Charged depth filter for treatment of biotechnological manufacturing processes

Abstract

A charged depth filter for removing cells and / or cell debris from a biopharmaceutical feedstock is provided, the depth filter having 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, the second functionalized nonwoven layer being located after the first functionalized nonwoven layer in the direction of biopharmaceutical feedstock flow, and wherein the first calculated pore size is greater than the second calculated pore size, and the first dynamic charge capacity is less than the second dynamic charge capacity.

Description

Background Technology

[0001] Due to their specificity for target diseases, monoclonal antibodies are the dominant form in the biopharmaceutical industry. The therapeutic antibody market is growing rapidly, with many drug phenotypes under regulatory review. Over the past 30 years, regulatory agencies in the US and EU have approved approximately 100 monoclonal antibodies, and the next generation of antibody therapies is expected to proceed at an even faster pace in the next decade. These include antibody-drug conjugates, biosimilars, engineered antibodies, bispecific antibodies, antibody fragments, antibody-like proteins, and more. Chinese hamster ovary (CHO) cells are the most commonly used cell line in the industry, based on their ability to adapt and grow in suspensions, their ability to grow in serum-free, chemically defined media, their high productivity, and their capacity for post-translational modification. CHO cells account for >70% of the protein therapeutics produced, but these biologics can be produced in several systems, including microbial, plant, insect, and other mammalian cells.

[0002] Proteins of interest in biopharmaceuticals include any of a wide range of naturally or recombinantly expressed proteins. Other biologics that can be used as therapeutic vectors include viral particles such as adenoviruses, adeno-associated viruses (AAVs), or lentiviruses; bacterial phages or viral particles; exosomes; or synthetic lipid nanoparticles. Besides 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 cells. Regardless of cell type or therapeutic vector, the clarification and purification challenges associated with isolating the biologics of interest from host cell components and other components produced by host cells can be similar. Summary of the Invention

[0003] In biopharmaceutical manufacturing, once cell culture medium is harvested from a bioreactor and sent downstream to a clarification process, it is necessary to separate the target biomolecules of interest, such as monoclonal antibodies (mAbs), viral particles, or other therapeutic vectors, from the feedstock containing cells, cell debris, and / or colloidal particles. Preliminary clarification steps, typically using centrifugation, deep filtration, microfiltration (tangential flow filtration), or combinations thereof, are used to remove whole cells and large cell debris from the harvested cell culture medium.

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

[0005] When used for initial clarification, centrifuges require extensive cleaning procedures between runs to ensure no cross-contamination between consecutive batches during production. Therefore, disposable, single-use devices are needed to replace the primary centrifugation clarification step, eliminating the risk of cross-contamination during batch-to-batch transfers and between therapeutic biomolecules of interest.

[0006] Tangential flow microfiltration can be used as a preliminary clarification step in place of centrifugation. However, tangential flow microfiltration membranes are generally sensitive to membrane fouling, and they also require extensive cleaning processes to prevent cross-contamination between runs and during transfers between therapeutic biomolecules of interest.

[0007] Alternatively, conventional depth filters (using size exclusion based solely on media pore size) can be used as a preliminary clarification step to remove cells and debris based on the size of the depth filter channels and filter aids in the depth filter media. However, as cell densities increase from 6 million cells / mL to greater than 50 million cells / mL, the flux achieved by conventional depth filtration becomes impractical in manufacturing environments. Therefore, what is needed is a single-use preliminary clarification step that can replace centrifugation, tangential flow microfiltration, and conventional depth filters as a preliminary clarification step.

[0008] The applicant has discovered that charged depth filters with at least two functionalized nonwoven layers, each with a different effective pore size and dynamic charge capacity, can accomplish this task, and are particularly effective for cell cultures with high cell densities. By carefully managing the gradients of both the effective pore size and dynamic charge capacity as feedstock moves through the layers of the depth filter, a depth filter can be constructed that does not clump with whole cells and large cell debris on the first layer, nor clog the first layer, while still effectively ensuring that the final layer of the depth filter (such as a membrane layer) is not clogged by debris. Both of these conditions lead to a significant reduction in flux, rendering the device unsuitable for use in biopharmaceutical manufacturing processes.

[0009] In particular, the applicant has discovered that the pore size of the continuous layers in the charged depth filter should be reduced, and the dynamic charge capacity of the continuous layers in the charged depth filter should be increased. If the pore size of the first layer of the functionalized nonwoven fabric encountered by the feed material in the depth filter is too small or the dynamic charge capacity is too large, it will easily agglomerate with whole cells and / or large cell debris, thereby significantly reducing the flux. Similarly, due to the failure to reduce the pore size of the continuous layers and increase the dynamic charge capacity of the continuous layers, too much debris will slide through the functionalized nonwoven layers, causing clogging of the downstream filter element, which may optionally be added as the final filter layer in the charged depth filter.

[0010] Therefore, in one aspect, the present invention relates to a charged depth filter for removing cells and / or cell debris from a biopharmaceutical feedstock, the depth filter having a first functionalized nonwoven layer having a first calculated pore size and a first dynamic charging capacity; a second functionalized nonwoven layer having a second calculated pore size and a second dynamic charging capacity, the second functionalized nonwoven layer being located after the first functionalized nonwoven layer in the direction of biopharmaceutical feedstock flow, and wherein the first calculated pore size is larger than the second calculated pore size, and the first dynamic charging capacity is smaller than the second dynamic charging capacity. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of a media stack for an electrically charged depth filter having four functionalized nonwoven layers (FNW-C / FNW-C / FNW-E / FNW-F), followed by a membrane layer, and then a nonwoven spunbond layer located between the inlet and outlet of the electrically charged depth filter.

[0012] Figure 2 This is an image of the functionalized nonwoven layer FNW-B. The nonwoven fabric before functionalization has an effective fiber diameter of 14µm, a density of 10%, and a density of 200g / m². 2 The basis weight and calculated pore size are 41.5 µm. After grafting, the effective fiber diameter is 21.6 µm, the density is 14.2%, and the basis weight is 302.0 g / m². 2 The calculated pore size was 50.5µm, and the MY DCC was 165.0mg / g.

[0013] Figure 3 This is an image of the functionalized nonwoven layer FNW-F. The nonwoven fabric before functionalization has an effective fiber diameter of 6µm, a density of 10%, and a density of 200g / m². 2 The basis weight and calculated pore size are 17.8 µm. After grafting, the effective fiber diameter is 9.1 µm, the density is 17.8%, and the basis weight is 355.8 g / m². 2 The calculated pore size was 17.9 µm, and the MY DCC was 407.4 mg / g.

[0014] Figure 4 This is an image of a stack of charged depth filter media, consisting of a larger pore functionalized nonwoven fabric, FNW-B, and a membrane, cut after cell culture clarification. The cell culture easily penetrates all four functionalized nonwoven layers, and cell and cell debris residue covers the membrane surface. This media stack performs poorly because too much debris contaminates the membrane layers.

[0015] Figure 5This is an image of the stack of charged depth filter media, consisting of a smaller pore functionalized nonwoven fabric, FNW-F, and a membrane, after the cell culture has clarified. The cell culture contaminates the upper layer and cannot penetrate all the functionalized nonwoven layers. The third and fourth layers are not utilized, and the membrane surface is clean with no residual cells or cell debris.

[0016] Figure 6A and Figure 6B The top surface of the first functionalized nonwoven fabric (FNW-C) in the media stack after filtering CHO cell culture is shown. Cells, debris, and / or DNA adhere to the charged fibers of this nonwoven layer and appear as spheres on the outer surface of the functionalized fibers.

[0017] Figure 6C The bottom surface of the first functionalized nonwoven fabric (FNW-C) in the media stack after filtering CHO cell culture is shown. Cells, debris, and / or DNA adhere to the charged fibers of this nonwoven layer and appear as spheres on the outer surface of the fibers.

[0018] Figure 6D The top surface of the second functionalized nonwoven fabric (FNW-C) in the media stack after filtering CHO cell culture is shown. Cells, debris, and / or DNA adhere to the charged fibers of this nonwoven layer and appear as spheres on the outer surface of the fibers.

[0019] Figure 6E The bottom surface of the second functionalized nonwoven fabric (FNW-C) in the media stack after filtering CHO cell cultures is shown. Cells, debris, and / or DNA adhere to the charged fibers of this nonwoven layer and appear as spheres on the outer surface of the fibers.

[0020] Figure 6F The top surface of the functionalized nonwoven fabric (FNW-E) in the media stack after filtering CHO cell culture is shown. Cells, debris, and / or DNA adhere to the charged fibers of this nonwoven layer and appear as spheres on the outer surface of the fibers.

[0021] Figure 6G The bottom surface of the functionalized nonwoven fabric (FNW-E) in the media stack after filtering CHO cell culture is shown. Cells, debris, and / or DNA adhere to the charged fibers of this nonwoven layer and appear as spheres on the outer surface of the fibers.

[0022] Figure 6H The top surface of the functionalized nonwoven fabric (FNW-F) in the media stack after filtering CHO cell culture is shown. Cells, debris, and / or DNA adhere to the charged fibers of this nonwoven layer and appear as spheres on the outer surface of the fibers.

[0023] Figure 6I The bottom surface of the functionalized nonwoven fabric (FNW-F) in the media stack after filtering CHO cell cultures is shown. Cells, debris, and / or DNA adhere to the charged fibers of this nonwoven layer and appear as spheres on the outer surface of the fibers.

[0024] Figure 6J The top surface of a 0.2 µm membrane layer in the media stack is shown after filtering CHO cell cultures. As can be seen, very few cells, debris, and / or DNA are present on the surface of this membrane.

[0025] Figure 7 A perspective view of an electrically charged depth filter with a housing having an inlet, an outlet, an optional vent, and a media stack (not shown) located between the inlet and the outlet for clarifying cell cultures. Detailed Implementation

[0026] Throughout this document, values ​​expressed in a range format should be interpreted flexibly to include not only the numerical values ​​explicitly listed as the limits of the range but also all individual numerical values ​​or subranges encompassed within that range, as if each numerical value and subrange were explicitly listed. For example, the range “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not only about 0.1% to about 5% but also 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 indicated range. Unless otherwise specified, the expression “about X to Y” has the same meaning as “about X to about Y”. Similarly, unless otherwise specified, the expression “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z”.

[0027] In this document, unless the context clearly indicates otherwise, the terms “an,” “a,” or “the / described” are used to include one or more (kinds). Unless otherwise specified, the term “or” is used to mean a non-exclusive “or.” The expressions “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.” Furthermore, it should be understood that the wording or terms used herein and not otherwise defined are for illustrative purposes only and are not restrictive. Section headings are intended to aid in reading the document and should not be construed as restrictive; information relating to a section heading may appear within or outside that particular section.

[0028] As used herein, the term “about” allows for a degree of variability in the value or range. For example, within 10%, 5%, or 1% of the limits of the value or range, and includes precisely stated values ​​or ranges.

[0029] As used herein, the term “substantially” means most or most, 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%. The term “substantially free” as used herein may mean none or present in negligible amounts such that the amount of material present does not affect the material properties of the composition containing the material, such that the composition contains about 0% to about 5% by weight of the material, or about 0% to about 1% by weight, or about 5% by weight or less, or less than or equal to about 4.5% by weight, 4% by weight, 3.5% by weight, 3% by weight, 2.5% by weight, 2% by weight, 1.5% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight, 0.4% by weight, 0.3% by weight, 0.2% by weight, 0.1% by weight, 0.01% by weight, or about 0.001% by weight or less.

[0030] As used herein, "layer" refers to the thickness of a material through which the fluid to be treated flows, wherein the material in the layer is entirely composed of the same material. A layer can be a monolithic layer formed of the same material thickness. Alternatively, a layer can have one or more discontinuous sheets of the same material, which are stacked within the layer to form its thickness. For example, a typical facial tissue layer is usually made of thin sheet material made of two separate sheets of paper placed face to face, and these two separate sheets can be easily separated from each other because they are usually held together by a weak mechanical bond in the form of a curled curve.

[0031] As used herein, "one or more sheets" refers to a single thickness of material that can be processed by conventional conversion processing operations, such as, but not limited to, winding, folding, cutting, or stacking. Typically, a sheet is the thickness of material after the forming process has been completed on a web-making machine. Subsequently, one or more sheets of the same material can be stacked to form a layer. For example, a nonwoven fabric can be made into a single sheet on a forming machine and wound into a roll. Then, as the nonwoven roll is passed longitudinally through a conversion processing machine, the nonwoven roll can be unfolded and folded laterally by a folding plate, and then the double-sheet layer is cut into discs by a cutting die to form a circular layer of nonwoven material with two discontinuous sheets.

[0032] As used herein, a “functionalized layer” is a layer that attracts target particles or molecules by an attractive force, such as electrostatic force, caused by the presence of one or more chemical moieties, ligands, or functional groups at the surface of the layer, which are different from the material forming the body of the layer, which primarily provides its structural shape and integrity. The chemical moieties, ligands, or functional groups are specifically designed to attract target particles or molecules to the surface of the functionalized layer. A functionalized layer can be formed by coating or grafting a porous layer with ligands, monomers, or polymers designed to molecularly attract target particles or molecules. Alternatively, a functionalized layer can be formed by providing a surface-modified polymer or chemical moieties in a formulation used to prepare such a layer, the surface-modified polymer or chemical moieties being located at the surface of the layer during its formation, resulting in the presence of chemical groups on the surface of the layer designed to attract target particles or molecules. In some embodiments, the attractive force between functional groups on the surface of the functionalized layer is electrostatic, and the chemical moieties, ligands, or polymers present on the surface of the functionalized layer are electrostatically charged. The functionalized layer may have a positive charge and attract negatively charged particles, i.e., anion exchange chromatography separation, or the functionalized layer may have a negative charge and attract positively charged particles, i.e., cation exchange chromatography separation. In other embodiments, the attractive force may be van der Waals forces, and target particles or molecules are attracted to functional groups on the surface of the functionalized layer through relatively concentrated or sparse polarizable or hydrogen-bonded portions (i.e., hydrophobic interactions). Furthermore, the attractive force may include a combination of electrostatic and van der Waals forces (i.e., a mixed mode). Functionalized materials suitable for functionalized layers in charged depth filter devices are manufactured by Pall, Millipore, and Sartorious and sold under the trademark Mustang. ® Q, NatriFlo ® HD-Q and Sartobind ® Q. The functionalized layer suitable for charged depth filter devices can be a nonwoven fabric, a membrane, or other suitable material. Preferred functionalized nonwoven materials are manufactured by 3M Company and disclosed in U.S. Patent No. 9,821,276, entitled "Nonwoven Article Grafted with Copolymer". Preferred functionalized membranes are manufactured by 3M Company and disclosed in U.S. Patent Nos. 9,650,470 and 1,001,7461, entitled "Method of Making Ligand Functionalized Substrates". The full text of all three mentioned patents is incorporated herein by reference.

[0033] As used herein, a “nonfunctionalized layer” is a layer that does not have an attractive chemical portion (e.g., a charged chemical portion, ligand, or functional group) that is coated, grafted, or surface-positioned with a material different from the host material of the forming layer.

[0034] As used in this article, "medium stack" refers to all the layers of material that the fluid to be treated flows through within the housing as it moves from the inlet through the housing of the charged depth filter to the outlet.

[0035] As used herein, "membrane" refers to a synthetic liquid-permeable membrane, comprising a sheet of material having multiple pores or an interconnected network of pores that allow fluid to pass through. Such membranes include polymer membranes typically prepared via phase inversion processes, 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 achieved by introducing the membrane of the homogeneous solution into a solvent-free bath (referred to as diffusion-induced phase separation) or a solvent-free atmosphere (referred to as vapor-induced phase separation) or by changing the temperature of the homogeneous solution (referred to as thermally induced phase separation). Alternatively, pores can be formed in the polymer sheet by a stretching process or by an irradiation process (track etching membrane). Membranes can have pore sizes ranging from about 0.1 micrometers to about 20 micrometers in diameter (microporous membranes) or smaller than about 0.1 micrometers (membranous membranes). Suitable polymers for forming the film include cellulose acetate, nitrocellulose, cellulose esters, polysulfones (including bisphenol A polysulfone and polyethersulfone), polyacrylonitrile, polyamides (e.g., nylon-6 and nylon-6,6), polyimide, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl chloride, and ethylene-trifluorochloroethylene copolymers.

[0036] Electrostatic Depth Filter

[0037] See Figure 1 and Figure 7 The charged depth filter 8 includes a housing 10 having an inlet 16, an outlet 18, an optional vent 20, and containing layers 25, 31, 33, 35, 37, and 39 located within the housing. Figure 1 The medium stack comprises at least two layers of functionalized nonwoven fabric disposed between the inlet and the outlet, such that the cell culture to be filtered passes from the inlet 16 through the medium stack to the outlet 18. For example, the edges of the medium stack are sealed by compression or thermoplastic welding to minimize or eliminate any leakage of cell culture to the outlet without first passing through the medium stack. Any suitable housing can be used for the charged depth filter to accommodate and seal the medium stack. Various housing and medium stack volumes are typically available to suit laboratory-scale research for commercial production.

[0038] The dielectric stack has at least a first functionalized nonwoven layer 25 having a first calculated pore size and a first dynamic charging capacity; and a second functionalized nonwoven layer 33 having a second calculated pore size and a second dynamic charging capacity, the second functionalized nonwoven layer being located after the first functionalized nonwoven layer in the direction of biopharmaceutical raw material flow; and wherein the first calculated pore size is larger than the second calculated pore size, and the first dynamic charging capacity is smaller than the second dynamic charging capacity.

[0039] The housing can be of any suitable size, appropriately scaled according to the surface area of ​​the medium within it. Typically, laboratory-scale devices will be relatively small and have a low retention volume for handling limited quantities of fluid. Pilot-scale and production-scale devices will contain correspondingly larger volumes of medium to handle larger volumes of fluid per run. For example, a laboratory-scale device might have a 3.2 cm³ / h² ... 2 Up to 25cm 2 The surface area of ​​the medium, for a pilot-scale device, is 340 cm². 2 Up to 1020cm 2 And the production scale of the equipment is 2300cm 2 Up to 16100cm 2 Other housing sizes and media volumes are available to suit specific application needs. Suitable housings are manufactured by 3M and are used in the 3M Emphaze AEX hybrid purifier product line. Housings of similar sizes and designs can be used to accommodate the media stacks of this invention.

[0040] A suitable housing is disclosed in U.S. Patent Application No. 62 / 792166, filed January 14, 2019, entitled “Sample Size Chromatography Device,” the entire contents of which are incorporated herein by reference. Figure 7As best shown, housing 10 is formed by joining upper housing 12 to lower housing 14. The housing has an inlet 16, an outlet 18, and an optional vent 20. A media stack in a chamber is disposed between inlet 16 and outlet 18, such that fluid from inlet 16 enters the internal chamber, passes through the media stack, and exits through outlet 18. The chamber is in fluid communication with inlet 16 and the optional vent 20, such that any air in the chamber can be purged out of vent 20. A Luer lock connector (not shown) can be attached to vent 20 and functions as a valve to purge air from the chamber until liquid from inlet 16 begins to exit vent 20 and the valve closes. A cylindrical protrusion 32 with opposing transverse tabs 80 extends from the housing and has a tapered hole for attaching the Luer lock connector to the inlet, outlet, and vent. Longitudinal ribs 58 are spaced along the periphery to provide enhanced grip when manipulating the housing.

[0041] Another suitable housing having a sealing membrane and a spacer ring is disclosed in U.S. Patent Application No. 63 / 023488, filed May 12, 2020, entitled “Membrane sealing layer and spacer ring for a virus removal chromatographic separation device”, the entire contents of which are incorporated herein by reference.

[0042] Dielectric stack

[0043] The dielectric 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 has a first calculated aperture and a first dynamic charging capacity; and the second functionalized nonwoven layer has a second calculated aperture and a second dynamic charging capacity, the second functionalized nonwoven layer being located after the first functionalized nonwoven layer in the direction of biopharmaceutical raw material flow, wherein the first calculated aperture is larger than the second calculated aperture, and the first dynamic charging capacity is smaller than the second dynamic charging capacity.

[0044] As used herein, the terms "first layer" and "second layer" do not necessarily mean that these layers must be exactly the first and second layers that the fluid passes through as it moves through the media stack. Rather, they indicate their relative position to each other, as the fluid will first flow through the first layer, then the second layer, and preceding and / or intermediate layers may also be present in the media stack. For example, the media stack may include layer A, then the first layer, layer B, layer C, followed by the second layer, and then layer D in the direction of fluid flow. Similarly, other identified layers, such as the third functionalized nonwoven layer, are treated in the same manner.

[0045] When the first functionalized nonwoven layer 25 has a first calculated pore size from 40.8µm to 65.0µm and a first dynamic charge capacity from 150 MYDCC mg / g to 300 MY DCC mg / g, and is combined with a second functionalized nonwoven layer 33 having a second calculated pore size from 5.0µm to less than 40.8µm and a second dynamic charge capacity from greater than 300 MY DCC mg / g to 650 MY DCC mg / g, the charged depth filter using two functionalized nonwoven layers showed better performance, as observed in the embodiments. Alternatively, when the first functionalized nonwoven layer 25 has a first calculated pore size from 55.0µm to 65.0µm and a first dynamic charge capacity from 150MY DCC mg / g to 300 MY DCC mg / g, the two-layer charged depth filter can achieve better performance when combined with a second functionalized nonwoven layer 233 having a second calculated pore size from 5.0µm to less than 55.0µm and a second dynamic charge capacity from greater than 300 MY DCC mg / g to 650 MY DCC mg / g.

[0046] When the first functionalized nonwoven layer 25 has a first calculated pore size from 40.8µm to 65.0µm and a first dynamic charge capacity from 150 MYDCC mg / g to 300 MY DCC mg / g, followed by a second functionalized nonwoven layer 33 having a second calculated pore size from 20.6µm to less than 40.8µm and a second dynamic charge capacity from 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 from 5.0µm to less than 20.6µm and a third dynamic charge capacity from greater than 300 MYDCC mg / g to 650 MY DCC mg / g, the charged depth filter using three functionalized nonwoven layers exhibits better performance, as observed in the embodiments. Alternatively, when the first functionalized nonwoven layer 25 has a first calculated pore size from 55.0µm to 65.0µm and a first dynamic charge capacity from 150 MY DCC mg / g to 300 MY DCC mg / g, and is combined with a second functionalized nonwoven layer 33 having a second calculated pore size from 20.6µm to less than 55.0µm and a second calculated pore size from 200 MY DCC mg / g to 475 MY DCC mg / g, and subsequently combined with a third functionalized nonwoven layer 35 having a third calculated pore size from 5.0µm to less than 20.6µm and a third dynamic charge capacity from greater than 300 MY DCC mg / g to 650 MY DCC mg / g, the three-layer charged depth filter can achieve better performance.

[0047] When using a three-layer functionalized nonwoven fabric, better performance is observed when the third functionalized nonwoven layer is permeable. If the pore size becomes too small due to grafting, the membrane becomes too closed. The permeability boundary of a functionalized nonwoven medium used in the embodiments can be plotted on an XY plot of dynamic charge capacity MY DCC mg / g versus calculated pore size in µm. The approximate location of this permeability line extends through points 1 and 2, with point 1 having a calculated pore size of 5.0 µm and a dynamic charge capacity of 300 MY DCC mg / g, and point 2 having a calculated pore size of 20.6 µm and a dynamic charge capacity of 525 MY DCC mg / g. Functionalized nonwoven fabrics with plotted data points above this line tend to be impermeable and are less preferred. Functionalized nonwoven fabrics with plotted data points below this line tend to be permeable and are more preferred.

[0048] Typically, the dielectric stack will include additional functionalized layers, non-functionalized layers, and / or membrane layers. The same layers can be repeated within the charged depth filter to increase the capacity for a specific fragment size before changing the pore size and / or dynamic charging capacity. Depending on the construction, the dielectric stack of the charged depth filter can have 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more layers, but typically fewer than 25 layers.

[0049] The charged depth filter may include an optional membrane layer. This membrane layer is located between the final functionalized layer and the housing outlet and can be used to increase capsule back pressure to enhance filtration uniformity. It may be selected from water-permeable membranes, including but not limited to polyethersulfone, polysulfone, cellulose, regenerated cellulose, and polyamide membranes.

[0050] The charged depth filter may include an optional nonfunctionalized nonwoven layer. This nonfunctionalized nonwoven layer is located between the optional membrane layer and the shell outlet and can be used to protect membrane integrity during capsule assembly and filtration. The nonfunctionalized nonwoven layer may be selected from nonwoven materials, including but not limited to polypropylene, polyethylene, polymethylpentene, and polyethylene terephthalate.

[0051] like Figure 1 As shown, the preferred structure of this dielectric laminate comprises six layers. The first functionalized nonwoven layer 25 after grafting has an effective fiber diameter of 18.9 µm and a fiber density of 272.7 g / m³. 2The first functionalized nonwoven layer has a basis weight, a density of 13.5%, a first calculated pore size of 45.6 µm, and a first dynamic charge capacity (MY DCC) of 291.7 mg / g. This first functionalized nonwoven layer is followed by a repeating first functionalized nonwoven layer 31 with the same properties, meaning there are two first functionalized nonwoven layers in this charged depth filter. This repeating first functionalized nonwoven layer 31 is followed by a second functionalized nonwoven layer 33. The grafted second functionalized nonwoven layer 33 has an effective fiber diameter of 12.1 µm and a density of 356.6 g / m². 2 The second functionalized nonwoven layer 33 has a basis weight, a density of 16.3%, a second calculated pore size of 25.5µm, and a second dynamic charge capacity (MY DCC) of 365.3mg / g. Following the second functionalized nonwoven layer 33 is a third functionalized nonwoven layer 35. The grafted third functionalized nonwoven layer 35 has an effective fiber diameter of 9.1µm and a density of 355.8g / m². 2 The basis weight, density of 17.8%, third calculated pore size of 17.9µm, and third dynamic charge capacity (MY DCC) of 407.4mg / g are shown. Following the third functionalized nonwoven layer 35 is membrane layer 37. This membrane layer is a 0.2µm PES membrane. Following membrane layer 37 is a nonfunctionalized nonwoven layer 39. The nonfunctionalized nonwoven layer 39 is a polypropylene spunbond layer.

[0052] Now refer to Figures 6A to 6J Micrographs of each layer in this six-layer structure were observed after clarifying CHO cell cultures with 3.2% PCV. As shown, cells, debris, and / or DNA adhere to the charged fibers of the functionalized nonwoven layer and appear as spheres on the outer surface of the fibers. The calculated pore size and dynamic charging capacity of each subsequent layer were controlled so that the surface of the functionalized nonwoven layer or membrane layer did not become clogged or agglomerated, while still ensuring that each layer in this charged depth filter removed debris of appropriate size, as demonstrated by the top and bottom surfaces of the layers with debris adhered to the functionalized grafted fibers. This structure ensures good flux and good debris removal.

[0053] Now refer to Figure 4 This illustrates the progression of "overly open" layers in the media stack of this charged depth filter. As seen, images of the media stack of the charged depth filter, including the larger-pore functionalized nonwoven fabric, FNW-B, and the membrane after cell culture clarification, are presented. Cell cultures readily penetrate all four functionalized nonwoven layers (stained portions of the discs), and cell and cell debris residues cover the surface of the membrane layers. This media stack performs poorly because too much debris contaminates the membrane layers (the rightmost disc), resulting in a significant reduction in flux.

[0054] Now refer to Figure 5This illustrates the progression of "too tight" layers in the media stack of this charged depth filter. As seen, images of the media stack of the charged depth filter, including the smaller pore functionalized nonwoven fabric, FNW-F, and the membrane after cell culture clarification, are presented. Cell culture contaminates the upper functionalized nonwoven layer (stained portions of the discs) and cannot penetrate all of the functionalized nonwoven layers (limited to unstained discs 3 and 4 from the left). Layers 3 and 4 are not utilized, and the membrane surface (the rightmost disc) is clean without any residual cells or cell debris. This media stack performs poorly because too much debris contaminates the initial functionalized nonwoven layer, resulting in a significant reduction in flux.

[0055] Single-stage approach to cell clarification

[0056] In biopharmaceutical manufacturing, clarification is an initial 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 microparticles before further downstream purification steps. For mammalian cell cultures (e.g., Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK-293) cells, young hamster kidney (BHK21) cells, NSO mouse myeloma cells, or PER.C6), clarification is crucial. ® The insoluble contaminants to be removed (human cells) range in size from over 10 micrometers for whole cells, from approximately 1 to 9 micrometers for cell debris, and less than 1 micrometer for colloidal debris. Other target molecules of interest can be produced by insect and bacterial cell lines, and the charged depth filter of this invention can also be used to clarify those materials.

[0057] Current clarification processes 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 via filtration are implemented in two or three stages: removing large particles in a first stage and then removing smaller particles in a second or third stage. Optimization of these processes or filtration stages for successfully clarifying biotherapeutic agents from cell cultures 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).

[0058] Recent advances in cell culture media, cell engineering, and bioreactor design have led to significant increases in cell density (e.g., greater than 100 million cells / mL or greater than approximately 20% of the collected cell volume in perfusion-based systems) and mAb titers (e.g., greater than 10 g / L). This significant increase in cell density presents challenges to clarification methods when using centrifugation and / or conventional depth filtration methods, resulting in lower yields and throughput.

[0059] The charged depth filter of this invention provides a mechanism for clarification through filtration that differs from the conventional size-based exclusion methods employed in conventional depth filters. In the charged depth filter of this invention, whole-cell and cell debris contaminants are removed through charge-based separation and size exclusion. Chromatographic separation techniques, such as packed resin column chromatography and membrane chromatography, are not designed for such applications based on their small porous matrix and operating device design. Figures 6A to 6J The SEM images shown demonstrate that the charge-based removal of cells and debris using the functionalized nonwovens presented in this invention is not diffusion-limited due to the high porosity within the functionalized nonwoven matrix. Negatively charged soluble and insoluble contaminants (e.g., cells, debris, DNA, and host cell proteins) in cell culture media are removed through electrostatic interactions with the positively charged surfaces of the functionalized nonwovens, resulting in a one-stage fiber chromatographic separation process.

[0060] The charged depth filter described in this disclosure is designed with a gradient structure based on both effective pore size and dynamic charge. This filter can clarify high-cell-density cultures in a single-stage process at 2% to 12% of the collection cell volume PCV (10 million to 60 million cells / mL), more preferably 3% to 11% PCV (15 million to 55 million cells / mL), or even more preferably 3% to 9% PCV (15 million to 45 million cells / mL). The flux can be 30 L / mL during this process. 2 Up to 200L / m 2 (liters / meter) 2 Flow rates range from 50 LMH to 600 LMH (liters per meter). 2 The throughput of high-cell-density cultures is 75 LMH to 400 LMH per hour, more preferably 100 LMH to 250 LMH per hour. Compared with conventional depth-filtering methods, the enhanced throughput of high-cell-density cultures reduces the manufacturing footprint.

[0061] High-density cell cultures containing whole cells and cell debris typically have a turbidity range of 1,000 to 10,000 turbidimetric units (NTU). The single-stage clarification method using the described charged depth filter can reduce the turbidity of high-density cell cultures to 50 NTU or less, 20 NTU or less, 15 NTU or less, or 10 NTU or less.

[0062] The charged depth filter of the present invention is preferably designed to be water-permeable, and the pretreatment rinsing requires only water. Using water for pretreatment reduces costs and is easy to operate.

[0063] The advantages of the single-stage clarification process of the charged depth filter of the present invention include, but are not limited to, increased product yield, reduced manufacturing footprint, a consistently low turbidity clarified fluid, and user-friendly operation. These combined benefits achieve optimal process economy for the manufacture of therapeutic drugs.

[0064] Parameters of nonfunctionalized and functionalized nonwovens

[0065] Properties of interest for both unfunctionalized and functionalized nonwovens (e.g., copolymer-grafted nonwovens) include basis weight, effective fiber diameter (EFD), density, and pore size. These properties can be determined for nonwovens before or after functionalization.

[0066] The fibers of nonfunctionalized nonwoven substrates typically have an effective fiber diameter of about 3 micrometers to 20 micrometers. The basis weight of the nonfunctionalized substrate is preferably about 10 g / m³. 2 Up to 400g / m 2 More preferably at approximately 80g / m 2 Up to 250g / m 2 Within the range. The average thickness of the nonfunctionalized substrate is preferably from about 0.1 mm to 10 mm, and more preferably from about 0.25 mm to 5 mm.

[0067] The bulk of functionalized or nonfunctionalized nonwovens is measured by density, which is a parameter defining the proportion of solids in the web volume. A lower density value indicates greater web bulk. Density is a dimensionless fraction, usually represented by α.

[0068]

[0069] Basis weight m f It is the mass per unit surface area (functionalized or unfunctionalized), and ρ f It refers to fiber density (functionalized or unfunctionalized). L 非织造物 The thickness of the nonwoven fabric (functionalized or unfunctionalized). The density of the nonwoven fabric can be measured before or after functionalization.

[0070] The fiber density (ρ) of the functionalized copolymer grafted fibers f The fiber density of the functionalized copolymer grafted fibers was determined by method A in the following examples. The fiber density can also be determined by a modified form of method A, wherein the molar ratio of the substrate and copolymer components is obtained entirely by solid-state carbon-13 NMR measurement, and the molar ratio is converted to a weight ratio. When the nonwoven substrate contains a mixture of two or more fibers, the same L... 非织造物 The density of each fiber was measured individually, and these individual densities were added together to obtain the density α of the web.

[0071] "Effective fiber diameter" or "EFD" refers to the apparent diameter of fibers in a nonwoven fiber web, as determined by an air permeation test. In the air permeation test, air is passed through a web sample of known thickness at a surface velocity of 5.3 cm / s at 1 atmosphere and room temperature, and the corresponding pressure drop is measured. Based on the measured pressure drop, the effective fiber diameter is calculated as shown below: Davies, CN, "Separation of Dust and Particles Carried by Air" The Separation of Airborne Dust and Particles The method proposed by the Institution of Mechanical Engineers (Proceedings 1B, 1952) can be used to calculate the EFD of nonwovens before or after functionalization.

[0072] The term "calculated aperture" is related to the arithmetic mean fiber diameter and the web tightness, and can be determined by the following formula: where D is the calculated aperture, d f Let α be the arithmetic mean fiber diameter, and α be the web density.

[0073]

[0074] The calculated pore size of the nonwoven fabric can be determined before or after functionalization. Before functionalization, the nonwoven substrate preferably has a calculated pore size of 1 micrometer to 50 micrometers.

[0075] The dynamic charge capacity (DCC) of the functionalized nonwoven substrate was determined using m-amino yellow test solution via method B in the examples and reported as MY DCC (m-amino yellow dynamic charge capacity).

[0076] nonwoven base mesh

[0077] The nonwoven substrate is a nonwoven web, which may include a nonwoven web manufactured by any generally known process for preparing a nonwoven web. As used herein, the term "nonwoven web" refers to a fabric having a structure in which single fibers or filaments are randomly and / or unidirectionally inserted in a felt-like manner. For example, fiber nonwoven webs can be made by methods such as carding, air spinning, wet spinning, jet spinning, spunbond, electrospinning, or meltblown techniques (such as melt spinning or meltblown), or combinations thereof. Spunbond fibers are typically small-diameter fibers that are formed by extruding molten thermoplastic polymer in filament form through multiple fine, typically circular capillaries via a spinneret, wherein the diameter of the extruded fibers decreases rapidly. Meltblown fibers are typically formed by extruding molten thermoplastic material as molten threads or filaments into a high-speed, typically heated gas stream (e.g., air) through multiple fine, usually circular, die capillaries. This gas stream thins the molten thermoplastic filaments to reduce their diameter. The meltblown fibers are then transported by the high-speed gas flow and deposited onto a collection surface to form a randomly distributed meltblown fiber web. Any of these nonwoven webs can be made from a single type of fiber or from two or more fibers that differ in type and / or thickness of thermoplastic polymer.

[0078] Suitable polyolefins for manufacturing nonwoven webs include, but are not limited to, polyethylene, polypropylene, poly(1-butene), copolymers of ethylene and propylene, α-olefin copolymers (such as 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.

[0079] Further details regarding the manufacturing method of the nonwoven web of the present invention can be found in Wente's "Superfine Thermoplastic Fibers" (48 INDUS. ENG. CHEM 1342(1956)) or Wente et al.'s "Manufacture of Superfine Organic Fibers" (Naval Research Laboratories Report No. 4364, 1954). Available methods for preparing the nonwoven substrate are described in US RE39399 (Allen), US Patent No. 3849241 (Butin et al.), US Patent No. 7374416 (Cook et al.), US Patent No. 4936934 (Buehning), and US Patent No. 6230776 (Choi).

[0080] Functionalized nonwoven layers

[0081] The functionalized nonwoven layer comprises the aforementioned nonwoven substrate and a graft copolymer comprising comonomer units, at least one of which is cationic or can be made cationic in a solution at a suitable pH (“cationically ionizable”). Suitable functionalized nonwoven webs are disclosed in U.S. Patent No. 9,821,276, entitled “Nonwoven Article Grafted with Copolymer,” published November 21, 2017, which is incorporated herein by reference.

[0082] Cationic or cationically ionizable monomers may include quaternary ammonium monomers and tertiary amine monomers. One or more cationic or cationically ionizable monomers may be used. Monomers typically contain polymerizable functional groups as well as cationic or cationically ionizable groups. In some monomers, the polymerizable group and the cationic group may be the same group. Polymerizable groups include vinyl, vinyl ether, (meth)acryloyl, (meth)acrylamido, allyl, cyclic unsaturated monomers, polyfunctional monomers, vinyl esters, and other readily polymerizable functional groups.

[0083] Useful (meth)acrylates include, for example, trimethylaminoethyl methacrylate, trimethylaminoethyl methacrylate, triethylaminoethyl methacrylate, triethylaminoethyl methacrylate, trimethylaminopropyl methacrylate, trimethylaminopropyl methacrylate, dimethylbutylaminopropyl methacrylate, diethylbutylaminopropyl methacrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, and 3-(dimethylamino)propyl methacrylate.

[0084] Exemplary (meth)acrylamides include, for example, 3-(trimethylamino)propylmethacrylamides, 3-(triethylamino)propylmethacrylamides, 3-(ethyldimethylamino)propylmethacrylamides, and n-[3-(dimethylamino)propyl]methacrylamides. Preferred quaternary salts of these (meth)acryloyl monomers include, but are not limited to, (meth)acrylamidoalkyltrimethylammonium salts (e.g., 3-methacrylamidopropyltrimethylammonium chloride and 3-acrylamidopropyltrimethylammonium chloride) and (meth)acryloyloxyalkyltrimethylammonium salts (e.g., 2-acryloyloxyethyltrimethylammonium chloride, 2-methacryloyloxyethyltrimethylammonium chloride, 3-methacryloyloxy-2-hydroxypropyltrimethylammonium chloride, 3-acryloyloxy-2-hydroxypropyltrimethylammonium chloride, and 2-acryloyloxyethyltrimethylammonium sulfate).

[0085] The graft copolymer also includes optional monomer units that can be copolymerized with cationic or cationically ionizable monomers. Although these monomers can be ionized under certain conditions, they are generally uncharged; they are neutral (“neutral monomers”). These neutral monomers have polymerizable groups used in the graft polymerization. These polymerizable groups may be the same as or different from the polymerizable groups on the cationic or cationically ionizable monomers. There may be one or more neutral monomers.

[0086] In addition to the polymerizable group, the neutral monomer may have one or more functional groups. In the case of a neutral monomer having more than one functional group, these functional groups may be the same or different. Some functional groups enable 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 oligomeric or polymeric functional groups. In some embodiments, the polymerizable group and the functional group may be the same group.

[0087] Examples of epoxy-neutral monomers include glycidyl (meth)acrylates, thioglycidyl (meth)acrylates, 3-(2,3-epoxypropoxy)phenyl (meth)acrylates, 2-[4-(2,3-epoxypropoxy)phenyl]-2-(4-(meth)acryloyloxy-phenyl)propane, 4-(2,3-epoxypropoxy)cyclohexyl (meth)acrylates, 2,3-epoxycyclohexyl (meth)acrylates, and 3,4-epoxycyclohexyl (meth)acrylates, and combinations thereof. Examples of hydroxyl-containing monomers include N-hydroxyethyl (meth)acrylates, poly(ethylene glycol) (meth)acrylates, poly(propylene glycol) (meth)acrylates, N-hydroxyethyl (meth)acrylamide, 2-hydroxypropyl (meth)acrylamide, N-hydroxypropyl (meth)acrylates, 2-hydroxy-3-phenoxypropyl (meth)acrylates, and combinations thereof. Examples of suitable amide monomers include N-vinylcaprolactam, N-vinylacetamide, N-vinylpyrrolidone, (meth)acrylamide, mono- or di-N-alkyl-substituted acrylamides, 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)acrylamide, tetrahydrofurfuryl(meth)acrylate, and combinations thereof.

[0088] The process of preparing the functionalized nonwoven layer includes the following steps: providing a nonwoven substrate, exposing the nonwoven substrate to ionizing radiation in an inert atmosphere, and subsequently contacting the exposed substrate with a solution or suspension containing grafted monomers to graft polymerize the monomers onto the nonwoven substrate.

[0089] In the first step, the nonwoven substrate is exposed to ionizing radiation in an inert atmosphere. Exemplary forms of ionizing radiation include electron beam radiation, gamma radiation, X-ray radiation, and other forms of electromagnetic radiation. The inert atmosphere is typically an inert gas such as nitrogen, carbon dioxide, helium, argon, etc., with a minimum amount of oxygen. The dose delivered by the ionizing radiation source can occur as a single dose or as multiple doses accumulated to a desired level. One or more nonwoven layers can be exposed to ionizing radiation.

[0090] Following the irradiation step, the irradiated nonwoven substrate is brought into contact with an aqueous or suspensionable monomer solution. "Contact" means bringing the irradiated nonwoven substrate into contact with the monomer solution or suspension. It can also be described as saturating, absorbing, or coating the irradiated nonwoven substrate with the monomer solution. The monomer solution may only partially fill the void volume of the nonwoven substrate, or it may contact the nonwoven substrate with a much larger amount of solution than is necessary to completely fill the void volume. The monomer contact step is also performed in an inert atmosphere. This atmosphere may be the same as or different from the atmosphere in the chamber where the substrate was irradiated. The chamber may be the same as or different from the chamber where the substrate was irradiated. The monomer solution is kept in contact with the nonwoven substrate for a sufficient time to graft polymerize some, most, or substantially all of the monomers in the monomer solution. After the required contact time has elapsed, the nonwoven substrate carrying the grafted polymer can be removed from the inert atmosphere.

[0091] Example

[0092] Table 1. Materials

[0093]

[0094] Grafting solution

[0095] Grafting solution A was prepared as a monomer solution containing 24.4% NVP, 8.8% GMA and 19.4% MAPTAC by weight in deionized water.

[0096] Grafting solution B was prepared as a monomer solution containing 18.3% NVP, 6.6% GMA and 14.6% MAPTAC by weight in deionized water.

[0097] Grafting solution C was prepared as a monomeric solution containing 12.2% NVP, 4.4% GMA and 9.7% MAPTAC by weight in deionized water.

[0098] Method A: Determination of basis weight, effective fiber diameter (EFD), density, and pore size of functionalized nonwovens

[0099] The basis weight, EFD, density, and pore size of functionalized nonwoven fabrics were determined according to the following procedure. Sample discs (13.33 cm in diameter) were obtained by punching holes in the functionalized nonwoven sheet, and each disc was individually rinsed by immersing it in a 2 L deionized water bath for 15 minutes. The rinsing process was repeated three times with the fresh deionized water used in each rinsing step. Each rinsed disc 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 top of each disc to prevent edge curling. The obtained dried functionalized nonwoven samples (basis weight, EFD, density, pore size) were characterized according to the methods and formulas described above. For each measurement or calculation, the results were reported as the average of three independent tests (n=3) with the calculated standard deviation (SD).

[0100] For the density formula (a), fiber density ( ρ f The measured value was the density of the polypropylene substrate (0.91 g / cm³). 3 The density of the graft copolymer (1.07 g / cm³) and the density of the graft copolymer. 3 The sum of the densities is adjusted by the weight ratio of the polypropylene substrate and the graft copolymer in the test sample (Formula 1). The weight ratio of the polypropylene substrate and the copolymer is 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.

[0101] By first using solid-state 13 ¹³C NMR (ssNMR) was used to measure the molar percentage of the monomer components (NVP, MAPTAC, GMA) in the graft copolymer. The molar percentage was then converted to weight percentage (wt.%) to determine the density (D) of the graft copolymer. GCP 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 Adjust (multiply) the corresponding component wt.% values ​​and add the three adjusted density values ​​together (Formula 2).

[0102] Formula 1:

[0103] Fiber density ( ρ f )=(0.91×wt.% 聚丙烯 )+(1.05×wt.% 接枝共聚物 )

[0104] Formula 2:

[0105] DGCP =(D NVP ×wt.% NVP )+(D MAPTAC ×wt.% MAPTAC )+(D GMA ×wt.% GMA )

[0106] Method B: Determination of the dynamic charge capacity (MY DCC) of m-amine yellow in functionalized nonwovens

[0107] Functionalized nonwoven discs were prepared according to Method A. The dynamic charging capacity of the discs was determined using the charged organic dye m-amino yellow as the target molecule in the test solution. The test solution used had a m-amino yellow concentration of 160 mg / L (160 ppm). The test solution was prepared by dissolving 3.2 g m-amino yellow, 93.98 g anhydrous disodium hydrogen phosphate, 46.64 g anhydrous disodium hydrogen phosphate, and 163.63 g NaCl in 20 L of deionized water. The test solution was used within 2 days of preparation. If necessary, the amount of m-amino yellow reagent used to prepare the test solution was adjusted based on reagent purity so that the test solution contained 160 ppm m-amino yellow. Analytical grade m-amino yellow (≥98.0%, product #44426 from Sigma-Aldrich, St. Louis, Missouri) was used to calibrate reagent purity. A buffer solution with the same formulation as the test solution, except that it did not contain m-amino yellow, was also prepared for pretreatment of the test components.

[0108] The filtration test assembly comprises a transparent polycarbonate body (47 mm inner diameter) with a threaded cap attached to its top. The cap contains an inlet port and an outlet port. The bottom of the body contains an outlet port with a piston. A pressure sensor is placed upstream of the inlet port. A polyamide membrane (0.2 micrometer scale) is placed at the bottom of the body. A stack comprising two functionalized nonwoven discs (each 47 mm in diameter and obtained by perforating a disc prepared according to method A) is placed on top of the membrane in the assembly. In this assembly, the nonwoven disc is sandwiched between two PTFE sealing rings, each containing a blade on its inner diameter to engage with the nonwoven fabric. The resulting sub-assembly is secured in place using O-rings. The front surface area of ​​the disc stack is 0.00097 m². 2Attach the cap to the main body and connect a standard flow filtration system from Pando Technology (Panto Technology, Princeton, NJ) to the inlet port. Connect a Hach 2100AN turbidimeter (Hach Corporation, Loveland, Colorado) with a 455nm light filter and flow cell to the outlet port to measure the concentration of m-aminoflavin in the filtrate. Prepare m-aminoflavin solutions at concentrations of 0.8 ppm, 4 ppm, and 8 ppm as test standards. The endpoint for charge capacity measurement was set at 5% breakthrough (8 ppm) of the m-aminoflavin solution. The flow rate was 15 mL / min. The pretreatment buffer was rinsed through the assembly for approximately 5 minutes before pumping the test solution.

[0109] The volume of the test solution passing through the test assembly to the endpoint (i.e., the breakthrough volume) is measured, and the dynamic charging capacity (mg / g) of the functionalized nonwoven sample is calculated according to Formula 3. For each functionalized nonwoven, MY DCC reports the average of three independent tests (n=3) with calculated standard deviations (SD).

[0110] Formula 3:

[0111]

[0112] Method C: Preparation of harvested cell culture medium (HCCF)-Chinese hamster ovary (CHO) cell culture

[0113] CHO cells were cultured in suspension from a frozen cell stock in a CO2 incubator to a series of flasks for seeding, followed by a fed-batch culture process using a Wave bioreactor (GE Healthcare, Chicago, Illinois) and 50L disposable cell bags with pH control and dissolved oxygen monitoring. Cell culture medium was obtained from Fujifilm Irvine Technology (Santa Ana, California). CHO cell cultures were typically harvested on day 12 during the stationary phase.

[0114] Live cell density and viability were measured using a hemocytometer. The harvested cell culture medium was mixed with 10% (vol / vol) trypan blue solution and loaded into a disposable hemocytometer. Live and dead cells were counted under a microscope. The percentage of collected cell volume (%PCV) was measured using PCV tubes (Sigma-Aldrich product #Z760986), to which 200 μL of harvested cell culture medium (HCCF) was added. The tube was centrifuged at 2500 relative centrifugation (rcf) for 1 minute. %PCV was calculated from the HCCF volume using three-dimensional volumetric analysis.

[0115] Method D: Clarification of the harvested cell culture medium (HCCF)

[0116] The filter housing capsule was tested using a standard flow filtration system from Pando Technology (Panto Technology Corporation) connected to the capsule via the Luer lock inlet. Figure 7 The HCCF of the filter capsule is clarified. The plastic filter capsule has an upper shell and a lower shell that are joined together in the final construction by ultrasonic welding. The upper shell has a Luerlock inlet port and a Luerlock vent. The lower shell has a Luerlock outlet port located in the center of the lower shell. A disc (2.54 cm in diameter) of TYPAR 3161L polypropylene spunbond nonwoven fabric (10 mil thick, from Fiberweb, Old Hickory, Tennessee) is placed at the bottom of the lower shell. A disc (2.54 cm in diameter) of MICRO-PES flat 2F polyethersulfone membrane (from 3M, St. Paul, Minnesota) with a nominal pore size of 0.2 microns is placed on top of the nonwoven layer. The nonwoven layer and the membrane layer are ultrasonically welded to the bottom inner surface of the lower shell at the edges. Then, a stack of four functionalized nonwoven layers (discs with a diameter of 2.54 cm) is placed on top of the membrane. A polypropylene spacer ring (25.4 mm OD, 21.84 mm ID, 50 mil thickness) was inserted between the second and third nonwoven layers. The upper and lower housings were fitted together and ultrasonically welded to form the final filter capsule. Ultrasonic welding was performed by placing the mating components in a fixture so that the outer surface of the lower housing contacted the ultrasonic welding head. A Biensen 20 kHz ultrasonic welding machine (Emerson Electric 2000xdt model, St. Louis, Missouri), a black intensifier, and a welding head with 2.5x gain were used. Fixed parameters were set: 80 psi pressure, 10% descent rate, 80%–60% stepped amplitude, 50 joule steps, 2-second welding time, and a 200 lbf initial welding trigger force. The welding energy was kept constant at 450 joules to produce a sample with a consistent level of compression. The housing assembly was positioned below the welding head such that the longitudinal axis of the housing was aligned with the axis of the ultrasonic welding head. When the welding process begins, the welding head compresses the shell and internal components downwards on the lower shell until a force of 200 lbf is reached. The finished capsule has a total outer diameter of approximately 3.7 cm and a total height including the inlet, outlet, and vent is approximately 4.8 cm. The front surface area of ​​the disc stack is 3.2 cm². 2 .

[0117] Stir HCCF throughout the process. At the start of filtration, fill the top space of the filter capsule with the specified flow rate of HCCF by opening the vent and closing the outlet. After filling the top space of the capsule with HCCF, close the vent and open the outlet to allow collection of clarified cell culture medium (CCCF). Monitor the differential pressure during clarification. Once the differential pressure reaches 5 psid (pounds per square inch), stop clarification. Record the collected CCCF volume and CCCF turbidity. Calculate the flux (L / m²) based on the CCCF volume collected per unit surface area of ​​the filter. 2The turbidity of the filtrate was measured in turbidity units (NTU) using an Orion AQ4500 turbidity meter (Thermo Fisher Scientific, Waltham, Massachusetts).

[0118] Method E: Preparation of AAV2 feed solution

[0119] HEK293-F cells suspended in Gibco LV-MAX production medium (Thermo Fisher Scientific, Waltham, Massachusetts) were grown in an incubator using 2.8L shake flasks with constant shaking at 90 rpm. The incubator was maintained at 37°C and 8% CO2. Cells were cultured when the cell density reached approximately 2 × 10⁻⁶ cells / min. 6 When the cell / mL ratio is 1, prepare the transfection mixture and apply it to a shake flask.

[0120] The transfection mixture consisted of plasmid pAAV2-RC2 vector (part number VPK-422), pHelper vector (part number 340202) (plasmid obtained from Cell Biolabs, San Diego, California), and FECTOVIR. ® The AAV transfection agent (Polyplus Transfection, New York City, NY) was prepared by first adding the pHelper vector and pAAV2-RC2 vector at a molar ratio of 62% to 38%, adjusting the total plasmid amount to 1 μg of plasmid mixture per million HEK cells for transfection. Next, DMEM (Dubor Modified Eagle Medium, Thermo Fisher Scientific) was added to the mixture to achieve a final concentration of 5% DMEM (volume / volume) after adding the mixture to the cell culture flasks (i.e., a volume / volume calculation of DMEM adjusted based on the total cell culture volume). After adding DMEM, the mixture was mixed, and then 1 μL of FectoVIR-AAV transfection agent was added for every μg of plasmid mixture in the mixture. The mixture was gently mixed and incubated at room temperature for 45 minutes. After the incubation step, the completed transfection mixture was gently mixed and then added dropwise to flasks containing cell culture. After adding the transfection mixture, the cells were grown in an incubator for 72 to 96 hours (37°C, 8% CO2) to induce AAV2 production.

[0121] Cell viability was measured using a hemocytometer. The harvested cell culture medium was mixed with 25% (v / v) trypan blue solution and then loaded into a disposable hemocytometer. Live and dead cells were counted under a microscope. The turbidity of the transfected cell cultures was measured in turbidimetric units (NTU) using an ORIONAQ 4500 turbidimeter (Thermo Fisher Scientific). AAV2 transfected cell cultures showed a viability of 6.2 × 10⁻⁶ cells / mL. 6Cell density of cells / mL, 74% cell viability, and turbidity of 560 NTU.

[0122] Triton X-100 detergent (available from Promega, Fitchburg, Wisconsin) was added to the transfected cell culture to achieve a final detergent concentration of 0.1 wt.%, and the culture was then incubated at 90 rpm for 2 hours (37°C, 8% CO2). The conductivity of the lysed samples was adjusted to 20 mS / cm using 5M sodium chloride solution. Conductivity was measured using a calibrated OrionStar A215 pH / conductivity benchtop multi-parameter instrument (Thermo Fisher Scientific). After cell lysis, the resulting AAV2 feed solution had a conductivity of 8.5 x 10⁻⁶ mS / cm. 11 The AAV2 capsid content was 4230 ng / mL, the total DNA content was 4230 ng / mL, and the turbidity was 165 NTU.

[0123] The AAV2 capsid content in the feed solution before filtration and the filtrate after filtration was measured using the ProGEN AAV2 Xpress ELISA kit (available from American Research Products, Waltham, .M.) according to the manufacturer's instructions. The DNA concentration in the feed solution before filtration and the filtrate after filtration was measured using the Quant-iT PicoGreen dsDNA assay (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0124] Preparation of Functionalized Nonwoven Fabric A (FNW-A)

[0125] Unfunctionalized meltblown polypropylene microfiber nonwoven web (with an effective fiber diameter (EFD) of 16 μm, a basis weight of 200 g / m², a density of 10%, and a calculated average pore size of 47.4 μm) was grafted onto a nitrogen-purged graft solution C. The nonwoven substrate was unwound and passed through an electron beam (Electrocure, Energy Science, Wilmington, MD) set to a potential of 300 kV, delivering a total dose of 7 Mrad. The environment in the electron beam chamber was purged with nitrogen. The web was then directly transferred to a saturation step purged with a monomer solution under nitrogen. The web was then rolled up in the purging atmosphere. The web was left in the purging atmosphere for at least 60 minutes, after which it was exposed to air. The web was then unwound and transferred to a deionized water bath at a rate of 10 ft / min for approximately 8 minutes. After leaving the bath, the web was rinsed multiple times by passing a brine solution (NaCl) through it using a vacuum belt. A small amount of glycerol was added to the brine solution in the final rinsing step. The unfolded web is dried until its moisture content is less than 14% (by weight). The web is then wound onto a mandrel. The grafted product is labeled as Functionalized Nonwoven A (FNW-A). The properties of FNW-A are summarized in Table 2. Perforations are punched from the web to obtain FNW-A discs (2.54 cm in diameter).

[0126] Preparation of Functionalized Nonwoven Fabric B (FNW-B)

[0127] Using the same grafting process described for FNW-A, an unfunctionalized meltblown polypropylene microfiber nonwoven web (with an effective fiber diameter of 14 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 41.5 μm) was grafted onto the web. The grafted product was labeled as functionalized nonwoven B (FNW-B). The properties of FNW-B are reported in Table 2. FNW-B discs (2.54 cm in diameter) were obtained by perforating the web.

[0128] Preparation of Functionalized Nonwoven Fabric C (FNW-C)

[0129] Using the same grafting process described for FNW-A, an unfunctionalized meltblown polypropylene microfiber nonwoven web (with an effective fiber diameter of 12 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 35.6 μm) was grafted, and the grafted product was designated as functionalized nonwoven C (FNW-C). The properties of FNW-C are reported in Table 2. FNW-C discs (2.54 cm in diameter) were obtained by perforating the web.

[0130] Preparation of Functionalized Nonwoven Fabric D (FNW-D)

[0131] Using the same grafting process described for FNW-A, an unfunctionalized meltblown polypropylene microfiber nonwoven web (with an effective fiber diameter of 10 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 29.6 μm) was grafted onto the web. The grafted product was designated as functionalized nonwoven fabric D (FNW-D). The properties of FNW-D are reported in Table 2. FNW-D discs (2.54 cm in diameter) were obtained by perforating the web.

[0132] Preparation of functionalized nonwoven fabric E (FNW-E)

[0133] Using the same grafting process described for FNW-A, an unfunctionalized meltblown polypropylene microfiber nonwoven web (with an effective fiber diameter of 8 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 23.7 μm) was grafted onto the web. The grafted product was designated as functionalized nonwoven E (FNW-E). The properties of FNW-E are reported in Table 2. FNW-E discs (2.54 cm in diameter) were obtained by perforating the web.

[0134] Preparation of functionalized nonwoven fabric F (FNW-F)

[0135] Using the same grafting process described for FNW-A, an unfunctionalized meltblown polypropylene microfiber nonwoven web (with an effective fiber diameter of 6 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 17.8 μm) was grafted, and the grafted product was designated as functionalized nonwoven F (FNW-F). The properties of FNW-F are reported in Table 2. FNW-F discs (2.54 cm in diameter) were obtained by perforating the web.

[0136] Preparation of functionalized nonwoven fabric G (FNW-G)

[0137] Using the same grafting process described for FNW-A, an unfunctionalized meltblown polypropylene microfiber nonwoven web (with an effective fiber diameter of 4.2 μm, a basis weight of 100 gsm, a density of 8.2%, and a calculated average pore size of 14.2 μm) was used to graft the product as functionalized nonwoven G (FNW-G). The properties of FNW-G are reported in Table 2. MY DCC was determined using method B with 4 sheets instead of 2 sheets of functionalized nonwoven. The discs (2.54 cm in diameter) of functionalized nonwoven G were obtained by perforating the web.

[0138] Table 2. Properties of functionalized nonwoven fabrics A to G

[0139]

[0140] Preparation of functionalized nonwoven fabric H (FNW-H)

[0141] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 14 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 41.5 μm) was grafted using the same process as described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted product was labeled as functionalized nonwoven fabric H (FNW-H). The properties of FNW-H are reported in Table 3. FNW-H discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0142] Preparation of Functionalized Nonwoven Fabric I (FNW-I)

[0143] The same grafting process described for FNW-A was used to graft unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 12 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 35.6 μm), except that grafting solution B was used instead of grafting solution C. The properties of FNW-I are reported in Table 3. FNW-I discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0144] Preparation of functionalized nonwoven fabric J (FNW-J)

[0145] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 10 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 29.6 μm) was grafted using the same process as described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted product was labeled as functionalized nonwoven fabric J (FNW-J). The properties of FNW-J are reported in Table 3. FNW-J discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0146] Preparation of functionalized nonwoven fabric K (FNW-K)

[0147] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 8 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 23.7 μm) was grafted using the same process as described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted product was labeled as functionalized nonwoven fabric K (FNW-K). The properties of FNW-K are reported in Table 3. FNW-K discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0148] Preparation of functionalized nonwoven fabric L (FNW-L)

[0149] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 6 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 17.8 μm) was grafted using the same process as described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted product was labeled as functionalized nonwoven fabric L (FNW-L). The properties of FNW-L are reported in Table 3. FNW-L discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0150] Preparation of functionalized nonwoven fabric M (FNW-M)

[0151] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 4.2 μm, a basis weight of 100 gsm, a density of 8.2%, and a calculated average pore size of 14.2 μm) was grafted using the same process as described for FNW-A, except that grafting solution B was used instead of grafting solution C. The grafted product was labeled as functionalized nonwoven fabric M (FNW-M). The properties of FNW-M are reported in Table 3. MY DCC was determined by method B using 4 sheets instead of 2 sheets of functionalized nonwoven fabric. A disc (2.54 cm in diameter) of functionalized nonwoven fabric M was obtained by punching holes in the mesh.

[0152] Table 3. Properties of functionalized nonwoven fabrics H to M

[0153]

[0154] Preparation of functionalized nonwoven fabric N (FNW-N)

[0155] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 14 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 41.5 μm) was grafted using the same process as described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted product was labeled as functionalized nonwoven fabric N (FNW-N). The properties of FNW-N are reported in Table 4. FNW-N discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0156] Preparation of Functionalized Nonwoven O (FNW-O)

[0157] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 12 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 35.6 μm) was grafted using the same process as described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted product was labeled as functionalized nonwoven O (FNW-O). The properties of FNW-O are reported in Table 4. FNW-O discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0158] Preparation of functionalized nonwoven fabric P (FNW-P)

[0159] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 10 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 29.6 μm) was grafted using the same process as described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted product was designated as functionalized nonwoven fabric P (FNW-P). The properties of FNW-P are reported in Table 4. FNW-P discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0160] Preparation of functionalized nonwoven fabric Q (FNW-Q)

[0161] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 8 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 23.7 μm) was grafted using the same process as described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted product was designated as functionalized nonwoven Q (FNW-Q). The properties of FNW-Q are reported in Table 4. FNW-Q discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0162] Preparation of functionalized nonwoven fabric R (FNW-R)

[0163] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 6 μm, a basis weight of 200 gsm, a density of 10%, and a calculated average pore size of 17.8 μm) was grafted using the same process as described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted product was designated as functionalized nonwoven fabric R (FNW-R). The properties of FNW-R are reported in Table 4. FNW-R discs (2.54 cm in diameter) were obtained by perforating the mesh.

[0164] Preparation of functionalized nonwoven fabric S (FNW-S)

[0165] Unfunctionalized meltblown polypropylene microfiber nonwoven mesh (with an effective fiber diameter of 4.2 μm, a basis weight of 100 gsm, a density of 8.2%, and a calculated average pore size of 14.2 μm) was grafted using the same process as described for FNW-A, except that grafting solution A was used instead of grafting solution C. The grafted product was labeled as functionalized nonwoven fabric S (FNW-S). The properties of FNW-S are reported in Table 4. MY DCC was determined by method B using 4 sheets instead of 2 sheets of functionalized nonwoven fabric. A disc (2.54 cm in diameter) of functionalized nonwoven fabric G was obtained by punching holes in this mesh.

[0166] Table 4. Properties of functionalized nonwovens from N to S

[0167]

[0168] Example (Ex 1)

[0169] The filter capsule was assembled using two FNW-B discs and two FNW-F discs as described in Method D. The discs were oriented from capsule inlet to outlet with two FNW-B discs followed by two FNW-F discs. Chinese hamster ovary (CHO) cell culture was prepared to evaluate the filtration performance of the assembled capsule (as described above). The harvested cell culture medium (HCCF) had a collection cell volume percentage (%PCV) of 3.2%, viability of 25.5%, and turbidity of 1879 NTU. The capsule was tested at a flow rate of 200 L / m² / h (LMH) according to Method D (as described above). The resulting clarified cell culture medium (CCCF) was collected until the pressure gradient across the capsule reached 5 psi. The flux was 44.4 L / m². 2 The CCCF turbidity is 3.15 NTU.

[0170] Example 2 (EX 2)

[0171] The filter capsule was assembled using two FNW-B discs and two FNW-G discs as described in Method D. The discs were oriented from capsule inlet to outlet with two FNW-B discs followed by two FNW-G discs. The filtration performance of the assembled capsule was determined using the procedure described in Example 1 and HCCF. The flux was 30.3 L / m³. 2 The CCCF turbidity was 2.98 NTU.

[0172] Example 3 (Ex 3)

[0173] The filter capsule was assembled using two FNW-B discs, one FNW-D disc, and one FNW-E disc as described in Method D. The disc orientation 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 described in Example 1 and HCCF. The flux was 37.8 L / m³. 2 The CCCF turbidity was 3.40 NTU.

[0174] Example 4 (Ex 4)

[0175] The filter capsule was assembled using two FNW-C discs and two FNW-E discs as described in Method D. The discs were oriented from capsule inlet to outlet with two FNW-C discs followed by two FNW-E discs. The filtration performance of the assembled capsule was determined using the procedure described in Example 1 and HCCF. The flux was 53.4 L / m³. 2 The CCCF turbidity was 3.08 NTU.

[0176] Example 5 (Ex 5)

[0177] The filter capsule was assembled using two FNW-C discs, one FNW-E disc, and one FNW-F disc as described in Method D. The disc orientation 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 described in Example 1 and HCCF. The flux was 54.4 L / m³. 2 The CCCF turbidity was 3.58 NTU.

[0178] Example 6 (Ex 6)

[0179] The filter capsule was assembled using three FNW-E discs and one FNW-G disc as described in Method D. The disc orientation 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 described in Example 1 and HCCF. The flux was 46.6 L / m³. 2 The CCCF turbidity was 3.03 NTU.

[0180] Comparative Example A (CEx A)

[0181] The filter capsule was assembled using four FNW-A discs stacked together as described in Method D. The filtration performance of the assembled capsule was determined using the procedure described in Example 1 and HCCF. The flux was 13.4 L / m³. 2 Insufficient CCCF volume was collected for turbidity measurement. Membrane fouling was observed.

[0182] Comparative Example B (CEx B)

[0183] The filter capsule was assembled using four FNW-B discs stacked together as described in Method D. The filtration performance of the assembled capsule was determined using the procedure described in Example 1 and HCCF. The flux was 22.2 L / m³. 2 The CCCF turbidity was 5.76 NTU. Membrane fouling was observed.

[0184] Comparative Example C (CEx C)

[0185] The filter capsule was assembled using four FNW-E discs stacked together as described in Method D. The filtration performance of the assembled capsule was determined using the procedure described in Example 1 and HCCF. The flux was 23.1 L / m³. 2 The CCCF turbidity was 2.79 NTU. Agglomeration of cell culture material was observed on the top surface of the filter stack.

[0186] Comparative Example D (CEx D)

[0187] The filter capsule was assembled using four FNW-G discs stacked together as described in Method D. The filtration performance of the assembled capsule was determined using the procedure described in Example 1 and HCCF. The flux was 0.6 L / m³. 2 Insufficient CCCF volume was collected for turbidity measurement. Agglomeration of cell culture material was observed on the top surface of the filter stack.

[0188] The filtration results of Examples 1 to 6 (Ex 1 to Ex 6) and Comparative Examples A to D (CEx A to CEx D) are summarized in Table 5. The filter capsules of Examples 1 to 6 exhibited low CCCF turbidity and significantly higher flux than the filter capsules of the comparative examples. Furthermore, the filter capsules of the comparative examples showed clumping of cell culture material on the top surface of the filter stack or contamination of the membrane portion downstream of the filter stack.

[0189] Table 5. HCCF Clarification

[0190]

[0191] NM = Not measured (insufficient CCCF volume collected for turbidity measurement)

[0192] Example 7 (Ex 7)

[0193] The filter capsule was assembled using two FNW-B discs, one FNW-D disc, and one FNW-E disc as described in Method D. The disc orientation from capsule inlet to outlet was FNW-B / FNW-B / FNW-D / FNW-E. Chinese hamster ovary (CHO) cell culture medium was prepared to evaluate the filtration performance of the assembled capsule. The harvested cell culture medium (HCCF) had 8.0% collected cell volume percentage (%PCV), 80.0% viability, and 2483 NTU turbidity. The capsule was tested at a flow rate of 200 L / m² / h (LMH) according to Method D as described above. The resulting clarified cell culture medium (CCCF) was collected until the pressure gradient across the capsule reached 5 psi. The flux was 59.4 L / m². 2 The CCCF turbidity was 4.81 NTU.

[0194] Example 8 (Ex 8)

[0195] The filter capsule was assembled using two FNW-C discs, one FNW-E disc, and one FNW-F disc as described in Method D. The disc orientation 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 described in Example 7 and HCCF. The flux was 61.6 L / m³. 2 The CCCF turbidity was 4.98 NTU.

[0196] Comparative Example E (CEx E)

[0197] The filter capsule was assembled using four FNW-A discs stacked together as described in Method D. The filtration performance of the assembled capsule was determined using the procedure described in Example 7 and HCCF. The flux was 15.3 L / m³. 2 Insufficient CCCF volume was collected for turbidity measurement. Membrane fouling was observed.

[0198] Comparative Example F (CEx F)

[0199] The filter capsule was assembled using four FNW-F discs stacked together as described in Method D. The filtration performance of the assembled capsule was determined using the procedure described in Example 7 and HCCF. The flux was 15.3 L / m³. 2 Insufficient CCCF volume was collected for turbidity measurement. Agglomeration of cell culture material was observed on the top surface of the filter stack.

[0200] Comparative Example G (CEx G)

[0201] The filter capsules were assembled using four FNW-G discs stacked together as described in Method D. The filtration performance of the assembled capsules was determined using the procedure described in Example 7 and HCCF. The flux was 0 L / m³. 2 Agglomeration of cell culture material was observed on the top surface of the filter stack.

[0202] The filtration results of Examples 7 to 8 (Ex 7 to Ex 8) and Comparative Examples E to G (CEx E to CEx G) are summarized in Table 6. The filter capsules of Examples 7 to 8 have low CCCF turbidity and significantly higher flux than the filter capsules of the comparative examples. In addition, the filter capsules of the comparative examples have agglomerates of cell culture material on the top surface of the filter stack or contamination of the membrane portion downstream of the filter stack.

[0203] Table 6. HCCF Clarification

[0204]

[0205] NM = Not measured (insufficient CCCF volume collected for turbidity measurement)

[0206] Example 9 (Ex 9)

[0207] A plastic filter capsule is used. The capsule consists of a sealed, circular shell. The capsule shell is made of two halves (upper and lower halves), which fit together and seal at the periphery after the filter element is inserted into the inner chamber of the lower shell. The fluid inlet and outlet are located in the upper part of the shell, and the fluid outlet is located in the lower part of the shell. The outlet port is located at the center of the lower shell surface.

[0208] Two discs (27 mm in diameter) of TYPAR 3161L polypropylene spunbond nonwoven fabric (10 mil thick, from Fiberweb, Old Hickory, Tennessee) were placed at the bottom of the lower housing. A single disc (27 mm in diameter) of MICRO-PES flat-plate 2F polyethersulfone membrane (from 3M) 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 to the bottom inner surface of the lower housing at the edges. A stack of four functionalized nonwoven layers (27 mm in diameter) was then placed on top of the membrane. The stack consisted of one functionalized nonwoven C disc, two functionalized nonwoven E discs, and two functionalized nonwoven G discs. The discs were oriented from capsule inlet to outlet as FNW-C / FNW-E / FNW-E / FNW-G / FNW-G. A polypropylene spacer ring (25.4 mm OD, 21.84 mm ID, 50 mil thickness) is inserted between the third and fourth nonwoven layers (i.e., between the FNW-E and FNW-G discs). The upper and lower housings are then assembled and ultrasonically welded using a Biensen 20 kHz ultrasonic welder (Emerson Electric 2000xdt model, St. Louis, Missouri) to form the finished filter capsule.

[0209] The finished capsule has a total outer diameter of approximately 4.3 cm and a total height including the inlet, outlet, and exhaust port of approximately 5.9 cm. The effective filtration area of ​​the capsule is 3.2 cm². 2 The volume of the nonwoven media bed is 2.1 mL.

[0210] Example 10 (Ex 10)

[0211] The finished capsules prepared according to Example 9 were attached to a standard flow filter system of Pando Technology (Pando Technology, Princeton, New Jersey) through the capsule inlet port. The capsules were washed with Tris acetate buffer (50 mM, pH 7.5, conductivity 4 mS / cm) at a constant flux of 200 LMH until reaching 54 L / m³. 2The fluid throughput was increased, and the medium disk was then flushed with air (up to a pressure differential of 5 psid) to dry it. Next, the cell lysate feed solution containing AAV2 prepared in Method E was pumped through the capsule at a constant flow rate of 140 LMH up to a pressure differential of 15 psid. The filtrate was collected and analyzed to determine flux, AAV2 capsid content, total DNA content, and turbidity. A total of two capsules were tested. The average flux was 249 L / m³. 2 (Standard deviation = 69). Tables 7 through 9 provide the results for AAV2 capsid content and total DNA content.

[0212] Table 7. Total content in the feed solution before filtration and the filtrate after filtration through the functionalized membrane capsule of Example 9 AAV2 capsid content

[0213]

[0214] Table 8. Total content in the feed solution before filtration and the filtrate after filtration through the functionalized membrane capsule of Example 9 DNA content

[0215]

[0216] Table 9. Turbidity in the feed solution before filtration and in the filtrate after filtration through the functionalized membrane capsule of Example 9 value

[0217]

Claims

1. An electrically charged depth filter for removing cells and / or cell debris from biopharmaceutical raw materials, the electrically charged depth filter comprising: A first functionalized nonwoven layer having a first calculated aperture and a first meta-amine yellow dynamic charge capacity (MY DCC), wherein the calculated aperture is determined by the arithmetic mean fiber diameter and the web tightness; A second functionalized nonwoven layer having a second calculated pore size and a second meta-amine yellow dynamic charging capacity (MY DCC) is located downstream of the first functionalized nonwoven layer in the direction of biopharmaceutical raw material flow. Wherein the first calculated aperture is larger than the second calculated aperture, and the first dynamic charging capacity of the meta-amine yellow is smaller than the second dynamic charging capacity of the meta-amine yellow, and The first functionalized nonwoven layer and / or the second functionalized nonwoven layer are cationic or cationically ionizable.

2. The charged depth filter according to claim 1, wherein for the first functionalized nonwoven layer, the first calculated pore size is 40.8µm to 65.0µm, and the first meta-amine yellow dynamic charging capacity is 150 MY DCC mg / g to 300 MY DCC mg / g; and wherein for the second functionalized nonwoven layer, the second calculated pore size is 5.0µm to less than 40.8µm, and the second meta-amine yellow dynamic charging capacity is greater than 300 MY DCC mg / g to 650 MY DCC mg / g.

3. The charged depth filter according to claim 1, wherein for the first functionalized nonwoven layer, the first calculated pore size is 55.0 µm to 65.0 µm, and the first meta-amine yellow dynamic charging capacity is 150 MY DCC mg / g to 300 MY DCC mg / g; and for the second functionalized nonwoven layer, the second calculated pore size is 5.0 µm to less than 55.0 µm, and the second meta-amine yellow dynamic charging capacity is 300 MY DCC mg / g to 650 MY DCC mg / g.

4. The charged depth filter according to claim 1, wherein the first functionalized nonwoven layer and the second functionalized nonwoven layer are grafted with a copolymer, the copolymer comprising comonomer units containing quaternary ammonium monomers, amide monomers and epoxy monomers.

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

6. The charged depth filter according to claim 1, wherein the charged depth filter further comprises a third functionalized nonwoven layer having a third calculated pore size and a third dynamic charging capacity of meta-amine yellow, the third functionalized nonwoven layer being located after the second functionalized nonwoven layer in the direction of flow of the biopharmaceutical raw material. Wherein the first calculated aperture is larger than the second calculated aperture, and the second calculated aperture is larger than the third calculated aperture; and the first meta-amine yellow dynamic charging capacity is smaller than the second meta-amine yellow dynamic charging capacity, and the second meta-amine yellow dynamic charging capacity is smaller than the third meta-amine yellow dynamic charging capacity, and The first functionalized nonwoven layer and / or the second functionalized nonwoven layer and / or the third functionalized nonwoven layer are cationic or cationically ionizable.

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

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

9. The charged depth filter according to claim 6, wherein the third functionalized nonwoven layer is permeable.

10. The charged depth filter according to claim 6, wherein on the graph of dynamic charge capacity versus calculated pore size, the permeability boundary line extends through points 1 and 2, point 1 having a calculated pore size of 5.0 µm and a dynamic charge capacity of 300 MY DCC mg / g, point 2 having a calculated pore size of 20.6 µm and a dynamic charge capacity of 525 MY DCC mg / g, and the third functionalized nonwoven layer having point 3 on the graph of third dynamic charge capacity versus third calculated pore size, point 3 being located below the permeability boundary line.

11. The charged depth filter according to claim 6, wherein the first functionalized nonwoven layer, the second functionalized nonwoven layer and the third functionalized nonwoven layer are grafted with copolymers, the copolymers comprising comonomer units containing quaternary ammonium monomers, amide monomers and epoxy monomers.

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, the copolymer comprising comonomer units of 3-methacryloylaminopropyltrimethylammonium chloride, N-vinylpyrrolidone and glycidyl methacrylate.

13. The charged depth filter according to claim 6, wherein a repeating first functionalized nonwoven layer is located between the first layer and the second layer.

14. The charged depth filter according to claim 6, wherein the membrane layer is located after the third functionalized nonwoven layer.

15. The charged depth filter according to claim 14, wherein the nonfunctionalized nonwoven layer is located after the membrane layer.

16. A method for clarifying a biopharmaceutical ingredient comprising whole cells and cell debris in a single stage, comprising passing a biopharmaceutical ingredient feed having a collected cell volume percentage between 2% and 12% through an charged depth filter as claimed in claim 1 to form a clarified biopharmaceutical ingredient.

17. The method of claim 16, wherein the clarified biopharmaceutical raw material has a turbidity of less than 50 NTU.

18. The method of claim 16, wherein the flux of the biopharmaceutical feedstock through the charged depth filter is 30 L / m³. 2 Up to 200L / m 2 between.

19. The method of claim 16, wherein the biopharmaceutical raw material has a turbidity of 1,000 NTU to 10,000 NTU.

20. The method of claim 16, wherein the flow rate of the biopharmaceutical raw material is 50 LMH to 600 LMH.

21. The method of claim 16, wherein the clarified biopharmaceutical feedstock has a turbidity of less than 50 NTU, and wherein the biopharmaceutical feedstock has a turbidity of 1,000 NTU to 10,000 NTU.

22. The method of claim 21, wherein the flow rate of the biopharmaceutical raw material is 50 LMH to 600 LMH.

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

24. The method of claim 23, wherein the mammalian cells are selected from the group consisting of Chinese hamster ovary cells, human embryonic kidney 293 cells, young hamster kidney cells, NSO mouse myeloma cells, or PER.C6® human cells.

Images