherein the medical device comprises a membrane according to anyof claims 1 to 13.

18. The medical device of claim 17, wherein the medical deviceis a hemodialyzer.

19. A method of increasing the selectivity of a membrane, com-prising the steps of (a) Contacting the membrane with an aqueous solution ofchondroitin having a chondroitin concentration of from 5 mg / mL to 160 mg / mL; (b) Irradiating the membrane of step (a) with e-beam at adose of from 12.5 to 110 kGy.

20. A method according to claim 19, wherein step (b) is car-ried out in the presence of the solution of step (a).

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