Heterocyclic acrylic acid derivatives as monomers for synthesizing polymers for use in ion exchange articles

Abstract

Described are monomers having nitrogen atoms that can be protonated at relatively low pH values ​​(e.g., below pH 9.5 or below pH 9), polymers comprising monomer units derived from these monomers, anion exchange separation articles having the polymers grafted to a porous substrate, and methods of using the anion exchange separation articles to separate mixtures of materials having different ionic contents. Advantageously, the anion exchange separation articles can be used to separate biological materials that cannot be subjected to high pH conditions (e.g., above 9 or 9.5) and / or high ionic strength conditions (e.g., above 0.5 M or 1 M). JPEG2026501205000051.jpg22128

Description

[Technical Field]

[0001] Ion exchange (IEX) chromatography is commonly used for the purification of biological species. Cation exchange (CEX) chromatography typically utilizes acidic ligands with pKa values ​​in the range of 0 to approximately 4 immobilized on a solid support. Anion exchange (AEX) chromatography typically uses ligands with pKa values ​​in the range of approximately 9 to 12. Relatively few ligands with pKa values ​​falling within these two ranges are available.

[0002] Anion exchange ligands commonly used in bioseparations have high pKa values. For example, the pKa of strong anion exchange quaternary amine ligands exceeds 12. Weak anion exchange ligands, such as those formed from monomers such as diethylaminopropylacrylamide and dimethylaminopropylacrylamide, have pKa values ​​of 10.3 and 9.3, respectively. When these ligands are used for binding and elution in AEX chromatographic purification, elution of captured biomaterials is typically achieved using buffers with high salt concentrations, e.g., 0.5 M or 1.0 M. Elution by pH change is usually not feasible because it requires an eluent with a pH above the pKa of the ligand. This can be problematic because many proteins, especially enzymes, cannot tolerate such high pH values. Furthermore, many biological species cannot tolerate high salt concentrations or low pH values, e.g., below 3.5 or 4. For example, many enzymes, viruses, and virus-like particles (VLPs) are stable only within a narrow pH and salt range. Furthermore, many viruses, especially enveloped viruses, are notoriously difficult to purify due to their low or near-neutral isoelectric points, and traditional IEX ligands have limited effectiveness in purifying viruses with low or near-neutral isoelectric points. Summary of the Invention

[0003] Monomers that can be used in the preparation of anion exchange separation articles are provided. The anion exchange separation articles have ligands formed from monomers with calculated pKa values ​​in the range of 3.5, or 4 to 9, or 9.5. These monomers can be used to form polymers grafted to solid supports to provide anion exchange (AEX) articles that can be used for bioseparations under relatively mild conditions. That is, the polymers can include monomers selected to have pKas that allow binding, washing, and elution (i.e., purification) within a range of pH and salt concentrations that maintain the stability of the target biological species.

[0004] In a first aspect, there is provided a monomer of formula (I):

[0005] [ka]

[0006] In formula (I), R 1 The group is hydrogen or methyl, and X 1 is -O- or -NH-, and R 2 is (hetero)alkylene. The Z group is -NH-(C=O)-, -NH-(C=O)-NH-, -NH-(C=O)-NH-R 3 -, -(C=O)-NH-, or -(C=O)-NH-R 3 -(In the formula, R 3 is an alkylene having at least two linked carbon atoms. 4 Groups and R 5 The groups are each alkylene having at least two carbon atoms, and the nitrogen, R 4 , Q, and R 5 The total number of ring atoms in the ring group consisting of is 6 or 7. The Q group has a single connecting atom, -O-, -N(R 6 )-, -S-, -S(=O)-, or -S(=O)2-, provided that when Z is -NH-(C=O)-, Q is -N(R 6 )-. R 6The groups are hydrogen, alkyl, or (hetero)aryl, where alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl, and (hetero)aryl is optionally further substituted with hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

[0007] In a second aspect, there is provided a polymer that is the polymerization product obtained from a monomer composition comprising a monomer of formula (I) described in the first aspect.

[0008] In a third aspect, an anion exchange separation article is provided that includes a porous substrate and a plurality of graft polymers attached to a surface of the porous substrate, the graft polymers being polymerization products obtained from a monomer composition that includes a monomer of formula (I) described in the first aspect.

[0009] In a fourth aspect, a method for separating a mixture of materials having different ionic contents is provided. The method includes preparing or providing an anion exchange separation article as described in the third aspect. The method further includes passing the mixture of materials through the anion exchange separation article at a first pH sufficiently low to protonate the monomer repeat units of the graft polymer derived from the monomer of Formula (I) and at a first ionic strength value that causes at least one component of the mixture of materials to bind to the anion exchange separation article as a binding moiety.

[0010] As used herein, the terms "a," "an," "the," and "at least one" are used interchangeably.

[0011] The term "and / or" means either one or both. For example, "A and / or B" means A alone, B alone, or both A and B.

[0012] The term "alkyl" refers to a monovalent group that is a radical of an alkane. An alkyl group can have 1 to 32 carbon atoms, 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. An alkyl can be linear, branched, cyclic, or a combination thereof. A linear alkyl has at least 1 carbon atom, and a cyclic or branched alkyl has at least 3 carbon atoms.

[0013] The term "alkylene" refers to a divalent group that is a radical of an alkane. An alkylene group can have 1 to 32 carbon atoms, 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. Alkylene can be linear, branched, cyclic, or a combination thereof. A linear alkylene has at least 1 carbon atom, and a cyclic or branched alkylene has at least 3 carbon atoms.

[0014] The term "heteroalkylene" refers to an alkylene in which one or more of the carbon atoms has been replaced with a heteroatom. The heteroatom is typically nitrogen (e.g., -NH-), oxygen (-O-), or sulfur (-S-). Typically, there are not two adjacent heteroatoms, such as in peroxide.

[0015] The term "(hetero)alkylene" refers to alkylene, heteroalkylene, or both.

[0016] The term "alkoxy" refers to a group of the formula -OR a (In the formula, R a refers to a monovalent radical of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 38,

[0017] The term "aryl" refers to a monovalent group that is a radical of an aromatic carbocyclic compound. An aryl group has at least one aromatic carbocyclic ring and can have 1 to 3 optional rings bonded or fused to the aromatic carbocyclic ring. Additional rings can be aromatic, aliphatic, or a combination thereof. Aryl groups typically have 5 to 20 carbon atoms or 6 to 10 carbon atoms.

[0018] The term "heteroaryl" refers to an aryl having at least one heteroatom in the ring. The term "heteroaryl" refers to an aryl in which one or more of the carbon rings has been replaced with a heteroatom. The heteroatom is typically selected from nitrogen, oxygen, or sulfur. The ring often has 1 to 3 heteroatoms and typically has 5 or 6 ring members. A heteroaryl can have 1 to 3 optional rings attached or fused to the heterocyclic ring. The additional rings can be aromatic, aliphatic, or a combination thereof, and may or may not contain heteroatoms.

[0019] The term "(hetero)aryl" refers to aryl or heteroaryl.

[0020] The term "linked" refers to atoms within the backbone and / or rings of a compound. In the compound CH3-CH2-CH2-CH(CH3)-CH(CH3)-CH(CH3)-CH2-CH2-CH3, for example, there are 12 carbon atoms, 9 of which are linked.

[0021] The term "grafted" is used to indicate that the polymer chains are covalently attached to the porous polymer substrate. In most embodiments, the polymer chains are grafted to carbon atoms in the polymer backbone of the porous polymer substrate.

[0022] The term "grafting density" refers to the number of millimoles of monomer units grafted to a substrate per gram. The number of millimoles is calculated by dividing the mass gain by the molecular weight of the monomer and multiplying by 1000. This value is then normalized by dividing by the original mass of the substrate in grams. Grafting density is expressed as the number of millimoles of monomer units grafted per gram of substrate (mmol / g). For clarity, grafted materials are typically polymeric materials containing multiple monomer units.

[0023] The term "pKa" refers to the acid dissociation constant. This term indicates the ease with which a proton is released from a molecule.

[0024] The terms "polymer" and "polymeric material" are used interchangeably and refer to a material formed by reacting one or more monomers. The terms include homopolymers, copolymers, terpolymers, etc. Similarly, the terms "polymerize" and "polymerizing" refer to the process of making a polymeric material, which may be a homopolymer, copolymer, terpolymer, etc.

[0025] The terms "in a range of" or "ranging from" are used interchangeably to include all values ​​within a range and the endpoints of that range. DETAILED DESCRIPTION OF THE INVENTION

[0026] Described herein are monomers having nitrogen atoms that can be protonated at relatively low pH values ​​(e.g., below pH 9.5 or below pH 9), polymers containing monomer units derived from these monomers, anion exchange separation articles having the polymers grafted to a porous substrate, and methods of using the anion exchange separation articles to separate mixtures of materials having different ionic contents. Advantageously, the anion exchange separation articles can be used at pHs ranging from about 3.5 or 4 to about 9 or 9.5, for example, and ionic strengths up to about 0.5 moles / liter (e.g., 50 millisiemens). That is, the pH and ionic strength can be selected to provide conditions that can be tolerated by various biomaterials of interest.

[0027] Anion exchange separation articles are formed by grafting multiple polymers (i.e., polymer chains) onto a porous polymer substrate, which is typically a solid material. The grafted polymer contains monomer units containing nitrogen-containing groups that can function as anion exchange ligands. That is, the nitrogen-containing groups are protonatable. The nitrogen-containing monomers used to form the grafted polymers have calculated pKa values ​​ranging from 3.5 or 4 to 9 or 9.5, allowing them to be deprotonated at lower pH values ​​compared to many nitrogen-containing monomers commonly used to prepare anion exchange separation articles. Anion exchange separation articles can be used in flow-through or bind-elute separation methods.

[0028] monomer Monomers having a protonatable nitrogen atom are provided. More specifically, the monomers have nitrogen atoms that can be protonated at low pH, and are typically selected to have a calculated pKa in the range of 3.5 or 4 to 9 or 9.5. The monomers are of formula (I):

[0029] [ka]

[0030] In formula (I), R 1 The group is hydrogen or methyl, and X1 is -O- or -NH-, and R 2 is (hetero)alkylene. The Z group is -NH-(C=O)-, -NH-(C=O)-NH-, -NH-(C=O)-NH-R 3 -, -(C=O)-NH-, or -(C=O)-NH-R 3 -(In the formula, R 3 is an alkylene having at least two linked carbon atoms. 4 Groups and R 5 The groups are each alkylene having at least two carbon atoms, and the nitrogen, R 4 , Q, and R 5 The total number of ring atoms in the ring group consisting of is 6 or 7. The Q group has a single connecting atom, -O-, -N(R 6 )-, -S-, -S(=O)-, or -S(=O)2-, provided that when Z is -NH-(C=O)-, Q is -N(R 6 )-. R 6 The groups are hydrogen, alkyl, or (hetero)aryl, where alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl, and (hetero)aryl is optionally further substituted with hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

[0031] The monomer has the formula CH2=CR 1 -(In the formula, R 1 is hydrogen or methyl).

[0032] X 1 The group is —O or —NH—. X 1 When X is —O—, the monomer of formula (I) is a (meth)acrylate, whereas when X is —NH—, the monomer is a (meth)acrylamide.

[0033] R 2 is (hetero)alkylene. In many embodiments, R 2is alkylene, e.g., having 1 to 20 carbon atoms. 2 has, for example, at least 1, at least 2, at least 3, at least 4, at least 6, at least 8, or at least 10 carbon atoms, and can have up to 20, up to 18, up to 16, up to 14, up to 12, up to 10, up to 8, up to 6, or up to 4 carbon atoms. 2 is of the formula -ROR, where each R is an alkylene having 2 to 10 linked carbon atoms. The alkylene R can have at least 2, at least 3, at least 4, up to 10, up to 8, up to 6, or up to 4 linked carbon atoms.

[0034] Z group is -NH-(C=O)-, -NH-(C=O)-NH-, -NH-(C=O)-NH-R 3 -, -(C=O)-NH-, and -(C=O)-NH-R 3 -(In the formula, R 3 is an alkylene having at least two linked carbon atoms. Suitable alkylene groups often have 2 to 10 carbon atoms, e.g., at least 2, at least 3, at least 4, and up to 10, up to 8, up to 6, or up to 4 carbon atoms. The Z group can form hydrogen bonds and can be beneficial in improving binding efficiency when monomers of Formula (I) are used to form, for example, anion exchange separation articles.

[0035] R 2 The total number of linked atoms in A, B, C, C, D, E, E, F, F, G, G, H, G, I ...

[0036] R 4and R 5 The groups are each alkylene having at least two carbon atoms. 4 , Q, and R 5 The ring group consisting of has 6 or 7 ring members. 4 and R 5 The R group typically has 2 or 3 carbon atoms included in atoms that form a ring (i.e., linked carbon atoms), but not ring atoms. 4 and R 5 There may be additional carbon atoms (i.e., unlinked carbon atoms) in R 4 and R 5 The total number of ring carbon atoms (i.e., connected atoms) in is 4 or 5. Nitrogen, R 4 , Q, and R 5 Ring groups consisting of are typically saturated (ie, there are no carbon-carbon double bonds).

[0037] The Q group is -O-, -N(R 6 )-, -S-, -S(=O)-, or -S(=O)2-, provided that when Z is -NH-(C=O)-, Q is -N(R 6 )-. When Q is -O-, -S-, -S(=O)-, or -S(=O)2-, the nitrogen atom that is part of the same ring and that is attached to the Z group is selected so that it can be protonated. 6 )-, there can be one or two protonated groups in the monomer of formula (I). The Q group has a single linked atom. That is, the Q group is selected from the group consisting of nitrogen, R 4 , Q, and R 5 For example, in the Q group, -N(R 6 )-, nitrogen is a ring atom, and in the cases of -S(=O) and -S(=O)2 in the Q group, sulfur is a ring atom.

[0038] When the nitrogen attached to the Z group is part of a urea group, the ring is -N(R 6 )-group, i.e., when Z is -NH-(C=O)-, Q is -N(R6 For example, in the case of the following monomer, 2-[[4-(2-hydroxyethyl)piperazine-1-carbonyl]amino]ethyl 2-methylprop-2-enoate (also known as IEM / N-(2-hydroxyethyl)piperazine):

[0039] [ka] The nitrogen attached to the -NH-(C=O)- group is part of the urea linking group, and this nitrogen atom is unlikely to be protonated. 6 )-(wherein, R 6 The other nitrogen atom in the ring, in the group (wherein is a hydroxy-substituted alkyl), is more likely to be protonated.

[0040] If the nitrogen attached to the Z group is not part of a urea group (i.e., Z is -NH-(C=O)-), the nitrogen atom bonded to the Z group and -N(R 6 The nitrogen in the )-group can be protonated. For example, in the case of the following monomer, 2-[(4-methylpiperazin-1-yl)carbamoylamino]ethyl 2-methylprop-2-enoate (also called IEM / 1-amino-4-methylpiperazine):

[0041] [ka] Both nitrogen atoms in the ring group are protonatable. The nitrogen bonded to Z(-NH-(C=O)-NH-) is not part of a urea bond but is adjacent to such a bond. The other nitrogen in the ring is -N(R 6 )-group, and R 6 is methyl (e.g., alkyl).

[0042] Q group-N(R 6 )-R in 6 The group R is hydrogen, alkyl, or (hetero)aryl. 6When R is alkyl, it may be optionally further substituted with hydroxy, alkoxy, or (hetero)aryl. 6 When R is (hetero)aryl, it may be optionally further substituted with hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy. 6 The alkyl group has 1 to 10 carbon atoms, e.g., at least 1, at least 2, at least 3, at least 4, and can have up to 10, up to 8, up to 6, or up to 4 carbon atoms. 6 Aryl groups typically have 6 carbon atoms, R 6 Heteroaryl groups often have either 5 or 6 ring atoms, one or two of which are heteroatoms, and the remaining ring atoms are carbon. The heteroatoms are usually nitrogen.

[0043] R 6 When is alkyl, it may be unsubstituted or substituted with hydroxy (—OH), alkoxy, or (hetero)aryl. Alkoxy groups suitable for substitution are of the formula —OR a (In the formula, R a is an alkyl having 1 to 10 carbon atoms, e.g., at least 1, at least 2, at least 3, at least 4, and up to 10, up to 8, up to 6, or up to 4 carbon atoms. (Hetero)aryl groups suitable for substitution can be aryl groups having 6 carbon atoms, or heteroaryl groups having 5 or 6 ring atoms, of which 1 or 2 are heteroatoms and the remainder are carbon. The heteroatom in a heteroaryl group is typically nitrogen.

[0044] R 6When a group is (hetero)aryl, it may be unsubstituted or substituted with hydroxy (-OH), halo (e.g., chloro or bromo), nitro (-NO), cyano (-CN), trifluoromethyl (-CF), alkyl, or alkoxy. Suitable alkyl groups often have 1 to 10 carbon atoms, e.g., at least 1, at least 2, at least 3, at least 4, and up to 10, up to 8, up to 6, or up to 4 carbon atoms. Suitable alkoxy groups for substitution have the formula -OR a (In the formula, R a is an alkyl having 1 to 10 carbon atoms, e.g., at least 1, at least 2, at least 3, at least 4, up to 10, up to 8, up to 6, or up to 4 carbon atoms.

[0045] Monomers of formula (I) can be prepared using any suitable method. Two methods are often used. In the first method, a (meth)acrylate monomer having an isocyanato group is reacted with a cyclic compound having a reactive -NH- or -NH2 group. In the second method, a cyclic compound having a reactive -NH- or -NH2 group is reacted with an alkenyl azlactone.

[0046] In some embodiments, a monomer of Formula (I) can be formed using Reaction Scheme A, in which compound (1), a (meth)acrylate monomer having an isocyanato group, is reacted with a compound having the ring structure shown in compound (2). The -NH- group in compound (2) reacts with the isocyanato group in compound (1) to form compound (3), a monomer having a cyclic nitrogen-containing group (i.e., the cyclic group is a group containing nitrogen, R 4 , Q, and R 5 (having ring members consisting of

[0047] Reaction Scheme A

[0048] [ka]

[0049] R in compounds (1), (2), and (3) 1 , X 1 , R 2 , R 4 , and R 5 The Q group in compounds (2) and (3) is typically -N(R 6 The Z group in formula (I) is —NH—(C═O)— in compound (3).

[0050] In many embodiments of compound (1), X 1 The group is -O- and R 2 is alkylene having 2 to 10 carbon atoms. The number of carbon atoms can be at least 2, at least 3, or at least 4, and up to 10, up to 8, up to 6, or up to 4. In some embodiments, R 2 has 2 or 3 carbon atoms.

[0051] In some methods of making compound (3), the Q group in compound (2) is -NH-, and this group is protected from reacting with compound (1). For example, the -NH group of Q is protected by reacting it with BocO, a compound of formula (CH)C-(C=O)-O-(C=O)-C(CH), to form -NR 7 (In the formula, R 7 can form an intermediate group Q1, which is -(C=O)-C(CH3)3). After reaction with compound (1), -NR 7 The - group can be reacted by heating in the presence of trifluoroacetic acid to reform the -N(H)- group of Q. For example, such a procedure can be used when compound (2) is a piperazine.

[0052] Examples of compound (2) include (A) Q is -N(R 6 )- and R 6 is an alkyl group substituted with a hydroxy group, (B) N-(2-hydroxyethyl)piperazine, (C) Q is -N(R 6 )- and R 6 is an alkyl group, (C) N-methylpiperazine, where Q is -N(R 6)- and R 6 is an aryl group, (D) N-phenylpiperazine, where Q is -N(R 6 )- and R 6 (E) piperazine where Q is —N(H)—; (F) piperazine where Q is —N(R 6 )- and R 6 N-methylhomopiperazine, (G) where Q is -N(R 6 )- and R 6 is a 6-membered heteroaryl having a nitrogen heteroatom, 1-(2-pyridyl)piperazine, (H)Q is —N(R 6 )- and R 6 is a 6-membered heteroaryl having two nitrogen heteroatoms; (I) Q is -N(R 6 )- and R 6 N-(4-hydroxyphenyl)piperazine, where Q is an aryl group substituted with hydroxy, (J) Q is -N(R 6 )- and R 6 N-(4-methoxyphenyl)piperazine, where Q is an aryl group substituted with alkoxy, (K) Q is -N(R 6 )- and R 6 is a nitro-substituted aryl group, N-(4-nitrophenyl)piperazine, (L) Q is -N(R 6 )- and R 6 N-(2-methoxyethyl)piperazine, wherein (M)Q is alkyl substituted with alkoxy, and (M)Q is —N(R 6 )- and R 6 is alkyl substituted with aryl.

[0053] In another embodiment, the monomer of Formula (I) can be formed using Reaction Scheme B, in which compound (1), a (meth)acrylate monomer having an isocyanato group, is reacted with a compound having an —NH group attached to a ring structure, such as shown in compound (4). The product is the monomer, compound (5).

[0054] Reaction Scheme B

[0055] [ka]

[0056] R in compounds (1), (4), and (5) 1 , X 1 , R 2 , R 4 , R 5 and Q groups are the same as those described above. The Z group in formula (I) is -NH-(C=O)-NH- in compound (5).

[0057] Examples of compound (4) include (A) Q is -N(R 6 )- and R 6 (B) 1-amino-4-methylpiperazine, where Q is -N(R 6 )- and R 6 is aryl, and (C) Q is —N(R 6 )- and R 6 is alkyl (i.e., cyclic alkyl).

[0058] In yet another embodiment, the monomer of formula (I) can be formed using Reaction Scheme C, in which compound (1), a (meth)acrylate monomer having an isocyanato group, is bonded to a ring structure such as shown in compound (6). 3 The compound (7) is reacted with a compound having a - group to produce a monomer.

[0059] Reaction Scheme C

[0060] [ka]

[0061] R in compounds (1), (6), and (7) 1, X 1 , R 2 , R 4 , R 5 and Q groups are the same as those described above. The Z group in formula (I) is the same as —NH—(C═O)—NH—R in compound (7). 3 -R 3 The group is alkylene and often has 2 to 10 carbon atoms. For example, R 3 has at least 2, at least 3, at least 4, or at least 6 carbon atoms, and can have up to 10, up to 8, up to 6, up to 4, or up to 2 carbon atoms.

[0062] Examples of compound (6) include (A) Q is -O- and R 3 (B) N-(2-aminoethyl)morpholine, where Q is —O— and R 3 N-(3-aminopropyl)morpholine, (C) Q is -S(O2)-, and R 3 N-(2-aminoethyl)thiomorpholine-1,1-dioxide, (D) Q is -S(O2)-, and R 3 N-(3-aminopropyl)thiomorpholine-1,1-dioxide, (E) Q is -S-, and R 3 N-(2-aminoethyl)thiomorpholine, (F) Q is —O—, and R 3 is alkylene, N-(2-aminoethyl)morpholine, (G) Q is -S(O)-, and R 3 N-(3-aminopropyl)thiomorpholine-1-oxide, (G) Q is —S(O)—, and R 3 N-(2-aminoethyl)thiomorpholine-1-oxide, (H) Q is -S-, and R 3 N-(3-aminopropyl)thiomorpholine, wherein Q is alkylene, and (I) N-(3-aminopropyl)thiomorpholine, wherein Q is —N(R 6 )- and R 6 is alkyl, and R 3 is alkylene.

[0063] In yet other embodiments, the monomer of Formula (I) can be formed using Reaction Scheme D, wherein the alkenyl azlactone, compound (8), has an NH—R bonded to the ring structure. 3 - group to produce the monomer compound (9). Compound (6) is the same as described above for use in Reaction Scheme (C).

[0064] Reaction Scheme D

[0065] [ka]

[0066] R in compounds (8), (6), and (10) 1 , R 3 , R 4 , R 5 The R and Q groups are the same as those described above. 2 The group is typically alkylene, such as, for example, -C(CH3)2- or -C(CH3)2CH2-. The Z group in formula (I) is the same as the -(C=O)-NH-R group in compound (9). 3 Examples of suitable compounds (6) are described above for Reaction Scheme C.

[0067] In a further embodiment, monomers of Formula (I) can be formed using Reaction Scheme E, in which alkenyl azlactone compound (8) is reacted with compound (4) having an NH- group attached to the ring structure to produce monomer compound (10).

[0068] Reaction Scheme E

[0069] [ka]

[0070] R in compounds (8), (4), and (10) 1 , R2 , R 4 , R 5 The R and Q groups are the same as those described above. 2 The group is typically alkylene, such as, for example, -C(CH)- or -C(CH)CH-. The Z group in formula (I) is -(C=O)-NH- in compound (10). Examples of suitable compounds (4) are described above for Reaction Scheme B.

[0071] R 2 The total number of linked atoms in the R and Z groups can be as low as three, and the combined groups preferably have at least four linked atoms, particularly when the monomer of formula (I) is used in forming anion exchange separation articles. 2 The sum of the linked atoms in R and Z determines the length of the spacer group between the alkenyl group (CH=CR- group) and the protonatable nitrogen atom. Typically, the spacer group will have at least five linked atoms, but when monomers of formula (I) are used to form anion exchange separation articles, R is often used to provide a longer spacer length. 2 and Z are selected. Often, R is selected to provide a spacer group having at least 6, at least 7, or at least 8 linked atoms. 2 and Z groups are selected. In some embodiments, the spacer length is at least 9, at least 10, at least 12 atoms, and up to 20, up to 18, up to 16, up to 14, or up to 12 linked atoms. If the spacer group is longer or R 2 A larger total number of linked atoms in the Z group tends to enhance binding to other materials, i.e., monomer units with longer spacer lengths tend to perform better in anion exchange separations.

[0072] In the following monomer, we show how to count the linked atoms.

[0073] [ka]

[0074] In this compound, 2-[(4-phenylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate (referred to herein as IEM / N-phenylpiperazine), there are nine connected atoms between the alkenyl group and the protonatable ring nitrogen. The nitrogen atom that is part of the urea bond, like the nitrogen atom at position 7 in the above compound, is not normally a protonatable nitrogen atom. In this compound, the protonatable nitrogen atom is the nitrogen atom at position 10. However, in the following compound, 2-(3-morpholinopropylcarbamoylamino)ethyl-2-methylprop-2-enoate (referred to herein as IEM / N-(3-aminopropyl)morpholine):

[0075] [ka] The nitrogen atom at position 11 is protonatable.

[0076] The monomers are often selected to have a calculated pKa in the range of 3 to 9.5, or 3.5, or 4 to 9, or 9.5. In some embodiments, the calculated pKa is at least 3.5, at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, or at least 8.0, and up to 9.5, up to 9.0, up to 8.5, up to 8.0, up to 7.5, up to 7.0, up to 6.5, or up to 6.0. The calculated pKa can be used to predict polymers that may be suitable for separating various mixtures of materials, such as, for example, materials containing biomaterials.

[0077] The method and software used to calculate pKa values ​​are presented in the Examples section below. This calculation allows rapid evaluation of whether the pH of a monomer is within an acceptable range prior to polymer chain synthesis and anion exchange separation article preparation. The estimated error in Hammett-Taft pKa calculations often depends on the structural similarity of a new substance to substances present in model databases. Estimates are useful in biological applications because they typically have smaller errors than the acceptable range of pH values ​​for the intended application. Thus, calculated pKa values ​​allow for the rapid selection of a limited number of potential ligands for evaluation for specific separation uses within a desired pH range. This selection can be performed in substantial time savings compared to random synthesis and evaluation of large ligand libraries.

[0078] Literature articles providing information on calculated pKa values ​​include, for example, J.R. Greenwood et al., "Towards the comprehensive, rapid, and accurate prediction of the favorable tautomeric states of drug-like molecules in aqueous solution," Journal of Computer-Aided Molecular Design, 2010, 24, 591-604, and J.C. Shelley et al., "Epik: a software program for pKa prediction and protonation state generation for drug-like molecules," Journal of Computer-Aided Molecular Design, 2007, 21, 681-691.

[0079] Articles with graft polymers Articles can be prepared having multiple polymer chains extending from the surface of a porous polymer substrate. The polymer chains are formed from monomers of formula (I). These articles can be used as anion exchange separation articles. The polymer chains can be homopolymers formed solely from monomers of formula (I), or copolymers formed from a mixture of monomers of formula (I) with other monomers not of formula (I). The other monomers in the mixture are often non-ionic hydrophilic monomers.

[0080] The polymer chains in the article are often grafted to a porous polymer substrate that is solid. The term "solid," when used in reference to a porous polymer substrate, means that the substrate is not a liquid and is not dissolved in a solution. Small particles suspended in a liquid are not considered to be dissolved in the liquid. That is, as used herein, suspensions are not considered solutions, and suspended particles are solids, as that term is used herein. However, in many embodiments, the solid substrate is selected to be not small particles, but larger forms, such as those described further below.

[0081] The pores of the porous polymer substrate may have any desired average size. In some embodiments, the pores are macroporous, mesoporous, microporous, or a mixture thereof. As used herein, the term "macroporous" refers to a polymer substrate having pores with diameters greater than 50 nanometers, the term "mesoporous" refers to a polymer substrate having pores with diameters ranging from 2 nanometers to 50 nanometers, and the term "microporous" refers to a material having pores with diameters less than 2 nanometers.

[0082] The terms "solid porous polymeric substrate," "porous polymeric substrate," "polymeric substrate," "substrate," and similar expressions may be used interchangeably herein.

[0083] The porous polymer substrate may have any desired size, shape, and form. For example, the porous polymer substrate may be in the form of particles, fibers, films, nonwoven webs, woven webs, membranes, sponges, or sheets. In some examples, the polymer substrate is a porous membrane or a porous nonwoven web. To prepare large or multiple separation articles and simplify manufacturing, the polymer substrate may be in the form of a roll of film, nonwoven web, woven web, membrane, sponge, or sheet, or may be formed from a roll. This allows the separation articles to be prepared using roll-to-roll processing. The porous polymer substrate may include a single layer or multiple layers of the same or different polymer materials.

[0084] Porous polymer substrates are often formed from thermoplastic materials. Suitable thermoplastics include, but are not limited to, polyolefins, poly(isoprene), poly(butadiene), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfones), poly(sulfones), poly(vinyl acetate) and its copolymers (e.g., poly(ethylene)-co-poly(vinyl acetate)), polyesters (e.g., poly(lactic acid)), poly(vinyl alcohol) and its copolymers (e.g., poly(ethylene)-co-poly(vinyl alcohol)), poly(vinyl esters), poly(vinyl ethers), poly(carbonates), polyurethanes, poly((meth)acrylates) and their copolymers, and combinations thereof.

[0085] Suitable polyolefins for the porous polymer substrate include poly(ethylene), poly(propylene), poly(1-butene), copolymers of ethylene and propylene, alpha-olefin copolymers (e.g., copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and / or 1-decene), poly(ethylene-co-1-butene), poly(ethylene-co-1-butene-co-1-hexene), poly(butadiene) and copolymers thereof, and combinations thereof.

[0086] Suitable fluorinated polymers for the porous polymer substrate include poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (e.g., poly(vinylidene fluoride-co-hexafluoropropylene)), copolymers of chlorotrifluoroethylene (e.g., poly(ethylene-co-chlorotrifluoroethylene)), and combinations thereof.

[0087] Suitable polyamides for the porous polymer substrate include various nylon compositions, such as poly(iminoadipoyliminohexamethylene), poly(iminoadipoyliminodecamethylene), polycaprolactam, and combinations thereof. Suitable polyimides include poly(pyromellitimide), and combinations thereof.

[0088] Suitable poly(ether sulfones) for the porous polymer substrate include poly(diphenyl ether sulfone), poly(diphenyl sulfone-co-diphenylene oxide sulfone), and combinations thereof.

[0089] Suitable copolymers of vinyl acetate for porous polymeric substrates include copolymers of ethylene and vinyl acetate, and terpolymers of vinyl acetate, vinyl alcohol, and ethylene.

[0090] In some embodiments, the porous polymer substrate is a porous membrane, often with an average pore size (average longest diameter of the pores) greater than 0.1 micrometers to minimize size exclusion separation, minimize diffusional limitations, and maximize surface area and separation. The average pore size can generally be in the range of 0.1 micrometer to 10 micrometers. For example, the average pore size is at least 0.2, at least 0.4, at least 0.6, or at least 0.8 micrometers, and at most 8, at most 6, at most 4, or at most 2 micrometers.

[0091] The porous polymer substrate can be a macroporous membrane, such as a thermally induced phase separation (TIPS) membrane. TIPS membranes are often prepared by forming a solution of a thermoplastic material and a second material above the melting point of the thermoplastic material. During cooling, the thermoplastic material crystallizes and phase separates from the second material. The crystallized material is often stretched. The second material is optionally removed either before or after stretching. Macroporous membranes are further described in U.S. Pat. Nos. 4,539,256 (Shipman), 4,726,989 (Mrozinski), 4,867,881 (Kinzer), 5,120,594 (Mrozinski), 5,260,360 (Mrozinski), and 5,962,544 (Waller, Jr.). Some exemplary TIPS membranes include poly(vinylidene fluoride) (PVDF), polyolefins such as poly(ethylene) or poly(propylene), vinyl-containing polymers or copolymers, such as ethylene-vinyl alcohol copolymers and butadiene-containing polymers or copolymers, and (meth)acrylate-containing polymers or copolymers. TIPS membranes including PVDF are further described in U.S. Pat. No. 7,338,692 (Smith et al.).

[0092] In some embodiments, the porous polymer substrate can include a nylon macroporous film or sheet (e.g., a macroporous membrane), such as those described in U.S. Pat. Nos. 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 (Marinaccio et al.), 3,928,517 (Knight et al.), 4,707,265 (Barnes, Jr. et al.), and 5,458,782 (Hou et al.).

[0093] In other embodiments, the porous polymeric substrate is a nonwoven web, which may include nonwoven webs made by any of the known methods for making nonwoven webs. As used herein, the term "nonwoven web" refers to a fabric having a structure of individual fibers or filaments arranged randomly and / or unidirectionally in a mat-like layered configuration.

[0094] For example, fibrous nonwoven webs can be produced by wet-laid, carded, air-laid, spunlace, spunbond, or meltblowing techniques, or a combination thereof. Spunbond fibers are typically small-diameter fibers formed by extruding a molten thermoplastic polymer in the form of filaments through multiple fine, usually circular, capillaries of a spinneret, where the diameter of the extruded fibers is rapidly reduced. Meltblown fibers are typically formed by extruding a molten thermoplastic material in the form of molten threads or filaments through multiple fine, usually circular, die capillaries into a high-velocity, usually heated, gas (e.g., air) stream, which attenuates the molten thermoplastic filaments and reduces their diameter. The meltblown fibers are then carried by the high-velocity gas stream and deposited on a collecting surface to form a web of randomly dispersed meltblown fibers. Any of the nonwoven webs can be produced from a single type of fiber or from two or more types of fibers differing in thermoplastic polymer type and / or thickness.

[0095] Further details regarding methods for making useful nonwoven webs are found in Wente, "Superfine Thermoplastic Fibers," Indus. Eng. Chem., 48, 1342 (1956) and Wente et al., "Manufacture of Superfine Organic Fibers," Naval Research Laboratories Report No. 4364 (1954).

[0096] The nonwoven web substrate may optionally further comprise one or more scrim layers. For example, either or both major surfaces of the nonwoven web may each optionally comprise a scrim layer. The scrim is typically a woven or nonwoven reinforcing layer made from fibers and is included to impart strength to the nonwoven web. Suitable scrim materials include, but are not limited to, nylon, polyester, fiberglass, polyethylene, polypropylene, and the like. The average thickness of the scrim can vary but is often in the range of about 25 micrometers to about 100 micrometers, preferably about 25 micrometers to about 50 micrometers. The scrim layer may optionally be bonded to the nonwoven article. Various adhesive materials may be used to bond the scrim to the nonwoven. Alternatively, the scrim may be thermally bonded to the nonwoven web.

[0097] The porosity of nonwoven substrates is typically characterized by properties such as fiber diameter, basis weight, or solidity, rather than pore size. The fibers of nonwoven substrates are typically microfibers with effective fiber diameters of at least 0.5, 1, 2, or even 4 micrometers, and up to 15, 10, 8, or even 6 micrometers, as calculated according to the method described in Davies, CN, "The Separation of Airborne Dust and Particles," Institution of Mechanical Engineers, London, Proceedings 1B, 1952. The nonwoven substrate preferably has a pore size of at least 5, 10, 20, or even 50 g / m 2 , and up to 800, 600, 400, 200, or even 100 g / m 2 The nonwoven web has a basis weight within the range of 0.01 to 0.01 mm. The minimum tensile strength of the nonwoven web is about 4.0 Newtons. It is generally recognized that the tensile strength of a nonwoven substrate in the machine direction is lower than the tensile strength in the cross-web direction because better fiber bonding and entanglement occurs in the cross-web direction.

[0098] Nonwoven web loft is measured by solidity, a parameter that defines the solid fraction within the web. Lower solidity values ​​tend to result in higher web loft. Solidity (α) is a unitless fraction and is usually expressed by the following formula: α = m f ÷ρ f ×L nonwoven where m f is the fiber mass per sample surface area, ρ f is the fiber density, L nonwoven is the thickness of the nonwoven. In this specification, solidity refers to the nonwoven substrate itself and does not include the functionalized nonwoven substrate. When the nonwoven substrate contains a mixture of two or more types of fibers, the same L nonwoven The individual solidities are determined using and added together to obtain the web solidity α.

[0099] The polymer chains grafted onto the porous polymer substrate can be homopolymers or copolymers (e.g., the term "copolymer" refers to a polymeric material having at least two different monomer units). To prepare a polymer with a high binding capacity for the desired entrapment material, the polymer chain is often a homopolymer composed of a monomer of formula (I). That is, the polymer chain can contain up to 100 weight percent of the first monomer of formula (I), based on the total weight of the monomers used to form the polymer chain. In some embodiments, another monomer (second monomer) can be copolymerized with the first monomer to adjust the binding capacity of the polymer chain and / or impart other desired properties. Any suitable second monomer can be used, but generally, a hydrophilic monomer is used. For example, these hydrophilic monomers are often water-soluble or water-miscible.

[0100] The amount of the first monomer of Formula (I) can be, for example, 10 to 100 weight percent or 20 to 100 weight percent, based on the total weight of the monomer units in the polymer chain. This amount can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 95 weight percent, based on the total weight of the monomer units in the polymer chain, and can be up to 100, up to 99, up to 98, up to 97, up to 95, up to 90, up to 85, up to 80, or up to 75 weight percent. A higher amount of the first monomer tends to increase the binding ability to various target compounds, such as biomaterials. In many embodiments, the amount of the first monomer of Formula (I) is in the range of 80 to 100, 85 to 100, 90 to 100, or 95 to 100 weight percent, based on the total weight of the monomer units.

[0101] The optional second monomer in the polymer chain can be a hydrophilic monomer, for example, to adjust the degree of hydrophilicity imparted to the substrate or the charge density of the anion exchange separation article. The hydrophilic monomer has an ethylenically unsaturated group and a hydrophilic group, such as a hydroxyl group, an ether group, or an amide group. Suitable hydrophilic monomers include, for example, acrylamide, dimethylacrylamide, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, ethoxyethyl methacrylate, diethylene glycol methyl ether methacrylate, 2-hydroxyethyl acrylamide, N-vinylpyrrolidone, and combinations thereof.

[0102] Other optional second monomers include monomers having two or more ethylenically unsaturated groups. Second monomers of this type are typically water-soluble and are used in relatively small amounts to impart some branching and / or relatively light crosslinking to the resulting copolymer. For example, these multifunctional monomers having two or more ethylenically unsaturated groups may be present in an amount ranging from 0.1 to 25 weight percent, based on the total weight of monomers in the polymerizable composition. The amount may be at least 0.1, at least 0.2, at least 0.5, or at least 1.0 weight percent, and may be up to 25, 20, 15, 10, 5, 4, 3, 2, or 1 weight percent. Crosslinking monomers are acceptable and may be beneficial in some applications, but tend to reduce binding capacity to some biomaterials.

[0103] Examples of suitable cross-linking monomers include, but are not limited to, poly(ethylene glycol di(meth)acrylate, methylene bisacrylamide, 3-acryloyloxy-2-hydroxypropyl methacrylate, glycerol dimethacrylate, glycerol diacrylate, diacryloylpiperazine, and 1,2-ethylene bisacrylamide.

[0104] The total amount of the second monomer can be up to 80 weight percent of the monomers used to form the polymer chain. A smaller amount of the second monomer usually enhances the binding ability to various target compounds, such as protein biomaterials. When present, its amount is usually equal to 100 - (weight percent of the first monomer of formula (I) relative to the total weight of the monomers in the polymerizable composition).

[0105] In some embodiments, the polymerizable composition contains 10 weight percent to 90 weight percent of a first monomer of Formula (I) and 90 weight percent to 10 weight percent of a second hydrophilic monomer. For example, the polymerizable composition can contain 20 to 90 weight percent of a first monomer and 80 to 10 weight percent of a second monomer, 10 to 80 weight percent of a first monomer and 90 to 20 weight percent of a second monomer, 30 to 90 weight percent of a first monomer and 70 to 10 weight percent of a second monomer, 30 to 80 weight percent of a first monomer and 70 to 20 weight percent of a second monomer, 30 to 70 weight percent of a first monomer and 70 to 30 weight percent of a second monomer, 40 to 90 weight percent of a first monomer and 60 to 10 weight percent of a second monomer, or 50 to 90 weight percent of a first monomer and 50 to 10 weight percent of a second monomer.

[0106] The polymer chains are grafted onto the porous polymer substrate. Any suitable grafting method can be used. In many embodiments, a type II photoinitiator is combined with a monomer composition to form a reaction mixture. When the reaction mixture is exposed to ultraviolet light, the type II photoinitiator abstracts hydrogen atoms from the porous polymer substrate, thereby generating free radicals on the porous polymer substrate. The free radicals react with the monomers present in the composition, resulting in the formation of polymer chains grafted to the porous polymer substrate. The polymer chains are often grafted to carbon atoms in the backbone of the polymer material contained in the porous polymer substrate.

[0107] Type II photoinitiators are usually aromatic ketone compounds. Examples include, but are not limited to, benzophenone, carboxybenzophenone (e.g., 3-carboxybenzophenone), 4-(3-sulfopropyloxy)benzophenone sodium salt, Michler's ketone, benzil, anthraquinone, 5,12-naphthacenequinone, aceanthracenequinone, benz(A)anthracene-7,12-dione, 1,4-chrysenequinone, 6,13-pentacenequinone, 5,7,12,14-pentacenetetrone, 9-fluorenone, anthrone, xanthone, thioxanthone, 2-(3-sulfopropyloxy)thioxanthen-9-one, acridone, dibenzosuberone, acetophenone, and chromone.

[0108] The ultraviolet (UV) light used to generate free radicals on the porous polymer substrate can be provided by a variety of light sources, such as light emitting diodes (LEDs), black lights, medium pressure mercury lamps, etc., or combinations thereof. Actinic radiation (e.g., UV radiation) can also be provided using high intensity light sources available, for example, from Fusion UV Systems Inc. Ultraviolet light sources typically have an intensity of 10 mW / cm. 2 The radiation source may be a relatively low intensity light source, such as a black light, that emits light over a wavelength range of 280 nanometers to 400 nanometers (e.g., as measured using a UVIMAP UM 365 LS radiometer manufactured by Electronic Instrumentation & Technology, Inc., Sterling, VA, using National Institute of Standards and Technology-approved procedures) or, typically, 10 mW / cm. 2 above 15 mW / cm 2 ~450mW / cm 2 A relatively high intensity light source can be used, such as a medium pressure mercury lamp providing an intensity of 1000 kJ / s. Exposure times can be up to about 30 minutes or even longer.

[0109] In another useful method for generating free radicals on the surface of a porous polymer substrate, the substrate itself is selected as the photoactive material, and no Type II photoinitiator is required. A monomer composition is exposed to actinic radiation, typically in the ultraviolet region of the electromagnetic spectrum. When exposed to actinic radiation, the polymer substrate absorbs enough energy to break some of its covalent bonds, generating free radicals that can react with the monomers to form polymer chains. Examples of photoactive polymer substrates include polysulfone and poly(ether sulfone). Other photoactive polymer substrates often contain aromatic groups, such as homopolymers and block copolymers of poly(methylphenylsilane) and various polyimides based on benzophenonetetracarboxylic dianhydride.

[0110] Other methods for generating free radicals on the surface of a polymer substrate use ionizing radiation rather than Type II photoinitiators and / or UV irradiation. As used herein, the term "ionizing radiation" refers to radiation of sufficient dose and energy to form free radical reaction sites on the surface and / or in the bulk of the polymer substrate. The radiation has sufficient energy if it is absorbed by the polymer substrate, resulting in the cleavage of chemical bonds in the substrate and the formation of free radicals. The ionizing radiation is often beta radiation, gamma radiation, electron beam radiation, X-ray radiation, plasma radiation, or other suitable types of electromagnetic radiation. Preferably, the ionizing radiation is administered in an inert environment to prevent oxygen from reacting with the radicals.

[0111] In many embodiments of this method, the ionizing radiation is electron beam radiation, gamma radiation, X-ray radiation, or plasma radiation, due to the readily available availability of suitable generators. Commercially available electron beam generators include the ESI ELECTROCURE EB SYSTEM manufactured by Energy Sciences, Inc. (Wilmington, MA, USA) and the BROADBEAM EB PROCESSOR manufactured by E-beam Technologies (Davenport, IA, USA). Commercially available gamma radiation generators include those manufactured by MDS Nordion that use a Cobalt-60 high-energy source.

[0112] For any given type of ionizing radiation, the delivered dose can be measured according to ISO / ASTM 52628-13, "Standard Practice for Dosimetry in Radiation Processing" by ASTM International (West Conshohocken, PA). Various dose rates can be obtained by varying the extractor grid voltage, beam diameter, exposure time, and distance from the irradiation source.

[0113] Multiple polymer chains are grafted to a porous polymer substrate. The term "ligand density" refers to the number of millimoles per gram of monomer units grafted to the substrate. The number of millimoles is calculated by dividing the mass gain by the molecular weight of the monomer and multiplying by 1000. This value is then normalized by dividing by the original mass (grams) of the porous polymer substrate. The ligand density (mmol / gram) is expressed as the number of millimoles of grafted monomer units per gram of substrate. For clarity, grafted materials are typically polymeric materials containing multiple monomer units.

[0114] When the substrate is a membrane, the anion exchange separation article often has a ligand density of about 0.02 mmol / gram to about 3 mmol / gram or more. The grafting density can be at least 0.02, at least 0.05, at least 0.1, at least 0.2, at least 0.5, or at least 1 mmol / gram, and can be up to 3, up to 2.5, up to 2, up to 1.5, up to 1, up to 0.8, up to 0.7, or up to 0.5 mmol / gram. The weight gain is calculated from the formula [100(wt2 - wt1) ÷ wt1], where wt1 is the weight of the substrate and wt2 is the weight of the substrate with the grafted polymer attached. The weight gain can range from 1 weight percent to 85 weight percent or more. The amount can be, for example, at least 1, at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 weight percent, and up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, or up to 30 weight percent.

[0115] When the substrate is a nonwoven or fibrous substrate, the weight gain upon grafting can often be higher than that of the membrane substrate. The weight gain can range from 20 weight percent to 400 weight percent or more. The amount can be, for example, at least 20, at least 50, at least 100, at least 150, at least 200, at least 250, or at least 300 weight percent, and up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 75, or up to 50 weight percent. For example, the weight gain can be in the range of 100 weight percent to 400 weight percent, 100 weight percent to 300 weight percent, or 100 weight percent to 200 weight percent.

[0116] Method for separating mixtures of materials using anion exchange separation articles An anion exchange separation article can be used to separate a mixture of materials having different ionic contents. The anion exchange separation article is the same as that described above. The method includes passing a mixture of materials through the anion exchange separation article at a first pH sufficiently low to protonate the monomer repeat units of the graft polymer derived from the monomer of formula (I) and at a first ionic strength value that causes at least one component of the mixture of materials to bind to the anion exchange separation article as a binding moiety.

[0117] The first pH is selected so that at least a portion of the monomer repeat units of the graft polymer derived from the monomer of Formula (I) are protonated. The protonated portion is often at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or at least 60 mole percent, and up to 100, up to 95, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, or up to 30 mole percent. The pH can be optimized using experimental approaches known to those skilled in the art and / or as demonstrated in the Examples section below. If desired, the percentage of protonated monomer units can be determined by first determining the actual pKa of the anion exchange separation article by potentiometric titration, and then substituting the pKa and pH values ​​into the Henderson-Hasselbalch equation to calculate the ratio of protonated to unprotonated units.

[0118] Typically, the pH is selected to be lower than the pKa value of the monomer of formula (I) used in preparing the graft polymer to be attached to the porous polymer substrate. When the pH is lower than the pKa value, the monomer units derived from the monomer of formula (I) are positively charged. Under these lower pH conditions, the graft polymer can bind to negatively charged species. When the pH is higher than the pKa value, the monomer units derived from the monomer of formula (I) in the graft polymer are neutralized, and the bound material can be released. The monomer of formula (I) often has a calculated pKa value ranging from about 3.5 or 4 to about 9 or 9.5. Therefore, the composition of the graft polymer can be adjusted to optimize the pKa value for the separation of different mixtures. That is, the composition and pH of the graft polymer can be adjusted to either (a) bind the substance of interest and not bind impurities, or (b) bind impurities and not bind the substance of interest.

[0119] As described above, the pH can be adjusted so that the substance of interest, but not the impurities, is retained as the sample passes through the anion exchange separation article. After the sample has passed through, the anion exchange separation article can optionally be washed to remove any remaining impurities. The substance of interest can then be released from the anion exchange separation article by increasing the pH and / or ionic strength of the composition passed through the anion exchange separation article. This method is sometimes referred to as a bind-and-release (i.e., elution) method. In some embodiments of the bind-and-release method, the pH and / or ionic strength can be increased gradually or in steps to further separate the mixture of bound materials.

[0120] Alternatively, the pH can be adjusted as the sample passes through the anion exchange separation article so that impurities are retained but the substance of interest is not. This method is sometimes referred to as a flow-through separation method. If desired, the retained impurities can be released from the anion exchange separation article by increasing the pH and / or ionic strength of the composition that passes through the anion exchange separation article after passing the substance of interest. Removal of the retained impurities allows the anion exchange separation article to be reused for separating other mixtures of materials. Alternatively, the impurities are not released and the anion exchange separation article is discarded after use. In this case, the anion exchange separation device is sometimes referred to as a single-use device.

[0121] Many substances that are desired to be separated are biological materials that have an isoelectric point (pI). At pH values ​​above the pI value, the biological material has a net negative charge and can bind to the positively charged grafted polymer; at pH values ​​below the pI value, the biological material has a net positive charge and is not attracted to the positively charged grafted polymer. Examples of biological materials that can be bound include, but are not limited to, proteins, nucleic acids, nucleic acid fragments, cells, viruses, and virus-like particles.

[0122] The anion exchange separation devices described herein are well suited for the separation of many biomaterials that cannot tolerate the high pH conditions (e.g., pH 10, 11, or higher, depending on the biomaterial) or low pH conditions (e.g., pH 5, 4, or lower, depending on the biomaterial) and / or high salt concentrations (e.g., 0.5 M or higher, e.g., 1.0 M) commonly used in many current anion exchange separation articles. Monomers of formula (I) can be used to form graft polymers on anion exchange separation articles that can be used under pH conditions of pH 11 or less than pH 10, e.g., pH 3.5 or in the range of 4 to 9 or 9.5. For example, the pH may be reduced to 3.5 or lower, or may be reduced to 4, 4.5, 5.0, 5.5, 6.0, 6.5, 7, 7.5, or 8, or may be increased to 11 or higher, or may be increased to 10.5, 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, or 5.

[0123] Furthermore, these anion exchange separation articles can bind and elute biological materials at salt concentrations of 0.5 M or less, e.g., in the range of 0.01 M to less than 0.5 M. The salt concentration can be, for example, at least 0.01 M, at least 0.02 M, at least 0.05 M, at least 0.07 M, at least 0.1 M, at least 0.15 M, or at least 0.2 M, or can be up to 0.5 M, up to 0.45 M, up to 0.4 M, up to 0.35 M, up to 0.3 M, up to 0.25 M, up to 0.2 M, up to 0.15 M, up to 0.1 M, or up to 0.05 M. Salt concentrations are often expressed in terms of conductivity. For example, conductivity is often 50 mS / cm (millisiemens per centimeter) or less, e.g., in the range of 1 mS / cm to 50 mS / cm. That is, the conductivity is at least 1, at least 2, at least 3, at least 5, or at least 10 mS / cm, and can be up to 50, up to 40, up to 30, up to 20, up to 10, or up to 5 mS / cm.

[0124] Non-grafted polymers having monomer units derived from monomers of formula (I) The polymer can be formed from a monomer of formula (I) that is not grafted to a porous polymer substrate as described above. The polymer can be a homopolymer or copolymer having additional monomer units that are not of formula (I). For example, the polymer can be prepared so that it is soluble in a polar solvent and can be coated onto a substrate, either porous or non-porous, as desired. The polymerization process can be carried out using any known method. In most polymerization processes, a radical initiator is used, which can be a thermal initiator or a photoinitiator. In many embodiments, a photoinitiator is used.

[0125] Useful photoinitiators include benzoin ethers, such as benzoin methyl ether and benzoin isopropyl ether, substituted acetophenones, such as 2,2-dimethoxyacetophenone available as Irgacure 651 photoinitiator (Ciba Specialty Chemicals), 2,2-dimethoxy-2-phenyl-1-phenylethanone available as Esacure KB-1 photoinitiator (IGM Resins, Charlette, NC), and 1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one available as Irgacure 2959 (Ciba Specialty Chemicals). Chemicals), dimethoxyhydroxyacetophenone, substituted α-ketols such as 2-methyl-2-hydroxypropiophenone, aromatic sulfonyl chlorides such as 2-naphthalene-sulfonyl chloride, and photoactive oximes such as 1-phenyl-1,2-propanedione-2-(O-ethoxy-carbonyl)oxime.

[0126] Other useful photoinitiators include, for example, hydrogen abstraction (Type II) photoinitiators such as benzophenone, 4-(3-sulfopropyloxy)benzophenone sodium salt, Michler's ketone, benzil, anthraquinone, 5,12-naphthacenequinone, aceanthracenequinone, benz(A)anthracene-7,12-dione, 1,4-chrysenequinone, 6,13-pentacenequinone, 5,7,12,14-pentacenetrone, 9-fluorenone, anthrone, xanthone, thioxanthone, 2-(3-sulfopropyloxy)thioxanthen-9-one, acridone, dibenzosuberone, acetophenone, and chromone.

[0127] Examples of suitable thermal initiators include peroxides, such as benzoyl peroxide, dibenzoyl peroxide, dilauryl peroxide, cyclohexane peroxide, methyl ethyl ketone peroxide, hydroperoxides (e.g., tert-butyl hydroperoxide and cumene hydroperoxide), dicyclohexyl peroxydicarbonate, t-butyl perbenzoate, 2,2-azo-bis(isobutyronitrile), and the like, or combinations thereof. Examples of commercially available thermal initiators include those available from Chemours (Wilmington, Delaware) under the VAZO trade name, including VAZO 67 (2,2'-azo-bis(2-methylbutyronitrile)), VAZO 64 (2,2'-azo-bis(isobutyronitrile)), and VAZO 52 (2,2'-azo-bis(2,2-dimethylvaleronitrile)), and those available from Elf Atochem North America (Philadelphia, PA) under the trade name Lucidol 70 (benzoyl peroxide).

[0128] The initiator can be used in an amount effective to initiate free radical polymerization of the monomers. Such an amount will vary depending, for example, on the type of initiator utilized and the polymerization conditions. The initiator can generally be used in an amount ranging from about 0.01 parts by weight to about 5 parts by weight per 100 parts of total monomers.

[0129] The polymerization solvent can be essentially any solvent capable of substantially dissolving (or dispersing or suspending, in the case of emulsion or suspension polymerization) the monomers (and comonomers, if used). In many embodiments, the solvent can be water or a water / water-miscible organic solvent mixture. The water to organic solvent ratio can vary widely, depending on the solubility of the monomers. For some monomers of Formula (I), the water to organic solvent ratio (volume / volume) can be greater than 1:1, e.g., greater than 5:1, greater than 7:1, or greater than 10:1. If desired, a higher proportion (in some cases, up to 100 percent) of organic solvent can be used, such as when the organic solvent is an alcohol.

[0130] Any such water-miscible organic solvent preferably does not have groups that retard polymerization. In some embodiments, the water-miscible solvent may be a protic group-containing organic liquid, such as a lower alcohol having 1 to 4 carbon atoms, a lower glycol having 2 to 6 carbon atoms, or a lower glycol ether having 3 to 6 carbon atoms and 1 to 2 ether linkages. In some embodiments, higher glycols such as poly(ethylene glycol) may be used. Specific examples include methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, ethylene glycol, methoxyethanol, ethoxyethanol, propoxyethanol, butoxyethanol, methyl carbitol, ethyl carbitol, and the like, and combinations thereof.

[0131] In other embodiments, aprotic water-miscible organic solvents can be used, including aliphatic esters (e.g., methoxyethyl acetate, ethoxyethyl acetate, propoxyethyl acetate, butoxyethyl acetate, and triethyl phosphate), ketones (e.g., acetone, methyl ethyl ketone, and methyl propyl ketone), and sulfoxides (e.g., dimethyl sulfoxide).

[0132] The monomer concentration in the polymerization solvent can vary depending on various factors, including, but not limited to, the nature of the monomer, the desired degree of polymerization, the reactivity of the monomer, and the solvent used. Typically, the monomer concentration can range from about 0.1 weight percent to about 60 weight percent or more, based on the total weight of the monomer and solvent. For example, the monomer concentration can be at least 0.1, at least 0.5, at least 1, at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 weight percent, and can be up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, or up to 20 weight percent, based on the total weight of the monomer and solvent.

[0133] Any of the optional second monomers described above for the grafted polymer can be included in the reaction mixture used to form the non-grafted polymer.

[0134] The times and temperatures used for polymerization reactions using thermal initiators can be any of those used in the art. Similarly, the times and radiation sources suitable for use with photoinitiators can be any of those used in the art.

[0135] The aqueous monomer mixture, optionally formulated with a relatively high level of multifunctional (crosslinking) monomer or comonomer (e.g., from about 5 weight percent (%) to about 90 weight percent based on the total weight of monomer and comonomer), can be polymerized as a suspension or dispersion in a non-polar, immiscible organic solvent, optionally in the presence of an added porogen, to produce crosslinked porous particles comprising monomer units derived from the monomer of Formula (I). Such methods are described, for example, in U.S. Patent Nos. 7,098,253 (Rasmussen et al.), 7,674,835 (Rasmussen et al.), 7,647,836 (Rasmussen et al.), and 7,683,100 (Rasmussen et al.). [Example]

[0136] pKa Calculation pKa values ​​were calculated using the EpiK pKa prediction tool with the Maestro GUI, both obtained from Schrodinger LLC (New York, NY). Calculations were performed on monomeric species, with HO adjusted to pH 9 as the solvent, using the "Sequential pKa Values" setting in Maestro version 2021-4, allowing for tautomerization. The EpiK pKa prediction tool used Hammett and Taft relationships to predict protonation states based on the structure of functional groups and their sensitivity to perturbations from other parts of the molecule. The model was empirically data-driven and trained based on a combination of publicly reported pKa values ​​and proprietary in-house measurement data from Schrodinger.

[0137] The Hammett and Taft relationship (Equation 1) states that each chemical group has a reference pKa

[0138]

number

[0139]

number

[0140] In the formula, n HA denotes the number of equivalent H atom removals from an acidic molecule, while n HR indicates the number of ways to add H to the conjugate base. RA is a correction term for the aliphatic ring. The uncertainty of the pKa value is

[0141]

number

[0142]

number

[0143] Calculated pKa values ​​for some representative monomers of the present disclosure are shown in Table 1.

[0144] [Table 1-1]

[0145] [Table 1-2]

[0146] [Table 1-3]

[0147] [Table 1-4]

[0148] material N-(2-hydroxyethyl)piperazine, 1-amino-4-methylpiperazine, N-phenethylpiperazine, 1-(2-pyridyl)piperazine, 1-(2-pyrimidyl)piperazine, and ethylenediaminetetraacetic acid (EDTA) were obtained from Alfa Aesar, Ward Hill, MA.

[0149] N-(2-aminoethyl)morpholine and N-(3-aminopropyl)morpholine were obtained from TCI America, Portland, OR.

[0150] N-phenylpiperazine, N-methylpiperazine, 2-hydroxyethylmethacrylate (HEMA), 2-morpholinoethane-1-sulfonic acid, 3-morpholinopropane-1-sulfonic acid, tris(hydroxymethyl)aminomethane, N-cyclohexyl-2-aminoethane-1-sulfonic acid, and 4-hydroxy-TEMPO were obtained from Sigma-Aldrich Company, St. Louis, MO.

[0151] Phosphate-buffered saline (PBS, 1×), Dulbecco's Modified Eagle Medium (DMEM), trypan blue (0.4% solution), and fetal bovine serum (FBS) were obtained from ThermoFisher Scientific, Waltham, MA.

[0152] 1-Boc-piperazine was obtained from Oakwood Chemical, Estill, SC.

[0153] 2-Vinyl-4,4-dimethylazlactone (VDM) was obtained from SNPE, Inc., Princeton, NJ and redistilled before use.

[0154] 2-Isocyanatoethyl methacrylate (IEM, Karenz MOI) and 2-isocyanatoethyl acrylate (IEA, Karenz AOI) were obtained from Showa Denko K.K., Tokyo, Japan.

[0155] 3-Carboxybenzophenone was obtained from Sigma-Aldrich Company. A solution of 3-carboxybenzophenone sodium salt (C-BP) (0.033 g / mL) was prepared by dissolving 3-carboxybenzophenone in 1 M sodium hydroxide and diluting with deionized water.

[0156] Method 1. Preparation of polymer-grafted membranes Monomer grafting solutions (5 grams each) were prepared at various monomer concentrations in deionized water relative to the measured percent solids of the monomer solution. Each monomer solution also contained 3-carboxybenzophenone sodium salt (62.5 microliters of an aqueous solution with a concentration of 0.033 g / mL). For each grafting solution, a nylon membrane substrate (#080ZN, reinforced nylon 6,6 membrane, nominal pore size 0.8 micrometers, obtained from 3M Company, St. Paul, MN) was placed on a sheet of polyester film, and enough grafting solution was pipetted onto the top surface of the substrate to completely wet it. The coating solution was allowed to soak into the substrate for approximately 1 minute, and then a second sheet of polyester film was placed on top of the substrate. A 2.28 kg cylindrical weight was rolled over the resulting three-layer sandwich to squeeze out excess coating solution. Ultraviolet (UV)-initiated grafting was performed by irradiating the sandwich using a UV stand (Classic Manufacturing, Inc., Oakdale, MN). The UV stand was equipped with 18 fluorescent lamps (Sylvania RG2 40W F40 / 350BL / ECO type), with 10 lamps positioned above the substrate and 8 below. Each lamp was 1.17 meters (46 inches) long, spaced 2 inches (5.1 cm) apart on center, and the exposure time was 15 minutes. The polyester sheet was removed, and the resulting grafted membrane was placed in a polyethylene bottle. The bottle was filled with 0.9% saline, sealed, and placed on a laboratory bottle roller for 30 minutes to wash away any residual monomer or ungrafted polymer. The saline was discarded and replaced with deionized water for another 30 minutes of washing. The washing was repeated with fresh 0.9% saline for 30 minutes, followed by two 30-minute washes with deionized water. After the washing process, the polymer-grafted membrane was air-dried. The graft density of the polymer-grafted membrane was estimated based on the mass gain.

[0157] Method 2: Bovine Serum Albumin (BSA) Static (Equilibrium) Binding Capacity Individual disks (16 mm in diameter) of polymer-grafted membrane were die-punched from sheets of polymer-grafted membrane. A single disk was placed in a 5 mL centrifuge tube containing 4.5 mL of bovine serum albumin (product number A-7906, Sigma-Aldrich) prepared at a concentration of approximately 4 mg / mL in buffer adjusted to pH 5.0, 6.0, 7.0, 8.0, or 9.0. Each tube was capped and rotated overnight (typically 14 h) on a rotating mixer. The resulting supernatant solution was analyzed using a UV-VIS spectrometer at 280 nm (background correction was applied at 325 nm). The static binding capacity of each disk was determined by comparing the absorbance of the supernatant solution with that of the starting BSA solution. Results were reported in mg / mL as the amount of BSA bound per mL of membrane volume and were reported as the average of three replicate measurements.

[0158] All buffers were prepared using deionized water. The pH 5.0 buffer was acetate buffer (10 mM). The pH 6.0 buffer was MES (2-morpholinoethane-1-sulfonic acid) buffer (25 mM). The pH 7.0 buffer was MOPS (3-morpholinopropane-1-sulfonic acid) buffer (10 mM). The pH 8.0 buffer was TRIS (tris(hydroxymethyl)aminomethane) buffer (25 mM). The pH 9.0 buffer was CHES (N-cyclohexyl-2-aminoethane-1-sulfonic acid) buffer (20 mM). Buffers were prepared by mixing the appropriate amounts of acid and base forms of buffer salts to achieve the desired pH. The formulas for the preparation of the individual buffer solutions were determined using the "Calculator for pH Buffers" software tool provided on the website of the Proteome Research Centre at the University of Liverpool (website: www.liverpool.ac.uk / pfg / Tools / BufferCalc / Buffer.html). To further increase the ionic strength, an appropriate amount of sodium chloride was added.

[0159] Method 3. Preparation of Filtration Capsules Plastic filtration capsules were used to test a portion of the grafted membrane. Each capsule consisted of a sealed circular housing. The capsule housing was prepared in two halves (top and bottom halves), which were joined together and the grafted membrane was inserted into the inner cavity of the lower housing, then the halves were joined together to seal the exterior. The fluid inlet and vent ports were located at the top of the housing, and the fluid outlet port was located at the bottom of the housing. The outlet port was centered in the middle of the lower housing surface.

[0160] Experimental capsules were prepared as follows: A single disk of grafted membrane (15.9 mm diameter) was placed at the bottom of the lower housing and covered with two polypropylene rings (15.9 mm outer diameter, 13.9 mm inner diameter, 1.3 mm thick) and a silicone gasket (15.9 mm outer diameter, 9.5 mm inner diameter, 3 mm thick). The upper and lower housings were mated together and ultrasonically welded using a Branson 20 kHz ultrasonic welder (Model 2000xdt, Emerson Electric Company, St. Louis, MO) to form the completed filtration capsule. The overall outer diameter of the completed capsule was approximately 4.3 cm, and the overall height, including the inlet, outlet, and vent ports, was approximately 4.8 cm. The effective filtration area of ​​the capsule was 1.0 cm. 2 , the bed volume of the medium was 0.3 mL.

[0161] Method 4. Preparation of 96-well centrifuge test devices for membrane samples Two 7.5 mm disks of grafted membrane (prepared as described in Method 1) were inserted into each well of a 96-well EMPORE filter plate (product number 6065, 3M Company) from which the original solid-phase extraction material had previously been removed. The filter disks were held in place by appropriately sized plastic O-rings to provide a robust seal such that when phosphate buffered saline (PBS) (1×) was applied to the top of the membrane layer in each well, liquid did not flow through the membrane when the plate was placed on a horizontal surface for 10 minutes. In operation, liquid flow through the membrane was achieved by centrifugation of the plate.

[0162] Method 5. Preparation of Phi6 Virus Stock Culture The Phi6 bacteriophage (DSMZ 21518) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany. Virus cultures were generated by inoculating 100 mL of tryptic soy broth and 5 mM magnesium sulfate (Hardy Diagnostics, Santa Maria, CA) with 1.5 mL of an overnight culture of the host bacteria Pseudomonas syringae (DSMZ 21482). The cultures were grown at 25°C with shaking at 210 revolutions per minute (rpm) for 2 hours. The cultures were then inoculated with 10 mL of Phi6 virus. 9 Plaque forming units (pfu) were inoculated. The inoculated culture was grown for an additional 3 hours at 25°C with shaking at 210 rpm. Cells were removed by centrifugation at 3700 x g and the supernatant was filtered through a 0.2 micron PES membrane filter.

[0163] Method 6. Determining Phi6 virus concentration by plaque assay Phi6 virus samples were serially diluted (10-fold). Fifty microliters of an overnight culture of Pseudomonas syringae host bacteria and 100 microliters of diluted Phi6 virus were mixed with molten tryptic soy top agar (2.5 mL of tryptic soy broth containing 5 mM MgSO4 and 0.9% agar). The mixture was poured onto a standard tryptic soy agar plate and incubated overnight at 25°C. After incubation, plaque-forming units (pfu) were counted. The number of pfu correlated with the number of virus particles. The virus particle concentration (particles / mL) was calculated from the pfu count adjusted for dilution.

[0164] Method 7. Preparation of Phi6 virus stock from agar plates An overnight culture of Pseudomonas syringae (DSMZ21482) was prepared by picking a single colony into 5-7 mL of liquid tryptic soy broth and incubating overnight at 25°C in a shaking incubator (210-250 rpm). A 50-microliter aliquot of the overnight culture broth was added to molten soft tryptic soy top agar (5 mL of tryptic soy broth containing 5 mM MgSO4 and 0.75% agar). The molten agar was poured onto a standard tryptic soy agar plate and allowed to solidify. 100 microliters of 1E+09 pfu / mL stock Phi6 bacteriophage stock solution (DSMZ PN21518) was spread onto the solidified top agar plate and incubated overnight at room temperature (approximately 22-25°C). Produced virus was recovered from the agar by scraping the top agar layer from the plate into a 50 mL conical tube, adding 20 mL of phage storage buffer (50 mM NaHPO, 22 mM KHPO, 85.5 mM NaCl, 1 mM MgSO, 1 mM CaCl), and vortexing for 15–30 min to release the virus into the buffer. The mixture was then centrifuged at 3,000 × g for 15 min and filtered through a 0.2 μm PES membrane.

[0165] Method 8. Determination of Phi6 virus concentration by surface drop method Phi6 virus samples were serially diluted (10-fold) in 96-well plates (up to a 100,000x dilution). Melted soft tryptic soy top agar supplemented with MgSO4 (5 mL of tryptic soy broth containing 5 mM MgSO4 and 0.9% agar) was mixed with 50 microliters of an overnight culture of Pseudomonas syringae host bacteria and poured onto a standard tryptic soy agar plate. After the agar solidified, 3 microliters of each dilution was spotted in an array onto the surface of the prepared plate. The plate was incubated overnight at 25°C. After incubation, virus was quantified by counting particle-forming units in the 3-microliter spots at the highest dilution at which plaques were countable. Virus particle concentrations (particles / mL) were calculated from pfu counts adjusted for dilution.

[0166] Method 9. Preparation and Characterization of Lentiviral Cultures GFP reporter gene-containing lentivirus was generated using LV-MAX virus-producing suspension cells (product number A35347, ThermoFisher Scientific) with the GIBCO LV-MAX Lentiviral Production System (product number A35684, ThermoFisher Scientific). Cells were transfected with the pLenti-GFP control vector (product number CALTV-400, Cell Biolabs San Diego) and the LV-MAX lentiviral packaging mix plasmid (product number A43237, ThermoFisher Scientific) according to the manufacturer's instructions for the LV-MAX lentiviral production system. Cells were maintained in LV-MAX culture medium, consisting of GIBCO LV-MAX Production Medium (product number A3583402, ThermoFisher Scientific) supplemented with GIBCO penicillin-streptomycin (product number 15140122, ThermoFisher Scientific) at the recommended 1x concentration. The cell density and viability of LV-MAX suspension cells were estimated by manually counting trypan blue-stained cell samples using a hemocytometer. Three days after transfection, the culture medium was centrifuged at 1300 × g for 15 minutes, and the lentivirus-containing supernatant was collected by filtering the supernatant through a 0.45-micron PES filter (product number 295-4545, ThermoFisher Scientific).

[0167] Method 10. Lentiviral Titer Determination Lentiviral titers were determined using a titration assay for transducing units / mL (TU / mL). LentiX-293T cells (product number 632180, Takara Bio USA, Mountain View, CA) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol / vol) fetal bovine serum (FBS) and GIBCO penicillin-streptomycin and seeded at a density of 10,000 cells / well in black-walled 96-well culture plates. After cell attachment, test samples were serially diluted in culture medium (product number TR1003G, Sigma-Aldrich) supplemented with 8 micrograms / mL of polybrene, and the cell medium was removed and replaced with medium containing the serial dilutions. The plates were then centrifuged at 900 × g for 30 minutes, followed by incubation for 96 hours. The "% GFP-positive cells" were measured using a BioTek Cytation 5 imaging reader (Agilent Technologies, Santa Clara, CA). Settings and focus for the GFP channel and gain were adjusted for the plate / cells used. All plates were scanned on the same day with the same settings. Using wells that read "% GFP-positive cells" between 1% and 20%, the formula TU / mL = [fraction of GFP-positive cells] was used. * The titer was calculated according to [10,000 cells seeded / well] / [original virus sample added to well (mL)].

[0168] Method 11. Determination of DNA concentration DNA was extracted from collected samples using a 96-well format DNA extraction kit (product number D100W, Cygnus Technologies, Southport, NC) according to the manufacturer's instructions. The extracted DNA was then quantified by fluorescence using the QUANT-IT PICOGREEN dsDNA Assay Kit (product number P7589, ThermoFisher Scientific) according to the manufacturer's instructions.

[0169] Method 12. Conductivity and pH Measurement Conductivity and pH measurements were performed using a calibrated Orion Star A215 pH / conductivity benchtop multiparameter meter (ThermoFisher Scientific). The instrument was calibrated for conductivity measurements using Oakton 1.413 mS / cm and 12.88 mS / cm standard solutions (Cole-Parmer, Vernon Hills, IL). The instrument was calibrated for pH measurements using ORION pH 4.01, 7.00, and 10.01 standard solutions (ThermoFisher Scientific).

[0170] Example 1 Preparation of 2-[[4-(2-hydroxyethyl)piperazine-1-carbonyl]amino]ethyl 2-methylprop-2-enoate (IEM / N-(2-hydroxyethyl)piperazine)

[0171] [ka]

[0172] N-(2-hydroxyethyl)piperazine (6.5 grams, 0.05 moles) was weighed into a 250 mL round-bottom flask equipped with a magnetic stirrer. Deionized water (50 mL) was added, and the stirred mixture was slowly placed in a nitrogen-purged ice-water bath. An aliquot (100 microliters) of 4-hydroxy-TEMPO solution (10,000 ppm in deionized water) was added, and the solution was stirred for 10 minutes, after which IEM (3.5 mL) was added. The solution was then stirred for 15 minutes, after which 3.5 mL of IEM (total amount of IEM = 7.0 mL, 0.05 moles) was added. The mixture was stirred for 15 minutes before being removed from the ice-water bath. Concentrated hydrochloric acid was then added dropwise with stirring to obtain a solution with a pH of 6.0, as determined by pH paper. The cloudy solution was filtered to give 2-[[4-(2-hydroxyethyl)piperazine-1-carbonyl]amino]ethyl 2-methylprop-2-enoate as a clear, colorless filtrate with % solids of 23.95%. 1H-NMR (DO): δ 1.74 (s, 3H), 3.13 (m, 6H), 3.33 (t, 2H), 3.51 (broad m, 4H), 3.76 (t, 2H), 4.09 (t, 2H), 5.55 (s, 1H), 5.95 (s, 1H). Percent solids were determined using an Ohaus moisture balance (Model No. MB35, obtained from Ohaus Corporation, Parsippany, NJ). A sample of the aqueous monomer solution was acidified with 0.1 N HCl to a pH of approximately 1 and then titrated with 0.1 N NaOH, yielding an experimental pKa of 6.8 for IEM / N-(2-hydroxyethyl)piperazine.

[0173] Example 2. Preparation of 2-[(4-methylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate (IEM / N-methylpiperazine)

[0174] [ka]

[0175] The general procedure described in Example 1 was followed, except that N-methylpiperazine (5.00 grams, 0.05 moles) was substituted for N-(2-hydroxyethyl)piperazine. The resulting filtrate of 2-[(4-methylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate was 21.65% solids. 1 H-NMR (D2O): δ1.73 (s, 3H), 2.71 (s, 3H), approximately 3.0 (wide, 8H), 3.32 (t, 2H), 4.08 (t, 2H), 5.54 (s, 1H), 5.94 (s, 1H).

[0176] Example 3 Preparation of 2-(2-morpholinoethylcarbamoylamino)ethyl 2-methylprop-2-enoate (IEM / N-(2-aminoethyl)morpholine)

[0177] [ka]

[0178] The general procedure described in Example 1 was followed, except that N-(2-aminoethyl)morpholine (6.51 grams, 0.05 moles) was substituted for N-(2-hydroxyethyl)piperazine. The resulting filtrate of 2-(2-morpholinoethylcarbamoylamino)ethyl 2-methylprop-2-enoate had a solids content of 23.55%. 1 H-NMR(D2O): δ1.75(s,3H),3.07(t,2H),3.19(wide s,4H),3.28(t,2H),3.35(t,2H),3.80(wide s,4H),4.07(t,2H),5.56(s,1H),5.96(s,1H).

[0179] Example 4. Preparation of 2-(3-morpholinopropylcarbamoylamino)ethyl 2-methylprop-2-enoate (IEM / N-(3-aminopropyl)morpholine)

[0180] [ka]

[0181] The general procedure described in Example 1 was followed, except that N-(2-hydroxyethyl)piperazine was replaced with N-(3-aminopropyl)morpholine (7.21 grams, 0.05 moles). The resulting filtrate of 2-(3-morpholinopropylcarbamoylamino)ethyl 2-methylprop-2-enoate had a solids content of 23.75%. 1 H-NMR (D2O): δ1.74 (s and m, 5H), 3.00 (m, 2H), 3.04 (t, 2H), 3.15 (wide s, 4H), 3.27 (t, 2H), 3.79 (wide s, 4H), 4.05 (t, 2H), 5.55 (s, 1H), 5.96 (s, 1H).

[0182] Example 5. Preparation of 2-[(4-methylpiperazin-1-yl)carbamoylamino]ethyl 2-methylprop-2-enoate (IEM / 1-amino-4-methylpiperazine)

[0183] [ka]

[0184] The general procedure described in Example 1 was followed, except that the total amount of IEM used was 3.5 mL (0.025 moles) and N-(2-hydroxyethyl)piperazine was replaced with 1-amino-4-methylpiperazine (2.88 grams, 0.025 moles). The resulting filtrate of 2-[(4-methylpiperazin-1-yl)carbomoylamino]ethyl 2-methylprop-2-enoate was 23.1% solids. 1 H-NMR (DO): δ 1.75 (s, 3H), 2.77 (s overlaps with another absorption, 5H), 3.02 (broad d, 2H), 3.12 (broad t, 2H), 3.32 (t, 2H), 3.39 (broad d, 2H), 4.11 (t, 2H), 5.56 (s, 1H), 5.96 (s, 1H).

[0185] Example 6 Preparation of 2-[(4-phenylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate (IEM / N-phenylpiperazine)

[0186] [ka]

[0187] N-Phenylpiperazine (4.05 grams, 0.025 moles) was weighed into a 250 mL round-bottom flask equipped with a magnetic stirrer. Diethyl ether (75 mL) was added, and the stirred mixture was slowly placed in a nitrogen-purged ice-water bath. The mixture was stirred for 10 minutes. IEM (3.5 mL) was added via pipette, and the mixture was stirred for 75 minutes. The colorless precipitate that formed was filtered and dried to yield 5.3 grams of 2-[(4-phenylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate. 1H-NMR(CDCl3): δ1.96(s,3H),3.18(m,4H),3.53(m,4H),3.59((q,2H),4.31 (t, 2H), 5.00 (wide t, 1H), 5.61 (s, 1H), 6.14 (s, 1H), 6.92 (m, 3H), 7.28 (m, 2H).

[0188] Example 7 Preparation of 2-[[4-(2-phenylethyl)piperazine-1-carbonyl]amino]ethyl 2-methylprop-2-enoate (IEM / N-phenethylpiperazine)

[0189] [ka]

[0190] N-phenethylpiperazine (9.5 grams, 0.05 moles) was weighed into a 250 mL round-bottom flask equipped with a magnetic stirrer. Dichloromethane (100 mL) was added, and the stirred mixture was slowly placed in a nitrogen-purged ice-water bath. The mixture was stirred for 10 minutes. IEM (3.5 mL) was added via pipette. The mixture was stirred for 10 minutes. A second portion of IEM (3.5 mL) was added via pipette, and the mixture was stirred for 30 minutes. The reaction mixture was then poured into a separatory funnel. A 50 mL portion of 1N HCl was added to the funnel, and the funnel was shaken. The funnel was placed on a stand, allowing the mixture to separate into three layers. The bottom layer was discarded, and the remaining layers were separated by collecting them in two containers. NMR analysis indicated that both layers contained the desired product. The layers were combined, and 50 microliters of 4-hydroxy-TEMPO solution (10,000 ppm in deionized water) was added. Most of the dichloromethane was removed by rotary evaporation to yield a clear, pale yellow aqueous solution of 2-[[4-(2-phenylethyl)piperazine-1-carbonyl]amino]ethyl 2-methylprop-2-enoate with a pH of 4-5 (measured with pH paper) and a % solids of 21.6%. 1H-NMR(D2O): δ1.73(s,3H),2.86(wide s,2H),2.92(m,2H),3.07(wide s,2H),3.24(m,2H),3.32(t,2H) , 3.42 (wide width s, 4H), 3.92 (wide width s, 2H), 4.07 (t, 2H), 5.54 (s, 1H), 5.95 (s, 1H), 7.17 (m, 3H), 7.23 (m, 2H).

[0191] Example 8 Preparation of 2-(piperazine-1-carbonylamino)ethyl 2-methylprop-2-enoate (IEM / piperazine)

[0192] [ka]

[0193] 1-Boc-piperazine (18.6 grams, 0.1 mole) was weighed into a 250 mL round-bottom flask equipped with a magnetic stirrer. Dichloromethane (75 mL) was added, and the stirred mixture was slowly placed in a nitrogen-purged ice-water bath. The mixture was stirred for 10 minutes. IEM (3.5 mL) was added via pipette, and the mixture was stirred for 1 minute, after which a second portion of IEM (3.5 mL) was added. Two additional 3.5 mL portions of IEM were added, spaced 5 minutes apart (a total of 14 mL of IEM was added in four equal portions). The resulting mixture was stirred for 30 minutes, and then the solvent was removed by rotary evaporation to give the BOC-protected IEM / piperazine intermediate as a colorless solid (33.5 grams). 1 H-NMR (CDCl3): δ1.39(s,9H),1.87(s,3H),3.28(m,4H),3.35(m,4H),3.47(q,2H),4.21(t,2H),5.12(wide t,1H),5.53(s,1H),6.06(s,1H).

[0194] The BOC-protected IEM / piperazine intermediate (5.0 grams) was placed in a round-bottom flask and dissolved in approximately 5 mL of trifluoroacetic acid, then heated for 1 hour at 50° C. The flask was placed on a rotary evaporator at 50° C. for 90 minutes to produce 2-(piperazine-1-carbonylamino)ethyl 2-methylprop-2-enoate, which contained some residual trifluoroacetic acid. 1 H-NMR (CD3OD): δ 1.91 (s, 3H), 3.19 (t, 4H), 3.45 (t, 2H), 3.64 (t, 4H), 4.21 (t, 2H), 5.61 (s, 1H), 6.10 (s, 1H). The residue was dissolved in 20 mL of deionized water to produce an approximately 0.5 M monomer solution, which was used to graft the membrane.

[0195] Example 9. Preparation of 2-[(4-methyl-1,4-diazepane-1-carbonyl)amino]ethyl 2-methylprop-2-enoate (IEM / N-methylhomopiperazine)

[0196] [ka]

[0197] The general procedure described in Example 1 was followed, except that N-methylhomopiperazine (5.71 grams, 0.05 moles) was substituted for N-(2-hydroxyethyl)piperazine. The resulting filtrate of 2-[(4-methyl-1,4-diazepane-1-carbonyl)amino]ethyl 2-methylprop-2-enoate was 22.45% solids. 1 H-NMR (D2O): δ1.69(m,2H), 1.71(s,3H), 2.08(s,3H), 2.37 and 2.41(2m,4H), 3.20(m,2H), 3.28(m,4H), 4.05(t,2H), 5.52(s,1H), 5.93(s,1H).

[0198] Example 10. Preparation of 2-[(4-methylpiperazine-1-carbonyl)amino]ethyl prop-2-enoate (IEA / N-methylpiperazine)

[0199] [ka]

[0200] N-Methylpiperazine (2.0 grams, 0.02 moles) was weighed into a 50 mL round-bottom flask equipped with a magnetic stirrer. Dichloromethane (20 mL) was added, and the stirred mixture was slowly placed in a nitrogen-purged ice-water bath. The mixture was stirred for 10 minutes. IEA (2.56 mL) was added via pipette, and the mixture was stirred for 15 minutes to produce 2-[(4-methylpiperazine-1-carbonyl)amino]ethyl prop-2-enoate as a solution in dichloromethane. 1 H-NMR (CDCl3): δ2.22(s,3H),2.31(t,4H),3.30(t,4H),3.46(q,2H),4.20(t,2H),4.93(wide t,1H),5.79(dd,1H),6.07(dd,1H),6.36(dd,1H).

[0201] Example 11 Preparation of 2-methyl-N-(2-morpholinopropyl)-2-(prop-2-enoylamino)propenamide (VDM / N-(3-aminopropyl)morpholine)

[0202] [ka]

[0203] The general procedure described in Example 4 was followed, except that IEM was replaced with VDM (6.95 g). The resulting VDM / N-(3-aminopropyl)morpholine filtrate had a solids content of 23.2%. 1 H-NMR(D2O): δ1.29(s,6H),1.73(p,2H),2.89(m,2H),3.05(wide s,4H),3.11(t,2H),3.76(wide s,4H),5.58(d,1H),5.99(d,1H),6.11(dd,1H).

[0204] Example 12 Preparation of 2-(4-(pyridin-2-yl)piperazine-1-carboxamido)ethyl methacrylate (IEM / N-(2-pyridyl)piperazine)

[0205] [ka]

[0206] A suspension of 1-(2-pyridyl)piperazine (4.00 g, 24.5 mmol) in 30 mL of water was cooled to 0° C. IEM (3.46 mL, 24.5 mmol) was added to the stirred solution over 3 minutes. A white solid formed. After stirring for 20 minutes, the white solid was isolated by filtration and washed several times with water. The solid was transferred to a crystallizing dish and air-dried for several days to yield 6.62 g of 2-(4-(pyridin-2-yl)piperazine-1-carboxamido)ethyl methacrylate as a white solid. 1 H NMR(500MHz,CD3OD)δ8.07-8.14(m,1H)7.59(ddd,J=8.7,7.1,2.0Hz,1H)6.84(d,J=8.7Hz,1H)6.71(dd,J=6.7 ,5.4Hz,1H)6.14(s,1H)5.61-5.68(m,1H)4.24(t,J=5.7Hz,2H)3.53(s,8H)3.49(t,J=5.7Hz,2H)1.94(s,3H).

[0207] Example 13 Preparation of 2-(4-(pyrimidin-2-yl)piperazine-1-carboxamido)ethyl methacrylate (IEM / N-(2-pyrimidyl)piperazine)

[0208] [ka]

[0209] A suspension of 1-(2-pyrimidyl)piperazine (3.60 g, 21.9 mmol) in 25 mL of water was cooled to 0° C. IEM (3.10 mL, 21.9 mmol) was added to the stirred solution over 3 minutes. A white solid formed. An additional 25 mL of water was added, and the mixture was stirred for 15 minutes. The white solid was isolated by filtration and washed with several portions of water. The solid was transferred to a crystallizing dish and air-dried overnight to give 6.11 g of 2-(4-(pyrimidin-2-yl)piperazine-1-carboxamido)ethyl methacrylate as a white solid. 1 H NMR(500MHz,CD3OD)δ8.35(d,J=4.8Hz,2H)6.63(t,J=4.8Hz,1H)6.14(s,1H)5.65(m,1H)4.24(t,J=5.7Hz,2H)3.81(m,4H)3.49(m,6H)1.95(m,3H).

[0210] Example 14. Preparation of 2-methyl-N-(4-methylpiperazin-1-yl)-2-(prop-2-enoylamino)propanamide (VDM / 1-amino-4-methylpiperazine)

[0211] [ka]

[0212] The general procedure described in Example 5 was followed, except that IEM was replaced with VDM (1.5 mL, 0.025 mol). The resulting filtrate of 2-methyl-N-(4-methylpiperazin-1-yl)-2-(prop-2-enoylamino)propanamide had a solids content of 21.85%. 1 H-NMR(D2O): δ1.22(s,6H),2.70(s,3H),3.5-2.5(br.m,8H),5.50(d,1H),5.92(d,1H),6.06(dd,1H).

[0213] Example 15. Nylon membranes were grafted with the monomer solutions of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) or Example 2 (IEM / N-methylpiperazine) according to the procedure described in Method 1. Each membrane was coated and grafted with a single monomer solution at a concentration of 0.25 M, 0.375 M, or 0.5 M. The grafted membranes were evaluated for BSA binding (mg / mL) at pH 6.0, pH 7.0, and pH 8.0 according to the procedure in Method 2. The results are reported in Table 2.

[0214] The results in Table 2 show that the polymer-grafted membrane prepared from IEM / N-(2-hydroxyethyl)piperazine monomer (calculated pKa 7.02) lost approximately 40% of its binding capacity for BSA when the buffer pH was increased from pH 6.0 to pH 7.0 and lost >80% of its binding capacity when the pH was increased from pH 6.0 to pH 8.0. In contrast, the polymer-grafted membrane prepared from IEM / N-methylpiperazine monomer (calculated pKa 7.46) lost only approximately 20% of its binding capacity for BSA when the buffer pH was increased from pH 6.0 to pH 7.0 and lost >80% of its binding capacity when the pH was increased from pH 6.0 to pH 8.0.

[0215] [Table 2] NT = Not Tested

[0216] Example 16. A polyethersulfone membrane (MacroPES, nominal pore size 5 microns, obtained from 3M Company) was grafted with the monomer from Example 1 (IEM / N-(2-hydroxyethyl)piperazine, pKa 7.02) at a 0.75 M monomer concentration and 20 min of irradiation time; no photoinitiator (C-BP) was included in the grafting solution. The resulting grafted membrane had a graft density of 1.04 mmol / g. The grafted substrate was tested for static BSA binding capacity in MES buffer at pH 6.0 according to the procedure in Method 2. The static BSA binding capacity was 114 mg / mL. The supernatant BSA solution was decanted from the centrifuge tube, and the membrane disk was washed three times with 4.5 mL of fresh MES buffer (pH 6.0). Each washing step was performed using a rotary mixer for 30 min. The final wash buffer was removed from the tube, and 4.5 mL of pH 8.0 TRIS buffer was added. The tube was then vortexed by end-over-end shaking for 30 minutes to elute the bound BSA from the disks. The eluate was assayed for BSA concentration, and the measured BSA concentration was used to calculate the eluted binding capacity (BSA elution binding capacity = 119 mg / mL). These results demonstrate that BSA protein was bound to the membrane using a near-neutral pH buffer and then quantitatively eluted from the membrane by gently adjusting the eluate pH.

[0217] Example 17. Nylon membranes were grafted with a monomer solution selected from Example 3 (IEM / N-(2-aminoethyl)morpholine), Example 4 (IEM / N-(3-aminopropyl)morpholine), Example 5 (IEM / 1-amino-4-methylpiperazine), Example 7 (IEM / N-phenethylpiperazine), or Example 8 (IEM / piperazine) according to the procedure described in Method 1. Each membrane was coated and grafted with a single monomer solution at a concentration of 0.375 M, 0.5 M, or 0.625 M. The grafted membranes were evaluated for BSA binding (mg / mL) at pH 5.0, pH 6.0, pH 7.0, pH 8.0, and pH 9.0 according to the procedure in Method 2. The results are reported in Table 3.

[0218] [Table 3] NT = Not Tested

[0219] Example 18. Nylon membranes were grafted with 0.5 M monomer solutions of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) or Example 2 (IEM / N-methylpiperazine) according to the procedure described in Method 1. The grafted membranes were evaluated for BSA binding capacity (mg / mL) using MOPS buffers of various ionic strengths at pH 7.0 according to the procedure in Method 2. The ionic strength of the individual MOPS buffers was adjusted to 50 mM and 150 mM by adding sodium chloride. The results are reported in Table 4.

[0220] [Table 4]

[0221] Example 19. Two buffers were prepared and designated as Buffer A and Buffer B. Buffer A was 25 mM MES buffer (pH 6.0), and Buffer B was 25 mM MES buffer (pH 6.0) containing 1 M NaCl. Test samples of BSA (0.56 grams of BSA in 50 milliliters of Buffer A) were prepared and then filtered using a 0.22 micron PES membrane filter (STERIFLIP sterile disposable vacuum filter unit, MilliporeSigma, Burlington, Mass.).

[0222] A nylon membrane was grafted with the monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine, calculated pKa of 7.02) according to the procedure described in Method 1. The grafting density was 0.36 mmol / g. Six 25 mm grafted membrane disks were stacked in a chromatography module, and O-rings were used to hold the stacked disks in place. The exposed front surface area of ​​the disks was 284 mm. 2The module contained a straight, cylindrical polycarbonate body with a cap attached to one end of the body. The cap included an inlet port and a vent port. The opposite end contained an outlet port equipped with a stopcock. The completed module was attached to an AKTA avant chromatography system (Cytiva, Marlborough, MA) equipped with a UV / VIS (280 nm absorbance setting) and a conductivity detector. The module was flushed with 5 mL of Buffer A at a flow rate of 1 mL / min. Next, the BSA test sample was pumped through the module at a flow rate of 0.5 mL / min, followed by 20 mL of Buffer A at a flow rate of 1 mL / min to wash the membrane stack. In the final elution step, BSA was eluted from the membrane stack by pumping a 20 mL gradient of 0% to 100% Buffer B:Buffer A (i.e., the gradient was from 100% Buffer A to 100% Buffer B by volume) at a flow rate of 1 mL / min. BSA bound to the grafted membrane stack was eluted at buffer conductivities of ≥ 1 mS / cm.

[0223] Example 20. The procedure of Example 19 was followed, except that the test sample was β-lactoglobulin from bovine milk (product number L3908, Sigma-Aldrich) in 50 mL of buffer A. β-lactoglobulin bound to the grafted membrane stack was eluted at a buffer conductivity of ≥ 1 mS / cm.

[0224] Example 21. Three buffer solutions were prepared using 50 mM TRIS buffer, and the pH was adjusted to 6.0, 7.0, or 8.0, with a conductivity of 5 mS / cm. The pH values ​​were adjusted using either 1 N HCl or 1 N NaOH. The conductivity values ​​were adjusted by adding 5 M sodium chloride. Phi6 virus challenge solutions with the virus concentrations listed in Table 5 were prepared by spiking each buffer solution with the Phi6 virus stock culture described in Method 5. Filtration capsules were prepared according to the procedure in Method 3 using nylon membranes grafted with the monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) according to the procedure in Method 1. The grafted membranes had a grafting density of 0.25 mmol / g. Each Phi6 virus challenge solution (7 mL) was pumped through an individual capsule at 0.5 mL / min. The resulting filtrate was collected. The input challenge solution and filtrate samples were measured for Phi6 content using the plaque assay described in Method 6. The log reduction value (LRV) from filtration was calculated by comparing the Phi6 virus concentration in the filtered sample with the Phi6 virus concentration in the corresponding input challenge solution before filtration (Equation 3). The results are reported in Table 5. These results indicate that when the pH of the challenge solution was between 6.0 and 7.0, a significant amount of Phi6 enveloped virus bound to the grafted membrane. However, when the pH value of the challenge solution was 8.0, the Phi6 virus flowed through the grafted membrane, and almost no Phi6 virus bound to the grafted membrane.

[0225]

number

[0226] [Table 5]

[0227] Example 22. Phi6 virus challenge solutions were prepared using 50 mM TRIS buffer adjusted to pH 7.0, 7.25, 7.5, 7.75, or 8.0 and conductivity 5 mS / cm according to the procedure described in Example 21. The results are reported in Table 6. These results indicate that when the pH of the challenge solution was between 7.0 and 7.5, some of the Phi6 enveloped virus bound to the grafted membrane. However, when the pH value of the challenge solution was between 7.75 and 8.0, the Phi6 virus flowed through the membrane, and almost no Phi6 virus bound to the grafted membrane.

[0228] [Table 6]

[0229] Example 23. Phi6 virus challenge solutions were prepared using 50 mM TRIS buffer adjusted to pH 7.0 and conductivity of 10 mS / cm, 12.5 mS / cm, 15 mS / cm, 17.5 mS / cm, or 20 mS / cm according to the procedure described in Example 21. Filtration capsules were prepared according to the procedure described in Method 3 using nylon membranes grafted with the monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) according to the procedure described in Method 1. The grafted membranes had a grafting density of 0.36 mmol / g. The results are reported in Table 7. These results indicate that when the conductivity of the challenge solution was 10 mS / cm, the majority of Phi6 enveloped virus bound to the grafted membrane. However, binding of Phi6 virus to the grafted membranes was substantially reduced when the conductivity of the challenge solution was increased from 12.5 mS / cm to 20 mS / cm.

[0230] [Table 7]

[0231] Example 24. Calf thymus DNA challenge solutions were prepared by dissolving 20 micrograms / mL of calf thymus DNA (Sigma-Aldrich Company) in 50 mM TRIS buffer (pH 7.0, 10 mS / cm) or 50 mM TRIS buffer (pH 8.0, 10 mS / cm). Filtration capsules were prepared according to the procedure in Method 3 using nylon membranes grafted with the monomer solution (IEM / N-(2-hydroxyethyl)piperazine) from Example 1 according to the procedure in Method 1. The grafted membranes had a grafting density of 0.36 mmol / g. Each DNA challenge solution (8 mL) was pumped through an individual capsule at 0.5 mL / min. The resulting filtrate was collected in four separate 2 mL fractions (i.e., fractions 1–4). The DNA concentrations (micrograms / mL) of the challenge solutions before and after filtration were determined using UV spectroscopy by measuring the absorbance at 260 nm and subtracting the background absorbance of the buffer. The results are reported in Table 8.

[0232] These results indicate that when the pH 7.0 challenge solution was used, a significant amount of DNA was bound by the grafted membrane (i.e., a small amount of DNA was collected in the filtrate fraction). However, when the pH 8.0 challenge solution was used, the DNA in the challenge solution did not bind to the membrane (i.e., most of the DNA was collected in the filtrate fraction). The increase in DNA levels in the fourth filtrate fraction from the pH 7.0 challenge solution likely occurred because the capacity of the grafted membrane was exceeded.

[0233] [Table 8]

[0234] Example 25. Membrane grafted with IEM / N-methylpiperazine:HEMA copolymer (75:25 molar ratio) A nylon membrane was grafted with a single comonomer solution containing Example 2 (IEM / N-methylpiperazine, calculated pKa of 7.46) and 2-hydroxyethyl methacrylate (HEMA) according to the procedure described in Method 1. The total monomer concentration of this solution was 0.35 M, and the molar ratio of the monomers in the solution was IEM / N-methylpiperazine:HEMA 75:25. The grafting density was 0.30 mmol / g.

[0235] Example 26. Membrane grafted with IEM / N-methylpiperazine:HEMA copolymer (50:50 molar ratio) A grafted membrane was prepared according to the procedure described in Example 25, except that the molar ratio of the monomers in the solution was IEM / N-methylpiperazine:HEMA 50:50, and the grafting density was 0.20 mmol / g.

[0236] Example 27. Membrane grafted with IEM / N-methylpiperazine:HEMA copolymer (25:75 molar ratio) A grafted membrane was prepared according to the procedure described in Example 25, except that the molar ratio of the monomers in the solution was IEM / N-methylpiperazine:HEMA 25:75, and the grafting density was 0.12 mmol / g.

[0237] Example 28. Membrane grafted with IEM / N-methylpiperazine:HEMA copolymer (75:25 molar ratio) A grafted membrane was prepared according to the procedure described in Example 25, except that the total monomer concentration of this solution was 0.50 M and the molar ratio of monomers in the solution was IEM / N-methylpiperazine:HEMA 75:25, resulting in a grafting density of 0.53 mmol / g.

[0238] Example 29. A 96-well EMPORE filter plate was modified with the grafted membranes of Example 2 (IEM / N-methylpiperazine, pKa 7.46) and Examples 25-27 (containing HEMA as a copolymer) as described in Method 4. Two disks of the same type of grafted membrane were added to each well, with nine wells prepared for each type of grafted membrane. The membrane disks of Example 2 had a grafting density of 0.375 mmol / g. A 500-microliter aliquot of 20 mM TRIS-acetate buffer (pH 7.4, supplemented with 100 mM NaCl) was added to each well. The pH of the buffer was adjusted with acetic acid. The EMPORE plate was placed on top of a 2 mL-deep 96-well collection plate, and the plate assembly was centrifuged at 1000 × g for 10 minutes using an ALLEGRA 25R centrifuge (Beckman Coulter, Indianapolis, IN). The flow-through collected in the collection plate was discarded. A Phi6 virus challenge solution was prepared by diluting a Phi6 virus stock sample (prepared according to Method 7) 100-fold with TRIS-acetate buffer to achieve a titer of approximately 1E+07 pfu / mL. A 300-microliter aliquot of the challenge solution was added to each well of the EMPORE plate, and the assembly was then centrifuged at 1000 x g for 10 minutes. The EMPORE plate was then removed from the collection plate and transferred onto a new collection plate.

[0239] Three separate elution buffers were prepared by adjusting the pH of 20 mM TRIS-acetate buffer to either pH 8.0, pH 8.5, or pH 9.0 with HCl. Phi6 virus was eluted from the grafted membranes using a two-step procedure. In the first step, 300-microliter aliquots of a single elution buffer were added to three wells containing each type of grafted membrane (n=3 for each elution buffer), and the plate was centrifuged at 1000 × g for 10 minutes. In the second step, a second 300-microliter aliquot of the same elution buffer used in the first step was added to each well, and the plate was centrifuged at 1000 × g for 10 minutes. Collected flow-through samples of the elution buffer were analyzed for Phi6 virus content using the procedure in Method 8. The results are shown in Table 9 as the mean percent Phi6 virus recovered from the challenge solution, along with the standard deviation (SD).

[0240] [Table 9]

[0241] Example 30. Clarified lentiviral cell culture challenge solution (approximately 1.25E+06 TU / mL lentivirus) was prepared according to Method 9 and spiked with approximately 1E+09 pfu / mL Phi6 virus (prepared according to Method 7) at a 1:1000 dilution. The pH of the clarified culture medium was 7.05 and the conductivity was 10 mS / cm.

[0242] A 96-well EMPORE filter plate was modified with the grafted membrane of Example 28 (prepared using a grafting solution with a 75:25 molar ratio of IEM / N-methylpiperazine:HEMA). The EMPORE plate was placed on top of a 2 mL deep 96-well collection plate, and 500 microliters of 1× phosphate buffered saline (PBS) was added to each well. The plate assembly was then centrifuged at 1000 × g for 5 minutes, and the collected PBS was discarded. Next, 500 microliters of clarified challenge solution (containing both Phi6 virus and lentivirus) was applied to each well, followed by centrifugation at 1000 × g for 5 minutes. The collected liquid was discarded.

[0243] A series of 20 elution buffers was prepared from 10 mM PBS with a pH of 7.25, 7.5, 7.75, 8.0, or 8.25 and a conductivity of 10, 20, 30, or 40 mS / cm. For each elution buffer, the pH was adjusted with 1 N HCl, and the conductivity was adjusted with NaCl. A 500 ml aliquot of a single elution buffer was added to each of three wells (n = 3), and the plate was centrifuged at 1000 × g for 5 minutes. Collected samples of the elution buffer were analyzed for lentivirus (TU / mL), Phi6 virus (pfu / mL), and DNA (nanograms / mL (ng / mL)) content according to the procedures in Methods 8–11. The results are presented in Tables 10–12 as mean concentration values ​​with standard deviations (SD).

[0244] The results, shown in Tables 10-12, demonstrate that the elution conditions of this method can be optimized to provide purified virus with reduced DNA content. Elution of material bound to the grafted membrane using buffers of pH 7.25-7.75 and conductivity 20 mS produced filtrates with significant amounts of lentivirus and Phi6 virus and low levels of DNA only.

[0245] [Table 10]

[0246] [Table 11]

[0247] [Table 12]

[0248] Example 31. pUC19 DNA was cloned using the Maximum Potency DH5α Competent Cell Kit (catalog number 18258012, ThermoFisher Scientific) according to the manufacturer's instructions. The kit contained Maximum Potency DH5α Competent Cells, pUC19 DNA, and SOC medium. E. coli cell cultures were grown at 37°C and agitated at 200 rpm for 18 hours. pUC19 DNA was purified using a Qiagen QIAprep Spin Miniprep Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. Nylon membranes were grafted with the monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine, calculated pKa of 7.02) according to the procedure described in Method 1. The grafting density was 0.36 mmol / g. EMPORE 96-well filter plates were prepared according to Method 4, except that only a single disk was added to each well. Three individual solutions of TE buffer (TE buffer = 50 mM TRIS buffer supplemented with 10 mM ethylenediaminetetraacetic acid (EDTA)) were prepared, with a pH of either 7.0, 8.0, or 9.0. Two individual solutions of 50 mM MES buffer were prepared, with a pH of either 5.0 or 6.0. The pH of the buffer was adjusted using 1 N HCl or 1 N NaOH. A 150 microliter aliquot of a single buffer was added to each of three wells (n = 3 for each buffer). The EMPORE plate was placed on top of a 96-well 2 mL deep-well collection plate, and the plate assembly was centrifuged at 1500 rpm for 5 minutes. The flow-through collected in the collection plate was discarded. The EMPORE plate was then placed on top of a new collection plate. A 150 microliter aliquot of pDNA (approximately 100 nanograms / microliter) dissolved in buffer was added to each well of the EMPORE plate. The pH of the pDNA solution added to the wells was chosen to match the pH of the initial 150 microliter aliquot of buffer added to the wells. The plate assembly was then centrifuged at 2000 rpm for 2 minutes.The collected flow-through samples were analyzed to determine the percent of bound pDNA using a NanoDrop ultramicrospectrophotometer (ThermoFisher Scientific) with an absorbance setting of 260 nm. Analysis of the flow-through samples showed that when buffers with pH values ​​between 5.0 and 8.0 were used, approximately >80% of the pDNA bound to the grafted membrane, whereas when a buffer at pH 9.0 was used, only approximately 3% of the pDNA bound to the grafted membrane.

[0249] The pDNA was then eluted from the grafted membranes by sequentially adding 100 microliter aliquots of buffer to each well. The pH of each subsequent aliquot in the sequence increased by 1.0 pH units. After each aliquot was added, the plate was centrifuged and the resulting flow-through sample was analyzed for pDNA as described above. This process was repeated until a pH 9.0 buffer was used as the eluent. Regardless of the initial buffer pH, the greatest percentage of pDNA was recovered from the grafted membranes using the pH 9.0 elution buffer. The average percent recovery of pDNA in the collected samples (n=3) is reported in Table 13, along with the standard deviation (SD).

[0250] [Table 13] NT = Not Tested ** = initial flow-through sample

Claims

1. Monomers of formula (I) 【Chemistry 1】 [In the formula, R １ is hydrogen or methyl, X １ is —O— or —NH—, R ２ is (hetero)alkylene, Z is -NH-(C=O)-, -NH-(C=O)-NH-, or -NH-(C=O)-NH-R ３ -, -(C=O)-NH-, and -(C=O)-NH-R ３ - and R ３ is an alkylene having at least two linked carbon atoms; R ４ and R ５ are each alkylene having at least two carbon atoms, nitrogen, R ４ , Q, and R ５ the total number of ring atoms in the ring group consisting of is 6 or 7; Q is -O-, -N(R ６ )-, -S-, -S(=O)-, or -S(=O) ２ -, provided that when Z is -NH-(C=O)-, Q is -N(R ６ )- and; R ６ is hydrogen, alkyl, or (hetero)aryl, wherein said alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl, and said (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

2. 2. The monomer of claim 1, having a calculated pKa in the range of 3.5 or 4 to 9 or 9.

5.

3. R ２ 2. The monomer of claim 1, wherein the total number of linked atoms in the Z group and the Z group is at least four.

4. A polymeric material comprising a first monomeric unit derived from a monomer of formula (I) according to any one of claims 1 to 3.

5. 5. The polymeric material of claim 4, further comprising a second monomeric unit not derived from a monomer of formula (I), wherein said second monomeric unit is hydrophilic.

6. The polymeric material of claim 5, wherein the polymeric material comprises 10 mole percent to 90 mole percent of the first monomeric units and 90 mole percent to 10 mole percent of the second monomeric units.

7. 1. An anion exchange separation article comprising: a solid porous polymer substrate; and The porous substrate comprises a plurality of graft polymers attached to a surface thereof, the graft polymers being represented by formula (I): 【Chemistry 2】 [In the formula, R １ is hydrogen or methyl, X １ is —O— or —NH—, R ２ is (hetero)alkylene, Z is -NH-(C=O)-, -NH-(C=O)-NH-, or -NH-(C=O)-NH-R ３ -, -(C=O)-NH-, and -(C=O)-NH-R ３ - and R ３ is an alkylene having at least two linked carbon atoms; R ４ and R ５ are each alkylene having at least two carbon atoms, nitrogen, R ４ , Q, and R ５ the total number of ring atoms in the ring group consisting of is 6 or 7; Q has a single linked atom and is not -O-, -N(R ６ )-, -S-, -S(=O)-, or -S(=O) ２ -, provided that when Z is -NH-(C=O)-, Q is -N(R ６ )- and; R ６ is hydrogen, alkyl, or (hetero)aryl, wherein said alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl, and said (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

8. For said monomers of formula (I), R ２ 8. The anion exchange separation article of claim 7, wherein the total number of linked atoms in the Z group and the Z group is at least four.

9. 9. The anion exchange separation article of claim 7 or 8, wherein the monomers of formula (I) include those having a calculated pKa in the range of 3.5 or 4 to 9 or 9.

5.

10. The anion exchange separation article of any one of claims 7 to 9, wherein the monomer composition further comprises a second monomer that is hydrophilic but is not of formula (I).

11. 11. The anion exchange separation article of claim 10, wherein the polymer composition comprises from 10 mole percent to 90 mole percent of the first monomeric units and from 90 mole percent to 10 mole percent of the second monomeric units.

12. 12. The anion exchange separation article of any one of claims 7 to 11, wherein each of the plurality of grafted polymers is grafted to a carbon atom of the solid porous polymer substrate.

13. The anion exchange separation article of any one of claims 7 to 12, wherein the porous polymeric substrate comprises particles, fibers, films, nonwoven webs, membranes, sponges, or sheets.

14. 1. A method for separating a mixture of materials having different ionic contents, comprising: a) an anion exchange separation article, a solid porous polymer substrate; and The porous substrate includes a plurality of graft polymers attached to a surface thereof, the graft polymers being the polymerization product of a monomer composition, the first monomer being a monomer of formula (I): 【Transformation 3】 [In the formula, R １ is hydrogen or methyl; X １ is —O— or —NH—, R ２ is (hetero)alkylene; Z is -NH-(C=O)-, -NH-(C=O)-NH-, or -NH-(C=O)-NH-R ３ -, -(C=O)-NH-, and -(C=O)-NH-R ３ - and R ３ is an alkylene having at least two linked carbon atoms; R ４ and R ５ are each alkylene having at least two carbon atoms, nitrogen, R ４ , Q, and R ５ the total number of ring atoms in the ring group consisting of is 6 or 7; Q has a single linked atom and is not -O-, -N(R ６ )-, -S-, -S(=O)-, or -S(=O) ２ -, provided that when Z is -NH-(C=O)-, Q is -N(R ６ )- and; R ６ is hydrogen, alkyl, or (hetero)aryl, wherein alkyl is optionally further substituted with hydroxy, alkoxy, or (hetero)aryl, and (hetero)aryl is optionally further substituted with one or more hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl, or alkoxy; and b) passing a mixture of materials through the anion exchange separation article at a first pH sufficiently low to protonate the monomer repeat units of the graft polymer derived from the monomer of formula (I) and at a first ionic strength value such that at least one component of the mixture of materials is bound to the anion exchange separation article as a binding component.

15. (c) optionally passing a wash solution through said anion exchange separation article; 15. The method of claim 14, further comprising: (d) passing the eluent composition through the anion exchange separation article at a second pH greater than or equal to the first pH and / or a second ionic strength value greater than the first ionic strength value, wherein the eluent removes bound components from the anion exchange separation article.

16. R ２ and the sum of the linked atoms in Z is at least 4.

17. 17. The method of any one of claims 14 to 16, wherein both the first pH and the second pH are in the range of 3.5 or 4 to 9 or 9.

5.

18. 18. The method according to any one of claims 14 to 17, wherein the first ionic strength and the second ionic strength are less than or equal to 0.5 moles / liter and / or less than or equal to 50 millisiemens.

19. The method of any one of claims 14 to 18, wherein the mixture of materials comprises proteins, nucleic acids, nucleic acid fragments, cells, viruses, or virus-like particles.

20. The method according to any one of claims 14 to 19, which is a flow-through method or a bind-elute method.

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