A filter system that can be used as a cell culture clarification filter in protein purification

Abstract

The present invention relates to a filter system that can be used as a chromatography for protein purification and / or a clarification filter / depth filter for cell culture clarification prior to ultrafiltration, and a method for separating cells and other contaminants from a fluid containing one or more target components by using the filter system of the present invention.

Description

Technical Field

[0001] The present invention relates to a filter system that can be used as a chromatography for protein purification and / or a clarification filter / depth filter for cell culture clarification prior to ultrafiltration, and a method for separating cells and other contaminants from a fluid containing one or more target components by using the filter system of the present invention.

Background Art

[0002] The production of recombinant proteins in cell-based and cell-free systems usually requires the more or less laborious purification of the protein of interest from a complex mixture. Related contaminants in this regard include host cell-derived contaminants such as whole cells, cell debris, host cell nucleic acids and host cell proteins, culture medium-derived contaminants such as proteinaceous and non-proteinaceous medium components, and vector-derived contaminants including vector nucleic acids and viral vectors.

[0003] The clarification of cell culture harvests and high-solid feedstocks can be a difficult task due to the high cell density that often requires primary clarification prior to large harvests from modern production batch bioreactors and subsequent chromatography operations. As the product molecule titers increase, higher cell mass and larger amounts of product pose challenges to downstream purification processes. High cell density leads to difficulties during clarification and sterile filtration. High product concentration generally increases the impurity load and requires larger chromatography equipment. Therefore, improvements in the form of increased efficiency and throughput are highly sought after.

[0004] Most of the cell components are removed from the liquid by filtration using a depth filter / clarification filter before chromatography and / or ultrafiltration. However, when the liquid is applied to the purification system, a surprisingly large amount of particularly small or soluble components remain in the liquid. Many of these compete with the desired protein, bind to the affinity substance in the column, or clog the ultrafilter.

[0005] Current clarification filters used for cell clarification in the market are cellulose-based depth filters, which consist of natural components such as diatomaceous earth, so pre-preventing and a large amount of flushing are required before filtration. Such depth filter materials release a large amount of TOC (total organic carbon) and particles. Furthermore, the consistency between batches is low for materials of natural origin. Such filters have a larger void volume due to the larger surface area required for a larger feed volume, which causes high product loss and high non-specific binding. Therefore, a large amount of post-flushing is required to be able to recover the target product from the depth filter. Since conventional depth filter materials are absorbent, not only many particles but also the product itself are retained. When a large amount of post-flushing is required, the final concentration of the product decreases due to dilution. Thereby, there may arise a problem that the product cannot be used for further analytical measurements, or an additional concentration step is required in the subsequent process.

[0006] In view of the above, the technical problem underlying the present invention is a filter system that can be used as a clarification filter / depth filter for cell culture clarification before chromatography and / or ultrafiltration, which exhibits high filterability (up to 40 million cells / mL), low TOC (total organic carbon) release, high throughput, high yield of the target component(s), and achieves a significant reduction in the turbidity of the fluid to be clarified. Summary of the Invention

[0007] The solution to the above technical problem is realized by the embodiments characterized in the claims.

[0008] In particular, the present invention a first synthetic nonwoven fabric layer having a first average pore size A; a second synthetic nonwoven fabric layer having a second average pore size B; a third synthetic nonwoven fabric layer having a third average pore size C; a fourth synthetic nonwoven fabric layer having a fourth average pore size D; a membrane layer having a fifth average pore size E; a filter system including them in this order, wherein the first average pore size A is 15.0 μm to 50.0 μm; the second average pore size B is 7.50 μm to 30.0 μm; the third average pore size C is 5.00 μm to 15.0 μm; the fourth average pore size D is 3.00 μm to 10.0 μm; the fifth average pore size E is 0.010 μm to 2.5 μm; and A > B > C > D > E, provides a filter system.

[0009] The filter system of the present invention is designed to be used before the capture step during protein purification, removes large particles, and protects the downstream membrane adsorber from fouling and clogging. By using synthetic nonwoven fabric layers in the filter system, it is possible to suppress the release of a large amount of TOC compared to conventional depth filters based on natural components such as diatomaceous earth. Therefore, the filter system of the present invention can be preferably used as a pre-filter system / clarification filter system, particularly a cell culture clarification filter system.

[0010] According to the present invention, the filter system includes four synthetic nonwoven fabric layers and a (final) membrane layer in this order from its upstream side to its downstream side.

[0011] According to the present invention, a "layer" is understood to mean a planar structure. "Planar" means that the structure has two opposing main surfaces that are mainly horizontal and substantially parallel to each other, and has a determined thickness between the opposing main surfaces. The "main surface" is the surface of the structure having the largest surface area.

[0012] The synthetic nonwoven fabric layers have different average pore diameters and are arranged such that the average pore diameter decreases from the upper (upstream) layer (i.e., the first synthetic nonwoven fabric layer) to the lower (downstream) layer (i.e., the fourth synthetic nonwoven fabric layer). That is, the first synthetic nonwoven fabric layer has a first average pore diameter A, the second synthetic nonwoven fabric layer has a second average pore diameter B, the third synthetic nonwoven fabric layer has a third average pore diameter C, and the fourth synthetic nonwoven fabric layer has a fourth average pore diameter D, and A > B > C > D. With this configuration, an excellent sieving effect and retention are achieved.

[0013] According to the present invention, the average pore diameter of the synthetic nonwoven fabric layer is determined in accordance with ASTM 1294-89 (1999) using a Topas PSM 165 (porometer).

[0014] According to the present invention, the average pore diameter of the membrane layer (s) (in some cases) is determined according to the following method. For an average pore size of at least 0.1 μm to a maximum of 10 μm, capillary flux porometry is used to determine the average pore size of the membrane layer(s). This is gas / liquid porosimetry, where the differences in gas pressure and flow rate passing through the membrane sample are measured first in the wet state and then in the dry state. Prior to measurement, the membrane sample is brought into contact with a wetting liquid so that all pores are filled with this liquid. After the pores are filled and the sample is introduced, the measurement cell must be closed and the measurement started. After the start of the measurement, the gas pressure automatically rises gradually, and the pore size corresponding to the applied pressure is emptied by the gas pressure. This is continued until the relevant pore range is covered, i.e., until there is no liquid left even in the smallest pores present within the measurement range. Next, the pressure is decreased again, and the measurement with the dry sample is automatically repeated. The pore size distribution is calculated from the difference between the two pressure-flow curves using the Young-Laplace equation (see also A. Shrestha, "Characterization of porous membranes via porometry", 2012, Mechanical Engineering Graduate Theses & Dissertations, Paper 38, University of Colorado at Boulder). For pore sizes less than 0.1 μm, a liquid-liquid displacement method similar to capillary flow porometry is used. However, instead of the gas flow rate, the flow rate of the displacement liquid is measured as a function of the differential pressure increase (see also R. Davila, "Characterization of ultra and nanofiltration commercial filters by liquid-liquid displacement porosimetry", 2013).

[0015] According to the present invention, the first average pore diameter A is 15.0 μm to 50.0 μm (preferably 17.0 μm to 30.0 μm, more preferably 20.0 μm to 30.0 μm), the second average pore diameter B is 7.50 μm to 30.0 μm (preferably 15.0 μm to 25.0 μm, more preferably 16.0 μm to 25.0 μm), the third average pore diameter C is 5.00 μm to 15.0 μm (preferably 7.50 μm to 12.5 μm or 7.00 μm to 9.00 μm), the fourth average pore diameter D is 3.00 μm to 10.0 μm (preferably 4.00 μm to 8.00 μm, more preferably 5.00 μm to 7.50 μm), and A > B > C > D. If any of the synthetic nonwoven fabric layers has an average pore diameter below its respective lower limit, the throughput of the filter system may be insufficient, and each synthetic nonwoven fabric layer having an average pore diameter below its respective lower limit will rapidly clog due to particle overload. If any of the synthetic nonwoven fabric layers has an average pore diameter above its respective upper limit, a significant reduction in the turbidity of the clarified fluid may not be achieved. Furthermore, a synthetic nonwoven fabric layer having an average pore diameter above its respective upper limit will allow fairly large particles to pass through, causing subsequent synthetic nonwoven fabric layers to clog prematurely. Therefore, the filtrate volume / m 2 , that is, the capacity of the filter system decreases.

[0016] The material used for each of the first, second, third, and fourth synthetic nonwoven fabric layers is not particularly limited. However, according to a preferred embodiment of the present invention, glass fibers are not used as the material for the synthetic nonwoven fabric layer due to potential high release amounts of TOC and fiber fragments, as well as the large amount of (post) flushing required.

[0017] Preferably, the material used for each of the first, second, third, and fourth synthetic nonwoven fabric layers is selected from the group consisting of polyolefins (e.g., polyethylene, polypropylene, polybutylene, etc.), fluoropolyolefins (e.g., PTFE, etc.), polyamides (e.g., nylon, etc.), and polyesters. According to a particularly preferred embodiment, the synthetic nonwoven fabric layers are each composed of (meltblown) polypropylene.

[0018] According to a preferred embodiment, the first, second, third and fourth synthetic nonwoven layers each have an average thickness of 20 μm to 20,000 μm, preferably 50 μm to 10,000 μm, 60 μm to 5,000 μm, 70 μm to 2,000 μm, 80 μm to 1,000 μm, 90 μm to 500 μm, and most preferably 100 μm to 300 μm. If any of the synthetic nonwoven layers have a thickness exceeding their respective upper limits, an internal chamber of the housing design of the filter system may be required, and as a result, the void volume of the filter system may increase. A low void volume is advantageous because it can reduce the capture of any generated volume within the filter system housing. If any of the synthetic nonwoven layers have a thickness below their respective lower limits, the filter system of the present invention may have a lower capacity for the particles removed from the feed solution. The average thickness of each of the first, second, third and fourth synthetic nonwoven layers can be measured in accordance with DIN EN ISO 12625-3 using a general-purpose thickness gauge S16502 (Frank-PTI).

[0019] Synthetic nonwoven layers having the above characteristics are commercially available.

[0020] The (final) membrane layer of the filter system of the present invention is not particularly limited as long as its average pore diameter E is from 0.010 μm to 2.5 μm as determined according to the above method. According to the present invention, the "membrane" for forming the membrane layer is a continuous solid matrix containing pores therebetween. In the context of the present invention, a fabric layer is also considered a membrane layer. A particulate bulk or a bundle of (unconnected) fibers, such as a nonwoven layer, is not regarded as a "membrane" in the present invention. The membrane layer has a first major surface and a second major surface opposite to the first major surface, and the first major surface is arranged (preferably directly) adjacent to the fourth synthetic nonwoven layer. The membrane layer may be a single membrane layer or a combination of two or more (separate) membrane layers, that is, the membrane layer may be composed of only one single membrane (layer), or may be composed of two or more different membranes (layers) each having different membrane properties (for example, pore diameter, material, thickness). When the membrane layer is a combination of two or more membranes (layers), each of the two or more membranes (layers) has an average pore diameter E of from 0.010 μm to 2.5 μm (or the preferred ranges described below).

[0021] Depending on the intended use of the filter system of the present invention, either a hydrophilic membrane or a hydrophobic membrane can be used. The membrane layer is considered a hydrophilic membrane when its water contact angle is 90° or less, whereas a hydrophobic membrane has a water contact angle of more than 90°. When the filter system of the present invention is used as a chromatography for protein purification and / or a clarification filter / depth filter for cell culture clarification before ultrafiltration, the membrane layer is preferably a hydrophilic membrane layer, that is, it has a water contact angle of 90° or less, preferably 80° or less, more preferably 70° or less. The water contact angle can be determined according to "Standard Test Method for Measurement of the Surface Tension of Solid Coatings, Substrates and Pigments using Contact Angle Measurements" defined in ASTM D7490-13.

[0022] According to a preferred embodiment of the present invention, the average pore size on the first main surface of the membrane layer (adjacent to the fourth synthetic nonwoven layer) is larger than the average pore size on the second main surface (opposite). Such a configuration of the membrane layer enables high throughput and achieves a significant reduction in the turbidity of the fluid to be clarified.

[0023] According to a particularly preferred membrane, the membrane layer is an asymmetric membrane layer. As used herein, an asymmetric membrane layer is a membrane layer in which the first main surface of the asymmetric membrane layer (adjacent to the fourth synthetic nonwoven layer) has an average pore size larger than the average pore size of the second main surface, and the average pore size decreases (continuously or discontinuously) from the first main surface to the second main surface of the asymmetric membrane layer. If the average pore size of the downstream section in the filtration direction is larger than the average pore size of the upstream section, since the medium has already flowed through the region with a smaller pore size, the region of the downstream section with a larger pore size remains substantially unused. The asymmetric membrane layer can advantageously avoid such so-called "waste" or "dead volume" that is only available for soil absorption (absorption of the components to be filtered) with limited efficiency.

[0024] As described above, the membrane layer may be a single membrane layer or a combination of two or more membrane layers. However, a single membrane layer is preferred due to its simpler manufacturing process and typically higher possible flow rate compared to multiple membrane layers.

[0025] The material used for the membrane layer is not particularly limited. Thus, the membrane layer can be composed of one or more polymers selected from the group consisting of cellulose and its derivatives (hydrated cellulose, cellulose esters, cellulose nitrate, cellulose acetate, etc.), regenerated cellulose, polyamides (nylon and perlon, etc.), fluoropolymers (polytetrafluoroethylene (PTFE) and polyvinylidene difluoride (PVDF), etc.), polyolefins (polyethylene and polypropylene, etc.), polysulfones (polysulfone, polyarylsulfone, and polyethersulfone, etc.), poly(meth)acrylic acid, and acrylic acid / methacrylic acid copolymers. According to a particularly preferred embodiment, the membrane layer is composed of regenerated cellulose or cellulose acetate due to its specific hydrophilicity.

[0026] The average pore size E of the membrane layer is 0.010 μm to 2.5 μm as determined according to the above method. When the membrane layer is a combination of two or more membrane layers, each of the two or more membrane layers has an average pore size E of 0.010 μm to 2.5 μm. With a specific average pore size E, the filter system of the present invention achieves both high throughput and a significant reduction in the turbidity of the clarified fluid. The average pore size E of the membrane layer is preferably 0.050 μm to 2.0 μm, more preferably 0.10 μm to 1.5 μm, still more preferably 0.15 μm to 1.0 μm, and most preferably 0.20 μm to 0.70 μm. According to a particularly preferred embodiment, the average pore size E of the membrane layer is 0.10 μm to 0.30 μm, even more preferably 0.15 μm to 0.25 μm, and most preferably 0.20 μm.

[0027] According to a preferred embodiment, the membrane layer has an average thickness of 20 μm to 400 μm, preferably 40 μm to 350 μm, 60 μm to 300 μm, 80 μm to 250 μm, 100 μm to 200 μm, 120 μm to 180 μm, and most preferably 140 μm to 170 μm. When the average thickness is below the lower limit, the filter system of the present invention may exhibit lower throughput and clogging earlier. When the average thickness exceeds the upper limit, the filter system of the present invention may only be able to reduce the flow rate. When the membrane layer is a combination of two or more membrane layers, the above average thickness range refers to the total average thickness of the two or more membrane layers.

[0028] Membrane layers having the above characteristics are commercially available.

[0029] According to a particularly preferred embodiment of the present invention, the pore diameters A to E are as follows. The first average pore diameter A is 17.0 μm to 30.0 μm, The second average pore diameter B is 16.0 μm to 25.0 μm, The third average pore diameter C is 7.00 μm to 9.00 μm, The fourth average pore diameter D is 4.00 μm to 8.00 μm, The fifth average pore diameter E is 0.10 μm to 0.30 μm (most preferably 0.20 μm), A > B > C > D > E.

[0030] Each of the first, second, third, and fourth synthetic non-woven fabric layers, as well as the shape (spatial form) of the membrane layer, is not particularly limited. Preferably, each of the filter layers is a flat filter layer. The term "flat" indicates that each filter material is substantially located within a single plane having a specific thickness. Preferably, all filter materials are located more or less within a plane that is substantially parallel to each other.

[0031] The type of connection between each of the first, second, third, and fourth synthetic nonwoven fabric layers and the membrane layer is not particularly limited. For example, the layers may be loosely connected within the housing, or the layers may be sealed by a clamp (at its edge), heat-sealing overmolding (plastic silicone), or the like. According to a preferred embodiment, the layers are loosely connected without lamination therebetween (within the housing, i.e., the connection between the layers is driven by the connection to the housing).

[0032] The filter system of the present invention can be composed of the first, second, third, and fourth synthetic nonwoven fabric layers and the membrane layer. Alternatively, the filter system of the present invention can include additional layers such as a support layer, etc., without impairing the effects of the present invention.

[0033] The present invention further provides a method for separating cells and other contaminants from a fluid containing one or more target components, providing the filter system according to the present invention, wherein the first synthetic nonwoven fabric layer is defined as the upstream side of the filter system and the membrane layer is defined as the downstream side of the filter system; including passing the fluid through the filter system from the upstream side to the downstream side of the filter system, retaining cells and other contaminants within the filter system, and eluting a fluid containing one or more target components from the filter system.

[0034] The target component is not particularly limited. For example, the target component can be any target biomolecule such as a protein, antibody, hormone, vaccine, nucleic acid, exosome, and virus, as well as virus-like particles. In a preferred embodiment, the target component is an antibody, more preferably a monoclonal antibody (mAb), or a fragment or derivative thereof, or a nanobody. Examples of monoclonal antibodies are adalimumab, cetuximab, rituximab, infliximab, omalizumab, and denosumab. The target component can be obtained, for example, from mammalian cells, bacterial cells or insect cells, media and cell lines such as "Chinese hamster ovary cells" (CHO cells), HeLa, or human umbilical vein endothelial cells (HUVEC).

[0035] The source of the fluid is not particularly limited. For example, the fluid can be obtained by applying any biological, biochemical, chemical, or pharmaceutical method. Thereby, the fluid can be obtained by previously performing other purification methods applying different purification units. For example, the target component can be produced by an appropriate cell line such as a CHO cell line, for example by perfusion culture.

[0036] The fluid contains a plurality of components and is not particularly limited as long as at least one of the plurality of components of the fluid is the target component. Further (impurity) components (inclusions) are not particularly limited and can depend on the preparation conditions of the target component. Examples of further components are aggregates, host cell proteins, deoxyribonucleic acids, and their fragments and charge variants.

[0037] The fluid medium is not particularly limited. In principle, any fluid is suitable, and water and salt aqueous solutions are preferred. For example, a buffer aqueous solution can be used as the fluid medium. Preferably, the fluid medium is selected from the group consisting of KPI buffer, sodium phosphate buffer, sodium acetate buffer, PBS, glycine, citrate buffer, Tris buffer, BIS-Tris buffer, HEPES buffer, and water. The concentration of the buffer in the aqueous solution is not particularly limited, and can be, for example, 1.0 mM to 5.0 M, preferably 5.0 mM to 1.0 M, more preferably 10 mM to 200 mM, and most preferably 25 mM to 100 mM. The fluid medium and its conditions can be appropriately selected according to the components of the fluid to be separated (inclusions and target components).

[0038] In the first step of the method of the present invention, a filter system according to the present invention as described above is provided. The first synthetic nonwoven layer is defined as the upstream side of the filter system, and the membrane layer is defined as the downstream side of the filter system.

[0039] In the second step, the fluid to be clarified is passed through the filter system from the upstream side to the downstream side of the filter system, while retaining cells and other inclusions in the filter system, and eluting a fluid containing one or more target components from the filter system.

[0040] In any third step, the fluid containing one or more target components eluted from the filter system is passed in-line through a further purification device such as a membrane adsorber (e.g., cellulose-based or agarose-based), monolith, resin material, etc., to further purify the one or more target components. Particularly preferred is anion exchanger, cation exchanger, hydrophobic interaction or mixed mode chromatography. Due to the excellent filtration performance of the filter system of the present invention, advantageously, the filter system can be used in-line as a depth filter / clarification filter before the chromatography step in the downstream purification process after cell culture.

[0041] The filter system of the present invention advantageously exhibits excellent performance in removing unwanted particles in cell culture, such as cells, lipids, large particles, etc., from a cell culture sample containing a target component, such as an antibody or virus particles, etc., and at the same time a high product yield can be obtained. The filter system has an improved filterability (up to 40 million cells / mL), releases less TOC, and can be used in a dry state, i.e., without the need for venting and pre-flushing, when compared with conventional depth filters known in the art. The filter system does not require a large amount of pre-flushing and post-filtering flushing, and has a very low void volume. Therefore, advantageously, the concentration of the target component in the filtrate is high, and further analytical measurements or processes can be carried out without an additional concentration step. The turbidity of the fluid to be clarified can be advantageously highly reduced (more than 90% or less than 50 NTU to less than 100 NTU) by the filter system, and the filter system of the present invention can be used as a depth filter / clarification filter before the chromatography step in the downstream purification process after cell culture. Due to the excellent performance (1 mL / cm 2 superior filtration capacity) of the filter system of the present invention in throughput, advantageously, the elution fluid can be directly (in-line) loaded from the filter system onto the chromatography material, and a bypass of the chromatography material is not required during pre-flushing.

Brief Description of the Drawings

[0042]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0043] The present invention will be further described by the following examples, but is not limited thereto.

Examples

[0044] Test apparatuses for the following Examples 1 to 6: In all cell culture filtration applications, forward flow filtration was applied at a constant flow rate of 10 mL / min up to an inlet pressure of 1.0 bar using a peristaltic pump and a pressure sensor. In Examples 4 and 5, filtration was carried out up to an inlet pressure of 2 bar. The experiments were carried out using the test apparatus shown in Figure 1. The product was filtered using a peristaltic pump up to an inlet pressure of 1 bar (Examples 1, 2, 3, and 6). The filtrate was collected on the output side of the filter system. Turbidity was measured using a turbidimeter to define the particle reduction in the filtrate. Further, the filtrate weight was measured using a laboratory balance to determine the product weight and the filter capacity.

[0045] The following nonwoven fabric layer and membrane layer were used.

[0046]

Table 1

[0047]

Table 2

[0048] Example 1: Performance tests were carried out using actual solutions commercially available from several vendors.

[0049] The following filter systems were used. DF-A: NW1 + NW2 + NW3 + Mem1 DF-B: NW1 + NW2 + NW5 + Mem1 DF-C: NW1 + NW2 + NW3 + NW4 + Mem1 DF-D: NW1 + NW2 + NW3 + NW6 + Mem1 DF-E: NW1 + NW2 + NW3 + NW7 + Mem1 FC-PES: A commercially available filter system based on polyethersulfone (Whatman GD / X syringe filter - prefilter, sterilized | Cytiva)

[0050] DF-C is the filter system according to the present invention. DF-A and DF-B are comparative filter systems because there is no fourth synthetic nonwoven layer. DF-D and DF-E are comparative filter systems because the average pore size of the fourth synthetic nonwoven layer is too small. FC-PES is a commercially available comparative filter system.

[0051] Sample 1 cell culture information: Test A: CHO K1A cell line, viability 72%, VCC 12 Mio / mL, turbidity 490 NTU Test B: CHO K1A cell line, viability 50%, VCC 9 Mio / mL, turbidity 370 NTU

[0052] Sample 2 cell culture information: Test C: ExpiCHO transient cell line, viability 85%, VCC 16 Mio / mL Test D: HEK293 transient cell line, viability 61%, VCC 9 Mio / mL Test E: CHO stable cell line, viability 94%, VCC 18 Mio / mL

[0053] Figure 2 shows the filtration results of Sample 1 and Sample 2 using different cell lines. As is clear from Figure 2, the filter system of the present invention (DF-C) shows the best performance in different cell cultures when considering both product yield and turbidity reduction.

[0054] Example 2: Three filtration systems according to the present invention (consisting of NW1 + NW2 + NW3 + NW4 combined with different membrane layers) and a comparative filtration system consisting of only NW1 + NW2 + NW3 + NW4 were evaluated in terms of their performance regarding turbidity reduction and filter capacity.

[0055] The results of filtering a model solution of 10×Caro / Ovo + 20 g / L dry yeast mixture are shown in the following table. The turbidity of the feed solution was about 600 NTU.

[0056] [Table 3]

[0057] The filter system of the present invention shows significant turbidity reduction and similar filter capacity when compared with a filter system without the final membrane layer.

[0058] Example 3: Five filtration systems according to the present invention (consisting of NW1 + NW2 + NW3 + NW4 combined with different membrane layers) were evaluated in terms of their performance regarding turbidity reduction and filter capacity.

[0059] The following table and Figure 3 show the filtration systems tested and the filtrate volume and turbidity achieved. The test medium was a CHO cell line with an average of 12 Mio / mL.

[0060] [Table 4]

[0061] Example 4: The final membrane layer (Mem1, an asymmetric membrane layer having an average pore size on the first major surface that is larger than the average pore size of the second major surface) was tested with respect to its orientation (A: the first major surface adjacent to the fourth synthetic nonwoven layer, B: the second major surface adjacent to the fourth synthetic nonwoven layer). The apparatus was NW1 + NW2 + NW3 + NW4 + Mem1. The test medium was a 10×Caro / Ovo + 20 g / L dry yeast mixture, a model solution with a turbidity of approximately 3000 NTU. The results are shown in Figure 4.

[0062] As is clear from Figure 4, orientation A (i.e., the first major surface having an average pore size larger than the average pore size of the second major surface of the final membrane layer is adjacent to the fourth synthetic nonwoven layer) functioned best with respect to the filtrate volume.

[0063] Example 5: The expandability of the final membrane layer combination was tested. The apparatus was NW1 + NW2 + NW3 + NW4 + Mem1. Figure 5 shows the normalized filtrate volume and turbidity for different filter areas. The medium was a 10×Caro / Ovo + 20 g / L dry yeast, model solution with a turbidity of 4500 NTU. The results are shown in Figure 5.

[0064] As is clear from Figure 5, the 20 cm 2 module also functioned well, enabling a filtration volume of more than 50 mL per module. Thus, expandability of the filter system of the present invention is possible.

[0065] Example 6: The amount of flushing / rinsing volume required to recover the target component from the filter system of the present invention was tested. The apparatus was NW1 + NW2 + NW3 + NW4 + Mem1.

[0066] In particular, Figure 6 shows the IgG yield / recovery rate after flushing. The cell culture was a CHO cell line, with a viability of 82%, an IgG titer of 2.3 g / L, and a WCW of 53 g / L. The flushing buffer was PBS (phosphate buffered saline) at pH 7.

[0067] A 2 mL flushing / rinsing volume was sufficient to achieve an 80% yield / recovery rate. Therefore, only a small flushing / rinsing volume is required to recover the target component from the filter system of the present invention.

[0068] Example 7: The filtration system according to the present invention (filter system 3, apparatus: NW1 + NW2 + NW3 + NW4 + Mem1) and three comparative filtration systems (filter system 1: NW1 + NW2 + NW3 + Mem1, filter system 2: NW5 + NW1 + NW3 + glass fiber nonwoven layer + Mem1, filter system 4: FC - PES) were evaluated in terms of their TOC release.

[0069] The TOC of the clarification filter was determined by flushing different fractions of purified water. Each fraction after flushing was collected, and the TOC was determined using a TOC LCPH system according to SOP: PD / SOP / TOC / 111. The procedure is as follows. Flush the test apparatus with Arium water for 2 to 3 hours Collect the Arium water from the apparatus (used as a blank) Connect the prototype (with the inlet facing upward) Maintain a constant flow rate of 10 mL / min, collect each 5 - mL fraction up to 50 mL (0 mL - 5 mL, 5 mL - 10 mL... 45 mL - 50 mL), dilute each sample to 50 mL to obtain a 1:3 dilution, and measure the TOC. From 50 mL to 100 mL, collect each 10 - mL fraction (50 mL - 60 mL, 60 mL - 70 mL... 90 mL - 100 mL), make the sample from 50 mL to 100 mL into a 1:2 dilution, and measure the TOC.

[0070] The results are shown in Figure 7.

[0071] As is clear from Figure 7, the filter system containing a glass fiber nonwoven layer releases the most TOC.

[0072] Example 8: Various synthetic nonwoven fabric layers and Mem1 were individually evaluated regarding their TOC release. That is, a 50-mm filter layer was tested for TOC by shaking the layer in 100 mL of purified water at 100 rpm for 2 hours.

[0073] The results are shown in the following table.

[0074]

Table 5

[0075] As is apparent from the above table, the glass fiber nonwoven fabric layer had no water resistance, whereas NW1 to NW4 and Mem1 released only a small amount of TOC.

Claims

1. A first synthetic nonwoven fabric layer having a first average pore diameter A, A second synthetic nonwoven fabric layer having a second average pore diameter B, A third synthetic nonwoven fabric layer having a third average pore diameter C, A fourth synthetic nonwoven fabric layer having a fourth average pore diameter D, A membrane layer having a fifth average pore diameter E, A filter system comprising them in this order, The first average pore diameter A is 15.0 μm to 50.0 μm, The second average pore diameter B is 7.50 μm to 30.0 μm, The third average pore diameter C is 5.00 μm to 15.0 μm, The fourth average pore diameter D is 3.00 μm to 10.0 μm, The fifth average pore diameter E is 0.010 μm to 2.5 μm, A filter system wherein A > B > C > D > E.

2. The filter system according to claim 1, wherein the first, second, third, and fourth synthetic nonwoven fabric layers are each composed of meltblown polypropylene.

3. The filter system according to claim 1 or 2, wherein the first, second, third, and fourth synthetic nonwoven fabric layers each have an average thickness of 20 μm to 20000 μm.

4. The filter system according to any one of claims 1 to 3, wherein the membrane layer is a hydrophilic membrane layer having a water contact angle of 90° or less.

5. The filter system according to any one of claims 1 to 4, wherein the membrane layer has a first main surface adjacent to the fourth synthetic nonwoven fabric layer and a second main surface opposite thereto, and the average pore diameter on the first main surface is larger than the average pore diameter on the second main surface.

6. The filter system according to any one of claims 1 to 5, wherein the membrane layer is an asymmetric membrane layer.

7. The filter system according to any one of claims 1 to 6, wherein the membrane layer is a single membrane layer.

8. The filter system according to any one of claims 1 to 7, wherein the membrane layer is composed of one or more polymers selected from the group consisting of cellulose and its derivatives, regenerated cellulose, polyamide, fluoropolymer, polyolefin, polysulfone, poly(meth)acrylic acid, and acrylic acid / methacrylic acid copolymer.

9. The filter system according to any one of claims 1 to 8, wherein the membrane layer is composed of regenerated cellulose or cellulose acetate.

10. The filter system according to any one of claims 1 to 9, wherein the fifth average pore diameter E is 0.20 μm to 0.70 μm.

11. The filter system according to any one of claims 1 to 10, wherein the film layer has a thickness of 20 μm to 400 μm.

12. A method for separating cells and other contaminants from a fluid containing one or more target components, providing the filter system according to any one of claims 1 to 11, wherein the first synthetic nonwoven layer is defined as the upstream side of the filter system and the film layer is defined as the downstream side of the filter system; passing the fluid through the filter system from the upstream side to the downstream side of the filter system, retaining the cells and other contaminants in the filter system while eluting the fluid containing the one or more target components from the filter system; comprising the steps of.

13. The method according to claim 12, further comprising passing the fluid containing the one or more target components eluted from the filter system in-line through a purification device to further purify the one or more target components.

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