Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/282—Porous sorbents
  • B01J20/283—Porous sorbents based on silica
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
  • B01D15/361—Ion-exchange
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/0203—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04
  • B01J20/0225—Compounds of Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt
  • B01J20/0229—Compounds of Fe
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/0203—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of metals not provided for in B01J20/04
  • B01J20/0248—Compounds of B, Al, Ga, In, Tl
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
  • B01J20/04—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising compounds of alkali metals, alkaline earth metals or magnesium
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
  • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
  • B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
  • B01J20/264—Synthetic macromolecular compounds derived from different types of monomers, e.g. linear or branched copolymers, block copolymers, graft copolymers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
  • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
  • B65B3/00—Packaging plastic material, semiliquids, liquids or mixed solids and liquids, in individual containers or receptacles, e.g. bags, sacks, boxes, cartons, cans, or jars
  • B65B3/04—Methods of, or means for, filling the material into the containers or receptacles
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
  • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
  • B65B7/00—Closing containers or receptacles after filling
  • B65B7/02—Closing containers or receptacles deformed by, or taking-up shape, of, contents, e.g. bags, sacks
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F222/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a carboxyl radical and containing at least one other carboxyl radical in the molecule; Salts, anhydrides, esters, amides, imides, or nitriles thereof
  • C08F222/36—Amides or imides
  • C08F222/38—Amides
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
  • B01J2220/44—Materials comprising a mixture of organic materials
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
  • B01J2220/46—Materials comprising a mixture of inorganic and organic materials
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
  • B01J2220/52—Sorbents specially adapted for preparative chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
  • B01J2220/58—Use in a single column
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2220/00—Aspects relating to sorbent materials
  • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
  • B01J2220/62—In a cartridge
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/02—Column chromatography
  • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
  • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
  • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
  • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

Definitions

• the present invention relates generally to functionalized particulate support material suitable for use in chromatography columns or cartridges, such as in a liquid chromatography column.
• the present invention further relates to chromatography columns or cartridges containing the functionalized particulate support material, methods of making functionalized particulate support material, and methods of using functionalized particulate support material, for example, as media in a chromatography column or cartridge.
• Cation exchange chromatographic materials typically contain media having surface attached anionic groups such as sulfonic acid groups (e.g., S strong anion exchange) and/or carboxylic methyl groups (e.g., CM weak cation exchange).
• Anion exchange chromatographic materials typically contain media having surface attached cationic groups such as quaternary ammonium (e.g., Q strong anion exchange) and/or diethylaminoethyl (e.g., DEAE weak anion exchange).
• tentacle concept was developed and found to be very useful and widely applied.
• tentacles comprising grafted polymer chains are grafted onto the surface of the media.
• the grafted polymer chains contain repeating units of ionic groups, connected from the end of the polymers to the surface of the media. These polymer chains can rotate freely, allowing interactions between protein molecules and polymeric stationary phase without requirements of the biomolecules to diffuse onto the surface of the media and thus enable high protein loading.
• the present invention is directed to cost-effective media suitable for use as cation and/or anionic exchange chromatographic materials
• the disclosed media referred to herein as "functionalized particulate support material”
• HPLC high performance liquid chromatography
• FPLC fast protein liquid chromatography
• the functionalized particulate support material comprises a particle having a particle surface; and a combination of functional groups extending from the particle surface, the combination of functional groups comprising (i) a first set of functional groups that enable polymerization of one or more monomers onto the particle surface via the first set of functional groups, and (ii) a second set of functional groups that increases the wettability of the particle surface.
• the particle comprises an inorganic metal oxide particle (or particles), such as a silica or silica gel particle (or particles).
• the functionalized particulate support material comprises a particle having a particle surface; and a combination of functional groups extending from the particle surface, the combination of functional groups comprising (i) a first set of functional groups on the particle surface bonded to at least one polymer that extends from the particle surface, and (ii) a second set of functional groups that increases the wettability of the particle surface.
• the functionalized particulate support material comprises a particle having a particle surface and a median pore size of at least 150 A; and a first set of functional groups extending from the particle surface ( said first set of functional groups on the particle surface bonded to at least one polymer that extends from the particle surface.
• the functionaSized support material includes a second set of functional groups that increases the wettability of said particle surface.
• the present invention is further directed to chromatography column or cartridge suitable for use in a chromatography apparatus, wherein the chromatography column or cartridge contains the herein-disclosed functionalized particulate support matenal of the present invention.
• the chromatography column or cartridge comprises a column structure having a column volume: and functionalized particulate support material positioned in the column volume of the column structure, wherein the functionalized particulate support material comprises a plurality of particles, wherein one or more particles within the plurality of particles comprise a particle having a particle surface; and a combination of functional groups extending from the particle surface, the combination of functional groups comprising (i) a first set of functional groups that enable polymerization of one or more monomers onto the particle surface via the first set of functional groups, and (ii) a second set of functional groups that increases the wettability of the particle surface.
• the present invention is even further directed to a chromatography apparatus comprising the herein-disclosed chromatography column or cartridge.
• the chromatography apparatus of the present invention comprises a chromatography column or cartridge containing functionalized particulate support material, wherein the functionalized particulate support materia! comprises a plurality of particles, wherein one or more particles within the plurality of particles comprise a particle having a particle surface; and a combination of functional groups extending from the particle surface, the combination of functional groups comprising (i) a first set of functional groups that enable polymerization of one or more monomers onto the particle surface via the first set of functional groups, and (ii) a second set of functional groups that increases the wettability of the particle surface,
• the present invention is also directed to methods of making functionalized particulate support material.
• the method of making functionalized particulate support material comprises treating a particle surface of a particle so as to result in a combination of functional groups extending from the particle surface, the combination of functional groups comprising (i) a first set of functional groups that enable polymerization of one or more monomers onto the particle surface via the first set of functional groups, and (ii) a second set of functional groups that increases the wettability of the particle surface.
• the treating step may comprise exposing the particle surface to (i) at least one sllane comprising a functional group from the first set of functional groups, and (ii) at least one silane comprising a functional group from the second set of functional groups.
• the present invention is even further directed to methods of making chromatography columns or cartridges containing functionalized particulate support material.
• the method of making a chromatography column or cartridge comprises: (1 ) sealing a first end of a tubular structure; (2) at least partially filling a column cavity of the tubular structure with functionalized particulate support material of the present invention; (3) at least partially filling the column cavity of the tubular structure with a slurry of the functionalized particulate support material; and, (4) sealing an opposite end of the tubular structure.
• the resulting chromatography column or cartridge may be incorporated into a chromatography apparatus and utilized to separate a sample,
• the present invention is also directed to methods of using functionalized particulate support material, columns or cartridges, and apparatus to detect the presence of one or more target molecules (e.g., one or more biomolecules) in a given sample.
• the method of separating a target molecule comprises separating a mixture potentially containing at least one target molecule (e.g., a biomolecule such as a protein or peptide), wherein the method comprises bringing the sample containing at least one target molecule (e.g., a biomolecule such as a protein or peptide) into contact with the herein- disclosed functionalized particulate support material of the present invention.
• the disclosed methods of separating samples may be used to isolate the presence of at least one biomolecule comprising an antibody, a protein, a peptide, a polypeptide, a norvpeptidyi compound, an oligonucleotide, a derivative thereof, an analogue thereof, or any combination thereof.
• FIG. 1 depicts a view of an exemplary chromatography device of the present invention
• FIG. 2 depicts a reaction scheme of an exemplary embodiment of the chromatography media of the present invention.
• FIG. 3 depicts a reaction scheme of an exemplary embodiment of the chromatography media of the present invention.
• FIG. 4 depicts a reaction scheme of an exemplary embodiment of the chromatography media of the present invention.
• FIG, 5 depicts a reaction scheme of an exemplary embodiment of the chromatography media of the present invention.
• the present invention is directed to functionalized particulate support material suitable for use in chromatography columns or cartridges, such as in liquid chromatography (e.g., high performance liquid chromatography (HPLC) or flash).
• the present invention is further directed to chromatography columns or cartridges comprising functionalized particulate support material.
• the present invention is even further directed to methods of making functionalized particulate support material and chromatography columns or cartridges, as well as methods of using functionalized particulate support material and chromatography columns or cartridges to analyze samples, including complex mixtures (e.g., mixtures containing biological components), which potentially contain one or more target molecules.
• the term “about” also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture. Whether modified by the term “about” the claims appended hereto include equivalents to these quantities.
• biomolecule means any molecule that is produced by a living organism, including large molecules such as proteins, polysaccharides, lipids, and nucleic acids; and small molecules such a primary metabolites, secondary metabolites, and natural products.
• biomolecules include ceils and cell debris; proteins and peptides; nucleic acids, such as DNA and RNA; endotoxins; viruses; vaccines and the like.
• Other examples of biomolecules include those recited in WO 2002/074791 and U.S. 5,451 ,880.
• inorganic oxides is defined as binary oxygen compounds where the inorganic component is the cation and the oxide is the anion.
• the inorganic material includes metals may also include metalloids. Metals include those elements on the left of the diagonal line drawn from boron to polonium on the periodic table. Metalloids or semi-metals include those elements that are on the right of this line. Examples of inorganic oxides include silica, alumina, titania, zirconia, etc., and mixtures thereof.
• particles includes particles comprised of inorganic materials, organic materials, or combinations of inorganic materials (e.g., metals, semi-metals, and their alloys; ceramics, including inorganic oxides; etc.) and organic materials (e.g., organic polymers), such as composite materials, which are heterogeneous or homogeneous in nature.
• heterogeneous composite materials include mere mixtures of materials, layered materials, core-shell, and the like.
• homogeneous composite materials include alloys, organic- inorganic polymer hybrid materials, and the like.
• the particles may be a variety of different symmetrical, asymmetrical or irreguiar shapes, including chain, rod or lath shape.
• the particles may have different structures including amorphous or crystalline, etc.
• the particles may include mixtures of particles comprising different compositions, sizes, shapes or physical structures, or that may be the same except for different surface treatments. Porosity of the particles may be intraparticle or inferparticle in cases where smaller particles are agglomerated to form larger particles.
• the particles are composed of inorganic materials such as inorganic oxides, sulfides, hydroxides, carbonates, silicates, phosphates, etc, but are preferably inorganic oxides, which may be formed via any known process including, but not limited to, solution polymerization such as for forming colloidal particles, continuous flame hydrolysis such as for forming fused particles, gelation such as for forming gelled particles, precipitation, spraying, templating, sol-gel, and the like.
• solution polymerization such as for forming colloidal particles
• continuous flame hydrolysis such as for forming fused particles
• gelation such as for forming gelled particles
• precipitation spraying, templating, sol-gel, and the like.
• the term "functionalized” means particles that have been surface modified by reaction with functional compound to alter the selectivity of at least a portion of the particle surface, including the surface area on the external portion of the particles, and/or on the surface area of the internal pores.
• the functionalized surface may be used to form a bonded phase (covalenfly or ionically), a coated surface (e.g., reverse phase C18 bonded), a clad surface (e.g., carbon clad as in EP8), a polymerized surface (e.g., ion exchange), an inherent surface (e.g., inorganic/organic hybrid material), or the like.
• reacting inorganic particles with octadecyltrichlorosilane forms a "reverse phase" by covalently bonding the silane to the inorganic surface (e.g., C4, C8, C18, etc.).
• reaction of the inorganic particles with aminopropyltrimethoxysiiane followed by quaternization of the amino group forms an "anion exchange phase".
• a bonded phase may be formed by reaction of the inorganic particles with aminopropyltrimethoxysiiane followed by formation of an amide with an acid chloride.
• bonded phases include diol, cyano, cation, affinity, chiral, amino, C18, hydrophilic interaction (HILIC), hydrophobic interaction (HIC), mixed mode, size exclusion, etc.
• a ligand may be used to show specific interaction with the target molecule or biomoiecule (e.g., ligate), such as those set forth in U.S. 4,895,808.
• biomoiecule e.g., ligate
• the term "average molecular weight” is defined as meaning the molar mass average of molecular weights of a polymer that possesses a distribution of molecular weights due to different numbers of repeating units in each polymer chain. This value is measured using gel permeation chromatography (GPC) analysis.
• chromatography means the process passing a mixture dissolved in a mobile phase through a stationary phase (i.e., chromatography media) housed in a column or cartridge or other container, which separates a target molecule from other molecules in the mixture and allows it to be isolated.
• a stationary phase i.e., chromatography media
• the target molecule may be adsorbed onto the stationary phase while the undesired components are passed through the device, or vice versa.
• liquid chromatography is a form of chromatography where a liquid is used as the mobile phase and a solid or a liquid on a solid support as the stationary phase.
• flash chromatography means liquid chromatography that is conducted under a positive pressure (e.g., up to 300 psi).
• high performance liquid chromatography HPLC
• HPLC high performance liquid chromatography
• preparatory chromatography means HPLC for the isolation and purification of a target compound or molecule.
• fast protein liquid chromatography FPLC is a form of HPLC useful for the separation of biomolecules.
• impurities means materials present in the inorganic particles, other than the inorganic.
• the term "irregular' as it applies to the inorganic particles means that the particle shape from one particle to the next is not uniform (i.e., random particle shape) with an aspect ratio of greater than 1.0,
• housing means vessel or container for holding a stationary phase for use in chromatography, and includes cartridges, columns, tubes, devices, beds, bags, and the like.
• Stationary phase or "chromatography media” or “chromatography support” means a material that shows different affinities for different components in a sample mixture, which is used in chromatography to separate a target molecule from a mixture of one or more other molecules.
• Stationary phases include organic and inorganic materials, or hybrids thereof, and may be in the form of particles, monoliths, membranes, coatings, and the like that have been functionalized, as defined herein.
• the functionalized particulate support material comprises a particle having a particle surface; and a combination of functional groups extending from the particle surface, the combination of functional groups comprising (i) a first set of functional groups that enable polymerization of one or more monomers onto the particle surface by forming a covalent bond between the first set of functional groups and said one or more monomers and (ii) a second set of functional groups that increases the wettability of the particle surface.
• the particle comprises an inorganic metal oxide particle (or particles), such as a silica or silica gel particle (or particles).
• the functionalized particulate support material comprises a particle having a particle surface; and a combination of functional groups extending from the particle surface, the combination of functional groups comprising (i) a first set of functional groups on the particle surface bonded to at least one polymer that extends from the particle surface, and (ii) a second set of functional groups that increases the wettability of the particle surface.
• the functionalized particulate support material comprises a particle having a particle surface and a median pore size of at least 150 A; and a first set of functional groups extending from the particle surface, said first set of functional groups on the particle surface bonded to at least one polymer that extends from the particle surface.
• the functionalized support material includes a second set of functional groups that increases the wettability of said particle surface.
• the second functional group of the functionalized particulate support material may be bonded to the one or more monomers or to the particle surface
• the first set of functional groups comprises unsaturated bonds, such as vinyl groups, ally! groups, aery! groups, methacryl groups, or any combination thereof.
• the second set of functional groups comprises at least one hydrophilic organic group, such as hydroxy! groups, diol groups, oxyethylene groups, polyethylene groups, carboxylic acid groups, amine groups, amide groups, or any combination thereof.
• the first set of functional groups includes vinyl groups, and the second set of functional groups includes dioi groups.
• the first and second set of functional groups may be part of a molecule including azo groups and carboxylic acid groups.
• at least a portion of said first set of functional groups is polymerized with one or more monomers and one or more spacer monomers so as to form polymer chains extending from the particle surface.
• the spacer monomer separates the one or more monomers from each other, which may assist in functional group orientation on the polymer.
• the one or more monomers may include anionic or cationic monomers.
• the one or more monomers may be an anionic monomer
• the monomer may be 2-acrylamido-2-methylpropane sulfonic acid
• the one or more optional spacer monomers may be methylenebisacrylamide.
• the one or more monomers may be a cationic monomer
• the one or more monomers may include 3-acrylamidopropyltrimethylammonium chloride or methylacrylamidopropyltrimethylammonium chloride
• the one or more optional spacer monomers may include diallyldimethylammonium chloride.
• the polymerization includes a chain transfer agent that reduces the chain Iength or molecular weight of the polymer, the chain transfer agent may include sulfur groups, thiol carbonyl groups, thiol ester groups, thiol carbonate groups and combinations thereof.
• the polymer chains comprise identical or different repeating units of the following formula
• R 1 is H or CH 3 ;
• R' and R ,! are each independently H or CH :3 ⁇ 4 ;
• R 2 and R 3 are each independently
• R 2 and R 3 are coordinated with one another so that either both radicals are acidic or basic, or one of the radicals is neutral and one is acidic or basic;
• R 4 is Ci.-io-alkyl, phenyl, phenyl-C-Mo-alkyl, cydoalkyl or C f ⁇ io-alkyl-cycloalkyl, or C 3 ⁇ 4 - c-cycloalkylphenyl, each monosubstituted or polysubstituted by each of amino, mono- or dialkylamino, trialkyiammonium, carboxyl, or suifony!; and
• n 2 to 1000
• the present invention is further directed to rigid support materials suitable for use in ion exchange columns, such as exemplary rigid support material 1S1 shown in RG. 1.
• the rigid support materials of the present invention preferably an inorganic support, comprise one or more of the following components.
• Inorganic supports suitable for use in the present invention include products commercially available as chromatographic media.
• the inorganic support may be prepared using methods known in the art
• the inorganic substrate provides support for one or more additional components applied to a surface of the inorganic substrate.
• the inorganic substrate is an inorganic oxide, more suitably an inorganic metal oxide, silicate or aluminosilicate or controlled pore glass.
• An inorganic metal oxide is more desirable.
• Inorganic oxides suitable for use in the present invention typically have free hydroxyl groups capable of bonding to or reacting with other chemical functionalities. Desirably, the inorganic oxide has about 1 to about 10 hydroxyl groups per square nanometer of solid inorganic oxide.
• Suitable inorganic oxides include, but are not limited to, silica such as chromatographic grade silica or silica gel, alumina, silica-alumina, zirconia, zirconate, controlled pore glass or titania.
• the inorganic metal oxide is silica, more desirably, chromatographic grade silica or silica gel.
• Magnetically responsive inorganic oxides such as siliceous oxide-coated magnetic particles disclosed in WO 98/31461 (the disclosure of which is incorporated herein in its entirety by reference) may also be used in the present invention.
• Mixed inorganic oxides e.g. co-gels of silica and alumina, or co- precipitates may also be used.
• the solid inorganic oxides may be in a physical form of particulates, fibers plates, or a combination thereof. Desirably, the solid inorganic oxides are in a physical form of particulates or particles having a substantially spherical shape.
• the solid inorganic oxides typically have a longest dimension (i.e., length, width or diameter) of up to about 150 micrometers ( ⁇ ).
• the solid inorganic metal oxide comprises a plurality of particles having a substantially spherical shape
• the plurality of particles desirably have a median particle diameter ranging from about 1 m to about 120 pm.
• the solid inorganic metal oxide comprises a plurality of silica or silica gel particles having a substantially spherical shape, wherein the plurality of silica or silica gel particles have a median particle diameter ranging from about 10 pm to about 130 ⁇ , or from about 20 ⁇ to about 120 ⁇ .
• Suitable solid inorganic oxides include, but are not limited to, silica particles commercially available from W. R. Grace & Co., (Columbia, D) under the trade designation DAVISIL 3 ⁇ 4 , which are irregular shaped with a median pore size of about 300 A to about 3000 A, desirably from about 500 A to about 1500 A, or VYDAC 3 ⁇ 4 silica having a spheriodai shape and a median pore size of about 300 A.
• VYDAC 3 ⁇ 4; silica having a spheriodai shape and an initial median pore size of about 300 A is used after being modified to increase the median pore size to about 800 A.
• Organic materials include agarose gel, polystyrene divinylbenzene (PSDVB) resins, poly (methylmethacrylate) resin or the like.
• Hybrid materials include ethylene-bridged silica hybrid particles made by the sol-gel method, such as Xterra 4 * particles available from Waters Corp.
• the surfaces of the above-described inorganic supports are treated or modified (i.e., functionalized) in order to reduce non-specific, non-selective binding and/or adsorption of non-target molecules (i.e., non-specific binding of mate ials other than the target molecule) and ligand-specific target molecules (i.e., nonspecific binding of the target molecule (i.e.
• the resulting modified support surface has (i) less affinity for non-target molecules (i.e., materials other than the target molecule) due to the presence of relatively inert R groups on the inorganic surface, and (ii) a controlled amount of reactive sites for selectively bonding to one or more ligands (described below) to the inorganic substrate surface directly or through a linker.
• the amount of reactive sites for selectively bonding to one or more ligands leads to selective, controlled binding of one or more molecules of interest to the one or more ligands attached to the inorganic support surface.
• the present invention is further directed to methods of making the above-described functionalized particulate support material.
• the present invention includes a method of making a functionalized particulate support material by treating a particle surface of a particle so as to result in a combination of functional groups extending from the particle surface, the combination of functional groups comprising (i) a first set of functional groups that enable polymerization of one or more monomers onto the particle surface via the first set of functional groups, and (ii) a second set of functional groups that increases the wettability of the particle surface.
• the treating step may include exposing the particie surface to (i) at least one silane having a functional group from the first set of functional groups, and (ii) at least one silane having a functional group from the second set of functional groups.
• the treating step bonds at least one of the first set of functional groups or second set of functional groups to the particle surface followed by the polymerization.
• the second functional group of the functionalized particulate support material may be bonded to the one or more monomers or to the particle surface.
• the first set of functional groups comprises unsaturated bonds, such as vinyl groups, ally! groups, acryl groups, methacryi groups, or any combination thereof.
• the second set of functional groups comprises at least one hydrophilic organic group, such as hydroxyl groups, diol groups, oxyefhylene groups, polyethylene groups, carboxylic acid groups, amine groups, amide groups, or any combination thereof, in a further embodiment, the first set of functional groups includes vinyl groups, and the second set of functional groups includes diol groups. In another embodiment, the first and second set of functional groups may be part of a molecule including azo groups and carboxylic acid groups.
• the particle surface is further treated prior to polymerization with a quaternary ammonium salt (e.g., tetramethy!ammonium chloride) or a quaternary ammonium cation (e.g., letramethylammonium cation), which is subsequently removed from the particle surface by washing.
• a quaternary ammonium salt e.g., tetramethy!ammonium chloride
• a quaternary ammonium cation e.g., letramethylammonium cation
• the one or more monomers may include anionic or cationic monomers.
• the one or more monomers may be an anionic monomer
• the monomer may be 2-acrylamido ⁇ 2 ⁇ methyipropane sulfonic acid
• the one or more optional spacer monomers may be methylenebisacrylamide.
• the one or more monomers may include 3 ⁇ acrylamidopropyltrimethylammonium chloride or methylacrylamidopropyltrimethylammonium chloride
• the one or more optional spacer monomers may include dial!yldimethylammonium chloride.
• the polymerization includes a chain transfer agent that reduces the chain length or molecular weight of the polymer, the chain transfer agent may include sulfur groups, thiol carbonyi groups, thiol ester groups, thiol carbonate groups and combinations thereof.
• the polymer chains comprise identical or different repeating units of the following formula
• R 1 is H or CH 3 ;
• R * and R" are each independently H or CH 3 ;
• R 2 and R 3 are each independently
• Ci-10-alk l phenyl, phenyl-C-Mo-alkyi, cydoaikyl, C t -io-alkyl-cycloalkyl or Ci,io ⁇ alkylphenyl,
• R 2 and R 3 are coordinated with one another so that either both radicals are acidic or basic, or one of the radicals is neutral and one is acidic or basic;
• R 4 is Ci- 0-aIky!, phenyl, phenyI-Ci-io-a!kyL cydoaikyl or Cno-a!kyl-cydoaSkyl, or d- io-cycloalkylphenyl, each monosubstituted or polysubstituted by each of amino, mono- or dialkylamino, trialkylammonium, carboxyi, or sulfonyl; and
• n 2 to 1000.
• the particle surface is further treated after polymerization to remove any unattached polymer or monomer by exposing the particle surface to a salt solution, such as a 5 wt% sodium chloride solution.
• the carbon content on the particle surface after the treating step is less than or equal to about 3.5%. or less than or equal to about 3.0% by weight, or less than or equal to about 2.5% by weight, or less than or equal to about 2.0% by weight, or less than or equal to about 1.5% by weight, based upon the total weight of the particle, after being dried overnight at 70°C.
• the carbon content on the particle surface after the polymerization step is at least about 3.5% by weight, or at least about 4.0% by weight, or at least about 4.5% by weight, or at least about 5.0% by weight, or at least about 5.5% by weight, based upon the total weight of the particle, after being dried overnight at 70°C.
• the ratio of carbon content on the particle surface after the polymerization step compared to the carbon content on the particle surface after the treating step is at least about 1.5, or at least about 2.0, or at least about 2,5, or at least about 3.0, or at least about 3.5, or at least about 4.0.
• the present invention is directed to ion exchange columns, such as exemplary ion exchange column 11 shown in HG. 1 , comprising one or more of the following components.
• ion exchange column includes columns having one or more of the following components, including ion exchange columns such as anion or cation exchange columns.
• the ion exchange columns of the present invention comprise a column structure having desired dimensions, column volume, and structural integrity.
• the column structure comprises a tubular structure having removable end caps on both ends of the tubular structure. End caps form a leak-proof seal with the tubular structure in order to prevent material from undesirably escaping the tubular structure.
• An exemplary ion exchange column 100 of the present invention is shown in FIG. 1 [0049]
• FIG. 1 provides a view of an exemplary chromatography column 100 of the present invention.
• exemplary chromatography column 100 comprises a column housing 150; and media bed space 151 positioned within column housing 150.
• media 151 comprises porous inorganic particles having a median pore size of at least 10 Angstroms (A).
• column housing 150 typically comprises a tubular housing member 156, a first tubular housing member end cap 152, a second tubular housing member end cap 153 opposite end cap 152, a column inlet 154, and a column outlet 155,
• the column 100 may be packed with porous inorganic particles in the form of a slurry through column inlet 154, the column inlet comprising a central bore 157 having a passageway therein, and nozzle 158 ⁇
• a wide range of nozzles may be used which facilitate the distribution and even packing of slurry within the bed space.
• Filters 159 are each positioned on the interior face of the end caps 152, 153 and act with the tubular member 156 to define the bed space 151 and also to prevent leakage of particulate medium from the bed space 151.
• a distribution channel 16 ⁇ is located transversely across the face of the first end cap 152 and/or second end cap 153, and is in fluid communication with filter 159.
• the fluid distribution channel 160 acts to facilitate radial distribution of the liquid.
• the distribution channel 160 comprises at least one circumferential and/or radial groove in the face of the first and/or second end caps 152 and 153. The groove is positioned such that it effects the circumferential and/or radial distribution of liquid emanating from nozzle 158 of inlet 154.
• Co!umn housing 150 may be formed from a variety of materials: Typically, column housing 150 comprises a polymeric material, a metal material, a glass material, a ceramic material, or a composite thereof, and desirably, comprises a polymeric material. Suitable polymeric materials for forming column housing 150 include, but are not limited to any synthetic or semi-synthetic organic solids, such as plastic, that are moldable, including polyolefins.
• Column housing 150 may be formed using conventional thermoforming techniques.
• tubular housing member 156, first tubular housing member end cap 152, and second tubuiar housing member end cap 153 of column housing 150 may each independently be formed via a molding step.
• tubular housing member 156 and one of (i) first tubular housing member end cap 152 and (ii) second tubular housing member end cap 153 of column housing 150 are formed via a single molding step (i.e., one of the end caps is integrally formed on one end of tubular housing member 158).
• Tubular structure 150 may be made from a variety of materials and have a wall construction so as to withstand relatively high performance within tubular structure 1S0. Desirably, tubular structure 150 has a structural integrity that withstands a constant pressure of up to about 6000 psi (400 bar), more desirably, from about 15 psi (1 bar) to about 4500 psi (300 bar). Suitable materials for forming tubular structure 150 include, but not limited to, polymers such as polyetherefherketone (PEEK) and polypropylene; metals such as stainless steel; and inorganic materials such as glass. In one desired embodiment of the present invention, tubular structure 150 comprises poSyefheretherketone (PEEK), or polycaprolactone.
• PEEK poSyefheretherketone
• polycaprolactone polycaprolactone
• Tubuiar structure 150 may have dimensions that vary depending on a number of factors including, but not limited to, particle size and geometry, flow rate, injection volume, number of required plates, etc. Typically, tubuiar structure 150 has a circular cross-sectional area, an outer diameter ranging from about 2 mm to about 5000 mm, an inner diameter ranging from about 1 mm to about 4000 mm, and an overall length ranging from about 2 mm to about 5000 mm.
• End caps 152 and 153 for use with tubular structure 150 are typically formed from PEEK, and have dimensions so as to form a leak-proof seal with ends of tubular structure 150.
• tubular structures having a circular cross-sectional area are desired, tubular structures having other cross-sectional area are also within the scope of the present invention.
• Suitable cross-sectional configurations for a variety of tubular structures include, but are not limited to, square, rectangular, triangular, oblong, pentagonal and hexagonal cross-sectional configurations.
• Chromatography columns and cartridges of the present invention may be prepared using the following steps: (1 ) sealing a first end of a tubular structure;
• the ion exchange column may be stored for future use or may be subsequently connected to an apparatus comprising one or all of the above-described apparatus components.
• the present invention is even further directed to methods of separating samples that potentially contain one or more molecules of interest.
• the method comprises the step of (a) introducing the sample into an ion exchange column containing a rigid support, wherein the rigid support comprises a plurality of inorganic metal oxide particles, wherein each particle comprises (i) a metal oxide substrate; (ii) a modified substrate surface that reduces non-specific binding of non-target molecules materials (i.e., non-specific binding of materials other than the target molecules) and ligand-specific target molecules (i.e., non-specific binding of the target molecules to reactive sites other than reactive sites provided by one or more ligands) to the inorganic substrate; and (iii) one or more ligands bonded to the inorganic substrate.
• the invention provides a method of isolating a target molecule from a mixture comprising: a) contacting the mixture containing the target molecule with an ion exchange chromatography matrix where the matrix comprises 1) a solid support comprising a silica particle having a median pore size greater than 150 A and less than 8000 A and a mean particle size greater than 20 micrometer and 2) ion exchange iigand having specificity for the target molecule linked to the solid support; and b) eluting the target molecule from the ion exchange chromatography matrix.
• the invention provides a method of isolating a target molecule from a mixture comprising: a) contacting the mixture containing the target molecule with an chromatography matrix, wherein the ion exchange chromatography matrix is contained within a housing, such as a column, and wherein the column packed with the ion exchange chromatography matrix has a maximum flow rate of at least 400 cm/hr.
• the matrix comprises 1) a silica particle and 2) an ion exchange ligand having specificity for the target molecuie, wherein the dynamic capacity of the matrix for the target molecule is at least 45 g/liter at 10% breakthrough; and optionally b) eiuting the target molecule from the ion exchange chromatography matrix.
• the invention provides a system for isolating a target molecule from a mixture comprising a) an ion exchange chromatography matrix where the matrix comprises a solid support comprising 1) a silica particle having a pore size greater than 150 A and less than 6000 A and a median particle size greater than 20 micrometers and less than 120 micrometers and 2) an ion exchange ligand having specificity for the target molecule linked to the solid support; and b) a housing for containing the ion exchange chromatography matrix.
• the system may optionally include a means to detect eiution of the target molecule from the ion exchange chromatography matrix.
• the invention provides a system for isolating a target molecule from a mixture comprising a) an ion exchange chromatography matrix wherein the ion exchange chromatography matrix is contained within a housing, such as a column, wherein the column packed with the ion exchange chromatography matrix has a maximum flow rate of at least 400 cm/hr, and wherein the matrix comprises 1) a silica particle and 2) an ion exchange ligand having specificity for the target molecule wherein the dynamic capacity of the matrix for the target molecule is at least 45 g/liter.
• the system may optionally include a means to detect eiution of the target molecule from the ion exchange chromatography matrix, [0062]
• the invention provides a container comprising an ion exchange chromatography matrix comprising a silica particle having a median pore size greater than 150 A and less than 8000 A and a median particle size greater than 20 micrometers and less than 120 micrometers and at least one container.
• the invention provides a device comprising an ion exchange chromatography matrix, wherein the ion exchange chromatography matrix is contained within a housing, such as a column, wherein the column packed with the ion exchange chromatography matrix has a maximum flow rate of at least
• the matrix comprises 1) a silica particle and 2) an ion exchange ligand having specificity for the target molecule wherein the dynamic capacity of the matrix for the target molecule is at least 45 g/liter and at least one container.
• the method of separating a sample may comprise the steps of (a) allowing the sample to come into contact with the rigid support and ligands thereon; (b) rinsing the rigid support to wash away any sample components that do not bond to the ligands; (c) introducing an eluent solution into the ion exchange column so that the eluent solution comes into contact with one or more molecules bound to the ligands on the rigid support; (d) allowing the eluent solution to remain in contact with the rigid support for a period of time so as to form an eluent sample potentially containing one or more molecules; and (e) separating contents of the column to determine the presence of one or more molecules in the sample.
• the method of separating an eluent sample wherein the ion exchange column is in fluid communication with the ion exchange column may further comprise one or more of the following steps:
• the eluent solution preferably remains in contact with the rigid support for a period of time so as to form the eluent sample.
• the eluent solution remains in contact with the rigid support for a period of time ranging from about 1 minute to about 4 hours.
• porous inorganic media based ion exchange material which was prepared by a process which consisted of two main steps: (1) bonding of large pore silica with two si!anes: (3-glycidyloxypropyl) trimethoxysilane and 3-(trimethoxysilyl) propyi me!hacryiate to form an initially bonded intermediate; and (2) solution polymerization of ionic monomer(s), with an azo initiator, in the presence of the initially bonded silica intermediate for either strong anion exchange media (Q-siiica) or strong cation exchange media (S-siiica).
• Q-siiica strong anion exchange media
• S-siiica strong cation exchange media
• Another embodiment of the invention shown in the examples was a process for the preparation Q-silica wherein, the monomers utilized were (3 ⁇ acrylamidopropyS) trirnethylammonium chloride, a small amount of diallyldimethylammonium chloride solution, and the initiator is 2,2'-azobis ⁇ 2- methylpropionamidine) dihydrochloride (V-50 initiator).
• the monomers utilized were (3 ⁇ acrylamidopropyS) trirnethylammonium chloride, a small amount of diallyldimethylammonium chloride solution, and the initiator is 2,2'-azobis ⁇ 2- methylpropionamidine) dihydrochloride (V-50 initiator).
• Another embodiment of the invention shown in the examples is a process for the preparation of S-silica.
• the process included an extra step of washing the initially bonded intermediate with tetramethySammonium chloride solution is added to aid the polymerization.
• the monomer is 2-acryamido-2-methy!-1-propanesuifonic acid (AMPS)
• the initiator is 4,4' ⁇ azobis (cyanovaleric acid) (V-501 initiator).
• AMPS 2-acryamido-2-methy!-1-propanesuifonic acid
• V-501 initiator 4,4' ⁇ azobis (cyanovaleric acid)
• This polymerization uses a chain transfer agent (CTA), e.g., S,S'-Bis ⁇ a,a'-dimethyi-a"-acetic acid)-trithiocarbonate, which is available from ABCR GmbH KG.
• the function of CTA is to control the chain length of the polymerization and help reduce any block
• This process is essentially a reverse addition fragmentation chain transfer (RAFT) polymerization, a living radical polymerization process.
• RAFT reverse addition fragmentation chain transfer
• Many different types of porous particles were functiona!ized by these processes.
• silica gel was utilized, which were silica gels having 75 micron particle size with median pore sizes of 250, 500, 800, 1000 A, The silica gels were prepared using the following procedure: 190g of a 19% sulfuric acid solution was placed in a reactor equipped with an overhead stirrer and chilled to 5 n C.
• the gel When the level fell below 1 % the gel was suspended in water and the pH of the liquid was adjusted to pH-9.7 and the solution heated to 67°C. The temperature was maintained for 20 hours and 20 minutes. At the end of the heating period the gel was recovered by filtration and dried in a 18G"C oven until the moisture content of the gel was less than about 5% by weight.
• the silica gel thus obtained had a nitrogen BET surface area of 325m 2 /g and a nitrogen pore volume of 1.24cc/g : Assuming cylindrical pores and using the equation: Pore Size (Angstroms) - 40000XPV/SA this material exhibits a pore size of 153 Angstroms, Subsequently, the gel is milled to the desired particle size (75 microns) using an ACM and then hydrothermaliy treated in an autoclave at 300" ⁇ until the desired pore size is achieved.
• BET using a Tristar 3000 also available from Micromefrics Instrument Cop. Pore volumes referenced herein represent mercury intrusion into pores 10,000 A and below. BET surface areas are also obtained from the nitrogen sorption analysis.
• Elemental analysis of carbon and sulfur content was conducted using a LECO Carbon and Sulfur Analyzer SG-832 available from LECO Corp. Average molecular weight was determined by GPC analysis using a GPCV 2000 with Rl and Viscometric Detection available from Waters Corp. The purity of the silica was measured by inductively coupled plasma (iCP) using an ICPE-90QG available from Shimadzu Corp.
• iCP inductively coupled plasma
• the supernatants were subjected to gel permeation chromatography (GPC) analysis using a GPCV 2000 with Rl and Viscometric Detection available from Waters Corp. that included Ultrahydrogel guard column and 120, 250, and 1000 columns.
• GPC gel permeation chromatography
• the solutions from above were injected into 1 % aqueous potassium nitrate in mobile phase with a Waters HPLC system equipped with an Rl defector.
• the molecule weights of the solutions were determined by using polyethylene glycol and polyethylene oxide as calibration standards. The molecular weights for the above polymers were below about 200- 300 KD.
• the static binding tests for Q were performed using BSA (25 mg/ml concentration in buffer) at a pH of 8.0 with 50 mM Tris HCi buffer.
• the binding/washing buffer was 50 m Tris-HCI at a pH of 8.0 and the elufion buffer was 50 mM Tris-HCI/1 M NaCI at a pH of 8.0.
• Dried silica samples were weighted into vials, and then protein solutions in binding buffer were added. After overnight adsorption, the samples were centrifuged and supernatant separated/discarded. The silica sample was washed three times with washing buffer with centrifugation and separation.
• the binding/washing buffer was 50 mM HOAc/NaOAc at a pH of 4.0 and the elution buffer was 1 M NaCl in 50 mM HOAc/NaOAc M at a pH of 4.0.
• Dried silica samples were weighted into vials, and then protein solutions in binding buffer were added. After overnight adsorption, the samples were centrifuged and supernatant separated/discarded. The silica sample was washed three times with washing buffer with centrifugation and separation. After the washing steps, elution buffer was added and the elution was repeated a second time.
• the UVA is adsorption was measured for the combined elution solution at 280 urn using a Genesys 10S Bio UV-Vis spectrophotometer available from Thermo Fisher Scientific Inc.
• FIG. 2 demonstrates general synthetic routes for making Q-silica and 8-silica materials.
• Samples of initially bonded porous silica particles were prepared by treating the particles with treating agent 1 (vinyl silane), which is 3- (trimethoxysilyl)propy! methacrylate, and/or treating agent 2 (epoxy siiane), which is (3-g!yc!doxypropy! rimethoxysi!ane.
• treating agent 1 vinyl silane
• epoxy siiane epoxy siiane
• the vinyl and epoxy silanes were premixed, A round bottom flask charged with porous particles, and the amount of treating agent mix was added into the flask. The mixture was allowed to roll overnight. 0.5 sulfuric acid in the amount of 1/10 of silica (by weight) was added. The mixture was rolled at room temperature for 1 hour, and then was heated up to 70°C for 1 hour.
• Example 3 Except for Example 3, e iqual amount of two silanes were used for these functionalizations and the amounts c ⁇ carbon obtained were in general proportional to the total amounts of silanes used. In example 3, only vinyl siiane was used for the dry bonding. As demonstrated in Table 1 the amount of carbon, measured by elemental analysis of the cleaned and dried silica samples after bonding process, was used as an indicator to determine the amount of surface functional groups after surface functionaiization.
• Examples 11-28 describe a process of preparing strong anion exchange materials (Table 2).
• the initially bonded silica from Examples 1-10 were surface treated using a first monomer: (3-Acrylamidopropyl)- trimethylammonum chloride (75% aqueous solution); an alternative monomer 1; [3- ( ethacryloylamino)propyl] trimethylammonium chloride (50% aqueous solution); an alternative monomer 2: [2- ⁇ Acryloyloxy)ethyl]trimethylammonium chloride (80% aqueous solution); a second monomer: Diallyldimethylammonium chloride (85% aqueous solution); V-50 initiator; and additional deionized water (DIW).
• DIW deionized water
• a three-necked round bottom flask was equipped with an overhead mechanical stirrer with gas tight fitting, a nitro gas inlet and outlet, and heating mantle with thermal couple feedback.
• the silica and all the reagents except initiator are first charged into the flask, The system was bubbled with nitrogen for 20 minutes. Then the initiator was introduced. Nitrogen was bubbled for another 20 min before the flask is gradually heated to 85°C. The mixture was kept at 85°C for 2 hours with overhead stirring, and then cooled down to room temperature. The mixture was poured into 5% NaCI solution in a beaker. The flask was rinsed with Dl water to completely move the residual silica inside the flask.
• Reagent ratio is the amount of reagent used in the reaction by weight. All the monomers used in Table 2 are aqueous solutions so the actual amounts are corrected by multiple by concentration.
• 0% ⁇ ,3 ⁇ 4 3 ⁇ bonding is the amount of carbon on the dried silica samples after the initial bonding step, as measured by elemental analysis.
• C%f ms ⁇ is the amount of carbon on the purified, dried silica samples, measured by elemental analysis, Cp o!y - C% fir , ai - 0% ⁇ onding is the amount of carbon contributed from polymeric groups on the surface of the silica.
• C oiy Cinittaibanding R ⁇ tio is the division of the two carbon numbers, which is a measure of carbon contributed by the polymer compared to that contributed by the initial bonding. While not wishing to be bound by theory, it is believed that higher ratio is an indication of longer chain polymer with fewer number of chains on the surface, and this is preferred against lower ratio indicating shorter chain with more chains on the surface for higher protein binding as longer chains give more flexibility for the bonded polymers.
• Bovine serum albumin was used as model protein for all the binding tests of samples. Higher binding values are preferred.
• S stands for Static binding (SBC) where the binding of BSA onto modified silica was measured in a static mode (see the procedure of the measurement below).
• D stands for dynamic binding (DBC) where the binding of BSA onto modified silica was measured in dynamic flow mode (see the procedure of the measurement below). Note that n/m means not measured.
• Example 13 As may be seen from Table 2, except for Example 13, all of the sampies provided acceptable binding results. In Example 13, no polymer attached onto the surface of silica. In Examples 14 and 15, the second monomer, diallyidimethylammonium chloride, provided higher BSA protein binding in general. In Example 18, increasing the ratio of the binding of BSA was improved. In Examples 17, 18 and 20, alternative monomers were tested. Alternative monomer 1 gave slightly higher BSA binding than a sample from the first monomer (Example 19), while alternative monomer 2 gave much lower protein binding than the first monomer. In Example 21 , the sample was made with silica having a pore diameter/size of 800 A, which yielded the highest BSA protein binding. Example 22 gave higher BSA binding than 23 because it had higher carbon number ratio In Example 24, lower protein binding was obtained.
• Examples 25-28 show another process for preparing strong anion exchange materials
• the general process procedure for Initial bonding samples for Examples 25-28 was as follows: 50 g of dried silica were mixed with 0.6 g of vinyl silane and 0.6 g of epoxy silane in a dried 1 L round bottom flask on a Rotavap at ambient temperature for overnight (16 hours), and then the silica was transferred to a 1 L beaker and soaked with 500 ml of 1 M sulfuric acid for 1 hour. Filtration and washing with 5 x 500 Dl water yielded initially bonded silica samples which were dried at 7Q a C overnight.
• Example 28 The polymerization process procedure for Example 28 was as follows: In a 250 ml Beaker the amount of reagents described for Example 28 in Table 3 were mixed. Stir to dissolve everything in water. The solution was poured into a 250 ml Erienmeyer flask containing 30g of initially bonded silica (0.76% Carbon). Nitrogen gas was bubbled into the flask for 30 mins (the flask was occasionally shaken to allow silica and aqueous solution mix well), and then the gas tubing was quickly removed and the top of the flasks were sealed with a tape. The flask was gradually heated to 65°C with a water bath ( ⁇ 3G minutes), and the temperature was kept at 65°C for 2 hours.
• the mixture was cooled down to room temperature.
• the mixture was poured into 400-500 ml 10% NaCI solution in a 1 L beaker with some Dl water rinsing to completely move the residual silica inside the flask.
• the silica was stirred with a spatula for a few minutes, and then particles were left to settle.
• the top liquid phase supernatant was decanted into waste, and the residual silica was mixed with 500 ml 5% NaCI solution.
• the silica sample was then washed with 3 x 500 ml of 5% NaCI solution with additional 3 x 500 ml Dl water, each washing was followed with filtration under vacuum.
• the final sample was left in air to dry except a small amount of sample was dried at 90°C for elemental analysis of carbon contents. .
• Examples 29-38 demonstrate a process for preparing strong cation exchange material S (see FIG. 2).
• a 500 mi three-necked round bottom flask was equipped with an overhead mechanical stirrer with gas tight fitting, a nitro gas inlet and outlet, and heating mantle with thermal couple feedback.
• the silica initially bonded and treated with tetramethylammonium chloride (30 g), and 37.5 g of AMPS, small amount of CTA and 200 ml of Dl water were first charged into the flask.
• the system was bubbled with nitrogen for 20 minutes.
• 0.15 g of V501 initiator was introduced. Nitrogen was bubbled for another 20 min before the flask is gradually heated 65°C.
• the mixture was kept at 65°C for 2 hours with overhead stirring, and then to 8G C C for another 2 hours.
• the flask was allowed to cool down to room temperature.
• the mixture was poured into 800 ml of 5% NaCI solution in a beaker.
• the flask was rinsed with Dl water to completely move the residual silica inside the flask.
• After the mixture was stirred with overhead stirrer for a few minutes, it was filtered and the washing was repeated three times with 500 ml 5% NaCI and three times with 500 ml Dl water
• the sample was left in air to dry except that a small amount of silica was dried at 90 a C overnight and then submitted for elemental analysis of carbon and sulfur content.
• Examples 29-34 chicken egg white lysozyme (Mw of about 17kD) and bovine gamma globulin ⁇ fvlvv of about 140kD) proteins were used for static binding capacity (SBC) studies for the cation exchange materials.
• the test procedure was the same as that for BSA for G-Si!ica described above in Examples 11-24, with the exception that different proteins (still 25 mg/ml concentrations) are used, and the binding and washing buffer was 50 m HOAc/NaOAc at pH 4.0.
• the elution buffer was 1 M NaCI in 50 m HOAc/NaOAc at pH 4.0.
• Examples 35 and 36 demonstrate the size of polymers with regard to the amount of CTA used in the polymerization (without involvement of silica).
• a three-necked round bottom flask was charged with 37.5 g (181 mmo! of AMPS, 1.4 g (18.1 mmol) of metbacrylic acid, 0.2 g (1 g for Example 32) of CTA, and 200 ml of Dl water.
• the polymerization was carried out (without silica) similar to the one described above. After the polymerization and sample was submitted for GCP analysis to determine the molecular weight of the polymers made. It was found that Mw for Example 31 was 87471 and Mw for Example 32 was 20878.
• the mixture was place in a 1 L round bottom flask, rolled on a rotovap at room temperature overnight, and then filtered and washed with 4 x 400 ml of methanol. The solids were allowed to dry in air overnight at room temperature. A small amount of sample was submitted for elemental analysis, and a carbon number of 2.03% was obtained for the sample.
• D1000 75 average particle size with 1000A average pore size
• 200 g was initially bonded with 20 g of aminopropyltrimethoxysilane with a procedure similar to that of Examples 1-10. After overnight rolling, the silica was soaked in 800 ml of 0.1 M HCI, and then filtered. Three times of washing with 1 L of Dl water were carried out with each step followed by filtration under vacuum. The silica filtration cake was dried at 70°C overnight and it was determined the amount of carbon with dried silica was at 0.80 %.
• epoxy porous resin (polymethacrylate polymer resin) particles were used (see Fig. 5). Since the particles (50 pm or 100 pm average particle size) have epoxy groups which will be hydrolyzed to give diol groups in aqueous media, onl vinyl groups will be needed for the modification with polymerization of Q polymers. Thus, 100 g of the particles were treated with 40 ml of ailylamine (available from Aldrich) in 400 ml of NfvlP at room temperature for 1 hour and 80°C for 1 hour.
• ailylamine available from Aldrich
• R R L + k(Ru -RJ, where k is a variable ranging from 1 % to 100% with a 1% increment, e.g., k is 1%, 2%, 3%, 4%, 5%. ... 50%, 51 %, 52%. ... 95%, 96%, 97%, 98%, 99%, or 100%.
• any numerical range represented by any two values of R, as calculated above is also specifically disclosed.

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