Anion exchange separation articles, methods of manufacture and methods of use

Abstract

An anion exchange separation article is provided that can be used to separate a composite sample containing a mixture of materials having different ionic charges. A separation article includes a plurality of polymer chains grafted to a porous polymer substrate, the porous polymer substrate being a solid. The plurality of polymer chains extend away from the surface of the porous polymer substrate, and the side chains of the monomer repeating units of the polymer chains are terminated with guanidinium groups or salts thereof. The separation articles may be used, for example, for separating biological material in a sample based on differences in charge of the biological material. Advantageously, the binding capacity of the anionic separation article is salt tolerant.

Description

Background Art

[0001] Detection, quantification, separation and purification of target biological materials such as viruses and biomacromolecules (e.g., components or products of living cells, such as proteins, carbohydrates, lipids and nucleic acids) have always been the goal of researchers. Detection and quantification are important in diagnosis, for example, as indicators of various physiological conditions such as diseases. Separation and purification of biomacromolecules are important for therapeutic use and biomedical research.

[0002] Polymeric materials have been widely used for separation and purification of various target biological materials. Such separation and purification methods can be based on any of a variety of binding factors or mechanisms including the presence of ionic groups, the size of the target biological material, hydrophobic interactions, affinity interactions, the formation of covalent bonds, etc.

[0003] Membrane-based technologies, especially in disposable form, are becoming increasingly important in biopharmaceutical and vaccine manufacturing processes. Membranes have been used for passive size-based separations (e.g., in virus removal applications) and more recently for active filtration (e.g., for removal of small amounts of contaminants in later stages of a purification process).

[0004] Functionalized membranes (e.g., membranes with functionalized polymers) generally suffer from relatively low binding capacity for biological materials, and this generally limits their use in large-scale purification. In addition, many of these functionalized membranes have reduced binding capacity for biological materials as the ionic strength of the sample increases. Therefore, porous chromatographic resins with ion exchange or other interacting ligand functional groups are generally used rather than functionalized membranes in "catch and elute" type purification processes (such as for protein purification). Summary of the invention

[0005] There is a need for new anion exchange separation articles having high binding capacity for various biological materials such as proteins and good salt tolerance. That is, the binding capacity does not decrease significantly with increasing ionic strength of the eluent and / or sample used during the separation process.

[0006] In a first aspect, an anion exchange separation article is provided, the anion exchange separation article comprising: (1) a porous polymer substrate, the porous polymer substrate being solid; and (2) a plurality of polymer chains grafted to the porous polymer substrate and extending away from the surface of the porous polymer substrate. The polymer chains comprise monomer units derived from a monomer of formula (I):

[0007] CH 2 =CR 1 -(C=O)-X 1 -R 2 -Z-NH-CH2 -Ph-CH 2 -NH-C(=NH)-NH 2

[0008] (I)

[0009] or a salt thereof. In formula (I), the group R 1 is hydrogen or methyl; X 1 is -O- or -NH-; R 2 is alkylene or heteroalkylene; Z is -NH-(C=O)- or -(C=O)-; and Ph is phenylene.

[0010] In a second aspect, a method for making an anion exchange separation article is provided. The method comprises providing a porous polymer substrate, the porous polymer substrate being solid, and grafting a plurality of polymer chains to the porous polymer substrate. The polymer chains comprise monomer units derived from monomers of formula (I) as described above in the first aspect.

[0011] In a third aspect, a method of separating a mixture of materials is provided. The method comprises providing an anion exchange separation article as described above in the first aspect and passing the mixture of materials through an anion exchange separation device, wherein the anion exchange separation device separates the mixture of materials based on their ionic charge.

[0012] In a fourth aspect, a monomer of formula (I) as described in the first aspect is provided. DETAILED DESCRIPTION

[0013] Provided is an anion exchange separation article that can be used to separate a composite sample containing a mixture of materials with different ionic charges. The separation article comprises a plurality of polymer chains grafted to a porous polymer substrate that is solid. The plurality of polymer chains extend away from the surface of the porous polymer substrate and contain a plurality of monomer units having a guanidinium group or a salt thereof. The separation article can be used, for example, to separate biological materials based on differences in anionic charge of biological materials in a sample or to separate anionic (i.e., negatively charged) materials from cationic (i.e., positively charged) materials. Advantageously, the binding capacity of the anion exchange separation article is salt-tolerant.

[0014] Salt tolerance means that the binding capacity of an anion exchange separation product generally does not substantially decrease when the ionic strength increases. For example, if the ionic strength increases from a low ionic strength of about 3 mM to 6 mM to a high ionic strength such as 50 millimolar (mM) or more, most conventional anion exchange media lose 50% or more of their binding capacity.

[0015] The salt tolerance of anion exchange media can be measured in comparison to the salt tolerance of conventional quaternary ammonium ligands (e.g., trimethylammonium or Q ligands), whose primarily electrostatic interactions with biological matter rapidly degrade at conductivities three to six times lower than the target range. For example, membranes functionalized with conventional Q ligands exhibit a conductivity of approximately 5 to 6 mS / cm from 1 mM NaCl to 50 mM NaCl (conductivity of approximately 5 to 6 mS / cm). Virus clearance dropped from six (6) log reduction values ​​(LRV) to one (1) LRV. Viruses with an isoelectric point (pI) close to 7 (neutral or near neutral) such as Very difficult to remove from process streams. Similar problems are observed when attempting to remove other biological substances from process fluids. For example, when attempting to remove positively charged proteins such as host cell proteins using a filtration device functionalized with conventional Q ligands, the process fluid may have to be diluted two or more times to reduce the conductivity to an acceptable range. This is expensive and can significantly increase the overall processing time.

[0016] Surprisingly, anion exchange separation devices described herein can effectively capture target biological substances even in the presence of high ionic strength conditions. For example, salt (e.g., NaCl) concentration can be up to 50mM, 75mM, 100mM, 125mM, 150mM, 175mM, 200mM, 225mM, 250mM, 275mM, or even 300mM or higher. In some embodiments, binding capacity remains constant or increases with ionic strength. In other embodiments, the binding capacity under 250mM ionic strength can be reduced by no more than 10%, 20%, 35% or 50% of the binding capacity measured in low ionic strength media (e.g., 3mM or 6mM). Surprisingly, the salt tolerance of anion exchange products described herein is very unusual. For example, the binding capacity for bovine serum albumin (BSA) can be maintained up to 250 mM ionic strength using the anion exchange articles described herein, while anion exchange articles with trimethylammonium ligands lose more than 40% of their capacity at 50 mM and 90% of their capacity at 250 mM ionic strength.

[0017] As used herein, the terms "a," "an," "the," and "at least one" are used interchangeably.

[0018] The term "and / or" means either or both. For example, "A and / or B" means A alone, B alone, or both A and B.

[0019] The term "guanidinium" refers to a guanidinium of the formula -NH-C(=NH)-NH 2A salt of a guanidinium group is a charged cationic group that is balanced with a counter anion. Any suitable counter anion may be used, such as a halide, sulfate, phosphate, and the like.

[0020] The term "(hetero)alkylene" refers to alkylene, heteroalkylene, or both.

[0021] The term "alkylene" refers to a divalent group that is a free radical of an alkane. The alkylene group can have 1 to 32 carbon atoms, 1 to 20 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. 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 chain alkylene group has at least 3 carbon atoms.

[0022] The term "heteroalkylene" refers to an alkylene group in which one or more of the linked carbon atoms are replaced by a heteroatom such as oxygen, nitrogen or sulfur. There are no two heteroatoms adjacent to each other as in peroxides. That is, if more than one heteroatom is present, the heteroatoms are separated from each other by at least one carbon atom.

[0023] The term "grafted" is used to indicate that polymer chains are covalently attached to the porous polymer substrate. In most embodiments, the polymer chains are grafted to carbon atoms in the polymer backbone of the porous polymer substrate.

[0024] Anion exchange separation products

[0025] Anion exchange separation products have a porous polymer substrate, which is solid. The term "solid" about a porous polymer substrate means that the substrate is not liquid and does not dissolve in a solution. The pores of a porous polymer substrate can have any desired average size. In some embodiments, the pores are macropores, mesopores, micropores, or mixtures thereof. As used herein, the term "macroporous" refers to a polymer substrate having a pore with a diameter greater than 50 nanometers, the term "mesoporous" refers to a polymer substrate having a pore with a diameter in the range of 2 nanometers to 50 nanometers, and the term "micropore" refers to a material having a pore with a diameter less than 2 nanometers.

[0026] The terms "solid porous polymeric substrate," "porous polymeric substrate," "polymeric substrate," "substrate" and similar variations are used interchangeably herein.

[0027] The porous polymer substrate can have any desired size, shape and form. For example, the porous polymer substrate can be in the form of particles, fibers, films, nonwoven webs, woven webs, membranes, sponges or sheets. In some examples, the polymer substrate is a porous membrane or a porous nonwoven web. In order to prepare large separation products or many separation products and for ease of manufacture, the polymer substrate can be in the form of a roll or formed by a roll, such as the roll of a film, nonwoven webs, woven webs, membranes, sponges or sheets. This allows the use of roll-to-roll processing to prepare separation products. The porous polymer substrate can include a monolayer or multilayer identical or different polymeric material.

[0028] 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(ethersulfones), poly(sulfones), poly(vinyl acetate) and copolymers thereof such as poly(ethylene)-co-poly(vinyl acetate), polyesters such as poly(lactic acid), poly(vinyl alcohol) and copolymers thereof such as poly(ethylene)-co-poly(vinyl alcohol), poly(vinyl esters), poly(vinyl ethers), poly(carbonates), polyurethanes, poly((meth)acrylates) and copolymers thereof, and combinations thereof.

[0029] Suitable polyolefins for use in the porous polymer substrate include poly(ethylene), poly(propylene), poly(1-butene), copolymers of ethylene and propylene, alpha-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 copolymers thereof, and combinations thereof.

[0030] 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.

[0031] Suitable polyamides for the porous polymer substrate include various nylon compositions such as, for example, poly(imidoadipimidohexamethylene), poly(imidoadipimidodecamethylene), polycaprolactam, and combinations thereof. Suitable polyimides include poly(pyromellitimide) and combinations thereof.

[0032] Suitable poly(ethersulfones) for use in the porous polymer substrate include poly(diphenyl ether sulfone), poly(diphenyl sulfone-co-diphenyl ether sulfone), and combinations thereof.

[0033] Suitable vinyl acetate copolymers for use in the porous polymeric substrate include copolymers of ethylene and vinyl acetate and terpolymers of vinyl acetate, vinyl alcohol and ethylene.

[0034] In some embodiments, the porous polymer substrate is a porous membrane having an average pore size (average longest diameter of the pores) generally greater than 0.1 microns to minimize size exclusion separation, minimize diffusion constraints and maximize surface area and separation. Typically, the average pore size can be in the range of 0.1 microns to 10 microns. For example, the average pore size 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.

[0035] The porous polymer substrate can be a macroporous membrane, such as a thermally induced phase separation (TIPS) membrane. TIPS membranes are generally prepared by forming a solution of a thermoplastic material and a second material having a melting point higher than the thermoplastic material. After cooling, the thermoplastic material crystallizes and separates from the second material. The crystallized material is generally stretched. The second material is optionally removed before or after stretching. Macroporous membranes are further described in U.S. Patents 4,539,256 (Shipman), 4726989 (Mrozinski), 4867881 (Kinzer), 5120594 (Mrozinski), 5260360 (Mrozinski) and 5962544 (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 comprising PVDF are further described in US Pat. No. 7,338,692 (Smith et al.).

[0036] In some embodiments, the porous polymer substrate may include a nylon macroporous film or sheet (e.g., a macroporous membrane), such as those described in U.S. Pat. Nos. 6,056,529 (Meyering et al.), 6,267,916 (Meyering et al.), 6,413,070 (Meyering et al.), 6,776,940 (Meyering et al.), 3,876,738 (Marinaccio et al.), 3,928,517 (Knight et al.), 4,707,265 (Barnes, Jr. et al.), and 5,458,782 (Hou et al.).

[0037] In other embodiments, the porous polymer substrate can be a nonwoven web, which can include a nonwoven web made by any known method for producing a nonwoven web. 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.

[0038] For example, fiber nonwoven webs can be made by wet-laid, carded, air-laid, jet-spun, spunbond or melt-blown technology or their combination. Spunbond fibers are usually small diameter fibers, which are formed by extruding a plurality of tiny, usually circular capillaries of a spinneret as filaments from a molten thermoplastic polymer, wherein the diameter of the extruded fibers is rapidly reduced. Meltblown fibers are usually formed as follows: molten thermoplastic material is extruded into a high speed, usually heated gas (e.g., air) stream in the form of a melt line or filament by a plurality of tiny, usually circular die capillaries, and the gas stream thins the filaments of the molten thermoplastic material to reduce their diameter. Then, meltblown fibers are carried by a high-speed gas stream and deposited on 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 having different types and / or thicknesses of thermoplastic polymers.

[0039] Further details of methods of making useful nonwoven webs are described in Wente, "Superfine Thermoplastic Fibers," Indus. Eng. Chem., 48, 1342 (1956) and Wente et al., "Manufacture of Superfine Organic Fibers," Naval Research Laboratories Report No. 4364 (1954).

[0040] The nonwoven web substrate may also optionally include one or more layers of scrim. For example, any one or two major surfaces of the nonwoven web may also optionally include a scrim layer. Scrim (usually a woven or nonwoven reinforcement layer made of fiber) is included to provide strength for 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 be variable, but the scope is typically about 25 microns to about 100 microns, preferably about 25 microns to about 50 microns. The scrim layer may optionally be bonded to the nonwoven article. A variety of adhesive materials can be used for bonding scrim to nonwoven material. Alternatively, scrim can be thermally bonded to the nonwoven web.

[0041] The porosity of nonwoven substrates is usually characterized by properties such as fiber diameter, or basis weight, or solidity, rather than by pore size. The fibers of nonwoven substrates are usually microfibers having an effective fiber diameter of at least 0.5 micron, 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 document: Davies, CN, "The Separation of Airborne Dust and Particles," Institution of Mechanical Engineers, London, Proceedings 1B, 1952. Nonwoven substrates preferably have a porosity of at least 5 g / m 2 , 10g / m 2 , 20g / m 2 Or even 50g / m 2 ; and up to 800g / m 2 , 600g / m 2 , 400g / m 2 , 200g / m 2 Or even 100g / m 2 The minimum tensile strength of a nonwoven web is about 4.0 Newtons. It is generally believed that the tensile strength of a nonwoven substrate is lower in the machine direction than in the cross-web direction due to better fiber bonding and entanglement in the latter.

[0042] Nonwoven web bulk is measured by Solidity, a parameter that defines the solid fraction in the web volume. Lower Solidity values ​​indicate greater web bulk. Solidity (α) is a unitless fraction, 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 L is the thickness of the nonwoven. 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 fibers, the same L is used. 非织造物 The individual stiffness of each fiber is determined and these individual stiffnesses are added together to obtain the stiffness of the web, a.

[0043] The polymer chains grafted to the porous polymer substrate comprise monomer units derived from the monomer of formula (I).

[0044] CH 2 =CR 1 -(C=O)-X 1 -R 2-Z-NH-CH 2 -Ph-CH 2 -NH-C(=NH)-NH 2

[0045] (I)

[0046] or a salt thereof. In formula (I), the group R 1 is hydrogen or methyl; X 1 is -O- or -NH-; R 2 is a (hetero)alkylene group; Z is -NH-(C=O)- or -(C=O)-; and Ph is a phenylene group. The monomer is typically grafted to a carbon atom in the polymer backbone of the polymer material contained in the porous polymer matrix. That is, if it is a homopolymer, the grafted polymer chain has the formula

[0047]

[0048] The variable q is the number of monomer units in the polymer chain.

[0049] Group R 2 Can be alkylene or heteroalkylene.Suitable alkylene usually has 1 to 20 carbon atoms, such as at least 1, at least 2, at least 3, at least 4 or at least 5 and up to 20, up to 18, up to 14, up to 12, up to 10, up to 8 or up to 6 carbon atoms.Suitable heteroalkylene usually has 2 to 20 carbon atoms and 1 to 5 heteroatoms.The number of carbon atoms in heteroalkylene can be at least 2, at least 3, at least 5, at least 6 and up to 20, up to 18, up to 14, up to 12, up to 10, up to 8 or up to 6 carbon atoms.Heteroatoms can be oxygen (e.g., group-O-) or nitrogen (e.g., group-NH-).In many embodiments, heteroatoms are oxygen.Some specific R 2 Groups include but are not limited to -C(CH 3 ) 2 -、-(CH 2 C(CH 3 ) 2 -、-CH 2 CH 2 -、-CH 2 CH 2 CH 2 -and-CH 2 CH 2 -(O-CH 2 CH 2 ) x -, wherein x is an integer in the range of 1 to 5 or 1 to 3.

[0050] The two methylene groups (-CH 2 -Ph-CH 2 -) can be in the meta or para configuration.

[0051] Monomers of formula (I) can be prepared, for example, by initially reacting xylene diamine (1) with O-methylisourea hemisulfate (2) as shown in Reaction Scheme A. For ease of explanation, the compound of formula (2) is shown without the hemisulfate counterion.

[0052] Reaction Scheme A

[0053]

[0054] The reaction product is a compound of formula (3). The intermediate compound can be reacted with an isocyanate-containing monomer, as shown in reaction scheme B, or with an alkene azlactone compound, as shown in reaction scheme C.

[0055] Reaction Scheme B is shown below for reacting the isocyanate-containing monomer of compound (5) with compound (3) of Reaction Scheme A to form compound (6).

[0056] Reaction Scheme B

[0057]

[0058] The group R in the isocyanate group-containing compound (5) 1 , X 2 and R 2 Same as described above. In many embodiments using Reaction Scheme B, R 1 is methyl and R 2 is an alkylene group, such as ethylene or propylene. Compound (6) corresponds to the compound of formula (I) wherein Z is equal to -NH-(C=O)-.

[0059] In an alternative method for preparing compounds of formula (I), compound (3) of Reaction Scheme A is reacted with an alkene azlactone compound. The alkene azlactone compound is typically compound (7) as shown in Reaction Scheme C.

[0060] Reaction Scheme C

[0061]

[0062] Compound (8) corresponds to formula (I), wherein R 1 is hydrogen, X 1 -NH-, R 2 -C(CH 3 ) 2-, and Z is -(C=O)-.

[0063] Exemplary monomers of formula (I) include, but are not limited to, the following compounds or salts thereof, wherein R 1 It is hydrogen or methyl.

[0064]

[0065]

[0066] The polymer chain grafted to the porous polymer substrate can be a homopolymer or a copolymer. The polymer chain is generally a homopolymer of a monomer of formula (I) to prepare a polymer with a high binding capacity for the material desired to be captured. That is, based on the gross weight of the monomers used to form the polymer chain, the polymer chain may contain up to 100% by weight of the first monomer of formula (I). In some embodiments, other monomers (second monomers) may be copolymerized with the first monomer to adjust the binding capacity and / or to achieve other desired properties of the polymer chain. Any suitable second monomer can be used, but they are generally hydrophilic monomers. For example, they are generally water-soluble or miscible with water.

[0067] Based on the gross weight of monomeric units in polymer chain, the amount of the first monomer of formula (I) can be, for example, in the range of 20 wt % to 100 wt %. Based on the gross weight of monomeric units in polymer chain, the amount can be at least 20 wt %, at least 30 wt %, at least 40 wt %, at least 50 wt %, at least 60 wt %, at least 70 wt %, at least 75 wt %, at least 80 wt %, at least 85 wt %, at least 90 wt % or at least 95 wt %, and up to 100 wt %, up to 99 wt %, up to 98 wt %, up to 97 wt %, up to 95 wt %, up to 90 wt %, up to 85 wt %, up to 80 wt % or up to 75 wt %. The first monomer of higher amount tends to increase the binding capacity to various target compounds (such as biomaterials). In many embodiments, the amount of the first monomer of formula (I) ranges from 80 wt% to 100 wt%, 85 wt% to 100 wt%, 90 wt% to 100 wt%, or 95 wt% to 100 wt%, based on the total weight of the monomer units.

[0068] The optional second monomer in the polymer chain can be, for example, a hydrophilic monomer to adjust the degree of hydrophilicity imparted to the substrate. The hydrophilic monomer has an ethylenically unsaturated group and a hydrophilic group, such as, for example, a hydroxyl group, an ether group or an acylamino group. Suitable hydrophilic monomers include acrylamide, dimethylacrylamide, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, ethoxyethyl methacrylate, diethylene glycol methyl ether methacrylate, 2-hydroxyethyl acrylamide, N-vinyl pyrrolidone, etc. and combinations thereof.

[0069] Other optional second monomers include those with more than one ethylenically unsaturated group. This type of second monomer is usually water-soluble and is only used in a relatively small amount, to give the resulting copolymer a certain degree of branching and / or relatively light crosslinking. For example, based on the gross weight of monomers in the first polymerizable composition, the amount of these multifunctional monomers with more than two ethylenically unsaturated groups can be present in an amount ranging from 0.1 wt % to 25 wt %. This 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 useful for some applications, they tend to reduce the binding capacity to some biomaterials.

[0070] Examples include, but are not limited to, poly(ethylene glycol di(meth)acrylate, methylenebisacrylamide, 3-acryloyloxy-2-hydroxypropyl methacrylate, glyceryl dimethacrylate, glyceryl diacrylate, diacryloylpiperazine, and 1,2-ethylenebisacrylamide.

[0071] The total amount of the second monomer can be up to 80% by weight of the monomers for forming the polymer chain. The second monomer in a lower amount generally enhances the binding ability to various target compounds (such as protein biomaterials). Based on the total weight of the monomers in the first polymerizable composition, this amount, if present, is generally equal to 100 minus the weight percent of the first monomer of formula (I).

[0072] The polymer chains are grafted to the porous polymer substrate. Any suitable grafting method can be used. In many embodiments, a Type II photoinitiator is combined with a monomer composition to form a reaction mixture. When the reaction mixture is exposed to ultraviolet 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.

[0073] Type II photoinitiators are typically aromatic ketone compounds. Examples include, but are not limited to, benzophenone, carboxybenzophenone (e.g., 3-carboxybenzophenone), 4-(3-sulfopropoxy)benzophenone sodium salt, Michler's ketone, benzil, anthraquinone, 5,12-tetraacenoquinone, aceanthracenequinone, benzo(A)anthraquinone-7,12-dione, 1,4-chrysenequinone, 6,13-pentaacenoquinone, 5,7,12,14-pentacenetetraone, 9-fluorenone, anthrone, xanthone, thioxanthone, 2-(3-sulfopropoxy)thioxanthene-9-one, acridone, dibenzosuberone, acetophenone, and chromone.

[0074] 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 / cm2 in the wavelength range of 280 nm to 400 nm. 2 or less (as measured according to procedures approved by the National Institute of Standards and Technology, such as with a UVIMAP manufactured by Electronic Instrumentation & Technology, Inc. in Sterling, VA). TM UM 365L-S radiometer). Alternatively, a relatively high light intensity source may be used, such as a medium pressure mercury lamp, which provides typically greater than 10 mW / cm 2 , preferably at 15 mW / cm 2 With 450mW / cm 2 The exposure time can be up to about 30 minutes or even longer.

[0075] In another method that can be used to generate free radicals on the surface of a porous polymer substrate, the substrate itself is selected to be photoactive and a Type II photoinitiator is not required. The monomer composition is exposed to actinic radiation, which is generally 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 polysulfones and poly(ethersulfones). Other photoactive polymer substrates typically contain aromatic groups, such as, for example, homopolymers and block copolymers of poly(methylphenylsilane) and various polyimides based on benzophenonetetracarboxylic dianhydride.

[0076] In other methods for generating free radicals on the surface of a polymer substrate, ionizing radiation is used instead of type II photoinitiators and / or UV radiation. As used herein, the term "ionizing radiation" refers to radiation with sufficient dose and energy to form free radical reaction sites on the surface and / or in the body of a polymer substrate. If the radiation is absorbed by the polymer substrate and causes the cleavage of chemical bonds in the substrate and the formation of free radicals, the radiation has sufficient energy. 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 free radicals.

[0077] In many embodiments of the 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, ESI ELECTROCURE EB SYSTEM from Energy Sciences, Inc., Wilmington, MA, USA and BROADBEAM EB PROCESSOR from E-beam Technologies, Davenport, IA, USA in Davenport, Georgia, USA. Gamma ray radiation generators using cobalt-60 high energy sources are commercially available from MDS Nordion.

[0078] For any given type of ionizing radiation, the delivered dose can be measured according to ISO / ASTM 52628-13, “Standard Practice for Dosimetry in Radiation Processing,” by ASTM International (West Conshohocken, PA). Various dose rates can be obtained by varying the extractor grid voltage, beam diameter, exposure time, and distance from the irradiation source.

[0079] A plurality of polymer chains are grafted to a porous polymer substrate. The term "ligand density" refers to the number of millimoles of monomer units grafted to the substrate per gram. The number of millimoles is calculated by dividing the mass gain by the molecular weight of the monomer and multiplying by 1000. The value is then normalized by dividing by the initial mass of the porous polymer substrate in grams. The ligand density (mmol / gram) is expressed as the number of millimoles of monomer units grafted per gram of substrate. For clarity, the grafted material is typically a polymer material containing multiple monomer units.

[0080] When the substrate is a membrane, the anion exchange separation article generally has a ligand density of about 0.02 mmol / g to about 3 mmol / g or even higher. The grafting density can be at least 0.02 mmol / g, at least 0.05 mmol / g, at least 0.1 mmol / g, at least 0.2 mmol / g, at least 0.5 mmol / g or at least 1 mmol / g, and up to 3 mmol / g, up to 2.5 mmol / g, up to 2 mmol / g, up to 1.5 mmol / g, up to 1 mmol / g, up to 0.8 mmol / g, up to 0.7 mmol / g or up to 0.5 mmol / g. The weight gain is calculated by the formula [100 (weight 2–weight 1) ÷ weight 1], where weight 1 is the weight of the substrate, and weight 2 is the weight of the substrate to which the grafted polymer is attached. The weight gain can be in the range of 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%.

[0081] When substrate is nonwoven or fiber substrate, weight gain during grafting can be higher than the weight gain of film substrate usually.Weight gain can be in the range of 20 wt % to 400 wt % or even higher.This amount can be for example at least 20 wt %, at least 50 wt %, at least 100 wt %, at least 150 wt %, at least 200 wt %, at least 250 wt % or at least 300 wt %, and up to 400 wt %, up to 350 wt %, up to 300 wt %, up to 250 wt %, up to 200 wt %, up to 150 wt %, up to 100 wt %, up to 75 wt % or up to 50 wt %.For example, weight gain can be in the range of 100 wt % to 400 wt %, 100 wt % to 300 wt % or 100 wt % to 200 wt %.

[0082] Binding efficiency (i.e., ligand efficiency) can be calculated by dividing the number of moles of ligand by the number of moles of adsorbed biomaterial (e.g., protein). The lower the number, the more effective the anion separation product for adsorbing biomaterial. The number generally depends on the size of the biomaterial. For example, the ligand efficiency for small biomaterials can be about 10 or higher, while the ligand efficiency for large biomaterials can be as much as 1000 or even greater. Surprisingly, the binding efficiency of an anion exchange separation device prepared, for example, by the VDM adduct of 1-(4-(aminomethyl)benzyl)guanidine sulfate for bovine serum albumin (BSA) is more than twice the binding efficiency of an anion exchange separation device prepared by the IEM adduct of agmatine, which is a compound without formula (I). The data are included in Tables 1 and 2 of the Examples section.

[0083] The anion exchange separation article is salt tolerant, which means that the anion exchange separation article can be used under conditions of high ionic strength. As used herein with respect to salt tolerance, the term "salt" includes all low molecular weight ionic species that contribute to the conductivity of a solution. Salt tolerance is important because many of the aqueous process solutions used in biopharmaceutical or enzyme manufacturing processes have a conductivity in the range of 15 mS / cm to 30 mS / cm (about 150 mM to 300 mM ionic strength) or higher.

[0084] In a typical ion exchange (IEX) process, the binding of proteins or other biological substances to the ionic ligands of the IEX support decreases as the ionic strength (salt concentration) increases. This is due to electrostatic screening caused by the salt ions in the solution. Such processes are not salt-tolerant.

[0085] Protein-based drugs, including monoclonal antibodies (mAbs), are typically purified by a series of chromatography steps. Typically, two or more of these steps are IEX chromatography steps. Typical IEX chromatography media require low ionic strength buffer solutions for protein to interact with IEX ligands. Therefore, before loading onto the IEX media, the protein solution collected from the previous chromatography step must usually be diluted to reduce the salt concentration. This can be very expensive (high buffer and purified water costs), may require larger or additional storage tanks to accommodate larger volumes of solution, and can be very time-consuming, resulting in an overall increased manufacturing cost. Therefore, the development of "salt-tolerant" ligands is very important, i.e., ligands that allow protein solutions to be loaded at relatively high ionic strengths without the need for dilution.

[0086] Example

[0087] Materials and methods

[0088] 2-Vinyl-4,4-dimethylazlactone (VDM) was purchased from SNPE, Inc. and redistilled before use.

[0089] 2-Isocyanatoethyl methacrylate (IEM) and 2-(2-isocyanatoethoxy)ethyl methacrylate (Karenz MOI-EG, CAS No. 107023-60-9) were obtained from Showa Denko KK, Tokyo, Japan.

[0090] Methacrylamidopropyltrimethylammonium chloride (MAPTAC) and 3-(N-morpholino)propanesulfonic acid (MOPS) were purchased from Sigma-Aldrich Company, St. Louis, MO.

[0091] 3-Carboxybenzophenone was purchased from Sigma-Aldrich Co. A solution of 3-carboxybenzophenone sodium salt (C-BP) (0.033 g / mL) was prepared by dissolving 3-carboxybenzophenone in 1 M sodium hydroxide and diluting with deionized water.

[0092] 4-Aminobenzylamine and p-xylylenediamine were purchased from TCI America, Portland, OR.

[0093] O-Methylisourea hydrochloride, O-methylisourea hemisulfate, and m-xylylenediamine were purchased from Thermo Fisher Scientific, Waltham, MA.

[0094] TRIS (tris(hydroxymethyl)aminomethane) was purchased from JT Baker, Phillipsburg, NJ.

[0095] Preparation of 1-(4-(aminomethyl)benzyl)guanidine sulfate :

[0096] The stirred solution of p-phenylenediamine (50.0g, 368mmol) dissolved in the methanol of 150mL is cooled in an ice bath, and it is treated with O-methylisourea hemisulfate (12.9g, 105mmol), and then the concentrated sulfuric acid (5.14g, 52.4mmol) is added dropwise. White precipitate is formed. The ice bath is removed, and stirring is continued overnight. The gained white solid is separated by filtration, and rinsed with some small portions of methanol. The white solid is treated with 200mL of water and the mixture is heated to reflux. The mixture is then stirred for another 15 minutes. The stirred mixture is slowly cooled, and then it is placed in an ice bath for 15 minutes. The gained solid is separated by filtration, rinsed with water, and air-dried, to provide 23.2g of 1-(4-(aminomethyl)benzyl)guanidine sulfate in the form of a white solid. 1 H-NMR (500 MHz, D 2 O, D 2 One drop of NaOD in O)d 7.15 (br s, 4H), 4.09 (s, 2H), 3.56 (s, 2H).

[0097] Preparation of 1-(3-(aminomethyl)benzyl)guanidine sulfate :

[0098] Meta-xylylenediamine (50.0g, 368mmol) is dissolved in the stirred solution in the methanol of 150mL and cooled in an ice bath, and treated with O-methylisourea hemisulfate (12.9g, 105mmol), and then concentrated sulfuric acid (5.14g, 52.4mmol) is added dropwise. White precipitate is formed. The ice bath is removed, and stirring is continued overnight. The gained white solid is separated by filtration, and rinsed with some small portions of methanol. By white solid crystallization (water / methanol), 1-(3-(aminomethyl)benzyl)guanidine sulfate in white crystals of 24.0g is provided. 1 H-NMR (500 MHz, D 2 O, D 2 O)d 7.35 (m, 1H), 7.30-7.25 (m, 3H), 4.32 (s, 2H), 4.05 (s, 2H).

[0099] General procedure for membrane coating and UV irradiation grafting

[0100] The coating solution was prepared by mixing the monomer solution as prepared with deionized water and C-BP photoinitiator (various amounts of 0.033 g / mL deionized water solution) to provide a mixture of the desired monomer and photoinitiator concentrations. The solids weight % of the prepared monomer solution was measured and used to calculate the dilution scheme for each coating experiment. A 9 cm x 12 cm section of nylon membrane substrate (nylon 6,6 membrane, single reinforced layer nylon three-zone membrane, nominal pore size 0.8 microns, #080ZN, purchased from 3M Company, St. Paul, Minnesota) was placed on a piece of polyester film and approximately 4.5 mL of the coating solution was pipetted onto the exposed surface of the membrane. The solution was allowed to soak into the membrane for about 1 minute, and then a second polyester film was placed on top of the substrate. A 2.28 Kg cylindrical weight was rolled over the 3-layer sandwich to squeeze out any excess coating solution. The grafting was performed by UV irradiation using a UV lamp stand (Classic Manufacturing, Inc., Oakdale, MN) equipped with 18 bulbs (Sylvania RG2 40W F40 / 350BL / ECO, 10 bulbs positioned above the membrane and 8 bulbs positioned below the membrane, 46 inches long, 2 inches on center) for 15 minutes. The polyester sheet was removed and the polymer-grafted membrane was placed in a 250 mL polyethylene bottle. The bottle was filled with a 0.9 wt % saline (NaCl) solution, sealed and shaken for 30 minutes to wash any residual monomer or ungrafted polymer from the membrane. In a second membrane washing step, the saline solution was decanted, the bottle was filled with deionized water, sealed, and then shaken for 30 minutes. The washing procedure was repeated three more times, washing once with a 0.9% saline solution and then washing twice with deionized water. The grafted membrane was removed from the bottle and allowed to air dry. Each grafted membrane was analyzed for polymer grafting density and static BSA binding capacity, from which ligand efficiency was calculated.

[0101] Static (equilibrium) BSA binding capacity method for functionalized membranes

[0102] The single disc (16mm diameter) of polymer-grafted film is punched out from the sheet die head of polymer-grafted film.Single disc is placed in the 5mL centrifuge tube containing 4.5mL bovine serum albumin (BSA, Sigma-Aldrich) prepared with the concentration of about 4mg / mL in 25mM TRIS buffer (pH8.0, 50mM NaCl).Each centrifuge tube is capped and rolled overnight (usually 14 hours) on a rotary mixer.Use UV-VIS spectrometer to analyze the gained supernatant solution (applying background correction at 325nm) at 280nm.Determine the static binding capacity for each disc by comparing with the absorption value of the initial BSA solution, and the result is recorded in units of mg / mL (that is, mL of the mg / film volume of the BSA combined with the film) and recorded as the mean value of three parallel determinations.

[0103] Determination of ligand density and ligand efficiency (molar ratio)

[0104] The ligand density was determined based on the mass gained by the membrane sample after the grafting procedure. First, the number of millimoles of ligand monomer grafted to each membrane sample was calculated by dividing the mass gain of the membrane sample by the molecular weight of the grafted monomer. Then, the ligand density (expressed as millimoles of ligand monomer grafted per gram of membrane substrate (mmol / g)) was calculated by dividing the number of millimoles of ligand monomer grafted to the membrane sample by the original mass of the membrane sample.

[0105] The ligand efficiency was determined by first converting the calculated ligand density to a volume basis using the measured bulk density of the membrane (0.415 g / mL) and then converting the calculated BSA binding capacity to a molar basis using the BSA molecular weight. The reported ligand efficiency (ligand molar ratio per BSA molecule) was expressed as the quotient of the ligand density and the BSA binding capacity.

[0106] Salt tolerance test method for BSA binding capacity

[0107] Prepare a buffered aqueous solution of 0.01M MOPS (pH 7.0). Adjust the ionic strength (IS) of the buffered solution by adding different amounts of sodium chloride. Use the buffered solution to prepare a BSA solution of about 3 mg / mL to provide a single BSA protein test solution at 6 mM, 50 mM, 150 mM, and 250 mM ionic strength. Test the membrane samples with the test solution according to the "Static (equilibrium) BSA binding capacity method for functionalized membranes" described above.

[0108] Example 1. Adduct of VDM and 1-(4-(aminomethyl)benzyl)guanidine

[0109]

[0110] 1-(4-(Aminomethyl)benzyl)guanidine sulfate (20.4 g, 74 mmol) was dissolved in 1 N NaOH (74 mL) with gentle heating and stirring. VDM (10.3 g, 74 mmol) was added dropwise to the stirred solution over a period of 4 minutes. After stirring for 6 hours, an aliquot of the slightly turbid solution was added. 1 H-NMR analysis indicated complete conversion to the desired monomer N-(1-((4-guanidinomethyl)benzyl)amino-2-methyl-1-oxopropan-2-yl)acrylamide, sodium bisulfate. 1 H-NMR (500MHz, D 2 O)d 7.17(br s,4H),6.16(m,1H),6.05(m,1H),5.62(m,1H),4.26(s,2H),4.23(s,2H),1.36(s,6H).

[0111] Example 2. Adduct of IEM and 1-(4-(aminomethyl)benzyl)guanidine

[0112]

[0113] IEM was substituted for the VDM reagent in the procedure of Example 1 to provide the monomer 2-(3-(4-(guanidinomethyl)benzyl)ureido)ethyl methacrylate, sodium bisulfate.

[0114] Example 3. Adduct of IEM and 1-(3-(aminomethyl)benzyl)guanidine

[0115]

[0116] 1-(3-(aminomethyl)benzyl)guanidine sulfate was substituted for 1-(4-(aminomethyl)benzyl)guanidine sulfate in the procedure of Example 2 to provide the monomer 2-(3-(3-(guanidinomethyl)benzyl)ureido)ethyl methacrylate, sodium bisulfate.

[0117] Example 4. Adduct of VDM and 1-(3-(aminomethyl)benzyl)guanidine

[0118]

[0119] 1-(3-(aminomethyl)benzyl)guanidine sulfate was substituted for 1-(4-(aminomethyl)benzyl)guanidine sulfate in the procedure of Example 1 to provide the monomer N-(1-((3-guanidinomethyl)benzyl)amino-2-methyl-1-oxopropan-2-yl)acrylamide, sodium bisulfate.

[0120] Example 5. Adduct of KarenzMOI-EG and 1-(4-(aminomethyl)benzyl)guanidine sulfate

[0121]

[0122] Karenz MOI-EG was substituted for the VDM reagent in the procedure of Example 1 to provide the monomer 2-(2-(3-(4-(guanidinomethyl)benzyl)ureido)ethoxy)ethyl methacrylate, sodium bisulfate.

[0123] Example 6. Karenz Adduct of MOI-EG and 1-(3-(aminomethyl)benzyl)guanidine sulfate

[0124]

[0125] 1-(3-(aminomethyl)benzyl)guanidine sulfate was substituted for 1-(4-(aminomethyl)benzyl)guanidine sulfate in the procedure of Example 5 to provide the monomer 2-(2-(3-(3-(guanidinomethyl)benzyl)ureido)ethoxy)ethyl methacrylate, sodium bisulfate.

[0126] Comparative Example A. Adduct of IEM and Agmatine

[0127]

[0128] 4-(2-(Methacryloyloxy)ethylaminocarbonylamino)butylguanidinium sodium sulfate (IEM-agmatine) was prepared according to the procedure of Example 99 of US Patent 9,272,246 (Rasmussen).

[0129] Comparative Example B. Adduct of IEM and 4-aminobenzylguanidine

[0130]

[0131] Under magnetic agitation, in 200mL round-bottomed flask, 4-aminobenzylamine (12.2 grams, 0.1 mole) is dissolved in deionized water (50mL).The O-methylisourea hydrochloride (11.61 grams, 0.1 mole) dissolved in deionized water (50mL) is added to flask.The gained mixture is stirred 24 hours at ambient temperature (about 21 ℃), and then cooled in ice-water bath for 15 minutes.IEM (5.0mL) is added to reaction mixture by syringe.Reaction mixture is stirred 20 minutes, and then add second portion of IEM (5.0mL).Mixture is stirred 15 minutes, and then add other portion of IEM (4.0mL) by syringe.Remove ice-water bath, and reaction mixture is stirred overnight.The product precipitated is filtered, washed with a small amount of deionized water, and dried, to provide the light yellow solid of 27.4 grams. 1 H-NMR analysis indicated complete conversion to the desired monomeric 2-[[4-(guanidinomethyl)phenyl]carbamoylamino]ethyl prop-2-enoate hydrochloride. 1 H-NMR (500MHz, CD 3OD)δ1.94(s,3H),3.50(t,2H),4.21(t,2H),4.31(s,2H),5.63(s,1H),6.14(s,1H),7.21(d,2H),7.38(d,2H).

[0132] Examples 7 to 12 and Comparative Example 1 .

[0133] The nylon membrane was coated and grafted with a single monomer selected from Examples 1 to 6 and Comparative Example A according to the "General Procedure for Membrane Coating and UV Radiation Grafting" described above. The coating solution was prepared at a monomer concentration of 0.25M and a C-BP photoinitiator concentration of 0.0625% w / w. The results for ligand density, BSA binding capacity, and ligand efficiency (molar ratio) are reported in Table 1.

[0134] Table 1. Results of the functionalized membranes of Examples 7 to 12 and Comparative Example 1

[0135]

[0136] Examples 13 to 16 and Comparative Example 2 .

[0137] The nylon membrane was coated and grafted with a single monomer selected from Example 1, Example 4 and Comparative Example A according to the description of Examples 7 to 12, except that a higher concentration of monomer was used in the coating solution. In Example 13, the concentration of the monomer of Example 1 in the coating solution was 0.375M, and for Example 14, the concentration of the monomer of Example 1 in the coating solution was 0.5M. In Example 15, the concentration of the monomer of Example 4 in the coating solution was 0.375M, and for Example 16, the concentration of the monomer of Example 4 in the coating solution was 0.5M. For Comparative Example 2, the concentration of the monomer of Comparative Example A in the coating solution was 0.375M. The results for ligand density, BSA binding capacity and ligand efficiency (molar ratio) are recorded in Table 2.

[0138] Table 2. Results of the functionalized membranes of Examples 13 to 16 and Comparative Example 2

[0139]

[0140] The results in Tables 1 and 2 show that the polymer grafted membranes of Examples 7 to 16 have better ligand efficiency when grafted to the membrane at similar or lower ligand density than the membrane grafted with IEM-agmatine (Comparative Example A).

[0141] Comparative Example 3 .

[0142] The nylon membrane was coated and grafted with the monomer of Comparative Example B according to the "General Procedure for Membrane Coating and UV Radiation Grafting" described above. The coating solution was prepared with the monomer of Comparative Example B at either a concentration of 0.375M or 0.5M and 0.0625% weight / weight C-BP photoinitiator. The results for ligand density, BSA binding capacity, and ligand efficiency (molar ratio) are reported in Table 3.

[0143] Table 3. Results of the functionalized membranes of Comparative Example 3

[0144]

[0145] The results in Table 3 show that the membranes grafted with the polymers of Comparative Example B monomers had lower BSA binding capacity and poorer ligand efficiency than the membranes grafted with the polymers of Examples 7 to 16.

[0146] Comparative Example 4 .

[0147] Nylon membranes were coated and grafted with methacrylamidopropyltrimethylammonium chloride (MAPTAC) at a monomer concentration of 0.5 M according to the "General Procedure for Membrane Coating and UV-Irradiation Grafting" described above. The resulting polymer-grafted membrane had a ligand density of 0.45 mmol / g.

[0148] Example 17. BSA binding capacity and salt tolerance

[0149] The membranes from Example 14, Example 15 and Comparative Example 4 were evaluated according to the "Salt Resistance Test Method for BSA Binding Capacity" described above. The results are reported in Table 4.

[0150] Table 4. Results of the functionalized membranes of Example 14, Example 15 and Comparative Example 4

[0151]

[0152] The results in Table 4 show that the BSA binding capacity is maintained or increased with increasing ionic strength of the test solution for the grafted membranes of Examples 14 and 15. However, for the grafted membrane of Comparative Example 4, the BSA binding capacity decreases with increasing ionic strength of the test solution.

Claims

1. An anion exchange separation product, comprising: a porous polymer substrate, the porous polymer substrate being a solid; and A plurality of polymer chains grafted to the porous polymer substrate and extending away from the surface of the porous polymer substrate, wherein the polymer chains comprise monomer units derived from a monomer of formula (I) or a salt thereof CH2=CR 1 -(C=O)-X 1 -R 2 -Z-NH-CH2-Ph-CH2-NH-C(=NH)-NH2 (I) in R 1 is hydrogen or methyl; X 1 is -O- or -NH-; R 2 is a (hetero)alkylene group; Z is -NH-(C=O)- or -(C=O)-; and Ph is phenylene.

2. The anion exchange separation article of claim 1, wherein the porous polymer substrate is a porous polymer membrane.

3. An anion exchange separation article according to claim 1 or 2, wherein the polymer chain comprises at least 20% by weight of monomer units derived from a monomer of formula (I) or a salt thereof.

4. The anion exchange separation article according to any one of claims 1 to 3, wherein the anion exchange separation article is salt tolerant.

5. The anion exchange separation product according to any one of claims 1 to 3, wherein the monomer of formula (I) is one or more of the following compounds: Where R 1 It is hydrogen or methyl.

6. A method for producing an anion separation product, the method comprising: providing a porous polymer substrate, wherein the porous polymer substrate is a solid; Grafting a plurality of polymer chains to the porous polymer substrate, wherein the polymer chains comprise monomer units derived from a monomer of formula (I) CH2=CR 1 -(C=O)-X 1 -R 2 -Z-NH-CH2-Ph-CH2-NH-C(=NH)-NH2 (I) in R 1 is hydrogen or methyl; X 1 is -O- or -NH-; R 2 is a (hetero)alkylene group; Z is -NH-(C=O)- or -(C=O)-; and Ph is phenylene.

7. A method for separating a mixture of materials, the method comprising: Providing an anion exchange separation product according to claim 1; The mixture of materials is passed through an anion exchange separation device, wherein the anion exchange separation device separates the mixture of materials based on the ionic charge of the materials.

8. The method of claim 7, wherein the anion exchange separation article is salt tolerant at an ionic strength of at least 50 millimolar.

9. A monomer of formula (I) or a salt thereof CH2=CR 1 -(C=O)-X 1 -R 2 -Z-NH-CH2-Ph-CH2-NH-C(=NH)-NH2 (I) in R 1 is hydrogen or methyl; X 1 is -O- or -NH-; R 2 is a (hetero)alkylene group; Z is -NH-(C=O)- or -(C=O)-; and Ph is phenylene.

10. The monomer according to claim 9, wherein the monomer of formula (I) is Where R 1 It is hydrogen or methyl.

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