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

Abstract

An anion exchange separation article is provided that is useful for separating complex samples containing a mixture of materials with different ionic charges. The separation article comprises a plurality of polymer chains grafted onto a solid porous polymer substrate. The polymer chains extend from the surface of the porous polymer substrate, and the side chains of the monomer repeat units of the polymer chains terminate with guanidinium groups or salts thereof. The separation article can be used, for example, to separate biological materials in a sample. Advantageously, the binding capacity of the anion separation article is salt-tolerant.

Description

[Technical Field]

[0001] The detection, quantification, isolation, and purification of target biomaterials, such as viruses and biopolymers (e.g., components or products of living cells, such as proteins, carbohydrates, lipids, and nucleic acids), have long challenged researchers. Detection and quantification are important diagnostically, for example, as indicators of various physiological conditions, such as disease. Isolation and purification of biopolymers are important in therapeutic applications and biomedical research.

[0002] Polymeric materials are widely used for the separation and purification of various target biomaterials. 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 biomaterial, hydrophobic interactions, affinity interactions, covalent bond formation, etc.

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

[0004] Functionalized membranes (e.g., functionalized polymer-supported membranes) typically suffer from relatively low biomaterial binding capacities, which generally limits their use in large-scale purification. Furthermore, many of these functionalized membranes exhibit decreasing biomaterial binding capacities as the ionic strength of the sample increases. Therefore, porous chromatography resins bearing ion-exchange or other interactive ligand functional groups have typically been used in "capture and elute"-type purification processes, such as protein purification, rather than functionalized membranes. Summary of the Invention

[0005] There is a need for new anion exchange separation articles that have high binding capacities for various biological materials, such as proteins, and good salt tolerance, i.e., the binding capacity does not decrease dramatically 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 that includes: (1) a solid porous polymer substrate; and (2) a plurality of polymer chains grafted to the porous polymer substrate and extending from a surface of the porous polymer substrate, the polymer chains comprising monomer units derived from a monomer of formula (I):

[0007] [ka] or a salt thereof. In formula (I), the group R 1 is hydrogen or methyl, and X 1 is -O- or -NH-, and R 2 is alkylene or heteroalkylene, Z is —NH—(C═O)— or —(C═O)—, and Ph is phenylene.

[0008] In a second aspect, a method of making an anion exchange separation article is provided, the method comprising providing a solid porous polymer substrate and grafting a plurality of polymer chains onto the porous polymer substrate, the polymer chains comprising monomer units derived from the monomer of formula (I) described above in the first aspect.

[0009] In a third aspect, a method of separating a mixture of materials is provided, the method comprising 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, where the anion exchange separation device separates the mixture of materials based on their ionic charge.

[0010] In a fourth embodiment, a monomer of formula (I) is provided as described in the first embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0011] An anion-exchange separation article is provided that is useful for separating complex samples containing mixtures of materials with different ionic charges. The separation article comprises a plurality of polymer chains grafted onto a solid, porous polymer substrate. The plurality of polymer chains extend 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 in a sample based on the difference in their anionic charge, 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.

[0012] Salt tolerance means that the binding capacity of an anion exchange separation article typically does not decrease substantially with increasing ionic strength. For example, most conventional anion exchange media lose 50 percent or more of their binding capacity when the ionic strength is increased from a low ionic strength of about 3 mM to 6 mM to a high ionic strength, such as 50 millimolar (mM) or higher.

[0013] The salt tolerance of anion exchange media can be measured relative to that of conventional quaternary ammonium ligands (e.g., trimethylammonium or Q ligands), whose primary electrostatic interactions with biological species rapidly decrease at conductivities between one-third and one-sixth of the target range. For example, membranes functionalized with conventional Q ligands exhibit a decrease in φX174 virus clearance from 6 log reduction values ​​(LRVs) to 1 LRV at 1 mM to 50 mM NaCl (conductivity of approximately 5 mS / cm to 6 mS / cm). Viruses such as φX174, which have an isoelectric point (pI) close to 7 (neutral or nearly neutral), are extremely difficult to remove from process streams. Similar problems are observed when attempting to remove other biological species from process fluids. For example, when using filtration devices functionalized with conventional Q ligands to remove positively charged proteins, such as host cell proteins, the process fluid may need to be diluted by more than two-fold to reduce the conductivity to an acceptable range. This is expensive and dramatically increases the overall processing time.

[0014] Surprisingly, the anion exchange separation devices described herein can be effective in capturing target biological species even in the presence of high ionic strength conditions. For example, salt (e.g., NaCl) concentrations can be as high as 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or even 300 mM or more. In some embodiments, binding capacity remains constant or increases with ionic strength. In other embodiments, binding capacity at 250 mM ionic strength can decrease by no more than 10%, 20%, 35%, or 50% of the binding capacity measured in low ionic strength media (e.g., 3 or 6 mM). Surprisingly, the salt tolerance of the anion exchange articles described herein is highly unusual. For example, binding capacity for bovine serum albumin (BSA) can be maintained up to 250 mM ionic strength using the anion exchange articles described herein, whereas anion exchange articles with trimethylammonium ligands lose more than 40 percent of their capacity at 50 mM and 90 percent of their capacity at 250 mM ionic strength.

[0015] As used herein, the terms "a," "an," "the," and "at least one" are used interchangeably.

[0016] The term "and / or" means either or both. For example, "A and / or B" means A alone, B alone, or both A and B.

[0017] The term "guanidinium" refers to a monovalent group of formula -NH-C(=NH)-NH. Salts of guanidinium groups are cationic groups with a counter-anion that balances the charge. Any suitable counter-anion can be used, such as halide, sulfate, phosphate, etc.

[0018] The term "(hetero)alkylene" refers to alkylene, heteroalkylene, or both.

[0019] The term "alkylene" refers to a divalent group that is a radical of an alkane. An alkylene group can have 1 to 32 carbon atoms, 1 to 20 carbon atoms, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Alkylene can be straight-chained, branched-chained, cyclic, or a combination thereof. A straight-chain alkylene has at least 1 carbon atom, while a cyclic or branched-chain alkylene has at least 3 carbon atoms.

[0020] The term "heteroalkylene" refers to an alkylene in which one or more of the carbon atoms in the chain is replaced with a heteroatom such as oxygen, nitrogen, or sulfur. There are no two heteroatoms adjacent to each other, as in peroxide. That is, if there are two or more heteroatoms, the heteroatoms are separated from each other by at least one carbon atom.

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

[0022] Anion Exchange Separation Articles The anion exchange separation article has a porous polymeric substrate that is solid. The term "solid" in reference to a porous polymeric substrate means that the substrate is not liquid and is not dissolved in a solution. The pores of the porous polymeric substrate can have any desired average size. In some embodiments, the pores are macroporous, mesoporous, microporous, or a mixture thereof. As used herein, the term "macroporous" refers to a polymeric substrate having pores with diameters greater than 50 nanometers, the term "mesoporous" refers to a polymeric substrate having pores with diameters in the range of 2 nanometers to 50 nanometers, and the term "microporous" refers to a material having pores with diameters less than 2 nanometers.

[0023] The terms "solid porous polymeric substrate," "porous polymeric substrate," "polymeric substrate," "substrate," and similar variations may be used interchangeably herein.

[0024] 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. To prepare large or numerous separation articles and for ease of manufacturing, the polymer substrate can be in the form of or formed from a roll, such as a roll of film, nonwoven web, woven web, membrane, sponge, or sheet. This allows the separation articles to be prepared using roll-to-roll processing. The porous polymer substrate can include a single layer or multiple layers of the same or different polymer materials.

[0025] Porous polymer substrates are often formed from thermoplastic materials. Suitable thermoplastics include, but are not limited to, polyolefins, poly(isoprene), poly(butadiene), fluorinated polymers, chlorinated polymers, polyamides, polyimides, polyethers, poly(ether sulfones), poly(sulfones), polyesters such as poly(vinyl acetate) and copolymers thereof, e.g., poly(ethylene)-co-poly(vinyl acetate), poly(lactic acid), poly(vinyl alcohol) and copolymers thereof, e.g., poly(ethylene)-co-poly(vinyl alcohol), poly(vinyl esters), poly(vinyl ethers), poly(carbonates), polyurethanes, poly((meth)acrylates) and copolymers thereof, and combinations thereof.

[0026] Suitable polyolefins for the porous polymer substrate include poly(ethylene), poly(propylene), poly(1-butene), copolymers of ethylene and propylene, alpha-olefin copolymers (e.g., copolymers of ethylene or propylene with 1-butene, 1-hexene, 1-octene, and / or 1-decene), poly(ethylene-co-1-butene), poly(ethylene-co-1-butene-co-1-hexene), poly(butadiene) and copolymers thereof, and combinations thereof.

[0027] Suitable fluorinated polymers for the porous polymer substrate include poly(vinyl fluoride), poly(vinylidene fluoride), copolymers of vinylidene fluoride (e.g., poly(vinylidene fluoride-co-hexafluoropropylene)), copolymers of chlorotrifluoroethylene (e.g., poly(ethylene-co-chlorotrifluoroethylene)), and combinations thereof.

[0028] Suitable polyamides for the porous polymer substrate include various nylon compositions, such as poly(iminoadipoyliminohexamethylene), poly(iminoadipoyliminodecamethylene), polycaprolactam, and combinations thereof. Suitable polyimides include poly(pyromellitimide), and combinations thereof.

[0029] Suitable poly(ether sulfones) for the porous polymer substrate include poly(diphenyl ether sulfone), poly(diphenyl sulfone-co-diphenylene oxide sulfone), and combinations thereof.

[0030] Suitable copolymers of vinyl acetate for porous polymeric substrates include copolymers of ethylene and vinyl acetate, and terpolymers of vinyl acetate, vinyl alcohol, and ethylene.

[0031] In some embodiments, the porous polymer substrate is a porous membrane having an average pore size (average longest diameter of the pores) often greater than 0.1 micrometers to minimize size exclusion separation, minimize diffusional limitations, and maximize surface area and separation. Generally, the average pore size can be in the range of 0.1 micrometer to 10 micrometers. For example, the average pore size is at least 0.2 micrometers, at least 0.4 micrometers, at least 0.6 micrometers, or at least 0.8 micrometers, and at most 8 micrometers, at most 6 micrometers, at most 4 micrometers, or at most 2 micrometers.

[0032] The porous polymer substrate can be a macroporous membrane, such as a thermally induced phase separation (TIPS) membrane. TIPS membranes are often prepared by forming a solution of a thermoplastic material and a second material above the melting point of the thermoplastic material. Upon cooling, the thermoplastic material crystallizes and phase separates from the second material. The crystallized material is often stretched. The second material is optionally removed either before or after stretching. Macroporous membranes are further described in U.S. Pat. Nos. 4,539,256 (Shipman), 4,726,989 (Mrozinski), 4,867,881 (Kinzer), 5,120,594 (Mrozinski), 5,260,360 (Mrozinski), and 5,962,544 (Waller, Jr.). Some exemplary TIPS membranes include poly(vinylidene fluoride) (PVDF), polyolefins such as poly(ethylene) or poly(propylene), vinyl-containing polymers or copolymers, such as ethylene-vinyl alcohol copolymers and butadiene-containing polymers or copolymers, and (meth)acrylate-containing polymers or copolymers. TIPS membranes including PVDF are further described in U.S. Pat. No. 7,338,692 (Smith et al.).

[0033] In some embodiments, the porous polymer substrate can include a nylon macroporous film or sheet (e.g., a macroporous membrane), such as those described in U.S. Pat. Nos. 6,056,529 (Meyering et al.), 6,267,916 (Meyering et al.), 6,413,070 (Meyering et al.), 6,776,940 (Meyering et al.), 3,876,738 (Marinaccio et al.), 3,928,517 (Knight et al.), 4,707,265 (Barnes, Jr. et al.), and 5,458,782 (Hou et al.).

[0034] In other embodiments, the porous polymeric substrate can be a nonwoven web, which can include nonwoven webs made by any of the commonly known methods for making nonwoven webs. As used herein, the term "nonwoven web" refers to a fabric having a structure of individual fibers or filaments that are randomly and / or unidirectionally interleaved in a mat-like manner.

[0035] For example, fibrous nonwoven webs can be made by wet-laid, carded, air-laid, spunlaced, spunbonded, or meltblowing techniques, or combinations thereof. Spunbond fibers are typically small-diameter fibers formed by extruding a molten thermoplastic polymer as filaments through multiple fine, usually circular, capillaries of a spinneret, where the diameter of the extruded fibers rapidly decreases. Meltblown fibers are typically formed by extruding a molten thermoplastic material as molten threads or filaments through multiple fine, usually circular, die capillaries into a high-velocity, usually heated, gas (e.g., air) stream, whereby the molten thermoplastic filaments weaken and decrease in diameter. The meltblown fibers are then carried by the high-velocity gas stream and deposited on a collecting surface to form a web of randomly dispersed meltblown fibers. Any of the nonwoven webs can be made from a single type of fiber or from two or more types of fibers differing in thermoplastic polymer type and / or thickness.

[0036] Further details regarding methods for making useful nonwoven webs are found in Wente, "Superfine Thermoplastic Fibers," Indus. Eng. Chem., 48, 1342 (1956) and Wente et al., "Manufacture of Superfine Organic Fibers," Naval Research Laboratories Report No. 4364 (1954).

[0037] The nonwoven web substrate may optionally further comprise one or more scrim layers. For example, either or both major surfaces of the nonwoven web may each optionally comprise a scrim layer. The scrim, typically a woven or nonwoven reinforcing layer made from fibers, is included to impart strength to the nonwoven web. Suitable scrim materials include, but are not limited to, nylon, polyester, fiberglass, polyethylene, polypropylene, and the like. The average thickness of the scrim can vary but is often in the range of about 25 micrometers to about 100 micrometers, preferably about 25 micrometers to about 50 micrometers. The scrim layer may optionally be bonded to the nonwoven article. Various adhesive materials can be used to bond the scrim to the nonwoven. Alternatively, the scrim can be thermally bonded to the nonwoven web.

[0038] The porosity of nonwoven substrates is typically characterized by properties such as fiber diameter, basis weight, or solidity, rather than pore size. The fibers of nonwoven substrates are typically microfibers having an effective fiber diameter of at least 0.5, 1, 2, or even 4 micrometers, and up to 15, 10, 8, or even 6 micrometers, as calculated according to the method described in Davies, CN, "The Separation of Airborne Dust and Particles," Institution of Mechanical Engineers, London, Proceedings 1B, 1952. The nonwoven substrate preferably has an effective fiber diameter of at least 5, 10, 20, or even 50 g / m 2 , and up to 800, 600, 400, 200, or even 100 g / m 2 The nonwoven web has a basis weight in the range of about 4.0 Newtons. The minimum tensile strength of the nonwoven web is about 4.0 Newtons. It is generally recognized that the tensile strength of a nonwoven substrate in the machine direction is lower than in the crossweb direction due to better fiber bonding and entanglement in the crossweb direction.

[0039] Nonwoven web loft is measured by solidity, a parameter that defines the solid fraction in the volume of the web. Lower solidity values ​​indicate greater web loft. Solidity (α) is determined by the formula α=m f ÷ρ f ×L nonwoven (In the formula, m f is the fiber mass per sample surface area, and ρ f is the fiber density and L nonwoven Solidity is a unitless fraction typically expressed by (where L is the nonwoven thickness). Solidity is used herein to refer to the nonwoven substrate itself, not to the functionalized nonwoven substrate. When a nonwoven substrate contains a mixture of two or more types of fibers, the individual solidities are determined by the same L nonwoven are used to determine the solidity of each type of fiber, and these individual solidities are added together to obtain the solidity α of the web.

[0040] The polymer chains grafted onto the porous polymer substrate are composed of monomer units derived from the monomer of formula (I)

[0041] [ka] or a salt thereof. In formula (I), the group R 1 is hydrogen or methyl, and X 1 is -O- or -NH-, and R 2 is a (hetero)alkylene, Z is -NH-(C=O)- or -(C=O)-, and Ph is phenylene. The monomer is often grafted to a carbon atom in the polymer backbone of the polymer material contained in the porous polymer substrate. That is, the grafted polymer chain, when it is a homopolymer, has the formula

[0042] [ka] and has a bond to a carbon atom in the substrate. The variable q is the number of monomer units in the polymer chain.

[0043] base R2 can be alkylene or heteroalkylene. Suitable alkylenes typically have 1 to 20 carbon atoms, e.g., 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 heteroalkylenes typically have 2 to 20 carbon atoms and 1 to 5 heteroatoms. The number of carbon atoms in the heteroalkylene can be at least 2, at least 3, at least 5, or 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. The heteroatom can be oxygen (e.g., -O-) or nitrogen (e.g., -NH-). In many embodiments, the heteroatom is oxygen. Some specific R 2 Examples of the group include -C(CH3)2-, -(CH2C(CH3)2-, -CH2CH2-, -CH2CH2CH2- and -CH2CH2-(O-CH2CH2) x - (wherein x is an integer ranging from 1 to 5 or 1 to 3), but are not limited to these.

[0044] The two methylene groups (-CH2-Ph-CH2-) attached to the phenylene in formula (I) can be in either the meta or para configuration.

[0045] Monomers of formula (I) can be prepared, for example, by first reacting xylylenediamine (1) with O-methylisourea hemisulfate (2), as shown in Reaction Scheme A. For ease of illustration, the compound of formula (2) is shown without the hemisulfate counterion.

[0046] [ka]

[0047] The reaction product is a compound of formula (3). This intermediate compound can be reacted with an isocyanate-containing monomer, as shown in Reaction Scheme B, or with an alkenyl azlactone compound, as shown in Reaction Scheme C.

[0048] 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).

[0049] [ka]

[0050] The group R in the isocyanate group-containing compound (5) 1 , X 2 and R 2 is as above. In many embodiments using Reaction Scheme B, R 1 is methyl and R 2 is an alkylene such as ethylene or propylene. Compound (6) corresponds to the compound of formula (I) in which Z is equal to -NH-(C=O)-.

[0051] An alternative method for preparing compounds of Formula (I) involves reacting compound (3) of Reaction Scheme A with an alkenyl azlactone compound, often compound (7) as shown in Reaction Scheme C.

[0052] [ka]

[0053] Compound (8) is R 1 is hydrogen and X 1 is -NH- and R 2 corresponds to formula (I) where is -C(CH3)2- and Z is -(C=O)-.

[0054] Examples of monomers of formula (I) include R 1is hydrogen or methyl, or a salt thereof, but is not limited to these.

[0055] [ka]

[0056] The polymer chains grafted onto the porous polymer substrate can be homopolymers or copolymers. The polymer chains are often homopolymers of the monomer of formula (I) to prepare polymers with high binding capacity for the material to be captured. That is, the polymer chains can contain up to 100 weight percent of the first monomer of formula (I), based on the total weight of the monomers used to form the polymer chain. In some embodiments, other monomers (second monomers) can be copolymerized with the first monomer to adjust the binding capacity of the polymer chain and / or achieve other desired properties. While any suitable second monomers can be used, they are typically hydrophilic monomers. For example, they are often water-soluble or water-miscible.

[0057] The amount of the first monomer of Formula (I) can range, for example, from 20 to 100 weight percent based on the total weight of the monomer units in the polymer chain. The amount can be at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 95, and up to 100, up to 99, up to 98, up to 97, up to 95, up to 90, up to 85, up to 80, or up to 75 weight percent based on the total weight of the monomer units in the polymer chain. A higher amount of the first monomer tends to increase the binding ability to various target compounds, such as biomaterials. In many embodiments, the amount of the first monomer of Formula (I) ranges from 80 to 100, 85 to 100, 90 to 100, or 95 to 100 weight percent based on the total weight of the monomer units.

[0058] The optional second monomer in the polymer chain can be a hydrophilic monomer, for example, to adjust the degree of hydrophilicity imparted to the substrate. The hydrophilic monomer has an ethylenically unsaturated group and a hydrophilic group, such as a hydroxyl group or an amide group. Suitable hydrophilic monomers include acrylamide, dimethylacrylamide, hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, ethoxyethyl methacrylate, diethylene glycol methyl ether methacrylate, 2-hydroxyethyl acrylamide, N-vinylpyrrolidone, and the like, and combinations thereof.

[0059] Other optional second monomers include those containing two or more ethylenically unsaturated groups. Second monomers of this type are typically water-soluble and are used in relatively small amounts to impart some branching and / or relatively light crosslinking to the resulting copolymer. For example, these polyfunctional monomers containing three or more ethylenically unsaturated groups can be present in an amount ranging from 0.1 to 25 weight percent, based on the total weight of the monomers in the first polymerizable composition. The amount can be at least 0.1, at least 0.2, at least 0.5, or at least 1.0 weight percent, and up to 25, 20, 15, 10, 5, 4, 3, 2, or 1 weight percent. Crosslinking monomers can be used and may be beneficial for some applications, but they tend to reduce binding capacity to some biomaterials.

[0060] Examples include, but are not limited to, poly(ethylene glycol di(meth)acrylate, methylene bisacrylamide, 3-acryloyloxy-2-hydroxypropyl methacrylate, glycerol dimethacrylate, glycerol diacrylate, diacryloylpiperazine, and 1,2-ethylene bisacrylamide.

[0061] The total amount of the second monomer can be up to 80 weight percent of the monomers used to form the polymer chain.The smaller the amount of the second monomer, the stronger the binding ability to various target compounds, such as protein biomaterials.When present, its amount is usually equal to 100 minus the weight percent of the first monomer of formula (I) based on the total weight of the monomers in the first polymerizable composition.

[0062] The polymer chains are grafted onto the porous polymer substrate. Any suitable grafting method can be used. In many embodiments, a Type II photoinitiator is combined with a monomer composition to form a reaction mixture. When the reaction mixture is exposed to ultraviolet light, the Type II photoinitiator abstracts hydrogen atoms from the porous polymer substrate, generating free radicals on the porous polymer substrate. The free radicals react with the monomers present in the composition, resulting in the formation of polymer chains grafted to the porous polymer substrate. The polymer chains are often grafted to carbon atoms in the backbone of the polymer material contained in the porous polymer substrate.

[0063] Type II photoinitiator is typically an aromatic ketone compound.Examples include, but are not limited to, benzophenone, carboxybenzophenone (for example, 3-carboxybenzophenone), 4-(3-sulfopropyloxy)benzophenone sodium salt, Michler's ketone, benzil, anthraquinone, 5,12-naphthacenequinone, aceanthracenequinone, benz(A)anthracene-7,12-dione, 1,4-chrysenequinone, 6,13-pentacenequinone, 5,7,12,14-pentacenetetrone, 9-fluorenone, anthrone, xanthone, thioxanthone, 2-(3-sulfopropyloxy)thioxanthen-9-one, acridone, dibenzosuberone, acetophenone, and chromone.

[0064] The ultraviolet (UV) light used to generate free radicals on the porous polymer substrate can be provided by a variety of light sources, such as light emitting diodes (LEDs), black lights, medium pressure mercury lamps, or combinations thereof. Actinic radiation can also be obtained using higher intensity light sources, such as those available from Fusion UV Systems Inc. Ultraviolet light sources typically provide irradiance of 10 mW / cm over a wavelength range of 280 nanometers to 400 nanometers. 2 Alternatively, it may be a relatively low intensity light source such as a black light that provides less than 10 mW / cm (e.g., as measured according to National Institute of Standards and Technology approved procedures using a UVIMAP™ UM365L-S radiometer manufactured by Electronic Instrumentation & Technology, Inc., Sterling, VA). 2 Greater than 15mW / cm 2 ~450mW / cm 2 A relatively high intensity light source can be used, such as a medium pressure mercury lamp providing an intensity of 1000 .mu.m. The exposure time can be up to about 30 minutes or even longer.

[0065] In another useful method for generating free radicals on the surface of a porous polymer substrate, the substrate itself is selected to be photoactive, and no type II photoinitiator is required. A monomer composition is exposed to actinic radiation, typically in the ultraviolet region of the electromagnetic spectrum. Upon exposure to actinic radiation, the polymer substrate absorbs enough 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 often contain aromatic groups, such as poly(methylphenylsilane) and various polyimide homopolymers and block copolymers based on benzophenonetetracarboxylic dianhydride.

[0066] Other methods for generating free radicals on the surface of a polymer substrate use ionizing radiation rather than Type II photoinitiators and / or UV irradiation. As used herein, the term "ionizing radiation" refers to radiation of sufficient dose and energy to form free radical reaction sites on the surface and / or in the bulk of the polymer substrate. The radiation has sufficient energy when absorbed by the polymer substrate, resulting in the cleavage of chemical bonds in the substrate and the formation of free radicals. The ionizing radiation is often beta radiation, gamma radiation, electron beam radiation, X-ray radiation, plasma radiation, or other suitable types of electromagnetic radiation. Preferably, the ionizing radiation is administered in an inert environment to prevent oxygen from reacting with the radicals.

[0067] In many embodiments of this method, the ionizing radiation is electron beam radiation, gamma radiation, X-ray radiation, or plasma radiation, as suitable generators are readily available. Electron beam generators are commercially available, such as 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 radiation generators that use a Cobalt-60 high-energy source are commercially available from MDS Nordion.

[0068] 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.

[0069] Multiple polymer chains are grafted to the porous polymer substrate. The term "ligand density" refers to the millimoles per gram of monomer units grafted to the substrate. Millimoles is calculated by dividing the mass increase by the molecular weight of the monomer and multiplying by 1000. This value is then normalized by dividing by the original mass (grams) of the porous polymer substrate. Ligand density is expressed as millimoles of grafted monomer units per gram of substrate (mmol / g). For clarity, the grafted material is typically a polymer material containing multiple monomer units.

[0070] When the substrate is a membrane, the anion exchange separation article often has a ligand density of about 0.02 mmol / gram to about 3 mmol / gram or even higher. The grafting density can be at least 0.02, at least 0.05, at least 0.1, at least 0.2, at least 0.5, or at least 1 mmol / gram, and up to 3, up to 2.5, up to 2, up to 1.5, up to 1, up to 0.8, up to 0.7, or up to 0.5 mmol / gram. The weight gain is calculated from the formula [100(wt2-wt1)÷wt1], where wt1 is the weight of the substrate and wt2 is the weight of the substrate with the grafted polymer attached. The weight gain can range from 1 weight percent to 85 weight percent or even higher. The amount can be, for example, at least 1, at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 weight percent, and up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, or up to 30 weight percent.

[0071] When the substrate is a nonwoven or fibrous substrate, the weight gain upon grafting can often be higher than that of the membrane substrate. The weight gain can range from 20 weight percent to 400 weight percent or even more. The amount can be, for example, at least 20, at least 50, at least 100, at least 150, at least 200, at least 250, or at least 300 weight percent, and up to 400, up to 350, up to 300, up to 250, up to 200, up to 150, up to 100, up to 75, or up to 50 weight percent. For example, the weight gain can be in the range of 100 to 400, 100 to 300, or 100 to 200 weight percent.

[0072] The 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 this number, the more effective the anion separation article is for adsorbing biomaterials. This number often depends on the size of the biomaterial. For example, the ligand efficiency for small biomaterials may be about 10 or greater, while the ligand efficiency for large biomaterials may be up to 1000 or even greater. Surprisingly, for example, anion exchange separation devices prepared from the VDM adduct of 1-(4-(aminomethyl)benzyl)guanidine sulfate had more than twice the binding capacity for bovine serum albumin (BSA) as anion exchange separation devices prepared from the IEM adduct of agmatine, a compound not of Formula (I). This data is included in Tables 1 and 2 in the Examples section.

[0073] Anion exchange separation articles are salt-tolerant, meaning that they 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 aqueous process solutions used in biopharmaceutical or enzyme manufacturing processes have conductivities in the range of 15 mS / cm to 30 mS / cm (approximately 150 mM to 300 mM ionic strength) or higher.

[0074] In a typical ion exchange (IEX) process, binding of proteins or other biological species to ionizable ligands on an IEX support decreases as the ionic strength (salt concentration) increases. This is due to electrostatic shielding by salt ions in solution. Such methods are not salt-tolerant.

[0075] Protein-based drugs, including monoclonal antibodies (mAbs), are typically purified through a series of chromatography steps. Often, two or more of these steps are IEX chromatography steps. Typical IEX chromatography media require low ionic strength buffer solutions for proteins to interact with IEX ligands. As a result, the protein solution recovered from the previous chromatography step must often be diluted to reduce the salt concentration before loading onto the IEX medium. This can be very expensive (high buffer and purified water costs), potentially requires larger or additional holding tanks to accommodate larger volumes of solution, and can be very time-consuming, increasing overall production costs. Therefore, the development of "salt-tolerant" ligands, i.e., ligands that allow loading of protein solutions at relatively high ionic strengths and eliminate the need for dilution, is of great importance. [Example]

[0076] Materials and Methods 2-vinyl-4,4-dimethylazlactone (VDM) was obtained from SNPE, Inc. and redistilled before use.

[0077] 2-Isocyanatoethylmethacrylate (IEM) and 2-(2-isocyanatoethoxy)ethyl methacrylate (KarenzMOI-EG, CAS number 107023-60-9) were obtained from Showa Denko KK (Tokyo, Japan).

[0078] Methacrylamidopropyltrimethylammonium chloride (MAPTAC) and 3-(N-morpholino)propanesulfonic acid (MOPS) were obtained from Sigma-Aldrich Company, St. Louis, MO.

[0079] 3-Carboxybenzophenone was obtained from Sigma-Aldrich. 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.

[0080] 4-aminobenzylamine and p-xylylenediamine were obtained from TCI America, Portland, OR.

[0081] O-Methylisourea hydrochloride, O-methylisourea hemisulfate, and m-xylylenediamine were obtained from Thermo Fisher Scientific, Waltham, MA.

[0082] TRIS (tris(hydroxymethyl)aminomethane) was obtained from JT Baker, Phillipsburg, NJ.

[0083] Preparation of 1-(4-(aminomethyl)benzyl)guanidine sulfate A stirred solution of p-xylylenediamine (50.0 g, 368 mmol) in 150 mL of methanol was cooled in an ice bath and treated with O-methylisourea hemisulfate (12.9 g, 105 mmol), followed by the dropwise addition of concentrated sulfuric acid (5.14 g, 52.4 mmol). A white precipitate formed. The ice bath was removed, and stirring was continued overnight. The resulting white solid was isolated by filtration and rinsed several times with small amounts of methanol. The white solid was treated with 200 mL of water, and the mixture was heated to reflux. The mixture was then stirred for an additional 15 minutes. The stirred mixture was gradually cooled and then placed in an ice bath for 15 minutes. The resulting solid was isolated by filtration, rinsed with water, and air-dried to provide 23.2 g of 1-(4-(aminomethyl)benzyl)guanidine sulfate as a white solid. 1 H-NMR (500 MHz, DO with 1 drop of NaOD in DO) d 7.15 (br s, 4H), 4.09 (s, 2H), 3.56 (s, 2H).

[0084] Preparation of 1-(3-(aminomethyl)benzyl)guanidine sulfate A stirred solution of m-xylylenediamine (50.0 g, 368 mmol) in 150 mL of methanol was cooled in an ice bath and treated with O-methylisourea hemisulfate (12.9 g, 105 mmol), followed by the dropwise addition of concentrated sulfuric acid (5.14 g, 52.4 mmol). A white precipitate formed. The ice bath was removed and stirring was continued overnight. The resulting white solid was isolated by filtration and rinsed several times with small amounts of methanol. The white solid was crystallized (water / methanol) to provide 24.0 g of 1-(3-(aminomethyl)benzyl)guanidine sulfate as white crystals. 1 H-NMR (500 MHz, DO with one drop of NaOD in DO) 7.35 (m, 1H), 7.30–7.25 (m, 3H), 4.32 (s, 2H), 4.05 (s, 2H).

[0085] General procedure for membrane coating and UV radiation grafting Coating solutions were prepared by mixing the prepared monomer solution with deionized water and C-BP photoinitiator (varying amounts of a 0.033 g / mL solution in deionized water) to provide mixtures with the desired monomer and photoinitiator concentrations. The weight percent solids of the prepared monomer solutions were measured and used to calculate the dilution protocol for each coating experiment. A 9 cm x 12 cm section of nylon membrane substrate (nylon 6,6 membrane, single-reinforced layer nylon 3-zone membrane, nominal pore size 0.8 microns, #080 ZN, obtained from 3M Company, St. Paul, MN) was placed on a sheet of polyester film, and approximately 4.5 mL of coating solution was pipetted onto the exposed surface of the membrane. Each solution was allowed to soak into the membrane 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 over the three-layer sandwich to squeeze out excess coating solution. UV irradiation grafting was performed using a UV light 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-inch center-to-center spacing) with a 15-minute irradiation time. The polyester sheet was removed, and the polymer-grafted membrane was placed in a 250 mL polyethylene bottle. The bottle was filled with 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 was decanted, and the bottle was filled with deionized water, sealed, and then shaken for 30 minutes. The washing procedure was repeated three more times, with one wash using 0.9% saline followed by two washes using 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 the ligand efficiency was calculated.

[0086] Static (equilibrium) BSA binding capacity method for functionalized membranes Individual disks (16 mm in diameter) of polymer-grafted membrane were die-punched from sheets of polymer-grafted membrane. A single disk was placed in a 5 mL centrifuge tube containing bovine serum albumin (BSA, Sigma-Aldrich) prepared at a concentration of approximately 4 mg / mL in 4.5 mL of 25 mM TRIS buffer (pH 8.0, 50 mM NaCl). Each tube was capped and rotated overnight (typically 14 h) on a rotary mixer. The resulting supernatant solution was analyzed using a UV-VIS spectrometer at 280 nm (background correction was applied at 325 nm). The static binding capacity for each disk was determined by comparing it to the absorbance value of the starting BSA solution, and results are reported in mg / mL (i.e., mg of BSA bound to the membrane / mL of membrane volume) and are reported as the average of three replicates.

[0087] Determination of ligand density and ligand efficiency (molar ratio) Ligand density was determined based on the mass gained by the membrane samples 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 grafting monomer. Then, the ligand density (expressed as millimoles of ligand monomer grafted per gram of membrane substrate (mmol / g)) was calculated by dividing the millimoles of ligand monomer grafted to the membrane sample by the original mass of the membrane sample.

[0088] Ligand efficiency was determined by first converting the calculated ligand density to a volumetric basis using the measured membrane bulk density (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 (molar ratio of ligand per BSA molecule) was expressed as the quotient of ligand density to BSA binding capacity.

[0089] Salt tolerance test method for BSA binding ability An aqueous buffer solution of 0.01 M MOPS (pH 7.0) was prepared. The ionic strength (IS) of the buffer was adjusted by adding various amounts of sodium chloride. Approximately 3 mg / mL BSA solutions were prepared using the buffer solution to provide individual BSA protein challenge solutions of 6, 50, 150, and 250 mM ionic strength. Membrane samples were tested with the challenge solutions according to the "Static (Equilibrium) Binding Capacity Method for Functionalized Membranes" described above.

[0090] Example 1. Adduct of VDM and 1-(4-(aminomethyl)benzyl)guanidine

[0091] [ka]

[0092] 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 4 minutes. After stirring for 6 hours, an aliquot of the slightly cloudy solution was 1 H-NMR analysis showed complete conversion to the desired monomer, N-(1-((4-guanidinomethyl)benzyl)amino-2-methyl-1-oxopropan-2-yl)acrylamide, sodium hydrogen sulfate. 1 H-NMR (500MHz, D2O)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).

[0093] Example 2. Adduct of IEM and 1-(4-(aminomethyl)benzyl)guanidine

[0094] [ka]

[0095] In the procedure of Example 1, the VDM reagent was replaced with IEM to provide the monomer 2-(3-(4-(guanidinomethyl)benzyl)ureido)ethyl methacrylate, sodium hydrogen sulfate.

[0096] Example 3. Adduct of IEM and 1-(3-(aminomethyl)benzyl)guanidine

[0097] [ka]

[0098] In the procedure of Example 2, 1-(4-(aminomethyl)benzyl)guanidine sulfate was replaced with 1-(3-(aminomethyl)benzyl)guanidine sulfate to provide the monomer 2-(3-(3-(guanidinomethyl)benzyl)ureido)ethyl methacrylate, sodium hydrogen sulfate.

[0099] Example 4. Adduct of VDM and 1-(3-(aminomethyl)benzyl)guanidine

[0100] [ka]

[0101] In the procedure of Example 1, 1-(4-(aminomethyl)benzyl)guanidine sulfate was replaced with 1-(3-(aminomethyl)benzyl)guanidine sulfate to give the monomer N-(1-((3-guanidinomethyl)benzyl)amino-2-methyl-1-oxopropan-2-yl)acrylamide, sodium hydrogen sulfate.

[0102] Example 5. Adduct of Karenz MOI-EG and 1-(4-(aminomethyl)benzyl)guanidine sulfate

[0103] [ka]

[0104] In the procedure of Example 1, the VDM reagent was replaced with KarenzMOI-EG to provide the monomer 2-(2-(3-(4-(guanidinomethyl)benzyl)ureido)ethoxy)ethyl methacrylate, sodium hydrogen sulfate.

[0105] Example 6. Adduct of Karenz MOI-EG and 1-(3-(aminomethyl)benzyl)guanidine sulfate

[0106] [ka]

[0107] In the procedure of Example 5, 1-(4-(aminomethyl)benzyl)guanidine sulfate was replaced with 1-(3-(aminomethyl)benzyl)guanidine sulfate to provide the monomer, 2-(2-(3-(3-(guanidinomethyl)benzyl)ureido)ethoxy)ethyl methacrylate, sodium hydrogen sulfate.

[0108] Comparative Example A. Adducts of IEM and Agmatine

[0109] [ka]

[0110] 4-(2-(methacryloyloxy)ethylaminocarbonylamino)butylguanidinium sulfate sodium (IEM-agmatine) was prepared according to the procedure of Example 99 of U.S. Pat. No. 9,272,246 (Rasmussen).

[0111] Comparative Example B. Adduct of IEM and 4-aminobenzylguanidine

[0112] [ka]

[0113] 4-Aminobenzylamine (12.2 grams, 0.1 mole) was dissolved in deionized water (50 mL) in a 200 mL round-bottom flask with magnetic stirring. O-Methylisourea hydrochloride (11.61 grams, 0.1 mole) dissolved in deionized water (50 mL) was added to the flask. The resulting mixture was stirred at ambient temperature (approximately 21°C) for 24 hours and then cooled in an ice-water bath for 15 minutes. IEM (5.0 mL) was added to the reaction mixture via syringe. The reaction mixture was stirred for 20 minutes, then a second portion of IEM (5.0 mL) was added. The mixture was stirred for 15 minutes, then an additional IEM (4.0 mL) was added via syringe. The ice-water bath was removed, and the reaction mixture was stirred overnight. The precipitated product was filtered, washed with a small amount of deionized water, and dried to provide 27.4 grams of a pale yellow solid. 1 H-NMR analysis showed complete conversion to the desired monomer 2-[[4-(guanidinomethyl)phenyl]carbamoylamino]ethyl prop-2-enoate hydrochloride. 1 H-NMR (500MHz, CD3OD) 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).

[0114] Examples 7 to 12 and Comparative Example 1 Nylon membranes were coated and grafted with single monomers selected from Examples 1-6 and Comparative Example A according to the "General Procedure for Membrane Coating and UV Irradiation Grafting" above. Coating solutions were prepared with a monomer concentration of 0.25 M 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.

[0115] [Table 1]

[0116] Examples 13 to 16 and Comparative Example 2. Nylon membranes were coated and grafted with a single monomer selected from Examples 1, 4, and Comparative Example A as described in Examples 7-12, except that a higher concentration of monomer was used in the coating solution. For Example 13, the concentration of the monomer of Example 1 in the coating solution was 0.375 M, and for Example 14, the concentration of the monomer of Example 1 in the coating solution was 0.5 M. For Example 15, the concentration of the monomer of Example 4 in the coating solution was 0.375 M, and for Example 16, the concentration of the monomer of Example 4 in the coating solution was 0.5 M. For Comparative Example 2, the concentration of the monomer of Comparative Example A in the coating solution was 0.375 M. The results of ligand density, BSA binding capacity, and ligand efficiency (molar ratio) are reported in Table 2.

[0117] [Table 2]

[0118] The results in Tables 1 and 2 show that compared to the membrane grafted with IEM-agmatine (Comparative Example A), the polymer-grafted membranes of Examples 7-16 have better ligand efficiency when grafted to the membrane at similar or lower ligand densities.

[0119] Comparative Example 3. Nylon membranes were coated and grafted with the monomer of Comparative Example B according to the "General Procedure for Membrane Coating and UV Irradiation Grafting" above. Coating solutions were prepared using 0.375 M or 0.5 M concentrations of the monomer of Comparative Example B and 0.0625% w / w of C-BP photoinitiator. The results for ligand density, BSA binding capacity, and ligand efficiency (molar ratio) are reported in Table 3.

[0120] [Table 3]

[0121] The results in Table 3 show that the polymer-grafted membrane prepared using Comparative Example B monomer has lower BSA binding capacity and inferior ligand efficiency than the polymer-grafted membranes of Examples 7-16.

[0122] Comparative Example 4. Following the "General Procedure for Membrane Coating and UV-Irradiation Grafting" described above, nylon membranes were coated and grafted with methacrylamidopropyltrimethylammonium chloride (MAPTAC) at a monomer concentration of 0.5 M. The resulting polymer-grafted membranes had a ligand density of 0.45 mmol / g.

[0123] Example 17. BSA binding capacity and salt tolerance The membranes from Examples 14, 15 and Comparative Example 4 were evaluated according to the above "Salt Tolerance Test Method for BSA Binding Ability." The results are reported in Table 4.

[0124] [Table 4]

[0125] The results in Table 4 show that the BSA binding capacity was maintained or increased with increasing ionic strength of the challenge solution for the grafted membranes of Examples 14 and 15. However, for the grafted membrane of Comparative Example 4, the BSA binding capacity decreased with increasing ionic strength of the challenge solution.

Claims

1. a porous polymer substrate that is a solid; a plurality of polymer chains grafted onto the porous polymer substrate and extending from a surface of the porous polymer substrate, the polymer chains comprising monomer units derived from a monomer of formula (I) or a salt thereof; 【Chemistry 1】 [In the formula, R １ is hydrogen or methyl, X １ is —O— or —NH—, R ２ is (hetero)alkylene, Z is —NH—(C═O)— or —(C═O)—; Ph is phenylene. and an anion exchange separation article comprising:

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

3. 3. The anion exchange separation article of claim 1 or 2, wherein the polymer chains comprise at least 20 weight percent of monomer units derived from a monomer of formula (I) or a salt thereof.

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

5. The anion-exchange separation article of any one of claims 1 to 3, wherein the monomer of formula (I) is one or more of the following compounds: 【Chemistry 2】 [In the formula, R １ is hydrogen or methyl.

6. 1. A method of making an anion exchange separation article, comprising: providing a porous polymeric substrate that is solid; grafting a plurality of polymer chains onto the porous polymer substrate, the polymer chains comprising monomer units derived from a monomer of formula (I). 【Transformation 3】 [In the formula, R １ is hydrogen or methyl, X １ is —O— or —NH—, R ２ is (hetero)alkylene, Z is —NH—(C═O)— or —(C═O)—; Ph is phenylene. and a method comprising:

7. 1. A method for separating a mixture of materials, comprising: providing an anion exchange separation article according to claim 1; passing the mixture of materials through an anion exchange separation device, the anion exchange device separating the mixture of materials based on their ionic charge. and

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. Monomer of formula (I) or a salt thereof 【Chemistry 4】 [In the formula, R １ is hydrogen or methyl, X １ is —O— or —NH—, R ２ is (hetero)alkylene, Z is —NH—(C═O)— or —(C═O)—; Ph is phenylene.

10. The monomer of formula (I) 【Transformation 5】 [In the formula, R １ is hydrogen or methyl. The monomer of claim 9 , wherein

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