Charged Depth Filters for Therapeutic Biotechnology Manufacturing Processes

Abstract

1. A charged depth filter for removing cells and / or cellular debris from a biopharmaceutical feedstock, comprising: a first functionalized nonwoven layer having a first calculated pore size and a first dynamic charge capacity; and a second functionalized nonwoven layer having a second calculated pore size and a second dynamic charge capacity disposed after the first functionalized nonwoven layer in a direction of flow of the biopharmaceutical feedstock, wherein the first calculated pore size is larger than the second calculated pore size and the first dynamic charge capacity is smaller than the second dynamic charge capacity.

Description

[Background technology]

[0001] Monoclonal antibodies are a major modality in the biopharmaceutical industry based on their specificity for target diseases. The therapeutic antibody market is growing rapidly with many drug candidates undergoing regulatory review. Approximately 100 monoclonal antibodies have been approved by regulatory agencies in the United States and the European Union over the last three decades, and next-generation antibody therapies are expected to increase at a higher rate over the next decade. These include antibody-drug conjugates, biosimilars, engineered antibodies, bispecific antibodies, antibody fragments, antibody-like proteins, etc. Chinese Hamster Ovary (CHO) cells are the most commonly used cell line in the industry based on their ability to adapt and grow in suspension, ability to grow in serum-free defined media, high production capacity, post-translational modifications, etc. CHO cells account for >70% of protein therapeutics produced, but these biologics can be produced in several systems including microbial, plant, insect, and other mammalian cells.

[0002] Proteins of biopharmaceutical interest include any of a number of naturally or recombinantly expressed proteins. Other biologics that can be used as therapeutic vectors include viral particles such as adenovirus, adeno-associated virus (AAV), or lentivirus, bacterial phages or viral particles, exosomes, or synthetic lipid nanoparticles. Apart from CHO cells, host cells that can be used to produce these biologics include other mammalian cell types (such as human embryonic kidney (HEK) cells, HeLa cells, or PER.C6 cells), bacteria (such as Escherichia coli or bacillus), insect cells such as Sf6, yeast cells, or plant cells such as tobacco. Regardless of the cell type or therapeutic vector, the clarification and purification challenges associated with isolating the biologic of interest from host cell components and from other components produced by the host cell are similar. Summary of the Invention

[0003] In biopharmaceutical manufacturing, when cell culture fluid is harvested from a bioreactor and sent to a downstream clarification process, the target biomolecules of interest, such as monoclonal antibodies (mAbs), viral particles, or other therapeutic vectors, must be separated from the feedstock, which contains cells, cell debris, and / or colloidal particles. Often, a primary clarification step is performed using a centrifugation step, depth filtration, microfiltration (tangential flow filtration), or a combination thereof to remove whole cells and large cell debris from the harvested cell culture fluid.

[0004] Significant advances in cell culture media, cell engineering, and bioreactor design have led to higher titers (e.g., 10 g / L) over the years. The resulting cultures also have increased cell densities from 6 million cells / mL to over 50 million cells / mL. This significant increase in cell density has impacted many primary clarification steps.

[0005] When used for primary clarification, centrifuges require extensive cleaning procedures between runs to ensure there is no cross-contamination between successive batches in the production process. Thus, there is a need for a disposable, single-use device to replace the primary centrifugation clarification step to eliminate the risk of cross-contamination when switching between batches and therapeutic biomolecules of interest.

[0006] Tangential flow microfiltration can be used as a primary clarification step instead of centrifugation, however, tangential flow microfiltration membranes are often susceptible to membrane fouling and require extensive cleaning procedures to prevent cross-contamination between runs and when switching between therapeutic biomolecules of interest.

[0007] Alternatively, conventional depth filters (which only use size exclusion based on the pore size of the media) can be used as a primary clarification step to remove cells and debris based on the size of the depth filter channel and filter the auxiliary in the depth filter media. However, as cell densities increase from 6 million cells / mL to over 50 million cells / mL, the throughput of conventional depth filtration has become unfeasible in a production manufacturing environment. Therefore, what is needed is a single-use primary clarification step that can replace centrifuges, tangential flow microfiltration, and conventional depth filters as a primary clarification step.

[0008] Applicants have found that a charged depth filter having at least two functionalized nonwoven layers, each layer having a different effective pore size and dynamic charge capacity, can accomplish such a task and is particularly effective for cell cultures with high cell densities. By carefully managing the gradients in both effective pore size and dynamic charge capacity as the feedstock moves through the layers of the depth filter, a depth filter can be constructed that does not clog the first layer with whole cells and large cell debris, yet is still effective in ensuring that the last layer of the depth filter, such as the membrane layer, is also not clogged with debris. Both situations significantly reduce throughput and make the device unacceptable for use in production biopharmaceutical manufacturing processes.

[0009] In particular, the applicants have found that the pore size of successive layers in the charged depth filter should decrease and the dynamic charge capacity of successive layers in the charged depth filter should increase. If the feedstock has too small a pore size or too large a dynamic charge capacity for the first layer of functionalized nonwoven found in the depth filter, it will easily clump with whole cells and / or large cell debris, significantly reducing throughput. Similarly, the inability to reduce the pore size and increase the dynamic charge capacity of successive layers will allow too much debris to slip through the functionalized nonwoven layer, resulting in clogging of the downstream filtration element, which can be optionally added as a final filter layer to the charged depth filter.

[0010] Thus, in one aspect, the invention relates to a charged depth filter for removing cells and / or cellular debris from a biopharmaceutical feedstock, comprising a first functionalized nonwoven layer having a first calculated pore size and a first dynamic charge capacity, and a second functionalized nonwoven layer having a second calculated pore size and a second dynamic charge capacity disposed after the first functionalized nonwoven layer in the direction of flow of the biopharmaceutical feedstock, wherein the first calculated pore size is larger than the second calculated pore size and the first dynamic charge capacity is smaller than the second dynamic charge capacity. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram of a media stack for a charged depth filter having four layers of functionalized nonwoven (FNW-C / FNW-C / FNW-E / FNW-F), followed by a membrane layer, and followed by a nonwoven spunbond layer, positioned between the inlet and outlet of the charged depth filter. [Diagram 2] 1 is an image of a functionalized nonwoven layer, FNW-B. The nonwoven before functionalization had an effective fiber diameter of 14 μm, a solidity of 10%, a basis weight of 200 g / m2, and a calculated pore size of 41.5 μm. After grafting, it has an effective fiber diameter of 21.6 μm, a solidity of 14.2%, a basis weight of 302.0 g / m2, a calculated pore size of 50.5 μm, and a MY DCC of 165.0 mg / g. [Diagram 3] 1 is an image of a functionalized nonwoven layer, FNW-F. Before functionalization, the nonwoven had an effective fiber diameter of 6 μm, a solidity of 10%, a basis weight of 200 g / m2, and a calculated pore size of 17.8 μm. After grafting, it had an effective fiber diameter of 9.1 μm, a solidity of 17.8%, a basis weight of 355.8 g / m2, a calculated pore size of 17.9 μm, and a MY DCC of 407.4 mg / g. [Figure 4]Image of a cut charged depth filter media stack of larger pore functionalized nonwoven, FNW-B, and membrane after cell culture clarification. The cell culture easily penetrated all four functionalized nonwoven layers and covered the membrane surface with cell residue and cell debris. This media stack did not work well because too much debris contaminated the membrane layer. [Diagram 5] 13 is an image of a cut charged depth filter media stack of smaller pore functionalized nonwoven, FNW-F, and membrane after cell culture clarification. The cell culture contaminates the top layer and is unable to penetrate all of the functionalized nonwoven layers. The third and fourth layers are not utilized and the membrane surface is clean without any cell residue and cell debris. [Figure 6A] 1 shows the top surface of the first functionalized nonwoven (FNW-C) in the media stack after filtering the CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven layer and appear as round balls on the outer surface of the functionalized fibers. [Figure 6B] 1 shows the top surface of the first functionalized nonwoven (FNW-C) in the media stack after filtering the CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven layer and appear as round balls on the outer surface of the functionalized fibers. [Figure 6C] The bottom surface of the first functionalized nonwoven (FNW-C) in the media stack after filtering the CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven layer and appear as round balls on the outer surface of the fibers. [Figure 6D] The top surface of the second functionalized nonwoven (FNW-C) in the media stack after filtering the CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven layer and appear as round balls on the outer surface of the fibers. [Figure 6E] The bottom surface of the second functionalized nonwoven (FNW-C) in the media stack after filtering the CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven layer and appear as round balls on the outer surface of the fibers. [Figure 6F]Shows the top surface of a functionalized nonwoven (FNW-E) in a media stack after filtering CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven layer and appear as round balls on the outer surface of the fibers. [Figure 6G] The bottom surface of a functionalized nonwoven fabric (FNW-E) in a media stack after filtering CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven fabric layer and appear as round balls on the outer surface of the fibers. [Figure 6H] Shows the top surface of a functionalized nonwoven fabric (FNW-F) in a media stack after filtering CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven fabric layer and appear as round balls on the outer surface of the fibers. [Figure 6I] The bottom surface of a functionalized nonwoven fabric (FNW-F) in a media stack after filtering CHO cell culture. Cells, debris, and / or DNA adhere to the charged fibers of the nonwoven fabric layer and appear as round balls on the outer surface of the fibers. [Figure 6J] Shows the top surface of the 0.2 μm membrane layer in the media stack after filtering a CHO cell culture. As can be seen, very little cells, debris, and / or DNA are present on the surface of the membrane. [Figure 7] FIG. 1 shows a perspective view of a charged depth filter having a housing with an inlet, an outlet, an optional vent, and a media stack (not shown) positioned between the inlet and outlet for clarifying cell culture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Throughout this document, values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​expressly set forth as the limits of that range, but also all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were expressly set forth. For example, the range "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted as including not only about 0.1% to about 5%, but also the individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the stated range. The statement "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise indicated.

[0013] In this document, the terms "a," "an," or "the" are used to include one or more, unless the context clearly indicates otherwise. The term "or" is used to refer to a nonexclusive "or," unless otherwise indicated. The statements "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B." In addition, any expressions or terms used herein that are not specifically defined are intended to be for descriptive purposes only and should not be construed as limiting. Any use of section headings is intended to aid in the reading of this document and should not be construed as limiting, and information associated with a section heading may be found within or outside that particular section.

[0014] As used herein, the term "about" can allow for variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of the limits of a stated range, including the stated value or range itself.

[0015] As used herein, the term "substantially" refers to a majority or majority, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99% or at least about 99.999% or more, or 100%. As used herein, the term "substantially free" can mean that the composition is present in such a small amount, or completely absent, that the amount of material present does not affect the material properties of the composition including the material, such that the composition is present in an amount of from about 0 wt% to about 5 wt%, or from about 0 wt% to about 1 wt%, or less than about 5 wt%, or less than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or less than about 0.001 wt%.

[0016] As used herein, "layer" refers to a thickness of material through which the fluid to be treated passes, and all of the materials in the layer are made of the same material. The layer can be a monolithic layer made of the same material of a thickness. Alternatively, the layer can have one or more individual plies of the same material that are stacked together in the layer to form the thickness of the layer. For example, a layer of a typical facial tissue is often a tissue paper material made from two individual plies of tissue paper placed in face-to-face contact, and the two individual plies are generally held together by a weak mechanical bond in the form of a crimp line, so that they can be easily pulled apart from each other.

[0017] As used herein, a "ply" or "plies" is a single material of a certain thickness that can be processed into a layer by conventional transformation operations, such as, but not limited to, rolling, folding, cutting, or stacking. In many cases, a ply is a material of a certain thickness after completing a forming process on a web-making machine. One or more plies of the same material can then be stacked to form a layer. For example, a nonwoven fabric can be made as a single ply on a former and wound into a roll. The nonwoven fabric roll can then be unwound and folded in half in the cross-machine direction by a folding plate as it passes longitudinally through a transformer, and the two-ply layer can then be cut into a disk by a cutting die to form a circular layer of nonwoven material having two individual plies.

[0018] As used herein, a "functionalized layer" is a layer that adsorbs target particles or molecules by attractive forces, such as electrostatic forces, resulting from the presence of one or more chemical moieties, ligands, or functional groups at the surface of the layer that are distinct from the material that forms the bulk of the layer, which primarily provides the structural shape and integrity of the layer. The chemical moieties, ligands, or functional groups are specifically intended to adsorb the target particles or molecules to the surface of the functionalized layer. A functionalized layer may be created by coating or grafting a ligand, monomer, or polymer designed to molecularly adsorb the target particles or molecules to the porous layer. Alternatively, a functionalized layer may be created such that the formulation used to create such a layer provides a surface-modifying polymer or chemical moiety that is localized to the surface of the layer during formation, such that chemical groups designed to adsorb the target particles or molecules are present at the surface of the layer. In some embodiments, the attractive forces between the functional groups on the surface of the functionalized layer are electrostatic forces, and the chemical moieties, ligands, or polymers present on the surface of the functionalized layer are electrostatically charged. The functionalized layer may be positively charged and adsorb negatively charged particles, i.e., anion exchange chromatography, or the functionalized layer may be negatively charged and adsorb positively charged particles, i.e., cation exchange chromatography. In other embodiments, the attraction may be van der Waals forces, and the target particles or molecules are adsorbed to the functional groups on the functionalized layer surface by their relative concentrations to each other, or by the lack of polarizable or hydrogen-bonding moieties (i.e., hydrophobic interactions). Additionally, the attraction may include a combination of electrostatic and van der Waals forces (i.e., mixed mode). Functionalized materials suitable for functionalized layers in charged depth filter devices are made by Pall, Millipore, and Sartorious and are sold under the following brands: Mustang® Q, NatriFlo® HD-Q, and Sartobind® Q. Functionalized layers suitable for use in charged depth filter devices may be nonwovens, membranes, or other suitable materials.A preferred functionalized nonwoven material is made by 3M Company and is disclosed in U.S. Patent No. 9,821,276, entitled "Nonwoven Article Grafted with Copolymer." A preferred functionalized membrane is made by 3M Company and is disclosed in U.S. Patent Nos. 9,650,470 and 10,017,461, entitled "Method of Making Ligand Functionalized Substrates." All three of the patents mentioned are incorporated herein by reference in their entirety.

[0019] As used herein, a "non-functionalized layer" is a layer that does not contain any coated, grafted, or surface-localized adsorbent chemical moieties (e.g., electrostatically charged chemical moieties, ligands, or functional groups) that are distinct from the material that forms the bulk of the layer.

[0020] As used herein, "media stack" refers to all of the layers of material through which the fluid being processed passes within the housing of a charged depth filter as the fluid moves from the inlet through the housing to the outlet.

[0021] As used herein, "membrane" refers to a synthetic liquid-permeable membrane comprising a sheet of material in which a plurality of pores or an interconnected network of pores are arranged that allow the passage of a fluid through the membrane. Such membranes generally include polymeric membranes made by a phase inversion process, in which a homogeneous solution of one or more polymers in a suitable solvent, or combination of solvents, undergoes phase separation to form a porous structure. Phase separation can be brought about by introducing a film of the homogeneous solution into a non-solvent bath (known as diffusion-induced phase separation), into a non-solvent atmosphere (known as vapor-induced phase separation), or by changing the temperature of the homogeneous solution (known as thermally-induced phase separation). Alternatively, pores can be formed in the polymer sheet by a stretching process or a radiation exposure process (track-etched membranes). Membranes can have pore sizes of about 0.1 micrometers to about 20 micrometers in diameter (microporous membranes) or less than about 0.1 micrometers (ultramicroporous membranes). Polymers suitable for forming membranes include cellulose acetate, nitrocellulose, cellulose esters, polysulfones, including bisphenol A polysulfone and polyethersulfone, polyacrylonitrile, polyamides (e.g., nylon-6 and nylon-6,6), polyimides, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl chloride, and ethylene-chlorotrifluoroethylene copolymers.

[0022] Charged Depth Filter With reference to Figures 1 and 7, the charged depth filter includes a housing 10 having an inlet 16, an outlet 18, an optional vent 20, and a media stack including layers 25, 31, 33, 35, 37, and 39 (Figure 1) located within the housing including at least two layers of functionalized nonwoven fabric disposed between the inlet and outlet such that the cell culture to be filtered passes through the media stack from the inlet 16 to the outlet 18. The edges of the media stack are sealed, for example by compression or thermoplastic welded seals, to minimize or eliminate any leakage of the cell culture to the outlet without first passing through the media stack. Any suitable housing can be used for the charged depth filter that can contain and seal the media stack. Often different sizes of housings and media stack volumes are provided that are suitable for laboratory bench scale studies to commercial production.

[0023] The media stack has at least a first functionalized nonwoven layer 25 having a first calculated pore size and a first dynamic charge capacity, and a second functionalized nonwoven layer 33 having a second calculated pore size and a second dynamic charge capacity disposed after the first functionalized nonwoven layer in the direction of flow of the biopharmaceutical feedstock, wherein the first calculated pore size is larger than the second calculated pore size and the first dynamic charge capacity is smaller than the second dynamic charge capacity.

[0024] The housing may be of any suitable size, with the size being appropriately scaled relative to the media surface area within the housing. Typically, laboratory scale devices are relatively small and have low hold-up volumes to process limited amounts of fluid. Pilot and production scale devices will have correspondingly larger amounts of media within them to process larger amounts of fluid for each run. For example, a laboratory scale device may have a 3.2 cm 2 ~25cm 2 and the pilot-scale device may have a media surface area of ​​340 cm 2 ~1,020cm 2 and a production scale device may have a media surface area of ​​2,300 cm 2~16,100cm 2 The media stack of the present invention may have a media surface area of ​​100 mm. Other housing sizes and media volumes may be provided as needed for a particular application. A suitable housing is made by 3M and used in the 3M Emphaze AEX Hybrid Purifier product line. See https: / / www.3m.com / 3M / en_US / company-us / all-3m-products / ~ / 3M-Emphaze-AEX-Hybrid-Purifier / ?N=5002385+3291555558&rt=rud. Similar housing sizes and designs can be used to house the media stack of the present invention.

[0025] A suitable housing is disclosed in U.S. Patent Application No. 62 / 792,166, entitled "Sample Size Chromatography Device," filed January 14, 2019, which is incorporated herein by reference in its entirety. As best seen in FIG. 7, the housing 10 is formed by joining an upper housing 12 to a lower housing 14. The housing has an inlet 16, an outlet 18, and an optional vent 20. A media stack is disposed within the chamber between the inlet 16 and the outlet 18 such that fluid from the inlet 16 enters the internal chamber and then passes through the media stack and exits through the outlet 18. The chamber is in fluid communication with the inlet 16 and the optional vent 20 such that air within the chamber can be purged through the vent 20. A luer lock connector (not shown) can be attached to the vent 20 and used as a valve to purge air from the chamber until liquid from the inlet 16 begins to exit the vent 20 and the valve is closed. A cylindrical projection 32 with opposed lateral tabs 80 extends from the housing and has tapered holes for attaching luer lock connectors to the inlets, outlets and vents. Longitudinal ribs 58 are spaced around the periphery to provide improved grip when handling the housing.

[0026] Another suitable housing having a sealing membrane and spacer ring is disclosed in U.S. Patent Application No. 63 / 023,488, filed May 12, 2020, and entitled "Membrane Sealing Layer and Spacer Ring for Viral Clearance Chromatography Device," which is incorporated by reference in its entirety.

[0027] Media Stack The media stack includes a first functionalized nonwoven layer 25 and a second functionalized nonwoven layer 33 disposed between the inlet and outlet of the housing, the first functionalized nonwoven layer having a first calculated pore size and a first dynamic charge capacity, the second functionalized nonwoven layer having a second calculated pore size and a second dynamic charge capacity, and disposed after the first functionalized nonwoven layer in the direction of flow of the biopharmaceutical feedstock, the first calculated pore size being larger than the second calculated pore size and the first dynamic charge capacity being smaller than the second dynamic charge capacity.

[0028] As used herein, "first" and "second" layers do not mean that they must be the first and second layers through which a fluid passes as it moves through the media stack. Rather, they refer to their relative positions relative to one another in that the fluid flows first through the first layer and then through the second layer, and there may be previous and / or intermediate layers in the media stack. For example, a media stack may include, in the direction of fluid flow, layer A, then the first layer, layer B, layer C, then the second layer, and layer D. Similarly, other specified numerical layers, such as a third functionalized nonwoven layer, are treated in the same manner.

[0029] From the examples, better performance of a charged depth filter using two functionalized nonwoven layers was observed when a first functionalized nonwoven layer 25 having a first calculated pore size of 40.8 μm to 65.0 μm and a first dynamic charge capacity of 150 MY DCC mg / g to 300 MY DCC mg / g was combined with a second functionalized nonwoven layer 33 having a second calculated pore size of 5.0 μm to less than 40.8 μm and a second dynamic charge capacity of greater than 300 MY DCC mg / g to 650 MY DCC mg / g. Alternatively, a first functionalized nonwoven layer 25 having a first calculated pore size of 55.0 μm to 65.0 μm and a first dynamic charge capacity of 150 MY DCC mg / g to 300 MY DCC mg / g in combination with a second functionalized nonwoven layer 233 having a second calculated pore size of 5.0 μm to less than 55.0 μm and a second dynamic charge capacity of greater than 300 MY DCC mg / g to 650 MY DCC mg / g may result in better performance of a two layer charged depth filter.

[0030] Better performance of a charged depth filter using three functionalized nonwoven layers was observed from the examples when a first functionalized nonwoven layer 25 had a first calculated pore size of 40.8 μm to 65.0 μm and a first dynamic charge capacity of 150 MY DCC mg / g to 300 MY DCC mg / g, followed by a second functionalized nonwoven layer 33 having a second calculated pore size of 20.6 μm to less than 40.8 μm and a second dynamic charge capacity of greater than 300 MY DCC mg / g to 475 MY DCC mg / g, followed by a third functionalized nonwoven layer 35 having a third calculated pore size of 5.0 μm to less than 20.6 μm and a third dynamic charge capacity of greater than 300 MY DCC mg / g to 650 MY DCC mg / g. Alternatively, a first functionalized nonwoven layer 25 having a first calculated pore size of 55.0 μm to 65.0 μm and a first dynamic charge capacity of 150 MY DCC mg / g to 300 MY DCC mg / g may result in better performance of a three layer charged depth filter when combined with a second functionalized nonwoven layer 33 having a second calculated pore size of 20.6 μm to less than 55.0 μm and a second dynamic charge capacity of 200 MY DCC mg / g to 475 MY DCC mg / g, followed by a third functionalized nonwoven layer 35 having a third calculated pore size of 5.0 μm to less than 20.6 μm and a third dynamic charge capacity of greater than 300 MY DCC mg / g to 650 MY DCC mg / g.

[0031] When using three layers of functionalized nonwoven fabric, better performance was observed when the third functionalized nonwoven fabric layer was water permeable. If the pore size becomes too small due to the amount of grafting, the membrane may be blocked too much. The water permeability boundary line of one functionalized nonwoven fabric medium used in the examples can be plotted on an XY graph of dynamic charge capacity MY DCC mg / g versus calculated pore size in μm. The approximate location of the water permeability line extends through point 1, which has a calculated pore size of 5.0 μm and a dynamic charge capacity of 300 MY DCC mg / g, and through point 2, which has a calculated pore size of 20.6 μm and a dynamic charge capacity of 525 MY DCC mg / g. Functionalized nonwoven fabrics with data points plotted above this line tend to be non-water permeable and are less preferred. Functionalized nonwoven fabrics with data points plotted below this line tend to be water permeable and are more preferred.

[0032] Often the media stack contains additional functionalized, non-functionalized, and / or membrane layers. The same layers may be repeated in the charged depth filter to increase the capacity for a particular debris size before the pore size and / or dynamic charge capacity is changed. The media stack of a charged depth filter can have 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more layers depending on the construction, but often has less than 25 layers.

[0033] The charged depth filter may include an optional membrane layer, which is disposed between the last functionalized layer and the housing outlet and can be used to increase the capsule back pressure to enhance filtration uniformity, which may be selected from water-permeable membranes including, but not limited to, polyethersulfone, polysulfone, cellulose, regenerated cellulose, and polyamide membranes.

[0034] The charged depth filter may include an optional non-functionalized non-woven layer, which is located between the optional membrane layer and the housing outlet and may be used to protect the integrity of the membrane during capsule assembly and filtration. The non-functionalized non-woven layer may be selected from non-woven materials, including, but not limited to, polypropylene, polyethylene, polymethylpentene, and polyethylene terephthalate materials.

[0035] As shown in Figure 1, the preferred structure of the media stack includes six layers. The first functionalized nonwoven layer 25 after grafting has an effective fiber diameter of 18.9 μm, a weight of 272.7 g / m 2 The first functionalized nonwoven layer has a basis weight of 1.0 μm, a solidity of 13.5%, and a first calculated pore size of 45.6 μm, and a first dynamic charge capacity MY DCC of 291.7 mg / g. The first functionalized nonwoven layer is followed by a repeated first functionalized nonwoven layer 31 having identical properties, i.e., there are two layers of the first functionalized nonwoven in the charged depth filter. The repeated first functionalized nonwoven layer 31 is followed by a second functionalized nonwoven layer 33. The second functionalized nonwoven layer 33 after grafting has an effective fiber diameter of 12.1 μm, a first calculated pore size of 356.6 g / m 2 The second functionalized nonwoven layer 33 has a basis weight of 1.0 μm, a solidity of 16.3%, and a second calculated pore size of 25.5 μm, and a second dynamic charge capacity MY DCC of 365.3 mg / g. The second functionalized nonwoven layer 33 is followed by a third functionalized nonwoven layer 35. The third functionalized nonwoven layer 35 after grafting has an effective fiber diameter of 9.1 μm, a second calculated pore size of 355.8 g / m 2 The third functionalized nonwoven layer 35 has a basis weight of 1.0%, a solidity of 17.8%, and a third calculated pore size of 17.9 μm, and a third dynamic charge capacity MY DCC of 407.4 mg / g. The third functionalized nonwoven layer 35 is followed by a membrane layer 37. The membrane layer is a 0.2 μm PES membrane. The membrane layer 37 is followed by a non-functionalized nonwoven layer 39. The non-functionalized nonwoven layer 39 is a polypropylene spunbond layer.

[0036] Referring now to Figures 6A-6J, micrographs of the various layers in the 6-layer structure can be observed after clarification of CHO cell cultures with 3.2% PCV. As can be seen, cells, debris, and / or DNA attach to the charged fibers of the functionalized nonwoven layer and appear as round balls on the outer surface of the fibers. Both the calculated pore size and dynamic charge capacity of each subsequent layer are controlled to avoid clogging or caking the surface of the functionalized nonwoven layer or membrane layer, while still ensuring that each layer in the charged depth filter removes debris of appropriate size, as evidenced by both the top and bottom surfaces of the layer having debris attached to the functionalized grafted fibers. This structure ensures good throughput and debris removal.

[0037] Referring now to FIG. 4, a series of layers in a media stack of a "too coarse" charged depth filter is shown. As can be seen, images of a media stack of a cut charged depth filter of larger pore functionalized nonwoven, FNW-B, and membrane after cell culture clarification are presented. The cell culture easily penetrates all four functionalized nonwoven layers (stained portions of the disks) and covers the surface of the membrane layer with a residue of cells and cell debris. This media stack did not perform well as too much debris contaminated the membrane layer (right-most circular disk) significantly reducing throughput.

[0038] Referring now to FIG. 5, a series of layers in a media stack of an "overcrowded" charged depth filter is shown. As can be seen, images of a media stack of a smaller pore functionalized nonwoven, FNW-F, and a dissociated charged depth filter of membrane after cell culture clarification are presented. The cell culture contaminates the top functionalized nonwoven layer (stained portion of the disk) and is unable to penetrate all of the functionalized nonwoven layers (limited to the absence of staining on disks 3 and 4 from the left). The third and fourth layers are not utilized and the surface of the membrane layer (rightmost circular disk) is clean without any residue of cells and cell debris. This media stack did not perform well as too much debris contaminated the first functionalized nonwoven layer, significantly reducing throughput.

[0039] A one-step method for cell clarification In biopharmaceutical manufacturing, clarification is the first processing step aimed at separating and recovering target biomolecules of interest, such as monoclonal antibodies (mAbs), viral particles, or other therapeutic vectors, from harvested cell culture feedstock by removing cells, cell debris, and / or colloidal particles prior to further downstream purification steps. In mammalian cell cultures (e.g., Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney 293 (HEK-293) cells, Baby Hamster Kidney (BHK21) cells, NS0 murine myeloma cells, or PER.C6® human cells), the size range of insoluble contaminants that need to be removed is greater than 10 microns for whole cells, approximately 1 micron to 9 microns for cell debris, and less than 1 micron for colloidal debris. Other target molecules of interest may be produced by insect and bacterial cell lines, and the charged depth filters of the present invention may also be used to clarify these feedstocks.

[0040] Current process techniques for clarification include, but are not limited to, centrifugation, depth filtration, microfiltration (e.g., tangential flow filtration), or combinations thereof. Due to the wide range of contaminant sizes, existing methods for clarification by filtration are accomplished in two or three stages, with the first stage removing larger size particles, followed by the second or third stage removing smaller particles. Optimization of these processes or filtration stages to successfully clarify biological therapeutics from cell cultures by filtration depends on the characteristics of the therapeutic product (e.g., isoelectric point) and the characteristics of the cell culture (e.g., cell density, viability, particle size distribution).

[0041] Recent advances in cell culture media, cell engineering, and bioreactor design have led to significant increases in both cell density (e.g., greater than 100 million cells / mL in perfusion-based systems, or greater than about 20% packed cell volume) and mAb titers (e.g., greater than 10 g / L). This significant increase in cell density poses challenges to the clarification process, resulting in lower yields and throughput when using centrifugation and / or traditional depth filtration processes.

[0042] Clarification by filtration using the charged depth filters of the present invention provides a different mechanism than the conventional size-based exclusion approach used by conventional depth filters. In the charged depth filters of the present invention, whole cell and cell debris contaminants are removed by both charge-based separation and size exclusion. Chromatographic separation techniques such as packed resin column chromatography and membrane chromatography are not designed for this type of application based on their small porous matrix and maneuverable device design. The charge-based removal of cells and debris using functionalized nonwovens, presented in this invention and shown in the SEM images of Figures 6A-6J, is not diffusion limited due to the high void volume within the functionalized nonwoven matrix. Negatively charged soluble and insoluble contaminants (e.g., cells, debris, DNA, and host cell proteins) in the cell culture fluid are removed by electrostatic interactions with the positively charged surface of the functionalized nonwoven, resulting in a one-step fiber chromatography process.

[0043] The charged depth filters described in this disclosure are designed to have a gradient structure based on both effective pore size and dynamic charge. The filters are capable of clarifying high cell density cultures with 2%-12% packed cell volume PCV (10 million cells / mL-60 million cells / mL), more preferably 3%-11% PCV (15 million cells / mL-55 million cells / mL), or more preferably 3%-9% PCV (15 million cells / mL-45 million cells / mL) in a single step process. During this process, the throughput can be as high as 30 L / m 2 (liters / meter 2 ~200L / m2 (liters / meter 2 ) The flow rate can be 50LMH (liters per meter 2 / hour) ~ 600LMH (liters / meter 2 / hr), more preferably 75 LMH to 400 LMH, or more preferably 100 LMH to 250 LMH. The enhanced throughput capacity for high cell density cultures reduces the manufacturing footprint compared to traditional depth filter processes.

[0044] High density cell cultures, including whole cells and cell debris, typically have turbidity ranging from 1,000 nephelometric turbidity units (NTU) to 10,000 nephelometric turbidity units (NTU). The described single-step clarification process using charged depth filters can reduce the turbidity of high density cell cultures to below 50 NTU, below 20 NTU, below 15 NTU, or below 10 NTU.

[0045] The charged depth filters of the present invention are preferably designed to be water permeable, so that only water is required for the preconditioning wash. Using water for preconditioning reduces costs and eases operation.

[0046] Advantages of the charged depth filter single-step clarification process of the present invention include, but are not limited to, increased product yield, reduced manufacturing footprint, clarified fluid with consistently low turbidity, and user-friendly operation. These combined advantages result in favorable process economics for therapeutic drug manufacturing.

[0047] Non-functionalized and functionalized nonwoven parameters Properties of interest for unfunctionalized and functionalized nonwovens (e.g., copolymer-grafted nonwovens) include basis weight, effective fiber diameter (EFD), solidity, and pore size, which can be determined for the nonwovens before or after functionalization.

[0048] The fibers of the non-functionalized nonwoven substrate typically have an effective fiber diameter of about 3 micrometers to 20 micrometers. The non-functionalized substrate preferably has an effective fiber diameter of about 10 g / m 2 ~400g / m 2 , more preferably about 80 g / m 2 ~250g / m 2 The average thickness of the non-functionalized substrate is preferably from about 0.1 mm to 10 mm, and more preferably from about 0.25 mm to 5 mm.

[0049] The loftiness of functionalized or non-functionalized nonwovens is measured by solidity, a parameter that defines the solid fraction in the volume of the web. A lower solidity value indicates a higher loftiness of the web. Solidity is a unitless fraction that is typically expressed as α.

number

[0050] The fiber density (ρ f ) is determined by Method A in the Examples described below. The fiber density of copolymer-grafted fibers after functionalization can also be determined by a modified version of Method A in which the molar ratios of the substrate and copolymer components are all obtained from solid-state carbon-13 NMR measurements and the molar ratios are converted to weight ratios. When a nonwoven substrate contains a mixture of two or more types of fibers, the same L 不織布 to obtain an individual solidity for each type of fiber, and these individual solidities are summed to obtain the web solidity α.

[0051] Effective fiber diameter (EFD) refers to the apparent diameter of the fibers in a nonwoven fibrous web as determined by an air permeability test in which air at 1 atmosphere and room temperature is passed through a web sample of known thickness at a face velocity of 5.3 cm / sec and the corresponding pressure drop is measured. Based on the measured pressure drop, the effective fiber diameter is calculated as described in CN, "The Separation of Airborne Dust and Particles", Institution of Mechanical Engineers, London, Proceedings 1B, 1952. EFD can be determined for nonwovens before or after functionalization.

[0052] The calculated pore size is related to the arithmetic mean fiber diameter and the web solidity and is determined by the following formula: where D is the calculated pore size and d f is the arithmetic mean fiber diameter and α is the web solidity.

number

[0053] The calculated pore size can be determined for the nonwoven fabric before or after functionalization. Preferably, the nonwoven fabric substrate has a calculated pore size of between 1 micrometer and 50 micrometers before functionalization.

[0054] The dynamic charge capacity (DCC) of the functionalized nonwoven substrate was determined using a metanil yellow challenge solution using Example Method B and is reported as MY DCC (Metanil Yellow Dynamic Charge Capacity).

[0055] Nonwoven base web The nonwoven substrate is a nonwoven web and may include nonwoven webs produced by any of the known processes for the manufacture of nonwoven webs. As used herein, the term "nonwoven web" refers to a fabric having a structure of individual fibers or filaments randomly and / or unidirectionally assembled into a mat. For example, fibrous nonwoven webs can be made by carding, airlaid, wetlaid, spunlace, spunbond, electrospinning, or meltblowing processes such as meltspun or meltblowing, or combinations thereof. Spunbond fibers are typically small diameter fibers formed by extruding molten thermoplastic polymer as filaments through multiple fine, usually circular spinneret capillaries with the diameter of the extruded fiber and then rapidly reducing the diameter. Meltblown fibers are typically formed by extruding molten thermoplastic material as molten threads or filaments through multiple fine, usually circular die capillaries into a high-velocity, usually heated gas (e.g., air) stream that attenuates the filaments of molten thermoplastic material and reduces their diameter. The meltblown fibers are then carried by the high velocity gas stream and deposited on a collecting surface forming a web of randomly distributed meltblown fibers. Any nonwoven web can be made from a single type of fiber or from two or more fibers differing in thermoplastic polymer type and / or thickness.

[0056] Suitable polyolefins for making the nonwoven web include, but are not limited to, polyethylene, polypropylene, poly(1-butene), copolymers of ethylene and propylene, alpha-olefin copolymers (e.g., copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and 1-decene), poly(ethylene-co-1-butene), poly(1-methylpentene), and poly(ethylene-co-1-butene-co-1-hexene). Preferably, the nonwoven substrate is polypropylene.

[0057] Further details of the method of making the nonwoven web of the present invention can be found in Wente, Superfine Thermoplastic Fibers, 48 ​​INDUS.ENG.CHEM.1342 (1956), or Wente et al. Manufacture of Superfine Organic Fibers, (Naval Research Laboratories Reort No. 4364, 1954). Useful methods of preparing nonwoven substrates are described in U.S. Reissue Patent No. 39,399 (Allen), U.S. Patent No. 3,849,241 (Butin et al.), U.S. Patent No. 7,374,416 (Cook et al.), U.S. Patent No. 4,936,934 (Buehning), and U.S. Patent No. 6,230,776 (Choi).

[0058] Functionalized nonwoven layer The functionalized nonwoven layer comprises a nonwoven substrate as described above and a grafted copolymer comprising interpolymerized monomer units, at least one of which is cationic or can be made cationic in a solution of appropriate pH ("cationically ionizable"). Suitable functionalized nonwoven webs are disclosed in U.S. Patent No. 9,821,276, entitled "Nonwoven Article Grafted with Copolymer," issued November 21, 2017, which is incorporated herein by reference.

[0059] Cationic or cationically ionizable monomers can include quaternary ammonium-containing monomers and tertiary amine-containing monomers. One or more cationic or cationically ionizable monomers can be used. The monomer typically contains a polymerizable functional group and a cationic or cationically ionizable group. In certain monomers, the polymerizable group and the cationic group can be the same group. Polymerizable groups include vinyl, vinyl ether, (meth)acryloyl, (meth)acrylamide, allyl, cyclic unsaturated monomers, multifunctional monomers, vinyl esters, and other easily polymerizable functional groups.

[0060] Useful (meth)acrylates include, for example, trimethylaminoethyl methacrylate, trimethylaminoethyl acrylate, triethylaminoethyl methacrylate, triethylaminoethyl acrylate, trimethylaminopropyl methacrylate, trimethylaminopropyl acrylate, dimethylbutylaminopropyl methacrylate, diethylbutylaminopropyl acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl acrylate, 2-(diethylamino)ethyl acrylate, and 3-(dimethylamino)propyl acrylate.

[0061] Exemplary (meth)acrylamides include, for example, 3-(trimethylamino)propyl methacrylamide, 3-(triethylamino)propyl methacrylamide, 3-(ethyldimethylamino)propyl methacrylamide, and n-[3-(dimethylamino)propyl]methacrylamide.Preferred quaternary salts of these (meth)acryloyl monomers include, but are not limited to, (meth)acrylamidoalkyltrimethylammonium salts, such as 3-methacrylamidopropyltrimethylammonium chloride and 3-acrylamidopropyltrimethylammonium chloride) and (meth)acryloxyalkyltrimethylammonium salts, such as 2-acryloxyethyltrimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride, 3-acryloxy-2-hydroxypropyltrimethylammonium chloride, and 2-acryloxyethyltrimethylammonium methyl sulfate.

[0062] The grafted copolymer further comprises optional monomer units that can be copolymerized with cationic or cationically ionizable monomers. Although it may be possible to ionize these monomers under certain conditions, they are typically uncharged and neutral ("neutral monomers"). These neutral monomers have a polymerizable group for use during graft polymerization. The polymerizable group may be the same as or different from the polymerizable group on the cationic or cationically ionizable monomer. There may be one or more neutral monomers.

[0063] The neutral monomer may have a functional group or more than one functional group in addition to the polymerizable group. For neutral monomers with more than one functional group, these functional groups may be the same or different. Some functional groups may allow the neutral monomer to dissolve or disperse in water. Some functional groups may be hydrophilic after polymerization. Useful functional groups include hydroxyl, alkyl, aryl, ether, ester, epoxy, amide, isocyanate, or cyclic functional groups. The neutral monomer may contain a spacer group between the polymerizable group and the functional group. The neutral monomer may contain an oligomeric or polymeric functional group. In some embodiments, the polymerizable group and the functional group may be the same group.

[0064] Examples of epoxy-containing neutral monomers include glycidyl (meth)acrylate, thioglycidyl (meth)acrylate, 3-(2,3-epoxypropoxy)phenyl (meth)acrylate, 2-[4-(2,3-epoxypropoxyl)phenyl]-2-(4-(meth)acryloyloxy-phenyl)propane, 4-(2,3-epoxypropoxyl)cyclohexyl (meth)acrylate, 2,3-epoxycyclohexyl (meth)acrylate, and 3,4-epoxycyclohexyl (meth)acrylate, and combinations thereof. Examples of hydroxyl-containing monomers include N-hydroxyethyl (meth)acrylate, poly(ethylene glycol) (meth)acrylate, poly(propylene glycol) (meth)acrylate, N-hydroxyethyl (meth)acrylamide, 2-hydroxypropyl (meth)acrylamide, N-hydroxypropyl (meth)acrylate, 2-hydroxy-3-phenoxypropyl (meth)acrylate, and combinations thereof. Examples of suitable amide monomers include N-vinylcaprolactam, N-vinylacetamide, N-vinylpyrrolidone, (meth)acrylamide, mono- or di-N-alkyl substituted acrylamide, and combinations thereof. Examples of suitable ether monomers include poly(ethylene glycol) (meth)acrylate, poly(propylene glycol) (meth)acrylate, 2-ethoxyethyl (meth)acrylate, ethylene glycol methyl ether (meth)acrylate, N-3-methoxypropyl (meth)acrylamide, di(ethylene glycol) methyl ether (meth)acrylate, poly(ethylene glycol) phenyl ether (meth)acrylate, 2-phenoxyethyl (meth)acrylate, other alkyl ether (meth)acrylates and alkyl ether (meth)acrylamides, tetrahydrofurfuryl (meth)acrylate, and combinations thereof.

[0065] The process for preparing the functionalized nonwoven layer includes providing a nonwoven substrate, exposing the nonwoven substrate to ionizing radiation in an inert atmosphere, and then contacting the exposed substrate with a solution or suspension containing a grafting monomer to graft polymerize the monomer onto the nonwoven substrate.

[0066] In the first step, the nonwoven substrate is exposed to ionizing radiation in an inert atmosphere. Exemplary forms of ionizing radiation include electron beam (e-beam), gamma radiation, X-rays, and other forms of electromagnetic radiation. The inert atmosphere is generally an inert gas, such as nitrogen, carbon dioxide, helium, argon, etc., with a minimal amount of oxygen. The dose delivered by the ionizing radiation source can occur in a single dose or can be multiple doses that build up to a desired level. One or more layers of the nonwoven substrate may be subjected to ionizing radiation.

[0067] After the irradiation step, the irradiated nonwoven substrate is contacted with an aqueous monomer solution or aqueous monomer suspension. By "contacting" it is meant contacting the irradiated nonwoven substrate with the monomer solution or monomer suspension. It can also be described as the irradiated nonwoven substrate being saturated, absorbed, or coated with the monomer solution. The monomer solution may only partially fill the void volume of the nonwoven substrate, or the nonwoven substrate may be contacted with much more solution than is necessary to completely fill the void volume. The monomer contact step is also carried out in an inert atmosphere. This atmosphere may be the same as or different from the atmosphere in the chamber in which the substrate is irradiated. The chamber may be the same as or different from the chamber in which the substrate is irradiated. The monomer solution remains in contact with the nonwoven substrate for a time sufficient to graft polymerize with some, most, or substantially all of the monomers in the monomer solution. Once the nonwoven substrate has been in contact for the desired time, the nonwoven substrate carrying the grafted polymer may be removed from the inert atmosphere. EXAMPLES

[0068] [Table 1] Grafting Solution Grafting solution A was prepared as a monomer solution containing 24.4 wt % NVP, 8.8 wt % GMA, and 19.4 wt % MAPTAC in deionized water.

[0069] Grafting solution B was prepared as a monomer solution containing 18.3 wt % NVP, 6.6 wt % GMA, and 14.6 wt % MAPTAC in deionized water.

[0070] Grafting solution C was prepared as a monomer solution containing 12.2 wt % NVP, 4.4 wt % GMA, and 9.7 wt % MAPTAC in deionized water.

[0071] Method A. Determination of Basis Weight, Effective Fiber Diameter (EFD), Solidity, and Pore Size of Functionalized Nonwovens The measurements of basis weight, EFD, solidity, and pore size of the functionalized nonwoven fabric were determined according to the following procedure. Sample disks (13.33 cm diameter) were punched out of the functionalized nonwoven sheet, and then each disk was individually rinsed by immersing it in a 2 L bath of deionized water for 15 minutes. The rinsing procedure was repeated three more times, using fresh deionized water for each rinsing step. Each rinsed disk was dried in an oven at 70° C. for at least 4 hours. During the drying step, a weight (approximately 100 g) was placed on each disk to prevent edge curling. The resulting dried functionalized nonwoven fabric samples were characterized (basis weight, EFD, solidity, pore size) according to the methods and formulas described above. For each measured or calculated value, the results were reported as the average of three independent trials (n=3), along with the calculated standard deviation (SD).

[0072] Regarding the solidity (a) formula, the fiber density (ρ f ) measured value was calculated based on the density of the polypropylene substrate (0.91 g / cm 3 ) and the density of the grafted copolymer (1.07 g / cm3) adjusted by the weight ratio of the polypropylene substrate to the grafted copolymer of the test sample. 3) (Equation 1). The weight ratio of polypropylene substrate to copolymer was determined by comparing the basis weight of the nonwoven fabric before the grafting step with the basis weight of the corresponding dried functionalized nonwoven fabric.

[0073] The density of the grafted copolymer (D GCP ) in the solid state 13 The mol% of the monomer components (NVP, MAPTAC, GMA) of the grafted copolymers were determined using C NMR (ssNMR) and converted to weight % (wt.%) values. The density values ​​of each monomer component (monomer density: D NVP =1.04g / cm 3 , D MAPTAC =1.067g / cm 3 , D GMA =1.07g / cm 3 ) were adjusted (multiplied) by the corresponding component wt.% values, and the three resulting adjusted density values ​​were summed (Equation 2). Formula 1:

number

number

[0074] Method B. Determination of the Metanil Yellow Dynamic Charge Capacity (MY DCC) of functionalized nonwoven fabrics. Functionalized nonwoven disks were prepared according to method A. The dynamic charge capacity of the disks was determined using the charged organic dye Metanil Yellow as the target molecule in the challenge solution. The challenge solution used had a Metanil Yellow concentration of 160 mg / L (160 ppm). The challenge solution was prepared by dissolving 3.2 g Metanil Yellow, 93.98 g Sodium phosphate anhydrous, 46.64 g Sodium phosphate monobasic, and 163.63 g NaCl in 20 L of deionized water. The challenge solution was used within 2 days of preparation. If necessary, the amount of Metanil Yellow reagent used to prepare the challenge solution was adjusted based on the purity of the reagent so that the challenge solution contained 160 ppm Metanil Yellow. Analytical standard grade Metanil Yellow (≧98.0%, product number 44426 from Sigma-Aldrich Company, St. Louis, MO) was used to calibrate the reagent purity. A buffer solution for preconditioning the test assembly was also prepared having the same formulation as the challenge solution, except that metanil yellow was not included.

[0075] The filtration test assembly included a clear polycarbonate body section (47 mm inner diameter) with a screw-on cap attached to the top of the body section. The cap included an inlet port and a vent port. The bottom of the body section included an outlet port with a stopcock. A pressure sensor was placed upstream of the inlet port. A polyamide membrane (0.2 micrometer grade) was placed at the bottom of the body section. A stack containing two functionalized nonwoven disks (each 47 mm in diameter and punched from disks prepared according to Method A) was placed in the assembly on top of the membrane. In the assembly, the nonwoven disk was sandwiched between two PTFE seal rings, each of which included a knife edge on the inner diameter to bite into the nonwoven. The resulting subassembly was secured in place using an O-ring. The disk stack had a frontal surface area of ​​0.00097 m 2The cap was attached to the body portion and a PendoTech normal flow filtration system (PendoTech Company, Princeton, NJ) was connected to the inlet port. A Hach Model 2100 AN turbidimeter (Hach Company, Loveland, CO) with a 455 nm light filter and a flow-through cell was connected to the outlet port and used to measure the Metanil Yellow concentration in the filtrate. Metanil Yellow solutions with concentrations of 0.8 ppm, 4 ppm, and 8 ppm were prepared as test standards. The end point of the charge capacity measurement was set at 5% breakthrough (8 ppm) of the Metanil Yellow solution. The fluid flow rate was 15 mL / min. The preconditioning buffer was allowed to flow through the assembly for approximately 5 minutes before pumping the challenge solution.

[0076] The volume of challenge solution that passed through the test assembly until the end point (i.e., the breakthrough volume) was measured, and the dynamic charge capacity (mg / g) of the functionalized nonwoven samples was calculated according to Equation 3. For each functionalized nonwoven, the MY DCC was reported along with the standard deviation (SD), calculated as the average value from three independent trials (n=3). Formula 3:

number

[0077] Method C. Preparation of Harvest Cell Culture Fluid (HCCF) - Chinese Hamster Ovary (CHO) Cell Cultures CHO cells were cultured in suspension from frozen cell stocks to a series of flask seed cultures in a CO2 incubator, followed by a fed-batch process using a Wave bioreactor (GE Healthcare, Chicago, IL) and 50 L disposable cell bags equipped with pH control and dissolved oxygen monitoring. Cell culture media was obtained from Fujifilm Irvine Scientific (Santa Ana, CA). CHO cell cultures were harvested during stationary phase, typically on day 12.

[0078] Viable cell density and viability were measured using a hemocytometer. The harvested cell culture fluid was mixed with 10% (vol / vol) trypan blue solution and then loaded into a disposable hemocytometer. Viable and dead cells were counted under a microscope. The packed cell volume percentage (PCV%) was measured using a PCV tube (product number Z760986, Sigma-Aldrich Company), and 200 microliters of harvested cell culture fluid (HCCF) was added to the PCV tube. The tube was centrifuged at 2500 relative centrifugal force (rcf) for 1 minute. The PCV% was calculated by the solid volume relative to the volume of HCCF.

[0079] Method D. Harvested Cell Culture Fluid (HCCF) Clarification The filter housing capsule (FIG. 7) was tested for HCCF clarification using a PendoTech normal flow filtration system (PendoTech Company) connected to the capsule via the capsule's luer lock inlet. The plastic filter capsule had an upper housing and a lower housing that were mated together in the final structure by ultrasonic welding. The upper housing had a luer lock inlet port and a luer lock vent. The lower housing had a luer lock outlet port centered in the middle of the lower housing. A disk (2.54 cm diameter) of TYPAR 3161L polypropylene spunbond nonwoven fabric (10 mil thick, obtained from Fiberweb, Inc., Old Hickory, TN) was placed at the bottom of the lower housing. A disk (2.54 cm diameter) of MICRO-PES Flat Type 2F polyethersulfone membrane (obtained from 3M Company, St. Paul, MN) with a nominal pore size of 0.2 micrometers was placed on top of the nonwoven fabric layer. The nonwoven and membrane layers were ultrasonically welded to the bottom inner surface of the lower housing at the edges. A stack of four functionalized nonwoven layers (2.54 cm diameter disks) was then placed on top of the membrane. A polypropylene spacer ring (25.4 mm OD, 21.84 mm ID, 50 mils thick) was inserted between the second and third nonwoven layers. The upper and lower housings were mated together and ultrasonically welded to form the completed filter capsule. Ultrasonic welding was accomplished by placing the mating assembly in a jig so that the outer surface of the lower housing was in contact with an ultrasonic horn. A Branson 20 kHz ultrasonic welder (Model 2000xdt, Emerson Electric Company, St. Louis, MO), black booster, and horn with 2.5x gain were used. Fixed parameters were set as follows: air pressure 80 psi, drop rate 10%, step amplitude 80%-60%, 50 Joule staging, weld time 2 seconds, and trigger force 200 lbf to initiate the weld. The weld energy was held constant at 450 Joules to produce samples with consistent compression levels.The housing assembly was placed under the ultrasonic horn such that the longitudinal axis of the housing was aligned with the axis of the horn. When the welding process was initiated, the horn lowered onto the lower housing, compressing the housing and internal components until a force of 200 lbf was reached. The overall outer diameter of the completed capsule was approximately 3.7 cm, and the overall height, including the inlet, outlet, and vent ports, was approximately 4.8 cm. The frontal surface area of ​​the disk stack was 3.2 cm. 2 It was.

[0080] The HCCF was stirred throughout the procedure. At the start of filtration, the filter capsule headspace was filled with HCCF at a specific flow rate by opening the vent and closing the outlet. After filling the capsule headspace with HCCF, the vent was closed and the outlet was opened to allow collection of clarified cell culture fluid (CCCF). The differential pressure was monitored during the clarification process. Clarification was stopped when the differential pressure reached 5 psid (pounds per square inch differential pressure). The collected CCCF volume and CCCF turbidity were recorded. The throughput (L / m ) was calculated based on the CCCF volume collected per unit surface area of ​​the filter. 2 ) was calculated. The turbidity of the filtrate was measured in nephelometric turbidity units (NTU) using an Orion AQ4500 turbidity meter (Thermo Fisher Scientific, Waltham, MA).

[0081] Method E. Preparation of AAV2 Feeding Solution HEK293-F cells suspended in Gibco LV-MAX production medium (Thermo Fisher Scientific, Waltham, MA) were grown in an incubator using 2.8 L shake flasks with constant shaking at 90 rpm (revolutions per minute). The incubator was maintained at 37°C and 8% CO2. Cell densities of approximately 2 × 10 6 When cells / mL was reached, the transfection cocktail was prepared and added to the shake flask.

[0082] The transfection cocktail consisted of plasmid pAAV2-RC2 vector (part number VPK-422), pHelper vector (part number 340202), (plasmid obtained from Cell Biolabs, San Diego, CA), and FECTOVIR®-AAV transfection reagent (Polyplus Transfection, New York, NY). The transfection cocktail was prepared by first adding pHelper vector and pAAV2-RC2 vector at a molar ratio of 62% to 38%, and the total plasmid amount was adjusted to be 1 microgram of plasmid mixture per million HEK cells used for transfection. DMEM (Dulbecco's Modified Eagle Medium, obtained from Thermo Fisher Scientific) was then added to the cocktail such that a final concentration of 5% DMEM (volume / volume) was achieved after the cocktail was added to the cell culture flask (i.e., the volume / volume calculation for DMEM was adjusted based on the total cell culture volume). After the addition of DMEM, the cocktail was mixed and then 1 μl of FectoVIR-AAV transfection reagent was added for every 1 μg of plasmid mixture in the cocktail. The cocktail was gently mixed and then incubated at room temperature for 45 minutes. Following the incubation step, the completed transfection cocktail was gently mixed and then added dropwise to the flask containing the cell culture. After the addition of the transfection cocktail, the cells were grown in an incubator (37°C and 8% CO2) for 72 to 96 hours to induce the production of AAV2.

[0083] Cell viability was measured using a hemocytometer. Harvested cell culture fluid was mixed with 25% (vol / vol) trypan blue solution and then loaded into a disposable hemocytometer. Viable and dead cells were counted under a microscope. Turbidity measurements of transfected cell cultures were determined in nephelometric turbidity units (NTU) using an ORION AQ4500 turbidity meter (Thermo Fisher Scientific). AAV2-transfected cell cultures were 6.2 × 10 6It had a cell density value of cells / mL, cell viability of 74%, and turbidity of 560 NTU.

[0084] TRITON X-100 detergent (obtained from Promega Corporation, Madison, WI) was added to the transfected cell cultures to achieve a final detergent concentration of 0.1 wt.% and then shaken at 90 rpm for 2 h in an incubator set at 37°C and 8% CO2. The conductivity of the lysed samples was adjusted to 20 mS / cm with 5 M sodium chloride solution. Conductivity was measured using a calibrated Orion Star A215 pH / Conductivity Benchtop Multiparameter Meter (Thermo Fisher Scientific). After cell lysis, the resulting AAV2 feeding solution contained 8.5 x 10 11 It had an AAV2 capsid content of capsids / mL, a total DNA content of 4230 ng / mL, and a turbidity of 165 NTU.

[0085] The AAV2 capsid content of both the pre-filtration feed solution and the filtrate obtained after filtration were measured using the ProGen AAV2 Xpress ELISA kit (obtained from American Research Products, Inc., Waltham, Mass.) according to the manufacturer's instructions. The DNA concentration of both the pre-filtration feed solution and the filtrate obtained after filtration were measured using the QUANT-IT PICOGREEN dsDNA assay (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0086] Preparation of functionalized nonwoven fabric A (FNW-A) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter (EFD) of 16 micrometers, a basis weight of 200 grams per square meter (gsm), a solidity of 10%, and a calculated average pore size of 47.4 micrometers) was grafted with nitrogen-purged grafting solution C. The nonwoven substrate was unwound and passed through an electron beam (Electrocure, Energy Science, Inc, Wilmington, MA) set at a potential of 300 kV to deliver a total dose of 7 Mrad. The environment in the electron beam chamber was purged with nitrogen. The web was then transported directly to a nitrogen-purged saturation step with monomer solution. The web was then wound in a purged atmosphere. The web was left in the purged atmosphere for a minimum of 60 minutes, after which the web was exposed to air. The web was then unwound and transported into a tank of deionized water at a speed of 10 feet per minute for approximately 8 minutes. After exiting the tank, the web was flushed multiple times by passing a salt water solution (NaCl) through the web using a vacuum belt. A small amount of glycerin was added to the salt water solution in the final flushing step. The unwound web was dried until the moisture content of the web was less than 14% by weight. The web was then wound on a spindle. The grafted article was labeled Functionalized Nonwoven A (FNW-A). The properties of FNW-A are reported in Table 2. Disks of FNW-A (2.54 cm diameter) were punched from the web.

[0087] Preparation of functionalized nonwoven fabric B (FNW-B) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 14 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 41.5 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was labeled Functionalized Nonwoven B (FNW-B). The properties of FNW-B are reported in Table 2. Disks of FNW-B (2.54 cm diameter) were punched from the web.

[0088] Preparation of functionalized nonwoven fabric C (FNW-C) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 12 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 35.6 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was labeled Functionalized Nonwoven C (FNW-C). The properties of FNW-C are reported in Table 2. Disks of FNW-C (2.54 cm diameter) were punched from the web.

[0089] Preparation of functionalized nonwoven fabric D (FNW-D) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 10 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 29.6 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was labeled Functionalized Nonwoven D (FNW-D). The properties of FNW-D are reported in Table 2. Disks of FNW-D (2.54 cm diameter) were punched from the web.

[0090] Preparation of functionalized nonwoven fabric E (FNW-E) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 8 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 23.7 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was labeled Functionalized Nonwoven E (FNW-E). The properties of FNW-E are reported in Table 2. Disks of FNW-E (2.54 cm diameter) were punched from the web.

[0091] Preparation of functionalized nonwoven fabric F (FNW-F) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 6 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 17.8 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was labeled Functionalized Nonwoven F (FNW-F). The properties of FNW-F are reported in Table 2. Disks of FNW-F (2.54 cm diameter) were punched from the web.

[0092] Preparation of functionalized nonwoven fabric G (FNW-G) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 4.2 micrometers, a basis weight of 100 gsm, a solidity of 8.2%, and a calculated average pore size of 14.2 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was labeled Functionalized Nonwoven G (FNW-G). The properties of FNW-G are reported in Table 2. The MY DCC was determined by Method B using four functionalized nonwoven discs instead of two discs. Discs (2.54 cm diameter) of Functionalized Nonwoven G were punched from the web. [Table 2]

[0093] Preparation of functionalized nonwoven fabric H (FNW-H) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 14 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 41.5 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven H (FNW-H). The properties of FNW-H are reported in Table 3. Disks of FNW-H (2.54 cm diameter) were punched from the web.

[0094] Preparation of functionalized nonwoven fabric I (FNW-I) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 12 micrometers, basis weight of 200 gsm, solidity of 10%, calculated average pore size of 35.6 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution B was used instead of grafting solution C. The properties of FNW-I are reported in Table 3. Discs of FNW-I (2.54 cm diameter) were punched from the web.

[0095] Preparation of functionalized nonwoven fabric J (FNW-J) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 10 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 29.6 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven J (FNW-J). The properties of FNW-J are reported in Table 3. Disks (diameter 2.54 cm) of FNW-J were punched from the web.

[0096] Preparation of functionalized nonwoven fabric K (FNW-K) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 8 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 23.7 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven K (FNW-K). The properties of FNW-K are reported in Table 3. Discs of FNW-K (2.54 cm diameter) were punched from the web.

[0097] Preparation of functionalized nonwoven fabric L (FNW-L) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 6 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 17.8 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven L (FNW-L). Properties of FNW-L are reported in Table 3. Disks of FNW-L (2.54 cm diameter) were punched from the web.

[0098] Preparation of functionalized nonwoven fabric M (FNW-M) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 4.2 micrometers, a basis weight of 100 gsm, a solidity of 8.2%, and a calculated average pore size of 14.2 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven M (FNW-M). The properties of FNW-M are reported in Table 3. The MY DCC was determined by Method B using four functionalized nonwoven discs instead of two discs. Discs (2.54 cm diameter) of Functionalized Nonwoven M were punched from the web. [Table 3]

[0099] Preparation of functionalized nonwoven fabric N (FNW-N) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 14 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 41.5 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven N (FNW-N). The properties of FNW-N are reported in Table 4. Discs of FNW-N (2.54 cm diameter) were punched from the web.

[0100] Preparation of functionalized nonwoven fabric O (FNW-O) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 12 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 35.6 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven O (FNW-O). The properties of FNW-O are reported in Table 4. Discs of FNW-O (2.54 cm diameter) were die-cut from the web.

[0101] Preparation of functionalized nonwoven fabric P (FNW-P) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 10 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 29.6 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven P (FNW-P). The properties of FNW-P are reported in Table 4. Discs of FNW-P (2.54 cm diameter) were punched from the web.

[0102] Preparation of functionalized nonwoven fabric Q (FNW-Q) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 8 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 23.7 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven Q (FNW-Q). The properties of FNW-Q are reported in Table 4. Disks of FNW-Q (2.54 cm diameter) were punched from the web.

[0103] Preparation of functionalized nonwoven fabric R (FNW-R) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 6 micrometers, basis weight of 200 gsm, solidity of 10%, and calculated average pore size of 17.8 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven R (FNW-R). The properties of FNW-R are reported in Table 4. Discs of FNW-R (2.54 cm diameter) were punched from the web.

[0104] Preparation of functionalized nonwoven fabric S (FNW-S) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 4.2 micrometers, a basis weight of 100 gsm, a solidity of 8.2%, and a calculated average pore size of 14.2 micrometers) was grafted using the same procedure described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted article was labeled Functionalized Nonwoven S (FNW-S). The properties of FNW-S are reported in Table 4. The MY DCC was determined by Method B using four functionalized nonwoven discs instead of two discs. Discs (2.54 cm diameter) of Functionalized Nonwoven G were punched from the web. [Table 4]

[0105] Example (Ex1). A filtration capsule was assembled as described in Method D using two disks of FNW-B and two disks of FNW-F. The orientation of the disks from capsule inlet to outlet was two disks of FNW-B followed by two disks of FNW-F. Chinese Hamster Ovary (CHO) cell culture fluid was prepared to evaluate the filtration performance of the assembled capsule (above). The harvested cell culture fluid (HCCF) had a packed cell volume percentage (PCV%) of 3.2%, a viability of 25.5%, and a turbidity of 1879 NTU. The capsule was tested according to Method D (above) at a flow rate of 200 liters per square meter per hour (LMH). The resulting clarified cell culture fluid (CCCF) was collected until the differential pressure cross capsule reached 5 psi. The throughput was 44.4 L / m 2 and the CCCF turbidity was 3.15 NTU.

[0106] Example 2 (Ex2). A filtration capsule was assembled as described in Method D using two disks of FNW-B and two disks of FNW-G. The orientation of the disks from the capsule inlet to the outlet was two disks of FNW-B followed by two disks of FNW-G. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 30.3 L / m 2 and the CCCF turbidity was 2.98 NTU.

[0107] Example 3 (Ex3). A filtration capsule was assembled as described in Method D using two disks of FNW-B, one disk of FNW-D, and one disk of FNW-E. The orientation of the disks from capsule inlet to outlet was FNW-B / FNW-B / FNW-D / FNW-E. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 37.8 L / m 2 and the CCCF turbidity was 3.40 NTU.

[0108] Example 4 (Ex4). A filtration capsule was assembled as described in Method D using two disks of FNW-C and two disks of FNW-E. The orientation of the disks from the capsule inlet to the outlet was two disks of FNW-C followed by two disks of FNW-E. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 53.4 L / m 2 and the CCCF turbidity was 3.08 NTU.

[0109] Example 5 (Ex5). A filtration capsule was assembled as described in Method D using two disks of FNW-C, one disk of FNW-E, and one disk of FNW-F. The orientation of the disks from capsule inlet to outlet was FNW-C / FNW-C / FNW-E / FNW-F. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 54.4 L / m 2 and the CCCF turbidity was 3.58 NTU.

[0110] Example 6 (Ex6). A filtration capsule was assembled as described in Method D using three disks of FNW-E and one disk of FNW-G. The orientation of the disks from capsule inlet to outlet was FNW-E / FNW-E / FNW-E / FNW-G. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 46.6 L / m 2 and the CCCF turbidity was 3.03 NTU.

[0111] Comparative example A (CExA). A four-disk stack of FNW-A was used to assemble a filtration capsule as described in Method D. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 13.4 L / m 2 An insufficient amount of CCCF was collected for turbidity measurements. Membrane fouling was observed.

[0112] Comparative example B (CExB). A four-disk stack of FNW-B was used to assemble a filtration capsule as described in Method D. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 22.2 L / m 2 and the CCCF turbidity was 5.76 NTU. Membrane fouling was observed.

[0113] Comparative example C (CExC). A four-disk stack of FNW-F was used to assemble a filtration capsule as described in Method D. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 23.1 L / m 2 and the CCCF turbidity was 2.79 NTU. Caking of the cell culture material was observed on the top surface of the filter stack.

[0114] Comparative Example D (CExD). A four-disk stack of FNW-G was used to assemble a filtration capsule as described in Method D. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 1. The throughput was 0.6 L / m 2 An insufficient amount of CCCF was collected for turbidity measurements. Caking of cell culture material was observed on the top surface of the filter stack.

[0115] The filtration results for Examples 1-6 (Ex1-Ex6) and Comparative Examples A-D (CExA-CExD) are summarized in Table 5. The filtration capsules of Ex1-Ex6 had lower CCCF turbidity and significantly higher throughput than the comparative filtration capsules. In addition, the comparative filtration capsules had either caking of cell culture material on the top surface of the filter stack or fouling of the membrane section downstream from the filter stack. [Table 5]

[0116] Example 7 (Ex7). A filtration capsule was assembled as described in Method D using two disks of FNW-B, one disk of FNW-D, and one disk of FNW-E. The orientation of the disks from capsule inlet to outlet was FNW-B / FNW-B / FNW-D / FNW-E. Chinese Hamster Ovary (CHO) cell culture fluid was prepared to evaluate the filtration performance of the assembled capsule. The harvested cell culture fluid (HCCF) had a packed cell volume percentage (PCV%) of 8.0%, a viability of 80.0%, and a turbidity of 2483 NTU. The capsule was tested according to Method D above at a flow rate of 200 liters per square meter per hour (LMH). The resulting clarified cell culture fluid (CCCF) was collected until the differential pressure cross capsule reached 5 psi. The throughput was 59.4 L / m 2 and the CCCF turbidity was 4.81 NTU.

[0117] Example 8 (Ex8). A filtration capsule was assembled as described in Method D using two disks of FNW-C, one disk of FNW-E, and one disk of FNW-F. The orientation of the disks from capsule inlet to outlet was FNW-C / FNW-C / FNW-E / FNW-F. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 7. The throughput was 61.6 L / m 2 and the CCCF turbidity was 4.98 NTU.

[0118] Comparative example E (CExE). A four-disk stack of FNW-A was used to assemble a filtration capsule as described in Method D. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 7. The throughput was 15.3 L / m 2 An insufficient amount of CCCF was collected for turbidity measurements. Membrane fouling was observed.

[0119] Comparative example F (CExF). A four-disk stack of FNW-F was used to assemble a filtration capsule as described in Method D. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 7. The throughput was 15.3 L / m 2 An insufficient amount of CCCF was collected for turbidity measurements. Caking of cell culture material was observed on the top surface of the filter stack.

[0120] Comparative example G (CExG). A four-disk stack of FNW-G was used to assemble a filtration capsule as described in Method D. The filtration performance of the assembled capsule was determined using the procedure and HCCF described in Example 7. The throughput was 0 L / m 2 Caking of the cell culture material was observed on the top surface of the filter stack.

[0121] The filtration results for Examples 7-8 (Ex7-Ex8) and Comparative Examples E-G (CExE-CExG) are summarized in Table 6. The filtration capsules of Ex7-Ex8 had lower CCCF turbidity and significantly higher throughput than the comparative filtration capsules. In addition, the comparative filtration capsules had either caking of cell culture material on the top surface of the filter stack or fouling of the membrane section downstream from the filter stack. [Table 6]

[0122] Example 9 (Ex9). A plastic filtration capsule was used. The capsule consisted of a sealed circular housing. The capsule housing was prepared from two halves (top and bottom halves) that were mated and sealed together around the periphery after the filtration element was inserted into the inner cavity of the lower housing. The fluid inlet and vent ports were located at the top of the housing and the fluid outlet port was located at the bottom of the housing. The outlet port was centered in the middle of the lower housing surface.

[0123] Two disks (27 mm diameter) of TYPAR 3161L polypropylene spunbond nonwoven (10 mil thick, obtained from Fiberweb, Inc., Old Hickory, TN) were placed at the bottom of the lower housing. A single disk (27 mm diameter) of MICRO-PES Flat Type 2F polyethersulfone membrane (obtained from 3M Company) with a nominal pore size of 0.2 micrometers was placed on top of the nonwoven layer. The nonwoven layer and membrane layer were ultrasonically welded at the edges to the bottom inner surface of the lower housing. A stack of four functionalized nonwoven layers (27 mm diameter) was then placed on top of the membrane. The stack included one disk of functionalized nonwoven C, two disks of functionalized nonwoven E, and two disks of functionalized nonwoven G. The orientation of the disks from the capsule inlet to the outlet was FNW-C / FNW-E / FNW-E / FNW-G / FNW-G. A polypropylene spacer ring (25.4 mm OD, 21.84 mm ID, 50 mils thick) was inserted between the third and fourth nonwoven layers (i.e., between the FNW-E and FNW-G disks). The upper and lower housings were mated and ultrasonically welded using a Branson 20 kHz ultrasonic welder (Model 2000xdt, Emerson Electric Company, St. Louis, MO) to form the finished filter capsule.

[0124] The overall outer diameter of the finished capsule was approximately 4.3 cm, and the overall height, including the inlet, outlet, and vent ports, was approximately 5.9 cm. The effective filtration area of ​​the capsule was 3.2 cm. 2 and the bed volume of the nonwoven media was 2.1 mL.

[0125] Example 10 (Ex10). The finished capsules prepared according to Example 9 were attached to a PendoTech Normal Flow Filter Screening System (PendoTech Company, Princeton, NJ) through the inlet port of the capsule. The capsules were then subjected to a constant flux of 200 LMH at 54 L / m 2The media disk was dried by flushing with Tris-acetate buffer (50 mM, pH 7.5, conductivity 4 mS / cm) to a throughput of 200 µL, then flushing with air (to a pressure difference of 5 psid). The AAV2-containing cell lysate feed solution prepared in method E was then pumped through the capsule at a constant flux of 140 LMH to a pressure difference of 15 psid. The filtrate was collected and analyzed for throughput, AAV2 capsid content, total DNA content, and turbidity. A total of two capsules were tested. The average throughput was 249 L / m 2 (standard deviation = 69). The results for AAV2 capsid content and total DNA content are provided in Tables 7 to 9. [Table 7] [Table 8] [Table 9]

Claims

1. 1. A charged depth filter for removing cells and / or cell debris from a biopharmaceutical feedstock, comprising: a first functionalized nonwoven layer having a first calculated pore size and a first dynamic charge capacity; a second functionalized nonwoven layer having a second calculated pore size and a second dynamic charge capacity, disposed after the first functionalized nonwoven layer in the direction of flow of the biopharmaceutical feedstock; A charged depth filter, wherein the first calculated pore size is larger than the second calculated pore size and the first dynamic charge capacity is smaller than the second dynamic charge capacity.

2. 10. The charged depth filter of claim 1, wherein for the first functionalized nonwoven layer, the first calculated pore size is from 40.8 μm to 65.0 μm and the first dynamic charge capacity is from 150 MY mg DCC / g to 300 MY mg DCC / g, and for the second functionalized nonwoven layer, the second calculated pore size is from 5.0 μm to less than 40.8 μm and the second dynamic charge capacity is from greater than 300 MY mg DCC / g to 650 MY mg DCC / g.

3. 10. The charged depth filter of claim 1, wherein for the first functionalized nonwoven layer, the first calculated pore size is from 55.0 μm to 65.0 μm and the first dynamic charge capacity is from 150 MY mg DCC / g to 300 MY mg DCC / g, and for the second functionalized nonwoven layer, the second calculated pore size is from 5.0 μm to less than 55.0 μm and the second dynamic charge capacity is from 300 MY mg DCC / g to 650 MY mg DCC / g.

4. 4. The charged depth filter of claim 1, 2, or 3, wherein the first functionalized nonwoven layer and the second functionalized nonwoven layer are grafted with a copolymer comprising interpolymerized monomer units that are quaternary ammonium-containing monomers, amide-containing monomers, and epoxy-containing monomers.

5. 5. The charged depth filter of claim 4, wherein the first functionalized nonwoven layer and the second functionalized nonwoven layer are grafted with a copolymer comprising interpolymerized monomer units that are 3-methacrylamidopropyltrimethylammonium chloride, N-vinylpyrrolidone, and glycidyl methacrylate.

6. 1. A charged depth filter for removing cells and / or cell debris from a biopharmaceutical feedstock, comprising: a first functionalized nonwoven layer having a first calculated pore size and a first dynamic charge capacity; a second functionalized nonwoven layer having a second calculated pore size and a second dynamic charge capacity, positioned after the first functionalized nonwoven layer in the direction of flow of the biopharmaceutical feedstock; a third functionalized nonwoven layer having a third calculated pore size and a third dynamic charge capacity, disposed after the second functionalized nonwoven layer in the direction of flow of the biopharmaceutical feedstock; a charged depth filter, wherein the first calculated pore size is larger than the second calculated pore size, the second calculated pore size is larger than the third calculated pore size, the first dynamic charge capacity is smaller than the second dynamic charge capacity, and the second dynamic charge capacity is smaller than the third dynamic charge capacity.

7. 7. The charged depth filter of claim 6, wherein for the first functionalized nonwoven layer, the first calculated pore size is from 40.8 μm to 65.0 μm and the first dynamic charge capacity is from 150 mg MY DCC / g to 300 mg MY DCC / g; for the second functionalized nonwoven layer, the second calculated pore size is from 20.6 μm to less than 40.8 μm and the second dynamic charge capacity is from greater than 300 mg MY DCC / g to 475 mg MY DCC / g; and for the third functionalized nonwoven layer, the third calculated pore size is from 5.0 μm to less than 20.6 μm and the third dynamic charge capacity is from greater than 300 mg MY DCC / g to 650 mg MY DCC / g.

8. 7. The charged depth filter of claim 6, wherein for the first functionalized nonwoven layer, the first calculated pore size is from 55.0 μm to 65.0 μm and the first dynamic charge capacity is from 150 mg DCC / g to 300 mg DCC / g; for the second functionalized nonwoven layer, the second calculated pore size is from 20.6 μm to less than 55.0 μm and the second dynamic charge capacity is from 200 mg DCC / g to 475 mg DCC / g; and for the third functionalized nonwoven layer, the third calculated pore size is from 5.0 μm to less than 20.6 μm and the third dynamic charge capacity is from greater than 300 mg DCC / g to 650 mg DCC / g.

9. 9. The charged depth filter of claim 6, 7, or 8, wherein the third functionalized nonwoven layer is water permeable.

10. 9. The charged depth filter of claim 6, 7, or 8, wherein on a plot of dynamic charge capacity versus calculated pore size, a water permeability line extends through point 1, which has a calculated pore size of 5.0 μm and a dynamic charge capacity of 300 MY DCC mg / g, through point 2, which has a calculated pore size of 20.6 μm and a dynamic charge capacity of 525 MY DCC mg / g, and wherein the third functionalized nonwoven layer has point 3 on the plot of third dynamic charge capacity and third calculated pore size, with point 3 located below the water permeability line.

11. 9. The charged depth filter of claim 6, 7, or 8, wherein the first functionalized nonwoven layer, the second functionalized nonwoven layer, and the third functionalized nonwoven layer are grafted with a copolymer comprising interpolymerized monomer units that are a quaternary ammonium-containing monomer, an amide-containing monomer, and an epoxy-containing monomer.

12. 12. The charged depth filter of claim 11, wherein the first functionalized nonwoven layer, the second functionalized nonwoven layer, and the third functionalized nonwoven layer are grafted with a copolymer comprising interpolymerized monomer units that are 3-methacrylamidopropyltrimethylammonium chloride, N-vinylpyrrolidone, and glycidyl methacrylate.

13. 10. A method for clarifying a biopharmaceutical feedstock containing whole cells and cell debris in one step, comprising feeding the biopharmaceutical feedstock having a packed cell volume (PCV) of 2% to 12% through a charged depth filter according to claim 1 or 6 to form a clarified biopharmaceutical feedstock.

14. The throughput of the biopharmaceutical feedstock through the charged depth filter is 30 L / m ２ Up to 200 L / m ２ The method of claim 13, wherein

15. 14. The method of claim 13, wherein the clarified biopharmaceutical feedstock has a turbidity of less than 50 NTU and the biopharmaceutical feedstock has a turbidity of between 1,000 NTU and 10,000 NTU.

16. The method of claim 13 , wherein the cell comprises a mammalian cell.

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