How biological products are collected

Abstract

Method. The method includes providing a filter medium comprising a functionalized nonwoven fabric; passing a first fluid containing cells through the filter medium, where at least a portion of the cells are captured by the functionalized nonwoven fabric; passing a second fluid, and optionally a third and / or fourth fluid, through the filter medium, where the second and / or the third and / or the fourth fluid disrupts at least one of the captured cells; and recovering intracellular biological products in the first and / or the second and / or the third and / or the fourth fluid.

Description

[Background technology]

[0001] Biologics such as recombinant proteins and gene therapy vectors are often produced using cell culture techniques. Host cells (i.e., mammalian, insect, bacterial or other cell lines) are utilized to produce the therapeutic of interest. Chinese Hamster Ovary (CHO) cells are the most commonly used cell line in the industry based on their ability to adapt and grow in suspension, ability to grow in serum-free chemically defined media, high production capacity, post-translational modifications, etc. Although CHO cells account for >70% of produced protein therapeutics, these biologics can be produced in several systems including microbial, plant, insect, and / or other mammalian cells. Whole cells and cell debris are typically removed, resulting in a clarified cell culture containing the desired biologic. The clarified cell culture product can then be subjected to further purification steps to increase purity and concentration. Summary of the Invention

[0002] In biopharmaceutical manufacturing, target biomolecules of interest, such as biomolecules expressed in cells, need to be separated from feedstocks containing cells. When the biomolecules of interest are expressed in cells, a cell disruption step is often required to release the desired product. When a lysis step is required for the recovery of the target biopharmaceutical molecules expressed in cells, the cells are generally suspended in cell culture fluid or placed in another buffer system, such as a lysis buffer. The resulting lysate then requires further purification to isolate the desired drug product from cell debris and other contaminants released during the lysis procedure. Therefore, a single-use primary clarification step is required that can replace centrifuges, tangential flow microfiltration, and traditional depth filters as a primary clarification step.

[0003] Thus, in one aspect, the present disclosure provides a method comprising providing a filter medium comprising a functionalized nonwoven fabric; passing a first fluid containing cells through the filter medium, where at least a portion of the cells are captured by the functionalized nonwoven fabric; passing a second fluid, and optionally a third and / or fourth fluid, through the filter medium, where the second and / or the third and / or the fourth fluid disrupts at least one of the captured cells; and recovering intracellular biological products in the first and / or the second and / or the third and / or the fourth fluid.

[0004] Various aspects and advantages of exemplary embodiments of the present disclosure have been summarized. The above summary is not intended to describe each exemplary embodiment or every implementation of the present disclosure. Additional features and advantages are disclosed in the following embodiments. The following detailed description more particularly illustrates certain embodiments that utilize the principles disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] Before any embodiment of the present disclosure is described in detail, it is understood that the present invention is not limited in its application to the details of use, structure, and arrangement of the components described in the following description. The present invention is capable of other embodiments and can be implemented or carried out in various ways that will be apparent to those skilled in the art upon reading this disclosure. It is also understood that the phraseology and terminology used herein are for the purpose of description and should not be construed as limiting. The use of "including," "comprising," or "having" and variations thereof herein means to include the items listed thereafter and their equivalents as well as additional items. It is understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope of the present disclosure.

[0006] Throughout this specification, values ​​expressed in the form of a range should be interpreted flexibly to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly stated. 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 ​​within the stated range (e.g., 1%, 2%, 3%, and 4%) and subranges within the stated range (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). The statement "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise indicated.

[0007] In this document, the terms "a," "an," or "the" are used to include one or more, unless the context makes clear otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, phraseology or terminology used herein and not otherwise defined should be understood to be for purposes of description only and not for purposes of limitation. Any use of section headings is intended to aid in the interpretation of this document and should not be construed as limiting, and information associated with a section heading may be found within or outside that particular section.

[0008] The term "about," as used herein, allows for some variability in values ​​or ranges, for example, within 10%, within 5%, or within 1% of the stated limits of a stated value or range, including the exactly stated value or range.

[0009] The term "substantially" as used herein refers to a large portion 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 can mean having no or insignificant amounts of a material present such that the amount of material present does not affect the material properties of a composition including the material, such that the composition is about 0% to about 5% by weight, or about 0% to about 1% by weight, or about 5% or less by weight, or about 4.5% or less by weight, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001% or less by weight of the material.

[0010] As used herein, "layer" refers to the thickness of material through which the fluid to be treated passes, and all of the materials in the layer are formed from the same material. The layer may be a monolithic layer formed from the same thickness of material. Alternatively, the layer may have one or more separate plies of the same material stacked on top of each other in the layer to form its thickness. For example, a layer of a typical facial tissue is often a tissue paper material made from two individual plies of tissue paper placed in face-to-face contact, and the two individual plies are held together by a weak mechanical bond, typically in the form of a crimp line, so that they can be easily separated from each other.

[0011] As used herein, a "functionalized nonwoven" is a nonwoven that attracts target particles or molecules by attractive forces, such as electrostatic forces, due to the presence of one or more chemical moieties, ligands, or functional groups on the surface of the nonwoven that are different from the material that forms the bulk of the nonwoven, which primarily provides its structural shape and integrity. The chemical moieties, ligands, or functional groups are specifically intended to attract the target particles or molecules to the surface of the functionalized nonwoven. Functionalized nonwovens can be made by coating or grafting a porous nonwoven with a ligand, monomer, or polymer designed to molecularly attract the target particles or molecules. Alternatively, functionalized nonwovens may be made by providing a surface-modifying polymer or chemical moiety in the formulation used to make such nonwovens that becomes localized to the surface of the nonwoven during its formation, and chemical groups designed to attract the target particles or molecules are present on the surface of the nonwoven. In some embodiments, the attraction between the functional groups on the surface of the functionalized nonwoven fabric is electrostatic, and the chemical moieties, ligands, or polymers present on the surface of the functionalized nonwoven fabric are electrostatically charged. The functionalized nonwoven fabric may have a positive charge and attract negatively charged particles (i.e., anion exchange chromatography), or the functionalized nonwoven fabric may have a negative charge and attract positively charged particles (i.e., cation exchange chromatography). In other embodiments, the attraction may be van der Waals forces, and the target particles or molecules are attracted to the functional groups on the surface of the functionalized nonwoven fabric by the relative concentration or lack of polarizable or hydrogen-bonding moieties (i.e., hydrophobic interactions). Additionally, the attraction may include a combination of electrostatic and van der Waals forces (i.e., mixed mode). Suitable functionalized materials for the functionalized nonwoven in the charged depth filtration device are manufactured by Pall, Millipore, and Sartorious and sold under the following brands: Mustang® Q, NatriFlo® HD-Q, and Sartobind® Q. Suitable functionalized nonwovens for use in the charged depth filtration device can be nonwovens, membranes, or other suitable materials.A preferred functionalized nonwoven material is manufactured by 3M Company and is disclosed in U.S. Patent No. 9,821,276, entitled "Nonwoven Article Grafted with Copolymer." A preferred functionalized membrane is manufactured by 3M Company and is disclosed in U.S. Patent Nos. 9,650,470 and 10,017,461, entitled "Method of Making Ligand Functionalized Substrates." All three of the patents mentioned are incorporated herein by reference in their entirety.

[0012] As used herein, "osmotic potential" refers to the potential for water molecules to move across a semipermeable membrane from a hypotonic solution (more water, less solute) to a hypertonic solution (less water, more solute). Osmotic potential can be calculated using the following formula: Osmotic potential = -C x R x T, where C is the concentration of the solute (i.e., sucrose, salt, etc.), R is the universal gas constant (i.e., 8.314472 JK-1mol-1), and T is the absolute temperature.

[0013] Clarification Method The present disclosure provides a method for harvesting a biological agent. The method includes providing a filter medium including a functionalized nonwoven fabric. A first fluid containing cells can be passed through the filter medium. At least a portion of the cells can be captured and immobilized by the functionalized nonwoven fabric. To disrupt the captured cells, a second fluid, and optionally a third and / or a fourth fluid, can be passed through the filter medium after the first fluid containing cells has passed through the filter medium. After the captured cells are disrupted, the target product, e.g., an intracellular biological product, can be collected in the first and / or second and / or third and / or fourth fluids. The disruption of the cells involves a decrease in the integrity of the cells, such that the internal contents of the cells at least partially or completely flow out of the cells into the fluid environment. This can include partial or complete degradation of the cell membrane. This can include an increase in the permeability of the cell membrane. In one embodiment, the cell disruption includes cell lysis.

[0014] The filter medium can include the filters disclosed in U.S. Patent Application No. 63 / 154,299, filed February 26, 2021, entitled "Charged Depth Filter for Therapeutic Biotechnology Manufacturing Process," which is incorporated herein by reference in its entirety. In some embodiments, the filter medium can have at least two layers of functionalized nonwoven fabric, each layer having the same or different calculated pore size and the same or different dynamic charge capacity (MY DCC). In some embodiments, the filter medium can have multiple layers of functionalized nonwoven fabric, each layer having the same or different calculated pore size and the same or different dynamic charge capacity. For example, the filter medium can have at least a first functionalized nonwoven layer having a first calculated pore size and a first dynamic charge capacity, and a second functionalized nonwoven layer having a second calculated pore size and a second dynamic charge capacity located behind the first functionalized nonwoven layer in the direction of the biologic feedstock flow, where the first calculated pore size is larger than the second calculated pore size and the first dynamic charge capacity is smaller than the second dynamic charge capacity.

[0015] In some embodiments, for the first functionalized nonwoven layer, the first calculated pore size is between 40.8 μm and 65.0 μm and the first dynamic charge capacity is between 150 MY DCC mg / g and 300 MY DCC mg / g, and for the second functionalized nonwoven layer, the second calculated pore size is between 5.0 μm and less than 40.8 μm and the second dynamic charge capacity is greater than 300 MY DCC mg / g and 650 MY DCC mg / g.

[0016] In some embodiments, for the first functionalized nonwoven layer, the first calculated pore size is from 55.0 μm to 65.0 μm and the first dynamic charge capacity is from 150 MY DCC mg / g to 300 MY DCC mg / g, and for the second functionalized nonwoven layer, the second calculated pore size is from 5.0 μm to less than 55.0 μm and the second dynamic charge capacity is from 300 MY DCC mg / g to 650 MY DCC mg / g.

[0017] In some embodiments, the filter media can have a third functionalized nonwoven layer having a third calculated pore size and a second dynamic charge capacity located after the second functionalized nonwoven layer in the direction of the biologic feedstock flow, where the second calculated pore size is larger than the third calculated pore size and the second dynamic charge capacity is smaller than the third dynamic charge capacity.

[0018] In some embodiments, for the first functionalized nonwoven layer, the first calculated pore size is between 40.8 μm and 65.0 μm and the first dynamic charge capacity is between 150 MY DCC mg / g and 300 MY DCC mg / g; for the second functionalized nonwoven layer, the second calculated pore size is between 20.6 μm and less than 40.8 μm and the second dynamic charge capacity is between greater than 300 MY DCC mg / g and 475 MY DCC mg / g; and for the third functionalized nonwoven layer, the third calculated pore size is between 5.0 μm and less than 20.6 μm and the third dynamic charge capacity is between greater than 300 MY DCC mg / g and 650 MY DCC mg / g.

[0019] In some embodiments, for the first functionalized nonwoven layer, the first calculated pore size is between 55.0 μm and 65.0 μm and the first dynamic charge capacity is between 150 MY DCC mg / g and 300 MY DCC mg / g; for the second functionalized nonwoven layer, the second calculated pore size is between 20.6 μm and less than 55.0 μm and the second dynamic charge capacity is between 200 MY DCC mg / g and 475 MY DCC mg / g; and for the third functionalized nonwoven layer, the third calculated pore size is between 5.0 μm and less than 20.6 μm and the third dynamic charge capacity is between 300 MY DCC mg / g and 650 MY DCC mg / g.

[0020] The same layer may be repeated within the filter media to increase capacity for a particular debris size before the pore size and / or dynamic charge capacity is altered. Charged depth filter media stacks can have 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more layers depending on the construction, but often have less than 25 layers.

[0021] In some embodiments, the first fluid can include cell culture feedstock, such as mammalian cell cultures (e.g., Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney 293 (HEK-293) cells, Baby Hamster Kidney (BHK-21) cells, NS0 mouse myeloma cells, or PER.C6® human cells), insect cell lines, and bacterial cell lines. In one embodiment, the first fluid can be a transfected cell culture. In one embodiment, the first fluid can be a HEK-293 cell culture that has been subjected to triple plasmid transfection for the production of adeno-associated virus.

[0022] In some embodiments, the second fluid can have an osmotic potential lower than that of the first fluid, and the change in osmotic potential within the filter media can destroy the trapped cells. For example, the second fluid can have an osmotic potential of less than -500 J, less than -620 J, or less than -750 J, and the first fluid can have an osmotic potential of -5 J to -1 J. In some of these embodiments, the second fluid can have a conductivity lower than that of the first fluid. For example, the second fluid can have a conductivity of less than 5 mS / cm, and the first fluid can have a conductivity of 5 mS / cm to 20 mS / cm. In these embodiments, the second fluid can be a sucrose solution. In some other embodiments, the second fluid can have a conductivity higher than that of the first fluid. In these embodiments, the second fluid may be a salt solution, such as NaCl solution, phosphate buffered saline, phosphate buffer, Tris-HCl buffer, Tris-acetate buffer, HEPES buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid].

[0023] In some embodiments, the second fluid can have an osmotic potential higher than that of the first fluid, and the change in osmotic potential within the filter medium can destroy the trapped cells. For example, the second fluid can have an osmotic potential of -5J to 0J, and the first fluid can have an osmotic potential of -5J to -1J. In some of these embodiments, the second fluid can have a conductivity less than that of the first fluid. In these embodiments, the second fluid can be water.

[0024] In some embodiments, the second fluid can be a detergent or chemical cell lysing agent. The detergent or chemical cell lysing agent can be any suitable detergent, such as non-ionic detergents, cationic detergents, detergents with a net charge of zero (zwitterionic detergents), and mixtures thereof. In some embodiments, the detergent or chemical cell lysing agent can include Triton® X1-100 or Tween 20, Tergitol NP9, and polysorbates.

[0025] In some embodiments, the conductivity of the third fluid is different from the conductivity of the first fluid and different from the conductivity of the second fluid. For example, the third fluid can have a conductivity in the range of 9 mS / cm to 60 mS / cm, the second fluid can have a conductivity less than 5 mS / cm, and the first fluid can have a conductivity of 5 mS / cm to 20 mS / cm. In some embodiments, the difference in conductivity between the first and third fluids is at least 2 mS / cm, 3 mS / cm, 5 mS / cm, 10 mS / cm, 20 mS / cm, 30 mS / cm, or 50 mS / cm.

[0026] In some embodiments, the third or fourth fluid may be a conductive salt solution having a conductivity in the range of 9 mS / cm to 60 mS / cm. In some embodiments, the third or fourth fluid may be selected from NaCl solution, phosphate buffered saline, phosphate buffer, Tris-HCl buffer, Tris-acetate buffer, HEPES buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid].

[0027] In some embodiments, the second fluid can have an osmotic potential lower than that of the first fluid and a lower conductivity than that of the first fluid, the third fluid can have an osmotic potential higher than that of the first fluid, and the fourth fluid can have an osmotic potential lower than that of the first fluid and a higher conductivity than that of the first fluid. For example, the second fluid can be a sucrose solution, the third fluid can be water, and the fourth fluid can be a NaCl solution. In these embodiments, the first fluid, cell culture medium as well as other soluble contaminants, can flow through the filter medium including the functionalized nonwoven fabric, resulting in a concentration of cells as they are captured. Application of the second fluid, a hypertonic solution such as 40 wt% sucrose or a high salt solution, serves to drive water away from the immobilized cells and increase the osmotic potential. Following a period of equilibration in the hypertonic solution, the environment can be quickly switched to hypotonic conditions without changing the cell concentration. A second fluid, a hypertonic liquid, can be drained from the functionalized nonwoven and a hypotonic medium, such as deionized water, can be applied to the cells captured on the functionalized nonwoven. The sudden change in environment can drive water into the cells, which increases the volume of the cells and results in lysis. Following the lysis step, a fourth liquid can be applied to the functionalized nonwoven to recover the residual products while retaining the impurities (i.e., soluble and insoluble species) by the functionalized nonwoven. Trapping of the cells / cell debris by the charge can allow the fourth liquid to be applied while maintaining a low differential pressure on the nonwoven.

[0028] In some embodiments, the second fluid can have a higher osmotic potential than the first fluid, and the third fluid can have a lower osmotic potential than the first fluid and a higher conductivity than the first fluid. For example, the second fluid can be water and the third fluid can be a NaCl solution. In these embodiments, it may not be necessary to pass the fourth fluid through a filter medium.

[0029] In some embodiments, the second fluid can be a surfactant or a chemical cell lysing agent, and the third fluid can have an osmotic potential lower than that of the first fluid and a conductivity higher than that of the first fluid. For example, the second fluid can be Triton® X1-100 or Tween 20, and the third fluid can be a NaCl solution. In these embodiments, it may not be necessary to pass the fourth fluid through a filter medium.

[0030] In some embodiments, the second fluid can have an osmotic potential lower than that of the first fluid and a conductivity higher than that of the first fluid. For example, the second fluid can be a NaCl solution. In these embodiments, it may not be necessary to pass the third and / or fourth fluid through a filter medium.

[0031] The biological products clarified by the methods of the present application can be intracellular biological products, such as adeno-associated virus (AAV) capsids, therapeutic / recombinant proteins, plasmids, DNA, RNA, viruses, virus-like particles, exosomes, and mixtures thereof.

[0032] Current biological product harvest protocols, especially for intracellular biological products, often require the addition of detergents followed by complex multi-stage primary clarification strategies to process the generated lysate. Highly variable particle size distributions often present challenges to traditional clarification approaches, resulting in multi-stage filter configurations with large effective filter areas. Measures must also be taken to remove the detergent in subsequent purification steps to ensure the final product is free of this contaminant.

[0033] The method offers a differentiated approach: the lysis and clarification steps occur sequentially within a single device. The technique also enables the use of osmotic pressure to lyse cells, eliminating the need to add any additional reagents. For example, in some embodiments, the method involves establishing an osmotic pressure difference between the interior of the cell and the surrounding environment. Lysis can occur when pressure changes occur rapidly, creating a large pressure difference across the cell membrane. Process limitations such as the time and challenges associated with buffer exchange (i.e., dilution volumes and capital investments required for large-scale equipment such as centrifuges) have limited osmotic lysis to small-scale applications. In addition, the method can be specifically tailored to accommodate the demands posed by in-situ cell lysis, such as high cell loads that generate large amounts of cell debris upon lysis. Rather than attempting to osmotically lyse cells in bulk solution, cells can be chromatographically captured and concentrated by charge-based interactions on the functionalized nonwoven fabric. The extracellular environment can then be easily manipulated to facilitate osmotic lysis. For example, the extracellular environment can be rapidly changed from hypertonic to hypotonic conditions to induce lysis of cells captured on the nonwoven fabric. As lysis occurs, the generated cellular debris is simultaneously captured and retained by the functionalized nonwoven, while the product of interest is collected in the flow-through / filtrate. Application of the functionalized nonwoven in this manner makes osmotic lysis a viable candidate for harvesting intracellularly expressed biologics and mitigates challenges posed by other lysis strategies, such as the large dilutions or process complications associated with buffer exchange. Because the functionalized nonwoven retains the debris generated during cell disruption, an additional clarification step to remove the debris generated during lysis can be eliminated using the present method. In addition to retaining large insoluble debris, the functionalized nonwoven can reduce the concentration of soluble impurities present in the lysate, such as DNA and host cell proteins. For example, the functionalized nonwoven may have the potential to reduce the DNA concentration in the first fluid to less than 10 ng / ml.

[0034] Nonwoven The nonwoven fabric may be a nonwoven web, which may include nonwoven webs made by any of the commonly known processes for making nonwoven webs. As used herein, the term "nonwoven web" refers to a fabric having a structure of individual fibers or filaments randomly and / or unidirectionally inserted in a mat-like manner. For example, fibrous nonwoven webs can be made by carding, airlaid, wetlaid, spunlace, spunbond, electrospinning, or meltblowing techniques, such as meltspun or meltblowing, or combinations thereof. Spunbond fibers are typically small diameter fibers formed by extruding molten thermoplastic polymers as filaments from multiple fine, usually circular, capillaries of a spinneret, causing the diameter of the extruded fibers to rapidly decrease. Meltblown fibers are typically formed by extruding molten thermoplastic material as molten threads or filaments through multiple fine, usually circular die capillaries into a high-velocity, usually heated, gas (e.g., air) stream that attenuates the filaments of molten thermoplastic material, reducing their diameter. The meltblown fibers are then carried by the high-velocity gas stream and deposited on a collecting surface to form a web of randomly distributed meltblown fibers. Any of the nonwoven webs may be made from a single type of fiber or two or more fibers that differ in thermoplastic polymer type and / or thickness.

[0035] Suitable polyolefins for making nonwoven webs include, but are not limited to, polyethylene, polypropylene, poly(1-butene), copolymers of ethylene and propylene, alpha-olefin copolymers (e.g., copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and 1-decene), poly(ethylene-co-1-butene), poly(1-methylpentene), and poly(ethylene-co-1-butene-co-1-hexene). Preferably, the nonwoven substrate is polypropylene.

[0036] Further details regarding the method of making the nonwoven webs of the present invention can be found in Wente, Superfine Thermoplastic Fibers, 48 ​​INDUS.ENG.CHEM.1342 (1956) and Wente et al., Manufacture of Superfine Organic Fibers, (Naval Research Laboratories Report No. 4364, 1954). Useful methods for preparing nonwoven substrates are described in U.S. Reissue Patent No. 39,399 (Allen), U.S. Patent No. 3,849,241 (Butin et al.), U.S. Patent No. 7,374,416 (Cook et al.), U.S. Patent No. 4,936,934 (Buehning), and U.S. Patent No. 6,230,776 (Choi).

[0037] functionalized nonwoven fabric A functionalized nonwoven may include a nonwoven substrate as described above and a graft copolymer including copolymerized monomer units, at least one of which is cationic or can be made cationic in a solution of an appropriate pH ("cationically ionizable"). Suitable functionalized nonwovens are disclosed in U.S. Patent No. 9,821,276, entitled "Nonwoven Article Grafted With Copolymer," issued Nov. 21, 2017, and incorporated herein by reference.

[0038] Cationic or cationically ionizable monomers can include quaternary ammonium-containing monomers and tertiary amine-containing monomers. One or more cationic or cationically ionizable monomers can be used. The monomer typically contains a polymerizable functional group and a cationic or cationically ionizable group. In a particular monomer, the polymerizable group and the cationic group can be the same group. Polymerizable groups include vinyl, vinyl ether, (meth)acryloyl, (meth)acrylamide, allyl, cyclic unsaturated monomers, multifunctional monomers, vinyl esters, and other easily polymerizable functional groups.

[0039] Useful (meth)acrylates include, for example, trimethylaminoethyl methacrylate, trimethylaminoethyl acrylate, triethylaminoethyl methacrylate, triethylaminoethyl acrylate, trimethylaminopropyl methacrylate, trimethylaminopropyl acrylate, dimethylbutylaminopropyl methacrylate, diethylbutylaminopropyl acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diethylamino)ethyl methacrylate, 2-(dimethylamino)ethyl acrylate, 2-(diethylamino)ethyl acrylate, and 3-(dimethylamino)propyl acrylate.

[0040] Exemplary (meth)acrylamides include, for example, 3-(trimethylamino)propyl methacrylamide, 3-(triethylamino)propyl methacrylamide, 3-(ethyldimethylamino)propyl methacrylamide, and n-[3-(dimethylamino)propyl]methacrylamide.Preferred quaternary salts of these (meth)acryloyl monomers include, but are not limited to, (meth)acrylamidoalkyltrimethylammonium salts (e.g., 3-methacrylamidopropyltrimethylammonium chloride and 3-acrylamidopropyltrimethylammonium chloride) and (meth)acryloxyalkyltrimethylammonium salts (e.g., 2-acryloxyethyltrimethylammonium chloride, 2-methacryloxyethyltrimethylammonium chloride, 3-methacryloxy-2-hydroxypropyltrimethylammonium chloride, 3-acryloxy-2-hydroxypropyltrimethylammonium chloride, and 2-acryloxyethyltrimethylammonium methyl sulfate).

[0041] The graft copolymer further comprises optional monomer units that can be copolymerized with cationic or cationically ionizable monomers. Although it may be possible to ionize these monomers under certain conditions, they are typically not charged; they are neutral ("neutral monomers"). These neutral monomers have a polymerizable group for use during graft polymerization. The polymerizable group may be the same as or different from the polymerizable group on the cationic or cationically ionizable monomer. There may be one or more neutral monomers.

[0042] The neutral monomer may have a functional group or more than one functional group in addition to the polymerizable group. For neutral monomers with more than one functional group, these functional groups may be the same or different. Some functional groups may allow the neutral monomer to dissolve or disperse in water. Some functional groups may be hydrophilic after polymerization. Useful functional groups include hydroxyl, alkyl, aryl, ether, ester, epoxy, amide, isocyanate, or cyclic functional groups. The neutral monomer may contain a spacer group between the polymerizable group and the functional group. The neutral monomer may contain an oligomeric or polymeric functional group. In some embodiments, the polymerizable group and the functional group may be the same group.

[0043] Examples of epoxy-containing neutral monomers include glycidyl (meth)acrylate, thioglycidyl (meth)acrylate, 3-(2,3-epoxypropoxy)phenyl (meth)acrylate, 2-[4-(2,3-epoxypropoxy)phenyl]-2-(4-(meth)acryloyloxy-phenyl)propane, 4-(2,3-epoxypropoxy)cyclohexyl (meth)acrylate, 2,3-epoxycyclohexyl (meth)acrylate, and 3,4-epoxycyclohexyl (meth)acrylate, and combinations thereof. Examples of hydroxyl-containing monomers include N-hydroxyethyl (meth)acrylate, poly(ethylene glycol) (meth)acrylate, poly(propylene glycol) (meth)acrylate, N-hydroxyethyl (meth)acrylamide, 2-hydroxypropyl (meth)acrylamide, N-hydroxypropyl (meth)acrylate, 2-hydroxy-3-phenoxypropyl (meth)acrylate, and combinations thereof. Examples of suitable amide monomers include N-vinylcaprolactam, N-vinylacetamide, N-vinylpyrrolidone, (meth)acrylamide, mono- or di-N-alkyl-substituted acrylamide, and combinations thereof. Examples of suitable ether monomers include poly(ethylene glycol) (meth)acrylate, poly(propylene glycol) (meth)acrylate, 2-ethoxyethyl (meth)acrylate, ethylene glycol methyl ether (meth)acrylate, N-3-methoxypropyl (meth)acrylamide, di(ethylene glycol) methyl ether (meth)acrylate, poly(ethylene glycol) phenyl ether (meth)acrylate, 2-pheoxyethyl (meth)acrylate, other alkyl ether (meth)acrylates and alkyl ether (meth)acrylamides, tetrahydrofurfuryl (meth)acrylate, and combinations thereof.

[0044] A method for preparing a functionalized nonwoven fabric includes providing a nonwoven substrate and exposing the nonwoven substrate to ionizing radiation in an inert atmosphere, and then contacting the exposed substrate with a solution or suspension containing a grafting monomer to graft polymerize the monomer onto the nonwoven substrate.

[0045] In the first step, the nonwoven substrate is exposed to ionizing radiation in an inert atmosphere. Exemplary forms of ionizing radiation include electron beam (e-beam), gamma radiation, X-rays, and other forms of electromagnetic radiation. The inert atmosphere is generally an inert gas, such as nitrogen, carbon dioxide, helium, argon, etc., with a minimal amount of oxygen. The dose delivered by the ionizing radiation source can occur in a single dose or can be multiple doses that build up to a desired level. One or more layers of the nonwoven substrate may be subjected to ionizing radiation.

[0046] After the irradiation step, the irradiated nonwoven substrate is contacted with an aqueous monomer solution or aqueous monomer suspension. By "contact" it is meant contacting the irradiated nonwoven substrate with the monomer solution or suspension. It can also be described as the irradiated nonwoven substrate being saturated, absorbed, or coated with the monomer solution. The monomer solution may only partially fill the void volume of the nonwoven substrate, or the nonwoven substrate may be contacted with much more solution than is necessary to completely fill the void volume. The monomer contact step is also carried out in an inert atmosphere. This atmosphere may be the same or different from the atmosphere in the chamber in which the substrate is irradiated. The chamber may be the same or different from the chamber in which the substrate is irradiated. The monomer solution remains in contact with the nonwoven substrate for a sufficient time to graft polymerize with some, most, or substantially all of the monomers in the monomer solution. Once the nonwoven substrate has been in contact for the desired time, the nonwoven substrate with the grafted polymer may be removed from the inert atmosphere.

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

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

[0049] The loft of functionalized or non-functionalized nonwovens is measured by solidity, a parameter that defines the solid fraction in the volume of the web. Lower solidity values ​​indicate greater web loft. Solidity is a unitless fraction commonly expressed as α: α=m f ÷(ρ f ×L 不織布 )

[0050] Basis weight m f is the mass per surface area (functionalized or non-functionalized), and ρ f is the fiber density (functionalized or non-functionalized). L 不織布 is the thickness of the nonwoven (functionalized or unfunctionalized). Solidity can be determined for the nonwoven before or after functionalization.

[0051] The fiber density (ρ f) is determined by Method A in the Examples set forth below. The fiber density of copolymer-grafted fibers after functionalization can also be determined by a modified version of Method A in which the molar ratios of the substrate and copolymer components are all obtained from solid-state carbon-13 NMR measurements and the molar ratios are converted to weight ratios. When a nonwoven substrate contains a mixture of two or more types of fibers, the individual solidities are determined by the same L 不織布 are used to determine for each type of fiber, and these individual solidities are added together to obtain the web solidity, α.

[0052] Effective fiber diameter (EFD) is the apparent diameter of the fibers in a nonwoven fibrous web, as determined by an air permeability test in which air at 1 atmosphere and room temperature is passed through a web sample of known thickness at a face velocity of 5.3 cm / sec and the corresponding pressure drop is measured. Based on the measured pressure drop, the effective fiber diameter is calculated as described in Davies, CN, "The Separation of Airborne Dust and Particles", Institution of Mechanical Engineers, London, Proceedings 1B, 1952. EFD can be determined for nonwovens before or after functionalization.

[0053] The calculated pore size is related to the arithmetic mean fiber diameter and the web solidity and is determined by the following formula, where D is the calculated pore size and d f is the arithmetic mean fiber diameter and α is the web solidity.

[0054]

number

[0055] The calculated pore size can be determined for the nonwoven fabric before or after functionalization. Preferably, the nonwoven fabric substrate has a calculated pore size of between 1 micrometer and 50 micrometers before functionalization.

[0056] The dynamic charge capacity (DCC) of the functionalized nonwoven substrate was determined using a metanil yellow challenge solution using Example Method B and is reported as MY DCC (Metanil Yellow Dynamic Charge Capacity).

[0057] The following examples are intended to illustrate, but not limit, the disclosure. EXAMPLES

[0058] Objects and advantages of this invention are further illustrated by the following examples, but the particular materials and amounts thereof recited in these examples, as well as other conditions and details, should not be construed to unduly limit this invention.

[0059] Conductivity measurements of cell cultures and eluents were performed at ambient temperature (approximately 20° C.) using an Accumet Excel XL50 conductivity meter (Fisher Scientific, Hampton, NH).

[0060] Grafting Solution

[0061] [Table 1]

[0062] The grafting solution was prepared as a monomer solution containing 12.2 wt % NVP, 4.4 wt % GMA, and 9.7 wt % MAPTAC in deionized water.

[0063] Method A. Determination of Basis Weight, Effective Fiber Diameter (EFD), Solidity, and Pore Size of Functionalized Nonwovens The measurements of basis weight, EFD, solidity, and pore size of the functionalized nonwoven fabric were determined according to the following procedure. Sample disks (13.33 cm diameter) were punched out of the functionalized nonwoven fabric sheet, and then each disk was individually rinsed by immersing it in a 2 L bath of deionized water for 15 minutes. The rinsing procedure was repeated three more times, using fresh deionized water for each rinsing step. Each rinsed disk was dried in an oven at 70° C. for at least 4 hours. During the drying step, a weight (approximately 100 g) was placed on top of each disk to prevent edge curling. The resulting dried functionalized nonwoven fabric samples were characterized (basis weight, EFD, solidity, pore size) according to the methods and formulas described above. For each measurement or calculation, the results were reported as the average of three independent trials (n=3), along with the calculated standard deviation (SD).

[0064] Regarding the solidity (a) formula, the fiber density (ρ f ) measured based on the density of the polypropylene substrate (0.91 g / cm 3 ) and the density of the graft copolymer (1.07 g / cm3) adjusted by the weight ratio of the polypropylene substrate and the graft copolymer of the test sample. 3 ) (Equation 1). The weight ratio of polypropylene substrate to copolymer was determined by comparing the basis weight of the nonwoven fabric before the grafting process with the basis weight of the corresponding dried functionalized nonwoven fabric.

[0065] The density of the graft copolymer (D GCP ) is first solid 13 The mol% of the monomer components (NVP, MAPTAC, GMA) of the graft copolymer was determined by using C NMR (ssNMR) and converting the mol% values ​​to weight% (wt.%) values. The density value of each monomer component (monomer density: D NVP =1.04g / cm 3 , D MAPTAC =1.067g / cm 3 , D GMA =1.07g / cm 3 ) were adjusted (multiplied) by the corresponding component weight percent values, and the three resulting adjusted density values ​​were summed (Equation 2). Formula 1: Fiber density (ρ f )=(0.91×weight% ポリプロピレン )+(1.05×weight% グラフトコポリマー ) Formula 2: D GCP =(D NVP ×weight% NVP )+(D MAPTAC ×weight% MAPTAC )+(D GMA ×weight% GMA )

[0066] Method B. Measuring the Metanil Yellow Dynamic Charge Capacity (MY DCC) of Functionalized Nonwovens Functionalized nonwoven disks were prepared for testing by Method A. The dynamic charge capacity of the disks was determined using the charged organic dye, Metanil Yellow, as the target molecule in the challenge solution. The challenge solution used had a Metanil Yellow concentration of 160 mg / L (160 ppm). The challenge solution was prepared by dissolving 3.2 g Metanil Yellow, 93.98 g Sodium phosphate dibasic anhydrous, 46.64 g Sodium phosphate monobasic monohydrate, and 163.63 g NaCl in 20 L of deionized water. The challenge solution was used within 2 days of preparation. If necessary, the amount of Metanil Yellow reagent used to prepare the challenge solution was adjusted based on the purity of the reagent so that the challenge solution contained 160 ppm Metanil Yellow. Analytical standard grade Metanil Yellow (≧98.0%, product number 44426 from Sigma-Aldrich Company, St. Louis, MO) was used to calibrate the reagent purity. A buffer solution for preconditioning the test assembly was also prepared having the same formulation as the challenge solution, except that it did not contain metanil yellow.

[0067] The filtration test assembly included a clear polycarbonate body section (47 mm inner diameter) with a screw-on cap attached to the top of the body section. The cap included an inlet port and a vent port. The bottom of the body section included an outlet port with a stopcock. A pressure sensor was placed upstream of the inlet port. A polyamide membrane (0.2 micrometer grade) was placed at the bottom of the body section. A stack containing two functionalized nonwoven disks (each 47 mm in diameter and punched from disks prepared according to Method A) was placed in the assembly on top of the membrane. In the assembly, the nonwoven disk was sandwiched between two PTFE seal rings, each of which included a knife edge on the inner diameter to bite into the nonwoven. The resulting subassembly was secured in place using an O-ring. The frontal surface area of ​​the disk stack was 0.00097 m 2 The cap was attached to the body portion, and a PendoTech normal flow filtration system (PendoTech Company, Princeton, NJ) was connected to the inlet port. A Hach Model 2100AN turbidimeter (Hach Company, Loveland, CO) with a 455 nm light filter and a flow-through cell was connected to the output port and used to measure the Metanil Yellow concentration in the filtrate. Metanil Yellow solutions with concentrations of 0.8 ppm, 4 ppm, and 8 ppm were prepared as test standards. The end point of the charge capacity measurement was set at 5% breakthrough (8 ppm) of the Metanil Yellow solution. The fluid flow rate was 15 mL / min. The preconditioning buffer was allowed to flow through the assembly for approximately 5 minutes before pumping the challenge solution.

[0068] The volume of challenge solution that passed through the test assembly to the endpoint (i.e., the breakthrough capacity) was measured, and the dynamic charge capacity (mg / g) of the functionalized nonwoven samples was calculated according to Equation 3. For each functionalized nonwoven, the MY DCC was reported as the average value from three independent trials (n=3) along with the calculated standard deviation (SD).

[0069]

number

[0070] Method C. Preparation of AAV2-transfected cell cultures HEK293-F cells suspended in Gibco LV-MAX Production Medium (Thermo Fisher Scientific, Waltham, MA) were grown in an incubator using 2.8 L shake flasks with constant shaking at 90 rpm (revolutions per minute). The incubator was maintained at 8% CO 2 The cells were incubated at 37°C for 1 h at a cell density of approximately 2 × 10 6 When the total cell number was reached, a transfection cocktail was prepared and added to the shake flasks.

[0071] The transfection cocktail consisted of three plasmids pAAV2-RC2 vector (part no. VPK-422), pHelper vector (part no. 340202), and pAAV2-GFP control vector (part no. AAV2-400) (all plasmids obtained from Cell Biolabs, San Diego, CA), as well as FECTOVIR-AAV2 transfection reagent (Polyplus Transfection, New York, NY). The transfection cocktail was prepared by first adding equimolar amounts of all three plasmids, and the total plasmid amount was adjusted to be 1 microgram of plasmid mixture per million HEK cells used for transfection. DMEM (Dulbecco's Modified Eagle Medium, obtained from Thermo Fisher Scientific) was then added to the cocktail such that a final concentration of 5% DMEM (volume / volume) was achieved after the cocktail was added to the cell culture flask (i.e., the volume / volume calculation for DMEM was adjusted based on the total cell culture volume). After adding DMEM, the cocktail was mixed and then one microliter of FectoVIR-AAV2 transfection reagent was added per microgram of plasmid mixture in the cocktail. The cocktail was gently mixed and then incubated at room temperature for 45 minutes. Following the incubation step, the completed transfection cocktail was gently mixed and then added dropwise to the flask containing the cell culture. After the addition of the transfection cocktail, the cells were placed in an incubator (8% CO 2 The cells were grown in a 37°C incubation containing 0.5% CO and 10% H2O for 72 to 96 hours to induce AAV2 production.

[0072] Cell viability was measured using a hemocytometer. The harvested cell culture fluid was mixed with 10% (vol / vol) trypan blue solution and then loaded into a disposable hemocytometer. Viable and dead cells were counted under a microscope.

[0073] Turbidity measurements of the transfected cell cultures and filtrates (after filtration of the cell culture fluid through a filtrate capsule) were determined in nephelometric turbidity units (NTU) using an ORION AQ4500 turbidity meter (Thermo Fisher Scientific).

[0074] The AAV2 transfected cell cultures used in the examples had conductivity values ​​of about 9 mS / cm to 10 mS / cm, and about 3×10 6 cells / mL~7×10 6 had cell density values ​​of cells / mL, cell viability values ​​of approximately 75%-90% at the time of harvest, and turbidity values ​​of approximately 270 NTU-540 NTU.

[0075] Preparation of functionalized nonwoven fabric A (FNW-A) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter (EFD) of 16 micrometers, a basis weight of 200 grams per square meter (gsm), a solidity of 10%, and a calculated average pore size of 47.4 micrometers) was grafted with a nitrogen-purged grafting solution. The nonwoven substrate was unwound and passed through an electron beam (Electrocure, Energy Science, Inc, Wilmington, MA) set at a potential of 300 kV to deliver a total dose of 7 Mrad. The environment in the electron beam chamber was purged with nitrogen. The web was then transported directly to a nitrogen-purged saturation step with monomer solution. The web was then wound in a purged atmosphere. The web was left in the purged atmosphere for a minimum of 60 minutes, after which the web was exposed to air. The web was then unwound and transported into a tank of deionized water at a speed of 10 feet per minute for approximately 8 minutes. After exiting the tank, the web was flushed multiple times by passing a salt water solution (NaCl) through the web using a vacuum belt. A small amount of glycerin was added to the salt water solution in the final flushing step. The unwound web was dried until the moisture content of the web was less than 14% by weight. The web was then wound on a spindle. The grafted article was designated Functionalized Nonwoven A (FNW-A). The properties of FNW-A are reported in Table 2. Disks of FNW-A (2.54 cm diameter) were punched from the web.

[0076] Preparation of functionalized nonwoven fabric B (FNW-B) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 14 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 41.5 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was designated Functionalized Nonwoven B (FNW-B). The properties of FNW-B are reported in Table 2. Disks of FNW-B (2.54 cm diameter) were punched from the web.

[0077] Preparation of functionalized nonwoven fabric C (FNW-C) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 12 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 35.6 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was designated Functionalized Nonwoven C (FNW-C). The properties of FNW-C are reported in Table 2. Disks of FNW-C (2.54 cm diameter) were punched from the web.

[0078] Preparation of functionalized nonwoven fabric D (FNW-D) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 10 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 29.6 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was designated Functionalized Nonwoven D (FNW-D). The properties of FNW-D are reported in Table 2. Disks of FNW-D (2.54 cm diameter) were punched from the web.

[0079] Preparation of functionalized nonwoven fabric E (FNW-E) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 8 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 23.7 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was designated Functionalized Nonwoven E (FNW-E). The properties of FNW-E are reported in Table 2. Disks of FNW-E (2.54 cm diameter) were die-cut from the web.

[0080] Preparation of functionalized nonwoven fabric F (FNW-F) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 6 micrometers, a basis weight of 200 gsm, a solidity of 10%, and a calculated average pore size of 17.8 micrometers) was grafted using the same procedure described for FNW-A. The grafted article was designated Functionalized Nonwoven F (FNW-F). The properties of FNW-F are reported in Table 2. Disks of FNW-F (2.54 cm diameter) were die-cut from the web.

[0081] Preparation of functionalized nonwoven fabric G (FNW-G) A non-functionalized meltblown polypropylene microfiber nonwoven web (having an effective fiber diameter of 4.2 micrometers, a basis weight of 100 gsm, a solidity of 8.2%, and a calculated average pore size of 14.2 micrometers) was grafted using the same procedure as described for FNW-A. The grafted article was designated Functionalized Nonwoven G (FNW-G). The properties of FNW-G are reported in Table 2. The MY DCC was determined by Method B using four functionalized nonwoven discs instead of two discs. Discs (2.54 cm diameter) of Functionalized Nonwoven G were punched from the web.

[0082] [Table 2]

[0083] Example 1 (Ex.1). A plastic filtration capsule was used. The capsule consisted of a sealed circular housing. The capsule housing was prepared from two halves (top and bottom halves) that were joined and sealed together at the periphery after the filtration element was inserted into the inner cavity of the lower housing. The fluid inlet and vent ports were located at the top of the housing, and the fluid outlet port was located at the bottom of the housing. The outlet port was located in the center of the lower housing surface.

[0084] Two disks (27 mm diameter) of TYPAR 3161L polypropylene spunbond nonwoven (10 mil thick, obtained from Fiberweb, Inc., Old Hickory, TN) were placed on the bottom of the lower housing. A single disk (27 mm diameter) of MICRO-PES Flat Type 2F polyethersulfone membrane (obtained from 3M Company) with a nominal pore size of 0.2 micrometers was placed on top of the nonwoven layer. The nonwoven layer and membrane layer were ultrasonically welded at the edges to the bottom inner surface of the lower housing. A stack of four functionalized nonwoven layers (27 mm diameter) was then placed on top of the membrane. The stack included two disks of functionalized nonwoven C, one disk of functionalized nonwoven E, and one disk of functionalized nonwoven F. The orientation of the disks from the capsule inlet to the outlet was FNW-C / FNW-C / FNW-E / FNW-F. A polypropylene spacer ring (25.4 mm outer diameter, 21.84 mm inner diameter, 50 mil thickness) was inserted between the second and third nonwoven layers. The upper and lower housings were joined and ultrasonically welded together using a Branson 20 kHz ultrasonic welder (Model 2000xdt, Emerson Electric Company, St. Louis, MO) to form the completed filter capsule.

[0085] The overall outer diameter of the finished capsule was approximately 4.3 cm, and the overall height, including the inlet, outlet, and vent ports, was approximately 5.9 cm. The effective filtration area of ​​the capsule was 3.2 cm. 2 and the bed volume of the nonwoven media was 2.1 mL.

[0086] Example 2 (Ex.2). The same procedure was followed as described in Example 1 for preparing a filtration capsule, except that a different stack of nonwoven layers was used. The stack included one disk of functionalized nonwoven C, one disk of functionalized nonwoven D, one disk of functionalized nonwoven E, and one disk of functionalized nonwoven F. The orientation of the disks from the capsule inlet to the outlet was FNW-C / FNW-D / FNW-E / FNW-F. A polypropylene spacer ring (25.4 mm outer diameter, 21.84 mm inner diameter, 50 mil thick) was inserted between the second and third nonwoven layers.

[0087] Example 3 (Ex.3). The same procedure was followed as described in Example 1 for preparing filtration capsules, except that a different stack of nonwoven layers was used. The stack included two disks of functionalized nonwoven A, one disk of functionalized nonwoven E, and one disk of functionalized nonwoven F. The orientation of the disks from the capsule inlet to the outlet was FNW-A / FNW-A / FNW-E / FNW-F. A polypropylene spacer ring (25.4 mm outer diameter, 21.84 mm inner diameter, 50 mil thick) was inserted between the second and third nonwoven layers.

[0088] Example 4 (Ex.4). The same procedure was followed as described in Example 1 for preparing a filtration capsule, except that a different stack of nonwoven layers was used. The stack included three disks of functionalized nonwoven C, one disk of functionalized nonwoven E, and one disk of functionalized nonwoven F. The orientation of the disks from the capsule inlet to the outlet was FNW-C / FNW-C / FNW-C / FNW-E / FNW-F. A polypropylene spacer ring (25.4 mm outer diameter, 21.84 mm inner diameter, 50 mil thick) was inserted between the third and fourth nonwoven layers.

[0089] Example 5 (Ex.5). The same procedure was followed as described in Example 1 for preparing filtration capsules, except that a different stack of nonwoven layers was used. The stack included one disk of functionalized nonwoven A, three disks of functionalized nonwoven C, and one disk of functionalized nonwoven F. The orientation of the disks from the capsule inlet to the outlet was FNW-A / FNW-C / FNW-C / FNW-C / FNW-F. A polypropylene spacer ring (25.4 mm outer diameter, 21.84 mm inner diameter, 50 mil thick) was inserted between the third and fourth nonwoven layers.

[0090] Example 6 (Ex.6) Osmotic lysis The reservoir containing the AAV2 transfected cell culture was connected using flexible tubing to the inlet port of the capsule prepared according to Example 1. A Masterflex L / S peristaltic pump (Masterflex, Vernon Hills, IL) was used to pump a constant flux of 200 LMH and 100 L / m 2 The cell culture was pumped through the capsules at a throughput of 1000 µL / well. The cell culture in the reservoir was stirred throughout the procedure. The resulting filtrate was collected and analyzed for AAV2 content (number of AAV2 capsids in the filtrate) using a ProGen AAV2 Xpress ELISA kit (obtained from American Research Products, Inc., Waltham, MA) according to the manufacturer's instructions.

[0091] After the capsule was filled with the cell culture, three separate fluids were pumped sequentially through the capsule via the inlet ports. The first fluid was a hypertonic aqueous sucrose solution (40% by weight). Approximately 54 L / m 2 The sucrose solution was pumped through the capsule at a constant flux of 200 LMH. The pump was then stopped and the functionalized filtration disks containing the bound HEK293 cells were allowed to equilibrate with the sucrose solution remaining within the capsule for 15-30 min. Following the equilibration period, deionized water (62.5 L / m 2) was pumped through the capsule at a constant flux of 200 LMH. The pump was stopped again to allow the functionalized filtration disk containing the bound HEK293 cells to equilibrate with the deionized water remaining in the capsule for 15-30 min. Following the equilibration period, a hyperosmolar aqueous NaCl solution (400 mM, 40 mS / cm) was pumped at a constant flux of 200 LMH and 62.5 L / m for the final elution of AAV2 capsids from the functionalized nonwoven disk. 2 The solution was pumped through the capsule at a throughput of 1000 ml.

[0092] After each of the three fluids was loaded, the collected filtrate was removed and analyzed for AAV2 capsid content (number of AAV2 capsids in each filtrate). A new collection vessel was used to collect each filtrate sample during the procedure. The AAV2 content of each filtrate was determined using the ProGen AAV2 Xpress ELISA kit. The results are shown in Table 3.

[0093] [Table 3]

[0094] Example 7 (Ex. 7). The cell cultures were incubated at a constant flow rate of 200 LMH and 300 L / m 2 The same procedure was followed as reported in Example 6, except that the loading was at a throughput of 1000 μl / min. The results are shown in Table 4.

[0095] [Table 4]

[0096] Comparative Example A (CEx.A). Alternative Detergent Dissolution Procedure A 2.8 L shake flask from the set used in preparing AAV2 transfected cell cultures (Method C procedure) was filled with TRITON® X-100 detergent (obtained from Promega Corporation, Madison, WI) to achieve a final detergent concentration of 0.1% by weight. To lyse the cells, the flask was placed in an incubator (37° C., 8% CO2 The lysed cell culture was then clarified using the following procedure.

[0097] A reservoir containing the cell culture fluid to be lysed was connected using flexible tubing to the inlet port of a capsule prepared according to Example 1. A Masterflex L / S peristaltic pump was used to provide a constant flow rate of 200 LMH and 100 L / m 2 The fluid was pumped through the capsule at a throughput of 4.45 × 10. The lysed cell culture fluid in the reservoir was stirred throughout the procedure. The resulting filtrate was collected and analyzed for AAV2 content using the ProGen AAV2 Xpress ELISA kit. A total of 4.45 × 10 10 AAV2 capsids were recovered in the filtrate. The number of capsids recovered by this procedure was approximately 200-fold lower than those recovered using the procedure of Example 6.

[0098] In this comparative example, the system differential pressure rose to about 15 psid (pounds per square inch differential) near the end of filtration, indicating increased fouling of the functionalized nonwoven media within the capsules. In contrast, loading the capsules with unlysed AAV2 transfected cell culture resulted in a maximum differential pressure of only about 1 psid, indicating that greater loading of the filtration capsules could be achieved using the filtration procedure of Example 6 instead of filtration procedure Comparative Example A.

[0099] Example 8 (Ex.8). The reservoir containing the AAV2 transfected cell culture was connected using flexible tubing to the inlet port of the capsule prepared according to Example 1. A Masterflex L / S peristaltic pump was used to deliver a constant flow of 200 LMH and 100 L / m 2The cell culture medium was pumped through the capsule at a throughput of 100 µL / m. The cell culture in the reservoir was stirred throughout the procedure. After loading the capsule with the cell culture, a hypertonic aqueous NaCl solution (conductivity of 20 mS / cm) was pumped at a constant flux of 200 LMH and a flow rate of 160 L / m for elution of AAV2 capsids from the functionalized nonwoven disks. 2 The solution was pumped through the capsule at a throughput of 1000 ml.

[0100] The resulting filtrate was collected and analyzed for AAV2 capsid content using the ProGen AAV2 Xpress ELISA kit. The turbidity of the culture was measured (using an ORION AQ4500 turbidity meter) before and after filtration through the filtration capsule to determine the percent reduction in turbidity resulting from the filtration procedure. The turbidity of the cell culture before filtration through the filtration capsule was 540 NTU. The results are shown in Table 5.

[0101] Example 9 (Ex. 9). The same procedure was followed as described in Example 8, except that the aqueous NaCl solution used for elution had a conductivity of 25 mS / cm. The AAV2 capsid content of the filtrate is shown in Table 5.

[0102] Example 10 (Ex. 10). AAV2-transfected cell cultures were cultured at 300 L / m 2 The same procedure was followed as described in Example 9, except that a throughput of 100 µL was used and a constant flux of 200 LMH was pumped through the capsules. The AAV2 capsid content of the filtrate is shown in Table 5.

[0103] Comparative Example B (CEx.B). Alternative detergent dissolution procedure TRITON®-X100 detergent solution was added to a small sample (50 mL) of AAV2 transfected cell culture (Method C procedure) to achieve a final detergent concentration of 0.1% (vol / vol). To lyse the cells, the flask was placed in an incubator (37° C., 8% CO 2The cells were placed on a shaking table in a 50-mL centrifuge (set to 35° C.) and shaken at 90 rpm for 2 hours. After the incubation period, aqueous sodium chloride (5 M) was added to adjust the conductivity of the cell culture to 20 mS / cm. The lysed cell culture was centrifuged at 2500×g for 1 minute. The resulting supernatant was then filtered through a 0.2 micron PES (polyethersulfone) membrane filter. The filtrate was collected and analyzed for AAV2 capsid content using a ProGen AAV2 Xpress ELISA kit. The number of AAV2 capsids measured in the filtrate was used to calculate the corresponding number of capsids in a sample having the same volume as the culture sample loaded in Example 8. The results are shown in Table 5.

[0104] [Table 5] N / A=Not Applicable ND=Not determined

[0105] Example 11 (Ex. 11). The reservoir containing the AAV2 transfected cell culture was connected using flexible tubing to the inlet port of the capsule prepared according to Example 1. A Masterflex L / S peristaltic pump was used to deliver a constant flow of 200 LMH and 183 L / m 2 The cell culture was pumped through the capsules at a throughput of 100 µL / min. The cell culture in the reservoir was stirred throughout the procedure. After loading the capsules with the cell culture, hypertonic phosphate buffered saline (PBS) (1×, pH 7.4, conductivity of 15 mS / cm) was pumped at a constant flux of 200 LMH (183 L / m 2 ) for elution of AAV2 capsids from the functionalized nonwoven disks. 2 The resulting filtrate was collected and analyzed for AAV2 capsid content. The AAV2 capsid content of the filtrate was determined using the ProGen AAV2 Xpress ELISA kit. The results are shown in Table 6.

[0106] Example 12 (Ex. 12). The same procedure was followed as described in Example 11, except that the conductivity of the PBS solution used to elute the AAV2 capsid from the functionalized nonwoven discs was 21 mS / cm. The AAV2 capsid content of the filtrate is shown in Table 6.

[0107] Example 13 (Ex. 13). The reservoir containing the AAV2 transfected cell culture was connected using flexible tubing to the inlet port of the capsule prepared according to Example 1. A Masterflex L / S peristaltic pump was used to deliver a constant flow of 200 LMH and 91 L / m 2 The cell culture was pumped through the capsules at a throughput of 100 µL / min. The cell culture in the reservoir was stirred throughout the procedure. After loading the capsules with the cell culture, a hypertonic 0.2 M sodium phosphate solution (conductivity of 20 mS / cm) was pumped at a constant flux of 200 LMH (91 L / m) for elution of AAV2 capsids from the functionalized nonwoven disks. 2 The resulting filtrate was collected and analyzed for AAV2 capsid content. The AAV2 capsid content of the filtrate was determined using the ProGen AAV2 Xpress ELISA kit. The results are shown in Table 6.

[0108] Example 14 (Ex. 14). The same procedure was followed as described in Example 13, except that 0.1 M sodium phosphate solution (conductivity: 11.5 mS / cm) was used to elute the AAV2 capsid from the functionalized nonwoven disks. The AAV2 capsid content of the filtrate is shown in Table 6.

[0109] [Table 6]

[0110] Example 15 (Ex. 15). The reservoir containing the AAV2 transfected cell culture was connected using flexible tubing to the inlet port of the capsule prepared according to Example 1. A Masterflex L / S peristaltic pump was used to deliver a constant flux of 200 LMH and 107 L / m 2 The cell culture was pumped through the capsule at a throughput of 100 µL / m. The cell culture in the reservoir was stirred throughout the procedure. After loading the capsule with the cell culture, hyperosmolar 50 mM Tris buffer (conductivity adjusted to 25 mS / cm using 5 M NaCl) was pumped at a constant flux of 200 LMH and 100 L / m for elution of AAV2 capsids from the functionalized nonwoven disks. 2 The resulting filtrate was collected and analyzed for AAV2 capsid content using the ProGen AAV2 Xpress ELISA kit. The results are shown in Table 7.

[0111] Example 16 (Ex. 16). The same procedure was followed as described in Example 15, except that a filtration capsule prepared according to Example 2 was used. A constant flux of 200 LMH and 101 L / m 2 The cell culture medium was pumped through the capsule at a constant flux of 200 LMH and a throughput of 100 L / m 2 Tris buffer was pumped through the capsule at a throughput of 1000 µL. The results are shown in Table 7.

[0112] Example 17 (Ex. 17). The same procedure was followed as described in Example 15, except for using the filtration capsule prepared according to Example 3. A constant flux of 200 LMH and 169 L / m 2 The cell culture medium was pumped through the capsule at a constant flux of 200 LMH and a throughput of 100 L / m 2 Tris buffer was pumped through the capsule at a throughput of 1000 µL. The results are shown in Table 7.

[0113] Example 18 (Ex. 18). The same procedure was followed as described in Example 15, except that a filtration capsule prepared according to Example 4 was used. A constant flux of 200 LMH and 76 L / m 2 The cell culture medium was pumped through the capsule at a constant flux of 200 LMH and a throughput of 100 / m 2 Tris buffer was pumped through the capsule at a throughput of 1000 µL. The results are shown in Table 7.

[0114] Example 19 (Ex. 19). The same procedure was followed as described in Example 15, except that a filtration capsule prepared according to Example 5 was used. A constant flux of 200 LMH and 125 L / m 2 The cell culture medium was pumped through the capsule at a constant flux of 200 LMH and a throughput of 100 L / m 2 Tris buffer was pumped through the capsule at a throughput of 1000 µL. The results are shown in Table 7.

[0115] [Table 7]

[0116] Example 20 (Ex. 20). The reservoir containing the AAV2 transfected cell culture was connected using flexible tubing to the inlet port of the capsule prepared according to Example 1. A Masterflex L / S peristaltic pump was used to deliver a constant flow of 200 LMH and 183 L / m 2 The cell culture was pumped through the capsules at a throughput of 100 µL / min. The cell culture in the reservoir was stirred throughout the procedure. After loading the capsules with the cell culture, an aqueous solution containing hypertonic 1× PBS and 0.1 wt % TRITON® X-100 (conductivity: 15 mS / cm, pH 7.4) was pumped at a constant flux of 200 LMH (183 L / m 2) for elution of AAV2 capsids from the functionalized nonwoven disks. 2The resulting filtrate was collected and analyzed for AAV2 capsid content. The AAV2 capsid content of the filtrate was determined using the ProGen AAV2 Xpress ELISA kit. The results are shown in Table 8.

[0117] Example 21 (Ex. 21). The same procedure was followed as described in Example 20, except that the conductivity of the solution used to elute the AAV2 capsid from the functionalized nonwoven discs was 21 mS / cm. The AAV2 capsid content of the filtrate is shown in Table 8.

[0118] Example 22 (Ex. 22). The reservoir containing the AAV2 transfected cell culture was connected using flexible tubing to the inlet port of the capsule prepared according to Example 1. A Masterflex L / S peristaltic pump was used to deliver a constant flow of 200 LMH and 91 L / m 2 The cell culture was pumped through the capsule at a throughput of 100 µL / min. The cell culture in the reservoir was stirred throughout the procedure. After loading the capsule with the cell culture, a hypertonic aqueous solution (conductivity: 20 mS / cm) containing 0.2 M sodium phosphate and 0.1 wt. % TRITON® X-100 was pumped at a constant flux of 200 LMH (91 L / m) for elution of AAV2 capsids from the functionalized nonwoven disks. 2 The resulting filtrate was collected and analyzed for AAV2 capsid content. The AAV2 capsid content of the filtrate was determined using the ProGen AAV2 Xpress ELISA kit. The results are shown in Table 8.

[0119] Example 23 (Ex. 23). The same procedure was followed as described in Example 22, except that AAV2 capsids were eluted from the functionalized nonwoven disks using an aqueous solution containing 0.1 M sodium phosphate and 0.1 wt % TRITON® X-100 (conductivity: 11.5 mS / cm). The AAV2 capsid content of the filtrate is shown in Table 8.

[0120] [Table 8]

[0121] Example 24 (Ex. 24). The reservoir containing the AAV2 transfected cell culture was connected using flexible tubing to the inlet port of the capsule prepared according to Example 1. A Masterflex L / S peristaltic pump was used to deliver a constant flow of 200 LMH and 100 L / m 2 The cell culture medium was pumped through the capsule at a throughput of 1000 rpm. The cell culture in the reservoir was stirred throughout the procedure.

[0122] After loading the capsules with the cell culture, hypotonic conditions were applied to the trapped cells by introducing deionized water. A constant flux of 200 LMH and a flow rate of 62.5 L / m 2 Deionized water was pumped through the capsules at a throughput of 100 µL / m. After application of the deionized water, the pump was stopped and the trapped cells were allowed to equilibrate and lyse with the water retained within the capsules for approximately 30 min. After equilibration with hypotonicity, a hypertonic solution of Tris buffer (50 mM, conductivity of 25 mS / cm) was pumped at a constant flux of 200 LMH (100 L / m) for elution of AAV2 capsids from the functionalized nonwoven disks. 2 The resulting filtrate was collected and analyzed for AAV2 capsid content using the ProGen AAV2 Xpress ELISA kit. The lysis process yielded a 2.42 × 10 11 This resulted in the recovery of AAV2 capsids.

[0123] All references and publications cited herein are expressly incorporated by reference in their entirety into this disclosure. Exemplary embodiments of the invention have been described and reference has been made to possible variations within the scope of the invention. For example, features shown in connection with one exemplary embodiment may be used in connection with other embodiments of the invention. These and other variations and modifications of the invention will be apparent to those skilled in the art without departing from the scope of the invention, and it should be understood that the invention is not limited to the exemplary embodiments described herein. Therefore, the invention should be limited only by the claims provided below and their equivalents.

Claims

**Claim 1** Providing a filter medium comprising a functionalized nonwoven fabric; Passing a first fluid containing cells through the filter medium, wherein at least a portion of the cells are captured by the functionalized nonwoven fabric; Passing a second fluid, and optionally a third and / or a fourth fluid, through the filter medium, wherein the second and / or third and / or fourth fluid destroys at least one of the captured cells; Recovering intracellular biological products in the first and / or second and / or third and / or fourth fluid, a method comprising. **Claim 2** The method according to claim 1, wherein the second fluid has an osmotic potential lower than that of the first fluid, and a change in the osmotic potential within the filter medium results in the destruction of at least one of the cells. **Claim 3** The method according to claim 1, wherein the second fluid has an osmotic potential higher than that of the first fluid, and a change in the osmotic potential within the filter medium results in the destruction of at least one of the cells. **Claim 4** The method according to claim 1, wherein the second fluid comprises a surfactant or a chemical cell lysing agent. **Claim 5** The method according to claim 1, wherein the conductivity of the third fluid is different from that of the first fluid and different from that of the second fluid. **Claim 6** The method according to claim 1, wherein the third or fourth fluid is a conductive salt solution having a conductivity in the range of 9 mS / cm to 60 mS / cm. **Claim 7** The method according to any one of claims 1 to 6, wherein the functionalized nonwoven fabric has a dynamic charge capacity. **Claim 8** The method according to any one of claims 1 to 6, wherein the functionalized nonwoven fabric is cationically charged. **Claim 9** The method according to any one of claims 1 to 6, wherein at least a portion of the cells are electrostatically captured by the functionalized nonwoven fabric. **Claim 10** The method according to any one of claims 1 to 6, wherein the functionalized nonwoven fabric is grafted with a copolymer comprising copolymerized monomer units containing a quaternary ammonium monomer, an amide monomer, and an epoxy monomer. **Claim 11** The method according to any one of claims 1 to 6, wherein the filter medium comprises a plurality of layers of functionalized nonwoven fabric, each layer having the same or different calculated pore sizes and the same or different dynamic charge capacities. **Claim 12** The method according to any one of claims 1 to 6, wherein the intracellular biological product comprises an AAV capsid, a therapeutic / recombinant protein, a plasmid, DNA, RNA, a virus, a virus-like particle, an exosome, and mixtures thereof.