Flow-through process and device for purifying target molecules - Patents.com

Abstract

A flow-through process for purifying target molecules (e.g., antibodies, enzymes, and hormones, particularly monoclonal antibodies) from a biological solution (e.g., a neutralized virus inactivated pool) in a sample containing the target molecules, and a device for carrying out such a process.

Description

[Background technology]

[0001] One of the most challenging areas in the manufacturing of biopharmaceuticals is the filtration and purification of monoclonal antibodies, which are crucial for therapeutic applications in a variety of diseases, including rheumatoid arthritis, Crohn's disease, hypercholesterolemia, and various cancers. Conventional downstream processing of therapeutic antibodies involves many steps. Purification schemes involve flow-through and binding and elution chromatography. Many of these processes require buffer exchange to enhance performance in reducing impurities such as DNA, HCP, and target protein aggregates (HMWs) while attempting to minimize product loss. For example, ion exchange chromatography processes are typically performed in binding and elution modes and often require buffer exchange, such as pH and salt adjustments. Simplifying these chromatographic steps to multifunctional ion exchange or hybrid devices by reducing or eliminating buffer exchange steps reduces processing time and materials during protein purification. The biopharmaceuticals industry is increasingly interested in the identification of continuous processing schemes, particularly flow-through processes, to help reduce processing time and steps. [Overview of the project]

[0002] This disclosure provides a flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, and a device for carrying out such a process.

[0003] In one embodiment, a flow-through process for purifying a target molecule from a biological solution in a sample optionally includes contacting the sample with an anion exchange adsorption depth filter (i.e., a filter element), optionally performing a buffer exchange with the sample before and / or after contacting the sample with the anion exchange adsorption depth filter, contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element (e.g., a membrane), and immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element (e.g., a membrane), wherein the flow-through process includes one or two buffer exchanges and does not include a buffer exchange between the contact of the sample with the salt-tolerant anion exchange non-fibrous porous filter element and the contact of the cation exchange non-fibrous porous filter element.

[0004] In another embodiment, a flow-through process for purifying a target molecule from a biological solution in a sample includes contacting the sample with an anion exchange adsorption depth filter, then immediately contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element, and then immediately contacting the sample with a cation exchange non-fibrous porous filter element, wherein the flow-through process does not involve buffer exchange.

[0005] In yet another embodiment, a flow-through process for purifying a target molecule from a biological solution in a sample includes contacting the sample with an anion exchange adsorption depth filter, performing buffer exchange with the sample after contacting the sample with the anion exchange adsorption depth filter, contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element, and immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element.

[0006] In another embodiment, a flow-through process for purifying a target molecule from a biological solution in a sample includes performing a buffer exchange with the sample, contacting the sample with a salt-tolerant anion-exchange non-fibrous porous filter element, and immediately thereafter contacting the sample with a cation-exchange non-fibrous porous filter element.

[0007] In further embodiments, the disclosure provides a filter cartridge comprising a salt-resistant anion-exchange non-fibrous porous filter element described herein and, preferably, a cation-exchange non-fibrous porous filter element described herein, positioned downstream from the salt-resistant filter element.

[0008] As used herein, “alkyl” refers to a monovalent group that is an alkane group, and includes linear, branched, cyclic, and bicyclic alkyl groups, as well as combinations thereof, including both unsubstituted and substituted alkyl groups. Unless otherwise indicated, alkyl groups typically contain 1 to 30 carbon atoms. In some embodiments, alkyl groups contain 1 to 20 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. Examples of “alkyl” groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl, t-butyl, isopropyl, n-octyl, n-heptyl, ethylhexyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, and others.

[0009] The term "alkylene" refers to a divalent group that is an alkane, and includes groups that are linear, branched, cyclic, bicyclic, or a combination thereof. Unless otherwise indicated, alkylene groups typically have 1 to 30 carbon atoms. In some embodiments, alkylene groups have 1 to 20 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In some embodiments, alkylene is a linear saturated divalent hydrocarbon having 1 to 12 carbon atoms, and in some embodiments, alkylene is a branched saturated divalent hydrocarbon having 3 to 12 carbon atoms, such as methylene, ethylene, propylene, 2-methylpropylene, pentylene, hexylene, 1,4-cyclohexylene, 1,4-cyclohexyldimethylene, etc.

[0010] The term "aryl" refers to a monovalent group that is aromatic and optionally a carbocyclic group. An aryl group has at least one aromatic ring. Any further rings may be unsaturated, partially saturated, saturated, or aromatic. Optionally, the aromatic ring may have one or more further carbocyclic groups fused to it. Unless otherwise indicated, an aryl group typically has 6 to 30 carbon atoms. In some embodiments, an aryl group has 6 to 20, 6 to 18, 6 to 16, 6 to 12, or 6 to 10 carbon atoms. Examples of aryl groups include phenyl, tolyl, benzyl, phenethyl, naphthyl, 2-naphthylethyl, biphenyl, phenanthryl, and anthracyl.

[0011] "Hydrocarbyl" contains aryl and alkyl compounds. "Hydrocarbylene" contains arylene and alkylene compounds.

[0012] "(hetero)hydrocarbyl" includes hydrocarbyl (alkyl and aryl) groups and heterohydrocarbyl (heteroalkyl and heteroaryl) groups. Heterohydrocarbyl groups include one or more suspended (in-chain) heteroatoms, or one or more substituents containing heteroatoms such as oxygen, sulfur, or nitrogen atoms. Heterohydrocarbyl groups may optionally contain one or more suspended (in-chain) functional groups, such as ester functional groups, amide functional groups, urea functional groups, urethane functional groups, and carbonate functional groups. Unless otherwise indicated, nonpolymeric (hetero)hydrocarbyl groups typically contain 1 to 60 carbon atoms, 1 to 40 carbon atoms, 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Some examples of such heterohydrocarbyls used herein include, but are not limited to, methoxyethyl, ethoxypropyl, propoxyethyl, 4-diphenylaminobutyl, 2-(2'-phenoxyethoxyl)ethyl, 3,6-dioxaheptyl, 3,6-dioxahexyl-6-phenyl, 2-imidazolyl, 3-furyl, and 3-indolemethyl.

[0013] "(hetero)hydrocarbylene" includes hydrocarbylene (alkylene and arylene) groups, and heterohydrocarbylene (heteroalkylene and heteroarylene) groups. A heterohydrocarbylene group includes one or more suspended (in-chain) heteroatoms, or one or more substituents containing heteroatoms such as oxygen, sulfur, or nitrogen atoms. A heterohydrocarbylene group may optionally contain one or more suspended (in-chain) functional groups, such as ester functional groups, amide functional groups, urea functional groups, urethane functional groups, and carbonate functional groups. Unless otherwise indicated, nonpolymeric (hetero)hydrocarbylene groups typically contain 1 to 60 carbon atoms, 1 to 40 carbon atoms, 1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms. Some examples of such heterohydrocarbylenes used herein include, but are not limited to, oxydiethylene, 3-thiabutylene, 3-diphenylaminobutylene, 2-(2'-phenoxyethyl)ethylene, 3,6-dioxaoctylene, 3,6-dioxahexyl-6-phenylene, 2,5-frylene, 2,6-pyridylene (also known as 2,6-pyridinediyl), and 2,5-thiophenedimethylene.

[0014] The term "ethylenically unsaturated group" refers to a group having a carbon-carbon double (or triple) bond that can be polymerized with free radicals, and includes (meth)acrylamide, (meth)acrylate, vinyl, vinyloxy groups, allyl and allyloxy groups, and acetylene groups.

[0015] The terms "polymer" and "polymer material" include, but are not limited to, organic homopolymers, copolymers (e.g., block copolymers, graft copolymers, random copolymers, and alternating copolymers), terpolymers, and blends and modifications thereof. Furthermore, unless otherwise specified, the term "polymer" encompasses all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic symmetry, syndiotactic symmetry, and atactic symmetry.

[0016] In this specification, the terms “including” and their variations are not restrictive when they appear in the specification and claims. Such terms are understood to suggest that they include one or more processes or elements described, but not that any other one or more processes or elements, or any other group of processes or elements, is excluded. “Consists of” means that it includes and is limited to everything that precedes the phrase “consists of.” Thus, the phrase “consists of” indicates that the enumerated elements are necessary or essential, and no other elements may be present. “Essentially consists of” means that it includes all elements that precede it, and is limited to other elements that do not interfere with or contribute to the function or role specified in this disclosure with respect to those enumerated elements. Thus, the phrase “essentially consists of” indicates that the enumerated elements are necessary or essential, but the other elements are optional and may or may not be present, depending on whether they substantially affect the function or role of the enumerated elements. Any element or combination of elements described herein in open-ended language (e.g., "contains" and its derivatives) may be further described in closed-ended language (e.g., "consists of" and its derivatives) and partially closed-ended language (e.g., "essentially consists of" and its derivatives).

[0017] The terms “preferred” and “preferably” refer to embodiments of the disclosure that can provide a particular benefit under certain circumstances. However, other claims may also be preferred under the same or other circumstances. Furthermore, descriptions of one or more preferred claims do not imply that the other claims are unhelpful, nor are they intended to exclude the other claims from the scope of the disclosure.

[0018] In this application, terms such as "a," "an," and "the" are not intended to refer only to singular entities, but include general classifications, and specific examples thereof may be used for illustrative purposes. The terms "a," "an," and "the" are used interchangeably with the term "at least one." The phrases "at least one of" and "including at least one of" following an enumeration refer to any one item in the enumeration, or any combination of two or more items in the enumeration.

[0019] As used herein, the term "or" is generally used in its ordinary sense, including "and / or," unless otherwise specified.

[0020] The term "and / or" means one or all of the listed elements, or any combination of two or more of the listed elements.

[0021] Furthermore, in this specification, all numbers are considered to be modified by the term “about,” and in certain embodiments, preferably by the term “exactly.” As used herein, in relation to a measured quantity, the term “about” refers to the variation of the measured quantity that can be predicted by a person skilled in the art who performs the measurement and exercises a level of care commensurate with the purpose of the measurement and the precision of the measuring instrument used. In this specification, the “maximum” number (e.g., maximum 50) includes that number (e.g., 50).

[0022] Furthermore, in this specification, the description of a numerical range by endpoints includes all numbers and their endpoints that fall within that range (for example, 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0023] As used herein, the term "room temperature" refers to a temperature of 20°C to 25°C or 22°C to 25°C.

[0024] The terms “in the range” or “within a range” (and similar phrases) include the endpoints of the range that is described.

[0025] The grouping of alternative elements or embodiments disclosed herein should not be construed as limiting. Each member of a group may be referred to and claimed individually or in any combination with other members of that group or other elements found within that group. For convenience and / or patentability reasons, it is anticipated that one or more members of a group may be included in or removed from a group. In the event of any such inclusion or removal, this specification shall be deemed to include the modified groups herein and thus realize the content of all Markush groups used in the appended claims.

[0026] If a group is present in multiple instances in a formula described herein, each group is selected "independently," regardless of whether it is specifically described or not. For example, if there are multiple Y groups in a formula, each Y group is selected independently. Furthermore, any subordinate groups contained within these groups are also selected independently. For example, if each Y group contains R, each R is also selected independently.

[0027] Throughout this specification, references to “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments” mean that the specific features, configurations, compositions, or properties described in relation to an embodiment are included in at least one embodiment of the present invention. Therefore, where such phrases appear in various places throughout this specification, they do not necessarily refer to the same embodiment of the present invention. Furthermore, specific features, configurations, compositions, or properties may be combined in any suitable manner in one or more embodiments.

[0028] The above “Summary of the Invention” in this disclosure is not intended to describe each or all of the disclosed embodiments of the Invention. The following description more specifically illustrates exemplary embodiments. Throughout this application, guidance is provided in several places through the enumeration of examples, which can be used in various combinations. In each example, the enumerated items described serve only as representative groups and should not be construed as an exclusive enumeration. Accordingly, the scope of this disclosure should not be limited to the specific exemplary structures described herein, but extends to at least the structures described by the language of the claims, and their equivalents. Any element that is actively described as an option herein may be expressly included in or excluded from the claims in any combination as desired. Various theories and possible mechanisms may be considered herein, but in no case shall such considerations limit the subject matter to which claims can be made. [Brief explanation of the drawing]

[0029] [Figure 1] This flowchart illustrates various flow-through processes for purifying target molecules from biological solutions as described herein. [Figure 2] This is a schematic diagram of a typical filter cartridge device for performing various flow-through processes for purifying target molecules from biological solutions as described herein. [Modes for carrying out the invention]

[0030] This disclosure provides a flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, and a device for carrying out such a process.

[0031] In certain embodiments, the target molecule includes a therapeutic agent. In certain embodiments, the target molecule includes viral vectors, proteins such as antibodies, and enzymes and hormones. In certain embodiments, the target molecule includes a monoclonal antibody.

[0032] In certain embodiments, the biological solution comprises a neutralized virus-inactivating pool. The virus-inactivating pool is a biological solution consisting of a protein A chromatography eluate, or a combination of several protein A eluates, which is then subjected to a virus-inactivating process involving acid titration, followed by a retention time at an appropriate retention pH, typically pH ≤ 3.5.

[0033] In one embodiment, the Disclosure provides a flow-through process for purifying a target molecule from a biological solution in a sample. As shown in Figure 1A, the process optionally includes contacting the sample (of the biological solution) with an anion exchange adsorption depth filter (AEX adsorption depth filter), optionally performing buffer exchange with the sample before and / or after contacting the sample with the anion exchange adsorption depth filter, contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element (salt-tolerant AEX FE), and immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element (CEX FE) (e.g., a membrane), wherein the flow-through process includes one or two buffer exchanges and does not include buffer exchange between the contact of the sample with the salt-tolerant anion exchange non-fibrous porous filter element (e.g., a membrane) and the contact with the cation exchange non-fibrous porous filter element.

[0034] Non-fibrous porous filter elements (e.g., membranes) and fibrous media forms allow for high flow rates and good adsorption capacity, but the resolution capabilities differ. For example, smaller pore sizes and better control of pore size distribution in non-fibrous porous filter elements provide better control in the separation of impurities, especially biological molecules. Furthermore, non-fibrous porous filter elements can offer higher ion exchange capacity due to their larger surface area. Therefore, non-fibrous porous filter elements may offer advantages over fibrous media in certain aspects of flow-through purification processes.

[0035] In the context of this disclosure, “immediately thereafter” means that there are no other separation steps / mediums used between the two listed media.

[0036] In the context of this disclosure, "flow-through" means that the target molecule passes through all filters.

[0037] The use of buffer exchange favorably optimizes the interaction between impurities and the membrane, providing higher target yields from compositions with low impurity levels, while reducing the number of buffer exchanges can lower processing time and costs.

[0038] In this context, "exchange" does not necessarily mean changing the buffer. Buffer exchange may include a complete change of buffer (i.e., a change from one buffer to another) by known methods (e.g., tangential flow filtration or cross-flow filtration), but "exchange" may also include modifying the buffer by, for example, changing the pH, changing the conductivity, and / or diluting the sample of interest. This may also be referred to as buffer modification or buffer preparation.

[0039] A "buffer solution" is a buffer solution that resists changes in pH caused by the action of organic acid-base conjugated components.

[0040] In one embodiment, examples of organic acids (in buffer solution) include, but are not limited to, formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, phosphoric acid, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), and MES (2-(N-morpholino)ethanesulfonic acid).

[0041] In one embodiment, the organic base (in the buffer solution) may include, but is not limited to, the group consisting of Tris base, arginine, bis-Tris, bis-Tris-propane, bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), and tricine (N-tris(hydroxymethyl)methylglycine).

[0042] In one embodiment, the conjugate base of the organic acid is the sodium, potassium, or ammonium salt of the conjugate base of the organic acid. In one embodiment, the organic acid is acetic acid, and the conjugate base of acetic acid is its sodium salt.

[0043] Typically, buffers include equilibration buffers, packing buffers, and elution buffers. In this specification, “equilibrium buffer” is used to prepare the solid phase of chromatography. “Packing buffer” is used to pack a mixture of protein and contaminants into the chromatography matrix. Equilibration buffer and packing buffer may be the same. “Elution buffer” is used to elute proteins from the chromatography matrix.

[0044] In a particular embodiment, the flow-through process includes contacting a sample (of a biological solution) with an anion exchange adsorption depth filter; optionally, performing buffer exchange with the sample before and / or after contacting the sample with the anion exchange adsorption depth filter; contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element; and immediately thereafter, contacting the sample with a cation exchange non-fibrous porous filter element.

[0045] In a particular embodiment, the flow-through process includes contacting a sample (of a biological solution) with an anion exchange adsorption depth filter; performing buffer exchange with the sample before and / or after contacting the sample with the anion exchange adsorption depth filter; contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element; and immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element.

[0046] In a particular embodiment, the flow-through process includes contacting a sample (of a biological solution) with an anion exchange adsorption depth filter, performing buffer exchange with the sample after contacting the sample with the anion exchange adsorption depth filter, contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element, and immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element.

[0047] In a particular embodiment, the flow-through process includes contacting the sample with an anion exchange adsorption depth filter, performing buffer exchange with the sample before contacting the sample with the anion exchange adsorption depth filter, contacting the sample with a salt-resistant anion exchange non-fibrous porous filter element, and immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element.

[0048] In a particular embodiment, the flow-through process includes contacting a sample with an anion exchange adsorption depth filter, performing buffer exchange with the sample before and after contacting the sample with the anion exchange adsorption depth filter, contacting the sample with a salt-resistant anion exchange non-fibrous porous filter element, and immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element.

[0049] In a particular embodiment, the flow-through process includes contacting the sample with a cation-exchange non-fibrous porous filter element, followed by performing a buffer exchange with the sample.

[0050] In a particular embodiment, as shown in Figure 1B, the flow-through process includes contacting a sample (of a biological solution) with an anion exchange adsorption depth filter (AEX adsorption depth filter), then immediately thereafter contacting the sample with a salt-resistant anion exchange non-fibrous porous filter element (salt-resistant AEX FE), and then immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element (CEX FE), wherein the flow-through process does not involve buffer exchange.

[0051] In a particular embodiment, as shown in Figure 1C, the flow-through process includes contacting a sample (of a biological solution) with an anion exchange adsorption depth filter (AEX adsorption depth filter), performing buffer exchange with the sample after contacting the sample with the anion exchange adsorption depth filter, contacting the sample with a salt-resistant anion exchange non-fibrous porous filter element (salt-resistant AEX FE), and immediately thereafter contacting the sample with a cation exchange non-fibrous porous filter element (CEX FE).

[0052] In a particular embodiment, as shown in Figure 1D, the flow-through process includes performing buffer exchange with the sample (of a biological solution), contacting the sample with a salt-tolerant anion-exchange non-fibrous porous filter element (salt-tolerant AEX FE), and immediately thereafter contacting the sample with a cation-exchange non-fibrous porous filter element (CEX FE).

[0053] Any anion exchange adsorption depth filter element A depth filter (i.e., a filter element) is a porous material that allows particles that may be present in a virus-inactivating pool (i.e., microparticles) to penetrate into it and then be trapped. In other words, a depth filter captures contaminants in a sample between the upstream and downstream surfaces of the filter substrate.

[0054] Exemplary depth filters include a porous base substrate and a grafted copolymer comprising an interpolymerized cationic nitrogen-containing ligand monomer (i.e., ligand) as disclosed in U.S. Patent No. 9,821,276 (Berrigan et al.), or a grafted ligand-functional polymer as disclosed in U.S. Patent No. 8,846,203 (Bothof et al.).

[0055] Porous base material. The porous base material (i.e., base material) of the depth filter may be a porous membrane, a porous nonwoven web, or a porous fibrous material.

[0056] In certain embodiments, it is a porous nonwoven web. As used herein, the terms “nonwoven web” or “nonwoven substrate” are used interchangeably and refer to a fabric having a structure of individual fibers or filaments irregularly and / or unidirectionally incorporated in a mat-like manner.

[0057] Nonwoven fiber webs can be manufactured by carding, airlaid, spunlace, spunbond, or meltblown methods, or a combination thereof. Spunbond fibers are typically small-diameter fibers formed by extruding molten thermoplastic polymer as filaments from multiple fine, usually circular, spinneret capillaries, rapidly reducing the diameter of the extruded fibers. 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-speed, usually heated gas (e.g., air) flow, which thins the filaments of the molten thermoplastic material and reduces their diameter. The meltblown fibers are then transported by the high-speed gas flow and deposited on a collection surface, forming a web of irregularly dispersed meltblown fibers. Any nonwoven web may be manufactured from one type of fiber, or from two or more types of fibers with different thermoplastic polymer types and / or thicknesses.

[0058] Nonwoven fabric substrates suitable for depth filters can be spunlaid, water-entangled, or meltblown. In certain embodiments, they have a tensile strength of at least 4.0 Newtons before grafting, and 15 m per square meter of nonwoven fabric substrate. 2 ~50m 2 It may have a surface area, an average pore diameter of 1 to 40 microns according to ASTMF 316-03, and a solidity of less than 20%.

[0059] Porous base materials can be formed from any suitable thermoplastic polymer material. Suitable polymer materials, but are not limited to, include polyolefins, poly(isoprene), poly(butadiene), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ethersulfone), poly(sulfone), poly(vinyl acetate), copolymers of vinyl acetate, such as poly(ethylene)-co-poly(vinyl alcohol), poly(phosphazene), poly(vinyl ester), poly(vinyl ether), poly(vinyl alcohol), and poly(carbonate).

[0060] Suitable polyolefins include, but are not limited to, poly(ethylene), poly(propylene), poly(1-butene), copolymers of ethylene and propylene, α-olefin copolymers (for example, copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and 1-decene), poly(ethylene-co-1-butene), and poly(ethylene-co-1-butene-co-1-hexene).

[0061] Suitable fluorinated polymers include, but are not limited to, poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (e.g., poly(vinylidene fluoride-co-hexafluoropropylene)), and copolymers of chlorotrifluoroethylene (e.g., poly(ethylene-co-chlorotrifluoroethylene)).

[0062] Suitable polyamides include, but are not limited to, poly(iminoadipoyliminohexamethylene), poly(iminoadipoyliminodecamethylene), and polycaprolactam. Suitable polyimides include, but are not limited to, poly(pyromellitimide).

[0063] Suitable poly(ethersulfones) include, but are not limited to, poly(diphenyl ethersulfone) and poly(diphenylsulfone-co-diphenylene oxidesulfone).

[0064] Suitable vinyl acetate copolymers include, but are not limited to, poly(ethylene-co-vinyl acetate) and copolymers in which at least some of the acetate groups in the copolymer are hydrolyzed to produce various poly(vinyl alcohols).

[0065] In some embodiments, the porous base substrate is formed from a propylene homopolymer or copolymer, most preferably a propylene homopolymer.

[0066] Immobilized cationic nitrogen-containing ligand. In certain embodiments, the porous substrate of the depth filter comprises a grafted copolymer containing a crosspolymerized cationic nitrogen-containing ligand monomer (i.e., ligand), as described in U.S. Patent No. 9,821,276 (Berrigan et al.). In other words, the porous substrate of the depth filter comprises a copolymer grafted thereon (thereby forming a copolymer-grafted article), the grafted copolymer comprising crosspolymerized monomer units containing a cationic nitrogen-containing ligand.

[0067] In certain embodiments, the anion-exchange ligand includes a cationic nitrogen-containing ligand. In certain embodiments, the cationic nitrogen-containing ligand includes a primary amine, a secondary amine, a tertiary amine, or a combination thereof. In certain embodiments, the cationic nitrogen-containing ligand includes a quaternary ammonium-containing ligand, a guanidinyl-containing ligand, or a combination thereof.

[0068] In certain embodiments, the grafted copolymer comprises crosspolymerized monomer units including cationic nitrogen-containing ligand monomers selected from the group consisting of quaternary ammonium-containing ligand monomers, guanidinyl-containing ligand monomers, and combinations thereof, amide monomers, epoxy-functional monomer units, alkyl ether-functional monomer units, and combinations thereof, as well as poly(alkylene oxide) monomers.

[0069] In a particular embodiment, the grafted copolymer comprises 10 to 50 parts by weight of cationic nitrogen-containing ligand monomers (selected from the group consisting of quaternary ammonium-containing ligand monomers, guanidinyl-containing ligand monomers, and combinations thereof), 10 to 80 parts by weight of amide monomers, and transpolymerized monomer units comprising 10 to 40 parts by weight of oxy monomers selected from the group consisting of epoxy-functional monomer units, alkyl ether-functional monomer units, and combinations thereof, and 0 to 30 parts by weight of poly(alkylene oxide) monomers, with a total monomer content of 100 parts by weight.

[0070] In a particular embodiment, the cationic nitrogen-containing ligand monomer is of formula (I): [ka] [In the formula, R 1 is H or CH3, X 1 -O- or -NR 3 - and R 3 is H or (hetero)hydrocarbyl, R 5 It is (hetero)hydrocarbylene, R Lig [This refers to a quaternary ammonium ligand group or a guanidinyl-containing ligand group.]

[0071] In certain embodiments, the cationic nitrogen-containing ligand monomer has the formula (II):

Chemical formula

[0072] The counterions of the quaternary ammonium salts include halide ions, sulfate ions, phosphate ions, nitrate ions, etc. Exemplary quaternary ammonium salt monomers include (meth)acrylamide alkyltrimethylammonium salts and (meth)acryloxyalkyltrimethylammonium salts, as described in U.S. Patent No. 9,821,276 (Berrigan et al.).

[0073] In certain embodiments, the cationic nitrogen-containing ligand monomer has the formula (III) or (IV):

Chemical formula

[0074] Examples of cationic nitrogen-containing ligand monomers include agmatine-containing ligands (e.g., isocyanatoethyl methacrylate-agmatine adducts), guanidine-containing ligands, biguanide-containing ligands, and combinations thereof, prepared as described in U.S. Patent No. 9,821,276 (Berrigan et al.).

[0075] In certain embodiments, the copolymer-grafted article is of formula (V) or (VI): [ka] [In the formula, R 1 is H or CH3, Each R 8 These are independently hydrogen, alkyl, or aryl. R 9 and R 10 The material comprises crosspolymerized amide monomers ((meth)acrylamide and N-vinylamide) which are alkyl groups or which can together form a 5- or 6-membered ring.

[0076] Examples of such monomers include N-vinylcaprolactam, N-vinylacetamide, N-vinylpyrrolidone, acrylamide, mono- or di-N-alkyl-substituted acrylamides, and combinations thereof.

[0077] In a particular embodiment, the copolymer-grafted article is of formula (VII): [ka] [In the formula, R 1 is H or CH3, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), R 16 This includes transpolymerized oxymonomers (epoxy-functional and monoether-functional (meth)acrylates and (meth)acrylamides) of epoxy-functional or ether-functional hydrocarbyl groups.

[0078] In a particular embodiment, the copolymer-grafted article is of formula (VIII): [ka] [In the formula, R 1 is H or CH3, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), R 7 It contains transpolymerized oxymonomers (epoxy monomers) of (hetero)hydrocarbylenes (e.g., C1-C6 alkylenes).

[0079] Examples of such monomers include glycidyl (meth)acrylate, thioglycidyl (meth)acrylate, 3-(2,3-epoxypropoxy)phenyl (meth)acrylate, 2-[4-(2,3-epoxypropoxyl)phenyl]-2-(4-(meth)acryloyloxy-phenyl)propane, 4-(2,3-epoxypropoxyl)cyclohexyl (meth)acrylate, 2,3-epoxycyclohexyl (meth)acrylate, and 3,4-epoxycyclohexyl (meth)acrylate, as well as others described in U.S. Patent No. 9,821,276 (Berrigan et al.).

[0080] As described in U.S. Patent No. 9,821,276 (Berrigan et al.), functionalized substrates may be prepared using the above monomers in a single reaction step or sequential reaction steps to provide polymers grafted onto the surface of a porous base substrate.

[0081] Grafted ligand-functional polymer. In certain embodiments, the porous substrate of the depth filter comprises a ligand-functional polymer grafted thereon, as disclosed in U.S. Patent No. 8,846,203 (Bothof et al.).

[0082] In a particular embodiment, the grafted ligand-functionalized polymer is expressed by formula (IX): -(M PI ) w -(M b ) x -(M c ) y -(M d ) z [In the formula, -(M PI ) w represents a residue of the grafted photoinitiator monomer, and w is 0 or at least 1. -(M b ) x represents a polymerized ligand monomer having "x" polymerized monomer units, where x is at least 1. -(M c ) y represents a polymerized crosslinked monomer having y polymerized monomer units, where y may be 0 or at least 1. -(M d ) z [where z represents a polymerized hydrophilic monomer having z polymerized monomer units, where z may be 0 or at least 1].

[0083] In certain embodiments, the functionalized substrate has grafted groups bonded to the surface of a base substrate, comprising: a) optionally at least one photoinitiator group (or a reaction product thereof); b) one or more ligand monomers; c) optionally one or more monomers having at least one acryloyl group and at least one additional free radical polymerizable group; and d) optionally one or more hydrophilic monomers.

[0084] Monomers grafted onto the surface of a base substrate typically have both an acryloyl group for electron beam grafting and at least one additional functional group thereon. Acryloyl groups, including acrylate and acrylamide groups, are preferred for direct grafting onto the substrate surface due to the high reactivity of such acryloyl groups to exposure to ionizing radiation, such as electron beam irradiation. All such acryloyl groups can be "directly grafted," i.e., they can form covalent bonds with the substrate surface. Some may remain free and then be "indirectly grafted" by being incorporated into the polymer chain upon exposure to UV ultraviolet light. Other ethylenically unsaturated groups, such as methacrylamide, methacrylate, vinyl and vinyloxy groups, allyl and allyloxy groups, and acetylene groups, are less reactive during electron beam grafting and are less likely to be directly grafted onto the base substrate. Therefore, some of these non-acryloyl groups can be directly grafted, but they mainly remain unreacted and are indirectly grafted onto the substrate by being incorporated into the polymer chain during UV-initiated polymerization.

[0085] The photoinitiator monomer "a)" can be directly grafted onto the surface of the base substrate, including the gaps and outer surface of the porous base substrate, to provide the grafted photoinitiator group via the acryloyl group.

[0086] The ligand "b)" monomer may have an acryloyl group for direct grafting or a non-acryloyl group such as a methacrylate group for subsequent incorporation into the polymer chain (indirect grafting) during UV-initiated polymerization.

[0087] The acryloyl group of monomer "c)" can typically be grafted (i.e., form covalent bonds) onto the surface of a base substrate when exposed to ionizing radiation, preferably an electron beam or gamma rays. In addition to the acryloyl group, the free radical polymerizable groups of monomer "c)" are typically other ethylenically unsaturated groups such as methacrylamide, methacrylate, vinyl groups, and acetylene groups, which have reduced reactivity during grafting and are therefore free and unreacted for subsequent UV-initiated polymerization and crosslinking.

[0088] A fourth graftable hydrophilic monomer "d)" may also be grafted via an acryloyl group to provide a hydrophilic or ionic group on the surface of the base substrate. In some embodiments, a hydrophilic monomer having an ionic group may be grafted directly or indirectly onto the substrate surface to provide secondary ionic interactions of the functionalized substrate.

[0089] Grafting photoinitiator monomer (M PI ) comprises an acryloyl group and a photoinitiator group, which may be hydrogen abstraction type or α-cleavage type photoinitiator groups. Such grafted photoinitiator monomers (M PI This is disclosed in U.S. Patent No. 8,846,203 (Bothof et al.).

[0090] In certain embodiments, ligand monomer (M b ) formula (X): [ka] [In the formula, R 1 is H or CH3, R 2 It is (hetero)hydrocarbylene, Each R 3 These are independently H or (hetero)hydrocarbyl, R 14 is H, (hetero)hydrocarbyl, or -N(R 3 )2(wherein, R 3(is independently H or (hetero)hydrocarbyl), X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), n is either 1 or 2.

[0091] In a particular embodiment, ligand monomer (M b ) is equation (XIV): [ka] [In the formula, R 1 is H or CH3, Each R 3 These are independently H or (hetero)hydrocarbyl, R 14 is H, (hetero)hydrocarbyl, or -N(R 3 )2(wherein, R 3 (is independently H or (hetero)hydrocarbyl), X 1 -O- or -NR 3 - and R 3 is H or (hetero)hydrocarbyl, R 6 and R 7 Each of these independently consists of (hetero)hydrocarbylenes (e.g., C1-C1). 10 Alkilen) Z 2 is an ester, amide, urea, or urethane group. n is either 1 or 2.

[0092] Such ligand monomers (M b ) can be produced using the condensation reaction described in U.S. Patent No. 8,846,203 (Bothof et al.).

[0093] In certain embodiments, the grafted ligand-functional polymer is a crosslinked monomer (M) having two or more free radical polymerizable groups.c )(where y is at least 1). In certain embodiments, the crosslinking monomer (M c ) has the formula (XI):

Chemical formula

[0094] In certain embodiments, the crosslinking monomer (M c ) comprises a poly(alkylene oxide) compound having at least one acryloyl group and at least one additional ethylenically unsaturated free - radical polymerizable group. In certain embodiments, the crosslinking monomer (M c ) has the formula (XII):

Chemical formula

[0095] Examples of suitable crosslinking (M c ) monomers include di (meth) acrylates of copolymers such as poly (ethylene oxide) polymers and poly (ethylene oxide - co - propylene oxide) copolymers, and partially acrylated polyols such as 3 - (acryloyloxy) - 2 - hydroxypropyl methacrylate).

[0096] In certain embodiments, the grafted ligand - functional polymer further comprises a hydrophilic monomer (M d )(z is at least 1), which has a free - radical polymerizable group and a hydrophilic group. The hydrophilic group can also include an ionic group that is positively charged, negatively charged, or neutral. In certain embodiments, the hydrophilic monomer (M d ) has the formula (XIII): [Chemical formula] [wherein each R 1 is independently H or CH3, X 1 is - O - or - NR 3 -(wherein R 3 is H or (hetero) hydrocarbyl), t is from 2 to 100] and is a neutral monomer.

[0097] Examples of suitable hydrophilic (M d ) monomers include poly (alkylene oxide) monomers.

[0098] Anion - exchange depth filters can be fabricated using standard techniques such as those described in U.S. Patent Nos. 9,821,276 (Berrigan et al.) and 8,846,203 (Bothof et al.).

[0099] Salt-resistant anion exchange non-fibrous porous filter element Salt-resistant anion-exchange non-fibrous porous filter elements are preferably porous membranes, but they are not depth filters, and their primary purpose is not to remove particulate matter. An exemplary salt-resistant anion-exchange non-fibrous porous filter element comprises a non-fibrous base substrate and a grafted copolymer containing crosspolymerized cationic nitrogen-containing ligand monomers (i.e., ligands) as disclosed in U.S. Patent No. 9,821,276 (Berrigan et al.), or a grafted ligand-functional polymer as disclosed in U.S. Patent No. 8,846,203 (Bothof et al.), and is used as the depth filter described above.

[0100] Salt-tolerant nitrogen-containing ligands typically contain a primary amine or a guanidinyl group, with guanidinyl-containing ligands being more salt-tolerant than primary amines. Secondary amines, tertiary amines, and quaternary ammonium groups are typically not salt-tolerant, as defined herein. In certain embodiments, the salt-tolerant ligand is a cationic nitrogen-containing ligand monomer comprising guanidinyl-containing ligand monomers of formulas (III) and (IV), along with monomers of formula (XIV) (described above), for example, as prepared in U.S. Patent No. 10,239,828 (Rasmussen et al.) and No. 8,846,203 (Bothof et al.).

[0101] Salt-tolerant anion exchange non-fibrous porous filter elements are useful under conditions of high salt concentration or high ionic strength; that is, they are "salt-tolerant." The term "salt" means including all low molecular weight ion species that contribute to the conductivity of a solution. Salt tolerance is important because many process solutions used in the manufacture of biopharmaceuticals or enzymes have conductivity in the range of 15 mS / cm to 30 mS / cm (approximately 150 to 300 mM salt) or higher. Salt tolerance can be measured in comparison to that of conventional quaternary amines or quart ligands (e.g., trimethylammonium ligand), whose electrostatic interactions with many biological species rapidly degrade conductivity to 3 to 6 times lower than the target range. For example, membranes derivatized with conventional quart ligands show a decrease in φX174 viral clearance from a 6-log decrease value (LRV) to (1)LRV, progressing to 0 mM to 50 mM NaCl (conductivity of approximately 5 mS / cm to 6 mS / cm). Viruses such as φX174, which have an isoelectric point close to 7 (meaning they are neutral or nearly neutral), are extremely difficult to remove from process flows. Similar problems are observed when attempting to remove other biological species from process fluids. For example, when attempting to remove positively charged proteins such as host cell proteins through the use of conventional quart-ligand-functionalized filtration devices, the process fluid may need to be diluted more than twice to reduce its conductivity to an acceptable level. This is expensive and dramatically increases the overall processing time.

[0102] Non-fibrous porous filter elements are advantageous for salt-tolerant membranes compared to fibrous porous substrates due to the larger membrane surface area and higher graft density of ligand-functionalized polymers that can be achieved, thus resulting in a higher capacity for removing impurities. Furthermore, the smaller pore size and better control of pore size distribution in non-fibrous porous filter elements provide better control in the separation of impurities, particularly biological molecules. Therefore, non-fibrous porous filter elements may offer advantages over fibrous media in salt-tolerant membranes in flow-through purification processes.

[0103] The non-fibrous porous filter element base material can be formed from any suitable thermoplastic polymer material, as described above for depth filters.

[0104] In some embodiments, the porous base material is a microporous membrane, such as a thermally induced phase separation (TIPS) membrane. TIPS membranes are often prepared by forming a homogeneous solution of a thermoplastic material and a second material exceeding the melting point of the thermoplastic material. Upon cooling, the thermoplastic material crystallizes and phase-separates from the second material. The crystallized thermoplastic material is often stretched. The second material is optionally removed either before or after stretching. Microporous membranes are further disclosed in U.S. Patents 4,539,256 (Shipman), 4,726,989 (Mrozinski), 4,867,881 (Kinzer), 5,120,594 (Mrozinski), 5,260,360 (Mrozinski et al.), and 5,962,544 (Waller). Furthermore, microporous films can be prepared from ethylene-vinyl alcohol copolymers, as described in U.S. Patent No. 5,962,544 (Waller).

[0105] Some exemplary TIPS films include poly(vinylidene fluoride) (PVDF), polyolefins, such as polyethylene homopolymers or copolymers or polypropylene homopolymers or copolymers, vinyl-containing polymers or copolymers, such as ethylene-vinyl alcohol copolymers, and butadiene-containing polymers or copolymers, as well as acrylate-containing polymers or copolymers. TIPS films containing PVDF are further described in U.S. Patent No. 7,338,692 (Smith et al.).

[0106] In another exemplary embodiment, the porous base substrate is a microporous membrane, such as a solvent-induced phase separation (SIPS) membrane. SIPS membranes are often prepared by forming a homogeneous solution of a thermoplastic material and a second material (solvent), which is then cast into a film or hollow fiber form and subsequently immersed in a non-solvent bath. The non-solvent solidifies or separates the thermoplastic material and extracts the solvent, leaving a porous polymer membrane. Examples of SIPS films prepared from polyamide include nylon microporous films or sheets described in U.S. Patents No. 6,056,529 (Meyering et al.), No. 6,267,916 (Meyering et al.), No. 6,413,070 (Meyering et al.), No. 6,776,940 (Meyering et al.), No. 3,876,738 (Marinacchio et al.), No. 3,928,517 (Knight et al.), No. 4,707,265 (Knight et al.), and No. 5,458,782 (Hou et al.). Other examples include microporous films prepared from polysulfone and polyethersulfone, many of which are commercially available from 3M Company, St. Paul, MN under the trade names MicroPES and DuraPES.

[0107] Salt-resistant membrane filters can be manufactured using standard techniques, such as those described in U.S. Patent No. 9,821,276 (Berrigan et al.), No. 8,846,203 (Bothof et al.), and No. 10,239,828 (Rasmussen et al.).

[0108] Cation exchange non-fibrous porous filter element The cation-exchange non-fibrous porous filter element comprises a non-fibrous base substrate disclosed above for salt-resistant anion-exchange non-fibrous porous filter element. It may be multimodal or mixed-mode. Multimodal or mixed-mode means that the filter element interacts with its target species by cation exchange and also interacts with at least one other interaction mode (e.g., hydrophobic interaction or hydrogen bonding interaction).

[0109] In a particular embodiment, the cation exchange non-fibrous porous filter element comprises a non-fibrous porous filter element and a polymer disposed on the non-fibrous porous filter element, the polymer comprising a hydrocarbon main chain and a plurality of pendant groups bonded to the hydrocarbon main chain, wherein each of the first plurality of pendant groups comprises at least one acidic group or a salt thereof, and includes a spacer group that directly links the at least one acidic group or salt thereof to the hydrocarbon main chain by a chain of at least six linearly linked atoms.

[0110] In certain embodiments, the spacer group comprises a chain having at least eight linearly linked atoms. In certain embodiments, the spacer group is a linearly linked heteroatom-containing hydrocarbon group. In certain embodiments, the spacer group comprises at least one hydrogen bonding moiety, which is defined as a moiety comprising at least one hydrogen bond donor and at least one hydrogen bond acceptor (both containing heteroatoms). In certain embodiments, the spacer group comprises at least two hydrogen bonding moieties, or at least one hydrogen bonding moiety and at least one hydrogen bond acceptor separate from this hydrogen bonding moiety (and not part of this hydrogen bonding moiety). In certain embodiments, the spacer group comprises at least two hydrogen bond donors, at least two hydrogen bond acceptors, or both.

[0111] In certain embodiments, at least one acidic group of a polymer placed on a non-fibrous porous filter element, or a salt thereof, is present at a density of at least 0.01 (or at least 0.02) millimoles / gram of the cation-exchange non-fibrous porous filter element. In certain embodiments, at least one acidic group of a polymer placed on a non-fibrous porous filter element, or a salt thereof, is present at a density of up to 0.6 millimoles / gram of the cation-exchange non-fibrous porous filter element.

[0112] In certain embodiments, at least one acidic group or salt thereof is selected from carboxyl groups, phosphono groups, phosphat groups, sulfono groups, sulfato groups, boronato groups, and combinations thereof.

[0113] In certain embodiments, the polymer comprises an interpolymerized unit of at least one monomer, each unit comprising at least one ethylenically unsaturated group, at least one acidic group or a salt thereof, and a spacer group that directly links the at least one ethylenically unsaturated group and the at least one acidic group or a salt thereof by a chain of at least six linearly linked atoms.

[0114] In certain embodiments, at least one ethylenically unsaturated group is selected from an ethenyl group, a 1-alkylethenyl group, and combinations thereof.

[0115] In certain embodiments, the polymer is covalently bonded to a non-fibrous porous filter element.

[0116] In certain embodiments, the polymer is a copolymer.

[0117] In a particular embodiment, the copolymer comprises a hydrocarbon main chain and a plurality of pendant groups bonded to the hydrocarbon main chain, each of the first plurality of pendant groups comprising at least one acidic group or a salt thereof and a spacer group that directly links at least one acidic group or a salt thereof to the hydrocarbon main chain by a chain of at least six linearly linked atoms, each of the second plurality of pendant groups comprising at least one acidic group or a salt thereof and a spacer group that directly links at least one acidic group or a salt thereof to the hydrocarbon main chain by a chain of at least six linearly linked atoms, and unlike the second plurality of pendant groups, the molar ratio of the first plurality of pendant groups to the second plurality of pendant groups is in the range of 95:5 to 5:95.

[0118] In a particular embodiment, the copolymer covalently bonded to a non-fibrous porous filter element comprises a reaction product of a monomer composition comprising: a first monomer comprising at least one ethylenically unsaturated group, at least one acidic group or a salt thereof, and a spacer group that directly links the at least one ethylenically unsaturated group and the at least one acidic group or a salt thereof by a chain of at least six linearly linked atoms; and a second monomer comprising at least one ethylenically unsaturated group, at least one acidic group or a salt thereof, and a spacer group that directly links the at least one ethylenically unsaturated group and the at least one acidic group or a salt thereof by a chain of at least six linearly linked atoms, wherein the second monomer differs from the first monomer, and the molar ratio of the first monomer to the second monomer is in the range of 95:5 to 5:95.

[0119] In certain embodiments, at least one ethylenically unsaturated group of the first monomer and / or the second monomer is selected from an ethenyl group, a 1-alkylethenyl group, and combinations thereof. In certain embodiments, the first monomer is of the following general formula (XV): [ka] [In the formula, R 1 is H or CH3, Each R 2 It is independently a (hetero)hydrocarbylene, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), Z 3 This is a heterohydrocarbylene group comprising at least one hydrogen bond donor, at least one hydrogen bond acceptor, or a combination thereof. r is either 0 or 1. L is one of the categories represented by a functional group that includes at least one acidic group or a salt thereof.

[0120] In this context, "hydrogen bond acceptor" refers to a heteroatom selected from oxygen, nitrogen, and sulfur that has a lone pair of electrons, and "hydrogen bond donor" refers to a portion consisting of hydrogen atoms covalently bonded to a heteroatom selected from oxygen, nitrogen, and sulfur. Examples of hydrogen bond donors include imino, thiol, or hydroxyl donors. Examples of hydrogen bond acceptors include acceptors in the form of carbonyl, carbonyloxy, or ether oxygen.

[0121] In a particular embodiment, the second monomer is one of the categories similarly represented by general formula (XV).

[0122] Cation exchange membrane filters can be fabricated using standard techniques, such as those described in U.S. Publication No. 2019 / 0194250 (Colak Atan et al.) and International Publication No. WO2018 / 048696 (Vail et al.).

[0123] device In further embodiments, the disclosure provides a filter cartridge comprising a salt-resistant anion-exchange non-fibrous porous filter element (salt-resistant AEX FE) as described herein and a cation-exchange non-fibrous porous filter element (CEX FE) as described herein, preferably positioned downstream from the salt-resistant filter element, as shown in Figure 2A. The device may also include an anion-exchange adsorption depth filter (AEX adsorption depth filter), preferably positioned upstream of the salt-resistant filter element, as shown in Figure 2B. The relative orientation of these filter elements may be for process purposes, as described herein. The device may also include other conventional filter elements, such as one or more microporous membranes, as shown in Figure 2C.

[0124] Exemplary Embodiments Embodiment 1 is a flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, wherein the process is: Optionally, the sample may be brought into contact with an anion exchange adsorption depth filter, Optionally, buffer exchange may be performed with the sample before and / or after contacting the sample with the anion exchange adsorption depth filter. The sample is brought into contact with a salt-resistant anion-exchange non-fibrous porous filter element, The process then includes bringing the sample into contact with a cation-exchange non-fibrous porous filter element, The flow-through process involves one or two buffer exchanges, and does not include buffer exchange between the contact of the sample with the salt-tolerant anion-exchange non-fibrous porous filter element and the contact with the cation-exchange non-fibrous porous filter element.

[0125] Embodiment 2 is, The sample is brought into contact with an anion exchange adsorption depth filter, Optionally, buffer exchange may be performed with the sample before and / or after contacting the sample with the anion exchange adsorption depth filter. The sample is brought into contact with a salt-resistant anion-exchange non-fibrous porous filter element, The flow-through process according to Embodiment 1 further comprises immediately thereafter contacting the sample with a cation-exchange non-fibrous porous filter element.

[0126] Embodiment 3 is, The sample is brought into contact with an anion exchange adsorption depth filter, Before and / or after contacting the sample with the anion exchange adsorption depth filter, perform buffer exchange with the sample. The sample is brought into contact with a salt-resistant anion-exchange non-fibrous porous filter element, The flow-through process according to Embodiment 2 further comprises immediately thereafter contacting the sample with a cation-exchange non-fibrous porous filter element.

[0127] Embodiment 4 is, The sample is brought into contact with an anion exchange adsorption depth filter, After contacting the sample with an anion exchange adsorption depth filter, buffer exchange is performed using the sample. The sample is brought into contact with a salt-resistant anion-exchange non-fibrous porous filter element, The flow-through process according to Embodiment 3 further comprises immediately thereafter contacting the sample with a cation-exchange non-fibrous porous filter element.

[0128] Embodiment 5 is, The sample is brought into contact with an anion exchange adsorption depth filter, Before contacting the sample with the anion exchange adsorption depth filter, perform a buffer exchange with the sample, The sample is brought into contact with a salt-resistant anion-exchange non-fibrous porous filter element, The flow-through process according to Embodiment 3 further comprises immediately thereafter contacting the sample with a cation-exchange non-fibrous porous filter element.

[0129] Embodiment 6 is, The sample is brought into contact with an anion exchange adsorption depth filter, Before and after contacting the sample with the anion exchange adsorption depth filter, buffer exchange should be performed with the sample. The sample is brought into contact with a salt-resistant anion-exchange non-fibrous porous filter element, The flow-through process according to Embodiment 3 further comprises immediately thereafter contacting the sample with a cation-exchange non-fibrous porous filter element.

[0130] Embodiment 7 is a flow-through process according to any one of Embodiments 1 to 6, which includes bringing the sample into contact with a cation-exchange non-fibrous porous filter element and then performing buffer exchange with the sample.

[0131] Embodiment 8 is a flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, wherein the process is: The sample is brought into contact with an anion exchange adsorption depth filter, Immediately thereafter, contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element, and Immediately thereafter, contacting the sample with a cation exchange non-fibrous porous filter element, and The flow-through process is a flow-through process that does not include buffer exchange.

[0132] Embodiment 9 is a flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, the process comprising Contacting the sample with an anion exchange adsorption depth filter, and After contacting the sample with the anion exchange adsorption depth filter, performing buffer exchange on the sample, and Contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element, and Immediately thereafter, contacting the sample with a cation exchange non-fibrous porous filter element, and

[0133] Embodiment 10 is a flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, the process comprising Performing buffer exchange on the sample, and Contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element, and Immediately thereafter, contacting the sample with a cation exchange non-fibrous porous filter element, and

[0134] Embodiment 11 is the flow-through process according to any one of Embodiments 1 to 10, wherein the target molecule comprises a monoclonal antibody.

[0135] Embodiment 12 is the flow-through process according to any one of Embodiments 1 to 11, wherein the biological solution comprises a neutralized virus inactivation pool.

[0136] Embodiment 13 is the flow-through process according to any one of Embodiments 2 to 9 and any one of 11 to 12 depending on any one of Embodiments 2 to 9, in which the anion exchange adsorption depth filter includes a porous substrate containing an immobilized anion exchange ligand.

[0137] Embodiment 14 is the flow-through process according to Embodiment 13, in which the anion exchange ligand of the anion exchange adsorption depth filter includes a cationic nitrogen-containing ligand.

[0138] Embodiment 15 is the flow-through process according to Embodiment 14, in which the cationic nitrogen-containing ligand of the anion exchange adsorption depth filter includes a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

[0139] Embodiment 16 is the flow-through process according to Embodiment 15, in which the cationic nitrogen-containing ligand of the anion exchange adsorption depth filter includes a quaternary ammonium-containing ligand, a guanidinyl-containing ligand, or a combination thereof.

[0140] Embodiment 17 is the flow-through process according to any one of Embodiments 14 to 16, in which the anion exchange adsorption depth filter includes a copolymer-grafted article including a porous substrate and a copolymer grafted thereto, and the grafted copolymer includes mutually polymerized monomer units containing a cationic nitrogen-containing ligand.

[0141] Embodiment 18 is such that the grafted copolymer of the anion exchange adsorption depth filter a cationic nitrogen-containing ligand monomer selected from the group consisting of a quaternary ammonium-containing ligand monomer, a guanidinyl-containing ligand monomer, and a combination thereof, an amide monomer, an oxy monomer selected from the group consisting of an epoxy-functional monomer unit, an alkyl ether-functional monomer unit, and a combination thereof, The flow-through process according to Embodiment 17 includes a poly(alkylene oxide) monomer and crosspolymerized monomer units.

[0142] Embodiment 19 describes a grafted copolymer of an anion exchange adsorption depth filter, A cationic nitrogen-containing ligand monomer in an amount of 10 to 50 parts by weight, wherein the cationic nitrogen-containing ligand monomer is selected from the group consisting of quaternary ammonium-containing ligand monomers, guanidinyl-containing ligand monomers, and combinations thereof, 10 to 80 parts by weight of amide monomer, 10 to 40 parts by weight of an oxymonomer selected from the group consisting of epoxy-functional monomer units, alkyl ether-functional monomer units, and combinations thereof, It contains a crosspolymerized monomer unit comprising 0 to 30 parts by weight of a poly(alkylene oxide) monomer, This is the flow-through process according to Embodiment 18, wherein the total amount of monomers is 100 parts by weight.

[0143] Embodiment 20 describes a cationic nitrogen-containing ligand monomer used to fabricate an anion exchange adsorption depth filter, wherein the monomer is of formula (I): [ka] [In the formula, R 1 is H or CH3, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), R 5 It is (hetero)hydrocarbylene, R Lig This is a flow-through process according to Embodiment 18 or 19, wherein [the ligand is a quaternary ammonium ligand group or a guanidinyl-containing ligand group].

[0144] Embodiment 21 describes a cationic nitrogen-containing ligand monomer used to fabricate an anion exchange adsorption depth filter, wherein the monomer is of formula (II): [ka] [In the formula, R 1 is H or CH3, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), R 5 It is (hetero)hydrocarbylene, Each R 4 The flow-through process described in Embodiment 20 is a quaternary ammonium monomer, which is independently alkyl or aryl.

[0145] Embodiment 22 is a cationic nitrogen-containing ligand monomer used to fabricate an anion exchange adsorption depth filter, wherein the monomer is of formula (III) or (IV): [ka] [In the formula, R 1 is H or CH3, R 2 is a (hetero)hydrocarbylene (for example, having 1 to 20 carbon atoms), Each R 3 These are independently H or (hetero)hydrocarbyl, R 14 is H, (hetero)hydrocarbyl, or -N(R 3 )2(wherein, R 3 (is independently H or (hetero)hydrocarbyl), R 15 is H or hydrocarbyl (e.g., C1-C4 alkyl group or aryl group), X 1 -O- or -NR 3 -(In the formula, R 3is H or (hetero)hydrocarbyl), and o is 0 or 1, n is 1 or 2] of the guanidinyl-containing ligand monomer, which is the flow-through process according to Embodiment 20.

[0146] Embodiment 23 is such that the amide monomer used for producing the anion exchange adsorption depth filter is of formula (V) or (VI):

Chemical formula

[0147] Embodiment 24 is such that the oxymonomer used for producing the anion exchange adsorption depth filter is of formula (VII):

Chemical formula

[0149] Embodiment 26 provides an anion exchange adsorption depth filter comprising a porous substrate and a ligand-functional polymer grafted thereon, wherein the grafted ligand-functional polymer is of formula: -(M PI ) w -(M b ) x -(M c ) y -(M d ) z [In the formula, -(M PI ) w represents a residue of the grafted photoinitiator monomer, and w is 0 or at least 1. -(M b ) x represents a polymerized ligand monomer having "x" polymerized monomer units, where x is at least 1. -(M c ) y represents a polymerized crosslinked monomer having y polymerized monomer units, where y may be 0 or at least 1. -(M d ) z This is a flow-through process according to Embodiment 13, where represents a polymerized hydrophilic monomer having z polymerized monomer units, where z may be 0 or at least 1.

[0150] Embodiment 27 describes a ligand monomer (M) used to fabricate an anion exchange adsorption depth filter. b ) is, equation (X): [ka] [In the formula, R 1 is H or CH3, R 2 It is (hetero)hydrocarbylene, Each R 3 These are independently H or (hetero)hydrocarbyl, R 14 is H, (hetero)hydrocarbyl, or -N(R 3 )2(in the formula, each R 3 (is independently H or (hetero)hydrocarbyl), X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), The flow-through process described in Embodiment 26 is one in which n is 1 or 2.

[0151] Embodiment 28 describes a ligand monomer (M) used to fabricate an anion exchange adsorption depth filter. b ) is equation (XIV): [ka] [In the formula, R 1 is H or CH3, Each R 3 These are independently H or (hetero)hydrocarbyl, R 14 is H, (hetero)hydrocarbyl, or -N(R 3 )2(in the formula, each R 3 (is independently H or (hetero)hydrocarbyl), X 1 -O- or -NR 3 -(In the formula, R 3(is H or (hetero)hydrocarbyl), R 6 and R 7 Each of these independently consists of (hetero)hydrocarbylenes (e.g., C1-C1). 10 Alkilen) Z 2 is an ester, amide, urea, or urethane group. The flow-through process described in Embodiment 27 is one in which n is 1 or 2.

[0152] Embodiment 29 describes a grafted ligand-functional polymer of an anion exchange adsorption depth filter, wherein the crosslinked monomer (M c The flow-through process according to any one of embodiments 26 to 28 further comprises (where y is at least 1), which has two or more free radical polymerizable groups.

[0153] Embodiment 30 uses a crosslinked monomer (M) to fabricate an anion exchange adsorption depth filter. c ) is given by equation (XI): [ka] [In the formula, Z 1 This is an ethylenically unsaturated polymerizable group of acryloyl or non-acryloyl, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), Q is a covalent bond, -O-, -NR 1 -, CO2-, and -C(O)NR 1 -(In the formula, R 1 (is a divalent linking group selected from H or CH3), R 11 This is an alkylene group with a valence of a+b, and optionally contains one or more suspended oxygen atoms and / or one or more hydroxyl groups. The flow-through process according to Embodiment 29, wherein a and b are each at least 1.

[0154] Embodiment 31 uses a crosslinked monomer (M) to fabricate an anion exchange adsorption depth filter. c ) is given by equation (XII): [ka] [In the formula, Z 1 This is a polymerizable ethylenically unsaturated group of acryloyl or non-acryloyl, R 1 is H or CH3, m is between 2 and 100. Q is a covalent bond, -O-, -NR 1 -, CO2-, and -C(O)NR 1 -(In the formula, R 1 This is a flow-through process according to Embodiment 29, in which the group is a divalent linking group selected from H or CH3.

[0155] Embodiment 32 describes a ligand-functionalized polymer grafted onto an anion exchange adsorption depth filter, wherein the polymer contains a hydrophilic monomer (M d The flow-through process described in any one of Embodiments 26 to 31 further comprises (where z is at least 1), which has a free radical polymerizable group and a hydrophilic group.

[0156] Embodiment 33 uses a hydrophilic monomer (M) to fabricate an anion exchange adsorption depth filter. d ) is given by equation (XIII): [ka] [In the formula, Each R 1 These are independently H or CH3, X 1 -O- or -NR 3 -(In the formula, R 3(is H or (hetero)hydrocarbyl), The flow-through process described in Embodiment 32 is such that t is between 2 and 100.

[0157] Embodiment 34 is a flow-through process according to any one of Embodiments 1 to 33, wherein the salt-resistant anion-exchange non-fibrous porous filter element comprises a non-fibrous porous filter element containing an immobilized anion-exchange ligand.

[0158] Embodiment 35 is the flow-through process described in Embodiment 34, wherein the anion exchange ligand of the salt-resistant anion exchange non-fibrous porous filter element includes a cationic nitrogen-containing ligand.

[0159] Embodiment 36 is the flow-through process according to Embodiment 35, wherein the cationic nitrogen-containing ligand of the salt-resistant anion-exchange non-fibrous porous filter element contains a guanidinyl-containing ligand.

[0160] Embodiment 37 is a flow-through process according to Embodiment 35 or 36, wherein the salt-resistant anion-exchange non-fibrous porous filter element comprises a copolymer-grafted article comprising a non-fibrous porous filter element and a copolymer grafted thereon, the grafted copolymer comprising crosspolymerized monomer units comprising a cationic nitrogen-containing ligand.

[0161] Embodiment 38 is a grafted copolymer of salt-resistant anion-exchange non-fibrous porous filter elements, Guanidinyl-containing ligand monomers, Amide monomers and An oxymonomer selected from the group consisting of epoxy-functional monomer units, alkyl ether-functional monomer units, and combinations thereof, This is a flow-through process according to Embodiment 37, comprising a poly(alkylene oxide) monomer and a crosspolymerized monomer unit.

[0162] Embodiment 39 is a grafted copolymer of salt-resistant anion-exchange non-fibrous porous filter elements, 10 to 50 parts by weight of a guanidinyl-containing ligand monomer, 10 to 80 parts by weight of amide monomer, 10 to 40 parts by weight of an oxymonomer selected from the group consisting of epoxy-functional monomer units, alkyl ether-functional monomer units, and combinations thereof, It contains a crosspolymerized monomer unit comprising 0 to 30 parts by weight of a poly(alkylene oxide) monomer, This is the flow-through process according to Embodiment 38, wherein the total amount of monomers is 100 parts by weight.

[0163] Embodiment 40 uses a cationic nitrogen-containing ligand monomer, which is used to produce a salt-resistant anion-exchange non-fibrous porous filter element, and is based on formula (I): [ka] [In the formula, R 1 is H or CH3, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), R 5 It is (hetero)hydrocarbylene, R Lig This is a flow-through process according to embodiment 38 or 39, wherein the ligand group is a guanidinyl-containing ligand group.

[0164] Embodiment 41 is a cationic nitrogen-containing ligand monomer used to produce a salt-resistant anion-exchange non-fibrous porous filter element, wherein the ligand monomer is of formula (III) or (IV): [ka] [In the formula, R 1 is H or CH3, R 2 is a (hetero)hydrocarbylene (for example, having 1 to 20 carbon atoms), Each R 3 These are independently H or (hetero)hydrocarbyl, R 14 is H, (hetero)hydrocarbyl, or -N(R 3 )2(in the formula, each R 3 (is independently H or (hetero)hydrocarbyl), R 15 is H or hydrocarbyl (e.g., C1-C4 alkyl group or aryl group), X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), o is either 0 or 1. The flow-through process according to Embodiment 40 is a guanidinyl-containing ligand monomer, where n is 1 or 2.

[0165] Embodiment 42 provides a salt-resistant anion-exchange non-fibrous porous filter element comprising a non-fibrous porous filter element and a ligand-functional polymer grafted thereon, wherein the grafted ligand-functional polymer has the formula: -(M PI ) w -(M b ) x -(M c ) y -(M d ) z [In the formula, -(M PI ) w represents a residue of the grafted photoinitiator monomer, and w is 0 or at least 1. -(M b ) x represents a polymerized ligand monomer having "x" polymerized monomer units, where x is at least 1. -(M c ) yrepresents a polymerized crosslinked monomer having y polymerized monomer units, where y may be 0 or at least 1. -(M d ) z This is a flow-through process according to Embodiment 34, where represents a polymerized hydrophilic monomer having z polymerized monomer units, where z may be 0 or at least 1.

[0166] Embodiment 43 describes a ligand monomer (M) used to produce a salt-resistant anion-exchange non-fibrous porous filter element. b ) is, equation (X): [ka] [In the formula, R 1 is H or CH3, R 2 It is (hetero)hydrocarbylene, Each R 3 These are independently H or (hetero)hydrocarbyl, R 14 is H, (hetero)hydrocarbyl, or -N(R 3 )2(in the formula, each R 3 (is independently H or (hetero)hydrocarbyl), X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), This is the flow-through process according to Embodiment 42, wherein n is 1 or 2.

[0167] Embodiment 44 describes a ligand monomer (M) used to produce a salt-resistant anion-exchange non-fibrous porous filter element. b )but, Formula (XIV): [ka] [In the formula, R 1is H or CH3, Each R 3 These are independently H or (hetero)hydrocarbyl, R 14 is H, (hetero)hydrocarbyl, or -N(R 3 )2(in the formula, each R 3 (is independently H or (hetero)hydrocarbyl), X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), R 6 and R 7 Each of these independently consists of (hetero)hydrocarbylenes (e.g., C1-C1). 10 Alkilen) Z 2 is an ester, amide, urea, or urethane group. The flow-through process described in Embodiment 43 is one in which n is 1 or 2.

[0168] Embodiment 45 describes a grafted ligand-functional polymer of a salt-resistant anion-exchange non-fibrous porous filter element, wherein the polymer is cross-linked monomer (M c The flow-through process described in any one of embodiments 42 to 44 further comprises (where y is at least 1), which has two or more free radical polymerizable groups.

[0169] Embodiment 46 describes a crosslinked monomer (M) used to produce a salt-resistant anion-exchange non-fibrous porous filter element. c ) is given by equation (XI): [ka] [In the formula, Z 1 This is an ethylenically unsaturated polymerizable group of acryloyl or non-acryloyl, X 1 -O- or -NR 3 -(In the formula, R 3(is H or (hetero)hydrocarbyl), Q is a covalent bond, -O-, -NR 1 -, CO2-, and -C(O)NR 1 -(In the formula, R 1 (is a divalent linking group selected from H or CH3), R 11 This is an alkylene group with a valence of a+b, and optionally contains one or more suspended oxygen atoms and / or one or more hydroxyl groups. The flow-through process according to Embodiment 45, wherein a and b are each at least 1.

[0170] Embodiment 47 describes a crosslinked monomer (M) used to produce a salt-resistant anion exchange non-fibrous porous filter element. c ) is given by equation (XII): [ka] [In the formula, Z 1 This is a polymerizable ethylenically unsaturated group of acryloyl or non-acryloyl, R 1 is H or CH3, m is between 2 and 100. Q is a covalent bond, -O-, -NR 1 -, -CO2-, and -C(O)NR 1 -(In the formula, R 1 This is a flow-through process according to Embodiment 46, in which the group is a divalent linking group selected from H or CH3.

[0171] Embodiment 48 describes a grafted ligand-functional polymer of a salt-resistant anion-exchange non-fibrous porous filter element, wherein the polymer contains a hydrophilic monomer (M d The flow-through process according to any one of embodiments 42 to 47 further comprises (where z is at least 1), which has a free radical polymerizable group and a hydrophilic group.

[0172] Embodiment 49 uses a hydrophilic monomer (M) to produce a salt-resistant anion-exchange non-fibrous porous filter element. d ) is given by equation (XIII): [ka] [In the formula, Each R 1 These are independently H or CH3, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), This is a flow-through process according to Embodiment 48, where t is between 2 and 100.

[0173] Embodiment 50 is a cation exchange non-fibrous porous filter element, Non-fibrous porous filter element, A polymer disposed on a non-fibrous porous filter element, A polymer comprising a hydrocarbon main chain and a plurality of pendant groups bonded to the hydrocarbon main chain, wherein each of the first plurality of pendant groups is At least one acidic group or a salt thereof, A flow-through process according to any one of Embodiments 1 to 49, comprising a spacer group that directly links at least one acidic group or a salt thereof to a hydrocarbon main chain by a chain of at least six linearly linked atoms.

[0174] Embodiment 51 is the flow-through process according to Embodiment 50, wherein the spacer group chain has at least eight chain-linked atoms.

[0175] Embodiment 52 is the flow-through process according to Embodiment 50 or 51, wherein the spacer group is a chain-linked heteroatom-containing hydrocarbon group.

[0176] Embodiment 53 is a flow-through process according to any one of Embodiments 50 to 52, wherein the spacer group includes at least one hydrogen bonding portion.

[0177] Embodiment 54 is a flow-through process according to any one of Embodiments 50 to 53, wherein at least one acidic group of a polymer disposed on a non-fibrous porous filter element, or a salt thereof, is present in the cation-exchange non-fibrous porous filter element at a density of at least 0.01 (or at least 0.02) millimoles / gram.

[0178] Embodiment 55 is a flow-through process according to any one of Embodiments 50 to 54, wherein at least one acidic group of a polymer disposed on a non-fibrous porous filter element, or a salt thereof, is present in the cation-exchange non-fibrous porous filter element at a density of up to 0.6 mmol / gram.

[0179] Embodiment 56 is a flow-through process according to any one of Embodiments 50 to 55, wherein at least one acidic group or salt thereof is selected from a carboxyl group, a phosphono group, a phosphat group, a sulfono group, a sulfato group, a boronato group, and combinations thereof.

[0180] Embodiment 57 is a flow-through process according to any one of Embodiments 50 to 56, wherein the polymer comprises an interpolymerized unit of at least one monomer, each unit comprising at least one ethylenically unsaturated group, at least one acidic group or a salt thereof, and a spacer group that directly links the at least one ethylenically unsaturated group and the at least one acidic group or a salt thereof by a chain of at least six linearly linked atoms.

[0181] Embodiment 58 is the flow-through process described in Embodiment 57, wherein at least one ethylenically unsaturated group is selected from an ethenyl group, a 1-alkylethenyl group, and a combination thereof.

[0182] Embodiment 59 is a flow-through process according to any one of Embodiments 50 to 58, wherein the polymer is covalently bonded to a non-fibrous porous filter element.

[0183] Embodiment 60 is a flow-through process according to any one of Embodiments 50 to 59, wherein the polymer is a copolymer.

[0184] Embodiment 61 comprises a copolymer comprising a hydrocarbon main chain and a plurality of pendant groups bonded to the hydrocarbon main chain. Each of the first multiple pendant bases is At least one acidic group or a salt thereof, It comprises a spacer group that directly links at least one acidic group or a salt thereof to a hydrocarbon main chain by a chain of at least six linearly linked atoms, Each of the second set of pendant bases is At least one acidic group or a salt thereof, It comprises a spacer group that directly links at least one acidic group or a salt thereof to a hydrocarbon main chain by a chain of at least six linearly linked atoms, The first set of pendant bases differs from the second set of pendant bases, This is the flow-through process according to Embodiment 60, wherein the molar ratio of the first plurality of pendant groups to the second plurality of pendant groups is in the range of 95:5 to 5:95.

[0185] Embodiment 62 is a copolymer covalently bonded to a non-fibrous porous filter element, The first monomer is At least one ethylenically unsaturated group, At least one acidic group or a salt thereof, A first monomer comprising a spacer group that directly links at least one ethylenically unsaturated group and at least one acidic group or a salt thereof by a chain of at least six linearly linked atoms, The second monomer is At least one ethylenically unsaturated group, At least one acidic group or a salt thereof, The reaction product of a monomer composition comprising a second monomer, which includes a spacer group that directly links at least one ethylenically unsaturated group and at least one acidic group by a chain of at least six linearly linked atoms, The second monomer is different from the first monomer, This is the flow-through process according to Embodiment 61, wherein the molar ratio of the first monomer to the second monomer is in the range of 95:5 to 5:95.

[0186] Embodiment 63 is the flow-through process described in Embodiment 62, wherein at least one ethylenically unsaturated group of the first monomer and / or the second monomer is selected from an ethenyl group, a 1-alkylethenyl group, and combinations thereof.

[0187] Embodiment 64 is a first monomer of the following general formula (XV): [ka] [In the formula, R 1 is H or CH3, Each R 2 It is independently a (hetero)hydrocarbylene, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), Z 3 This is a (hetero)hydrocarbylene group comprising at least one hydrogen bond donor, at least one hydrogen bond acceptor, or a combination thereof. r is either 0 or 1. The flow-through process according to Embodiment 62 or 63 is one of the categories represented by [a functional group comprising at least one acidic group or a salt thereof].

[0188] Embodiment 65 is a second monomer of the following general formula (XV): [ka] [In the formula, R 1 is H or CH3, Each R 2 It is independently a (hetero)hydrocarbylene, X 1 -O- or -NR 3 -(In the formula, R 3 (is H or (hetero)hydrocarbyl), Z 3 This is a (hetero)hydrocarbylene group comprising at least one hydrogen bond donor, at least one hydrogen bond acceptor, or a combination thereof. r is either 0 or 1. A flow-through process according to any one of embodiments 62 to 64, where L is one of the categories represented by a functional group comprising at least one acidic group or a salt thereof.

[0189] Embodiment 66 is a filter cartridge comprising a salt-resistant anion-exchange non-fibrous porous filter element and a cation-exchange non-fibrous porous filter element as described herein.

[0190] Embodiment 67 is the filter cartridge according to Embodiment 66, wherein the cation exchange non-fibrous porous filter element is located downstream of the salt-resistant anion exchange non-fibrous porous filter element.

[0191] Embodiment 68 is a filter cartridge according to Embodiment 66 or 67, wherein the salt-resistant anion-exchange non-fibrous porous filter element includes a non-fibrous porous filter element containing an immobilized cationic nitrogen-containing ligand.

[0192] Embodiment 69 provides a salt-resistant anion-exchange non-fibrous membrane comprising a non-fibrous porous filter element and a ligand-functional polymer grafted thereon, wherein the grafted ligand-functional polymer is of formula: -(MPI ) w -(M b ) x -(M c ) y -(M d ) z , [In the formula, -(M PI ) w represents a residue of the grafted photoinitiator monomer, and w is 0 or at least 1. -(M b ) x represents a polymerized ligand monomer having "x" polymerized monomer units, where x is at least 1. -(M c ) y represents a polymerized crosslinked monomer having y polymerized monomer units, where y may be 0 or at least 1. -(M d ) z The filter cartridge according to any one of embodiments 66 to 68, wherein represents a polymerized hydrophilic monomer having z polymerized monomer units, where z may be 0 or at least 1.

[0193] Embodiment 70 is a cation exchange non-fibrous porous filter element, Non-fibrous porous filter element, A polymer disposed on a non-fibrous porous filter element, A polymer comprising a hydrocarbon main chain and a plurality of pendant groups bonded to the hydrocarbon main chain, wherein each of the first plurality of pendant groups is At least one acidic group or a salt thereof, A filter cartridge according to any one of embodiments 66 to 69, comprising a spacer group that directly links at least one acidic group or a salt thereof to a hydrocarbon main chain by a chain of at least six linearly linked atoms. [Examples]

[0194] These examples are for illustrative purposes only and are not intended to unduly limit the scope of the attached claims. While the broad ranges and parameters of this disclosure are approximations, the values ​​shown in the specific examples are reported as accurately as possible. However, each value inherently contains certain errors that inevitably arise from the standard deviation found in the respective test measurements. At a minimum, each numerical parameter should be interpreted by applying common rounding techniques in light of the number of significant figures reported, but this is not intended to limit the application of the doctrine of equivalents to the claims.

[0195] Unless otherwise stated, all parts, percentages, ratios, etc., in the examples and other parts of the specification are by weight, and all reagents used in the examples are obtained or available from general chemical suppliers, such as Sigma-Aldrich Company (Saint Louis, Missouri), or can be synthesized by conventional methods.

[0196] [Table 1]

[0197] Changes or adjustments to the buffer solution containing monoclonal antibodies Conductivity adjustment was performed using 4 moles (4 M) NaCl. pH adjustment was achieved using acetic acid (200 mmol (mM) or 500 mM) and Tris base (2 M). Conductivity was determined using an Accumet Excel XL50 conductivity meter (Fisher Scientific, Hampton, NH). pH was determined using a VWR Symphony benchtop pH meter (VWR International, Radnor, PA).

[0198] Challenge plate for filter elements The completed filter elements (FE-A to FE-L) were cut into 7.5 mm diameter discs. For the anion exchange filter element (FE-L), a single disc was loaded into each well of a 96-well EMPORE filter plate (Model 6065, 3M Corporation, St. Paul, MN). For each type of cation exchange filter element, two discs of the same filter element were loaded into each well of the 96-well EMPORE filter plate. The filter elements were held in place with plastic O-rings. The total working filter volume of each well was approximately 8.6 microliters. Each challenge plate loaded with filter elements was placed on a 96-well deep-well collection plate (Thermo Fisher Scientific, Waltham, MA) before centrifugation for sample collection. Centrifuge was performed using an Allegra 25R centrifuge (Beckman Coulter, Brea, CA). I received a commercially available challenge plate (Sartorius SARTOBIND 96-well plate) from the manufacturer and used it according to the manufacturer's instructions.

[0199] Methods for analyzing target molecules and impurities The concentration of Chinese hamster ovary (CHO) host cell protein (HCP) was determined using the CHO HCP ELISA Kit, 3G (Cygnus Technologies, Southport, NC) according to the manufacturer's protocol.

[0200] The protein concentration of the mAb was determined by Beer's Law, using absorbance at 280 nanometers and an extinction coefficient of 1.36. Absorbance was determined using a SpectraMax M5 spectrophotometer (Molecular Devices, San Jose, CA).

[0201] The monomer yield ("monomer mAb yield %) and the composition of high molecular weight (HMW) species in the filtered solution are measured using TOSOH TSKgel G3000SW. XLAnalysis was performed by size exclusion chromatography (SEC) using a Shimadzu Prominence HPLC system (Shimadzu Scientific Instruments, Columbia, MD) equipped with a column (Tosoh BioScience LLC, Griesheim, Germany) (analytical conditions: injection volume of 20 microliters; flow rate of 1 mL / min; mobile phase: 100 mM sodium phosphate, 300 mM NaCl, pH 6.9; detection at 280 nanometers). Monomer yield and high molecular weight (HMW) species composition were determined by comparing the peak areas of the starting solution and the filtered solution, and the results were reported as the average of two replicate tests.

[0202] The percentage of HMW species present in the mAb solution was determined using the SEC chromatography method described above. The peak area of ​​the HMW species component in the sample (peak A) was compared with the total peak area of ​​the sample. The "HMW composition %" was calculated using Equation 1.

[0203] The removal rate of HMW species was determined using the SEC chromatography method described above. The peak area of ​​the aggregated components was measured before the separation process (peak B) and after the separation process using the depth filter or filter element challenge plate described in the examples (peak C). The "removed HMW%" was calculated using Equation 2.

[0204] The yield percentage of monomer monoclonal antibodies (mAbs) was determined by measuring the peak area of ​​the monomer component before performing the separation process using the SEC chromatography method described above (peak D), and by measuring the peak area of ​​the monomer component after performing the separation process using the depth filter or filter element challenge plate described in the examples (peak E). The "percentage of monomer mAb yield" was calculated using Equation 3.

[0205] The HCP removal rate was determined by measuring the HCP concentration before ([pre-HCP]) and after ([post-HCP]) the separation process using the HCP quantification method described above. The "HCP removal %" was calculated using Equation 4.

number

[0206] Preparation of cation exchange non-fibrous porous filter element A (FE-A) The coating solution was prepared by mixing 4-aminobutyrate sodium salt / IEM monomer solution (IEM-GABA) (as described in Monomer Example B of International Publication WO2018 / 048698) (Vail et al.). (20.8% w / w solution in 6.73 g of deionized water) and sulfonated benzophenone (S-BP) (0.1 g / ml solution in 250 microliters of deionized water). Deionized water (13.02 g) was added to obtain a mixture of approximately 0.25 M in monomer. A nylon membrane substrate (18 cm × 23 cm, nylon 66 membrane, single reinforced layer nylon 3 zone membrane, nominal pore size 1.8 micrometers (μm or micron), #080ZN, obtained from 3M Purification, Inc., Meriden, CT) was placed on a polyester film sheet, and the coating solution was pipetteed onto the top surface of the substrate. After immersing the substrate in the coating solution for approximately 1 minute, a second sheet of polyester film was placed on top of the substrate. A 2.28 kg cylindrical weight was rolled over the resulting three-layer sandwich-like sample to squeeze out excess coating solution. Using a UV stand (Classic Manufacturing, Inc., Oakdale, MN) equipped with 18 valves [Sylvania RG2 40W F40 / 350BL / ECO, 10 on top of the substrate, 8 below, 1.17 m (46 inch) in length, with a central spacing of 5.1 cm (2 inch)], the sandwich-like sample was irradiated for 15 minutes to perform ultraviolet (UV) initiation grafting. The polyester sheets were removed, and the resulting functionalized substrate was placed in a 1000 mL polyethylene bottle. The bottle was filled with 0.9% (w / w) saline solution, sealed, and placed on a roller for 30 minutes to wash off any residual monomers or ungrafted polymers. The functionalized substrate was then washed with fresh saline solution for 30 minutes, followed by washing with deionized water for 30 minutes (twice), and then dried. The graft density was 0.266 mmol / g (mmol / g) of the membrane, as determined by the mass increase.

[0207] Preparation of cation exchange non-fibrous porous filter element B (FE-B) A nylon membrane (#080ZN) was grafted with an IEM-glycine sodium salt monomer solution (0.25 M) as described in Example 30 of U.S. Patent Application No. 2019 / 0194250 (Colak Atan et al.). The graft density was 0.28 mmol / g of the membrane.

[0208] Preparation of cation exchange non-fibrous porous filter element C(FE-C) A nylon membrane (#080ZN) was grafted with an IEM-glycine sodium salt monomer solution at a concentration of 0.375 M, as described in Example 30 of U.S. Patent Application No. 2019 / 0194250 (Colak Atan et al.). The graft density was 0.38 mmol / g of the membrane.

[0209] Preparation of cation exchange non-fibrous porous filter element D (FE-D) A nylon membrane (#080Zn) was grafted with a 0.25 M solution of 2-[[2-methyl-2-(propa-2-enoylamino)propanoyl]amino]ethyl phosphate disodium (VDM-O-phosphorylethanolamine disodium) monomer using the procedure described for FE-A. The graft density was 0.12 mmol / g of the membrane.

[0210] The monomer was prepared as follows: O-phosphorylethanolamine (21.15 g) was weighed into a 500 ml (mL) round-bottom (RB) flask and placed in an ice bath with magnetic stirring. NaOH (2N (2 equivalents), 150 mL) was added and the mixture was stirred until dissolved. VDM (10 mL) was added by pipette. The initial turbid suspension was stirred for 10 minutes to homogenize it. A second 10 mL of VDM was added (a total of 20 mL was added). The mixture was stirred for a further 50 minutes. The reaction mixture was adjusted to pH 7 by adding a few drops of concentrated hydrochloric acid and filtered. Solids content % = 25.0%. 1H-NMR (D2O): δ1.30(s,6H),3.20(t,2H),3.59(q,2H),5.56(dd,1H),5.99(dd,1H),6.09(dd,1H).

[0211] Preparation of cation exchange non-fibrous porous filter element E (FE-E) A nylon membrane (#080Zn) was grafted with a 0.25 M solution of [2-carboxy-2-[[2-methyl-2-(propa-2-enoylamino)propanoyl]amino]ethyl]phosphate disodium (VDM-O-phosphoserine disodium) monomer, using the procedure described for FE-A. The graft density was 0.07 mmol / g of the membrane.

[0212] The monomer was prepared as follows: LO-phosphoserine (18.5 g) was added to a 500 mL RB flask containing a magnetic stirring rod. The flask was placed in an ice bath, and deionized water (40 mL) and 5N NaOH (60 mL) were added to the flask. The contents were stirred until all solids were dissolved. After cooling in the ice bath for about 30 minutes, VDM (5.0 mL) was added by syringe. The mixture was stirred for 10 minutes, and then a further 8.33 mL of VDM was added (total addition of 13.33 mL). Stirring was continued for 55 minutes to complete the reaction. The mixture was adjusted to pH 7 by adding 6 drops of concentrated hydrochloric acid. The reaction product was filtered to obtain the product. Solid content % = 28.4%. 1 H-NMR(D2O): δ1.34(s,3H),1.38(s,3H),3.81(2m,2H),4.07(m,1H),5.56(dd,1H),5.99(dd,1H),6.11(dd,1H).

[0213] Preparation of cation exchange non-fibrous porous filter element F(FE-F) A nylon membrane (#080ZN) was grafted with a 50:50 (mol / mol) VDM-GABA sodium salt (VDM-4-aminobutyrate sodium salt) / VDM-phenylalanine sodium salt monomer solution at a concentration of 0.25 M, as described in Example 5 of International Publication No. WO2018 / 048696 (Vail et al.). The graft density was 0.16 mmol / g of the membrane.

[0214] Preparation of cation exchange non-fibrous porous filter element G (FE-G) A nylon membrane (#080ZN) was grafted with a 0.25 M solution of VDM-7-aminoheptanate sodium salt monomer, as described in Example 21 of U.S. Patent Application No. 2019 / 0194250 (Colak Atan et al.). The graft density was 0.18 mmol / g of the membrane.

[0215] Preparation of cation exchange non-fibrous porous filter element H(FE-H) A nylon membrane (#080ZN) was grafted with a 50:50 (mol / mol) IEM-glycine sodium salt / VDM-phenylalanine sodium salt monomer solution at a concentration of 0.25 M, as described in Example 11 of International Publication No. WO2018 / 048696 (Vail et al.). The graft density was 0.14 mmol / g of the membrane.

[0216] Preparation of cation exchange non-fibrous porous filter element I (FE-I) A nylon membrane (#080ZN) was grafted with a 50:50 (mol / mol) VDM-GABA sodium salt / VDM-4-aminomethyl-cyclohexanecarboxylate sodium salt monomer solution at a concentration of 0.25 M, as described in Example 2 of International Publication No. WO2018 / 048696 (Vail et al.). The graft density was 0.19 mmol / g of the membrane.

[0217] Preparation of cation exchange non-fibrous porous filter element J (FE-J) A nylon membrane (#080Zn) was grafted with a 50:50 (mol / mol) IEM-glycine sodium salt / IEM-phenylalanine sodium salt monomer solution at a concentration of 0.25 M, according to the procedure described for FE-H. The IEM-phenylalanine sodium salt monomer was prepared as described in International Publication WO2018 / 048696 (Vail et al.), except that racemic phenylalanine was used. The graft density was 0.42 mmol / g of the membrane.

[0218] Preparation of cation exchange non-fibrous porous filter element K(FE-K) A nylon membrane (#080Zn) was grafted with an IEM-glycine sodium salt monomer solution (0.25 M) as described in Example 30 of U.S. Patent Application No. 2019 / 0194250 (Colak Atan et al.). The graft density was 0.26 mmol / g of the membrane.

[0219] Preparation of salt-resistant anion-exchange non-fibrous porous filter element L(FE-L) IEM-agmatine sulfate sodium was radiation-graft polymerized on a nylon membrane (#080Zn) to produce a guanidinyl ligand-functionalized microporous membrane with similar dynamic binding ability to bovine serum albumin (BSA) as described in U.S. Patent No. 10,471,398 (Bothof et al.), Example 28.

[0220] Example 1. A flow-through purification process using sequential processing of a monoclonal antibody solution with an anion exchange adsorption depth filter, a salt-tolerant anion exchange non-fibrous porous filter element, and a cation exchange non-fibrous porous filter element.

[0221] A solution of a virus-inactivated pool (VIP) (IgG1, pI 8.0, 10.5 mg / mL, pH 6.27, 5.3 mS / cm) of monoclonal antibody mAbA was filtered through an anion exchange adsorption depth filter prepared from the internal media components of an EMPHAZE AEX hybrid purifier (EMPHAZE AEX HP, obtained from 3M Corporation) assembled in a 25 mm holder [10 bed volumes / min (BVM)]. The challenge load was 180 liters / square meter (L / m²). 2 The throughput was 1700 grams / liter (g / L). The challenge load was determined as the amount of mAbA per unit volume of filter media (grams / liter, g / L). Throughput was the amount of liquid passing through a given surface area of ​​filter media (liters / square meter, L / m 2 The depth filter was determined to reduce the turbidity of the VIP solution from >60 NTU (specific turbidity units) to <5 NTU, and the differential pressure did not exceed 5 psi (pounds per square inch). Table 2 shows the characteristics of the obtained depth-filtered solution.

[0222] The FE-L load challenge plate (above) was washed with 0.9% sodium chloride (1 mL) by centrifugation at 3000 rcf (relative centrifugal force) for 5 minutes. After washing, the depth-filtered solution was filtered using the FE-L challenge plate with a 1200 g / L challenge load by centrifugation at 300 rcf for 5 minutes. The FE-L filtration solutions were pooled together. Aliquots of the pooled solutions with different challenge loads [333 microliters (400 g / L challenge load) or 450 microliters (550 g / L challenge load)] were prepared for addition to the wells of a series of second challenge plates. Each second challenge plate contained two discs of a single type of cation exchange filter element selected from FE-A to FE-F (as above). The second challenge plates were pre-washed with saline according to the method described for the FE-L challenge plate. The plates were centrifuged continuously at 300, 600, 1200, and 3000 rcf for 5 minutes each. The filtered sample was analyzed for product and impurities.

[0223] The VIP solution before treatment had a 4.2% HMW composition and an HCP concentration of 3899 nanograms / liter (ng / L).

[0224] The solution obtained after the depth filtration process step had an HMW composition of 4.2%, a monomer mAb yield of 98.4%, and an HCP concentration of 3366 nanograms / milliliter (ng / mL).

[0225] The pooled solution obtained after the filtration process step using FE-L had an HMW composition of 3.6%, a monomer mAb yield of 100.1%, and an HCP concentration of 1717 ng / mL.

[0226] Table 3 reports the HMW composition percentage, monomer mAb yield, and HCP concentration values ​​obtained after the final process step of filtering the solution through a cation exchange filter element (selected from FE-A to FE-F). Results are reported for experiments using either a 400 g / L challenge load or a 550 g / L challenge load.

[0227] [Table 2]

[0228] [Table 3]

[0229] Example 2. A flow-through purification process using sequential processing of a monoclonal antibody solution with an anion exchange adsorption depth filter, a buffer exchange step, a salt-tolerant anion exchange non-fibrous porous filter element, and a cation exchange non-fibrous porous filter element.

[0230] A VIP (IgG1, pI 8.0, 7.1 mg / mL, pH 6.27, 4.0 mS / cm) solution of monoclonal antibody mAbA was filtered through a depth filter according to the process described in Example 1. Individual aliquots from the depth-filtered solution were adjusted to a target pH of 5.5, 6.25, or 7.0 and a conductivity of 8, 16, or 24 mS / cm. The properties of the buffer-prepared mAbA solutions are provided in Table 4. The equilibration buffer was prepared starting with 20 mM acetate buffer (pH 5.5) and adjusted with 2 M Tris. The conductivity of the buffer was adjusted using 4 M sodium chloride. The equilibration buffer was matched to the pH and conductivity properties of the buffer-prepared mAb solution.

[0231] In this process, the SARTOBIND STIC plate (primary amine functional group) was used as the anion exchange filter element challenge plate, and the SARTOBIND S plate (sulfonic acid functional group) was used as the cation exchange filter element challenge plate. Each plate was pretreated by filtration with one of the equilibration buffers (selected from the equilibration buffers listed above). The pretreatment filtration condition was 500 microliters of buffer centrifuged at 1,000 rcf for 2 minutes. This process was repeated a total of four times. The equilibration buffer-treated plates were matched with mAbA solutions prepared using the same buffer composition.

[0232] After equilibration, individual aliquots of the buffer-exchanged mAbA solution were filtered using a SARTOBIND STIC plate. The filtration conditions were 500 microliters of solution centrifuged at 1,000 rcf for 2 minutes. This process was repeated a total of two times for a challenge load of approximately 400 g / L (membrane with a bed volume of 19 microliters). The resulting filtrate was then filtered using a SARTOBIND S plate. The filtration conditions were the same as for the SARTOBIND STIC plate. The final filtrate was collected and analyzed. The HCP concentration, HMW composition%, removed HMW%, and monomer mAb yield% of the final filtrate were determined, and the results are reported in Tables 5-8.

[0233] Comparative Example 2. During the process of Example 2, an intermediate process sample of the solution was collected before filtration through a SARTOBIND S plate, although filtration through a SARTOBIND STIC plate had already been performed. The intermediate sample was analyzed for HCP concentration, HMW composition %, removed HMW %, and monomer mAb yield %. The results are shown in Tables 5-8.

[0234] Example 2a. The same process as described in Example 2 was followed, except that the FE-L loaded plate was used as the anion exchange filter element challenge plate and the FE-J loaded plate was used as the cation exchange filter element challenge plate. The process conditions were modified to use 1,000 microliters of equilibration buffer centrifuged at 1,500 rcf for 5 minutes for each FE-L and FE-J filter plate. After equilibration, individual aliquots (500 microliters) of the buffer-exchanged mAb solution were successively filtered through the FE-L loaded plate and then the FE-J loaded plate using centrifugation at 1,500 rcf for 5 minutes. The challenge load for each well was approximately 400 g / L (membrane with a bed volume of 9 microliters). The HCP concentration, HMW composition%, removed HMW%, and monomer mAb yield% of the final filtrate were determined, and the results are reported in Tables 5-8.

[0235] Comparative example 2a. During the process in Example 2a, an intermediate process sample of the solution was collected after filtration through the FE-L loaded plate, but before filtration through the FE-J loaded plate. The intermediate sample was analyzed for HCP concentration, HMW composition%, removed HMW%, and monomer mAb yield. The results are shown in Tables 5-8.

[0236] Example 2b. The same process as described in Example 2a was followed, except that the FE-K loaded plate was used instead of the FE-J plate as the cation exchange filter element challenge plate. The HCP concentration, HMW composition%, removed HMW%, and monomer mAb yield% of the final filtrate were determined, and the results are reported in Tables 5-8.

[0237] Example 2c. The same process as described in Example 2 was followed, except that the FE-L loaded plate was used as the anion exchange filter element challenge plate and the FE-F loaded plate was used as the cation exchange filter element challenge plate. The processing conditions were modified to use 1,000 microliters of equilibration buffer centrifuged at 1,500 rcf for 5 minutes for each FE-L and FE-F filter plate. After equilibration, individual aliquots (500 microliters) of the buffer-exchanged mAb solution were successively filtered through the FE-L loaded plate and then the FE-F loaded plate using centrifugation at 1,500 rcf for 5 minutes. An additional centrifugation cycle (5 minutes at 3,000 rcf) was used for the FE-F loaded plate. The challenge load for each well was approximately 400 g / L (membrane with a bed volume of 9 microliters). The HCP concentration, HMW composition%, removed HMW%, and monomer mAb yield% of the final filtrate were determined, and the results are reported in Tables 5-8.

[0238] [Table 4]

[0239] [Table 5]

[0240] [Table 6]

[0241] [Table 7]

[0242] [Table 8]

[0243] Example 3. A flow-through purification process using sequential processing of a monoclonal antibody solution with an anion exchange adsorption depth filter, a buffer exchange step, a salt-tolerant anion exchange non-fibrous porous filter element, and a cation exchange non-fibrous porous filter element.

[0244] A VIP (IgG1, pI 8.0, 7.1 mg / mL, pH 6.27, 4.0 mS / cm) solution of monoclonal antibody mAbA was filtered through a depth filter according to the process described in Example 1. The VIP solution before treatment had a 4.2% HMW composition and an HCP concentration of 4098 ng / L. The solution obtained after the depth filtration process step had a 4.2% HMW composition, a 97.9% monomer mAb yield, and an HCP concentration of 3546 ng / mL.

[0245] Buffer-adjusted mAbA solutions were prepared, and their properties are provided in Table 9. Twelve different buffer-adjusted mAbA solutions were prepared by adjusting the pH with 500 mM acetic acid or 2 M Tris and adjusting the conductivity with 4 M sodium chloride. The solutions had a target pH of 6.0, 6.5, or 7.0 and a target conductivity of 8, 14, 19, or 24 mS / cm.

[0246] In the process, FE-L loaded plates were used as anion exchange filter element challenge plates, and FE-C loaded plates were used as cation exchange filter element challenge plates. Each plate was pretreated by filtering with one of the equilibration buffers (selected from the equilibration buffers described above in Example 2). The pretreatment filtration condition was 1000 microliters of buffer centrifuged at 3000 rcf for 5 minutes. The equilibration buffer-treated plates were used in conjunction with mAbA solutions prepared using the same buffer composition. After equilibration, individual aliquots of the buffer-exchanged mAbA solutions were filtered using FE-L loaded plates. The filtration condition was 1000 microliters of solution centrifuged at 300 rcf for 5 minutes. The challenge load was approximately 1200 g / L (membrane with a bed volume of 9 microliters), and the collected filtrates were pooled for each individual condition.

[0247] Next, the obtained filtrate was filtered using an FE-C loaded plate. The filtration conditions were aliquots of pooled solutions with different challenge loads [333 microliters (400 g / L challenge load) or 450 microliters (550 g / L challenge load)], centrifuged for 5 minutes at 100, 200, 300, 600, 1200, and 3000 rcf. The final filtrate was collected and analyzed. The HCP concentration, HMW composition%, monomer mAb yield%, removed HMW%, and HCP removal percentage of the final filtrate were determined, and the results are reported in Tables 11-12.

[0248] Comparative Example 3. During the process of Example 3, an intermediate process sample of the solution was collected after filtration through the FE-L loaded plate, but before filtration through the FE-C loaded plate. The intermediate sample was analyzed for HCP concentration, HMW composition %, and monomer mAb yield %. The results are reported in Table 10.

[0249] Example 3a. The same process as described in Example 3 was followed, except that the FE-G loaded plate was used instead of the FE-C plate as the cation exchange filter element challenge plate. The HCP concentration, HMW composition %, monomer mAb yield %, removed HMW %, and HCP removal % of the final filtrate were determined, and the results are reported in Tables 13-14.

[0250] Example 3b. A VIP (IgG1, pI 8.0, 7.1 mg / mL, pH 6.27, 4.0 mS / cm) solution of monoclonal antibody mAbA was filtered through a depth filter according to the process described in Example 1. The VIP solution before treatment had a 4.2% HMW composition and an HCP concentration of 4098 ng / L. The solution obtained after the depth filtration process step had a 4.2% HMW composition, a 97.9% monomer mAb yield, and an HCP concentration of 3255 ng / mL. The same process as described in Example 3 was followed, except that an FE-F loaded plate was used instead of an FE-C plate as a cation exchange filter element challenge plate. The HCP concentration, HMW composition, monomer mAb yield, removed HMW, and HCP removal percentage of the final filtered solution were determined, and the results are reported in Tables 15-16.

[0251] Comparative example 3b. During the process of Example 3b, an intermediate process sample of the solution was collected after filtration through the FE-L loaded plate, but before filtration through the FE-F loaded plate. The intermediate sample was analyzed for HCP concentration, HMW composition %, and monomer mAb yield %. The results are reported in Table 10.

[0252] [Table 9]

[0253] [Table 10]

[0254] [Table 11]

[0255] [Table 12]

[0256] [Table 13]

[0257] [Table 14]

[0258] [Table 15]

[0259] [Table 16]

[0260] Example 4. A flow-through purification process using sequential processing of a monoclonal antibody solution with a buffer exchange step, a salt-tolerant anion exchange non-fibrous porous filter element, and a cation exchange non-fibrous porous filter element.

[0261] Pooled monoclonal antibody mAb2 (IgG1, pI approx. 8) was purified using a ZetaPlus ZP90 depth filter (3M Corporation), followed by a protein A chromatography step using acetic acid elution (mAbSelect ProA resin, GE Healthcare Life Sciences, Pittsburgh, PA), and held at pH 3.5 for 30 minutes for virus inactivation. Following virus inactivation, the resulting low pH solution was neutralized with 2 M Tris. The neutralized solution had an initial pH of 7.4 and a conductivity of 10.2 (mS / cm).

[0262] Buffer-adjusted mAb2 solutions were prepared, and their properties are provided in Table 17. Nine different buffer-adjusted mAb2 solutions were prepared by adjusting the pH with 500 mM acetic acid and adjusting the conductivity using 4 M sodium chloride. The solutions had a target pH of 6.0, 6.5, or 7.0 and a target conductivity of 12, 17, or 22 mS / cm.

[0263] In the process, FE-L loaded plates were used as anion exchange filter element challenge plates, and FE-C loaded plates were used as cation exchange filter element challenge plates. Each plate was pretreated by filtration with one of the equilibration buffers (selected from the equilibration buffers described above in Example 2). The pretreatment filtration condition was 1000 microliters of buffer centrifuged at 3000 rcf for 5 minutes. The equilibration buffer-treated plates were used in conjunction with mAb2 solutions prepared using the same buffer composition. After equilibration, individual aliquots of the buffer-exchanged mAb2 solutions were filtered using FE-L loaded plates. The filtration condition was 1000 microliters of solution centrifuged at 300 rcf for 5 minutes. The challenge load was approximately 350 g / L (membrane with a bed volume of 9 microliters), and the collected filtrates were pooled for each individual condition.

[0264] Next, the obtained filtrate was filtered using an FE-C loaded plate. The filtration conditions were 1000 microliters of solution centrifuged for 5 minutes at 300, 600, 1200, and 3000 rcf. The final filtrate was collected and analyzed. The HCP concentration, HMW composition %, removed HMW %, and monomer mAb yield % of the final filtrate were determined, and the results are reported in Tables 18-21.

[0265] Comparative Example 4. During the process of Example 4, an intermediate process sample of the solution was collected after filtration through the FE-L loaded plate, but before filtration through the FE-C loaded plate. The intermediate sample was analyzed for HCP concentration, HMW composition%, removed HMW%, and monomer mAb yield. The results are shown in Tables 18-21.

[0266] Example 4a. The same process as described in Example 4 was followed, except that the FE-F loaded plate was used instead of the FE-C plate as the cation exchange filter element challenge plate. The HCP concentration, HMW composition%, removed HMW%, and monomer mAb yield% of the final filtrate were determined, and the results are reported in Tables 18-21.

[0267] Example 4b. The same process as described in Example 4 was followed, except that the FE-E loaded plate was used instead of the FE-C plate as the cation exchange filter element challenge plate. The HCP concentration, HMW composition %, removed HMW %, and monomer mAb yield % of the final filtrate were determined, and the results are reported in Tables 18-21.

[0268] Example 4c. The same process as described in Example 4 was followed, except that the FE-H loaded plate was used instead of the FE-C plate as the cation exchange filter element challenge plate. The HCP concentration, HMW composition%, removed HMW%, and monomer mAb yield% of the final filtrate were determined, and the results are reported in Tables 18-21.

[0269] Example 4d. The same process as described in Example 4 was followed, except that the FE-I loaded plate was used instead of the FE-C plate as the cation exchange filter element challenge plate. The HCP concentration, HMW composition %, removed HMW %, and monomer mAb yield % of the final filtrate were determined, and the results are reported in Tables 18-21.

[0270] [Table 17]

[0271] [Table 18]

[0272] [Table 19]

[0273] [Table 20]

[0274] [Table 21]

[0275] All disclosures of patents, patent documents, and publications referenced herein are incorporated by reference as if each were incorporated individually. In the event of any inconsistency or conflict between the statements herein and the disclosures of any of the documents incorporated herein by reference, the statements herein shall prevail. Those skilled in the art will see various modifications and changes to this disclosure that do not deviate from the scope and intent of this disclosure. It should be understood that this disclosure is not intended to be unduly limited by the exemplary embodiments and examples described herein, and that such embodiments and examples are presented only as examples within the scope of this disclosure, which is intended to be limited only by the claims described herein as follows.

Claims

1. A flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, the process comprising: Optionally, contacting the sample with an anion exchange adsorption depth filter; Optionally, performing a buffer exchange on the sample before and / or after contacting the sample with the anion exchange adsorption depth filter; Contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element; Immediately thereafter, contacting the sample with a cation exchange non-fibrous porous filter element, The flow-through process includes one or two buffer exchanges, and does not include a buffer exchange between the contact of the sample with the salt-tolerant anion exchange non-fibrous porous filter element and the contact with the cation exchange non-fibrous porous filter element. Flow-through process.

2. A flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, the process comprising: Contacting the sample with an anion exchange adsorption depth filter; Immediately thereafter, contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element; Immediately thereafter, contacting the sample with a cation exchange non-fibrous porous filter element, The flow-through process does not include a buffer exchange. Flow-through process.

3. A flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, the process comprising: Contacting the sample with an anion exchange adsorption depth filter; After contacting the sample with the anion exchange adsorption depth filter, performing a buffer exchange on the sample; Contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element; Immediately thereafter, contacting the sample with a cation exchange non-fibrous porous filter element. Flow-through process.

4. A flow-through process for purifying a target molecule from a biological solution in a sample containing the target molecule, the process comprising: Performing a buffer exchange on the sample; Contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element; Immediately thereafter, contacting the sample with a cation exchange non-fibrous porous filter element. Flow-through process.

5. The flow-through process according to any one of claims 1 to 4, wherein the target molecule comprises a monoclonal antibody.

6. The flow-through process according to any one of claims 1 to 5, wherein the anion exchange adsorption depth filter comprises a porous substrate containing an immobilized anion exchange ligand.

7. The flow-through process according to claim 6, wherein the anion exchange ligand of the anion exchange adsorption depth filter comprises a cationic nitrogen-containing ligand.

8. The anion exchange adsorption depth filter comprises a porous substrate and a ligand-functional polymer grafted thereto, and the grafted ligand-functional polymer has the formula: -(M ＰＩ ) ｗ -(M ｂ ) ｘ -(M ｃ ) ｙ -(M ｄ ) ｚ 、 [wherein, -(M ＰＩ ) ｗ represents the residue of the grafted photoinitiator monomer, w is 0 or at least 1, and -(M ｂ ) ｘ represents a polymerized ligand monomer having "x" polymerized monomer units, where x is at least 1, and the ligand monomer has the formula (X): 【Chemical 1】 [wherein, R １ is H or CH ３ and R ２ is a (hetero)hydrocarbylene, Each R ３ is independently H or (hetero)hydrocarbyl, R １４ is H, (hetero)hydrocarbyl, or -N(R ３ ), ２ (wherein each R ３ is independently H or (hetero)hydrocarbyl). X １ is -O- or -NR ３ -(wherein R ３ is H or (hetero)hydrocarbyl). n is 1 or 2], -(M ｃ ) ｙ represents a polymerized cross-linked monomer having y polymerized monomer units, where y may be 0 or at least 1, -(M ｄ ) ｚ represents a polymerized hydrophilic monomer having z polymerized monomer units, where z may be 0 or at least 1], the flow-through process according to claim 6.

9. The flow-through process according to any one of claims 1 to 8, wherein the salt-tolerant anion exchange non-fibrous porous filter element comprises a porous membrane containing an immobilized anion exchange ligand.

10. The flow-through process according to claim 9, wherein the anion exchange ligand of the salt-tolerant anion exchange non-fibrous porous filter element comprises a cationic nitrogen-containing ligand.

11. The flow-through process according to claim 10, wherein the cationic nitrogen-containing ligand of the salt-tolerant anion exchange non-fibrous porous filter element comprises a guanidinyl-containing ligand.

12. The salt-tolerant anion exchange non-fibrous porous filter element comprises a non-fibrous porous filter element and a ligand-functional polymer grafted thereto, and the grafted ligand-functional polymer has the formula: -(M ＰＩ ) ｗ -(M ｂ ) ｘ -(M ｃ ) ｙ -(M ｄ ) ｚ , [wherein, -(M ＰＩ ) ｗ - represents the residue of the grafted photoinitiator monomer, and w is 0 or at least 1, -(M ｂ ) ｘ represents a polymerized ligand monomer having "x" polymerized monomer units, where x is at least 1, and the ligand monomer has the formula (X): 【Chemistry 2】 [wherein, R １ is H or CH ３ and R ２ is a (hetero)hydrocarbylene, Each R ３ is, independently, H or (hetero)hydrocarbyl, R １４ is H, (hetero)hydrocarbyl, or -N(R ３ ), ２ where each R ３ is independently H or (hetero)hydrocarbyl), X １ is -O- or -NR ３ -(wherein R ３ is H or (hetero)hydrocarbyl). n is 1 or 2], -(M ｃ ) ｙ represents a polymerized crosslinked monomer having y polymerized monomer units, where y may be 0 or at least 1, -(M ｄ ) ｚ represents a polymerized hydrophilic monomer having z polymerized monomer units, where z may be 0 or at least 1], the flow-through process according to claim 9.

13. The cation exchange non-fibrous porous filter element comprises a non-fibrous porous substrate, a polymer disposed on the porous non-fibrous membrane, the polymer comprising a hydrocarbon main chain and a plurality of pendant groups bonded to the hydrocarbon main chain, and each of the first plurality of pendant groups comprises at least one acidic group or a salt thereof, and a spacer group that directly links the at least one acidic group or a salt thereof to the hydrocarbon main chain by a chain of at least six atoms linked in series, The flow-through process according to any one of claims 1 to 12. The polymer covalently bonded to the non-fibrous porous filter element is a copolymer covalently bonded to the non-fibrous porous filter element, at least one ethylenically unsaturated group, at least one acidic group or a salt thereof, a spacer group that directly connects the at least one ethylenically unsaturated group and the at least one acidic group or a salt thereof by a chain of at least six atoms connected in series, a first monomer containing, at least one ethylenically unsaturated group, at least one acidic group or a salt thereof, a spacer group that directly connects the at least one ethylenically unsaturated group and the at least one acidic group by a chain of at least six atoms connected in series, a second monomer containing, a reaction product of a monomer composition containing, wherein the second monomer is different from the first monomer, The flow-through process according to claim 13, wherein the molar ratio of the first monomer to the second monomer is in the range of 95:5 to 5:

95.

15. A filter cartridge comprising a salt-resistant anion exchange non-fibrous porous filter element and a cation exchange non-fibrous porous filter element, wherein the salt-resistant anion exchange non-fibrous porous filter element is a non-fibrous porous filter element containing an immobilized cationic nitrogen-containing ligand, or a non-fibrous porous filter element and a ligand-functional polymer grafted thereto, wherein the grafted ligand-functional polymer has the formula: -(M ＰＩ ) ｗ -(M ｂ ) ｘ -(M ｃ ) ｙ -(M ｄ ) ｚ 、 [wherein, -(M ＰＩ ) ｗ - represents the residue of the grafted photoinitiator monomer, w is 0 or at least 1, -(M ｂ ) ｘ represents a polymerized ligand monomer having "x" polymerized monomer units, where x is at least 1, and the ligand monomer has the formula (X): [Chemical 3] [wherein, R １ is H or CH ３ and R ２ is a (hetero)hydrocarbylene, Each R ３ is, independently, H or (hetero)hydrocarbyl, R １４ is H, (hetero)hydrocarbyl, or -N(R ３ ), ２ where each R ３ is independently H or (hetero)hydrocarbyl), X １ is -O- or -NR ３ -(wherein R ３ is H or (hetero)hydrocarbyl). n is 1 or 2], - (M ｃ ) ｙ represents a polymerized crosslinking monomer having y polymerized monomer units, where y may be 0 or at least 1; -(M ｄ ) ｚ represents a polymerized hydrophilic monomer having z polymerized monomer units, where z may be 0 or at least 1], and wherein the cation exchange non-fibrous porous filter element is a non-fibrous porous filter element, and a polymer disposed on the non-fibrous porous filter element, a polymer containing a hydrocarbon main chain and a plurality of pendant groups bonded to the hydrocarbon main chain, and each of the first plurality of pendant groups contains at least one acidic group or a salt thereof, and a spacer group that directly connects the at least one acidic group or a salt thereof to the hydrocarbon main chain by a chain of at least six atoms connected in series. A filter cartridge.

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