Filter system useful as cell culture clarification filter in protein purification

Abstract

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

Description

Technical Field

[0001] The present application relates to a filter system which can be used as a clarification filter / depth filter for clarifying cell culture prior to chromatography and / or ultrafiltration for protein purification. The present application also relates to a method for separating cells and other contaminants from a fluid comprising one or more target components by using the filter system of the present application. Background Art

[0002] In both cell-based and cell-free systems, the production of recombinant proteins often requires more or less laborious purification of the protein of interest from a complex mixture. In this regard, relevant contaminants 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 protein and non-protein medium components; and vector-derived contaminants, including vector nucleic acids and viral vectors.

[0003] Clarification of cell culture harvests and high solids feedstocks can be a troubling task due to the large harvests from modern production batch bioreactors and the high cell density that often requires primary clarification prior to subsequent chromatography operations. With the increase in product molecule titer, higher cell mass and larger amounts of product challenge downstream purification steps. Higher cell density leads to difficulties during clarification and sterile filtration. Higher product concentrations typically lead to increased impurity loads and the need for larger chromatography facilities. Like this, improvements in the form of gains in efficiency and throughput are greatly sought.

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

[0005] The clarification filter currently used for cell clarification on the market is a cellulose-based depth filter, which requires pre-ventilation and a large amount of rinsing before filtering because they are composed of natural components such as diatomaceous earth. This depth filter material releases a large amount of TOC (total organic carbon) and particles. In addition, for natural-based materials, the consistency between batches is low. Since a larger feed volume requires a larger surface area, this filter has a larger void volume, which leads to high product loss and high non-specific binding. Thus, a large amount of post-rinsing is required to be able to recover the target product from the depth filter. Since conventional depth filter materials are absorbent, a large amount of particles and the product itself are retained. When a large amount of post-rinsing is required, the final concentration of the product is reduced due to dilution. This may cause problems, and the product cannot be used for further analytical measurements, or the process will require additional concentration steps subsequently. Summary of the invention

[0006] In view of the above problems, the technical problem of the present application is to provide a filter system that can be used as a clarification filter / depth filter for cell culture clarification prior to chromatography and / or ultrafiltration, which filter system exhibits high filter capacity (up to 40 million cells / mL), low TOC (total organic carbon) release, high throughput, high yield of target components and achieves a high reduction in turbidity of the fluid to be purified.

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

[0008] In particular, the present application provides a filter system, which comprises, in order:

[0009] a first synthetic nonwoven layer having a first average pore size A;

[0010] a second synthetic nonwoven layer having a second average pore size B;

[0011] a third synthetic nonwoven layer having a third average pore size C;

[0012] a fourth synthetic nonwoven layer having a fourth average pore size D; and

[0013] A membrane layer having a fifth average pore size E,

[0014] in,

[0015] The first average pore diameter A is 15.0 μm to 50.0 μm,

[0016] The second average pore diameter B is 7.50 μm to 30.0 μm,

[0017] The third average pore diameter C is 5.00 μm to 15.0 μm,

[0018] The fourth average pore diameter D is 3.00 μm to 10.0 μm,

[0019] The fifth average pore diameter E is 0.010 μm to 2.5 μm, and

[0020] A>B>C>D>E.

[0021] The filter system of the present application is designed to be used before the capture step during protein purification, and to remove large particles and protect the downstream membrane adsorber from scaling and clogging. Since a synthetic nonwoven layer is used in the filter system, the release of a large amount of TOC can be suppressed compared to conventional depth filters based on natural components such as diatomaceous earth. Thus, the filter system of the present application can be preferably used as a pre-filter system / clarification filter system, in particular a cell culture clarification filter system.

[0022] According to the present application, the filter system comprises, in order from its upstream side to its downstream side, four synthetic nonwoven layers and a (finally) membrane layer.

[0023] According to the present application, "layer" is understood to mean a planar structure. "Planar" means that the structure has two opposing major surfaces, which are mainly horizontal and substantially parallel to each other and have a defined thickness between the opposing major surfaces. A "major surface" is the surface of the structure having the largest surface area.

[0024] The synthetic nonwoven layers differ in average pore size and have been aligned so that the average pore size decreases from the top (upstream) layer (i.e., the first synthetic nonwoven layer) to the bottom (downstream) layer (i.e., the fourth synthetic nonwoven layer). That is, the first synthetic nonwoven layer has a first average pore size A, the second synthetic nonwoven layer has a second average pore size B, the third synthetic nonwoven layer has a third average pore size C, and the fourth synthetic nonwoven layer has a fourth average pore size D, wherein A>B>C>D. This configuration achieves excellent screening effects and retention.

[0025] According to the present application, the average pore size of the synthetic nonwoven layer is determined according to ASTM 1294-89 (1999) using a Topas PSM 165 (porometer).

[0026] According to the present application, the average pore size of one or more membrane layers is determined according to the following method:

[0027] For average pore diameters of at least 0.1 μm and at most 10 μm, the average pore diameter of the one or more membrane layers is determined using capillary flux porosimetry. This is a gas / liquid porosimetry method in which the gas pressure difference and the flow rate through the membrane sample are measured first in the moist state and then in the dry state. Before the measurement, the membrane sample is brought into contact with the moist liquid so that all pores are filled with this liquid. After the pores have been filled and the sample has been introduced, the measuring cell must be closed and the measurement started. After the start of the measurement, the gas pressure is automatically gradually increased and the pores corresponding to the applied pressure are emptied by the gas pressure. This continues until the relevant pore range has been covered, i.e. until even the smallest pores present in the measuring range are free of liquid. The pressure is then reduced again and the measurement is automatically repeated on the now dry sample. The pore size distribution is calculated from the difference between the two pressure-flow rate curves using the Young-Laplace equation (see also A.

[0028] Shrestha, “Characterization of porous membranes via porometry”,

[0029] 2012, Mechanical Engineering Graduate Theses&Dissertation, Paper 38, University of Colorado at Boulder).

[0030] For pore sizes smaller than 0.1 μm, a liquid-liquid displacement method is used that is similar to capillary flow porosity. However, instead of the gas flow rate, the flow rate of the displacing liquid is measured as a function of the pressure difference increase (see also R. Dávila,

[0031] "Characterization of ultra and nanofiltration commercial filters by liquid-liquid displacement porosimetry", 2013).

[0032] According to the present application, the first average pore size A is 15.0μm to 50.0μm (preferably 17.0μm to 30.0μm, more preferably 20.0μm to 30.0μm), the second average pore size B is 7.50μm to 30.0μm (preferably 15.0μm to 25.0μm, more preferably 16.0μm to 25.0μm), the third average pore size C is 5.00μm to 15.0μm (preferably 7.50μm to 12.5μm or 7.00μm to 9.00μm), and the fourth average pore size D is 3.00μm to 10.0μm (preferably 4.00μm to 8.00μm, more preferably 5.00μm to 7.50μm), and A>B>C>D. In the case where any layer in the synthetic nonwoven layer has an average pore size lower than the corresponding lower limit, the throughput of the filter system may be insufficient, and the corresponding synthetic nonwoven layer with an average pore size lower than the corresponding lower limit is quickly closed due to particle overload. In the case where any layer of the synthetic nonwoven layer has an average pore size higher than the corresponding upper limit, a high reduction in the turbidity of the fluid to be purified can not be achieved. In addition, the synthetic nonwoven layer with an average pore size higher than its corresponding upper limit allows quite large particles to pass, so that the subsequent synthetic nonwoven layer is closed early. Thus, the filtrate volume / m 2 , that is, the capacity of the filter system is reduced.

[0033] The material for each of the first synthetic nonwoven layer, the second synthetic nonwoven layer, the third synthetic nonwoven layer and the fourth synthetic nonwoven layer is not particularly limited. However, according to a preferred embodiment of the present application, glass fiber is not used as the material for the synthetic nonwoven layer due to the potential high release of TOC and fiber fragments and the need for a large amount of (post) washing.

[0034] Preferably, the material for each of the first synthetic nonwoven layer, the second synthetic nonwoven layer, the third synthetic nonwoven layer and the fourth synthetic nonwoven layer is selected from the group consisting of polyolefins (e.g. polyethylene, polypropylene, polybutylene, etc.), fluoropolyolefins (e.g. PTFE), polyamides (e.g. nylon) and polyesters. According to a particularly preferred embodiment, the synthetic nonwoven layers are each composed of (melt-blown) polypropylene.

[0035] According to a preferred embodiment, the first synthetic nonwoven layer, the second synthetic nonwoven layer, the third synthetic nonwoven layer and the fourth synthetic nonwoven layer each have an average thickness of 20 μm-20,000 μm, preferably each have an average thickness of 50 μm-10,000 μm, 60 μm-5,000 μm, 70 μm-2,000 μm, 80 μm-1,000 μm, 90 μm-500 μm and most preferably 100 μm-300 μm. In the case where any layer in the synthetic nonwoven layer has a thickness higher than the corresponding upper limit, the internal space of the housing design for the filter system may be required, which may result in a larger void volume of the filter system. Low void volume is advantageous because the capture of any product volume in the filter system housing can be reduced. In the case where any layer in the synthetic nonwoven layer has a thickness lower than the corresponding lower limit, the filter system of the present application can have a smaller ability to remove particles from the feed solution. The average thickness of each of the first synthetic nonwoven layer, the second synthetic nonwoven layer, the third synthetic nonwoven layer, and the fourth synthetic nonwoven layer may be measured according to DIN EN ISO 12625-3 using a universal thickness gauge S16502 (Frank-PTI).

[0036] Synthetic nonwoven layers having the above-described characteristics are commercially available.

[0037] The (final) membrane layer of the filter system of the present application is not particularly limited, as long as its average pore size E is 0.010 μm to 2.5 μm, as determined according to the method described above. According to the present application, the "membrane" for forming the membrane layer is a continuous solid matrix including holes therebetween. In the context of the present application, the woven layer is also considered to be a membrane layer. In the present application, particle bodies or (unconnected) fiber bundles, such as nonwoven layers, are not considered to be "membranes". 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 (preferably directly) adjacent to the fourth synthetic nonwoven layer arrangement. The membrane layer can be a single membrane layer or can be a combination of two or more (separated) membrane layers, that is, the membrane layer can be composed of only a single membrane (layer) or two or more different membranes (layers), and each membrane (layer) can have different membrane properties (such as pore size, material, thickness). In the case where 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 0.010 μm to 2.5 μm (or the preferred range mentioned below).

[0038] According to the expected application of the filter system of the present application, a hydrophilic membrane or a hydrophobic membrane can be used. When its water contact angle is 90 ° or less, the rete is considered to be a hydrophilic membrane, and the hydrophobic membrane has a water contact angle greater than 90 °. The filter system of the present application is used as a clarification filter / depth filter for clarifying cell culture before chromatography and / or ultrafiltration, and in the case of protein purification, the rete is preferably a hydrophilic rete, i.e., 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.

[0039] According to a preferred embodiment of the present application, the average pore size on the first major surface of the membrane layer (which is adjacent to the fourth synthetic nonwoven layer) is greater than the average pore size at the (opposite) second major surface. This construction of the membrane layer allows high throughput and achieves a high reduction in the turbidity of the fluid to be purified.

[0040] According to particularly preferred membrane, the membrane layer is an asymmetric membrane layer. As used herein, an asymmetric membrane layer is such a membrane layer, wherein the first major surface (it is adjacent to the fourth synthetic nonwoven layer) of the asymmetric membrane layer has an average pore size greater than the average pore size of the second major surface, and the average pore size (continuously or discontinuously) is reduced from the first major surface of the asymmetric membrane layer toward the second major surface. If the average pore size of the section downstream in the filtering direction is greater than the average pore size of the upstream section, the region of the downstream section with a larger aperture is basically kept unused, because the medium has flowed through the region with a smaller aperture. Asymmetric membrane layers can advantageously avoid this so-called "waste" or "dead volume", which can only be used for soil absorption (absorption of components to be filtered) with limited efficiency.

[0041] As mentioned above, the membrane layer can 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 generally higher possible flow rates compared to multiple membrane layers.

[0042] The material 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 (such as cellulose hydrate, cellulose ester, cellulose nitrate, cellulose acetate), regenerated cellulose, polyamide (such as nylon and polyamide fibers), fluoropolymers (such as polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF), polyolefins (such as polyethylene and polypropylene), polysulfones (such as polysulfone, polyarylsulfone and polyethersulfone), poly (meth) acrylic acid and acrylic acid / methacrylic acid copolymers. According to a particularly preferred embodiment, due to the specified hydrophilicity of regenerated cellulose or cellulose acetate, the membrane layer is composed of regenerated cellulose or cellulose acetate.

[0043] As determined according to the method described above, the average pore size E of the membrane layer is 0.010 μm to 2.5 μm. In the case where 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. Due to the specified average pore size E, the filter system of the present application achieves both high throughput and high reduction in turbidity of the fluid to be purified. 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, 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.

[0044] According to a preferred embodiment, the average thickness of the membrane layer is 20 μm-400 μm, preferably 40 μm-350 μm, 60 μm-300 μm, 80 μm-250 μm, 100 μm-200 μm, 120 μm-180 μm, and most preferably 140 μm-170 μm. If the average thickness is below the lower limit, the filter system of the present application may exhibit lower production volume and early closure. If the average thickness is above the upper limit, the filter system of the present application may only allow a reduced flow rate. If 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.

[0045] Film layers having the above-described characteristics are commercially available.

[0046] According to a particularly preferred embodiment of the present application, the apertures A to E are as follows:

[0047] The first average pore diameter A is 17.0 μm to 30.0 μm,

[0048] The second average pore diameter B is 16.0 μm to 25.0 μm,

[0049] The third average pore diameter C is 7.00 μm to 9.00 μm,

[0050] The fourth average pore diameter D is 4.00 μm to 8.00 μm,

[0051] The fifth average pore diameter E is 0.10 μm to 0.30 μm (most preferably 0.20 μm), and

[0052] A>B>C>D>E.

[0053] The shape (spatial form) of each in the first synthetic nonwoven layer, the second synthetic nonwoven layer, the third synthetic nonwoven layer and the fourth synthetic nonwoven layer and the shape (spatial form) of the membrane layer are not subject to any specific restrictions.Preferably, each in the filter layer is a flat filter layer.Term " flat " indicates that the corresponding filter material is basically located in a single plane with a specified thickness.Preferably, all filter materials are more or less located in a plane substantially parallel to each other.

[0054] The connection type between each of the first synthetic nonwoven layer, the second synthetic nonwoven layer, the third synthetic nonwoven layer and the fourth synthetic nonwoven layer and the film layer is not particularly limited. For example, these layers can be loosely connected in the housing, or these layers can be sealed by clamping (at its edge), heat-sealed overmolding (plastic silicone resin), etc. According to a preferred embodiment, these layers are loosely connected (in the housing, that is, the connection between the layers id is driven to the housing by the connection), without lamination between them.

[0055] The filter system of the present application may be composed of a first synthetic nonwoven layer, a second synthetic nonwoven layer, a third synthetic nonwoven layer, a fourth synthetic nonwoven layer and a membrane layer. Alternatively, the filter system of the present application may include additional layers, such as a support layer that does not impair the effect of the present application.

[0056] The present application also provides a method for separating cells and other contaminants from a fluid comprising one or more target components, the method comprising:

[0057] A filter system of the present application is provided, wherein the first synthetic nonwoven layer is defined as an upstream side of the filter system, and the membrane layer is defined as a downstream side of the filter system, and

[0058] Fluid is passed through the filter system from an upstream side of the filter system to a downstream side of the filter system to retain cells and other contaminants in the filter system while eluting fluid including one or more target components from the filter system.

[0059] The target component is not particularly limited. For example, the target component can be any biomolecule of interest and virus-like particles, wherein the biomolecule is such as protein, antibody, hormone, vaccine, nucleic acid, exosome and virus. 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. The example of a monoclonal antibody is adalimumab, cetuximab, rituximab, infliximab, omalizumab and denosumab. The target component can be obtained from, for example, mammalian cells, bacterial cells or insect cells, culture medium and cell lines such as "Chinese hamster ovary cells" (CHO cells), HeLa cells (HeLa) or human umbilical vein endothelial cells (HUVEC).

[0060] 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. Thus, the fluid can be obtained by other purification methods using different purification units previously performed. For example, the target component can be produced by an appropriate cell line, such as produced by a CHO cell line, for example, produced by perfusion culture.

[0061] The fluid is not particularly limited as long as it includes multiple components, wherein at least one of the multiple components of the fluid is a target component. Other (impurity) components (contaminants) are not particularly limited and may depend on the preparation conditions of the target component. Examples of other components are aggregates, host cell proteins, deoxyribonucleic acids and fragments thereof, and charge variants.

[0062] The medium of fluid is not particularly limited. In principle, any fluid is suitable, wherein preferably water and aqueous saline solution. For example, aqueous buffer solution can be used as the medium of fluid. Preferably, the medium of fluid 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 buffer in aqueous solution is not particularly limited, for example, can be 1.0mM to 5.0M, preferably 5.0mM to 1.0M, more preferably 10mM to 200mM, most preferably 25mM to 100mM. The medium of fluid and its condition can be suitably selected according to the component (pollutant and target component) of fluid to be separated.

[0063] In the first step of the method of the present application, a filter system according to the present application 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.

[0064] In the second step, the fluid to be purified passes through the filter system from the upstream side to the downstream side of the filter system to retain cells and other contaminants in the filter system while eluting the fluid including the one or more target components from the filter system.

[0065] In an optional third step, the fluid comprising one or more target components eluted from the filter system is passed through another purification device online, such as a membrane adsorber (e.g., cellulose-based or agarose-based), a monolith, a resin material, etc., to further purify the one or more target components. Particularly preferred are anion exchangers, cation exchangers, hydrophobic interaction or mixed mode chromatography. Since the filter system of the present application has excellent filtration performance, it is advantageous that the filter system can be used online as a depth filter / clarification filter before the chromatography step in the downstream purification process after cell culture.

[0066] The filter system of the present application advantageously shows excellent performance in removing unwanted particles in cell culture from a cell culture sample containing a target component, and at the same time obtains a high product yield, wherein the target component is such as an antibody or a viral particle, and the unwanted particles are, for example, cells, lipids and large particles. Compared with conventional depth filters known in the art, the filter system has an improved filter capacity (up to 4 million cells / mL), releases less TOC, and can be used dry, i.e., does not require exhaust and pre-rinsing. Since the filter system does not require pre-ventilation and a large amount of flushing before and after filtration, and has a very low void volume, the target component in the filtrate has an advantageous high concentration, so that further analytical measurements or processing can be performed without an additional concentration step. The turbidity of the fluid to be purified can be advantageously reduced to a high degree (>90% or <50 to 100 NTU) by the filter system, and the filter system of the present application can be used as a depth filter / clarification filter before the chromatography step in the downstream purification process after cell culture. Since the filter system of the present application has a high production volume (filtration capacity, such as>1mL / cm 2 ), it is advantageous that the eluting fluid can be loaded directly (online) from the filter system to the chromatography material and no by-pass of the chromatography material is required during the pre-flushing. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The test setups for Examples 1 to 6 are shown.

[0068] Figure 2 The filtering results obtained in Example 1 are shown.

[0069] Figure 3 A comparison of the final membrane layer of Example 3 with respect to filtrate volume and turbidity reduction is shown.

[0070] Figure 4 The test results of Example 4 are shown.

[0071] Figure 5 The filtering results of Example 5 are shown.

[0072] Figure 6 It is shown that in Example 6, after 20 cm of rinsing the filter with buffer after filtration 2 Product yield of the filter system.

[0073] Figure 7 The TOC release results for Example 7 are shown.

[0074] The present application will be further illustrated in the following examples, but is not limited thereto. DETAILED DESCRIPTION

[0075] Test setup for Examples 1 to 6 below:

[0076] In all cell culture filtration applications, forward flow filtration was applied at a constant flow rate of 10 mL / min to 1.0 bar inlet pressure using a peristaltic pump and a pressure sensor. In Examples 4 and 5, filtration was performed up to 2 bar inlet pressure. Figure 1 The experiment was carried out using the test device depicted. The product was filtered using a peristaltic pump up to 1 bar inlet pressure (Example 1, Example 2, Example 3 and Example 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. In addition, the filtrate weight was measured using a laboratory balance to determine the product weight and filter capacity.

[0077] The following nonwoven and film layers were used:

[0078]

[0079] *Average thickness measured according to DIN EN ISO 12625-3 using universal thickness gauge S16502 (Frank-PTI).

[0080] ** Average pore size measured according to ASTM E 1294-89 (1999) using a Topas PSM 165 (porometer).

[0081]

[0082]

[0083] RC...Regenerated Cellulose

[0084] CA……Calcium acetate cellulose

[0085] ** Determined as above

[0086] Example 1:

[0087] Performance testing was performed using actual solutions available from several distributors.

[0088] Use the following filter system:

[0089] DF-A: NW1+NW2+NW3+Mem1

[0090] DF-B: NW1+NW2+Nw5+Mem1

[0091] DF-C: NW1+NW2+NW3+Nw4+Mem1

[0092] DF-D: NW1+NW2+NW3+Nw6+Mem1

[0093] DF-E: NW1+NW2+NW3+NW7+Mem1

[0094] FC-PES: Commercially available polyethersulfone-based filter system (Whatman GD / X Syringe Filters-Prefilter, Sterile|Cytiva)

[0095] DF-C is a filter system according to the present application. 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.

[0096] Sample 1 cell culture information:

[0097] Test A: CHO K1A cell line, 72% viability, 12 Mio / mL VCC, 490 NTU turbidity

[0098] Test B: CHO K1A cell line, 50% viability, 9 Mio / mL VCC, 370 NTU turbidity

[0099] Sample 2 cell culture information:

[0100] Test C: ExpiCHO transient cell line 85% viability, VCC 16 Mio / mL

[0101] Test D: HEK293 transient cell line 61% viability, VCC 9 Mio / mL

[0102] Test E: CHO stable cell line 94% viability, VCC 18 Mio / mL

[0103] Figure 2 The results of filtration of sample 1 and sample 2 using different cell lines are shown. Figure 2 It can be clearly seen that the filter system of the present application (DF-C) shows the best performance for different cell cultures when considering both product yield and turbidity reduction.

[0104] Example 2:

[0105] The performance of three filter systems according to the present application (each consisting of NW1+NW2+NW3+NW4 with a different membrane layer combination) and a comparative filter system consisting of only NW1+NW2+NW3+NW4 were evaluated in terms of turbidity reduction and filter capacity.

[0106] The results of filtering the model solution of 10xCaro / Ovo + 20g / L dry yeast mixture are shown in the table below. The turbidity of the feed solution was about 600 NTU.

[0107] Table 1:

[0108]

[0109] The filter system of the present application exhibits high turbidity reduction and similar filter capacity compared to a filter system that does not include a final membrane layer.

[0110] Example 3:

[0111] Five filter systems according to the present application (each consisting of NW1+NW2+NW3+NW4 with a different membrane layer combination) were evaluated for their performance in terms of turbidity reduction and filter capacity.

[0112] table below and Figure 3 The filter systems tested and the filtrate volumes and turbidities achieved are shown.

[0113] The test medium was an average of 12 Mio / mL CHO cell line.

[0114] Filter Code Film Average pore size of membrane layer [μm] V6.23 Mem1 0.41 V6.25 Mem4+Mem5 1.0+0.40 V6.26 Mem6+Mem7 1.2+0.63 V6.27 Mem2+Mem3 0.68+0.46 V6.28 Mem8+Mem6 2.1+1.2

[0115] Example 4:

[0116] Regarding the orientation of the final membrane layer, the final membrane layer (Mem1; an asymmetric membrane layer in which the average pore size of the first major surface is greater than the average pore size of the second major surface) was tested (A: the first major surface adjacent to the fourth synthetic nonwoven layer; B: the second major surface adjacent to the fourth synthetic nonwoven layer). It was set to NW1+NW2+NW3+NW4+Mem1. The test medium was a model solution of 10xCaro / Ovo+20g / L dry yeast mixture with a turbidity of about 3000NTU. The results are shown in Figure 4 middle.

[0117] from Figure 4 It can be clearly seen that orientation A (ie, a first major surface having an average pore size greater than the average pore size of the second major surface of the final membrane layer adjacent to the fourth synthetic nonwoven layer) performs best in terms of filtrate volume.

[0118] Example 5:

[0119] Test the scalability of the final membrane layer combination. Set to NW1+NW2+NW3+NW4+Mem1. Figure 4 The amount and turbidity of the normalized filtrate for different filter areas are shown. The medium is a model solution of 10xCaro / Ovo+20g / L dry yeast, turbidity 4500NTU. The results are shown in Figure 5 middle.

[0120] from Figure 5 It can be clearly seen that 20cm 2 The modules also performed well and allowed filtration volumes of >50 mL per module. Thus, scalability of the filter system of the present application is possible.

[0121] Example 6:

[0122] The amount of flushing / rinsing volume required to recover target components from the filter system of the present application was tested. Set up as NW1+NW2+NW3+NW4+Mem1.

[0123] In particular, Figure 6 IgG yield / recovery after washing is shown. Cell culture was a CHO cell line, 82% viability, IgG titer 2.3 g / L, WCW 53 g / L. Wash buffer was PBS (phosphate buffered saline) at pH 7.

[0124] A wash / rinse volume of 2 mL was sufficient to achieve a yield / recovery of 80%. Thus, only a small wash / rinse volume was required to recover the target component from the filter system of the present application.

[0125] Example 7:

[0126] The filter system according to the present application (Filter System 3; setup: NW1+NW2+NW3+NW4+Mem1) and three comparative filter 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 their TOC release.

[0127] Determine the TOC of the clarification filter by flushing the purified water with different fractions. Collect the flushed fractions and determine the TOC using the TOC LCPH system as per SOP: PD / SOP / TOC / 111. The steps are given below:

[0128] ● Rinse the test device with Arium water for 2-3 hours

[0129] ● Collect water Arium through the device-take it as blank

[0130] ●Connection prototype - upward facing entrance

[0131] • Maintain a constant flow rate of 10 mL / min and collect each 5 mL fraction until 50 mL (0-5, 5-10 ... 45-50 mL) Dilute each sample 1:3 to 50 mL and measure TOC.

[0132] ● Collect each 10 mL after 50 mL until 100 mL (50-60, 60-70, ..., 90-100 mL) Dilute the 50-100 mL sample 1:2 and measure TOC.

[0133] The results are shown in Figure 7 middle.

[0134] from Figure 7 It can be clearly seen that the filter system including the glass fiber nonwoven layer releases the most TOC.

[0135] Example 8:

[0136] Each synthetic nonwoven layer and Mem 1 were evaluated for their TOC release. That is, the TOC of a 50 mm filter layer was tested by shaking the layer at 100 rpm for 2 hours in 100 mL of purified water.

[0137] The results are shown in the following table.

[0138]

[0139] It is obvious from the table that the glass fiber nonwoven layer is not resistant to water, while NW1 to NW4 and Mem1 only release a small amount of TOC.

Claims

1. A filter system, comprising: a first synthetic nonwoven layer having a first average pore size A; a second synthetic nonwoven layer having a second average pore size B; a third synthetic nonwoven layer having a third average pore size C; a fourth synthetic nonwoven layer having a fourth average pore size D; as well as A membrane layer having a fifth average pore size E, in 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, and A>B>C>D>E.

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

3. The filter system according to claim 1 or 2, wherein: The first synthetic nonwoven layer, the second synthetic nonwoven layer, the third synthetic nonwoven layer, and the fourth synthetic nonwoven layer each have an average thickness of 20 μm to 20,000 μm.

4. The filter system according to any one of claims 1 to 3, wherein: The film layer is a hydrophilic film 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 film layer has a first major surface adjacent to the fourth synthetic nonwoven layer and an opposing second major surface, and an average pore size at the first major surface is greater than an average pore size at the second major 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 film layer is a single film layer.

8. The filter system according to any one of claims 1 to 7, wherein: The film layer is composed of one or more polymers selected from the group consisting of cellulose and its derivatives, regenerated cellulose, polyamide, fluorine-containing polymer, 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 film 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 comprising one or more target components, the method comprising: Providing a filter system according to any one of claims 1-11, wherein the first synthetic nonwoven layer is defined as an upstream side of the filter system, and the membrane layer is defined as a downstream side of the filter system; and The fluid is passed through the filter system from the upstream side of the filter system to the downstream side of the filter system to retain the cells and the other contaminants in the filter system while eluting the fluid including the one or more target components from the filter system.

13. The method according to claim 12, further comprising: The fluid including the one or more target components eluted online from the filter system is passed through a purification device to further purify the one or more target components.

Images