Heterocyclic acrylic acid derivatives as monomers for synthesis of polymers for ion exchange articles

Abstract

The present invention relates to monomers of formula (I) having nitrogen atoms that can be protonated at relatively low pH values (e.g., less than pH 9.5 or less than pH 9), polymers containing monomer units derived from these monomers, anion exchange separation articles having the polymer grafted to a porous substrate, and methods of making the same. And a method for separating a mixture of materials having different ion contents using the anion separation article. Advantageously, the anion exchange separation article can be used to separate biological materials that are not subject to high pH conditions (e.g., greater than 9 or 9.5) and / or high ionic strength conditions (e.g., greater than 0.5 M or 1 M). # imgabs0 #

Description

[0001] Ion exchange (IEX) chromatography is commonly used to purify biological substances. Cation exchange (CEX) chromatography typically utilizes acidic ligands immobilized on a solid support, with pKa values in the range of 0 to about 4. Anion exchange (AEX) chromatography typically uses ligands with pKa values in the range of about 9 to 12. Ligands with pKa values between these two ranges are relatively few.

[0002] Commonly used anion exchange ligands for bioseparation have high pKa values. For example, the pKa of a strong anion exchange quaternary amine ligand is greater than 12. Weak anion exchange ligands (such as those formed from monomeric diethylaminopropylacrylamide and dimethylaminopropylacrylamide) have pKa values of 10.3 and 9.3, respectively. When these ligands are used for binding and elution in AEX chromatography purification, buffer solutions with relatively high salt concentrations (such as 0.5 M or 1.0 M) are typically used to elute the captured biological material. Elution by changing the pH is usually not possible because an eluent with a pH higher than the ligand pKa is required. This can be a problem because many proteins, especially enzymes, cannot tolerate such high pH values. In addition, many biological species cannot tolerate high salt concentrations or low pH values, such as below 3.5 or 4. For example, many enzymes, viruses, and virus-like particles (VLPs) are stable only within a narrow range of pH and salt conditions. In addition, many viruses, especially enveloped viruses, are difficult to purify due to their low or near-neutral isoelectric points. Conventional IEX ligands have limited effectiveness in purifying viruses with low or near-neutral isoelectric points. Summary of the Invention

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

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

[0005]

[0006] In formula (I), the group R 1 is hydrogen or methyl, X 1 is -O- or -NH-, and R 2 is (hetero)alkylene. The group Z is -NH-(C=O)-, -NH-(C=O)-NH-, -NH-(C=O)-NH-R3 -, -(C=O)-NH-, or -(C=O)-NH-R 3 -, where R 3 is an alkylene group having at least 2 linked carbon atoms. Group R 4 and R 5 are each an alkylene group having at least 2 carbon atoms, where the total number of ring atoms in the ring group composed of nitrogen, R 4 , Q, and R 5 is 6 or 7. Group Q has a single linking atom and is -O-, -N(R 6 ), -S-, -S(=O)-, or -S(=O)2-, provided that when Z is -NH-(C=O)-, Q is equal to -N(R 6 ). Group R 6 is hydrogen, alkyl, or (hetero)aryl, where the alkyl is optionally further substituted by hydroxy, alkoxy, or (hetero)aryl, and where the (hetero)aryl is optionally further substituted by hydroxy, halogen, nitro, cyano, trifluoromethyl, alkyl, or alkoxy.

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

[0008] In a third aspect, there is provided an anion exchange separation article comprising a porous substrate and a plurality of graft polymers attached to the surface of the porous substrate, wherein the graft polymer is a polymerization product of a monomer composition comprising a monomer of formula (I) as described in the first aspect.

[0009] In a fourth aspect, there is provided a method of separating a mixture of materials having different ion contents. The method comprises preparing or providing an anion exchange separation article as described in the third aspect. The method further comprises passing the material mixture through the anion exchange separation article at a first pH and a first ionic strength value, the first pH being low enough to protonate the monomer repeating units of the graft polymer derived from the monomer of formula (I), the first ionic strength value binding at least one component of the material mixture as a bound component to the anion exchange separation article.

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

[0011] The term "and / or" means either 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 the radical of an alkane. The alkyl group may 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. The alkyl group can be straight-chain, branched, cyclic, or a combination thereof. A straight-chain alkyl group has at least one carbon atom, while a cyclic or branched alkyl group has at least 3 carbon atoms.

[0013] The term "alkylene" refers to a divalent group that is the radical of an alkane. The alkylene group may 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. The alkylene group can be straight-chain, branched, cyclic, or a combination thereof. A straight-chain alkylene group has at least one carbon atom, while a cyclic or branched alkylene group has at least 3 carbon atoms.

[0014] The term "heteroalkylene" refers to an alkylene group in which one or more of the carbon atoms are replaced by heteroatoms. The heteroatoms are typically nitrogen (e.g., -NH-), oxygen (-O-), or sulfur (-S-). Generally, there are no two adjacent heteroatoms such as in peroxides.

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

[0016] The term "alkoxy" refers to a monovalent group of the formula -OR a wherein R a is an alkyl group as defined above.

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

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

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

[0020] The term "link" refers to the atoms in 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, and 9 of them are links.

[0021] The term "grafting" is used to indicate that a polymer chain is covalently attached to a porous polymer substrate. In most embodiments, the polymer chain is grafted to a carbon atom in the polymer backbone of the porous polymer substrate.

[0022] The term "grafting density" refers to the millimoles of monomer units grafted per gram of substrate. The millimoles are 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 initial mass (grams) of the substrate. The grafting density is expressed as millimoles of monomer units grafted per gram of substrate (mmol / g). For clarity, the material being grafted is typically a polymeric material containing multiple monomer units.

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

[0024] The terms "polymer" and "polymeric material" are used interchangeably and refer to materials formed by reacting one or more monomers. The term includes homopolymers, copolymers, terpolymers, etc. Similarly, the terms "polymerization" and "polymerize" refer to the process of making polymeric materials that can be homopolymers, copolymers, terpolymers, etc.

[0025] The term "in a range of or ranging from" is used interchangeably to refer to all values within the range plus the end values of the range. Detailed Description

[0026] Monomers having nitrogen atoms that can be protonated at relatively low pH values (e.g., less than pH 9.5 or less than pH 9), polymers containing monomer units derived from these monomers, anion exchange separation articles having polymers grafted to a porous substrate, and methods of using the anion separation articles to separate mixtures of materials having different ionic contents are described. Advantageously, the anion exchange separation articles can be used, for example, in a pH range of about 3.5 or 4 to about 9 or 9.5 and at an ionic strength of up to about 0.5 molar / liter (e.g., 50 millisiemens). That is, the pH and ionic strength can be selected to provide conditions that various biomaterials of interest can tolerate.

[0027] Anion exchange separation articles are formed by grafting a plurality of polymers (i.e., polymer chains) onto a porous polymer substrate, which is typically a solid material. The grafted polymers contain monomer units having nitrogen-containing groups that can be used as anion exchange ligands. That is, the nitrogen-containing groups can be protonated. The calculated pKa value of the nitrogen-containing monomers used to form the grafted polymers is in the range of 3.5 or 4 to 9 or 9.5, and can be deprotonated at a lower pH value compared to many nitrogen-containing monomers commonly used to prepare anion exchange separation articles. The anion exchange separation articles can be used in flow-through separation methods or binding-elution separation methods.

[0028] Monomer

[0029] Monomers having protonatable nitrogen atoms are provided. More specifically, monomers are selected in which the nitrogen atom can be protonated at low pH, and the calculated pKa value of the monomers is generally in the range of 3.5 or 4 to 9 or 9.5. The monomers have formula (I).

[0030]

[0031] In formula (I), the group R 1 is hydrogen or methyl, X 1 is -O- or -NH-, and R 2 is (hetero)alkylene. The group Z is -NH-(C=O)-, -NH-(C=O)-NH-, -NH-(C=O)-NH-R 3 -, -(C=O)-NH- or -(C=O)-NH-R 3 -, where R 3 is alkylene having at least 2 linked carbon atoms. The groups R 4 and R 5 are each alkylene having at least 2 carbon atoms, where the total number of ring atoms in the ring group composed of nitrogen, R 4 , Q and R 5 is 6 or 7. The group Q has a single linking atom and is -O-, -N(R 6 )-, -S-, -S(=O)- or -S(=O)2-, provided that when Z is -NH-(C=O)-, Q is equal to -N(R 6 ). The group R 6 is hydrogen, alkyl or (hetero)aryl, where the alkyl is optionally further substituted by hydroxy, alkoxy or (hetero)aryl, and where the (hetero)aryl is optionally further substituted by hydroxy, halo, nitro, cyano, trifluoromethyl, alkyl or alkoxy.

[0032] The monomer has a (meth)acryloyl group of formula CH2=CR 1 -(C=O)-, where R 1is hydrogen or methyl.

[0033] Group X 1 is -O- or -NH-. If X 1 is -O-, the monomer of formula (I) is (meth)acrylate, but if X is -NH-, the monomer is (meth)acrylamide.

[0034] Group R 2 is (hetero)alkylene. In many embodiments, R 2 is alkylene, such as alkylene having 1 to 20 carbon atoms. For example, alkylene R 2 may have 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 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. In other embodiments, R 2 has the formula -R-O-R-, where each R is an alkylene having 2 to 10 linked carbon atoms. The alkylene R may have at least 2, at least 3, at least 4, and up to 10, up to 8, up to 6 or up to 4 linked carbon atoms.

[0035] Group Z is -NH-(C=O)-, -NH-(C=O)-NH-, -NH-(C=O)-NH-R 3 -, -(C=O)-NH- and -(C=O)-NH-R 3 -, where R 3 is an alkylene having at least 2 linked carbon atoms. Suitable alkylene groups generally have 2 to 10 carbon atoms, such as 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. Group Z can form hydrogen bonds, which can be beneficial for improving binding efficiency when using the monomer of formula (I) to form, for example, an anion exchange separation article.

[0036] Although for some uses of the monomer of formula (I), the total number of linked atoms in R 2 plus Z can be as low as 3, if the monomer of formula (I) is used to form an anion exchange article, the total number is preferably greater. The total number is preferably at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10. The total number can be, for example, up to 20 or more, up to 18, up to 16, up to 14, up to 12, up to 10, up to 8, up to 6 or up to 5.

[0037] Group R 4 and R 5 are each an alkylene having at least two carbon atoms. The nitrogen, R 4 、Q and R5 The ring group formed has 6 or 7 ring members. Each group R 4 and R 5 usually has 2 or 3 carbon atoms, which are included in the atoms forming the ring (e.g., the linking carbon atoms), but additional carbon atoms that are not ring atoms (e.g., non-linking carbon atoms) may be present in R 4 and R 5 . The total number of ring carbon atoms (i.e., linking atoms) in R 4 and R 5 is 4 or 5. The ring group composed of nitrogen, R 4 , Q and R 5 is usually saturated (i.e., without carbon-carbon double bonds).

[0038] Group Q is -O-, -N(R 6 )-, -S-, -S(=O)- or -S(=O)2-, provided that when Z is -NH-(C=O)-, Q is equal to -N(R 6 ). When Q is -O-, -S-, -S(=O)- or -S(=O)2-, the nitrogen atom selected as part of the same ring and attached to the group Z is such that it can be protonated. When Q is -N(R 6 ), one or two protonated groups may be present in the monomer of formula (I). Group Q has a single linking atom. That is, group Q contributes a single ring atom (i.e., a single linking atom) to the ring formed by nitrogen, R 4 , Q and R 5 . For example, in the Q group -N(R 6 ), nitrogen is the ring atom; and in the Q groups -S(=O) and -S(=O)2, sulfur is the ring atom.

[0039] If the nitrogen bonded to the group Z is part of a urea group, the ring has a single group that can be protonated, and this single group is generated by the group -N(R 6 ). That is, if Z is equal to -NH-(C=O)-, then Q is equal to -N(R 6 ). For example, in the following monomer, which is ethyl 2-[[4-(2-hydroxyethyl)piperazine-1-carbonyl]amino]2-methylprop-2-enoate (also known as IEM / N-(2-hydroxyethyl)piperazine),

[0040]

[0041] the nitrogen bonded to the -NH-(C=O)- group is part of a urea bond group, and this nitrogen atom is less likely to be protonated. The other nitrogen atom in the ring, which is in the group of formula -N(R 6 ), where R 6is a hydroxy-substituted alkyl group and is more likely to be protonated.

[0042] If the nitrogen bonded to group Z is not part of a urea group (i.e., Z is not equal to -NH-(C=O)-), then the nitrogen atom bonded to group Z plus the nitrogen in the group -N(R 6 )- can be protonated. For example, in the following monomer, which is ethyl 2-[(4-methylpiperazin-1-yl)carbamoyl amino]-2-methylprop-2-enoate (also known as IEM / 1-amino-4-methylpiperazine),

[0043]

[0044] both nitrogen atoms in the ring group can be protonated. The nitrogen attached 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 the group -N(R 6 )-, where R 6 is methyl (e.g., an alkyl group).

[0045] The group R in the Q group -N(R 6 )- 6 is hydrogen, an alkyl group, or a (hetero)aryl group. When R 6 is an alkyl group, it can optionally be further substituted by a hydroxy group, an alkoxy group, or a (hetero)aryl group. When R 6 is a (hetero)aryl group, it can optionally be further substituted by a hydroxy group, a halogen group, a nitro group, a cyano group, a trifluoromethyl group, an alkyl group, or an alkoxy group. Suitable R 6 alkyl groups can have 1 to 10 carbon atoms, such as 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 R 6 aryl groups typically have six carbon atoms, while R 6 heteroaryl groups typically have 5 or 6 ring atoms, where 1 or 2 of these ring atoms are heteroatoms and the remaining ring atoms are carbon. The heteroatoms are typically nitrogen.

[0046] When the group R 6 is an alkyl group, it can be unsubstituted or substituted by a hydroxy group (-OH), an alkoxy group, or a (hetero)aryl group. Suitable alkoxy groups for substitution have the formula -OR a , where R ais an alkyl group having 1 to 10 carbon atoms, such as 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 (hetero)aryl groups for substitution can be aryl groups having six carbon atoms or heteroaryl groups having 5 or 6 ring atoms, where 1 or 2 of these ring atoms are heteroatoms and the remaining ring atoms are carbon. The heteroatoms in the heteroaryl group are usually nitrogen.

[0047] When the group R 6 is (hetero)aryl, it can be unsubstituted or substituted with a hydroxyl group (-OH), a halogen group (such as chlorine or bromine), a nitro group (-NO2), a cyano group (-CN), a trifluoromethyl group (-CF3), an alkyl group or an alkoxy group. Suitable alkyl groups usually have 1 to 10 carbon atoms, such as 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 , where R a is an alkyl group having 1 to 10 carbon atoms, such as 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.

[0048] The monomer of formula (I) can be prepared using any suitable method. Usually two methods are used. In the first method, a (meth)acrylate monomer having an isocyanate 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 vinyl azlactone.

[0049] In some embodiments, the monomer of formula (I) can be formed using reaction scheme A, where compound (1) (which is a (meth)acrylate monomer having an isocyanate group) is reacted with a compound having a ring structure as shown in compound (2). The -NH- group in compound (2) reacts with the isocyanate group in compound (1) to form compound (3), which is a monomer having a cyclic nitrogen-containing group (i.e., the cyclic group has ring members composed of nitrogen, R 4 , Q and R 5 ).

[0050] Reaction Scheme A

[0051]

[0052] The groups R 1 , X 1 , R 2 , R4 and R 5 is the same as above. The group Q in compound (2) and compound (3) is generally -N(R 6 )-. The group Z in formula (I) is equal to -NH-(C=O)- in compound (3).

[0053] In many embodiments of compound (1), the group X 1 is -O-, and R 2 is an alkylene group 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.

[0054] In some methods for preparing compound (3), the group Q in compound (2) is -NH-, and this group is protected so that it does not react with compound (1). For example, the -NH- group of Q can be protected by reacting with Boc2O, which is a compound of formula (CH3)3C-(C=O)-O-(C=O)-C(CH3)3, to form an intermediate group Q 1 , which is -NR 7 -, where R 7 is -(C=O)-C(CH3)3. After reacting with compound (1), the group -NR 7 - can react by heating in the presence of trifluoroacetic acid to reform the -N(H)- group of Q. For example, if compound (2) is piperazine, such a method can be used.

[0055] Examples of compound (2) include but are not limited to: (A) N-(2-hydroxyethyl)piperazine, where Q is -N(R 6 )-, where R 6 is an alkyl group substituted by a hydroxyl group, (B) N-methylpiperazine, where Q is -N(R 6 )-, and R 6 is an alkyl group, (C) N-phenylpiperazine, where Q is -N(R 6 )-, and R 6 is an aryl group, (D) N-phenethylpiperazine, where Q is -N(R 6 )-, and R 6 is an alkyl group substituted by an aryl group, (E) piperazine, where Q is -N(H)-, (F) N-methylhomopiperazine, where Q is -N(R 6 )-, and R 6 is an alkyl group, (G) 1-(2-pyridyl)piperazine, where Q is -N(R 6 )-, and R6 is a 6 - membered heteroaryl having a nitrogen heteroatom, (H) 1-(2 - pyrimidinyl) piperazine, where Q is -N(R 6 )-, and R 6 is a 6 - membered heteroaryl having 2 nitrogen heteroatoms, (I) N-(4 - hydroxyphenyl) piperazine, where Q is -N(R 6 )-, and R 6 is an aryl group substituted by a hydroxyl group, (J) N-(4 - methoxyphenyl) piperazine, where Q is -N(R 6 )-, and R 6 is an aryl group substituted by an alkoxy group, (K) N-(4 - nitrophenyl) piperazine, where Q is -N(R 6 )-, and R 6 is an aryl group substituted by a nitro group, (L) N-(2 - methoxyethyl) piperazine, where Q is -N(R 6 )-, and R 6 is an alkyl substituted by an alkoxy group, and (M) N - benzylpiperazine, where Q is -N(R 6 )-, and R 6 is an alkyl substituted by an aryl group.

[0056] In other embodiments, the monomer of formula (I) can be formed using reaction scheme B, where compound (1) (which is a (meth) acrylate monomer having an isocyanate group) reacts with a compound having an -NH2 group attached to a ring structure as shown in compound (4). The product is compound (5), which is the monomer.

[0057] Reaction Scheme B

[0058]

[0059] The groups R 1 , X 1 , R 2 , R 4 , R 5 and Q in compounds (1), (4) and (5) are the same as above. The group Z in formula (I) is equal to -NH-(C = O)-NH- in compound (5).

[0060] Examples of compound (4) include but are not limited to: (A) 1 - amino - 4 - methylpiperazine, where Q is -N(R 6 )-, where R 6 is an alkyl; (B) 1 - amino - 4 - phenylpiperazine, where Q is -N(R 6 )-, where R 6is an aryl; and (C) 1-amino-4-cyclopentylpiperazine, wherein Q is -N(R 6 ), wherein R 6 is an alkyl (i.e., cycloalkyl).

[0061] In still other embodiments, the monomer of formula (I) can be formed using reaction scheme C, wherein compound (1) (which is a (meth)acrylate monomer having an isocyanate group) reacts with a compound having an NH2-R 3 - group to produce compound (7) which is a monomer, and the NH2-R 3 - group is attached to the ring structure as shown in compound (6).

[0062] Reaction Scheme C

[0063]

[0064] The groups R 1 , X 1 , R 2 , R 4 , R 5 and Q in compound (1), compound (6) and compound (7) are the same as those described above. The group Z in formula (I) is equal to -NH-(C=O)-NH-R 3 - in compound (7). The group R 3 is an alkylene group, usually having 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.

[0065] Examples of compound (6) include but are not limited to: (A) N-(2-aminoethyl)morpholine, wherein Q is -O-, and R 3 is an alkylene group; (B) N-(3-aminopropyl)morpholine, wherein Q is -O-, and R 3 is an alkylene group; (C) N-(2-aminoethyl)thiomorpholine-1,1-dioxide, wherein Q is -S(O2)-, and R 3 is an alkylene group; (D) N-(3-aminopropyl)thiomorpholine-1,1-dioxide, wherein Q is -S(O2)-, and R 3 is an alkylene group; (E) N-(2-aminoethyl)thiomorpholine, wherein Q is -S-, and R 3 is an alkylene group; (F) N-(2-aminoethyl)morpholine, wherein Q is -O-, and R 3 is an alkylene group; (G) N-(3-aminopropyl)thiomorpholine-1-oxide, wherein Q is -S(O)-, and R 3is an alkylene group; (G) N-(2-aminoethyl) thiomorpholine-1-oxide, where Q is -S(O)-, and R 3 is an alkylene group; (H) N-(3-aminopropyl) thiomorpholine, where Q is -S-, and R 3 is an alkylene group; and (I) 1-(3-aminopropyl)-4-methylpiperazine, where Q is -N(R 6 )-, R 6 is an alkyl group, and R 3 is an alkylene group.

[0066] In other embodiments, the monomer of formula (I) can be formed using reaction scheme D, where compound (8) (which is an alkenyl azlactone) reacts with compound (6) having an NH2-R 3 - group to produce compound (9) which is the monomer, and the NH2-R 3 - group is attached to the ring structure. Compound (6) is the same as the compound used for reaction scheme (C) above.

[0067] Reaction Scheme D

[0068]

[0069] The groups R 1 , R 3 , R 4 , R 5 and Q in compound (8), compound (6) and compound (10) are the same as above. The group R 2 is usually an alkylene group, such as -C(CH3)2- or -C(CH3)2CH2-. The group Z in formula (I) is equal to -(C=O)-NH-R 3 - in compound (9). Examples of suitable compound (6) are described above for reaction scheme C.

[0070] In a further embodiment, the monomer of formula (I) can be formed using reaction scheme E, where compound (8) (which is an alkenyl azlactone) reacts with compound (4) having an NH2- group to produce compound (10) which is the monomer, and the NH2- group is attached to the ring structure.

[0071] Reaction Scheme E

[0072]

[0073] The groups R 1 , R 2 , R 4 , R 5 and Q in compound (8), compound (4) and compound (10) are the same as above. The group R2 is usually an alkylene group such as -C(CH3)2- or -C(CH3)2CH2-. The group Z in formula (I) is equal to -(C=O)-NH- in compound (10). Examples of suitable compounds (4) are described above for Reaction Scheme B.

[0074] Although the total number of linking atoms in the groups R 2 and Z can be as low as three, the combined groups preferably have at least 4 linking atoms, especially when the monomer of formula (I) is used to form an anion exchange separation article. R 2 and the total number of linking atoms in Z determine the length of the spacer group between the alkenyl group (CH2=CR 1 - group) and the nitrogen atom that can be protonated. Generally, the spacer group has at least 5 linking atoms, but when the monomer of formula (I) is used to form an anion exchange separation article, R 2 and Z are usually selected to provide a longer spacer length. The groups R 2 and Z are usually selected to provide a spacer group having at least 6, at least 7, or at least 8 linking atoms. 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 linking atoms. A longer spacer group or a larger total number of linking atoms in the groups R 2 and Z tends to enhance the binding to other materials. That is, monomer units having a longer spacer length tend to function better in anion exchange separation methods.

[0075] The method of counting linking atoms is shown in the following monomers.

[0076]

[0077] For the compound (ethyl 2-[(piperazine-1-carbonyl)phenylamino]prop-2-enoate (referred to herein as IEM / N-phenylpiperazine)), there are 9 linking atoms between the alkenyl group and the protonatable ring nitrogen. The nitrogen atom as part of the urea bond, such as the nitrogen atom at position 7 in the above compound, generally cannot be protonated. In this compound, the nitrogen at position 10 can be protonated. However, for the following compound

[0078] ">

[0079] (ethyl 2-(3-morpholinopropylcarbamoylamino)prop-2-enoate (referred to herein as IEM / N-(3-aminopropyl)morpholine)), the nitrogen atom at position 11 can be protonated.

[0080] The calculated pKa values of the monomers are typically selected in the range of from 3 to 9.5, or in the range of from 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 values can be used to predict which monomers may be suitable for separating various material mixtures, such as mixtures containing biological materials.

[0081] Methods and software for calculating pKa values are provided in the Examples section below. These calculations can be used to quickly evaluate whether the pH of the monomer is within an acceptable range prior to synthesizing a polymer chain and preparing an anion exchange separation article. The error estimate for calculating the pKa value based on the Hammett-Taft method typically depends on the structural similarity of the new material to those present in the model database. The estimate is useful for biological applications because the error is generally less than the range of acceptable pH values for the application. Thus, by calculating the pKa value, a limited number of potential ligands can be quickly selected to evaluate their suitability for a particular separation in the desired pH range. As opposed to randomly synthesizing and evaluating a large library of ligands, this selection can significantly save time.

[0082] Literature articles providing pKa value calculation information 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 pK a"Prediction and Protonation State Generation for Drug-like Molecules)", Journal of Computer-Aided Molecular Design, 2007, 21, 681-691.

[0083] Article with graft polymer

[0084] The following articles can be prepared, which have a plurality of 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 or copolymers, the homopolymer being formed only from monomers of formula (I), and the copolymer being formed from a mixture of monomers of formula (I) and other monomers that are not of formula (I). The other monomers in the mixture are typically hydrophilic monomers that are not ionic.

[0085] The polymer chains in the article are typically grafted onto a porous polymer substrate that is solid. The term "solid" with respect to the porous polymer substrate means that the substrate is not a liquid and is insoluble in solution. Small particles suspended in a liquid are not considered to be dissolved in the liquid. That is, as used herein, a suspension is not considered to be a solution in this context, and the suspended particles are solids. However, in many embodiments, the solid substrate is not small particles, but rather a larger form is selected, such as those described further below.

[0086] The pores of the porous polymer substrate can have any desired average size. In some embodiments, the pores are macropores, mesopores, micropores, or a mixture thereof. As used herein, the term "macropore" refers to a polymer substrate having pores with a diameter greater than 50 nanometers, the term "mesopore" refers to a polymer substrate having pores with a diameter in the range of 2 to 50 nanometers, and the term "micropore" refers to a material having pores with a diameter less than 2 nanometers.

[0087] The terms "solid porous polymer substrate", "porous polymer substrate", "polymer substrate", "substrate", and similar variant forms are used interchangeably herein.

[0088] The porous polymer substrate can have any desired size, shape, and form. For example, the porous polymer substrate can be in the form of granules, 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 separation articles or many separation articles and for ease of manufacture, the polymer substrate can be in the form of a roll or formed from a roll, such as a roll of film, nonwoven web, woven web, membrane, sponge, or sheet. This allows for roll-to-roll processing to prepare the separation articles. The porous polymer substrate can include a single layer or multiple layers of the same or different polymer materials.

[0089] The porous polymer substrate is typically formed from a thermoplastic material. Suitable thermoplastics include, but are not limited to, polyolefins, poly(isoprene), poly(butadiene), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfone), poly(sulfone), poly(vinyl acetate) and its copolymers such as poly(ethylene)-co-poly(vinyl acetate), polyesters such as poly(lactic acid), poly(vinyl alcohol) and its copolymers such as poly(ethylene)-co-poly(vinyl alcohol), poly(vinyl esters), poly(vinyl ethers), poly(carbonates), polyurethanes, poly((meth)acrylates) and their copolymers, and combinations thereof.

[0090] Suitable polyolefins for the porous polymer substrate include poly(ethylene), poly(propylene), poly(1-butene), copolymers of ethylene and propylene, α-olefin copolymers (such as copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and / or 1-decene), poly(ethylene-co-1-butene), poly(ethylene-co-1-butene-co-1-hexene), poly(butadiene) and its copolymers, and combinations thereof.

[0091] Suitable fluorinated polymers for the porous polymer substrate include poly(vinyl fluoride), poly(vinylidene fluoride), vinylidene fluoride copolymers (such as poly(vinylidene fluoride-co-hexafluoropropylene)), chlorotrifluoroethylene copolymers (such as poly(ethylene-co-chlorotrifluoroethylene)), and combinations thereof.

[0092] Suitable polyamides for the porous polymer substrate include various nylon compositions, such as, for example, poly(iminoadipoyliminohexamethylene), poly(iminoadipoyliminodecamethylene), polycaprolactam, and combinations thereof. Suitable polyimides include poly(pyromellitimide) and combinations thereof.

[0093] Suitable poly(ether sulfone) for the porous polymer substrate include poly(diphenyl ether sulfone), poly(diphenyl sulfone-co-diphenyl ether sulfone), and combinations thereof.

[0094] Suitable vinyl acetate copolymers for the porous polymer substrate include copolymers of ethylene and vinyl acetate and terpolymers of vinyl acetate, vinyl alcohol, and ethylene.

[0095] In some embodiments, the porous polymer substrate is a porous membrane having an average pore diameter (average longest diameter of the pores) typically greater than 0.1 microns to minimize size exclusion separation, minimize diffusional constraints, and maximize surface area and separation. Generally, the average pore diameter can range from 0.1 microns to 10 microns. For example, the average pore diameter is at least 0.2 microns, at least 0.4 microns, at least 0.6 microns, or at least 0.8 microns, and up to 8 microns, up to 6 microns, up to 4 microns, or up to 2 microns.

[0096] The porous polymer substrate can be a macroporous membrane, such as a thermally induced phase separation (TIPS) membrane. TIPS membranes are typically prepared by forming a solution of a thermoplastic material and a second material above the melting point of the thermoplastic material. After cooling, the thermoplastic material crystallizes and phase separates from the second material. The crystallized material is typically stretched. The second material is optionally removed before or after stretching. Macroporous membranes are further described in U.S. Patent 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. Patent No. 7,338,692 (Smith et al.).

[0097] 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. Patent 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.).

[0098] In other embodiments, the porous polymer substrate can be a nonwoven web, which can include nonwoven webs made by any known method for producing nonwoven webs. As used herein, the term "nonwoven web" refers to a fabric having a structure of individual fibers or filaments that are randomly and / or unidirectionally interlaced in a mat-like manner.

[0099] For example, fibrous nonwoven webs can be made by wet-laying, carding, air-laying, spunlacing, spunbonding, or meltblowing techniques, or combinations thereof. Spunbond fibers are generally small-diameter fibers formed by extruding a molten thermoplastic polymer as filaments from a plurality of fine, generally circular capillaries of a spinneret, where the diameter of the extruded fibers rapidly decreases. Meltblown fibers are generally formed by extruding a molten thermoplastic material through a plurality of fine, generally circular die capillaries in the form of molten threads or filaments into a high-velocity, generally heated gas stream (e.g., air), which attenuates the filaments of the molten thermoplastic material to reduce their diameter. The meltblown fibers are then carried by the high-velocity gas stream and deposited onto a collecting surface to form a web of randomly dispersed meltblown fibers. Any nonwoven web can be made of a single type of fiber or of two or more fibers of different types and / or thicknesses of thermoplastic polymers.

[0100] Further details of available methods for manufacturing nonwoven webs are described in the following references: 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).

[0101] The nonwoven web substrate can also optionally include one or more scrim layers. For example, either or both major surfaces of the nonwoven web can each optionally include a scrim layer. The scrim (which is typically a woven or nonwoven reinforcing layer made of fibers) is included to provide strength to the nonwoven web. Suitable scrim materials include, but are not limited to, nylon, polyester, fiberglass, polyethylene, polypropylene, etc. The average thickness of the scrim can vary, but generally ranges from about 25 microns to about 100 microns, preferably from about 25 microns to about 50 microns. The scrim layer can optionally be bonded to the nonwoven article. A variety of adhesive materials can be used to bond the scrim to the nonwoven material. Alternatively, the scrim can be thermally bonded to the nonwoven web.

[0102] The porosity of the nonwoven substrate is typically characterized by properties such as fiber diameter, or basis weight, or density, rather than by pore diameter. The fibers of the nonwoven substrate are typically microfibers having an effective fiber diameter of at least 0.5 microns, 1 micron, 2 microns or even 4 microns and at most 15 microns, 10 microns, 8 microns or even 6 microns, as calculated according to the method listed in the following literature: Davies, C.N., “The Separation of Airborne Dust and Particles,” Institution of Mechanical Engineers, London, Proceedings 1B, 1952. The nonwoven substrate preferably has a basis weight in the range of at least 5 g / m 2 、10 g / m 2 、20 g / m 2 or even 50 g / m 2 ; and at most 800 g / m 2 、600 g / m 2 、400 g / m 2 、200 g / m 2 or even 100 g / m 2 . The minimum tensile strength of the nonwoven web is about 4.0 Newtons. It is generally believed that the tensile strength of the nonwoven substrate is lower in the longitudinal direction than in the cross-direction, due to better fiber bonding and entanglement in the latter.

[0103] The bulkiness of the nonwoven web is measured by density, which is a parameter defining the solid fraction in the volume of the web. A lower density value indicates a greater bulkiness of the web. The density (α) is a unitless fraction and is usually expressed as: α = m f ÷ρ f ×L 非织造物 , where m f is the fiber mass per sample surface area, ρ f is the fiber density, and L 非织造物 is the nonwoven thickness. The density used herein refers to the nonwoven substrate itself rather than the functionalized nonwoven substrate. When the nonwoven substrate contains a mixture of two or more types of fibers, the same L 非织造物 is used to determine the individual hardness of each fiber, and these individual hardnesses are added together to obtain the hardness α of the web.

[0104] The polymer chains grafted to 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). The polymer chains are typically homopolymers of monomers of formula (I) to prepare polymers having a high binding capacity for the material to be trapped as desired. That is, based on the total weight of the monomers used to form the polymer chains, the polymer chains can contain up to 100% by weight of the first monomer of formula (I). In some embodiments, other monomers (second monomers) can be copolymerized with the first monomer to adjust the binding capacity and / or achieve other desired properties of the polymer chains. Any suitable second monomers can be used, but they are typically hydrophilic monomers. For example, they are typically water-soluble or miscible with water.

[0105] Based on the total weight of the monomer units in the polymer chains, the amount of the first monomer of formula (I) can be, for example, in the range of 10% to 100% by weight or in the range of 20% to 100% by weight. Based on the total weight of the monomer units in the polymer chains, the amount can be at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight or at least 95% by weight, and up to 100% by weight, up to 99% by weight, up to 98% by weight, up to 97% by weight, up to 95% by weight, up to 90% by weight, up to 85% by weight, up to 80% by weight or up to 75% by weight. Higher amounts of the first monomer tend to increase the binding capacity for various target compounds such as biomaterials. In many embodiments, based on the total weight of the monomer units, the amount of the first monomer of formula (I) is in the range of 80% to 100% by weight, 85% to 100% by weight, 90% to 100% by weight or 95% to 100% by weight.

[0106] The optional second monomers in the polymer chains can be, for example, hydrophilic monomers to adjust the degree of hydrophilicity imparted to the substrate or to adjust the charge density of the anion exchange separation article. The hydrophilic monomers have an ethylenically unsaturated group and a hydrophilic group such as, for example, a hydroxyl group, an ether group or an amido group. Suitable hydrophilic monomers include, for example, acrylamide, dimethylacrylamide, 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, ethoxyethyl methacrylate, diethylene glycol methyl ether methacrylate, 2-hydroxyethyl acrylamide, N-vinylpyrrolidone, etc. and combinations thereof.

[0107] Other optional second monomers include those having more than one ethylenically unsaturated group. Second monomers of this type are generally water-soluble and are used only in relatively small amounts to impart a degree of branching and / or relatively light crosslinking to the resulting copolymer. For example, based on the total weight of the monomers in the polymerizable composition, the amount of these polyfunctional monomers having more than two ethylenically unsaturated groups can be present in an amount ranging from 0.1 wt% to 25 wt%. The amount can be at least 0.1 wt%, at least 0.2 wt%, at least 0.5 wt% or at least 1.0 wt%, and up to 25 wt%, up to 20 wt%, up to 15 wt%, up to 10 wt%, up to 5 wt%, up to 4 wt%, up to 3 wt%, up to 2 wt% or up to 1 wt%. Although crosslinking monomers can be used and can be beneficial for some applications, they tend to reduce the binding ability to some biomaterials.

[0108] Examples of suitable crosslinking monomers include, but are not limited to, poly(ethylene glycol di(meth)acrylate), methylenebisacrylamide, 3-acryloyloxy-2-hydroxypropyl methacrylate, glycerol dimethacrylate, glycerol diacrylate, diacryloyl piperazine, and 1,2-ethylenebisacrylamide.

[0109] The total amount of the second monomer can be up to 80 wt% of the monomers used to form the polymer chains. Lower amounts of the second monomer generally enhance the binding ability to various target compounds such as protein biomaterials. Based on the total weight of the monomers in the polymerizable composition, this amount, if present, is generally equal to 100 minus the weight percentage of the first monomer of formula (I).

[0110] In some embodiments, the polymerizable composition comprises 10 wt% to 90 wt% of the first monomer of formula (I) and 90 wt% to 10 wt% of the second hydrophilic monomer. For example, the polymerizable composition can comprise 20 wt% to 90 wt% of the first monomer and 80 wt% to 10 wt% of the second monomer, 10 wt% to 80 wt% of the first monomer and 90 wt% to 20 wt% of the second monomer, 30 wt% to 90 wt% of the first monomer and 70 wt% to 10 wt% of the second monomer, 30 wt% to 80 wt% of the first monomer and 70 wt% to 20 wt% of the second monomer, 30 wt% to 70 wt% of the first monomer and 70 wt% to 30 wt% of the second monomer, 40 wt% to 90 wt% of the first monomer and 60 wt% to 10 wt% of the second monomer, or 50 wt% to 90 wt% of the first monomer and 50 wt% to 10 wt% of the second monomer.

[0111] Polymer chains are grafted to a 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 radiation, the type II photoinitiator extracts hydrogen atoms from the porous polymer substrate, resulting in the generation of 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 typically grafted to carbon atoms in the backbone of the polymer material contained in the porous polymer substrate.

[0112] Type II photoinitiators are typically aromatic ketone compounds. Examples include, but are not limited to, benzophenone, carboxybenzophenone (e.g., 3-carboxybenzophenone), sodium 4-(3-sulfopropoxy)benzophenone, Michler's ketone, benzil, anthraquinone, 5,12-tetracenequinone, acetylanthracenequinone, benz[a]anthracene-7,12-dione, 1,4-chrysenequinone, 6,13-pentacenequinone, 5,7,12,14-pentacene tetrone, 9-fluorenone, anthrone, xanthone, thioxanthone, 2-(3-sulfopropoxy)thioxanthen-9-one, acridone, dibenzocycloheptanone, acetophenone, and chromone.

[0113] The ultraviolet (UV) light used to generate free radicals on the porous polymer substrate can be provided by various 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 higher-intensity light sources, such as those available from Fusion UV Systems Inc. The UV light source can be a relatively low-light-intensity source, such as a black light, which provides typically 10 mW / cm 2 or less of light in the wavelength range of 280 nanometers to 400 nanometers (as measured according to procedures approved by the National Institute of Standards and Technology, e.g., using a UVIMAP UM 365L-S radiometer manufactured by Electronic Instrumentation & Technology, Inc. of Sterling, VA). Alternatively, a relatively high-light-intensity source, such as a medium-pressure mercury lamp, can be used, which provides typically greater than 10 mW / cm 2 and preferably in the range of 15 mW / cm 2 to 450 mW / cm 2 The exposure time can be up to about 30 minutes or even longer.

[0114] In another method that can be used to generate free radicals on the surface of a porous polymer substrate, the substrate itself is chosen to be photoactive and does not require a type II photoinitiator. The monomer composition is exposed to actinic radiation, which is typically in the ultraviolet region of the electromagnetic spectrum. Upon exposure to actinic radiation, the polymer substrate absorbs sufficient energy to break some of its covalent bonds, resulting in the generation of 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 typically contain aromatic groups, such as, for example, poly(methylphenylsilane) and homopolymers and block copolymers of various polyimides based on benzophenone tetracarboxylic dianhydride.

[0115] In other methods for generating free radicals on the surface of a polymer substrate, ionizing radiation is used instead of a type II photoinitiator and / or UV radiation. As used herein, the term "ionizing radiation" refers to radiation having a 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 and causes the cleavage of chemical bonds in the substrate and the formation of free radicals. Ionizing radiation is typically beta radiation, gamma radiation, electron beam radiation, x-ray radiation, plasma radiation, or other suitable types of electromagnetic radiation. Preferably, the ionizing radiation is carried out in an inert environment to prevent oxygen from reacting with the free radicals.

[0116] In many embodiments of this method, the ionizing radiation is electron beam radiation, gamma ray radiation, x-ray radiation, or plasma radiation because suitable generators are readily available. Electron beam generators are commercially available, such as, for example, the ESI ELECTROCURE EB SYSTEM from Energy Sciences, Inc., Wilmington, MA, USA and the BROADBEAM EB PROCESSOR from E-beam Technologies, Davenport, IA, USA. Gamma ray radiation generators using cobalt-60 high energy sources are commercially available from MDS Nordion.

[0117] 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,” of ASTM International (West Conshohocken, PA). By varying the extractor grid voltage, beam diameter, exposure time, and distance from the irradiation source, various dose rates can be obtained.

[0118] Multiple polymer chains are grafted to a porous polymer substrate. The term “ligand density” refers to the millimoles of monomer units grafted to the substrate per gram. The millimoles are 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 initial mass (grams) of the porous polymer substrate. The ligand density (millimoles / gram) is expressed as the millimoles of monomer units grafted per gram of substrate. For clarity, the grafted material is typically a polymeric material containing multiple monomer units.

[0119] When the substrate is a membrane, the anion exchange separation article typically has a ligand density of from about 0.02 millimoles / gram to about 3 millimoles / gram or even higher. The graft density can be at least 0.02 millimoles / gram, at least 0.05 millimoles / gram, at least 0.1 millimoles / gram, at least 0.2 millimoles / gram, at least 0.5 millimoles / gram, or at least 1 millimole / gram, and up to 3 millimoles / gram, up to 2.5 millimoles / gram, up to 2 millimoles / gram, up to 1.5 millimoles / gram, up to 1 millimole / gram, up to 0.8 millimoles / gram, up to 0.7 millimoles / gram, or up to 0.5 millimoles / gram. The weight gain is calculated by the formula [100 (weight2 – weight1) ÷ weight1], where weight1 is the weight of the substrate and weight2 is the weight of the substrate with the grafted polymer attached. The weight gain can range from 1 wt% to 85 wt% or even higher. The amount can be, for example, at least 1 wt%, at least 2 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, at least 35 wt%, at least 40 wt%, at least 45 wt%, or at least 50 wt%, and up to 85 wt%, up to 80 wt%, up to 75 wt%, up to 70 wt%, up to 65 wt%, up to 60 wt%, up to 55 wt%, up to 50 wt%, up to 45 wt%, up to 40 wt%, up to 35 wt%, or up to 30 wt%.

[0120] When the substrate is a nonwoven or fibrous substrate, the weight gain during grafting is typically higher than that of a membrane substrate. The weight gain can range from 20% to 400% by weight or even higher. The amount can be, for example, at least 20% by weight, at least 50% by weight, at least 100% by weight, at least 150% by weight, at least 200% by weight, at least 250% by weight or at least 300% by weight, and up to 400% by weight, up to 350% by weight, up to 300% by weight, up to 250% by weight, up to 200% by weight, up to 150% by weight, up to 100% by weight, up to 75% by weight or up to 50% by weight. For example, the weight gain can range from 100% to 400% by weight, from 100% to 300% by weight or from 100% to 200% by weight.

[0121] Method for separating a mixture of materials using an anion exchange separation article

[0122] Anion exchange separation articles can be used to separate mixtures of materials having different ionic contents. The anion exchange separation articles are the same as those described above. The method includes passing the mixture of materials through the anion exchange separation article at a first pH and a first ionic strength value, the first pH being low enough to protonate the monomer repeating units of the graft polymer of the monomer derived from formula (I), the first ionic strength value binding at least one component of the mixture of materials as a bound component to the anion exchange separation article.

[0123] A first pH is selected to protonate at least a portion of the monomer repeating units of the graft polymer of the monomer derived from formula (I). The protonated portion is typically at least 5 mol%, at least 10 mol%, at least 20 mol%, at least 30 mol%, at least 40 mol%, at least 50 mol% or at least 60 mol%, and up to 100 mol%, up to 95 mol%, up to 90 mol%, up to 80 mol%, up to 70 mol%, up to 60 mol%, up to 50 mol%, up to 40 mol% or up to 30 mol%. The pH can be optimized using experimental methods known to those skilled in the art and / or as demonstrated in the Examples section below. If desired, the proportion of protonated monomer units can be calculated 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 units to unprotonated units.

[0124] Typically, the pH selected is lower than the pKa value of the monomer of formula (I) used to prepare the graft polymer attached to the porous polymeric substrate. If the pH is lower than the pKa value, the monomer units derived from the monomer of formula (I) will carry a positive charge. Under these lower pH conditions, the graft polymer can bind to negatively charged substances. If the pH is increased above the pKa value, the monomer units in the graft polymer derived from the monomer of formula (I) are neutralized, and the bound material can be released. The calculated pKa values of the monomers of formula (I) are generally in the range of about 3.5 or 4 to about 9 or 9.5. Thus, the composition of the graft polymer can be adjusted to optimize the pKa value for separating different mixtures. That is, the composition of the graft polymer and the pH can be adjusted to (a) bind the material of interest rather than the impurities, or (b) bind the impurities rather than the material of interest.

[0125] As described above, the pH can be adjusted such that the material of interest is retained rather than the impurities when the sample passes through the anion exchange separation article. After the sample has passed through, the anion separation article can optionally be washed to remove any residual impurities. Then, by increasing the pH of the composition passing through the anion exchange separation article and / or increasing its ionic strength, the material of interest can be released from the anion exchange separation article. This method can be referred to as a binding and release (i.e., elution) process. In some embodiments of the binding and release process, the pH and / or ionic strength can be increased gradually or in a stepwise manner to further separate the mixture of bound materials.

[0126] Alternatively, the pH can be adjusted such that impurities are retained but the material of interest is not retained when the sample passes through the anion exchange separation article. This method can be referred to as a flow-through separation method. If desired, after the material of interest has passed through, by increasing the pH of the composition passing through the anion exchange separation article and / or increasing its ionic strength, the retained impurities can be released from the anion exchange separation article. After removing the retained impurities, the anion exchange separation article can be used again to separate 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 can be referred to as a disposable device.

[0127] Many materials that need to be separated are biological materials with an isoelectric point (pI). At a pH value above its pI value, the biological material carries a net negative charge and can bind to the positively charged graft polymer; however, at a pH value below its pI value, the biological material carries a net positive charge and is not attracted to the positively charged graft 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.

[0128] The anion exchange separation devices described herein are well-suited for separating many biological materials that cannot tolerate the high pH conditions (e.g., pH 10, 11 or higher, depending on the biological material) or low pH conditions (e.g., pH 5, 4 or lower, depending on the biological material) and / or high salt concentrations (e.g., 0.5 M or higher, such as 1.0 M) commonly used in many current anion exchange separation articles. The monomers of formula (I) can be used to form graft polymers on anion exchange separation articles, which can be used at pH conditions less than pH 11 or pH 10, such as in the range of pH 3.5 or 4 to 9 or 9.5. For example, the pH can be as low as 3.5 or even lower, as low as 4, as low as 4.5, as low as 5.0, as low as 5.5, as low as 6.0, as low as 6.5, as low as 7, as low as 7.5 or as low as 8, and as high as 11 or even higher, as high as 10.5, as high as 10, as high as 9.5, as high as 9, as high as 8.5, as high as 8, as high as 7.5, as high as 7, as high as 6.5, as high as 6, as high as 5.5 or as high as 5.

[0129] In addition, these anion exchange separation articles can bind and elute biological materials at salt concentrations less than or equal to 0.5 M, such as in the range of 0.01 M to less than 0.5 M. The 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, and as high as 0.5 M, as high as 0.45 M, as high as 0.4 M, as high as 0.35 M, as high as 0.3 M, as high as 0.25 M, as high as 0.2 M, as high as 0.15 M, as high as 0.1 M or as high as 0.05 M. The salt concentration is typically expressed based on conductivity. For example, the conductivity is typically less than or equal to 50 mS / cm (millisiemens per centimeter), such as in the range of 1 mS / cm to 50 mS / cm. That is, the conductivity can be at least 1 mS / cm, at least 2 mS / cm, at least 3 mS / cm, at least 5 mS / cm or at least 10 mS / cm, and as high as 50 mS / cm, as high as 40 mS / cm, as high as 30 mS / cm, as high as 20 mS / cm, as high as 10 mS / cm or as high as 5 mS / cm.

[0130] Non-grafted polymer having monomer units of monomers derived from formula (I)

[0131] The polymer can be formed from monomers of formula (I) that are not grafted to the 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 to be soluble in polar solvents and, if desired, can be coated onto a porous or non-porous substrate. The polymerization method can be carried out using any known method. In most polymerization methods, a radical initiator is used, which can be a thermal initiator or a photoinitiator. In many embodiments, a photoinitiator is used.

[0132] Useful photoinitiators include, but are not limited to, 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); 1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one available as Irgacure 2959 (Ciba Specialty Chemicals); dimethoxyhydroxyacetophenone; substituted α-ketols such as 2-methyl-2-hydroxypropiophenone; aromatic sulfonyl chlorides such as 2-naphthalenesulfonyl chloride; and photosensitive oximes such as 1-phenyl-1,2-propanedione-2-(O-ethoxy-carbonyl)oxime.

[0133] Other useful photoinitiators also include, for example, hydrogen abstraction type (type II) photoinitiators such as benzophenone, sodium 4-(3-sulfopropyl)benzophenone, Michler's ketone, benzil, anthraquinone, 5,12-tetracenequinone, acetylanthraquinone, benz(A)anthracene-7,12-dione, 1,4-chrysenequinone, 6,13-pentacenequinone, 5,7,12,14-pentacenetetrone, 9-fluorenone, anthrone, xanthone, thioxanthone, 2-(3-sulfopropyl)thioxanthen-9-one, acridone, dibenzocycloheptanone, acetophenone, and chromone.

[0134] Examples of suitable thermal initiators include peroxides such as benzoyl peroxide, dibenzoyl peroxide, dilauroyl peroxide, cyclohexanone peroxide, methyl ethyl ketone peroxide, hydroperoxides (e.g., tert-butyl hydroperoxide and cumene hydroperoxide), dicyclohexyl peroxydicarbonate, tert-butyl perbenzoate, 2,2'-azobis(isobutyronitrile), etc., or combinations thereof. Examples of commercially available thermal initiators include initiators available under the VAZO trade name from Chemours Company (Chemours (Wilmington, Delaware)), Wilmington, Delaware, including VAZO 67 (2,2'-azobis(2-methylbutyronitrile)), VAZO 64 (2,2'-azobis(isobutyronitrile)), and VAZO 52 (2,2'-azobis(2,2-dimethylvaleronitrile)); and also initiators available as Lucidol 70 (benzoyl peroxide) from Elf Atochem North America (Elf Atochem North America (Philadelphia, PA)), Philadelphia, Pennsylvania.

[0135] The initiator can be used in an effective amount sufficient to initiate free radical polymerization of the monomers. This amount will vary depending on, for example, the type of initiator used and the polymerization conditions. Based on 100 parts of total monomers, the amount of initiator used is typically in the range of about 0.01 parts by weight to about 5 parts by weight.

[0136] The polymerization solvent can be substantially any solvent that is capable of dissolving (or, in the case of emulsion or suspension polymerization, dispersing or suspending) the monomers (and comonomers, if used). In many embodiments, the solvent can be water or a water / water-miscible organic solvent mixture. The ratio of water to organic solvent can vary widely depending on monomer solubility. For some monomers of formula (I), the ratio of water to organic solvent can be greater than 1:1 (volume / volume), such as greater than 5:1, greater than 7:1, or even greater than 10:1. If desired, a higher proportion of organic solvent (even up to 100%) can be used, such as when the organic solvent is an alcohol.

[0137] Any such water-miscible organic solvent preferably does not have groups that would retard polymerization. In some embodiments, the water-miscible solvent can be an organic liquid containing a proton group, such as a lower alcohol having 1 to 4 carbon atoms, a lower diol having 2 to 6 carbon atoms, and a lower diol ether having 3 to 6 carbon atoms and 1 to 2 ether bonds. In some embodiments, higher diols such as poly(ethylene glycol) can be used. Specific examples include methanol, ethanol, isopropanol, n-butanol, tert-butanol, ethylene glycol, methoxyethanol, ethoxyethanol, propoxyethanol, butoxyethanol, methyl carbitol, ethyl carbitol, etc., and combinations thereof.

[0138] In other embodiments, aprotic water-miscible organic solvents can be used. Such solvents include 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).

[0139] The monomer concentration in the polymerization solvent can vary depending on various factors, including but not limited to the nature of one or more monomers, the desired degree of polymerization, the reactivity of the monomers, and the solvent used. Generally, based on the total weight of the monomers and the solvent, the monomer concentration can range from about 0.1 wt% to about 60 wt% or even higher. For example, based on the total weight of the monomers and the solvent, the concentration of the monomers can be at least 0.1 wt%, at least 0.5 wt%, at least 1 wt%, at least 2 wt%, at least 5 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 25 wt%, or at least 30 wt%, and up to 60 wt%, up to 55 wt%, up to 50 wt%, up to 45 wt%, up to 40 wt%, up to 35 wt%, up to 30 wt%, up to 25 wt%, or up to 20 wt%.

[0140] Any of the above optional second monomers for the graft polymer can be included in the reaction mixture for forming the non-graft polymer.

[0141] The time and temperature used for the polymerization reaction with a thermal initiator can be any amounts used in the art. Similarly, the time and radiation source suitable for use with a photoinitiator can be any amounts used in the art.

[0142] The aqueous monomer mixture can optionally be formulated with a relatively high level of polyfunctional (crosslinking) monomers or comonomers (e.g., about 5 wt% to about 90 wt% based on the total weight of the monomers and comonomers), and optionally in the presence of an added porogen, polymerized as a suspension or dispersion in a nonpolar immiscible organic solution to produce crosslinked porous particles comprising monomer units derived from the monomers of formula (I). Such methods are described, for example, in U.S. Pat. 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.).

[0143] Example

[0144] Calculation of pKa

[0145] The pKa values were calculated using the EpiK pKa prediction tool and the Maestro GUI, both obtained from Schrödinger, LLC (New York, NY). The calculations were performed on monomeric substances using Maestro version 2021-4, set to "Sequential pKa values", with H2O as the solvent up to pH 9 and allowing for tautomerization. The EpiK pKa prediction tool uses Hammett and Taft relationships to predict protonation states based on functional group structure and sensitivity to perturbations from the rest of the molecule. The model is empirically driven and trained using publicly reported pKa values and proprietary internal measurements performed by Schrödinger. The Hammett and Taft equations (Equation 1) assume that each chemical group has a base pKa

[0146] value which is then modulated by a correction factor (CF, Equation 2) and the perturbing term (ρ) for the target substituent and the influence constant (σ) of other substituents in the molecule. Equation 1

[0147] Equation 2

[0148] In the equations, n

[0149] represents the number of ways to remove an equivalent H atom from the acidic molecule, while n HA represents the number of ways to add H to the conjugate base. RA is a correction term for aliphatic rings. The uncertainty in the pKa value is calculated from the HR uncertainty in and ρ. Novel molecules with low coverage of and ρ have a default uncertainty of 2.0 pKa units. and ρ Calculated pKa values for some representative monomers of this disclosure are provided in Table 1.

[0150]

[0151] Table 1. Monomers and calculated pKa values

[0152]

[0153]

[0154]

[0155]

[0156] Material

[0157] ​N-(2-Hydroxyethyl)piperazine, 1-amino-4-methylpiperazine, N-phenethylpiperazine, 1-(2-pyridyl)piperazine, 1-(2-pyrimidinyl)piperazine, and ethylenediaminetetraacetic acid (EDTA) were purchased from Alfa Aesar (Alfa Aesar, Ward Hill, MA).

[0158] N-(2-Aminoethyl)morpholine and N-(3-aminopropyl)morpholine were purchased from TCI America (TCI America, Portland, OR).

[0159] N-Phenylpiperazine, N-methylpiperazine, 2-hydroxyethyl methacrylate (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 purchased from Sigma-Aldrich Company (Sigma-Aldrich Company, St. Louis, MO).

[0160] Phosphate-buffered saline (PBS, 1X), Dulbecco's modified Eagle's medium (DMEM), trypan blue (0.4% solution), and fetal bovine serum (FBS) were purchased from ThermoFisher Scientific (ThermoFisher Scientific, Waltham, MA).

[0161] 1-Boc-piperazine was purchased from Oakwood Chemical (Oakwood Chemical, Estill, SC).

[0162] 2-Vinyl-4,4-dimethylazlactone (VDM) was purchased from SNPE, Inc. (SNPE, Inc., Princeton, NJ) and redistilled before use.

[0163] 2-Isocyanatoethyl methacrylate (IEM, Karenz MOI) and 2-isocyanatoethyl acrylate (IEA, Karenz AOI) were from Showa Denko KK (Showa Denko KK, Tokyo, Japan).

[0164] 3-Carboxybenzophenone was purchased from Sigma-Aldrich. A solution (0.033 g / mL) of 3-carboxybenzophenone sodium salt (C-BP) was prepared by dissolving 3-carboxybenzophenone in 1 M sodium hydroxide and diluting with deionized water.

[0165] Method 1 . Preparation of Polymer Grafted Membrane

[0166] Based on the measured solids % of the monomer solution, grafting solutions of the monomers (5 g each) were prepared in deionized water at various monomer concentrations. Each monomer solution also contained sodium 3-carboxybenzophenone (62.5 μL of a 0.033 g / mL aqueous solution). For each grafting solution, a nylon membrane substrate (#080ZN, reinforced nylon 6,6 membrane, 0.8 micron nominal pore size, purchased from 3M Company, St. Paul, MN) was placed on a polyester film sheet, and sufficient grafting solution was pipetted onto the top surface of the substrate to completely wet the substrate. The coated solution was immersed into the substrate for approximately 1 minute, and then a second polyester film was placed on top of the substrate. A 2.28 kg cylindrical weight was rolled on top of the resulting three-layer sandwich structure to squeeze out the excess coated solution. Ultraviolet (UV)-induced grafting was carried out by irradiating the sandwich with a UV table (Classic Manufacturing, Inc., Oakdale, MN) equipped with 18 bulbs (Sylvania RG2 40W F40 / 350BL / ECO, 10 above and 8 below the substrate, 1.17 m (46 inches) long, spaced 5.1 cm (2 inches) center-to-center), with an irradiation time of 15 minutes. The polyester sheets were removed, and the resulting grafted membrane was placed in a polyethylene bottle. The bottle was filled with 0.9% saline solution, sealed, and placed on a laboratory bottle roller for 30 minutes to wash away any residual monomers or ungrafted polymers. The saline solution was poured out, and the membrane was washed with deionized water for another 30 minutes. The washing with fresh 0.9% saline solution was repeated for 30 minutes, and then the membrane was washed with deionized water for 30 minutes (twice). After the washing steps, the polymer grafted membrane was air-dried. The grafting density of the polymer grafted membrane was estimated based on the mass gain.

[0167] Method 2 . Bovine Serum Albumin (BSA) Static (Equilibrium) Binding Capacity Method

[0168] Individual discs (16 mm diameter) of the polymer-grafted membrane are punched out from a sheet die head of the polymer-grafted membrane. The individual discs are placed in 5 mL centrifuge tubes containing 4.5 mL of bovine serum albumin (product number A-7906, Sigma Aldrich) prepared at a concentration of approximately 4 mg / mL in a buffer solution, and the pH of the selected buffer solution is 5.0, 6.0, 7.0, 8.0, or 9.0. Each centrifuge tube is capped and tumbled overnight (usually 14 hours) on a rotary mixer. The resulting supernatant solution is analyzed using a UV-VIS spectrometer at 280 nm (background correction is applied at 325 nm). The static binding capacity for each disc is determined by comparing the absorbance value of the supernatant with the absorbance value of the starting BSA solution, and the results are recorded in mg / mL (i.e., mg of BSA bound to the membrane per mL of membrane volume) and recorded as the average of three parallel determinations.

[0169] All buffer solutions are prepared using deionized water. The pH 5.0 buffer is an acetate buffer (10 mM). The pH 6.0 buffer is a MES (2-morpholinoethane-1-sulfonic acid) buffer (25 mM). The pH 7.0 buffer is a MOPS (3-morpholinopropane-1-sulfonic acid) buffer (10 mM). The pH 8.0 buffer is a TRIS (tris(hydroxymethyl)aminomethane) buffer (25 mM). The pH 9.0 buffer is a CHES (N-cyclohexyl-2-aminoethane-1-sulfonic acid) buffer (20 mM). The buffer solutions are prepared by mixing appropriate amounts of the acid and base forms of the buffer salts to achieve the desired pH. The formulation for preparing each individual buffer is determined using the "pH Buffer Calculator" software tool available on the website of the Proteomics Research Centre, University of Liverpool (website: www.liverpool.ac.uk / pfg / Tools / BufferCalc / Buffer.html). To obtain a higher ionic strength, an appropriate amount of sodium chloride is added.

[0170] Method 3 . Preparation of Filter Capsules

[0171] Some of the grafted membranes are tested using plastic filter capsules. Each capsule consists of a sealed circular housing. The capsule housing is prepared from two halves (an upper half and a lower half) that mate and seal together at the perimeter after the grafted membrane is inserted into the inner cavity of the lower housing. The fluid inlet and vent are located at the upper part of the housing, and the fluid outlet is located at the lower part of the housing. The outlet port is centered in the middle of the lower housing surface.

[0172] The experimental capsules were prepared as follows. A single disk of the 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 thickness) and a silicone gasket (15.9 mm outer diameter, 9.5 mm inner diameter, 3 mm thickness). 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 finished filtration capsule. The overall outer diameter of the finished capsule was approximately 4.3 cm, and the overall height including the inlet port, outlet port, and vent port was approximately 4.8 cm. The effective filtration area of the capsule was 1.0 cm 2 , and the bed volume of the media was 0.3 mL.

[0173] Method 4 . Preparation of 96-well centrifugation test device for membrane samples

[0174] Two 7.5-mm disks of the 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 been previously removed. The filter disks were held in place by appropriately sized plastic O-rings to provide a tight seal such that when phosphate-buffered saline (PBS, 1X) was applied to the top of the membrane layer in each well, no liquid flowed 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.

[0175] Method 5 . Preparation of Phi6 virus stock culture

[0176] Phi6 bacteriophage (DSMZ 21518) was purchased from the DSMZ German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. The virus culture was prepared by adding 5 mM magnesium sulfate to 100 mL of tryptic soy broth (Hardy Diagnostics, Santa Maria, CA, USA) and inoculating with 1.5 mL of an overnight culture of the host bacterium Pseudomonas syringae (DSMZ 21482). The culture was incubated at 25 °C with shaking at 210 revolutions per minute (rpm) for 2 hours. Then, the culture was inoculated with 10 9Phi 6 virus with a titer of

[0177] Method 6 . Determine the concentration of Phi6 virus by plaque assay

[0178] Serial dilute (10-fold) the Phi6 virus sample. Mix the molten tryptone soya top agar (2.5 mL tryptone soya broth containing 5 mM MgSO4 and 0.9% agar) with 50 μL of an overnight culture of Pseudomonas syringae host bacteria and 100 μL of the diluted Phi 6 virus. Pour the mixture on top of a standard tryptone soya agar plate and incubate overnight at 25 °C. After incubation, count the plaque-forming units (pfu). The number of pfu is related to the number of virus particles. Calculate the virus particle concentration (particles / mL) by adjusting the pfu count used for dilution.

[0179] Method 7 . Prepare the stock solution of Phi6 virus from the agar plate

[0180] Prepare an overnight culture of Pseudomonas syringae (DSMZ 21482) by picking a single colony into 5 mL to 7 mL of liquid tryptone soya broth and incubating overnight at 25 °C in an orbital shaker (210 rpm to 250 rpm). Add 50 μL aliquots of the overnight culture broth to the molten soft tryptone soya top agar (5 mL tryptone soya broth containing 5 mM MgSO4 and 0.75% agar). Pour the molten agar onto a standard tryptone soya agar plate and allow it to solidify. Spread 100 μL of the Phi6 phage stock solution at 1E+09 pfu / mL (DSMZ PN21518) on top of the solidified top agar plate and incubate overnight at room temperature (about 22 °C to 25 °C). Harvest the resulting virus from the agar by scraping the top agar on the plate into a 50 mL conical tube, adding 20 mL of phage storage buffer (50 mM NaHPO4, 22 mM KH2PO4, 85.5 mM NaCl, 1 mM MgSO4, 1 mM CaCl2), and vortexing for 15 minutes to 30 minutes to release the virus into the buffer. Then centrifuge the mixture at 3000 × g for 15 minutes and filter through a 0.2 μm PES membrane.

[0181] Method 8 . Determine the concentration of Phi6 virus by surface spotting method

[0182] Serial dilute (10-fold) the Phi6 virus sample in a 96-well plate (down to 100,000x dilution). Mix molten soft tryptone soy top agar supplemented with MgSO4 (5 mL of tryptone soy broth containing 5 mM MgSO4 and 0.9% agar) with 50 μL of an overnight culture of the Pseudomonas syringae host bacterium and pour it on top of a standard tryptone soy agar plate. After the agar has solidified, spot 3 μL of each dilution in an array onto the surface of the prepared plate. Incubate the plate overnight at 25 °C. After incubation, quantify the virus by counting the plaque-forming units in the 3 μL spots at the highest dilution where plaques can be counted. Calculate the virus particle concentration (particles / mL) by adjusting the pfu count used for dilution.

[0183] Method 9 . Preparation and Characterization of Lentiviral Cultures

[0184] Generate lentivirus containing the GFP reporter gene in LV-MAX virus production suspension cells (product number A35347, Thermo Fisher Scientific) equipped with the GIBCO LV-MAX lentivirus production system (product number A35684, Thermo Fisher Scientific). Transfect the cells with the pLenti-GFP control vector (product number CALTV-400, Cell Biolabs San Diego) and the LV-MAX lentivirus packaging mix plasmid (product number A43237, Thermo Fisher Scientific) according to the manufacturer's instructions for the LV-MAX lentivirus production system. Maintain the cells in LV-MAX medium, which consists of GIBCO LV-MAX production medium (product number A3583402, Thermo Fisher Scientific) supplemented with the recommended 1X concentration of GIBCO penicillin-streptomycin (product number 15140122, Thermo Fisher Scientific). Estimate the cell density and viability of the LV-MAX suspension cells by manually counting a trypan blue-stained cell sample using a hemocytometer. On day 3 post-transfection, harvest the supernatant containing lentivirus by centrifuging the culture at 1300 × g for 15 minutes and then filtering the supernatant through a 0.45 μm filter PES filter (product number 295-4545, Thermo Fisher Scientific).

[0185] Method 10 . Determination of Lentiviral Titer

[0186] The lentivirus titer was determined using a titration assay for transduction units per milliliter (TU / mL). LentiX-293T cells (product number 632180, Takara Bio USA, Mountain View, CA) were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10 volume % fetal bovine serum (FBS) and GIBCO penicillin-streptomycin and seeded in a black-walled 96-well culture plate at a density of 10,000 cells / well. After cell attachment, the test samples were serially diluted in medium (product number TR1003G, Sigma-Aldrich) supplemented with 8 μg / mL polybrene, and then the original medium of the cells was removed and replaced with fresh medium containing the serial dilutions. The plate was then centrifuged at 900×g for 30 minutes, followed by incubation for 96 hours, at which time the "% GFP positive cells" were determined using a BioTek Cytation 5 imaging reader (Agilent Technologies, Santa Clara, CA). The settings and gain of the GFP channel and focus were adjusted for the plates / cells used. All plates were scanned on the same day with the same settings. The titer was calculated using the wells with "% GFP positive cells" readings between 1% and 20% according to the equation TU / mL = [fraction of GFP positive cells] * [10,000 seeded cells / well] / [mL of original virus sample added to the well].

[0187] Method 11 .Determination of DNA concentration

[0188] DNA was extracted from the 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, Thermo Fisher Scientific) according to the manufacturer's instructions.

[0189] Method 12 .Measurement of conductivity and pH

[0190] Conductivity and pH measurements were performed using a calibrated Orion Star A215 benchtop multi-parameter pH / conductivity meter (Thermo Fisher Scientific). The conductivity measurements of the instrument were calibrated using Oakton 1.413 mS / cm and 12.88 mS / cm standard solutions (Cole-Parmer, Vernon Hills, IL). The pH measurements of the instrument were calibrated using ORION pH 4.01, 7.00, and 10.01 standard solutions (Thermo Fisher Scientific).

[0191] Example 1 .

[0192] Preparation of 2-methylprop-2-enoic acid 2-[[4-(2-hydroxyethyl)piperazine-1-carbonyl]amino]ethyl ester (IEM / N-(2-hydroxyethyl)piperazine)

[0193]

[0194] N-(2-Hydroxyethyl)piperazine (6.5 g, 0.05 mol) 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 placed in an ice-water bath under a slow nitrogen purge. An aliquot (100 μL) of a 4-hydroxy-TEMPO solution (10,000 ppm in deionized water) was added, and the solution was stirred for 10 minutes, then IEM (3.5 mL) was added. The solution was then stirred for 15 minutes, followed by the addition of 3.5 mL of IEM (total amount of IEM = 7.0 mL, 0.05 mol). The mixture was stirred for 15 minutes and then 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 measured by pH paper. The cloudy solution was filtered to obtain 2-methylprop-2-enoic acid 2-[[4-(2-hydroxyethyl)piperazine-1-carbonyl]amino]ethyl ester as a clear, colorless filtrate with a solid percentage of 23.95%. 1 1H-NMR (D2O): 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). The solid percentage was determined using an Ohaus moisture balance (model MB35, available from Ohaus Corporation, Parsippany, NJ). Samples of the monomer aqueous solution were acidified to a pH of approximately 1 with 0.1 N HCl and then titrated with 0.1 N NaOH, and the experimentally estimated pKa of IEM / N-(2-hydroxyethyl)piperazine was 6.8.

[0195] Example 2 .

[0196] Preparation of 2-[(4-methylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate (IEM / N-methylpiperazine)

[0197]

[0198] According to the general procedure described in Example 1, except that N-methylpiperazine (5.00 g, 0.05 mol) was used instead of N-(2-hydroxyethyl)piperazine. The filtrate of the obtained 2-[(4-methylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate had 21.65% solids. 1 H-NMR (D2O): δ 1.73 (s, 3H), 2.71 (s, 3H), ca. 3.0 (broad, 8H), 3.32 (t, 2H), 4.08 (t, 2H), 5.54 (s, 1H), 5.94 (s, 1H).

[0199] Example 3 .

[0200] Preparation of 2-(2-morpholinoethylcarbamoylamino)ethyl 2-methylprop-2-enoate (IEM / N-(2-aminoethyl)morpholine)

[0201]

[0202] According to the general procedure described in Example 1, except that N-(2-aminoethyl)morpholine (6.51 g, 0.05 mol) was used instead of N-(2-hydroxyethyl)piperazine. The filtrate of the obtained 2-(2-morpholinoethylcarbamoylamino)ethyl 2-methylprop-2-enoate had 23.55% solids. 1 H-NMR (D2O): δ 1.75 (s, 3H), 3.07 (t, 2H), 3.19 (broad s, 4H), 3.28 (t, 2H), 3.35 (t, 2H), 3.80 (broad s, 4H), 4.07 (t, 2H), 5.56 (s, 1H), 5.96 (s, 1H).

[0203] Example 4 .

[0204] Preparation of 2-(3-morpholinopropylcarbamoylamino)ethyl 2-methylprop-2-enoate (IEM / N-(3-aminopropyl)morpholine)

[0205]

[0206] According to the general procedure described in Example 1, except that N-(3-aminopropyl)morpholine (7.21 g, 0.05 mol) was used instead of N-(2-hydroxyethyl)piperazine. The filtrate of 2-methylprop-2-enoic acid 2-(3-morpholinopropylcarbamoylamino)ethyl ester obtained had 23.75% solids. 1 H-NMR (D2O): δ 1.74 (s and m, 5H), 3.00 (m, 2H), 3.04 (t, 2H), 3.15 (broad s, 4H), 3.27 (t, 2H), 3.79 (broad s, 4H), 4.05 (t, 2H), 5.55 (s, 1H), 5.96 (s, 1H).

[0207] Example 5 .

[0208] Preparation of 2-methylprop-2-enoic acid 2-[(4-methylpiperazin-1-yl)carbamoylamino]ethyl ester (IEM / 1-amino-4-methylpiperazine)

[0209]

[0210] According to the general procedure described in Example 1, except that the total amount of IEM used was 3.5 mL (0.025 mol), and 1-amino-4-methylpiperazine (2.88 g, 0.025 mol) was used instead of N-(2-hydroxyethyl)piperazine. The filtrate of 2-methylprop-2-enoic acid 2-[(4-methylpiperazin-1-yl)carbamoylamino]ethyl ester obtained had 23.1% solids. 1 H-NMR (D2O): δ 1.75 (s, 3H), 2.77 (s overlapping with another absorption peak, 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).

[0211] Example 6 .

[0212] Preparation of 2-methylprop-2-enoic acid 2-[(4-phenylpiperazine-1-carbonyl)amino]ethyl ester (IEM / N-phenylpiperazine)

[0213]

[0214] N-Phenylpiperazine (4.05 g, 0.025 mol) was weighed into a 250 mL round-bottom flask equipped with a magnetic stirrer. Ether (75 mL) was added and the stirred mixture was placed in an ice-water bath under a slow nitrogen purge. 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 formed was filtered and dried to give 5.3 g of 2-[(4-phenylpiperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate. 1 H-NMR (CDCl3): δ 1.96 (s, 3H), 3.18 (m, 4H), 3.53 (m, 4H), 3.59 ((q, 2H), 4.31 (t, 2H), 5.00 (broad t, 1H), 5.61 (s, 1H), 6.14 (s, 1H), 6.92 (m, 3H), 7.28 (m, 2H).

[0215] Example 7 .

[0216] Preparation of 2-[(4-(2-phenylethyl)piperazine-1-carbonyl)amino]ethyl 2-methylprop-2-enoate (IEM / N-phenethylpiperazine)

[0217]

[0218] N-Phenethylpiperazine (9.5 g, 0.05 mol) was weighed into a 250 mL round-bottom flask equipped with a magnetic stirrer. Dichloromethane (100 mL) was added and the stirred mixture was placed in an ice-water bath under a slow nitrogen purge. 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. 50 mL portions of 1N HCl were added to the funnel and the funnel was shaken. The funnel was placed in a stand to allow the mixture to separate into 3 layers. The lower layer was discarded and the remaining layers were separated by collecting into two containers. NMR analysis showed that both layers contained the desired product. The layers were combined and 50 μL of 4-hydroxy-TEMPO solution (10,000 ppm in deionized water) was added. Most of the dichloromethane was removed by rotary evaporation to give 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 to 5 (measured with pH paper) and a solids % of 21.6%. 1H-NMR (D2O): δ 1.73 (s, 3H), 2.86 (broad s, 2H), 2.92 (m, 2H), 3.07 (broad s, 2H), 3.24 (m, 2H), 3.32 (t, 2H), 3.42 (broad s, 4H), 3.92 (broad s, 2H), 4.07 (t, 2H), 5.54 (s, 1H), 5.95 (s, 1H), 7.17 (m, 3H), 7.23 (m, 2H).

[0219] Example 8 .

[0220] Preparation of 2-(Piperazine-1-carbonylamino)ethyl 2-methylprop-2-enoate (IEM / Piperazine)

[0221]

[0222] Weigh 1-Boc-piperazine (18.6 g, 0.1 mol) into a 250 mL round-bottom flask equipped with a magnetic stirrer. Add dichloromethane (75 mL), and place the stirred mixture in an ice-water bath under a slow nitrogen purge. Stir the mixture for 10 minutes. Add IEM (3.5 mL) via pipette, stir the mixture for 1 minute, then add a second portion of IEM (3.5 mL). Add two additional 3.5 mL portions of IEM at 5-minute intervals (a total of 14 mL of IEM added in 4 equal portions). Stir the resulting mixture for 30 minutes, then remove the solvent by rotary evaporation to obtain the BOC-protected IEM / piperazine intermediate as a colorless solid (33.5 g). 1 H-NMR (CDCl3): δ 1.39 (s, 9H), 1.87 (s, 3H), 3.28 (m, 4H), 3.35 (m, 4H), 3.47 (q, 2H), 4.21 (t, 2H), 5.12 (broad t, 1H), 5.53 (s, 1H), 6.06 (s, 1H).

[0223] Place the BOC-protected IEM / piperazine intermediate (5.0 g) in a round-bottom flask, dissolve it in approximately 5 mL of trifluoroacetic acid, and then heat at 50 °C for 1 hour. Place the flask on a rotary evaporator at 50 °C for 90 minutes to obtain 2-(Piperazine-1-carbonylamino)ethyl 2-methylprop-2-enoate containing 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). Dissolve the residue in 20 mL of deionized water to provide an approximately 0.5 M monomer solution for membrane grafting.

[0224] Example 9 .

[0225] Preparation of 2-[(4-Methyl-1,4-diazepane-1-carbonyl)amino]ethyl 2-methylprop-2-enoate (IEM / N-methylhomopiperazine)

[0226]

[0227] According to the general procedure described in Example 1, except that N-methylhomopiperazine (5.71 g, 0.05 mol) was used instead of N-(2-hydroxyethyl)piperazine. The filtrate of the obtained 2-[(4-Methyl-1,4-diazepane-1-carbonyl)amino]ethyl 2-methylprop-2-enoate had 22.45% solids. 1 1H-NMR (D2O): δ 1.69 (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).

[0228] Example 10 .

[0229] Preparation of 2-[(4-Methylpiperazine-1-carbonyl)amino]ethyl prop-2-enoate (IEA / N-methylpiperazine)

[0230]

[0231] N-methylpiperazine (2.0 g, 0.02 mol) was weighed into a 50 mL round bottom flask equipped with a magnetic stirrer. Dichloromethane (20 mL) was added and the stirred mixture was placed in an ice-water bath under a slow nitrogen purge. The mixture was stirred for 10 minutes. IEA (2.56 mL) was added via pipette and the mixture was stirred for 15 minutes to obtain 2-[(4-Methylpiperazine-1-carbonyl)amino]ethyl prop-2-enoate as a dichloromethane solution. 1 1H-NMR (CDCl3): δ 2.22 (s, 3H), 2.31 (t, 4H), 3.30 (t, 4H), 3.46 (q, 2H), 4.20 (t, 2H), 4.93 (broad t, 1H), 5.79 (dd, 1H), 6.07 (dd, 1H), 6.36 (dd, 1H).

[0232] Example 11 .

[0233] Preparation of 2-Methyl-N-(2-morpholinopropyl)-2-(prop-2-enoylamino)acrylamide (VDM / N-(3-aminopropyl)morpholine)

[0234]

[0235] According to the general procedure described in Example 4, except that VDM (6.95 g) was used instead of IEM. The resulting filtrate of VDM / N-(3-aminopropyl)morpholine had 23.2% solids. 1 H-NMR (D2O): δ 1.29 (s, 6H), 1.73 (p, 2H), 2.89 (m, 2H), 3.05 (broad s, 4H), 3.11 (t, 2H), 3.76 (broad s, 4H), 5.58 (d, 1H), 5.99 (d, 1H), 6.11 (dd, 1H).

[0236] Example 12 .

[0237] Preparation of 2-(4-(pyridin-2-yl)piperazine-1-carboxamido)ethyl methacrylate (IEM / N-(2-pyridyl)piperazine)

[0238]

[0239] 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 dropwise to the stirred solution over 3 minutes. A white solid formed. After stirring for 20 minutes, the white solid was separated by filtration and washed with several portions of water. The solid was transferred to a crystallization dish and allowed to air dry for several days to give 6.62 g of 2-(4-(pyridin-2-yl)piperazine-1-carboxamido)ethyl methacrylate as a white solid. 1 H NMR (500 MHz, CD3OD) δ 8.07 - 8.14 (m, 1H) 7.59 (ddd, J = 8.7, 7.1, 2.0 Hz, 1H) 6.84 (d, J = 8.7 Hz, 1H) 6.71 (dd, J = 6.7, 5.4 Hz, 1H) 6.14 (s, 1H) 5.61 - 5.68 (m, 1H) 4.24 (t, J = 5.7 Hz, 2H) 3.53 (s, 8H) 3.49 (t, J = 5.7 Hz, 2H) 1.94 (s, 3H).

[0240] Example 13 .

[0241] Preparation of 2-(4-(pyrimidin-2-yl)piperazine-1-carboxamido)ethyl methacrylate (IEM / N-(2-pyrimidinyl)piperazine)

[0242]

[0243] A suspension of 1-(2-pyrimidinyl)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 separated 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 ethyl 2-(4-(pyrimidin-2-yl)piperazine-1-carboxamido)acrylate as a white solid. 1 H NMR (500 MHz, CD3OD) δ 8.35 (d, J = 4.8 Hz, 2H) 6.63 (t, J = 4.8 Hz, 1H) 6.14 (s, 1H) 5.65 (m, 1H) 4.24 (t, J = 5.7 Hz, 2H) 3.81 (m, 4H) 3.49 (m, 6H) 1.95 (m, 3H).

[0244] Example 14 .

[0245] Preparation of 2-methyl-N-(4-methylpiperazin-1-yl)-2-(prop-2-enoylamino)propanamide (VDM / 1-amino-4-methylpiperazine)

[0246]

[0247] Following the general procedure described in Example 5, except that VDM (1.5 mL, 0.025 mol) was used in place of IEM. The filtrate of the resulting 2-methyl-N-(4-methylpiperazin-1-yl)-2-(prop-2-enoylamino)propanamide had 21.85% solids. 1 H-NMR (D2O): δ 1.22 (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).

[0248] Example 15.

[0249] According to the procedure described in Method 1, nylon membranes were grafted with the monomer solutions of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) or Example 2 (IEM / N-methylpiperazine). 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. According to the procedure of Method 2, the BSA binding (mg / mL) of the grafted membranes was evaluated at pH 6.0, pH 7.0 and pH 8.0. The results are recorded in Table 2.

[0250] The results in Table 2 show that for the polymer grafted membrane prepared from the IEM / N-(2-hydroxyethyl)piperazine monomer (calculated pKa of 7.02), when the pH of the buffer increased from pH 6.0 to pH 7.0, the binding ability of the polymer grafted membrane to BSA decreased by about 40%; when the pH increased from pH 6.0 to pH 8.0, the binding ability of the polymer grafted membrane decreased by >80%. In contrast, for the polymer grafted membrane prepared from the IEM / N-methylpiperazine monomer (calculated pKa of 7.46), when the pH of the buffer increased from pH 6.0 to pH 7.0, the binding ability of the polymer grafted membrane to BSA only decreased by about 20%; when the pH increased from pH 6.0 to pH 8.0, the binding ability of the polymer grafted membrane decreased by >80%.

[0251] Table 2.

[0252]

[0253] NT = Not tested

[0254] Example 16 .

[0255] The polyethersulfone membrane (MacroPES, nominal pore size of 5 microns, purchased from 3M) was grafted using the monomer in Example 1 (IEM / N-(2-hydroxyethyl)piperazine, pKa 7.02) under grafting conditions of a monomer concentration of 0.75 M, an irradiation time of 20 minutes, and no photoinitiator (C-BP) added to the grafting solution. The resulting grafted membrane had a grafting density of 1.04 mmol / g. According to the steps of Method 2, the static BSA binding ability of the grafted substrate was tested in MES buffer at pH 6.0. The static BSA binding ability was 114 mg / mL. The supernatant BSA solution was decanted from the centrifuge tube, and the membrane disk was washed 3 times with 4.5 mL of fresh MES buffer (pH 6.0) each time. Each washing step was carried out using a rotary mixer for 30 minutes. The final washing buffer was decanted from the centrifuge tube, and the bound BSA was eluted from the disk by adding 4.5 mL of TRIS buffer at pH 8.0 and rolling the centrifuge tube over for 30 minutes. The BSA concentration of the eluate was measured, and then the eluted binding ability was calculated using the measured BSA concentration (BSA eluted binding ability = 119 mg / mL). The results show that the BSA protein can be bound to the membrane using a buffer with a near-neutral pH, and then it can be quantitatively eluted from the membrane by gently adjusting the pH of the eluate.

[0256] Example 17.

[0257] According to the steps described in Method 1, nylon membranes were grafted with monomer solutions 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). 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. According to the steps of Method 2, the BSA binding capacity (mg / mL) of the grafted membranes was evaluated at pH 5.0, pH 6.0, pH 7.0, pH 8.0, and pH 9.0. The results were recorded in Table 3.

[0258] Table 3.

[0259]

[0260] NT = Not tested

[0261] Example 18 .

[0262] According to the steps described in Method 1, nylon membranes were grafted with a 0.5 M monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) or Example 2 (IEM / N-methylpiperazine). According to the steps of Method 2, the BSA binding capacity (mg / mL) of the grafted membranes was evaluated at pH 7.0 using MOPS buffer solutions with different ionic strengths. The ionic strength of each MOPS buffer solution was adjusted to 50 mM and 150 mM by adding sodium chloride. The results were recorded in Table 4.

[0263] Table 4.

[0264]

[0265] Example 19.

[0266] Two buffer solutions were prepared and named Buffer A and Buffer B. Buffer A was 25 mM MES buffer (pH 6.0), and Buffer B was 25 mM MES buffer containing 1 M NaCl (pH 6.0). A test sample of BSA (0.56 grams of BSA in 50 mL of Buffer A) was prepared and then filtered using a 0.22 μm PES membrane filter (STERIFLIP Sterile Disposable Vacuum Filter Unit, MilliporeSigma, Burlington, MA).

[0267] According to the steps described in Method 1, graft the nylon membrane with the monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine, calculated pKa is 7.02). The grafting density is 0.36 mmol / g. Stack six 25-mm grafted membrane discs in the chromatography module, where an O-ring is used to hold the disc stack in place. The exposed front surface area of the disc is 284 mm 2 . The module comprises a straight cylindrical polycarbonate body with a cap attached to one end. The cap contains an inlet port and an exhaust port. The opposite end contains an outlet port with a stopcock. Attach the completed module to an AKTA avant chromatography system (Cytiva, Marlborough, MA) equipped with UV / VIS (280 nm absorbance setting) and conductivity detectors. Flush the module with 5 mL of buffer A at a flow rate of 1 mL / min. Next, pump the BSA test sample through the module at a flow rate of 0.5 mL / min, and then pump 20 mL of buffer A at a flow rate of 1 mL / min to wash the membrane stack. In the final elution step, elute BSA from the membrane stack by pumping a gradient of 20 mL of 0% to 100% buffer B: buffer A (i.e., the gradient is based on volume from 100% buffer A to 100% buffer B) at a flow rate of 1 mL / min. BSA bound to the grafted membrane stack elutes at a buffer conductivity of ≥1 mS / cm.

[0268] Example 20.

[0269] Follow the steps of Example 19, except that the test sample is β-lactoglobulin from cow milk (product number L3908, Sigma-Aldrich) in 50 mL of buffer A. β-Lactoglobulin bound to the grafted membrane stack elutes at a buffer conductivity of ≥1 mS / cm.

[0270] Example 21.

[0271] Three buffer solutions were prepared using 50 mM TRIS buffer with the pH adjusted to 6.0, 7.0, or 8.0 and the conductivity adjusted to 5 mS / cm. The pH value was adjusted using 1N HCl or 1N NaOH. The conductivity value was adjusted by adding 5M sodium chloride. Phi6 virus challenge solutions were prepared by incorporating the original Phi6 virus culture broth described in Method 5 into each buffer solution, and the Phi6 virus challenge solutions had the virus concentrations listed in Table 5. According to the steps of Method 3, filtration capsules were prepared using nylon membranes that had been grafted with the monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) according to the steps described in Method 1. The grafted membrane had a graft density of 0.25 mmol / g. Each Phi6 virus challenge solution (7 mL) was pumped through a single capsule at 0.5 mL / min. The resulting filtrate was collected. The Phi6 content of the input challenge solution and the filtrate samples was measured using the plaque assay described in Method 6. The log reduction value (LRV) of filtration was calculated by comparing the Phi6 virus concentration in the filtrate sample with the Phi6 virus concentration in the corresponding input challenge solution before filtration (Equation 3). The results were recorded in Table 5. The results showed that when the pH of the challenge solution was from 6.0 to 7.0, a large amount of Phi 6 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.

[0272] Equation 3:

[0273]

[0274] Table 5.

[0275]

[0276] Example 22.

[0277] Phi6 virus challenge solutions were prepared according to the steps described in Example 21 using 50 mM TRIS buffer solutions with the pH adjusted to pH 7.0, 7.25, 7.5, 7.75, or 8.0 and the conductivity adjusted to 5 mS / cm. The results were recorded in Table 6. The results showed that when the pH of the challenge solution was from 7.0 to 7.5, a portion of the Phi 6 enveloped virus bound to the grafted membrane. However, when the pH value of the challenge solution was from 7.75 to 8.0, the Phi6 virus flowed through the membrane and almost no Phi6 virus bound to the grafted membrane.

[0278] Table 6.

[0279]

[0280] Example 23.

[0281] Prepare the Phi6 virus attack solution using a 50 mM TRIS buffer solution adjusted to pH 7.0 and conductivity adjusted to 10 mS / cm, 12.5 mS / cm, 15 mS / cm, 17.5 mS / cm, or 20 mS / cm according to the steps described in Example 21. Prepare the filtration capsule using a nylon membrane according to the steps of Method 3, which has been grafted with the monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) according to the steps described in Method 1. The grafted membrane has a grafting density of 0.36 mmol / g. The results are recorded in Table 7. The results show that when the conductivity of the attack solution is 10 mS / cm, most of the Phi6 enveloped viruses bind to the grafted membrane. However, when the conductivity of the attack solution increases from 12.5 mS / cm to 20 mS / cm, the binding of Phi6 virus to the grafted membrane decreases significantly.

[0282] Table 7.

[0283]

[0284] Example 24.

[0285] Prepare the calf thymus DNA attack solution by dissolving 20 micrograms / mL of calf thymus DNA (Sigma-Aldrich) in 50 mM TRIS buffer (pH 7.0, 10 mS / cm) or 50 mM TRIS buffer (pH 8.0, 10 mS / cm). Prepare the filtration capsule using a nylon membrane according to the steps of Method 3, which has been grafted with the monomer solution of Example 1 (IEM / N-(2-hydroxyethyl)piperazine) according to the steps described in Method 1. The grafted membrane has a grafting density of 0.36 mmol / g. Pump each DNA attack solution (8 mL) through a single capsule at 0.5 mL / minute. Collect the resulting filtrate in four separate 2 mL fractions (i.e., fractions 1 to 4). The DNA concentration (micrograms / mL) of the attack solution before and after filtration is determined by UV spectroscopy by measuring the 260 absorbance and subtracting the background absorbance of the buffer. The results are recorded in Table 8.

[0286] The results show that when using the attack solution with pH 7.0, the grafted membrane binds a large amount of DNA (i.e., only a small amount of DNA is recovered in the filtrate fraction). However, when using the attack solution with pH 8.0, the DNA in the attack solution does not bind to the membrane (i.e., most of the DNA is recovered in the filtrate fraction). It is believed that the DNA level in the 4th filtrate fraction from the attack solution with pH 7.0 is elevated due to exceeding the capacity of the grafted membrane.

[0287] Table 8.

[0288]

[0289] Example 25. Membrane grafted with IEM / N-methylpiperazine:HEMA copolymer (75:25 molar ratio)

[0290] According to the steps described in Method 1, 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). The total monomer concentration of the solution was 0.35 M, and the molar ratio of the monomers in the solution was 75:25 IEM / N-methylpiperazine:HEMA. The grafting density was 0.30 mmol / g.

[0291] Example 26. Membrane grafted with IEM / N-methylpiperazine:HEMA copolymer (50:50 molar ratio)

[0292] A grafted membrane was prepared according to the steps described in Example 25, except that the molar ratio of the monomers in the solution was 50:50 IEM / N-methylpiperazine:HEMA and the grafting density was 0.20 mmol / g.

[0293] Example 27. Membrane grafted with IEM / N-methylpiperazine:HEMA copolymer (25:75 molar ratio)

[0294] A grafted membrane was prepared according to the steps described in Example 25, except that the molar ratio of the monomers in the solution was 25:75 IEM / N-methylpiperazine:HEMA and the grafting density was 0.12 mmol / g.

[0295] Example 28. Membrane grafted with IEM / N-methylpiperazine:HEMA copolymer (75:25 molar ratio)

[0296] A grafted membrane was prepared according to the steps described in Example 25, except that the total monomer concentration of the solution was 0.50 M, the molar ratio of the monomers in the solution was 75:25 IEM / N-methylpiperazine:HEMA, and the grafting density was 0.53 mmol / g.

[0297] Example 29.

[0298] As described in Method 4, 96-well EMPORE filter plates were modified with the grafted membranes of Example 2 (IEM / N-methylpiperazine, pKa 7.46) and Examples 25 to 27 (including HEMA as a copolymer). Disks of two identical types of grafted membranes were added to the wells, and nine wells were prepared for each type of grafted membrane. The membrane disks of Example 2 had a grafting density of 0.375 mmol / g. An aliquot of 500 μL of 20 mM TRIS-acetate buffer (added with 100 mM NaCl, pH 7.4) was added to each well. The pH of the buffer was adjusted using acetic acid. The EMPORE plate was placed on top of a 96-well 2 mL deep-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. An attack solution of Phi6 virus was prepared by diluting the Phi6 virus stock solution sample (prepared according to Method 7) 100-fold with TRIS-acetate buffer to achieve a titer of approximately 1E+07 pfu / mL. An aliquot of 300 μL of the attack solution was added to each well of the EMPORE plate, and then the assembly was centrifuged at 1000×g for 10 minutes. Next, the EMPORE plate was removed from the collection plate and transferred to the top of a new collection plate.

[0299] Three separate elution buffers were prepared by adjusting the pH of 20 mM TRIS-acetate buffer to pH 8.0, pH 8.5, or pH 9.0 using HCl. Phi6 virus was eluted from the grafted membranes using a 2-step procedure. In the first step, an aliquot of 300 μL of a single elution buffer was added to three wells of 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 aliquot of 300 μL 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. The Phi6 virus content of the flow-through samples of the collected elution buffers was analyzed using the procedure of Method 8. The results are presented in Table 9, showing the average percentage and standard deviation (SD) of Phi6 virus recovered from the attack solution.

[0300] Table 9.

[0301]

[0302] Example 30.

[0303] Prepare a clarified lentiviral cell culture challenge solution (approximately 1.25E+06 TU / mL lentivirus) according to Method 9 and incorporate it at a 1:1000 dilution into approximately 1E+09 pfu / mL Phi6 virus (prepared according to Method 7). The pH of the clarified culture is 7.05 and the conductivity is 10 mS / cm.

[0304] Modify a 96-well EMPORE filter plate with the grafted membrane of Example 28 (prepared using a grafting solution with a 75:25 IEM / N-methylpiperazine:HEMA molar ratio). Place the EMPORE plate on top of a 96-well 2 mL deep well collection plate and add 500 microliters of 1X phosphate buffered saline (PBS) to each well. Then centrifuge the plate assembly at 1000×g for 5 minutes and discard the collected PBS. Next, apply 500 microliters of the clarified challenge solution (containing both Phi6 virus and lentivirus) to each well and then centrifuge at 1000×g for 5 minutes. Discard the collected liquid.

[0305] Prepare a series of 20 elution buffers from 10 mM PBS with pH values of 7.25, 7.5, 7.75, 8.0, or 8.25 and conductivities of 10 mS / cm, 20 mS / cm, 30 mS / cm, or 40 mS / cm. For each elution buffer, adjust the pH using 1N HCl and the conductivity using NaCl. Add 500 aliquots of a single elution buffer to each of 3 wells (n = 3), then centrifuge the plate at 1000×g for 5 minutes. Analyze the lentivirus (TU / mL), Phi6 virus (pfu / mL), and DNA (nanograms / mL (ng / mL)) content of the collected elution buffer samples according to the procedures of Methods 8 to 11. The results are presented as mean concentration values with standard deviation (SD) in Tables 10 to 12.

[0306] The results presented in Tables 10 to 12 show that the elution conditions of the method can be optimized to obtain purified virus with a lower DNA content. Eluting the material bound to the grafted membrane with a buffer solution having a pH of 7.25 to 7.75 and a conductivity of 20 mS results in a filtrate with a large amount of lentivirus and Phi6 virus and only a low level of DNA.

[0307] Table 10.

[0308]

[0309]

[0310] Table 11.

[0311]

[0312] Table 12.

[0313]

[0314] Example 31.

[0315] Clone pUC19 DNA using the MAX EFFICIENCY DH5α Competent Cell Kit (Catalog No. 18258012, Thermo Fisher Scientific) according to the manufacturer's instructions. The kit contains MAX EFFICIENCY DH5α competent cells, pUC19 DNA, and S.O.C. medium. Culture the Escherichia coli cell culture at 37 °C and stir at 200 rpm for 18 hours. Purify pUC19 DNA using the Qiagen QIAprep Spin Miniprep Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions. Graft the nylon membrane 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 is 0.36 mmol / g. Prepare the EMPORE 96-well filter plates according to Method 4, except that only a single disk is added to each well. Prepare three separate TE buffer solutions (TE buffer = 50 mM TRIS buffer supplemented with 10 mM ethylenediaminetetraacetic acid (EDTA)) at pH 7.0, 8.0, or 9.0. Prepare two separate solutions of 50 mM MES buffer at pH 5.0 or 6. It is 0. Adjust the pH of the buffer solutions using 1 N HCl or 1 N NaOH. Add 150 μL aliquots of the single buffer solution to each of the 3 wells (n = 3 for each buffer solution). Place the EMPORE plate on top of a 96-well 2 mL deep well collection plate and centrifuge the plate assembly at 1500 rpm for 5 minutes. Discard the flow-through liquid collected in the collection plate. Then place the EMPORE plate on top of a new collection plate. Add 150 μL aliquots of pDNA (about 100 ng / μL) dissolved in the buffer to each well of the EMPORE plate. The pH of the pDNA solution added to the wells is chosen to match the pH of the 150 μL aliquot buffer solution initially added to the wells. Then centrifuge the plate assembly at 2000 rpm for 2 minutes. Analyze the collected flow-through samples at an absorbance setting of 260 nm using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher Scientific) to determine the percentage of bound pDNA. Analysis of the flow-through samples showed that when using buffer solutions with pH values from 5.0 to 8.0, more than about 80% of the pDNA bound to the grafted membrane, but when using a buffer at pH 9.0, only about 3% of the pDNA bound to the grafted membrane.

[0316] Subsequently, pDNA was eluted from the grafted membranes by sequentially adding 100 μL aliquots of buffer solution into each well. The pH of each subsequent aliquot in the sequence was increased by 1.0 pH unit. After adding each aliquot, the plate was centrifuged and the pDNA in the resulting flow-through sample was analyzed as described above. This process was repeated until a buffer solution with pH 9.0 was used as the eluent. Regardless of the initial buffer pH, the maximum percentage of pDNA was recovered from the grafted membranes using a pH 9.0 elution buffer. The average recovery percentage and standard deviation (SD) of pDNA (n = 3) in the collected samples are reported in Table 13.

[0317] Table 13.

[0318]

[0319] NT = Not tested

[0320] ** = Initial flow-through sample

Claims

1. A monomer of formula (I) wherein 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 -, where R 3 is an alkylene group having at least two linked carbon atoms; R 4 and R 5 each is an alkylene group having at least 2 carbon atoms, wherein the total number of ring atoms in the ring group composed of nitrogen, R 4 , Q and R 5 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 equal to -N(R 6 )-; and R 6 is hydrogen, alkyl or (hetero)aryl, where the alkyl is optionally further substituted by hydroxy, alkoxy or (hetero)aryl, and where the (hetero)aryl is optionally further substituted by one or more hydroxy, halogen, nitro, cyano, trifluoromethyl, alkyl or alkoxy.

2. The monomer according to claim 1, wherein the calculated pKa value of the monomer is in the range of 3.5 or 4 to 9 or 9.

5.

3. The monomer according to claim 1, wherein the group R 2 plus the total number of linking atoms in the group Z is equal to at least 4.

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

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

6. The polymeric material according to claim 5, wherein the polymeric material comprises 10 mol% to 90 mol% of the first monomer unit and 90 mol% to 10 mol% of the second monomer unit.

7. An anion exchange separation article, the anion exchange separation article comprising: a solid porous polymeric substrate; and a plurality of graft polymers attached to the surface of the porous substrate, wherein the graft polymers are the polymerization product of a monomer composition comprising a first monomer of formula (I) wherein R1 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 -, wherein R 3 is an alkylene group having at least two linked carbon atoms; R 4 and R 5 each is an alkylene group having at least 2 carbon atoms, wherein the total number of ring atoms in the ring group composed of nitrogen, R 4 , Q and R 5 is 6 or 7; Q has a single linking atom and is -O-, -N(R 6 )-, -S-, -S(=O)- or -S(=O)2-, provided that when Z is -NH-(C=O)-, Q is equal to -N(R 6 )-; and R 6 is hydrogen, alkyl or (hetero)aryl, wherein said alkyl is optionally further substituted by hydroxy, alkoxy or (hetero)aryl, and wherein said (hetero)aryl is optionally further substituted by one or more hydroxy, halogen, nitro, cyano, trifluoromethyl, alkyl or alkoxy.

8. The anion exchange separation article according to claim 7, wherein for the monomer of formula (I), the group R 2 plus the total number of linking atoms in the group Z is equal to at least 4.

9. The anion exchange separation article according to any one of claims 7 or 8, wherein the calculated pKa value of the monomer of formula (I) is in the range of 3.5 or 4 to 9 or 9.

5.

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

11. The anion exchange separation article according to claim 10, wherein the monomer composition comprises 10 mol% to 90 mol% of the first monomer unit and 90 mol% to 10 mol% of the second monomer unit.

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

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

14. A method of separating a mixture of materials having different ionic contents, the method comprising: a) preparing or providing an anion exchange separation article, the anion exchange separation article comprising a solid porous polymeric substrate; and a plurality of graft polymers attached to the surface of the porous substrate, wherein the graft polymers are the polymerization product of a monomer composition, the first monomer having formula (I) wherein R1 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 -, where R 3 is an alkylene group having at least two linked carbon atoms; R 4 and R 5 each is an alkylene group having at least 2 carbon atoms, wherein the total number of ring atoms in the ring group composed of nitrogen, R 4 , Q and R 5 is 6 or 7; Q has a single linking atom and is -O-, -N(R 6 )-, -S-, -S(=O)- or -S(=O)2-, provided that when Z is -NH-(C=O)-, Q is equal to -N(R 6 )-; R 6 is hydrogen, alkyl or (hetero)aryl, wherein the alkyl is optionally further substituted by hydroxy, alkoxy or (hetero)aryl, and wherein the (hetero)aryl is optionally further substituted by one or more hydroxy, halogen, nitro, cyano, trifluoromethyl, alkyl or alkoxy; and (b) passing the material mixture through the anion exchange separation article at a first pH and a first ionic strength value, the first pH being low enough to protonate the monomer repeat units of the graft polymers derived from the monomer of formula (I), the first ionic strength value binding at least one component of the material mixture as a bound component to the anion exchange separation article.

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

16. The method according to claim 14 or 15, wherein the sum of R 2 plus the total number of linking atoms in Z is equal to at least 4.

17. The method according to 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. 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 mol / L and / or less than or equal to 50 mS.

19. The method according to any one of claims 14 to 18, wherein the material mixture 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, wherein the method is a flow-through method or a bind-and-elute method.

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