Flow method and apparatus for purifying target molecules

Abstract

The present invention provides a flow method for purifying a target molecule (e.g., an antibody, enzyme, or hormone, particularly a monoclonal antibody) from a biological solution (e.g., a neutralized virus-inactivated mixture sample) containing the target molecule, and an apparatus for performing such a method.

Description

Background Technology

[0001] One of the most challenging areas of biopharmaceutical drug production is the filtration / purification of monoclonal antibodies, which are important for therapeutic use in a variety of diseases, including rheumatoid arthritis, Crohn's disease, hypercholesterolemia, and various cancers. Traditional downstream processing of therapeutic antibodies consists of numerous steps. Purification protocols involve flow-through and bind-elution chromatography. Many of these methods require buffer exchange, which enhances the reduction of impurities such as DNA, HCP, and target protein aggregates (HMWs) while attempting to mitigate product loss. For example, ion exchange chromatography methods typically operate in bind-elution mode and often require buffer exchange, such as pH and salt adjustment. Condensing these chromatographic steps into multifunctional ion exchange or mixing devices by reducing or eliminating buffer exchange steps would reduce processing time and materials during protein purification. The biopharmaceutical industry is increasingly interested in identifying continuous processing protocols, particularly flow-through methods, to help reduce processing time and steps. Summary of the Invention

[0002] This disclosure provides a flow method for purifying a target molecule from a biological solution in a sample containing the target molecule, and an apparatus for performing such a method.

[0003] In one embodiment, a flow-through method for purifying a target molecule from a biological solution in a sample includes: optionally, 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 then immediately contacting the sample with a cation exchange non-fibrous porous filter element (e.g., a membrane); wherein the flow-through method includes one or two buffer exchanges, and there is no buffer exchange between the sample contacting the salt-tolerant anion exchange non-fibrous porous filter element and the cation exchange non-fibrous porous filter element.

[0004] In another embodiment, a flow-through method 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 method does not include buffer exchange.

[0005] In yet another embodiment, a flow method for purifying a target molecule from a biological solution in a sample includes: 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 with the sample; 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.

[0006] In another embodiment, a flow method for purifying a target molecule from a biological solution in a sample includes: buffer exchange with the sample; 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.

[0007] In another embodiment, this disclosure provides a filter cartridge comprising a salt-resistant anion exchange non-fibrous porous filter element as described herein and a cation exchange non-fibrous porous filter element as described herein, preferably located downstream of the salt-resistant filter element.

[0008] As used herein, "alkyl" means a monovalent group that is an alkane group and includes straight-chain, branched, cyclic, and bicyclic alkyl groups, as well as combinations thereof, including both unsubstituted and substituted alkyl groups. Unless otherwise specified, 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, tert-butyl, isopropyl, n-octyl, n-heptyl, ethylhexyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, norbornyl, and the like.

[0009] The term "alkylene" refers to a divalent group that is a free radical of an alkane and includes straight-chain groups, branched groups, cyclic groups, bicyclic groups, or combinations thereof. Unless otherwise specified, alkylene groups typically have 1 to 30 carbon atoms. In some embodiments, the alkylene group has 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, the alkylene is a straight-chain saturated divalent hydrocarbon having 1 to 12 carbon atoms, and in some embodiments, the 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 has a carbon ring. An aryl group has at least one aromatic ring. Any additional rings may be unsaturated, partially saturated, saturated, or aromatic. Optionally, the aromatic ring may have one or more additional carbon rings fused to the aromatic ring. Unless otherwise specified, an aryl group typically contains 6 to 30 carbon atoms. In some embodiments, the aryl group contains 6 to 20 carbon atoms, 6 to 18 carbon atoms, 6 to 16 carbon atoms, 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Examples of aryl groups include phenyl, tolyl, benzyl, phenethyl, naphthyl, 2-naphthylethyl, biphenyl, phenanthrene, and anthracene.

[0011] "Hydrocarbon group" includes aryl and alkyl groups. "Hydrocarbon subunit" includes aryl subunit and alkyl subunit.

[0012] "(Hetero)alkyl" includes alkyl (alkyl and aryl) groups and heteroalkyl (heteroalkyl and heteroaryl) groups. A heteroalkyl group includes one or more in-chain (inter-chain) heteroatoms, or one or more substituents containing heteroatoms such as oxygen, sulfur, or nitrogen atoms. The heteroalkyl group may optionally include one or more in-chain (inter-chain) functional groups, including ester, amide, urea, carbamate, and carbonate functional groups. Unless otherwise specified, non-polymeric (hetero)alkyl 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. Examples of such heteroalkyl groups used herein include, but are not limited to, methoxyethyl, ethoxypropyl, propoxyethyl, 4-diphenylaminobutyl, 2-(2'-phenoxyethoxy)ethyl, 3,6-dioxaheptyl, 3,6-dioxahexyl-6-phenyl, 2-imidazolyl, 3-furanyl, and 3-indolemethyl.

[0013] "(Hetero)hydrocarbon subunit" includes hydrocarbon subunit (alkane and aromatic subunit) groups and heterohydrocarbon subunit (heteroalkyl and heteroaromatic subunit) groups. A heterohydrocarbon subunit group includes one or more in-chain (inter-chain) heteroatoms, or one or more substituents including heteroatoms such as oxygen, sulfur, or nitrogen atoms. The heterohydrocarbon subunit group may optionally contain one or more in-chain (inter-chain) functional groups, including ester, amide, urea, carbamate, and carbonate functional groups. Unless otherwise specified, non-polymeric (hetero)hydrocarbon subunit 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. Examples of such heteroalkyl subunits used herein include, but are not limited to, oxadiethyl subunit, 3-thiobutane subunit, 3-diphenylaminobutane subunit, 2-(2'-phenoxyethyl)ethyl subunit, 3,6-dioxaoctane subunit, 3,6-dioxahexyl-6-phenylene subunit, 2,5-furan subunit, 2,6-pyridinium subunit (also known as 2,6-pyridindiyl) and 2,5-thiophene dimethyl subunit.

[0014] The term "olefinic unsaturated group" refers to those groups that are free radical polymerizable and have carbon-carbon double (or triple) bonds, and includes (meth)acrylamide, (meth)acrylate, vinyl and ethoxy groups, allyl and allyloxy groups, and alkynyl groups.

[0015] The terms "polymer" and "polymer material" include, but are not limited to, organic homopolymers, copolymers such as, for example, block, graft, random and alternating copolymers, trimers, etc., and blends and modifications thereof. Furthermore, unless otherwise expressly limited, the term "polymer" shall include all possible geometries of the material. These geometries include, but are not limited to, isotactic, syndiotactic, and atactic symmetries.

[0016] In this document, the term "comprising" and its variations are not intended to be limiting wherever they appear in the specification and claims. Such terms are to be understood as implying the inclusion of the stated steps or elements or groups of steps or elements, but not excluding any other steps or elements or groups of steps or elements. The phrase "consisting of..." means including and limited to what follows the phrase "consisting of...". Thus, the phrase "consisting of..." indicates that the listed elements are required or mandatory, and that no other elements may be present. The phrase "substantially consisting of..." means including any elements listed after this phrase, and is limited to other elements that do not impede or contribute to the activity or effect specified for the listed elements in this disclosure. Thus, the phrase "substantially consisting of..." indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present depending on whether they substantially affect the activity or effect of the listed elements. Any element or combination of elements referenced in this specification in open language (e.g., including and its derivatives) is considered to be referenced in closed language (e.g., consisting of and its derivatives) and additionally in partially closed language (e.g., substantially consisting of and its derivatives).

[0017] The terms "preferred" and "ideally" refer to embodiments of this disclosure that may provide certain beneficial effects in certain circumstances. However, other claims may also be preferred in the same or other circumstances. Furthermore, the formulation of one or more preferred claims does not imply that other claims are unusable, nor is it intended to exclude other claims from the scope of this disclosure.

[0018] In this application, terms such as “a,” “an,” and “the” are not intended to refer only to a single entity, but to encompass general categories, with specific examples provided for illustration. The terms “a,” “an,” “the,” and “the” are used interchangeably with the term “at least one.” The phrases “at least one of…” and “containing at least one of…” followed by a list refer to any item in the list and any combination of two or more items in the list.

[0019] As used herein, the term “or” is generally used in its usual sense, including “and / or”, unless the context clearly indicates otherwise.

[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, throughout this document, all numerical values ​​are assumed to be modified by the term “about,” and in some embodiments preferably by the term “precisely.” As used herein, with respect to the quantity measured, the term “about” refers to a deviation in the quantity measured that is commensurate with the target of the measurement and the accuracy of the measuring equipment used, as a technician who would expect to perform the measurement with a certain degree of care. Throughout this document, “at most” a number (e.g., at most 50) includes that number (e.g., 50).

[0022] In addition, in this article, the numerical range expressed by endpoints includes all numbers contained in the range as well as endpoint values ​​(e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0023] As used in this article, the term "room temperature" refers to a temperature between 20°C and 25°C or between 22°C and 25°C.

[0024] The terms “in the range” or “within the scope” (and similar expressions) include the endpoints of the range.

[0025] The grouping of alternative elements or embodiments disclosed herein should not be construed as restrictive. Each member of a group may be individually referenced and protected by the claims or in any combination with other members of the group or other elements found therein. One or more members of a group may be included in or removed from the group for convenience and / or patentability reasons. In the event of any such inclusion or removal, the specification herein is deemed to contain the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0026] When a group appears more than once in the formula described herein, each group is chosen "independently," whether explicitly stated or not. For example, when there is more than one Y group in the formula, each Y group is chosen independently. Furthermore, the subgroups contained within these groups are also chosen independently. For example, when each Y group contains R, each R is also chosen independently.

[0027] Throughout this specification, references to "an embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., mean that a specific feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Therefore, such phrases appearing throughout this specification do not necessarily refer to the same embodiment of the invention. Furthermore, specific features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0028] The above description of the invention is not intended to describe every disclosed embodiment or every implementation of the invention. The following description illustrates exemplary embodiments in more detail. Guidance is provided throughout this application by a list of examples, which may be used in various combinations. In each case, the cited list is used only as a representative group and should not be construed as an exclusive list. Therefore, the scope of this disclosure should not be limited to the specific illustrative structures described herein, but should extend at least to the structures described by the language of the claims and their equivalents. Any element positively referenced as an alternative in this specification may be expressly included in or excluded from the claims in any combination as desired. While various theories and possible mechanisms may have been discussed herein, such discussion should in no way be used to limit the subject matter protected by the claims. Attached Figure Description

[0029] Figure 1 (AD) is a flowchart illustrating various flow methods for purifying target molecules from biological solutions as described herein.

[0030] Figure 2 (AC) is a schematic diagram of a representative filter cartridge device used for various flow methods to purify target molecules from biological solutions as described herein. Detailed Implementation

[0031] This disclosure provides a flow method for purifying a target molecule from a biological solution in a sample containing the target molecule, and an apparatus for performing such a method.

[0032] In some embodiments, the target molecule includes a therapeutic agent. In some embodiments, the target molecule includes a viral vector, proteins such as antibodies and enzymes, and hormones. In some embodiments, the target molecule includes a monoclonal antibody.

[0033] In some embodiments, the biosolution comprises a neutralized virus-inactivated mixture sample. The virus-inactivated mixture sample is a biosolution consisting of a protein A chromatographic eluent or a combination of several protein A eluents, which is then subjected to a virus inactivation process involving acid titration followed by holding at an appropriate holding pH (typically pH ≤ 3.5) for a period of time.

[0034] In one embodiment, this disclosure provides a flow method for purifying a target molecule from a biological solution in a sample. For example... Figure 1As shown in A, the method includes: optionally, contacting the sample (of a biological solution) with an anion exchange adsorption depth filter (AEX adsorption depth filter); 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 (salt-tolerant AEX FE); and then immediately contacting the sample with a cation exchange non-fibrous porous filter element (CEX FE) (e.g., a membrane); wherein the flow method includes one or two buffer exchanges, and there is no buffer exchange between the sample contacting the salt-tolerant anion exchange non-fibrous porous filter element (e.g., a membrane) and the cation exchange non-fibrous porous filter element.

[0035] Non-fibrous porous filter elements (e.g., membranes) and fibrous media allow for high flow rates and good adsorption capacity, but differ in resolution. For example, the smaller pore size and better control over pore size distribution of non-fibrous porous filter elements provide better control over the separation of impurities, especially biomolecules. Additionally, non-fibrous porous filter elements offer higher ion exchange capacity due to their higher surface area. Therefore, non-fibrous porous filter elements can offer advantages over fibrous media at certain points in flow-through purification methods.

[0036] In the context of this disclosure, “then immediately” means that no other separation step / medium is used between the two media.

[0037] In the context of this disclosure, “circulation” means that the target molecule passes through all filters.

[0038] The use of buffer exchange is advantageous in optimizing the interaction between impurities and the membrane, and provides higher target yields with compositions having low impurity levels; however, reducing the amount of buffer exchange can reduce processing time and cost.

[0039] In this context, "exchange" does not require alteration of the buffer solution. While buffer exchange may include completely changing the buffer solution (i.e., changing one buffer solution to another) by known methods (e.g., tangential flow filtration or cross-flow filtration), "exchange" may also include altering the buffer solution by, for example, changing the pH, changing the conductivity, and / or diluting the sample of interest. This may also be referred to as buffer change or buffer adjustment.

[0040] A "buffer solution" is a buffer solution that resists pH changes through the action of organic acid-base conjugate components.

[0041] In one embodiment, the organic acids (in the 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).

[0042] In one embodiment, the organic base (in the buffer solution) includes, 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-piperazine ethanesulfonic acid), TAPS (3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), and Tricine (N-tris(hydroxymethyl)methylglycine).

[0043] In one embodiment, the conjugate base of the organic acid is a 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 a sodium salt.

[0044] Typically, buffer solutions include equilibration buffers, loading buffers, and elution buffers. In this article, "equilibration buffer" refers to a buffer used to prepare the solid phase for chromatography. "Loading buffer" is a buffer used to load a mixture of proteins and contaminants onto the chromatographic matrix. The equilibration buffer and loading buffer can be the same. "Elution buffer" is used to elute proteins from the chromatographic matrix.

[0045] In some embodiments, the flow method includes: contacting the sample (of a biological solution) with an anion exchange adsorption depth filter; optionally, before and / or after contacting the sample with the anion exchange adsorption depth filter, performing a buffer exchange with the sample; 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.

[0046] In some embodiments, the flow method includes: contacting the 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 then immediately contacting the sample with a cation exchange non-fibrous porous filter element.

[0047] In some embodiments, the flow method includes: contacting the sample (of a biological solution) with an anion exchange adsorption depth filter; after contacting the sample with the anion exchange adsorption depth filter, performing a buffer exchange with the sample; 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.

[0048] In some embodiments, the flow method includes: contacting the sample with an anion exchange adsorption depth filter; performing a buffer exchange with the sample before 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 then immediately contacting the sample with a cation exchange non-fibrous porous filter element.

[0049] In some embodiments, the flow method includes: contacting the 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-tolerant anion exchange non-fibrous porous filter element; and then immediately contacting the sample with a cation exchange non-fibrous porous filter element.

[0050] In some embodiments, the flow method includes: after contacting the sample with a cation-exchange non-fibrous porous filter element, performing a buffer exchange with the sample.

[0051] In some implementations, such as Figure 1 As shown in B, the flow method includes: contacting the sample (of a biological solution) with an anion exchange adsorption depth filter (AEX adsorption depth filter); then immediately contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element (Salt-tolerant AEX FE); and then immediately contacting the sample with a cation exchange non-fibrous porous filter element (CEX FE); wherein the flow method does not include buffer exchange.

[0052] In some implementations, such as Figure 1 As shown in C, the flow method includes: contacting the sample (of a biological solution) with an anion exchange adsorption depth filter (AEX adsorption depth filter); after contacting the sample with the anion exchange adsorption depth filter, performing buffer exchange with the sample; contacting the sample with a salt-tolerant anion exchange non-fibrous porous filter element (salt-tolerant AEX FE); and then immediately contacting the sample with a cation exchange non-fibrous porous filter element (CEX FE).

[0053] In some implementations, such as Figure 1As shown in D, the flow method includes: 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 then immediately contacting the sample with a cation exchange non-fibrous porous filter element (CEX FE).

[0054] Optional anion exchange adsorption depth filtration element

[0055] A depth filter (i.e., a filter element) is a porous material that allows particles (i.e., microparticles) in a sample, such as a virus-inactivated mixture, to permeate and subsequently be trapped therein. Specifically, a depth filter traps contaminants within the sample between the upstream and downstream surfaces of the filter substrate.

[0056] Exemplary depth filters include a porous base substrate and a graft copolymer comprising interpolymerized cationic nitrogen-containing ligand monomers (i.e., ligands) as disclosed in U.S. Patent 9,821,276 (Berrigan et al.) or graft ligand-functionalized polymers as disclosed in U.S. Patent 8,846,203 (Bothof et al.).

[0057] Porous foundation substrate. The porous substrate (i.e., the base substrate) of the depth filter can be a porous membrane, a porous nonwoven fiber web, or a porous fiber substrate.

[0058] In some embodiments, it is a porous nonwoven fiber web. As used herein, the terms “nonwoven web fiber” or “nonwoven substrate” are used interchangeably and refer to a fabric having a structure in which monofibers or filaments are randomly and / or unidirectionally inserted in a felt-like manner.

[0059] Nonwoven fiber webs can be prepared using techniques such as carding, air spinning, jet spinning, spunbond, meltblown, or combinations thereof. Spunbond fibers are typically small-diameter fibers formed by extruding molten thermoplastic polymers as filaments through multiple fine, typically circular capillaries via a spinneret, where the diameter of the extruded fibers rapidly decreases. Meltblown fibers are typically formed by extruding molten thermoplastic material as molten wires or filaments into a high-speed, typically heated gas (e.g., air) stream through multiple fine, typically circular molding capillaries, which refines the filaments of the molten thermoplastic material to reduce their diameter. The meltblown fibers are then transported by the high-speed gas stream and deposited on a collecting surface to form randomly distributed meltblown fibers. Any nonwoven web can be made from a single type of fiber or from two or more fibers that differ in type and / or thickness of thermoplastic polymer.

[0060] Suitable nonwoven substrates for depth filters can be spunbonded, hydroentangled, or meltblown. In some embodiments, they may have a tensile strength of at least 4.0 Newtons prior to grafting, with 15 to 50 m² of nonwoven substrate per square meter. 2 The surface area, average pore size of 1 micrometer to 40 micrometers according to ASTM F 316-03, and density of less than 20%.

[0061] The porous substrate can be made of any suitable thermoplastic polymer material. Suitable polymer materials include, but are not limited to, polyolefins, poly(isoprene), poly(butadiene), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfone), 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).

[0062] Suitable polyolefins include, but are not limited to, poly(ethylene), poly(propylene), poly(1-butene), copolymers of ethylene and propylene, α-olefin copolymers (such as copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene and 1-decene), poly(ethylene-co-1-butene), and poly(ethylene-co-1-butene-co-1-hexene).

[0063] Suitable fluorinated polymers include, but are not limited to, poly(vinylidene fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (such as poly(vinylidene fluoride-co-hexafluoropropylene)), and copolymers of trifluorochloroethylene (such as poly(ethylene-co-trifluorochloroethylene)).

[0064] Suitable polyamides include, but are not limited to, poly(iminoadiminohexamethylene), poly(iminoadiminodecamethylene), and polycaprolactam. Suitable polyimides include, but are not limited to, poly(pyromellitictetramide).

[0065] Suitable poly(ether sulfone) includes, but is not limited to, poly(diphenyl ether sulfone) and poly(diphenyl sulfone-co-diphenyl sulfone oxide).

[0066] Suitable vinyl acetate copolymers include, but are not limited to, poly(ethylene-co-vinyl acetate) and those copolymers in which at least some of the acetate groups have been hydrolyzed to provide a variety of poly(vinyl alcohols).

[0067] In some embodiments, the porous base substrate is formed of propylene homopolymer or copolymer, most preferably propylene homopolymer.

[0068] Fixed cationic nitrogen-containing ligands.In some embodiments, the porous substrate of the depth filter includes a graft copolymer comprising interpolymerized cationic nitrogen-containing ligand monomers (i.e., ligands), as disclosed in U.S. Patent 9,821,276 (Berrigan et al.). In other words, the porous substrate of the depth filter comprises a copolymer grafted onto the porous substrate (thereby forming a copolymer-grafted article), wherein the graft copolymer comprises interpolymerized monomer units containing cationic nitrogen-containing ligands.

[0069] In some embodiments, the anion exchange ligand comprises a cationic nitrogen-containing ligand. In some embodiments, the cationic nitrogen-containing ligand comprises a primary amine, a secondary amine, a tertiary amine, or a combination thereof. In some embodiments, the cationic nitrogen-containing ligand comprises a quaternary ammonium-containing ligand, a guanidinium-containing ligand, or a combination thereof.

[0070] In some embodiments, the graft copolymer includes interpolymer monomer units, said interpolymer monomer units comprising: cationic nitrogen-containing ligand monomers selected from the group consisting of quaternary ammonium ligand monomers, guanidine ligand monomers, and combinations thereof; amide monomers; oxomonomers selected from the group consisting of epoxy functional monomer units, alkyl ether functional monomer units, and combinations thereof; and poly(epoxyalkylene) monomers.

[0071] In some embodiments, the graft copolymer comprises interpolymerizable monomer units, said interpolymerizable monomer units comprising: 10 to 50 parts by weight of a cationic nitrogen-containing ligand monomer, wherein the cationic nitrogen-containing ligand monomer is selected from the group consisting of quaternary ammonium ligand monomers, guanidine ligand monomers, and combinations thereof; 10 to 80 parts by weight of an amide monomer; 10 to 40 parts by weight of an oxomonomer, 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 a poly(epoxyalkylene) monomer; wherein the total amount of monomers is 100 parts by weight.

[0072] In some embodiments, the cationic nitrogen-containing ligand monomer has formula (I):

[0073]

[0074] in:

[0075] R 1 It is H or CH3;

[0076] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0077] R 5 It is a (hetero)hydrocarbon subunit; and

[0078] R LigIt is a quaternary ammonium ligand group or a guanidine ligand group.

[0079] In some embodiments, the cationic nitrogen-containing ligand monomer is a quaternary ammonium monomer (salt) of formula (II):

[0080]

[0081] in:

[0082] R 1 It is H or CH3;

[0083] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0084] R 5 It is a (hetero)hydrocarbon subunit; and

[0085] Each R 4 It can be alkyl or aryl independently.

[0086] Counterions of quaternary ammonium salts include halides, sulfates, phosphates, nitrates, and the like. Exemplary quaternary ammonium salt monomers include (meth)acrylamidoalkyltrimethylammonium salt and (meth)acryloyloxyalkyltrimethylammonium salt, described in U.S. Patent 9,821,276 (Berrigan et al.).

[0087] In some embodiments, the cationic nitrogen-containing ligand monomer is a guanidine-containing ligand monomer of formula (III) or (IV):

[0088]

[0089] in:

[0090] R 1 It is H or CH3;

[0091] R 2 It is a (hetero)hydrocarbon subunit (e.g., having 1 to 20 carbon atoms);

[0092] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0093] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0094] R 15 It is an H or hydrocarbon group (e.g., a C1-C4 alkyl group or an aryl group);

[0095] X1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0096] o is 0 or 1; and

[0097] n is 1 or 2.

[0098] Examples of cationic nitrogen-containing ligand monomers include guanidinobutylamine ligands (e.g., isocyanate ethyl methacrylate-guanidinobutylamine adducts), guanidinoligands, biguanide ligands, and combinations thereof, prepared as described in U.S. Patent 9,821,276 (Berrigan et al.).

[0099] In some embodiments, the copolymer-grafted article comprises a cross-polymerized amide monomer of formula (V) or (VI) ((meth)acrylamide and N-vinylamide):

[0100]

[0101] in:

[0102] R 1 It is H or CH3;

[0103] Each R 8 Independently hydrogen, alkyl, or aryl; and

[0104] R 9 and R 10 It is an alkyl group, or it can form a 5- or 6-membered ring together.

[0105] Examples of such monomers include N-vinylcaprolactam, N-vinylacetamide, N-vinylpyrrolidone, acrylamide, mono- or di-N-alkyl substituted acrylamides, and combinations thereof.

[0106] In some embodiments, the copolymer-grafted article comprises interpolymerized oxymonomers of formula (VII) (epoxy-functionalized and monoether-functionalized (meth)acrylates and (meth)acrylamides):

[0107]

[0108] in:

[0109] R 1 It is H or CH3;

[0110] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; and

[0111] R 16It is a hydrocarbon group that is epoxy-functional or ether-functional.

[0112] In some embodiments, the copolymer-grafted article comprises interpolymerized oxymonomers (epoxy monomers) of formula (VIII):

[0113]

[0114] in:

[0115] R 1 It is H or CH3;

[0116] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; and

[0117] R 7 It is a (hetero)hydrocarbon subunit (e.g., C1-C6 alkyl subunit).

[0118] Examples of such monomers include glycidyl (meth)acrylate, thioglycidyl (meth)acrylate, 3-(2,3-epoxypropoxy)phenyl (meth)acrylate, 2-[4-(2,3-epoxypropoxy)phenyl]-2-(4-(meth)acryloyloxy-phenyl)propane, 4-(2,3-epoxypropoxy)cyclohexyl (meth)acrylate, 2,3-epoxycyclohexyl (meth)acrylate, and 3,4-epoxycyclohexyl (meth)acrylate, as well as other monomers described in U.S. Patent 9,821,276 (Berrigan et al.).

[0119] As described in U.S. Patent 9,821,276 (Berrigan et al.), the above-described monomers can be used in a single reaction step or in a sequential reaction step to prepare a functionalized substrate to provide a grafted polymer on the surface of a porous substrate.

[0120] Grafted ligands-functionalized polymers. In some embodiments, the porous substrate of the depth filter includes a ligand-functionalized polymer grafted onto the porous substrate, as disclosed in U.S. Patent 8,846,203 (Bothof et al.).

[0121] In some embodiments, the grafted ligand-functional polymer has formula (IX):

[0122] -(M PI ) w -(M b ) x -(M c ) y -(M d ) z

[0123] in:

[0124] -(M PI ) w Represents the residues of the grafted photoinitiator monomer, where w is 0 or at least 1;

[0125] -(M b ) x This represents a polymeric ligand monomer having "x" polymeric monomer units, where x is at least 1;

[0126] -(M c ) y This represents a polymeric crosslinking monomer having y polymeric monomer units, where y can be 0 or at least 1; and

[0127] -(M d ) z This represents a polymeric hydrophilic monomer having z polymeric monomer units, where z can be 0 or at least 1.

[0128] In some embodiments, the functionalized substrate has graft groups attached to the surface of the base substrate, said graft groups comprising: a) optionally, at least one photoinitiator group (or its reaction product); 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.

[0129] Monomers grafted onto the surface of a base substrate typically have 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 directly grafting monomers onto the substrate surface because they are more reactive when exposed to ionizing radiation, such as electron beam radiation. Not all such acryloyl groups can be “directly grafted,” i.e., forming covalent bonds with the substrate surface. Some acryloyl groups remain free and are subsequently “indirectly grafted” by incorporation into the polymer chain upon exposure to ultraviolet radiation. Other olefinically unsaturated groups (such as methacrylamide, methacrylate, vinyl and ethoxy groups, allyl and allyloxy groups, and alkynyl groups) are less reactive during electron beam grafting and are unlikely to be directly grafted onto the base substrate. Therefore, some of these non-acryloyl groups can be directly grafted, but most remain unreacted and are indirectly grafted onto the substrate by incorporation into the polymer chain during UV-initiated polymerization.

[0130] The photoinitiator monomer “a)” can be directly grafted onto the surface of the base substrate (including the interstitial and outer surfaces of porous base substrates) to provide grafted photoinitiator groups via acryloyl groups.

[0131] The monomer of ligand “b)” may have an acryloyl group or a non-acryloyl group, such as a methacrylate group, for direct grafting, to be subsequently incorporated (indirectly grafted) into the polymer chain during UV-initiated polymerization.

[0132] When exposed to ionizing radiation, preferably electron beams or gamma radiation, the acryloyl groups of monomer “c)” can typically be directly grafted (i.e., covalently bonded) to the surface of the base substrate. In addition to the acryloyl groups, the radically polymerizable groups of monomer “c)” are typically other olefinically unsaturated groups (such as methacrylamide, methacrylate, vinyl groups, and alkynyl groups, which are low-reactivity during grafting) and are therefore free and unreacted for subsequent UV-initiated polymerization and crosslinking.

[0133] The fourth grafted hydrophilic monomer "d)" can also be grafted via an acryloyl group and can provide hydrophilic or ionic groups to the substrate surface. In some embodiments, the hydrophilic monomer having ionic groups can be grafted directly or indirectly onto the substrate surface to provide a second ionic interaction to the functionalized substrate.

[0134] Grafted photoinitiator monomer (M) PI This includes an acryloyl group and a photoinitiator group, which can be a hydrogen-abstracting or α-cleavage type photoinitiator group. Such grafted photoinitiator monomers (M...) PI It is disclosed in U.S. Patent 8,846,203 (Bothof et al.).

[0135] In some implementations, the ligand monomer (M b It has the following formula (X):

[0136]

[0137] in:

[0138] R 1 It is H or CH3;

[0139] R 2 It is a (hetero)hydrocarbon subunit;

[0140] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0141] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0142] X 1 -O- or -NR 3 -, where R 3It is an H or (hetero)hydrocarbon group, and

[0143] n is 1 or 2.

[0144] In some implementations, the ligand monomer (M b It has the formula (XIV):

[0145]

[0146] in:

[0147] R 1 It is H or CH3;

[0148] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0149] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0150] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0151] R 6 and R 7 Each is independently a (hetero)hydrocarbon subunit (e.g., C1-C). 10 Alkyl subunit);

[0152] Z 2 It is an ester, amide, urea, or carbamate group, and

[0153] n is 1 or 2.

[0154] This type of ligand monomer (M b It can be prepared using a condensation reaction as described in U.S. Patent 8,846,203 (Bothof et al.).

[0155] In some embodiments, the grafted ligand-functional polymer further comprises a crosslinking monomer (M c (y is at least 1), which has two or more radically polymerizable groups. In some embodiments, the crosslinking monomer (M) c It has the formula (XI):

[0156]

[0157] in:

[0158] Z 1 It is an unsaturated polymerizable group of an acrylate or non-acryloyl olefin.

[0159] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0160] Q is selected from covalent bonds, -O-, and -NR. 1 -, -CO2- and -C(O)NR 1 - a divalent linker, wherein R 1 It is H or CH3;

[0161] R 11 It is an alkylene group with a valence of a+b, and optionally contains one or more oxygen atoms in the chain and / or one or more hydroxyl groups; and

[0162] a and b are each at least 1.

[0163] In some implementations, the crosslinking monomer (M c The compound comprises a poly(epoxide) compound having at least one acryloyl group and at least one additional olefinically unsaturated, free-radical polymerizable group. In some embodiments, the crosslinking monomer (M... c It has the formula (XII):

[0164]

[0165] in:

[0166] Z 1 It is a polymerizable olefinic unsaturated group, either acryloyl or non-acryloyl.

[0167] R 1 It is H or CH3;

[0168] m ranges from 2 to 100; and

[0169] Q is selected from covalent bonds, -O-, and -NR. 1 -, -CO2- and -C(O)NR 1 - a divalent linker, wherein R 1 It is H or CH3;

[0170] Suitable crosslinking (M) c Examples of monomers include poly(ethylene oxide) polymers and copolymers (such as di(meth)acrylates of poly(ethylene oxide-co-propylene oxide) copolymers) and partially acrylated polyols (such as 3-(acryloyloxy)-2-hydroxypropyl methacrylate).

[0171] In some 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 may also include positively charged, negatively charged, or neutral ionic groups. In some embodiments, the hydrophilic monomer (M) d ) is the neutral monomer of formula (XIII):

[0172]

[0173] in:

[0174] Each R 1 Independently, it can be H or CH3;

[0175] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; and

[0176] t ranges from 2 to 100.

[0177] Suitable hydrophilicity (M) d Examples of monomers include poly(epoxy) monomers.

[0178] Anion exchange depth filters can be made using standard techniques, such as those described, for example, in U.S. Patents 9,821,276 (Berrigan et al.) and 8,846,203 (Bothof et al.).

[0179] Salt-resistant anion exchange non-fiber porous filter element

[0180] Although salt-resistant anion exchange non-fibrous porous filter elements are preferably porous membranes, they are not depth filters, and their primary purpose is not particle removal. Exemplary salt-resistant anion exchange non-fibrous porous filter elements comprise a non-fibrous base substrate and a graft copolymer containing interpolymerized cationic nitrogen-containing ligand monomers (i.e., ligands) as disclosed in U.S. Patent 9,821,276 (Berrigan et al.), or graft ligand-functionalized polymers as disclosed in U.S. Patent 8,846,203 (Bothof et al.), and are used as described in the aforementioned depth filters.

[0181] Salt-resistant nitrogen-containing ligands typically comprise a primary amine or guanidine group, wherein the guanidine-containing ligand is more salt-resistant than the primary amine. Secondary amines, tertiary amines, and quaternary ammonium groups are generally not salt-resistant as defined herein. In some embodiments, the salt-resistant ligand is a cationic nitrogen-containing ligand monomer comprising guanidine-containing ligand monomers of formulas (III) and (IV) (described above), and monomers of formula (XIV) (described above) prepared, for example, as described in U.S. Patents 10,239,828 (Rasmussen et al.) and 8,846,203 (Bothof et al.).

[0182] Salt-resistant anion exchange non-fibrous porous filter elements are useful under conditions of high salt concentration or high ionic strength; that is, they are “salt-resistant.” The term “salt” is intended to include all low molecular weight ions that contribute to the conductivity of the solution. Salt resistance is important, at least because many process solutions used in the manufacture of biopharmaceuticals or enzymes have conductivity in the range of 15 mS / cm–30 mS / cm (approximately 150 mM–300 mM salt) or greater. Salt resistance can be measured by comparison with the salt resistance of conventional quaternary ammonium or Quat ligands (e.g., trimethylammonium ligands), which rapidly degrade at conductivitys three to six times lower than the target range due to their primary electrostatic interactions with many biomolecules. For example, membranes derivatized with conventional Quat ligands exhibit significantly lower conductivity as NaCl increases from 0 to 50 mM (approximately 5 mS / cm–6 mS / cm). The logarithmic decrease in viral clearance was reduced from six (LRV) to one (1) LRV. Viruses with an isoelectric point close to 7 (neutral or near-neutral), such as It is extremely difficult to remove from the processing fluid stream. Similar problems have been observed when attempting to remove other biological material from the processing fluid stream. For example, when attempting to remove positively charged proteins such as host cell proteins using a filter device functionalized with conventional Quat ligands, the processing fluid stream may have to be diluted two-fold or more to reduce the conductivity to an acceptable level. This is costly and significantly increases the overall processing time.

[0183] Non-fibrous porous filter elements offer superior impurity removal capabilities compared to fibrous porous substrates for salt-tolerant membranes due to the higher membrane surface area and grafting density of achievable ligand-functionalized polymers. Furthermore, the smaller pore size and better control over pore size distribution of non-fibrous porous filter elements provide better control over the separation of impurities, particularly biomolecules. Therefore, in flow-through purification methods, non-fibrous porous filter elements offer advantages over fibrous media in salt-tolerant membranes.

[0184] The base substrate of the non-fibrous porous filter element can be formed from any suitable thermoplastic polymer material as described above for depth filters.

[0185] In some embodiments, the porous substrate is a microporous membrane, such as a thermally induced phase separation (TIPS) membrane. TIPS films are often prepared by forming a homogeneous solution of a thermoplastic material and a second material with a melting point greater than that of the thermoplastic material. Upon cooling, the thermoplastic material crystallizes and undergoes phase separation with the second material. The crystallized thermoplastic material is typically stretched. Optionally, the second material is removed before or after stretching. Microporous membranes are also 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). Alternatively, the microporous membrane can be prepared from an ethylene-vinyl alcohol copolymer as described in U.S. Patent 5,962,544 (Waller).

[0186] Some exemplary TIPS films include poly(vinylidene fluoride) (PVDF), polyolefins (e.g., polyethylene homopolymers or copolymers or polypropylene homopolymers or copolymers), vinyl-containing polymers or copolymers (e.g., ethylene-vinyl alcohol copolymers), and butadiene-containing polymers or copolymers, as well as acrylate-containing polymers or copolymers. TIPS films containing PVDF are also described in U.S. Patent 7,338,692 (Smith et al.).

[0187] In another exemplary embodiment, the porous substrate is a microporous membrane, such as a solvent-induced phase separation (SIPS) membrane. SIPS membranes are typically prepared by forming a homogeneous solution of a thermoplastic material and a second material (solvent), casting the solution into the form of a membrane or hollow fiber, and then immersing it in a non-solvent bath. The non-solvent causes the thermoplastic material to solidify, or undergo phase separation, and also extracts the solvent, leaving a porous polymer membrane. Examples of SIPS membranes made from polyamides include nylon microporous membranes or sheets, such as those described in U.S. Patents 6,056,529 (Meyering et al.), 6,267,916 (Meyering et al.), 6,413,070 (Meyering et al.), 6,776,940 (Meyering et al.), 3,876,738 (Marinacchio et al.), 3,928,517 (Knight et al.), 4,707,265 (Knight et al.), and 5,458,782 (Hou et al.). Other examples include microporous membranes made of polysulfone and polyethersulfone, many of which are commercially available from 3M Company, St. Paul, MN, under the trade names MicroPES and DuraPES.

[0188] Salt-resistant membrane filters can be manufactured using standard techniques, such as those described, for example, in U.S. Patents 9,821,276 (Berrigan et al.), 8,846,203 (Bothof et al.), and 10,239,828 (Rasmussen et al.).

[0189] Cation exchange non-fiber porous filter element

[0190] Cation exchange non-fibrous porous filter elements comprise a non-fibrous base substrate as disclosed above for salt-tolerant anion exchange non-fibrous porous filter elements. This substrate can be multimodal or hybrid. Multimodal or hybrid means that the filter element interacts with its target material through cation exchange and at least one other interaction mode (e.g., hydrophobic interaction or hydrogen bonding).

[0191] In some embodiments, the cation exchange non-fibrous porous filter element includes: a non-fibrous porous filter element; and a polymer disposed on the non-fibrous porous filter element, the polymer comprising: a hydrocarbon backbone and a plurality of side groups connected to the hydrocarbon backbone, wherein each of the first plurality of side groups comprises: at least one acid group or a salt thereof; and a spacer group that directly connects at least one acid group or a salt thereof to the hydrocarbon backbone via a chain of at least six atoms in the chain.

[0192] In some embodiments, the spacer group comprises a chain having at least eight in-chain atoms. In some embodiments, the spacer group is a hydrocarbon group containing heteroatoms in the chain. In some 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 some embodiments, the spacer group comprises at least two hydrogen-bonding moietyes or comprises at least one hydrogen-bonding moiety and at least one hydrogen bond acceptor different from the hydrogen-bonding moiety (not part of the hydrogen-bonding moiety). In some embodiments, the spacer group comprises at least two hydrogen bond donors, at least two hydrogen bond acceptors, or both.

[0193] In some embodiments, at least one acidic group or a salt thereof of the polymer disposed on the non-fibrous porous filter element is present at a density of at least 0.01 (or at least 0.02) mmol / g cation exchange non-fibrous porous filter element. In some embodiments, at least one acidic group or a salt thereof of the polymer disposed on the non-fibrous porous filter element is present at a density of up to 0.6 mmol / g cation exchange non-fibrous porous filter element.

[0194] In some embodiments, at least one acidic group or a salt thereof is selected from carboxyl groups, phosphonyl groups, phosphate groups, sulfonyl groups, sulfate groups, borate groups, and combinations thereof.

[0195] In some embodiments, the polymer comprises interpolymerization units of at least one monomer, the monomer comprising: at least one olefinic unsaturated group; at least one acidic group or a salt thereof; and a spacer group, the spacer group being directly linked to at least one olefinic unsaturated group and at least one acidic group or a salt thereof by a chain of at least six atoms.

[0196] In some embodiments, at least one olefinic unsaturated group is selected from vinyl groups, 1-alkyl vinyl groups, and combinations thereof.

[0197] In some implementations, the polymer is covalently attached to the non-fibrous porous filter element.

[0198] In some embodiments, the polymer is a copolymer.

[0199] In some embodiments, the copolymer comprises a hydrocarbon backbone and a plurality of side groups attached to the hydrocarbon backbone, wherein: each of the first plurality of side groups comprises: at least one acidic group or a salt thereof; and a spacer group that directly attaches at least one acidic group or a salt thereof to the hydrocarbon backbone via a chain of at least six atoms; and each of the second plurality of side groups comprises: at least one acidic group or a salt thereof; and a spacer group that directly attaches at least one acidic group or a salt thereof to the hydrocarbon backbone via a chain of at least six atoms; wherein the first plurality of side groups are different from the second plurality of side groups; and wherein the molar ratio of the first plurality of side groups to the second plurality of side groups is in the range of 95:5 to 5:95.

[0200] In some embodiments, the copolymer covalently attached to the non-fibrous porous filter element comprises the reaction product of a monomer composition comprising: a first monomer comprising: at least one olefinic unsaturated group; at least one acidic group or a salt thereof; and a spacer group directly linked to at least one olefinic unsaturated group and at least one acidic group or a salt thereof via a chain of at least six atoms; and a second monomer comprising: at least one olefinic unsaturated group; at least one acidic group or a salt thereof; and a spacer group directly linked to at least one olefinic unsaturated group and at least one acidic group via a chain of at least six atoms; wherein the second monomer is different from the first monomer; and wherein the molar ratio of the first monomer to the second monomer is in the range of 95:5 to 5:95.

[0201] In some embodiments, at least one olefinic unsaturated group of the first monomer and / or the second monomer is selected from vinyl groups, 1-alkyl vinyl groups, and combinations thereof. In some embodiments, the first monomer is one of the categories represented by the following general formula (XV):

[0202]

[0203] in:

[0204] R 1 It is H or CH3;

[0205] Each R 2 Independently a (hetero)hydrocarbon subunit;

[0206] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0207] Z 3 It is a heterohydrocarbon subunit group, which contains at least one hydrogen bond donor, at least one hydrogen bond acceptor, or a combination thereof;

[0208] r is 0 or 1; and

[0209] L is a functional group containing at least one acidic group or a salt thereof.

[0210] 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. Hydrogen bond donors include, for example, donors such as imino, thiol, or hydroxyl groups. Hydrogen bond acceptors include acceptors in the form of, for example, carbonyl, carbonyloxy, or etheroxy groups.

[0211] In some implementations, the second monomer is also one of the categories represented by the general formula (XV).

[0212] Cation exchange membrane filters can be made using standard techniques, such as those described, for example, in U.S. Publication 2019 / 0194250 (Colak Atan et al.) and International Publication WO 2018 / 048696 (Vail et al.).

[0213] Device

[0214] In another embodiment, this 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 located downstream of the salt-resistant filter element, such as... Figure 2 As shown in Figure A. The device may also include anion exchange adsorption depth filter (AEX adsorption depth filter), preferably located upstream of the salt-resistant filter element, such as... Figure 2 As shown in B. The relative orientation of these filter elements may be as described herein with respect to the method. The device may include other conventional filter elements, such as one or more microporous membranes, such as Figure 2As shown in C.

[0215] Exemplary Implementation

[0216] Implementation Scheme 1 is a flow-through method for purifying a target molecule from a biological solution containing the target molecule, the method comprising:

[0217] Optionally, the sample is brought into contact with an anion exchange adsorption depth filter;

[0218] Optionally, the sample is buffer-exchanged before and / or after contacting the sample with the anion exchange adsorption depth filter;

[0219] The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and

[0220] Then immediately bring the sample into contact with a cation exchange non-fibrous porous filter element;

[0221] The flow method described therein includes one or two buffer exchanges, with no buffer exchange between samples in contact with the salt-resistant anion-exchange non-fibrous porous filter element and the cation-exchange non-fibrous porous filter element.

[0222] Implementation Scheme 2 is the distribution method according to Implementation Scheme 1, the distribution method comprising:

[0223] The sample is brought into contact with the anion exchange adsorption depth filter;

[0224] Optionally, the sample is buffer-exchanged before and / or after contacting the sample with the anion exchange adsorption depth filter;

[0225] The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and

[0226] The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

[0227] Implementation scheme 3 is a distribution method according to implementation scheme 2, the distribution method comprising:

[0228] The sample is brought into contact with the anion exchange adsorption depth filter;

[0229] Before and / or after contacting the sample with the anion exchange adsorption depth filter, the sample is exchanged with a buffer solution.

[0230] The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and

[0231] The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

[0232] Implementation scheme 4 is the distribution method according to implementation scheme 3, the distribution method comprising:

[0233] The sample is brought into contact with the anion exchange adsorption depth filter;

[0234] After the sample is brought into contact with the anion exchange adsorption depth filter, a buffer exchange is performed with the sample.

[0235] The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and

[0236] The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

[0237] Implementation scheme 5 is a distribution method according to implementation scheme 3, the distribution method comprising:

[0238] The sample is brought into contact with the anion exchange adsorption depth filter;

[0239] Before contacting the sample with the anion exchange adsorption depth filter, the sample is subjected to buffer exchange.

[0240] The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and

[0241] The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

[0242] Implementation Scheme 6 is a distribution method according to Implementation Scheme 3, the distribution method comprising:

[0243] The sample is brought into contact with the anion exchange adsorption depth filter;

[0244] Before and after contacting the sample with the anion exchange adsorption depth filter, the sample is exchanged with a buffer solution.

[0245] The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and

[0246] The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

[0247] Implementation scheme 7 is a flow method according to any of the foregoing implementation schemes, the flow method comprising, after contacting the sample with a cation exchange non-fibrous porous filter element, performing buffer exchange with the sample.

[0248] Implementation Scheme 8 is a flow-through method for purifying said target molecule from a biological solution containing the target molecule, the flow-through method comprising:

[0249] The sample is brought into contact with the anion exchange adsorption depth filter;

[0250] Then immediately bring the sample into contact with a salt-resistant anion exchange non-fibrous porous filter element; and

[0251] Then immediately bring the sample into contact with a cation exchange non-fibrous porous filter element;

[0252] The flow method described herein does not include buffer exchange.

[0253] Implementation Scheme 9 is a flow-through method for purifying a target molecule from a biological solution containing the target molecule, the flow-through method comprising:

[0254] The sample is brought into contact with the anion exchange adsorption depth filter;

[0255] After the sample is brought into contact with the anion exchange adsorption depth filter, a buffer exchange is performed with the sample.

[0256] The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and

[0257] The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

[0258] Implementation Scheme 10 is a flow-through method for purifying a target molecule from a biological solution containing the target molecule, the flow-through method comprising:

[0259] Buffer exchange with the sample;

[0260] The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and

[0261] The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

[0262] Implementation scheme 11 is a distribution method according to any of the foregoing implementation schemes, wherein the target molecule includes a monoclonal antibody.

[0263] Implementation scheme 12 is a circulation method according to any of the foregoing implementation schemes, wherein the biological solution comprises a neutralized virus-inactivated mixture sample.

[0264] Implementation scheme 13 is a flow method according to any one of implementation schemes 2 to 9, and as described in implementation schemes 11 to 12 which are dependent on any one of implementation schemes 2 to 9, wherein the anion exchange adsorption depth filter includes a porous substrate containing fixed anion exchange ligands.

[0265] Implementation scheme 14 is the flow method according to implementation scheme 13, wherein the anion exchange ligand of the anion exchange adsorption depth filter includes a cationic nitrogen-containing ligand.

[0266] Implementation scheme 15 is the flow method according to implementation scheme 14, wherein the cationic nitrogen-containing ligand of the anion exchange adsorption depth filter includes primary amines, secondary amines, tertiary amines, or combinations thereof.

[0267] Implementation scheme 16 is the flow method according to implementation scheme 15, wherein the cationic nitrogen-containing ligand of the anion exchange adsorption depth filter includes quaternary ammonium ligands, guanidinium ligands, or combinations thereof.

[0268] Implementation Scheme 17 is a flow method according to any one of Implementation Schemes 14 to 16, wherein the anion exchange adsorption depth filter comprises a copolymer grafted article, the copolymer grafted article comprising a porous substrate and a copolymer grafted onto the porous substrate, wherein the grafted copolymer comprises interpolymerized monomer units, the interpolymerized monomer units comprising cationic nitrogen-containing ligands.

[0269] Implementation scheme 18 is the flow method according to implementation scheme 17, wherein the graft copolymer of the anion exchange adsorption depth filter comprises interpolymerizable monomer units, the interpolymerizable monomer units comprising:

[0270] The cationic nitrogen-containing ligand monomer is selected from the group consisting of quaternary ammonium ligand monomers, guanidinium ligand monomers, and combinations thereof;

[0271] Amide monomers;

[0272] Oxymonomers, wherein the oxymonomers are selected from the group consisting of epoxy-functional monomer units, alkyl ether-functional monomer units, and combinations thereof; and

[0273] Poly(epoxy) monomers.

[0274] Implementation Scheme 19 is the flow method according to Implementation Scheme 18, wherein the graft copolymer of the anion exchange adsorption depth filter comprises interpolymerizable monomer units, the interpolymerizable monomer units comprising:

[0275] From 10 to 50 parts by weight of a cationic nitrogen-containing ligand monomer, wherein the cationic nitrogen-containing ligand monomer is selected from the group consisting of quaternary ammonium ligand monomers, guanidinium ligand monomers, and combinations thereof;

[0276] 10 to 80 parts by weight of amide monomer;

[0277] From 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; and

[0278] 0 to 30 parts by weight of poly(epoxy) monomer;

[0279] The total amount of monomers is 100 parts by weight.

[0280] Implementation scheme 20 is a flow method according to implementation scheme 18 or 19, wherein the cationic nitrogen-containing ligand monomer used to manufacture the anion exchange adsorption depth filter has formula (I):

[0281]

[0282] in:

[0283] R 1 It is H or CH3;

[0284] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0285] R 5 It is a (hetero)hydrocarbon subunit; and

[0286] R Lig It is a quaternary ammonium ligand group or a guanidine ligand group.

[0287] Implementation scheme 21 is the flow method according to implementation scheme 20, wherein the cationic nitrogen-containing ligand monomer used to manufacture the anion exchange adsorption depth filter is a quaternary ammonium monomer of formula (II):

[0288]

[0289] in:

[0290] R 1 It is H or CH3;

[0291] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0292] R 5 It is a (hetero)hydrocarbon subunit; and

[0293] Each R 4 It can be alkyl or aryl independently.

[0294] Implementation scheme 22 is the flow method according to implementation scheme 20, wherein the cationic nitrogen-containing ligand monomer used to manufacture the anion exchange adsorption depth filter is a guanidine-containing ligand monomer of formula (III) or (IV):

[0295]

[0296]

[0297] in:

[0298] R 1 It is H or CH3;

[0299] R 2 It is a (hetero)hydrocarbon subunit (e.g., having 1 to 20 carbon atoms);

[0300] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0301] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0302] R 15 It is an H or hydrocarbon group (e.g., a C1-C4 alkyl group or an aryl group);

[0303] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0304] o is 0 or 1; and

[0305] n is 1 or 2.

[0306] Embodiment 23 is a flow method according to any one of embodiments 18 to 22, wherein the amide monomer used to manufacture the anion exchange adsorption depth filter has formula (V) or (VI):

[0307]

[0308]

[0309] in:

[0310] R 1 It is H or CH3;

[0311] Each R 8 Independently hydrogen, alkyl, or aryl; and

[0312] R 9 and R 10 It is an alkyl group, or it can form a 5- or 6-membered ring together.

[0313] Implementation scheme 24 is a flow method according to any one of embodiments 18 to 23, wherein the oxomonomer used to manufacture the anion exchange adsorption depth filter has formula (VII): wherein:

[0314] R 1 It is H or CH3;

[0315] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; and

[0316] R 16 It is a hydrocarbon group that is epoxy-functional or ether-functional.

[0317] Implementation scheme 25 is the flow method according to implementation scheme 24, wherein the oxomonomer used to manufacture the anion exchange adsorption depth filter has formula (VIII):

[0318]

[0319] in:

[0320] R 1 It is H or CH3;

[0321] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; and

[0322] R 7 It is a (hetero)hydrocarbon subunit (e.g., C1-C6 alkyl subunit).

[0323] Implementation Scheme 26 is the flow method according to Implementation Scheme 13, wherein the anion exchange adsorption depth filter comprises a porous substrate and a ligand-functional polymer grafted onto the porous substrate, wherein the grafted ligand-functional polymer has the following formula:

[0324] -(M PI ) w -(M b ) x -(M c ) y -(M d ) z

[0325] in:

[0326] -(M PI ) w Represents the residues of the grafted photoinitiator monomer, where w is 0 or at least 1;

[0327] -(M b ) x This represents a polymeric ligand monomer having "x" polymeric monomer units, where x is at least 1;

[0328] -(M c ) y This represents a polymeric crosslinking monomer having y polymeric monomer units, where y can be 0 or at least 1; and

[0329] -(M d ) z This represents a polymeric hydrophilic monomer having z polymeric monomer units, where z can be 0 or at least 1.

[0330] Implementation scheme 27 is the flow method according to implementation scheme 26, wherein the ligand monomer (M) used to manufacture the anion exchange adsorption depth filter b It has the following formula (X):

[0331]

[0332] in:

[0333] R 1 It is H or CH3;

[0334] R 2 It is a (hetero)hydrocarbon subunit;

[0335] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0336] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0337] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group, and

[0338] n is 1 or 2.

[0339] Implementation scheme 28 is the flow method according to implementation scheme 27, wherein the ligand monomer (M) used to manufacture the anion exchange adsorption depth filter b It has the formula (XIV):

[0340]

[0341] in:

[0342] R 1 It is H or CH3;

[0343] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0344] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0345] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0346] R 6 and R 7 Each is independently a (hetero)hydrocarbon subunit (e.g., C1-C). 10 Alkyl subunit);

[0347] Z 2 It is an ester, amide, urea, or carbamate group, and

[0348] n is 1 or 2.

[0349] Embodiment 29 is a flow method according to any one of Embodiments 26 to 28, wherein the grafted ligand-functional polymer of the anion exchange adsorption depth filter further comprises a crosslinking monomer (M c (y is at least 1), the crosslinking monomer has two or more free radical polymerizable groups.

[0350] Implementation scheme 30 is the flow method according to implementation scheme 29, wherein the crosslinking monomer (M) used to manufacture the anion exchange adsorption depth filter c It has the formula (XI):

[0351]

[0352] in:

[0353] Z 1 It is an unsaturated polymerizable group of an acrylate or non-acryloyl olefin.

[0354] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0355] Q is selected from covalent bonds, -O-, and -NR. 1 -, -CO2- and -C(O)NR 1 - a divalent linker, wherein R 1 It is H or CH3;

[0356] R 11 It is an alkylene group with a valence of a+b, and optionally contains one or more oxygen atoms in the chain and / or one or more hydroxyl groups; and

[0357] a and b are each at least 1.

[0358] Implementation scheme 31 is the flow method according to implementation scheme 29, wherein the crosslinking monomer (M) used to manufacture the anion exchange adsorption depth filter c It has the formula (XII):

[0359]

[0360] in:

[0361] Z 1 It is a polymerizable olefinic unsaturated group, either acryloyl or non-acryloyl.

[0362] R 1 It is H or CH3;

[0363] m is between 2 and 100; and

[0364] Q is selected from covalent bonds, -O-, and -NR. 1 -, -CO2- and -C(O)NR 1 - a divalent linker, wherein R 1 It is H or CH3;

[0365] Implementation scheme 32 is a flow method according to any one of embodiments 26 to 31, wherein the grafted ligand-functional polymer of the anion exchange adsorption depth filter further comprises a hydrophilic monomer (M d (z is at least 1), the hydrophilic monomer has a free radical polymerizable group and a hydrophilic group.

[0366] Implementation scheme 33 is the flow method according to implementation scheme 32, wherein the hydrophilic monomer (M) used to manufacture the anion exchange adsorption depth filter d It has formula (XIII):

[0367]

[0368] in:

[0369] Each R 1Independently, it can be H or CH3;

[0370] X 1 -O- or -NR 3 -, where R 3 It is H or (hetero)hydrocarbon group; and

[0371] t ranges from 2 to 100.

[0372] Implementation scheme 34 is a flow method according to any of the foregoing implementation schemes, wherein the salt-resistant anion exchange non-fibrous porous filter element comprises a non-fibrous porous filter element containing a fixed anion exchange ligand.

[0373] Implementation scheme 35 is the flow method according to implementation scheme 34, wherein the anion exchange ligand of the salt-resistant anion exchange non-fibrous porous filter element comprises a cationic nitrogen-containing ligand.

[0374] Implementation scheme 36 is the flow method according to implementation scheme 35, wherein the cationic nitrogen-containing ligand of the salt-resistant anion exchange non-fibrous porous filter element includes a guanidine-containing ligand.

[0375] Implementation scheme 37 is a flow method according to implementation scheme 35 or 36, wherein the salt-resistant anion exchange non-fibrous porous filter element comprises a copolymer grafted article, the copolymer grafted article comprising a non-fibrous porous filter element and a copolymer grafted onto the non-fibrous porous filter element, wherein the grafted copolymer comprises interpolymerized monomer units, the interpolymerized monomer units comprising cationic nitrogen-containing ligands.

[0376] Implementation scheme 38 is the flow method according to implementation scheme 37, wherein the graft copolymer of the salt-resistant anion exchange non-fiber porous filter element comprises interpolymerizable monomer units, the interpolymerizable monomer units comprising:

[0377] Monomers containing guanidine ligands;

[0378] Amide monomers;

[0379] Oxymonomers, wherein the oxymonomers are selected from the group consisting of epoxy-functional monomer units, alkyl ether-functional monomer units, and combinations thereof; and

[0380] Poly(epoxy) monomers.

[0381] Implementation scheme 39 is the flow method according to implementation scheme 38, wherein the graft copolymer of the salt-resistant anion exchange non-fiber porous filter element comprises interpolymerizable monomer units, the interpolymerizable monomer units comprising:

[0382] 10 to 50 parts by weight of guanidine-containing ligand monomer;

[0383] 10 to 80 parts by weight of amide monomer;

[0384] From 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; and

[0385] 0 to 30 parts by weight of poly(epoxy) monomer;

[0386] The total amount of monomers is 100 parts by weight.

[0387] Implementation scheme 40 is a flow method according to implementation scheme 38 or 39, wherein the cationic nitrogen-containing ligand monomer used to manufacture the salt-resistant anion exchange non-fibrous porous filter element has formula (I):

[0388]

[0389] in:

[0390] R 1 It is H or CH3;

[0391] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0392] R 5 It is a (hetero)hydrocarbon subunit; and

[0393] R Lig It contains a guanidine ligand group.

[0394] Implementation scheme 41 is the flow method according to implementation scheme 40, wherein the cationic nitrogen-containing ligand monomer used to manufacture the salt-resistant anion exchange non-fibrous porous filter element is a guanidine-containing ligand monomer of formula (III) or (IV):

[0395]

[0396] in:

[0397] R 1 It is H or CH3;

[0398] R 2 It is a (hetero)hydrocarbon subunit (e.g., having 1 to 20 carbon atoms);

[0399] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0400] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R3 Independently, it is an H or (hetero)hydrocarbon group;

[0401] R 15 It is an H or hydrocarbon group (e.g., a C1-C4 alkyl group or an aryl group);

[0402] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0403] o is 0 or 1; and

[0404] n is 1 or 2.

[0405] Implementation scheme 42 is the flow method according to implementation scheme 34, wherein the salt-resistant anion exchange non-fibrous porous filter element comprises a non-fibrous porous filter element and a ligand-functional polymer grafted onto the non-fibrous porous filter element, wherein the grafted ligand-functional polymer has the following formula:

[0406] -(M PI ) w -(M b ) x -(M c ) y -(M d ) z

[0407] in:

[0408] -(M PI ) w Represents the residues of the grafted photoinitiator monomer, where w is 0 or at least 1;

[0409] -(M b ) x This represents a polymeric ligand monomer having "x" polymeric monomer units, where x is at least 1;

[0410] -(M c ) y This represents a polymeric crosslinking monomer having y polymeric monomer units, where y can be 0 or at least 1; and

[0411] -(M d ) z This represents a polymeric hydrophilic monomer having z polymeric monomer units, where z can be 0 or at least 1.

[0412] Implementation scheme 43 is the flow method according to implementation scheme 42, wherein the ligand monomer (M) used to manufacture the salt-resistant anion exchange non-fibrous porous filter element b It has the following formula (X):

[0413]

[0414] in:

[0415] R 1 It is H or CH3;

[0416] R 2 It is a (hetero)hydrocarbon subunit;

[0417] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0418] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0419] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group, and

[0420] n is 1 or 2.

[0421] Implementation scheme 44 is the flow method according to implementation scheme 43, wherein the ligand monomer (M) used to manufacture the salt-resistant anion exchange non-fibrous porous filter element b It has the formula (XIV):

[0422]

[0423] in:

[0424] R 1 It is H or CH3;

[0425] Each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0426] R 14 It is H, (hetero)hydrocarbon group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group;

[0427] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0428] R 6 and R 7 Each is independently a (hetero)hydrocarbon subunit (e.g., C1-C). 10 Alkyl groups); and

[0429] Z 2 It is an ester, amide, urea, or carbamate group, and

[0430] n is 1 or 2.

[0431] Embodiment 45 is a flow method according to any one of Embodiments 42 to 44, wherein the grafted ligand-functional polymer of the salt-resistant anion exchange non-fibrous porous filter element further comprises a crosslinking monomer (M c (y is at least 1), the crosslinking monomer has two or more free radical polymerizable groups.

[0432] Implementation scheme 46 is the flow method according to implementation scheme 45, wherein the crosslinking monomer (M) used to manufacture the salt-resistant anion exchange non-fiber porous filter element c It has the formula (XI):

[0433]

[0434] in:

[0435] Z 1 It is an unsaturated polymerizable group of an acrylate or non-acryloyl olefin.

[0436] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0437] Q is selected from covalent bonds, -O-, and -NR. 1 -, -CO2- and -C(O)NR 1 - a divalent linker, wherein R 1 It is H or CH3;

[0438] R 11 It is an alkylene group with a valence of a+b, and optionally contains one or more oxygen atoms in the chain and / or one or more hydroxyl groups; and

[0439] a and b are each at least 1.

[0440] Implementation scheme 47 is the flow method according to implementation scheme 46, wherein the crosslinking monomer (M) used to manufacture the salt-resistant anion exchange non-fiber porous filter element c It has the formula (XII):

[0441]

[0442] in:

[0443] Z 1It is a polymerizable olefinic unsaturated group, either acryloyl or non-acryloyl.

[0444] R 1 It is H or CH3;

[0445] m ranges from 2 to 100; and

[0446] Q is selected from covalent bonds, -O-, and -NR. 1 -, -CO2- and -C(O)NR 1 - a divalent linker, wherein R 1 It is H or CH3;

[0447] Embodiment 48 is a flow method according to any one of Embodiments 42 to 47, wherein the grafted ligand-functional polymer of the salt-resistant anion exchange non-fibrous porous filter element further comprises a hydrophilic monomer (M d (z is at least 1), the hydrophilic monomer has a free radical polymerizable group and a hydrophilic group.

[0448] Implementation scheme 49 is the flow method according to implementation scheme 48, wherein the hydrophilic monomer (M) used to manufacture the salt-resistant anion exchange non-fibrous porous filter element d It has formula (XIII):

[0449]

[0450] in:

[0451] Each R 1 Independently, it can be H or CH3;

[0452] X 1 -O- or -NR 3 -, where R 3 It is H or (hetero)hydrocarbon group; and

[0453] t ranges from 2 to 100.

[0454] Embodiment 50 is a flow method according to any of the foregoing embodiments, wherein the cation exchange non-fibrous porous filter element comprises:

[0455] Non-fiber porous filter elements; and

[0456] The polymer disposed on the non-fibrous porous filter element, the polymer comprising:

[0457] A hydrocarbon backbone and a plurality of side groups connected to the hydrocarbon backbone, wherein each of the first plurality of side groups comprises:

[0458] At least one acidic group or a salt thereof; and

[0459] A spacer group, wherein the spacer group directly connects the at least one acidic group or its salt to the hydrocarbon backbone via a chain of at least six atoms in the chain.

[0460] Embodiment 51 is the flow method according to Embodiment 50, wherein the chain of the spacer group has at least 8 chain atoms.

[0461] Implementation scheme 52 is a circulation method according to implementation scheme 50 or 51, wherein the spacer group is a hydrocarbon group containing heteroatoms in the chain.

[0462] Embodiment 53 is a flow method according to any one of embodiments 50 to 52, wherein the spacer group comprises at least one hydrogen-bonded portion.

[0463] Embodiment 54 is a flow method according to any one of embodiments 50 to 53, wherein the at least one acidic group or salt thereof of the polymer disposed on the non-fibrous porous filter element is present at a density of at least 0.01 (or at least 0.02) mmol / g cation exchange non-fibrous porous filter element.

[0464] Embodiment 55 is a flow method according to any one of embodiments 50 to 54, wherein at least one acidic group or a salt thereof of the polymer disposed on the non-fibrous porous filter element is present at a density of up to 0.6 mmol / g cation exchange non-fibrous porous filter element.

[0465] Embodiment 56 is a circulation method according to any one of Embodiments 50 to 55, wherein the at least one acidic group or a salt thereof is selected from carboxyl groups, phosphonyl groups, phosphate groups, sulfonyl groups, sulfate groups, borate groups, and combinations thereof.

[0466] Embodiment 57 is a circulation method according to any one of Embodiments 50 to 56, wherein the polymer comprises interpolymerization units of at least one monomer, the monomer comprising: at least one olefinic unsaturated group; at least one acidic group or a salt thereof; and a spacer group, the spacer group being directly linked to at least one olefinic unsaturated group and at least one acidic group or a salt thereof by a chain of at least six atoms in the chain.

[0467] Embodiment 58 is the circulation method according to Embodiment 57, wherein the at least one olefinic unsaturated group is selected from vinyl groups, 1-alkyl vinyl groups, and combinations thereof.

[0468] Embodiment 59 is a flow method according to any one of embodiments 50 to 58, wherein the polymer is covalently attached to the non-fibrous porous filter element.

[0469] Embodiment 60 is a distribution method according to any one of embodiments 50 to 59, wherein the polymer is a copolymer.

[0470] Embodiment 61 is a flow method according to Embodiment 60, wherein the copolymer comprises a hydrocarbon backbone and a plurality of side groups connected to the hydrocarbon backbone, wherein:

[0471] Each of the first plurality of side groups contains:

[0472] At least one acidic group or a salt thereof; and

[0473] A spacer group, wherein the spacer group directly connects the at least one acidic group or its salt to the hydrocarbon backbone via a chain of at least six atoms; and

[0474] Each of the second plurality of side groups contains:

[0475] At least one acidic group or a salt thereof; and

[0476] A spacer group, wherein the spacer group directly connects the at least one acidic group or its salt to the hydrocarbon backbone through a chain of at least six atoms in the chain;

[0477] Wherein the first plurality of side groups are different from the second plurality of side groups; and

[0478] The molar ratio of the first plurality of side groups to the second plurality of side groups is in the range of 95:5 to 5:95.

[0479] Embodiment 62 is the flow method according to Embodiment 61, wherein the copolymer covalently linked to the non-fibrous porous filter element comprises a reaction product of a monomer composition, the monomer composition comprising:

[0480] The first monomer comprises:

[0481] At least one olefinic unsaturated group;

[0482] At least one acidic group or a salt thereof; and

[0483] A spacer group, wherein the spacer group is directly linked to the at least one olefinic unsaturated group and the at least one acidic group or a salt thereof via a chain of at least six atoms; and

[0484] The second monomer, the second monomer comprising:

[0485] At least one olefinic unsaturated group;

[0486] At least one acidic group or a salt thereof; and

[0487] A spacer group, wherein the spacer group is directly connected to the at least one olefinic unsaturated group and the at least one acidic group through a chain of at least six atoms in the chain;

[0488] The second monomer is different from the first monomer; and

[0489] The molar ratio of the first monomer to the second monomer is in the range of 95:5 to 5:95.

[0490] Embodiment 63 is a circulation method according to Embodiment 62, wherein the at least one olefinic unsaturated group of the first monomer and / or the second monomer is selected from vinyl groups, 1-alkyl vinyl groups, and combinations thereof.

[0491] Implementation scheme 64 is the circulation method according to implementation scheme 62 or 63, wherein the first monomer is one of the categories represented by the following general formula (XV):

[0492]

[0493] in:

[0494] R 1 It is H or CH3;

[0495] Each R 2 Independently a (hetero)hydrocarbon subunit;

[0496] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0497] Z 3 It is a (hetero)hydrocarbon subunit group, which contains at least one hydrogen bond donor, at least one hydrogen bond acceptor, or a combination thereof;

[0498] r is 0 or 1; and

[0499] L is a functional group containing at least one acidic group or a salt thereof.

[0500] Implementation scheme 65 is a circulation method according to any one of implementation schemes 62 to 64, wherein the second monomer is one of the categories represented by the following general formula (XV):

[0501]

[0502] in:

[0503] R 1 It is H or CH3;

[0504] Each R 2 Independently a (hetero)hydrocarbon subunit;

[0505] X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group;

[0506] Z 3 It is a (hetero)hydrocarbon subunit group, which contains at least one hydrogen bond donor, at least one hydrogen bond acceptor, or a combination thereof;

[0507] r is 0 or 1; and

[0508] L is a functional group containing at least one acidic group or a salt thereof.

[0509] Implementation scheme 66 is a filter cartridge comprising a salt-resistant anion exchange non-fibrous porous filter element as described herein and a cation exchange non-fibrous porous filter element as described herein.

[0510] Implementation scheme 67 is a filter cartridge according to implementation scheme 66, wherein the cation exchange non-fiber porous filter element is located downstream of the salt-resistant anion exchange non-fiber porous filter element.

[0511] Implementation scheme 68 is a filter cartridge according to implementation scheme 66 or 67, wherein the salt-resistant anion exchange non-fiber porous filter element includes a non-fiber porous filter element containing a fixed nitrogen-containing ligand.

[0512] Embodiment 69 is a filter cartridge according to any one of Embodiments 66 to 68, wherein the salt-resistant anion exchange non-fibrous membrane comprises a non-fibrous porous filter element and a ligand-functional polymer grafted onto the non-fibrous porous filter element, wherein the grafted ligand-functional polymer has the following formula:

[0513] -(M PI ) w -(M b ) x -(M c ) y -(M d ) z ,

[0514] in:

[0515] -(M PI ) w Represents the residues of the grafted photoinitiator monomer, where w is 0 or at least 1;

[0516] -(M b ) xThis represents a polymeric ligand monomer having "x" polymeric monomer units, where x is at least 1;

[0517] -(M c ) y This represents a polymeric crosslinking monomer having y polymeric monomer units, where y can be 0 or at least 1; and

[0518] -(M d ) z This represents a polymeric hydrophilic monomer having z polymeric monomer units, where z can be 0 or at least 1.

[0519] Embodiment 70 is a filter cartridge according to any one of embodiments 66 to 69, wherein the cation exchange non-fibrous porous filter element comprises:

[0520] Non-fiber porous filter elements; and

[0521] The polymer disposed on the non-fibrous porous filter element, the polymer comprising:

[0522] A hydrocarbon backbone and a plurality of side groups connected to the hydrocarbon backbone, wherein each of the first plurality of side groups comprises:

[0523] At least one acidic group or a salt thereof; and

[0524] A spacer group, wherein the spacer group directly connects the at least one acidic group or its salt to the hydrocarbon backbone via a chain of at least six atoms in the chain.

[0525] Example

[0526] These embodiments are for illustrative purposes only and are not intended to unduly limit the scope of the appended claims. While the numerical ranges and parameters illustrating the broad scope of this disclosure are approximations, the values ​​shown in the specific examples are recorded as precisely as possible. However, any numerical value inherently contains some error, which is necessarily caused by the standard deviation present in the respective test measurements. At a minimum, and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should at least be interpreted according to the number of significant digits reported and by applying customary rounding.

[0527] Unless otherwise specified, all parts, percentages, ratios, etc., in the examples and the remainder of the description are by weight, and all reagents used in the examples are derived from or purchased from common chemical suppliers, such as, for example, Sigma-Aldrich Company, Saint Louis, Missouri, or can be synthesized by conventional methods.

[0528] Table 1. Materials

[0529]

[0530]

[0531] Changes or adjustments to the buffer solution containing monoclonal antibody solutions

[0532] Conductivity was adjusted using 4 mol (4M) NaCl. pH was adjusted using acetic acid (200 mM or 500 mM) and Tris base (2M). Conductivity measurements were taken using an Accumet Excel XL50 conductivity meter (Fisher Scientific, Hampton, NH, New Hampton). pH measurements were taken using a VWR Symphony benchtop pH meter (VWR International, Radnor, PA, PA).

[0533] Filter Element Challenge Board

[0534] Cut the finished filter elements (FE-A to FE-L) into 7.5 mm diameter discs. For anion exchange filter elements (FE-L), load a single disc 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, load two discs of the same filter element into each well of a 96-well EMPORE filter plate. Hold the filter elements in place with plastic O-rings. The total working filtration volume per well is approximately 8.6 μL. Before centrifugation for sample collection, place each challenge plate loaded with filter elements onto a 96-well deep-well collection plate (Thermo Fisher Scientific, Waltham, MA). Centrifuge using an Allegra 25R centrifuge (Beckman Coulter, Brea, CA). The business challenge board (Sartorius SARTOBIND 96-hole board) is received from the manufacturer and used according to the manufacturer's instructions.

[0535] Target molecule and impurity analysis methods

[0536] Using the CHO HCP ELISA kit, 3G (Cygnus Technologies, Southport, NC) determined the concentration of host cell protein (HCP) in Chinese hamster ovaries (CHO) according to the manufacturer's protocol.

[0537] Protein concentration of mAb was determined using the Beer law with absorbance at 280 nm and an extinction coefficient of 1.36. Absorbance was measured using a SpectraMax M5 spectrophotometer (Molecular Instruments, San Jose, CA).

[0538] Use with TOSOH TSKgel G3000SW XL The monomer yield ('monomer mAb yield %') and high molecular weight (HMW) composition of the filtered solution were analyzed by size exclusion chromatography (SEC) using a Shimadzu Prominence HPLC system (Shimadzu Scientific Instruments, Columbia, MD) from Tosoh BioScience LLC, Griesheim, Germany (column). Analytical conditions: 20 μL injection volume; 1 mL / min flow rate; mobile phase: 100 mM sodium phosphate, 300 mM NaCl, pH 6.9; detection at 280 nm). The monomer yield and HMW composition were determined by comparing the peak areas of the starting and filtered solutions, and the results were reported as the average of two replicates.

[0539] The percentage of HMW components present in the mAb solution was determined using the SEC chromatography method described above. The peak area of ​​the HMW component (peak A) of the sample was compared with the sum of the peak areas of the sample (total peak area). The "HMW composition %" was calculated according to Equation 1.

[0540] The percentage of HMW removal was determined using the SEC chromatography method described above, and the peak area of ​​the aggregated components was measured before the separation method (peak B) and after the separation method using the depth filter or filter element challenge plate described in the examples (peak C). The "HMW removal rate %" was calculated according to Equation 2.

[0541] The percentage yield of monomeric monoclonal antibodies (mAbs) was determined using the SEC chromatography method described above, and the peak area of ​​the monomeric components was measured before the separation method (peak D) and after the separation method using the depth filter or filter element challenge plate described in the examples (peak E). The "monomeric mAb yield %" was calculated according to Equation 3.

[0542] The percentage of HCP removal was determined using the quantitative HCP method described above, and the HCP concentration was measured before (before HCP removal) and after (after HCP removal). The "HCP removal rate %" was calculated according to Equation 4.

[0543] Equation 1:

[0544] Equation 2:

[0545] Equation 3:

[0546] Equation 4:

[0547] Preparation of cation exchange non-fibrous porous filter element A (FE-A)

[0548] The coating solution was prepared by mixing a sodium 4-aminobutyrate / IEM monomer solution (IEM-GABA) (prepared as described in Example B of monomer in International Publication WO 2018 / 048698) (Vail et al.) (6.73 g (g) of a 20.8% w / w solution in deionized water) with sulfonated benzophenone (S-BP) (250 μL of a 0.1 g / mL solution in deionized water). Deionized water (13.02 g) was added to provide approximately 0.25 M of the monomer mixture. A nylon membrane substrate (18 cm × 23 cm; nylon 66 membrane, single-reinforced nylon tri-zone membrane, nominal pore size 1.8 μm or micrometer, #080ZN, obtained from 3M Purification, Inc., Meriden, CT) was placed on a polyester membrane, and the coating solution was drawn onto the top surface of the substrate. Immerse the coating solution into the substrate for approximately 1 minute, and then place a second polyester film on top of the substrate. Roll over the resulting three-layer sandwich structure with a 2.28 kg cylindrical weight to expel excess coating solution. Perform UV-induced grafting on the sandwich structure by irradiating it with a UV stage (Classic Manufacturing, Inc., Oakdale, MN) equipped with 18 bulbs [Sylvania RG2 40W F40 / 350BL / ECO, 10 bulbs above the substrate and 8 bulbs below the substrate, 1.17 m (46 in) long, 5.1 cm (2 in) center-to-center spacing] for 15 minutes. Remove the polyester film and place the resulting functionalized substrate in a 1000 mL polyethylene bottle. The bottle was filled with 0.9% (w / w) brine, sealed, and placed on a roller for 30 minutes to wash away any residual monomers or ungrafted polymers. The brine solution was poured off, and the functionalized substrate was washed again with fresh brine solution for 30 minutes, followed by washing with deionized water for 30 minutes (twice), and then dried. The grafting density was 0.266 mmol / g membrane, as determined by mass gain.

[0549] Preparation of cation exchange non-fibrous porous filter element B (FE-B)

[0550] As described in Example 30 of U.S. Patent Application 2019 / 0194250 (Colak Atan et al.), a nylon membrane (#080ZN) was grafted with an IEM-sodium glycinate monomer solution (0.25M). The grafting density was 0.28 mmol / g membrane.

[0551] Preparation of cation exchange non-fibrous porous filter element C (FE-C)

[0552] As described in Example 30 of U.S. Patent Application 2019 / 0194250 (Colak Atan et al.), but with a nylon membrane (#080ZN) grafted using an IEM-sodium glycinate monomer solution at a concentration of 0.375 M. The grafting density was 0.38 mmol / g membrane.

[0553] Preparation of cation exchange non-fibrous porous filter element D (FE-D)

[0554] Using the procedure described for FE-A, nylon membranes (#080ZN) were grafted with a 0.25 M solution of disodium 2-[[2-methyl-2-(propenoylamino)propionyl]amino]ethyl phosphate (VDM-O-phosphoethanolamine disodium salt) monomer. The grafting density was 0.12 mmol / g membrane.

[0555] The monomer was prepared as follows: O-phosphoethanolamine (21.15 g) was weighed into a 500 mL round-bottom (RB) flask and placed in an ice-water bath with magnetic stirring. NaOH (2 N (2 equivalents), 150 mL) was added, and the mixture was stirred until dissolved. VDM (10 mL) was added via pipette. The initially turbid suspension was stirred for 10 minutes until homogeneous. A second 10 mL of VDM was added (totaling 20 mL). The mixture was stirred for another 50 minutes. The reaction mixture was adjusted to pH 7 by adding a few drops of concentrated hydrochloric acid and then filtered. Solids % = 25.0%. 1 H-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).

[0556] Preparation of cation exchange non-fibrous porous filter element E (FE-E)

[0557] Using the procedure described for FE-A, nylon membranes (#080ZN) were grafted with a monomer solution of [2-carboxy-2-[[2-methyl-2-(propenoylamino)propionyl]amino]ethyl]phosphate disodium salt (VDM-O-phosphoserine disodium salt) at a concentration of 0.25 M. The grafting density was 0.07 mmol / g membrane.

[0558] The monomer was prepared as follows: 18.5 g of LO-phosphoserine was added to a 500 mL RB flask containing a magnetic stir bar. The flask was placed in an ice-water bath, and 40 mL of deionized water and 60 mL of 5N NaOH were added. The contents were stirred until all solids dissolved. After cooling in an ice bath for approximately 30 minutes, 5.0 mL of VDM was added via syringe. The mixture was stirred for 10 minutes, and then another 8.33 mL of VDM was added (totaling 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 was filtered to provide the product. Solids % = 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).

[0559] Preparation of cation exchange non-fibrous porous filter element F (FE-F)

[0560] As described in Example 5 of International Publication WO2018 / 048696 (Vail et al.), 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. The grafting density was 0.16 mmol / g membrane.

[0561] Preparation of cation exchange non-fibrous porous filter element G (FE-G)

[0562] As described in Example 21 of U.S. Patent Application 2019 / 0194250 (Colak Atan et al.), a nylon membrane (#080ZN) was grafted with a 0.25 M concentration of VDM-7-aminoheptanate sodium salt monomer solution. The grafting density was 0.18 mmol / g membrane.

[0563] Preparation of cation exchange non-fibrous porous filter element H (FE-H)

[0564] As described in Example 11 of International Publication WO 2018 / 048696 (Vail et al.), a nylon membrane (#080ZN) was grafted with a 50:50 (mol / mol) IEM-sodium glycinate / VDM-sodium phenylalanine monomer solution at a concentration of 0.25 M. The grafting density was 0.14 mmol / g membrane.

[0565] Preparation of cation exchange non-fibrous porous filter element I (FE-I)

[0566] As described in Example 2 of International Publication WO 2018 / 048696 (Vail et al.), 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. The grafting density was 0.19 mmol / g membrane.

[0567] Preparation of cation exchange non-fibrous porous filter element J (FE-J)

[0568] Nylon membranes (#080ZN) were grafted at a concentration of 0.25 M with a 50:50 (mol / mol) IEM-sodium glycinate / IEM-sodium phenylalanine monomer solution. The IEM-sodium phenylalanine monomer was prepared as described in International Publication WO 2018 / 048696 (Vail et al.), except that racemic phenylalanine was used. The grafting density was 0.42 mmol / g membrane.

[0569] Preparation of cation exchange non-fibrous porous filter element K (FE-K)

[0570] As described in Example 30 of U.S. Patent Application 2019 / 0194250 (Colak Atan et al.), a nylon membrane (#080ZN) was grafted with an IEM-sodium glycinate monomer solution (0.25M). The grafting density was 0.26 mmol / g membrane.

[0571] Preparation of salt-resistant anion exchange non-fibrous porous filter element L (FE-L)

[0572] IEM-guanidinobutylamine sulfate sodium was radiation-grafted onto a nylon membrane (#080ZN) to prepare a guanidinoligand-functionalized microporous membrane with a similar procedure to that described in Example 28 of U.S. Patent 10,471,398 (Bothof et al.) and exhibiting similar bovine serum albumin (BSA) dynamic binding capacity.

[0573] Example 1 A flow-through purification method is employed, which involves sequentially processing a monoclonal antibody solution with an anion exchange adsorption depth filter, a salt-resistant anion exchange non-fibrous porous filter element, and a cation exchange non-fibrous porous filter element.

[0574] Virus-inactivated mixture samples (VIP) containing monoclonal antibody mAbA (IgG1, pI 8.0, 10.5 mg / mL, pH 6.27, 5.3 mS / cm) were filtered using an anion-exchange adsorption depth filter prepared from internal culture medium components of an EMPHAZE AEX hybrid purifier (EMPHAZE AEX HP, 3M) mounted in a 25 mm holder [10 bed volumes / min (BVM)]. The challenge load was 1700 g / L, and the throughput was 180 L / m². 2 Challenge load was determined as the amount of mAbA per given volume of filter media (g / L). Throughput was determined as the amount of liquid volume passing through a given surface area of ​​the filter media (L / m²). 2 The depth filter reduces the turbidity of VIP solutions from >60 NTU (specific turbidimetric units) to <5 NTU with a pressure differential not exceeding 5 psi (pounds per square inch). Table 2 shows the characteristics of the solutions treated by the resulting depth filter.

[0575] The FE-L loaded challenge plate (as described above) was washed with 0.9% sodium chloride (1 mL) by centrifugation at 3000 rcf (relative centrifugal force) for 5 minutes. After washing, the solution treated by the depth filter was filtered through the FE-L challenge plate at a challenge load of 1200 g / L and centrifuged at 300 rcf for 5 minutes. The FE-L filtered solutions were combined. Aliquots of the combined solutions with different challenge loads [333 μL (400 g / L challenge load) or 450 μL (550 g / L challenge load)] were prepared and added to the wells of a series of second challenge plates. Each second challenge plate contained two disks of a single type of cation exchange filter element selected from FE-A to FE-F (as described above). The second challenge plates were prewashed with saline solution according to the method described for the FE-L challenge plate. The plates were centrifuged sequentially at 300 rcf, 600 rcf, 1200 rcf, and 3000 rcf for 5 minutes each. The filtered samples were analyzed for product and impurity analysis.

[0576] The VIP solution prior to treatment had an HMW composition of 4.2% and an HCP concentration of 3899 ng / L.

[0577] The solution obtained after the deep filtration process had an HMW composition of 4.2%, a monomer mAb yield of 98.4%, and an HCP concentration of 3366 ng / mL.

[0578] The combined solution obtained after the filtration process 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.

[0579] Table 3 reports the HMW composition (%), monomer mAb yield (%), and HCP concentration values ​​obtained after the final process step of filtering the solution through cation exchange filter elements (selected from FE-A to FE-F). Results of experiments using 400 g / L challenge loading or 550 g / L challenge loading are reported.

[0580] Table 2. Characterization of VIP solutions treated by depth filtration

[0581]

[0582] Table 3. HMW composition (%), monomer mAb yield (%), and HCP concentration of the mAbA solution purified according to the method of Example 1.

[0583]

[0584] Example 2 A flow-through purification method is employed, which involves sequential processing of a monoclonal antibody solution with an anion exchange adsorption depth filter, a buffer exchange step, a salt-resistant anion exchange non-fibrous porous filter element, and a cation exchange non-fibrous porous filter element.

[0585] Following the method described in Example 1, a VIP of a monoclonal antibody mAbA (IgG1, pI 8.0, 7.1 mg / mL, pH 6.27, 4.0 mS / cm) solution was filtered through a depth filter. Aliquots of the solution from the depth filter treatment were adjusted to target pH values ​​of 5.5, 6.25, or 7.0 and conductivities of 8 mS / cm, 16 mS / cm, or 24 mS / cm. The properties of the buffer-regulated mAbA solutions are provided in Table 4. An 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 pH and conductivity characteristics of the equilibration buffer were matched to those of the buffer-regulated mAb solutions.

[0586] In this method, SARTOBIND STIC plates (primary amine functional groups) are used as challenge plates for anion exchange filtration elements, and SARTOBIND S plates (sulfonic acid functional groups) are used as challenge plates for cation exchange filtration elements. Each plate is pretreated by filtration with one of the equilibration buffers (selected from the above-mentioned equilibration buffers). The pretreatment filtration conditions are 500 μL buffer, centrifuged at 1,000 rcf for 2 minutes. This process is repeated 4 times. Plates treated with the equilibration buffer are matched with mAbA solutions adjusted using the same buffer composition.

[0587] After equilibration, single aliquots of the buffer-exchanged mAbA solution were filtered using a SARTOBIND STIC plate. Filtration conditions were 500 μL solution centrifuged at 1,000 rcf for 2 min. This process was repeated twice for a challenge load of approximately 400 g / L (19 μL bed volume membrane). The resulting filtrate was then filtered using a SARTOBIND S plate under the same filtration conditions as the SARTOBIND STIC plate. The final filtrate was collected and analyzed. The HCP concentration, HMW composition (%), HMW removal (%), and monomer mAb yield (%) of the final filtrate were determined, and the results are reported in Tables 5 through 8.

[0588] Comparative Example 2 .

[0589] In Example 2, an intermediate process sample was recovered after filtration through a SARTOBIND STIC plate but before filtration through a SARTOBIND S plate. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the intermediate sample were analyzed. The results are shown in Tables 5 to 8.

[0590] Example 2a .

[0591] The procedure was the same as described in Example 2, except that the FE-L supported plate was used as the challenge plate for the anion exchange filter element, and the FE-J supported plate was used as the challenge plate for the cation exchange filter element. The process conditions were modified to equilibrate with 1000 μL of equilibration buffer for each FE-L and FE-J filter element by centrifugation at 1,500 rcf for 5 min. After equilibration, single aliquots (500 μL) of the buffer-exchanged mAb solution were filtered sequentially using the FE-L supported plate, followed by the FE-J supported plate, by centrifugation at 1,500 rcf for 5 min. The challenge loading per well was approximately 400 g / L (9 μL bed volume membrane). The HCP concentration, HMW composition % (%), HMW removal % (%), and monomer mAb yield % (%) of the final filtered solution were determined, and the results are reported in Tables 5 through 8.

[0592] Comparative Example 2a .

[0593] In Example 2a, an intermediate process sample was recovered after filtration through an FE-L supported plate but before filtration through an FE-J supported plate. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the intermediate sample were analyzed. The results are shown in Tables 5 to 8.

[0594] Example 2b .

[0595] The same method as described in Example 2a was followed, except that an FE-K supported plate was used instead of an FE-J plate as the challenge plate for the cation exchange filter element. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the final filtered solution were determined, and the results are reported in Tables 5 to 8.

[0596] Example 2c .

[0597] The same method as described in Example 2 was followed, except that the FE-L supported plate was used as the challenge plate for the anion exchange filter element, and the FE-F plate was used as the challenge plate for the cation exchange filter element. The processing conditions were modified to 1000 μL of equilibration buffer for each FE-L and FE-F filter element, centrifuged at 1,500 rcf for 5 min. After equilibration, single aliquots (500 μL) of the buffer-exchanged mAb solution were filtered sequentially using the FE-L supported plate, followed by the FE-F supported plate, centrifuged at 1,500 rcf for 5 min. Additional centrifugation cycles (3,000 rcf, 5 min) were used for the FE-F supported plate. The challenge loading per well was approximately 400 g / L (9 μL bed volume membrane). The HCP concentration, HMW composition % (%), HMW removal % (%), and monomer mAb yield % (%) of the final filtered solution were determined, and the results are reported in Tables 5 through 8.

[0598] Table 4. pH, conductivity, and mAbA concentration of the buffer-adjusted solutions for Examples 2, 2a-c, Comparative Examples 2 and 2a. degree, HCP concentration and HMW composition %

[0599]

[0600] Table 5. Host cells purified by the method described in Examples 2, 2a-c and Comparative Examples 2 and 2a Protein content

[0601]

[0602] Table 6. Composition of HMW purified by the method described in Examples 2, 2a-c and Comparative Examples 2 and 2a

[0603]

[0604] Table 7. HMW removal after purification by the method described in Examples 2, 2a-c and Comparative Examples 2 and 2a Rate

[0605]

[0606]

[0607] Table 8. Monomer yields purified by the methods described in Examples 2, 2a-c and Comparative Examples 2 and 2a

[0608]

[0609] Example 3A flow-through purification method is employed, which involves sequential processing of a monoclonal antibody solution with an anion exchange adsorption depth filter, a buffer exchange step, a salt-resistant anion exchange non-fibrous porous filter element, and a cation exchange non-fibrous porous filter element.

[0610] According to the method described in Example 1, a VIP solution of monoclonal antibody mAbA (IgG1, pI 8.0, 7.1 mg / mL, pH 6.27, 4.0 mS / cm) was filtered through a depth filter. The VIP solution prior to treatment had a HMW composition of 4.2% and an HCP concentration of 4098 ng / L. The solution obtained after the depth filtration process had a HMW composition of 4.2%, a monomer mAb yield of 97.9%, and an HCP concentration of 3546 ng / mL.

[0611] Buffer-regulated mAbA solutions were prepared, and the properties of these solutions are provided in Table 9. Twelve different buffer-regulated 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. These solutions had target pH values ​​of 6.0, 6.5, or 7.0 and target conductivity values ​​of 8 mS / cm, 14 mS / cm, 19 mS / cm, or 24 mS / cm.

[0612] In this method, the FE-L supported plate is used as the challenge plate for the anion exchange filter element, and the FE-C supported plate is used as the challenge plate for the cation exchange filter element. Each plate is pretreated by filtration with one of the equilibration buffers (selected from those described above in Example 2). The pretreatment filtration conditions are 1000 μL buffer, centrifuged at 3000 rcf for 5 min. The plate treated with the equilibration buffer is matched with a mAbA solution adjusted using the same buffer composition. After equilibration, a single aliquot of the buffer-exchanged mAbA solution is filtered using the FE-L supported plate. The filtration conditions are 1000 μL solution, centrifuged at 300 rcf for 5 min. The challenge load is approximately 1200 g / L (9 μL bed volume membrane), and the collected filtrates are combined for each individual condition.

[0613] The resulting filtrate was then filtered using an FE-C loaded plate. Filtration conditions included aliquots of combined solutions with different challenge loads [333 μL (400 g / L challenge load) or 450 μL (550 g / L challenge load)] centrifuged at 100 rcf, 200 rcf, 300 rcf, 600 rcf, 1200 rcf, and 3000 rcf for 5 minutes. The final filtrate was collected and analyzed. The HCP concentration, HMW composition (%), monomer mAb yield (%), HMW removal (%), and HCP removal (%) of the final filtrate were determined, and the results are reported in Tables 11 and 12.

[0614] Comparative Example 3 .

[0615] In Example 3, an intermediate process sample was recovered after filtration through an FE-L supported plate but before filtration through an FE-C supported plate. The HCP concentration, HMW composition (%), and monomer mAb yield (%) of the intermediate sample were analyzed. The results are reported in Table 10.

[0616] Example 3a .

[0617] The same method as described in Example 3 was followed, except that an FE-G supported plate was used instead of an FE-C plate as the challenge plate for the cation exchange filter element. The HCP concentration, HMW composition (%), monomer mAb yield (%), HMW removal rate (%), and HCP removal rate (%) of the final filtered solution were determined, and the results are reported in Tables 13 and 14.

[0618] Example 3b .

[0619] According to the method described in Example 1, a VIP solution of monoclonal antibody mAbA (IgG1, pI 8.0, 7.1 mg / mL, pH 6.27, 4.0 mS / cm) was filtered through a depth filter. The VIP solution prior to treatment had a HMW composition of 4.2% and an HCP concentration of 4098 ng / L. The solution obtained after the depth filtration process had a HMW composition of 4.2%, a monomer mAb yield of 97.9%, and an HCP concentration of 3255 ng / mL. The same method as described in Example 3 was followed, except that an FE-F supported plate was used instead of an FE-C plate as the cation exchange filter element challenge plate. The HCP concentration, HMW composition (%), monomer mAb yield (%), HMW removal (%), and HCP removal (%) of the final filtered solution were determined, and the results are reported in Tables 15 and 16.

[0620] Comparative Example 3b .

[0621] In Example 3b, an intermediate process sample was recovered after filtration through an FE-L supported plate but before filtration through an FE-F supported plate. The HCP concentration, HMW composition (%), and monomer mAb yield (%) of the intermediate sample were analyzed. The results are reported in Table 10.

[0622] Table 9. Conductivity and pH adjustment of mAbA solution

[0623]

[0624] Table 10. HMW composition (%), HCP concentration and monomer mAb yield (%) of Comparative Example 3 and Comparative Example 3b

[0625]

[0626]

[0627] Table 11. HMW composition (%), HCP concentration, and monomeric mAb production after purification of mAbA solution according to the method of Example 3. Rate%

[0628]

[0629] Table 12. HMW removal rate and HCP removal rate by the method of Example 3

[0630]

[0631] Table 13. HMW composition (%), HCP concentration, and monomeric mAb production after purification of mAbA solution according to the method of Example 3a Rate%

[0632]

[0633] Table 14. HMW removal rate and HCP removal rate by the method of Example 3a

[0634]

[0635] Table 15. HMW composition (%), HCP concentration, and monomeric mAb production after purification of mAbA solution according to the method in Example 3b Rate%

[0636]

[0637] Table 16. HMW removal rate and HCP removal rate by the method of Example 3b

[0638]

[0639]

[0640] Example 4 A flow-through purification method is employed, which involves sequential processing of a monoclonal antibody solution with a buffer exchange step, a salt-resistant anion-exchange non-fibrous porous filter element, and a cation-exchange non-fibrous porous filter element.

[0641] The conjoined monoclonal antibody mAb2 (IgG1, pI approx. 8) was purified using a ZetaPlus ZP90 depth filter (3M), followed by a protein A chromatography step with acetic acid elution (mAbSelect ProA resin, GE Healthcare Life Sciences, Pittsburgh, PA) and incubation at pH 3.5 for 30 min for viral inactivation. Following viral inactivation, the resulting low-pH solution was neutralized with 2M Tris. The neutralized solution had an initial pH of 7.4 and a conductivity of 10.2 (mS / cm).

[0642] Buffer-regulated mAb2 solutions were prepared, and the properties of these solutions are provided in Table 17. Nine different buffer-regulated mAb2 solutions were prepared by adjusting the pH with 500 mM acetic acid and the conductivity with 4 M sodium chloride. These solutions had target pH values ​​of 6.0, 6.5, or 7.0 and target conductivity values ​​of 12 mS / cm, 17 mS / cm, or 22 mS / cm.

[0643] In this method, the FE-L loaded plate is used as the challenge plate for the anion exchange filter element, and the FE-C loaded plate is used as the challenge plate for the cation exchange filter element. Each plate is pretreated by filtration with one of the equilibration buffers (selected from those described above in Example 2). The pretreatment filtration conditions are 1000 μL buffer, centrifuged at 3000 rcf for 5 min. The plate treated with the equilibration buffer is matched with a mAb2 solution adjusted using the same buffer composition. After equilibration, a single aliquot of the buffer-exchanged mAb2 solution is filtered using the FE-L loaded plate. The filtration conditions are 1000 μL solution, centrifuged at 300 rcf for 5 min. The challenge load is approximately 350 g / L (9 μL bed volume membrane), and the collected filtrates are combined for each individual condition.

[0644] The resulting filtrate was then filtered using an FE-C supported plate. Filtration conditions were: 1000 μL solution centrifuged at 300 rcf, 600 rcf, 1200 rcf, and 3000 rcf for 5 minutes. The final filtrate was collected and analyzed. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the final filtrate were determined, and the results are reported in Tables 18 to 21.

[0645] Comparative Example 4 .

[0646] In Example 4, an intermediate process sample was recovered after filtration through an FE-L supported plate but before filtration through an FE-C supported plate. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the intermediate sample were analyzed. The results are shown in Tables 18 to 21.

[0647] Example 4a .

[0648] The same method as described in Example 4 was followed, except that an FE-F supported plate was used instead of an FE-C plate as the challenge plate for the cation exchange filter element. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the final filtered solution were determined, and the results are reported in Tables 18 to 21.

[0649] Example 4b .

[0650] The same method as described in Example 4 was followed, except that an FE-E supported plate was used instead of an FE-C plate as the challenge plate for the cation exchange filter element. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the final filtered solution were determined, and the results are reported in Tables 18 to 21.

[0651] Example 4c .

[0652] The same method as described in Example 4 was followed, except that an FE-H supported plate was used instead of an FE-C plate as the challenge plate for the cation exchange filter element. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the final filtered solution were determined, and the results are reported in Tables 18 to 21.

[0653] Example 4d .

[0654] The same method as described in Example 4 was followed, except that an FE-I supported plate was used instead of an FE-C plate as the challenge plate for the cation exchange filter element. The HCP concentration, HMW composition (%), HMW removal rate (%), and monomer mAb yield (%) of the final filtered solution were determined, and the results are reported in Tables 18 to 21.

[0655] Table 17. Characterization of mAb2 solution after deep filtration and protein A purification (Example 4)

[0656]

[0657] Table 18. Host cell protein content purified by the methods described in Examples 4, 4a-4d and Comparative Example 4 quantity

[0658]

[0659] Table 19. Composition of HMW purified by the method described in Examples 4, 4a-4d and Comparative Example 4

[0660]

[0661]

[0662] Table 20. HMW removal rates using the methods described in Examples 4, 4a-4d and Comparative Example 4

[0663]

[0664] Table 21. Monomer yield following the methods described for Examples 4, 4a-4d and Comparative Example 4

[0665]

[0666] All patents, patent documents, and publications cited herein are incorporated herein by reference in their entirety as if each document were cited individually. In the event of any conflict or contradiction between the disclosures in this written specification and any documents incorporated herein by reference, the written specification shall prevail. Various modifications and alterations to this disclosure will become apparent to those skilled in the art without departing from its scope and spirit. It should be understood that this disclosure is not intended to be unduly limited to the exemplary embodiments and examples shown herein, and such embodiments and examples are presented by way of example only. The scope of this disclosure is intended to be limited only by the claims shown herein.

Claims

1. A flow-through method for purifying said monoclonal antibody from a biological solution in a sample containing the monoclonal antibody, the method comprising: Optionally, the sample is brought into contact with an anion exchange adsorption depth filter; Optionally, the sample is buffer-exchanged 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-fiber porous filter element; and Then immediately bring the sample into contact with a cation exchange non-fibrous porous filter element; The flow method described herein includes one or two buffer exchanges, with no buffer exchange between samples contacting the salt-resistant anion-exchange non-fibrous porous filter element and the cation-exchange non-fibrous porous filter element; and The salt-resistant anion exchange non-fiber porous filter element is: A non-fibrous porous filter element comprising a porous membrane, the porous membrane comprising a graft copolymer, and the graft copolymer comprising interpolymerizable monomer units, the interpolymerizable monomer units comprising: 10 to 50 parts by weight of guanidine-containing ligand monomer; 10 to 80 parts by weight of amide monomer; From 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; and 0 to 30 parts by weight of poly(epoxy) monomer; The total amount of monomers is 100 parts by weight; or A non-fibrous porous filter element and a ligand-functional polymer grafted onto the non-fibrous porous filter element, wherein the grafted ligand-functional polymer has the following formula: -(M PI ) w -(M b ) x -(M c ) y -(M d ) z , in: -(M PI ) w - represents a residue of the grafted photoinitiator monomer, where w is 0 or at least 1; -(M b ) x This represents a polymeric ligand monomer having "x" polymeric monomer units, where x is at least 1, and the ligand monomer has the formula (X): in: R 1 It is H or CH3; R 2 It is a (hetero)hydrocarbon subunit; Each R 3 Independently, it is an H or (hetero)hydrocarbon group; R 14 It is H, (hetero)hydro group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group; X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group, and n is 1 or 2; -(M c ) y This represents a polymeric crosslinking monomer having y polymeric monomer units, where y can be 0 or at least 1, and the (M) c ) y For formula (XI): in: Z 1 It is an unsaturated polymerizable group of an acrylate or non-acryloyl olefin. X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; Q is selected from covalent bonds, -O-, and -NR. 1 -, -CO2- and -C(O)NR 1 - a divalent linker, wherein R 1 It is H or CH3; R 11 It is an alkylene group with a oxidation state of a+b, and contains one or more oxygen atoms in the chain and / or one or more hydroxyl groups; and a and b are each at least 1; and -(M d ) z This represents a polymeric hydrophilic monomer having z polymeric monomer units, where z can be 0 or at least 1, and the (M) d ) z For equation (XIII): in: Each R 1 Independently, it can be H or CH3; X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; and t is between 2 and 100; and The cation exchange non-fibrous porous filter element comprises the following: Non-fibrous porous substrates; and The copolymer disposed on the non-fibrous porous substrate, the copolymer comprising the reaction product of a monomer composition comprising: The first and second monomers are represented by the following general formula (XV): in: R 1 It is H or CH3; Each R 2 Independently a (hetero)hydrocarbon subunit; X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; Z 3 It is a heterohydrocarbon subunit group, which contains at least one hydrogen bond donor, at least one hydrogen bond acceptor, or a combination thereof; r is 0 or 1; and L is a functional group containing at least one acidic group or a salt thereof; The second monomer is different from the first monomer; The molar ratio of the first monomer to the second monomer is in the range of 95:5 to 5:95; The at least one acidic group or its salt is selected from carboxyl groups, phosphonyl groups, phosphate groups, sulfonyl groups, sulfate groups, borate groups, and combinations thereof.

2. A flow-through method for purifying said monoclonal antibody from a biological solution in a sample containing the monoclonal antibody, the method comprising: The sample is brought into contact with the anion exchange adsorption depth filter; Then immediately bring the sample into contact with a salt-resistant anion exchange non-fiber porous filter element; as well as Then immediately bring the sample into contact with a cation exchange non-fibrous porous filter element; The flow method described herein does not include buffer exchange; and The salt-resistant anion exchange non-fiber porous filter element is: A non-fibrous porous filter element comprising a porous membrane, the porous membrane comprising a graft copolymer, and the graft copolymer comprising interpolymerizable monomer units, the interpolymerizable monomer units comprising: 10 to 50 parts by weight of guanidine-containing ligand monomer; 10 to 80 parts by weight of amide monomer; From 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; and 0 to 30 parts by weight of poly(epoxy) monomer; The total amount of monomers is 100 parts by weight; or A non-fibrous porous filter element and a ligand-functional polymer grafted onto the non-fibrous porous filter element, wherein the grafted ligand-functional polymer has the following formula: -(M PI ) w -(M b ) x -(M c ) y -(M d ) z , in: -(M PI ) w - represents a residue of the grafted photoinitiator monomer, where w is 0 or at least 1; -(M b ) x This represents a polymeric ligand monomer having "x" polymeric monomer units, where x is at least 1, and the ligand monomer has the formula (X): in: R 1 It is H or CH3; R 2 It is a (hetero)hydrocarbon subunit; Each R 3 Independently, it is an H or (hetero)hydrocarbon group; R 14 It is H, (hetero)hydro group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group; X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group, and n is 1 or 2; -(M c ) y This represents a polymeric crosslinking monomer having y polymeric monomer units, where y can be 0 or at least 1, and the (M) c ) y For formula (XI): in: Z 1 It is an unsaturated polymerizable group of an acrylate or non-acryloyl olefin. X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; Q is selected from covalent bonds, -O-, and -NR. 1 -, -CO2- and -C(O)NR 1 - a divalent linker, wherein R 1 It is H or CH3; R 11 It is an alkylene group with a oxidation state of a+b, and contains one or more oxygen atoms in the chain and / or one or more hydroxyl groups; and a and b are each at least 1; and -(M d ) z This represents a polymeric hydrophilic monomer having z polymeric monomer units, where z can be 0 or at least 1, and the (M) d ) z For equation (XIII): in: Each R 1 Independently, it can be H or CH3; X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; and t is between 2 and 100; and The cation exchange non-fibrous porous filter element comprises the following: Non-fibrous porous substrates; and The copolymer disposed on the non-fibrous porous substrate, the copolymer comprising the reaction product of a monomer composition comprising: The first and second monomers are represented by the following general formula (XV): in: R 1 It is H or CH3; Each R 2 Independently a (hetero)hydrocarbon subunit; X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group; Z 3 It is a heterohydrocarbon subunit group, which contains at least one hydrogen bond donor, at least one hydrogen bond acceptor, or a combination thereof; r is 0 or 1; and L is a functional group containing at least one acidic group or a salt thereof; The second monomer is different from the first monomer; The molar ratio of the first monomer to the second monomer is in the range of 95:5 to 5:95; The at least one acidic group or its salt is selected from carboxyl groups, phosphonyl groups, phosphate groups, sulfonyl groups, sulfate groups, borate groups, and combinations thereof.

3. The circulation method according to claim 1 further includes: The sample is brought into contact with the anion exchange adsorption depth filter; After the sample is brought into contact with the anion exchange adsorption depth filter, a buffer exchange is performed with the sample. The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; and The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

4. The distribution method according to claim 1 includes: Buffer exchange with the sample; The sample is brought into contact with a salt-resistant anion exchange non-fiber porous filter element; as well as The sample is then immediately brought into contact with a cation exchange non-fibrous porous filter element.

5. The circulation method according to claim 1, wherein the biological solution comprises a neutralized virus-inactivated mixture sample.

6. The flow method according to claim 1, wherein the anion exchange adsorption depth filter comprises a porous substrate, the porous substrate containing fixed anion exchange ligands.

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

8. The flow method according to claim 7, wherein the cationic nitrogen-containing ligand of the anion exchange adsorption depth filter comprises a primary amine, a secondary amine, a tertiary amine, or a combination thereof.

9. The flow method according to claim 8, wherein the cationic nitrogen-containing ligand of the anion exchange adsorption depth filter comprises a quaternary ammonium ligand, a guanidine ligand, or a combination thereof.

10. The flow method according to claim 6, wherein the anion exchange adsorption depth filter comprises a porous substrate and a ligand-functional polymer grafted onto the porous substrate, wherein the grafted ligand-functional polymer has the following formula: -(M PI ) w -(M b ) x -(M c ) y -(M d ) z , in: -(M PI ) w Represents the residues of the grafted photoinitiator monomer, where w is 0 or at least 1; -(M b ) x This represents a polymeric ligand monomer having "x" polymeric monomer units, where x is at least 1, and the ligand monomer has the formula (X): in: R 1 It is H or CH3; R 2 It is a (hetero)hydrocarbon subunit; Each R 3 Independently, it is an H or (hetero)hydrocarbon group; R 14 It is H, (hetero)hydro group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group; X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group, and n is 1 or 2; -(M c ) y This represents a polymeric crosslinking monomer having y polymeric monomer units, where y can be 0 or at least 1; and -(M d ) z This represents a polymeric hydrophilic monomer having z polymeric monomer units, where z can be 0 or at least 1.

11. The flow method according to claim 1, wherein the salt-resistant anion exchange non-fibrous porous filter element is a porous membrane.

12. A filter cartridge for use in purifying a monoclonal antibody from a biological solution containing a sample according to the method of claim 1, the filter cartridge comprising a salt-resistant anion-exchange non-fibrous porous filter element and a cation-exchange non-fibrous porous filter element; The salt-resistant anion exchange non-fiber porous filter element includes: Non-fibrous porous filter elements containing fixed cationic nitrogen-containing ligands, or A non-fibrous porous filter element and a ligand-functional polymer grafted onto the non-fibrous porous filter element, wherein the grafted ligand-functional polymer has the following formula: -(M PI ) w -(M b ) x -(M c ) y -(M d ) z , in: -(M PI ) w - represents a residue of the grafted photoinitiator monomer, where w is 0 or at least 1; -(M b ) x This represents a polymeric ligand monomer having "x" polymeric monomer units, where x is at least 1, and the ligand monomer has the formula (X): in: R 1 It is H or CH3; R 2 It is a (hetero)hydrocarbon subunit; Each R 3 Independently, it is an H or (hetero)hydrocarbon group; R 14 It is H, (hetero)hydro group or -N(R) 3 )2, where each R 3 Independently, it is an H or (hetero)hydrocarbon group; X 1 -O- or -NR 3 -, where R 3 It is an H or (hetero)hydrocarbon group, and n is 1 or 2; -(M c ) y This represents a polymeric crosslinking monomer having y polymeric monomer units, where y can be 0 or at least 1; and -(M d ) z This represents a polymeric hydrophilic monomer having z polymeric monomer units, where z can be 0 or at least 1; and The cation exchange non-fiber porous filter element includes: Non-fiber porous filter elements; and The polymer disposed on the non-fibrous porous filter element, the polymer comprising: A hydrocarbon backbone and a plurality of side groups connected to the hydrocarbon backbone, wherein each of the first plurality of side groups comprises: At least one acidic group or a salt thereof; and A spacer group, wherein the spacer group directly connects the at least one acidic group or its salt to the hydrocarbon backbone via a chain of at least six atoms in the chain.

13. The use according to claim 12, wherein the cation exchange non-fibrous porous filter element is located downstream of the salt-resistant anion exchange non-fibrous porous filter element.

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