Multi-layered membrane

Abstract

The present invention relates to a multi-layered membrane comprising at least one layer of a porous pre-filter material, a track-etched membrane and a layer comprising a porous flow distribution material, a process for preparation of such a multi-layered membrane as well as the use of such membranes for the separation of cells or other larger items from fluid, especially from body fluids and more specifically for the separation of blood into blood cells and serum or plasma.

Description

[0001] 222647PEP / HCath 1

[0002] Multi-layered Membrane

[0003] The present invention relates to a multi-layered membrane comprising at least one layer of a porous pre-filter material, a track-etched membrane and a layer comprising a porous flow distribution material, a process for preparation of such a multi-layered membrane as well as the use of such membranes for the separation of cells or other larger items from fluid, especially from body fluids and more specifically for the separation of blood into blood cells and serum or plasma.

[0004] Background and Prior Art

[0005] Track-etched membranes (TEM) have been considered suitable for cell separation due to their uniform pore size and smooth surface for a long time. The even surface of these membranes minimizes cell damage, as cells are gently retained on the surface rather than being trapped in rough structures or narrow pores. The uniform pore size of TEM can be precisely selected according to the size of the particles to be separated, ensuring the retention of such particles and cells on the membrane surface enabling effective separation.

[0006] Unlike other filter materials such as expanded PTFE or sintered materials with sponge-like or tapered pores that potentially allow particles (e.g. cells) to enter and clog, the structure of TEM prevents particles from lodging in the pores, which reduces clogging and facilitates cleaning, thus enabling long-lasting use.

[0007] To facilitate the efficient passage of liquids through the membrane, the surface must be wettable. Materials such as polyethylene terephthalate (PET) are naturally somewhat hydrophilic, while polycarbonate (PC) can be made hydrophilic by applying a coating. In any case, applying a hydrophilic coating onto the membrane supports the wetting and, thus, the fast separation of blood cells from whole blood. 222647PEP / HCath 2

[0008] Another challenge separating blood components is blood clotting or drying of body fluids. If the separation process takes too long, clots or residues may block membrane pores, thereby stopping the flow of plasma, serum, or other fluids through the membrane.

[0009] WO 2024 / 056252 proposes membrane filters that promise reduced clotting. However, these or technically similar filters would greatly benefit from an improved flow of liquid during filtration. The objective of the present invention is therefore to provide materials that facilitate a rapid and even fluid flow through the filter membrane, preferably without relying on external forces such as pressure or vacuum.

[0010] Summary of the Invention

[0011] The object has been solved by the present invention as defined in the appended claims.

[0012] According to a first aspect, the present invention provides a multi-layered membrane comprising:

[0013] (a) a top layer comprising at least one layer of porous pre-filter material having pores having a pore size equal to or larger than the pore size of the track-etched membrane (b);

[0014] (b) a middle layer comprising a track-etched membrane; and

[0015] (c) a bottom layer comprising a porous flow distribution material, wherein the multi-layered membrane includes preferably a hydrophilic coating on at least the track-etched membrane.

[0016] The top layer (a) diffuses the whole blood sample and retains red and white blood cells, the middle layer (b) separates the sample by size, and the bottom layer (c) holds and / or distributes the obtained plasma, optionally transferring the plasma to a detection device, e.g., a microfluidic device or lateral flow detection device. 222647PEP / HCath 3

[0017] According to a second aspect a process for the preparation of a multi-layered membrane is provided, wherein the process comprises: a) providing a middle layer (b) comprising a track-etched membrane including a hydrophilic coating, a top layer (a) comprising at least one layer of porous pre-filter material with pores having a pore size equal to or larger than the pore size of the track-etched membrane, and a bottom layer (c) comprising a porous flow distribution material; b) laminating the top layer (a) comprising at least one layer of a porous pre-filter material, the middle layer (b) comprising the track-etched membrane, and the bottom layer (c) comprising a porous flow distribution material together; c) optionally treating the obtained multi-layered membrane with a hydrophilic coating solution; and optionally drying the multi-layered membrane; or, alternatively, d) providing a track-etched membrane including a hydrophilic coating, a porous pre-filter material with pores having a pore size equal to or larger than the pore size of the track-etched membrane, and a porous flow distribution material; e) optionally treating the porous pre-filter material and / or the porous flow distribution material with a hydrophilic coating solution and drying the treated materials; f) stacking together at least one layer of porous pre-filter material from d) or e), a track-etched membrane from e), and a porous flow distribution material from d) or e); and g) laminating the stacked layers from f) together.

[0018] In a third aspect of the present invention, the inventive multi-layered membrane is used for separation of cells or other larger items from fluids, especially from body fluids. Preferably, this aspect includes the separation of blood into blood cells and serum or plasma, especially for diagnostic and point of care or home applications.

[0019] A fourth aspect of the present invention relates to a device for vertical blood separation comprising the multi-layered membrane as herein described. 222647PEP / HCath 4

[0020] A further aspect of the present invention pertains to an in vitro method for obtaining pure blood plasma from a whole blood sample, comprising the use of a multi-layered membrane or a device as described herein.

[0021] Detailed Description of the Invention

[0022] The present invention is based on the realization that the problems encountered by the prior art can be avoided by providing and using a multi-layered membrane of the present invention.

[0023] Thus, a first aspect of the present invention relates to a multi-layered membrane comprising:

[0024] (a) a top layer comprising at least one layer of porous pre-filter material having pores having a pore size equal to or larger than the pore size of the track-etched membrane (b), herein also called prefilter layer;

[0025] (b) a middle layer comprising a track-etched membrane; and

[0026] (c) a bottom layer comprising a porous flow distribution material, herein also called release aid layer, wherein the multi-layered membrane includes preferably a hydrophilic coating on at least the track-etched membrane.

[0027] Multi-layered membrane including a hydrophilic coating on at least the track-etched membrane are in particular preferred. The hydrophilic coating on the track-etched membrane usually faces the top layer (a).

[0028] The inventive multi-layered membrane preferably consists of the 3-layers (a), (b) and (c).

[0029] Preferably layer (a) comprises two layers of porous pre-filter material having pores having a pore size equal to or larger than the pore size of the track-etched membrane (b) which can rest directly on top of each other, ie. have no air gap between them, or which can be separated by an air gap. 222647PEP / HCath 5

[0030] As used herein, an air gap between 2 prefilter layers may be described as follows. The fibers of the 2 prefilter layers are not fused together by any means (e.g. by lamination) but rather placed on top of each other during production. Thus, according to preferred embodiments, the 2 prefilter layers are not laminated. Preferably the 2 prefilter layers are spaced at least 1 nm apart. It may be presumed that an air gap is formed while sealing the 2 prefilter layers at their perimeter.

[0031] As used herein, "lamination”, "laminating” or "laminated” refers to the process of bonding the membrane to a permeable and porous material to increase the robustness and resistance of the membrane. PET with different densities (60 and 100 g / m2) is preferably used as the backing material for this application. The laminated TEM may be manufactured using heated rollers, which apply uniform pressure and temperature to the materials so that they bond thermally. This process can also bond several layers of backing material together without the need for a TEM in between.

[0032] Embodiments comprising two prefilter layers are in particular preferred. According to such embodiments the two prefilter layers may be of the same material or may differ with regard to the material and / or functional features such as pore size and / or thickness. Having different prefilter layers materials and / or functional features, these may be independently selected from the ones described. Preferably, the two prefilter layers are identical.

[0033] Inventive multi-layered membranes having two prefilter layers and an air gap between them are especially preferred. Such air gap provides a better yield, allows better scaling as well as reduces hemolysis.

[0034] The multi-layered membranes show superior filtering efficiency, especially when filtering samples like body fluids and especially whole blood.

[0035] While experimenting with a two-layered membrane consisting of a porous pre-filter and a TEM for size exclusion, the inventors observed that adding an additional layer 222647PEP / HCath 6 of porous material beneath the TEM facilitated passaging of plasma through the layers above.

[0036] In the resulting multi-layered membrane, the pre-filter material of the multi-layered membrane absorbs the fluid, e.g., the blood sample, and spreads the fluid inside the filter material. The pre-filter material also at least partly absorbs larger items and possibly also compounds which lead to clotting of blood. As the fluid passes through the pre-filter material, the wider distribution of the fluid in the pre-filter also leads to a broader application and wetting of the track-etched membrane area, while the 3rdof porous material helps to wick out the plasma for improved collection.

[0037] The asymmetric cross-sectional structure of the multi-layered setup, featuring a more open porous pre-filter layer above a micron-rated track-etched membrane (TEM), followed by a third layer of porous material as a flow distribution layer, enhances capillary forces, which facilitate the spontaneous separation and collection of plasma from whole blood samples.

[0038] This inventive multilayer design enables plasma flow without the need for external forces such as pressure or vacuum, wherein the flow is additionally improved by enhancing the hydrophilicity or surface energy of each layer through dip-and-dry coating with the same surfactant, salt, and sugar mixture.

[0039] With the described multi-layered structure, more of the track-etched membrane’s pores are covered by the fluid compared to when a blood drop is applied directly to the membrane surface, while the capillary forces of the additional third layer help wick and channel the plasma through the membrane, efficiently distributing it for storage or further analysis.

[0040] The multi-layered membrane is preferably formed or aligned in that the porous prefilter material is to receive the sample to be separated, in particular body fluid, and even more preferably blood while the flow distribution layer at the bottom of the stack enhances capillary forces through all layers and distributes the plasma for further use or storage. 222647PEP / HCath 7

[0041] Sealing the edges of the multi-layered filter and shaping the filter material into a concave form (such as shown in Fig. 12A) focuses the capillary forces across all three layers of porous material, thereby further enhancing flow and plasma separation. Such sealing provides a spontaneous and direct flow. Embodiments having a sealing at the edge are in particular preferred, i.e. the multi-layered membrane is preferably bound by seals, preferably ultrasonic, heat or adhesive seals. As used herein, “seal” or “sealing” preferably relate to a liquid impermeable seals or liquid impermeable sealings. Accordingly, especially preferred embodiments, the edges of the inventive multilayer membrane are liquid impermeable sealed.

[0042] Thus, the inventive multi-layered filter have an upper side (top layer (a)), a middle layer consisting of the TEM (b) and a lower side (bottom layer (c)), wherein the upper side, i.e. the porous pre-filter material, receives the sample to be separated, such as body fluids and in particular blood.

[0043] The track-etched membrane (middle layer (b)) may face an absorber as herein described. The pre-filter material on the upper side serves for pre-separation of larger items and also compounds which might lead to clotting of blood. By using the correspondingly arranged pre-filter material, improved filtration results can be achieved as compared to a track etched membrane alone, while the flow distribution layer (bottom layer) enhances the flow of liquid through the filter. Therefore, first the porous pre-filter material comes into contact with the liquid to be filtrated, followed the track-etched membrane and finally the porous flow-distribution layer.

[0044] Furthermore, the hydrophilic coating applied onto at least the track-etched membrane enhances the wetting and the spreading of the fluid on the membrane. Thus, especially for filtration of whole blood, the liquid parts of the blood, i.e., serum or plasma can be readily absorbed by the pores and channeled through the TEM before the clotting process sets in and / or the blood sample dries out. 222647PEP / HCath 8

[0045] Larger particles and especially blood cells are retained not only on the membrane surface as with some prior art products, but also within the pre-filter material. Thus, blocking of the pores by retained blood cells is minimized while enhanced flow also minimizes dry out effects for the inventive membranes.

[0046] Summarizing the above detailed description of the inventive membranes and their properties and effectivity, the invention provides a multi-layered filter material which exhibits superior properties and is especially suitable for separation of blood cells from whole blood to obtain blood plasma or serum in an efficient manner.

[0047] Within the context of the invention, the porous pre-filter material can either be made of a porous solid or from suitable fibers in the form of a woven or a non-woven material. The pores or the porous structure of the pre-filter material have to be larger or equal to the pore size of the track-etched membrane to generate the pre-filter effect. Thus, the pre-filter material absorbs larger constituents of a fluid sample whereas the fluid itself and items smaller than the pore size of the pre-filter material can accumulate on the surface of the track-etched membrane and, depending on the pore size, pass through the track-etched membrane. To enable the pre-filter to absorb for instance whole blood and pass the plasma or serum to the track-etched membrane, which performs the final separation step, the blood preferably also effectively wets the pre-filter. This can either be achieved by using a hydrophilic or other suitable base material for the pre-filter or by providing a hydrophilic coating not only on the track-etched membrane, but also on the pre-filter material. Red blood cells are preferably captured in the prefilter-layer, rather be absorption than by size exclusion. The prefilter layer is therefore preferably made of materials that absorb red blood cells such as PET and in particular non-woven PET.

[0048] Within the context of the invention, the porous flow distribution material can be made of a porous solid or from suitable fibers in the form of a woven or a non-woven material. The pores or the porous structure of the flow distribution material are preferably of the same size as the track-etched membrane thereby generating additional capillary force which enhances flow of the sample through the filter. This can be achieved by using a hydrophilic or other suitable base material for flow 222647PEP / HCath 9 distribution material or by providing a hydrophilic coating not only on the track- etched membrane, but also on the flow distribution material.

[0049] In a preferred embodiment of the invention, the porous flow distribution material is a natural or synthetic, woven or non-woven polymeric fiber material. In an especially preferred embodiment of the invention, the porous flow distribution material itself has hydrophilic properties, but is usually not water-soluble. Alternatively, if a porous pre-filter material is used exhibiting hydrophobic properties, such materials are preferably treated by applying a hydrophilic coating onto such material.

[0050] In preferred embodiments of the invention, the porous pre-filter material and / or porous flow distribution layer material is a natural or synthetic, woven or non-woven polymeric fiber material or other porous media.

[0051] In an especially preferred embodiment of the invention, the porous pre-filter material and / or porous flow distribution layer material itself has hydrophilic properties but is usually not water-soluble.

[0052] Alternatively, if a porous pre-filter material and / or porous flow distribution layer material is used, which is a respective natural or synthetic, woven or non-woven polymeric fiber material exhibiting hydrophobic properties, such materials are preferably treated by applying a hydrophilic coating onto such material.

[0053] It is particular preferred that the porous pre-filter material and / or porous flow distribution material are a non-woven material comprising bi-component polymeric fibers.

[0054] The bi-component polymeric fibers are preferably selected from the group comprising coPET / PET (co-extruded Polyethylene Terephthalate I Polyethylene Terephthalate), PET / PP (Polyethylene Terephthalate I Polypropylene), PE / PP (Polyethylene I Polypropylene), PET / PBT (Polyethylene Terephthalate I Polybutylene Terephthalate), and PET / Nylon (Polyethylene Terephthalate I Nylon). This selection is not intended to limit the invention, but to serve as an example so 222647PEP / HCath 10 that the person skilled in the art can supplement it as required without additional inventive activity.

[0055] The porous pre-filter material and the flow distribution material can be selected independently. The materials can be the same or different.

[0056] According to a preferred embodiment, the pre-filter material and the flow distribution material are both a PET non-woven porous material.

[0057] As indicated, the multi-layered membrane comprises one layer of a track-etched membrane (b), one layer of a flow distribution material (c) and one or several layers of a porous pre-filter material (a). The layers of the inventive membrane can either be loosely stacked and held together in an appropriate manner or in a suitable device and appropriate measures can be taken to avoid separation of the layers of such stack. However, in preferred embodiments of the present invention, at least one layer of a pre-filter material (a) is laminated onto the track-etched membrane (b) and the flow distribution layer (c). In preferred embodiments, at least one layer of a non-woven pre-filter material (a) the track-etched membrane (b) and the flow distribution layer (c) are stacked and laminated together. According to further preferred embodiments of the invention, the membrane contains two or more layers of a pre-filter material, and all layers (a-c) are joined by lamination.

[0058] Within the context of the present invention, the more than one layers of pre-filter material can include multiple layers of the same pre-filter material or multiple layers of different materials or any combinations thereof. In some instances, it is preferred to use layers of different pre-filter materials, e.g., with, towards the track-etched membrane, decreasing pore sizes. Furthermore, it is possible and a further preferred embodiment, to use a pre-filter material which in one layer includes different pore sizes, especially decreasing pore sizes relative to the track-etched membrane. While also in such specific embodiments of the present invention, the pore size of all existing pre-filter material layers is equal to or larger than the pore size of the track-etched membrane, an even higher filtering efficiency can be 222647PEP / HCath 11 achieved by using a pre-filter material with decreasing pore sizes. Also, an enhanced spreading of the fluid and wetting of the membrane is obtained due to the fact that clogging of the various layers is minimized due to the continuously decreasing pore size and the fact that the largest items are retained in a different layer than smaller items.

[0059] The track-etched membrane layer of the inventive multi-layered membranes, can be made of any material known to the skilled person as applicable in this context. Track-etched membranes are commercially available or can be obtained by treating a membrane base material under conditions forming pores of the desired size and density by a track-etching treatment. Such track-etching treatment conditions are well known to the skilled person.

[0060] According to preferred embodiments of the present invention, the material of the track-etched membrane is selected from polyethylene terephthalate (PET), polycarbonate (PC), polyimide (PE), polyvinylidine fluoride (PVDF), polyetheretherketon (PEEK), ethylenetetrafluoroethylene (ETFE) or from other polymers having similar properties. In especially preferred embodiments, the material of the track-etched membrane is PET or PC.

[0061] As mentioned above, a hydrophilic coating can be applied to the track-etched membrane only, to the track-etched membrane and at least one layer of porous prefilter material, and / or to the flow distribution material or to all layers of the inventive membrane.

[0062] In preferred embodiments of the invention, the hydrophilic coating is applied to all layers of the inventive membrane.

[0063] The coating can be applied to some of the layers before assembling the multilayered membrane. In preferred embodiments, however, the coating is applied to all layers of the membrane, and in especially preferred embodiments, the coating is applied to an already assembled multi-layered membrane. Preferably, the layers of the assembled multi-layered membrane are joined by lamination before applying the 222647PEP / HCath 12 coating. In such case, the coating will also cover the intersection(s) between the track-etched membrane and the pre-filter material(s) respectively the flow distribution material.

[0064] In case of lamination of an already coated track-etched membrane, a pre-filter material, and a flow distribution material, the coating of the track-etched membrane could be damaged. Also, in some instances, using different precoated materials of the membrane and the other layers could lead to an inhomogeneous coating and, thus, inhomogeneous wetting of the surfaces. A slower passage of the plasma or serum through the membrane could result from such inhomogeneous coating.

[0065] Depending on the intended use and application of the inventive multi-layered membrane, the thickness of the track-etched membrane can be adapted as desired and as explained above for materials of the other layers.

[0066] In preferred embodiments of the invention, the track-etched membrane has a thickness of 1 to 100 pm, preferably 2 to 50 pm and more preferably 5 to 25 pm.

[0067] The pore size of the track-etched membrane is also selected as best suited for the intended use of the membrane.

[0068] Pore size may be a particular critical feature. Increasing the pore size provides a greater number of pinholes. Such pinholes can be easily spotted because the blood travels through the TEM and instead of a plasma a blood spot is provided. As the pore size increases so do the pinholes. A double layer laminated did not change the presence of pinholes.

[0069] In preferred embodiments of the invention and for well-established uses like separation of whole blood into blood cells and plasma or serum, the pore size of the track-etched membrane is 0.1 to 5 pm, preferably 0.125 to 4.5 pm, more preferably 0.15 to 3.5 pm, more preferably 0.2 to 3.0 pm, more preferably 0.225 to 2.8 pm, more preferably 0.3 to 2.5 pm, and even more 1 .0 to 1 .75 pm. 222647PEP / HCath 13

[0070] Pore sizes mentioned in the context for the present invention are defined as the pore diameter, especially the mean pore diameter.

[0071] The pore density can also be varied according to the intended use, however, for most purposes a preferred pore density lies between 10,000 to 1 ,000,000,000 pores per cm2, preferably 2,000,000 to 100,000,000 pores per cm2.

[0072] As mentioned above, the porous pre-filter material and the flow distribution material can be made of any material that is suitable for achieving the intended filtration effect. In preferred embodiments, the pre-filter material is a woven or a non-woven material, non-woven materials being especially preferred. Such materials can be produced from any polymeric fiber, with polyester; polyolefin, polyimide, cellulose, and nylon being fiber materials, which are considered as preferred within the context of the present invention.

[0073] Further preferred porous pre-filter or flow distribution materials are phase-inverted membranes or stretched membranes.

[0074] Phase-inverted membranes are made through a process called phase inversion, where a polymer solution transforms from a liquid to a solid state, typically by immersing it in a nonsolvent bath, causing the polymer to precipitate and form a porous membrane.

[0075] In contrast, a stretched membrane is made by mechanically stretching a polymer film, which creates uniform pores and increases the surface area, resulting in a membrane that filters particles while allowing fluid or gas to pass through.

[0076] According to the invention, the pore size of the porous pre-filter material is equal to or larger than the pore size of the track-etched membrane.

[0077] Preferred pore sizes of the pre-filter material are 5 to 10,000 pm, more preferably between 10 and 500 pm, and especially preferably between 20 and 100 pm. 222647PEP / HCath 14

[0078] The pore size of the flow distribution material may be smaller or wider than the pore size of the pre-filter layer. Preferably, the pore size of the flow distribution material is in the same range as the pore size of the pre-filter layer. More preferably the flow distribution material has essentially the same pore size as the pre-filter layer has.

[0079] The thickness of the layer or layers of porous pre-filter material can be adapted to the intended use and should especially take into account the volumes to be treated per unit of filter area. Also, the nature of the fluid applied onto the filter and the proportion or mass of substances or cells to be removed from the fluid by filtration need to be considered for determining the appropriate size and thickness of the prefilter material in the inventive membranes.

[0080] The thickness of the combined layers (a) - (c) of the multi-layered filter is preferably between 200 pm to 1000 pm, wherein the top layer (a) is preferably from 100 pm to 800 pm, more preferably from 200 pm to 600 pm the middle layer (b) is preferably from 1 pm to 100 pm, more preferably from 25 pm to 75 pm and the bottom layer (c) is preferably from 100 pm to 800 pm, more preferably from 200 pm to 600 pm.

[0081] In general, the thickness of the layers (a) - (c) can be selected independently.

[0082] Hydrophilic coating solutions, which can be applied to the multi-layered membrane of the present invention, can be selected from commercially available coating solutions or custom made for the intended use by selecting appropriate hydrophilic materials and preparing a respective solution in a suitable solvent.

[0083] According to preferred embodiments of the invention, the hydrophilic coating comprises polyvinylpyrrolidone (PVP), is a sulphonated coating, or a coating comprising alcoholic or carboxylic groups or any other coating that supports the wetting process of the membrane. A hydrophilic coating may be on the top and / or the bottom side of the multi-layered membrane and / or independently each layer of the membrane. If the hydrophilic coating is only on one side of a layer of the inventive membrane, the top side is preferred. 222647PEP / HCath 15

[0084] The multi-layered membranes according to the present invention may contain or be coated with agents that serve for stabilizing components in the liquid to be filtered, in particular blood. Examples for such agents include alkyl polyethoxylates, i.e. mild tensides, which are suitable for isolating functional membrane complexes, such as Brij® 35 (polyoxyethylene(23)lauryl ether) or tocopherol. The use of tocopherol and Brij® 35 is in particular preferred.

[0085] In addition to the above detailed description regarding the inventive multi-layered membrane itself, also processes for producing such membranes are subjects of the present invention. All features mentioned above which include or refer to process steps for obtaining the respective materials, are also considered disclosed within the manufacturing process. Basic and additional information regarding the process is provided in the following:

[0086] In a second aspect of the present invention, a process for the preparation of an inventive multi-layered membrane is disclosed. Such process comprises provision of the various layers of the multi-layered membrane as defined above and joining the layers in an appropriate manner. According to a preferred embodiment of the present invention, such process comprises a) providing a middle layer comprising a track-etched membrane including a hydrophilic coating, a top layer comprising at least one layer of porous prefilter material with pores having a pore size equal to or larger than the pore size of the track-etched membrane, and a bottom layer comprising a porous flow distribution material; b) laminating the top layer comprising at least one layer of a porous pre-filter material, the middle layer comprising the track-etched membrane, and the bottom layer comprising a porous flow distribution material together; c) optionally treating the obtained multi-layered membrane with a hydrophilic coating solution; and optionally drying the multi-layered membrane; or, alternatively, d) providing a track-etched membrane including a hydrophilic coating, a porous pre-filter material with pores having a pore size equal to or larger than the 222647PEP / HCath 16 pore size of the track-etched membrane, and a porous flow distribution material; e) optionally treating the porous pre-filter material and / or the porous flow distribution material with a hydrophilic coating solution and drying the treated materials; f) stacking together at least one layer of porous pre-filter material from d) or e), a track-etched membrane from e), and a porous flow distribution material from d) or e); and

[0087] While in principle the layers can be stacked and held together by appropriate means, in preferred embodiments of the inventive process, the laminating steps b) or g) are performed. These steps include stacking the membrane layers and applying a pressure of 1 to 5 bars, preferably 2 to 3 bars. The temperature applied in preferred embodiments of this lamination step is 60 to 180°C, preferably 120 to 180°C, more preferably 130 to 160°C and most preferably 135 to 155°C.

[0088] In further preferred embodiments, the treating steps c) or e), respectively, are performed by applying an aqueous solution of polyvinylpyrrolidone or a sulphonated coating, a coating containing alcoholic and / or carboxylic groups or another suitable coating, such as, for example, agents that serve for stabilizing components in the liquid to be filtered, in particular blood. Examples for such agents include alkyl polyethoxylates, i.e. mild tensides, which are suitable for isolating functional membrane complexes, such as Brij® 35 (polyoxyethylene(23)lauryl ether) and / or tocopherol. Most preferably, a polyvinylpyrrolidone solution is applied at a concentration of 0.1 to 50 g / L. The use of tocopherol and Brij® 35 is in particular preferred.

[0089] Further conditions and process steps can be adapted as appropriate for the production of multi-layered membranes, and especially further characteristics of the materials to be employed can be chosen based on the definitions provided above. More specifically, the material of the porous pre-filter material, its thickness and its pore size, the nature of the flow distribution material its thickness and its pore size and the nature of the track-etched membrane material, its thickness, its pore size 222647PEP / HCath 17 and pore density are as defined above. Furthermore, instead of laminating the track- etched membrane to at least one pre-filter material layer and one layer of flow distribution material, which is preferred, in alternative embodiments also a stacking of the layers and an attachment or mounting of the layers can also be achieved by securing or pinning or clamping the layers together in an appropriate frame or device.

[0090] A third aspect of the present invention is the use of an inventive multi-layered membrane or of a membrane prepared according to the inventive process for separation of cells or other items from fluids.

[0091] The particularly improved fluid flow through, which can be achieved by the inventive combination of the various layers and the resulting superior capillary forces which leads to a highly efficient separation of material and is especially useful in the application of separating cells from body fluids. Considering the problems encountered in the prior art with clotting and slow flow rates frequently leading to dry out effects during the separation processes, the inventive multi-layered membrane provides a novel and superior concept for such applications.

[0092] Preferably the multi-layered membrane described herein is used for diffusing a whole blood sample, retaining red and white blood cells, separating the sample by size and / or obtaining plasma, wherein the top layer (a) diffuses the whole blood sample and retains red and white blood cells, the middle layer (b) separates the sample by size, and the bottom layer (c) holds and / or distributes the obtained plasma, optionally transferring the plasma to a detection device, e.g., a microfluidic device or lateral flow detection device. Thus, according to this aspect, the invention relates also to the use of a multi-layered membrane as herein described for diffusing a whole blood sample, retaining red and white blood cells, separating the sample by size and / or obtaining plasma.

[0093] The inventive membranes can be applied in larger separation units for diagnostic purposes; however, it can also be effectively used in smaller devices for point of care and home applications. One example of such point of care or home 222647PEP / HCath 18 applications is a lateral flow analysis test system, in which the inventive membrane can be used as a first filter, which separates the cellular components form the fluid, especially from blood, while the fluid moves through the test system for determination of the presence or absence of certain substances and molecules.

[0094] Thus, a further aspect of the present invention relates to a device for vertical blood separation comprising a multi-layered membrane as herein described.

[0095] Such device preferably comprises an optional absorber and / or a means for even distribution of the sample to be separated, in particular blood.

[0096] The device may comprise a collection substrate, preferably a porous adsorber or a non-porous plate, in particular a microfluidic chip. It is especially preferred that the collection substrate is in close and / or good contact, providing effective capillary forces, with the inventive multilayered membrane.

[0097] In a preferred embodiment, the multi-layered membrane, the absorber layer and the means for distribution or collection are aligned in vertical direction as follows from bottom to top: a) an optional absorber layer, b) a multi-layered membrane as herein described, and c) optional means for even distribution, and d) a collection substrate as herein described.

[0098] Suitable absorber materials are known to the person skilled in the art and, for example, comprise cellulose-based materials, materials comprising microfluidic channels, and / or suitable foams, in particular foams made of polymeric bonded fibers and / or polyurethane such as Porex HRM (high-release media) fiber media.

[0099] The means for distribution can be, for example, a grid and / or the cover plate of the device itself. Such cover plate preferably comprises a sample receiving hole. 222647PEP / HCath 19

[0100] In such a device the multi-layered membrane is preferably formed or aligned in that the porous pre-filter material is to receive the sample to be separated, in particular body fluid, and even more preferably blood while the flow distribution layer at the bottom of the stack enhances capillary forces through all layers and distributes the plasma for further use or storage.

[0101] Sealing the edges of the multi-layered filter and shaping the filter material into a concave form (Fig. 12A) focuses the capillary forces across all three layers of porous material, thereby further enhancing flow and plasma separation. Thus, according to preferred embodiments, the multi-layered membrane is bounded by seals, preferably heat or adhesive seals. Devices having a multi-layered filter with sealed edges are in particular preferred. Sealed edges may be provided, for example, by ultrasonic and / or heat based sealing as well as adhesive seals.

[0102] Preferably, the concave form of the multi-layered membrane enables focused contact with collection substrate.

[0103] Devices according to the present invention are, for example, illustrated in FIG. 12.

[0104] Besides the multi-layered membrane, the optional absorber, the optional distribution means and collection substrate the device can further comprise a base plate, which preferably uptakes the absorber, and a cover plate as well as screws to tighten the assembly.

[0105] Furthermore, the multi-layered membranes of the present invention can also be used in other automated analysis systems.

[0106] Yet another aspect of the invention relates to an in vitro methods for obtaining pure blood plasma from a whole blood sample, comprising the multi-layered membrane or device as described herein. 222647PEP / HCath 20

[0107] Figures and Examples:

[0108] EXAMPLE 1

[0109] Figure 1 : Figure 1 shows a multi-layered membrane as a 12 mm disk filter that was perimeter heat sealed at 260 C.

[0110] Figure 2: Upstream surface of the disk filter of Figure 1 after 5 microliters of whole blood were applied to the top of the filter and plasma was collected on a porous hydrophilic polyethylene absorber.

[0111] Figure 3: Downstream Surface of the disk filter of Figure 1 after application of

[0112] 5pl of whole blood to the upstream surface and collection of spontaneously released plasma onto the adsorber. The downstream surface is free of red blood cells.

[0113] EXAMPLE 2

[0114] Figure 4: Figure 4 shows a filter comprised of a prefilter on TEM, without the

[0115] 3rd layer on the bottom of the TEM or heat sealing of the perimeter with 5 microliters of whole blood applied to the surface. Blue tape was used to hold the filter on the porous hydrophilic polyethylene adsorber.

[0116] Figure 5: Figure 5 depicts the back of the filtration setup of Figure 4. Whole blood leaked around the perimeter and no clean plasma separation was achieved toward the center of the collection substrate via capillary forces.

[0117] EXAMPLE 3

[0118] Figures 6 & 7: Figures 6 & 7 show the membrane of Figures 4 & 5 where the prefilter was sealed on the perimeter using heat sealing (Fig. 6) or epoxy around the perimeter (Fig. 7). 222647PEP / HCath 21

[0119] Figure 8: Figure 8 depicts the back of the filtration setups from Figure 6. The perimeter seal prevented whole blood leakage from the top to the bottom onto the porous hydrophilic polyethylene adsorber. But, without the 3rd layer on the bottom of the TEM, the plasma was not spontaneously wicked out for collection as in Example 1 .

[0120] Figure 9: Figure 9 depicts the back of the filtration setups from Figure 7. The perimeter seal prevented whole blood leakage from the top to the bottom onto the porous hydrophilic polyethylene adsorber. But, without the 3rd layer on the bottom of the TEM, the plasma was not spontaneously wicked out for collection as in Example 1 .

[0121] EXAMPLE 4

[0122] Figure 10: Top view of two TEMs, each with flow distribution layer but without a non-woven pre-filter; 5 microliters of whole blood applied to the top of the filter and plasma was collected on a porous hydrophilic polyethylene absorber.

[0123] Figure 11 : Bottom view of two TEMs, each with flow distribution layer but without a non-woven pre-filter; 5 microliters of whole blood applied to the top of the filter and plasma that has collected on a porous hydrophilic polyethylene absorber. Even without the prefilter, the bottom porous distribution layer helps wick out plasma via capillary forces onto the porous hydrophilic polyethylene adsorber

[0124] Figure 12: Schematic structure of a device for the separation and collection of plasma from whole blood. Shown is A) an embodiment with a concave, multi-layered membrane for focused transfer of plasma to collection substrate, and B) an embodiment with a straight, multilayered membrane.

[0125] EXAMPLE 5: A 4-layered membrane reduces hemolysis 222647PEP / HCath 22

[0126] Figure 13: Figure 13 shows 3 types of prefilter layer, either 1 prefilter layer, or 2 prefilter layers laminated, or 2 prefilter layers separated by an air gap.

[0127] Figure 14: Figure 14 shows quantification of average plasma yield and lysed blood in pL from 10 pL of EDTA whole blood input sample for three membrane types. Samples were tested in triplicate.

[0128] An extra layer of PET was added to decrease hemolysis. The additional layer captures more red blood cells (RBCs) which means fewer RBCs will impact the TEM, thus reducing hemolysis. There are two variations. Either an extra layer laminated onto the original design (4-layers) or adding an extra layer on top postlam ination and sealing together (4-layers, air gap). Figure 14 shows that the 4-layers did not affect hemolysis but did slightly increase yield compared 3-layers. However, 4-layers, gap significantly decreased the rate of hemolysis. The air gap created by sealing an extra layer on causes the decrease in hemolysis. It’s possible that the RBC’s have more room and can be capture better with that added airgap.

[0129] Additionally, we noted a higher STDEV with the 4-layers, gap which could indicate that the size of the air gap can vary among samples. This is not a significant concern because in each replicate the hemolysis is always much lower than the original prototype. This indicates that although both 4-layers and 4-layers, gap have an extra layer, the order in which you laminate, and seal has the greatest impact on hemolysis. Taken together the data in Figure 14 suggests that adding an additional prefilter layer will increase yield but to decrease hemolysis the additional layer needs to be added after lamination.

[0130] We found that ultrasonic welding was the preferred method. This is partially due to the thickness of the membrane. When the prototype is heat sealed, sometime sealing was not complete or an increase in melted material was seen. The other concern with heat sealing was keeping the integrity of the TEM. Melting might be disadvantageous. Ultrasonic welding was much faster, more energy efficient, more precise. One does not need to worry about the sample melting and the seal 222647PEP / HCath 23 spreading out. Another benefit with ultrasonic welding was the ability to customize the settings. Since ultrasonic welding also uses vibration instead of heat sealing for a more delicate sealing process.

[0131] The sealing is critical for the function of the membrane. When the blood encounters our membrane, it will travel the path of least resistance. If there is no seal, the blood will travel wider and horizontally rather than vertically through the TEM. Blood also contains small compounds which can travel through any gap in a seal. For example, if the back of the prototype is not properly sealed, the plasma that was separated by the TEM will travel through the gaps in the seal rather than through the transfer membrane. In the presence of a strong seal, the blood will travel down in the direction of the TEM and yield a higher plasma yield compared to an unsealed sample.

[0132] Figure 15: Figure 15 shows average plasma yield and lysed blood volumes for donor samples with various hematocrit values. Input volume was 10pL of EDTA whole blood. (Functionality of membrane type 60-1 - 60, a symmetric 3-layer membrane consisting of a prefilter layer (facing the the whole blood side) with lower density (PET nonwoven 60g / cm2), TEM layer with pore size 1 um, release aid layer (facing the the plasma side) with lower density (PET non-woven 60g / cm2.)

[0133] Based on the data in Figure 15, 3-layers consistently yields 2ul of plasma from 10ul of EDTA whole blood despite donors being different hematocrits. The initial hypothesis was that a higher hematocrit would cause an increase in hemolysis. What we found was that the rate of hemolysis was consistent between all the donors with the except of Donor E which also didn’t yield 2ul of plasma. In fact, donors C and H in Figure 15 yielded very similar results despite having 10% difference in RBC count. The yield did vary between donors but did not always increase when hematocrit decreases. What this shows is that yield is very dependent on the donor. 222647PEP / HCath 24

[0134] This can be explained, because there are many other factors that would affect the viscosity and separate rate of blood.

[0135] Another important characteristics is that just because a hematocrit is high does not mean that the rate of hemolysis will increase. This indicates that the rate of hemolysis is not directly proportional to hematocrit. Taken together this demonstrates that 3-layers works under a variety of conditions and the variation between yields is more due to the donors and not their hematocrit.

[0136] Figure 16: Figure 16 shows average plasma yield on volume of lysed blood by membrane types. Membrane types differ in density of prefilter layer and release aid layer.

[0137] We found that having a 100-backing increased hemolysis. In Figure 16, both the 100-1 -100 and the 60up had a 100 on the back it increased hemolysis. Whereas when there was a 60-backing material, we saw the lowest rates of hemolysis. Both the 60-1 -60 and the 100 up had the lower rates of hemolysis. Based on figure 16, the 60-1 -60 is the favored prototype because it has a lower STDEV, lowest rate of hemolysis and the highest yield compared to the asymmetric 60-1 -100.

[0138] The membrane types mentioned above are defined as follows. The 60-1 -60 membrane is a symmetric 3-layer membrane consisting of a prefilter layer (facing the the whole blood side) with lower density (PET non-woven 60g / cm2), TEM layer with pore size 1 urn, release aid layer (facing the the plasma side) with lower density (PET non-woven 60g / cm2). The 100-1 -100 membrane type is a symmetric 3-layer membrane consisting of a prefilter layer (facing the the whole blood side) with higher density (PET non-woven 100g / cm2), TEM layer with pore size 1 um, release aid layer (facing the the plasma side) with higher density (PET non-woven 100g / cm2). The membrane type 60-1 -100 (60 up) is a assymmetric 3-layer membrane consisting of a prefilter layer (facing the the whole blood side) with lower density (PET non-woven 60g / cm2), TEM layer with pore size 1 um, release aid layer facing the the plasma side with higher density (PET non-woven 100g / cm2). The 60-1 -100 (100 up) membrane type is assymmetric 3-layer membrane consisting of a prefilter layer 222647PEP / HCath 25

[0139] (facing the the whole blood side) with higher density (PET non-woven 100g / cm2), TEM layer with pore size 1 um, release aid layer (facing the the plasma side) with lower density (PET non-woven 60g / cm2).

[0140] Figure 17: Figure 17 shows a multi-layered membrane disk filter after whole blood filtration, top view of whole blood and top view of a collection substrate after plasma transfer, once with good contact of membrane to collection substrate, and once with less good contact to the collection substrate.

Claims

222647PEP / HCath 26Claims1 . A multi-layered membrane comprising:(a) a top layer comprising at least one layer of porous pre-filter material having pores having a pore size equal to or larger than the pore size of the track-etched membrane (b);(b) a middle layer comprising a track-etched membrane; and(c) a bottom layer comprising a porous flow distribution material, wherein the multi-layered membrane includes a hydrophilic coating on at least the track-etched membrane.

2. The multi-layered membrane according to claim 1 , which consists of layers (a), (b) and (c).

3. The multi-layered membrane according to claim 1 or 2, wherein layer (a) comprises two layers of porous pre-filter material having pores having a pore size equal to or larger than the pore size of the track- etched membrane (b) which can rest directly on top of each other, or which can be separated by an air gap.

4. The multi-layered membrane according to claim 1 to 3, wherein a hydrophilic coating is additionally provided on at least one side of the top layer (a) and / or bottom layer (c).

5. The multi-layered membrane according to any of claims 1 to 4, wherein the porous pre-filter material (a) and / or porous flow distribution layer material (c) is a hydrophilic, natural, synthetic, woven or non-woven polymeric fiber material or other porous media.

6. The multi-layered membrane according to any of claims 1 to 5, wherein the porous pre-filter material (a) and / or porous flow distribution material (c) is a non-woven material comprising bi-component polymeric fibers.222647PEP / HCath 277. The multi-layered membrane according to any of claims 1 to 6, wherein the bi-component polymeric fibers are selected from the group comprising coPET / PET (co-extruded Polyethylene Terephthalate I Polyethylene Terephthalate), PET / PP (Polyethylene Terephthalate I Polypropylene), PE / PP (Polyethylene I Polypropylene), PET / PBT (Polyethylene Terephthalate I Polybutylene Terephthalate), and PET / Nylon (Polyethylene Terephthalate I Nylon).

8. The multi-layered membrane according to any of claims 1 to 7, wherein the pre-filter material (a) and the flow distribution material (c) are the same or different, wherein the materials optionally differ in pore size, pore density, material thickness, or hydrophilic coating.

9. The multi-layered membrane according to any of claims 1 to 8, wherein the pre-filter material (a) and the flow distribution material (c) are a PET non-woven porous material.

10. The multi-layered membrane according to any of claims 1 to 9, wherein the material of the track-etched membrane is selected from polyethylene terephthalate (PET), polycarbonate (PC), polyimide (PI), polyvinylidene fluoride (PVDF), polyether ether ketone (PEEK), ethylene tetrafluoro ethylene (ETFE) and other polymers having similar properties, preferably PET or PC.11 . The multi-layered membrane according to any of claims 1 to 10, wherein the pore size of the track-etched membrane is 0.1 to 5 pm, preferably 0.3 to 2.5 pm, more preferably 0.3 to 1 .75 pm, and wherein the pore density is 10,000 to 1 ,000,000,000 pores per cm2.

12. The multi-layered membrane according to any of claims 1 to 11 , wherein the thickness of the track-etched membrane is 1 to 100 pm, preferably 2 to 50 pm and more preferably 5 to 25 pm.222647PEP / HCath 2813. The multi-layered membrane according to any of claims 1 to 12, wherein the overall thickness of the multi-layered membrane is between 200 pm to 1000 pm, wherein the top layer (a) is preferably from 100 pm to 800 pm, more preferably from 200 pm to 600 pm the middle layer (b) is preferably from 1 pm to 100 pm, more preferably from 25 pm to 75 pm and the bottom layer (c) is preferably from 100 pm to 800 pm, more preferably from 200 pm to 600 pm.

14. The multi-layered membrane according to any of claims 1 to 13, wherein the pore size of porous pre-filter material is between 5 and 1000 pm, preferably between 10 and 500 pm and more preferably between 20 and 100 pm.

15. The multi-layered membrane according to any of claims 1 to 14, wherein all layers are joined, preferably by lamination.

16. The multi-layered membrane according to any of claims 1 to 15, wherein the hydrophilic coating comprises polyvinyl pyrrolidone (PVP), a sulfonated coating, or a coating containing alcoholic and carboxylic groups.

17. The multi-layered membrane according to any of claims 1 to 16, wherein the top layer (a) diffuses the whole blood sample and retains red and white blood cells, the middle layer (b) separates the sample by size, and the bottom layer (c) holds and / or distributes the obtained plasma, optionally transferring the plasma to a detection device, e.g., a microfluidic device or lateral flow detection device.

18. Process for the preparation of a multi-layered membrane according to any of claims 1 to17, wherein the process comprises: a) providing a middle layer (b) comprising a track-etched membrane including a hydrophilic coating, a top layer (a) comprising at least one layer of porous222647PEP / HCath 29 pre-filter material with pores having a pore size equal to or larger than the pore size of the track-etched membrane, and a bottom layer (c) comprising a porous flow distribution material; b) laminating the top layer (a) comprising at least one layer of a porous pre-filter material, the middle layer (b) comprising the track-etched membrane, and the bottom layer (c) comprising a porous flow distribution material together; c) optionally treating the obtained multi-layered membrane with a hydrophilic coating solution; and optionally drying the multi-layered membrane; or, alternatively, d) providing a track-etched membrane including a hydrophilic coating, a porous pre-filter material with pores having a pore size equal to or larger than the pore size of the track-etched membrane, and a porous flow distribution material; e) optionally treating the porous pre-filter material and / or the porous flow distribution material with a hydrophilic coating solution and drying the treated materials; f) stacking together at least one layer of porous pre-filter material from d) or e), a track-etched membrane from e), and a porous flow distribution material from d) or e); and g) laminating the stacked layers from f) together.

19. The process according to claim 18, wherein laminating of steps b) or g) comprises applying pressure of 1 to 5 bar, preferably 2 to 3 bar, and a temperature of 120 to 180°C, preferably 130 to 160°C, more preferably 135 to 155°C.

20. The process according to 18 or 19, wherein in steps c) or e), respectively, an aqueous solution of polyvinyl pyrrolidone or a sulfonated coating, a coating containing alcoholic and carboxylic groups is applied, preferably a polyvinyl pyrrolidone solution at a concentration of 0.1 to 50 g / L.222647PEP / HCath 3021. Use of a multi-layered membrane according to any of claims 1 to 17 or prepared according to any of claims 18 to 20, for separation of cells or other larger items from fluids, especially from body fluids, preferably for separation of blood into blood cells and serum or plasma, especially for diagnostic and point of care or home applications.

22. The use according to claim 21 , wherein the multi-layered membrane is included in an automated analysis system, especially a lateral flow analysis test system.

23. Device for vertical blood separation comprising a multi-layered membrane according to any of claims 1 to 18.

24. The device according to claim 23 comprising a means for even distribution.

25. The device according to claim 23 or 24 comprising collection substrate, preferably a porous adsorber or a non-porous plate, in particular a microfluidic chip.

26. The device according to any of claims 23 to 25, wherein the multi-layered membrane has a concave shape, preferably enabling a focused contact with the collection substrate.

27. The device according to any of claims 23 to 26, wherein the multi-layered membrane is bounded by seals, preferably ultrasonic, heat or adhesive seals.

28. In vitro method for obtaining pure blood plasma from a whole blood sample, comprising the use of a multi-layered membrane according to any of claims 1 to 18 or a device according to any of claims 23 to 27.

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