WO2023102505A1 - Process for preparation of functionalized fiber - Google Patents
Process for preparation of functionalized fiber Download PDFInfo
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- WO2023102505A1 WO2023102505A1 PCT/US2022/080786 US2022080786W WO2023102505A1 WO 2023102505 A1 WO2023102505 A1 WO 2023102505A1 US 2022080786 W US2022080786 W US 2022080786W WO 2023102505 A1 WO2023102505 A1 WO 2023102505A1
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- nanoweb
- protein
- ether
- glycidyl
- polymer
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
- B01J20/28007—Sorbent size or size distribution, e.g. particle size with size in the range 1-100 nanometers, e.g. nanosized particles, nanofibers, nanotubes, nanowires or the like
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- B01J20/28023—Fibres or filaments
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- B01J20/28033—Membrane, sheet, cloth, pad, lamellar or mat
- B01J20/28038—Membranes or mats made from fibers or filaments
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- B01J20/28078—Pore diameter
- B01J20/28085—Pore diameter being more than 50 nm, i.e. macropores
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- B01J20/28088—Pore-size distribution
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- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
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- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3206—Organic carriers, supports or substrates
- B01J20/3208—Polymeric carriers, supports or substrates
- B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
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- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
- B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
- B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
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- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3289—Coatings involving more than one layer of same or different nature
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D1/00—Processes for applying liquids or other fluent materials
- B05D1/62—Plasma-deposition of organic layers
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- B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D3/00—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials
- B05D3/14—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials by electrical means
- B05D3/141—Plasma treatment
- B05D3/142—Pretreatment
- B05D3/144—Pretreatment of polymeric substrates
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- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M10/00—Physical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. ultrasonic, corona discharge, irradiation, electric currents, or magnetic fields; Physical treatment combined with treatment with chemical compounds or elements
- D06M10/02—Physical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. ultrasonic, corona discharge, irradiation, electric currents, or magnetic fields; Physical treatment combined with treatment with chemical compounds or elements ultrasonic or sonic; Corona discharge
- D06M10/025—Corona discharge or low temperature plasma
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- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M10/00—Physical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. ultrasonic, corona discharge, irradiation, electric currents, or magnetic fields; Physical treatment combined with treatment with chemical compounds or elements
- D06M10/04—Physical treatment combined with treatment with chemical compounds or elements
- D06M10/08—Organic compounds
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- D06M14/00—Graft polymerisation of monomers containing carbon-to-carbon unsaturated bonds on to fibres, threads, yarns, fabrics, or fibrous goods made from such materials
- D06M14/18—Graft polymerisation of monomers containing carbon-to-carbon unsaturated bonds on to fibres, threads, yarns, fabrics, or fibrous goods made from such materials using wave energy or particle radiation
- D06M14/26—Graft polymerisation of monomers containing carbon-to-carbon unsaturated bonds on to fibres, threads, yarns, fabrics, or fibrous goods made from such materials using wave energy or particle radiation on to materials of synthetic origin
- D06M14/28—Graft polymerisation of monomers containing carbon-to-carbon unsaturated bonds on to fibres, threads, yarns, fabrics, or fibrous goods made from such materials using wave energy or particle radiation on to materials of synthetic origin of macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
- D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
- D06M15/15—Proteins or derivatives thereof
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D3/00—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials
- B05D3/04—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials by exposure to gases
- B05D3/0486—Operating the coating or treatment in a controlled atmosphere
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- D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
- D06M2101/16—Synthetic fibres, other than mineral fibres
- D06M2101/18—Synthetic fibres consisting of macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
- D06M2101/22—Polymers or copolymers of halogenated mono-olefins
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- D06M2400/00—Specific information on the treatment or the process itself not provided in D06M23/00-D06M23/18
- D06M2400/01—Creating covalent bondings between the treating agent and the fibre
Definitions
- This invention relates generally to a method for producing a functionalized nanoweb useful in chromatography.
- an affinity capture and elute purification step is frequently employed. This step most typically involves the binding of the desired therapeutic by a protein ligand conjugated to a chromatography support. Some support materials require surface functionalization prior to attachment of the protein ligand to the functionalized support surface. Fibers have been used as support materials, e.g., in Z. Ma et al., J. Chromatogr. B, 877 (2009) 3686-3694, fibers are exposed to an air plasma in the presence of methacrylic acid to produce a functionalized fiber. In a separate step, in a liquid-phase reaction, a protein is bound to the functionalized fiber via amino groups on the protein.
- the present invention is directed to a method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb; said method comprising steps of: a) exposing said nanoweb and a polyepoxide material to an atmospheric plasma to produce a coated nanoweb; and b) exposing the coated nanoweb to a molecule capable of forming covalent bonds, preferably a protein.
- the present invention is further directed to a functionalized nanoweb comprising a polymer and a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume %; wherein the polymer is an ether or ester of an epoxy alcohol.
- (meth)acrylate acrylate or methacrylate, acrylic or methacrylic, and acrylamide or methacrylamide, respectively (collectively “acrylic monomers”).
- An acrylic polymer is a polymer comprising at least 50 wt% polymerized units of (meth)acrylic acid, alkyl, glycidyl or hydroxyalkyl (meth)acrylates, alkyl, glycidyl and/or hydroxyalkyl (meth)acrylamides, or a combination thereof.
- a carbohydrate polymer is a polymer comprising polymerized units of sugar molecules, i.e., a polysaccharide.
- a web of randomly distributed fibers is commonly referred to as a “nonwoven.”
- the fibers can be bonded to each other or unbonded, preferably unbonded.
- a “nanoweb” is a nonwoven web comprising at least one nanofiber.
- a nanoweb may also be referred to as a “nanofiber mat.”
- the nanofibers in the nanoweb are “continuous,” i.e., having been laid down in one continuous stream to form the web.
- Fiber diameters may be determined by SEM picture examination.
- the fiber has a diameter from 0.1 to 1 pm.
- at least 95% of the fiber has a diameter in the stated range, based on length of the fiber.
- fiber diameter is the arithmetic average of at least 50 measurements, preferably at least 100 measurements.
- the nanoweb has a mean flow pore size of at least 0.15 pm, preferably at least 0.2 pm, preferably at least 0.25 pm; preferably no more than 2 pm, preferably no more than 1 pm, preferably no more than 0.5 pm.
- Mean flow pore size is a calculated quantity from material porometry measurements, where the dry sample is subjected to airflow at various flow rates, then wetted with a fluid of known surface tension and air flows are returned at steadily increasing flow rate until the last wetted pore of the material is evacuated with air.
- a mean flow pore is determined from a /i slope of the dry air flow curve intersecting the wet flow curve. Relationships between fiber diameter and mean flow pore size have been determined.
- the nanoweb has a porosity from 40 to 90 volume %, preferably at least 50 volume%, preferably at least 60 volume %, preferably at least 65 volume%; preferably no more than 85 volume%, preferably no more than 80 volume%, preferably no more than 75 volume%.
- the fiber comprises a synthetic polymer; preferably poly(vinylidene fluoride) (PVDF), copolymers of PVDF such as poly(vinylidene fluoride-co-trifluoroethylene), polyamide, polyethersulfone (PES), polyethylene, polypropylene, polyester, polyimide or a combination thereof; preferably the fiber comprises PVDF, polyethersulfone, nylon or a combination thereof.
- PVDF polymer has a number average molecular weight is from 100,000 to 2,000,000 daltons, preferably from 200,000 to 500,000.
- a polyamide has a number average molecular weight from 5,000 to 40,000, preferably from 10,000 to 20,000.
- a polyethersulfone has a number average molecular weight from 20,000 to 80,000, preferably from 40,000 to 60,000.
- the fiber comprises PVDF; preferably the fiber comprises at least 50 wt% PVDF, preferably at least 80 wt%, preferably at least 90 wt%, preferably at least 95 wt%.
- the polyepoxide material comprises at least two epoxy groups; preferably at least three.
- the polyepoxide material is an ether or ester of an epoxy alcohol, preferably an ester of glycidyl alcohol.
- the epoxide material is a polymeric poly epoxide which is an ester of a polymer comprising carboxylic acid groups, preferably poly((meth)acrylic acid) or an ether of a polymer comprising hydroxyl groups.
- the polymer is an acrylic polymer (preferably one comprising at least 75 wt% acrylic polymers, preferably at least 90 wt%) or an ether of a non-acrylic vinyl polymer (e.g., poly(allyl glycidyl ether, poly(vinylglycidyl ether).
- polymeric polyepoxide materials include, but are not limited to: poly(glycidyl (meth)acrylate), poly(allyl glycidyl ether), poly(vinyl glycidyl ether), poly(glycidyloxyalkyl (meth)acrylate) and poly(glycidyl (meth)acrylate)-co-poly(hydroxy ethyl (meth)acrylate) (preferably having from 24 to 75% poly(glycidyl (meth)acrylate); preferably poly(glycidyl methacrylate) (polyGMA).
- the polymeric polyepoxide has a number- average molecular weight from 5,000 to 50,000, preferably 5,000 to 40,000, preferably 8,000 to 30,000, preferably 8,000 to 25,000.
- at least 50% of the polymerized monomer units have an epoxy group, preferably at least 75%, preferably at least 85%.
- the polyepoxide material is an aliphatic ether or ester compound having from two to four glycidyl groups, preferably two to four.
- the glycidyl groups are attached via ether or ester linkages.
- the aliphatic ether or ester compound has a molecular weight no greater than 1000 Daltons, preferably no greater than 700 Daltons, preferably no greater than 600 Daltons, preferably no greater than 500 Daltons.
- Preferred examples of aliphatic ethers having from one to four glycidyl groups include, but are not limited to: pentaerythritol tetraglycidyl ether, trimethylolpropane triglycidyl ether (TMPTE), glycerol triglycidyl ether and 1,4-butanediol di glycidyl ether.
- TMPTE trimethylolpropane triglycidyl ether
- glycerol triglycidyl ether 1,4-butanediol di glycidyl ether.
- Preferred examples of aliphatic esters having from two to four glycidyl groups include, but are not limited to: l,2,4-tris(2- oxiranylmethyl) 1,2,4-butanetricarboxylate (CAS# 93164-66-0), 3-butene-l,2,3-tricarboxylic acid, l,2,3-tris(2-oxiranylmethyl)ester (CAS# 2639793-39-6), 2,2’-[2-ethyl-2-[[(2- oxiranylcarbonyl)oxy]methyl]-l,3-propanediyl] bis(2-oxiranecarboxylate) (CAS# 98306-86-6), l,4-bis(2-oxiranylmethyl)-2-(2-oxiranylmethoxy)butanedioate (CAS# 100620-09-5), 1 ,2,3-tris(2- oxiranylmethyl)-2-hydroxy-l,2,3-propanetricarboxylate (CAS
- the molecule capable of forming covalent bonds can include, but are not limited to, molecules that include nucleophilic groups. These nucleophilic groups can include, but are not limited to, hydroxyl, amine or thiol groups. These groups can be found in amino acids, peptides, proteins, nucleic acids, polynucleic acids, viruses and cells among others.
- the molecule capable of forming a covalent bond is a protein.
- the coated nanoweb is contacted with the protein in an aqueous medium.
- the protein is capable of binding other proteins to an active site and comprises a free thiol group.
- the aqueous medium comprising the protein also comprises inorganic salts in a concentration from 1 to 2.25M, preferably from 1.25 to 2.25M.
- Preferred salts are those recognized in the literature as having antichaotropic activity. Examples of antichaotropic salts include sodium sulfate, ammonium sulfate, and magnesium or zinc salts, including chloride, phosphate and carbonate.
- the present invention is further directed to a functionalized nanoweb comprising a polymer and a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume %; wherein the polymer is an ether or ester of an epoxy alcohol; preferably an ether or ester of glycidyl alcohol; preferably an ester of a polymer comprising carboxylic acid groups, preferably poly((meth)acrylic acid).
- Preferences for mean flow pore size and porosity are the same as those stated previously in the description of the method.
- the fiber comprises PVDF, preferably the fiber comprises at least 50 wt% PVDF, preferably at least 80 wt%, preferably at least 90 wt%, preferably at least 95 wt%.
- the polymer forms a continuous coating covering at least 25% of the fiber surface, preferably at least 50%, preferably at least 75%.
- the protein comprises a free cysteine thiol group, i.e., a thiol group (-SH) which is not part of a disulfide linkage, and said protein is active with respect to binding other proteins to an active site.
- a free cysteine thiol group i.e., a thiol group (-SH) which is not part of a disulfide linkage
- the protein comprising a free cysteine thiol group is obtained by cleaving a disulfide bond in the protein which is not required for maintaining the integrity of the protein binding site, i.e., the protein’s activity for binding other proteins.
- the disulfide is reduced using Tris(2-carboxyethylphosphine) hydrochloride (TCEP.HC1) which can be used at a pH between 1.5 and 9.0 depending upon the stability of the protein being reduced at that pH.
- TCEP.HC1 Tris(2-carboxyethylphosphine) hydrochloride
- a typical pH for the reduction is pH 3 to pH 8, using nonphosphate buffers e.g. TRIS, HEPES, Borate.
- Additional reducing agents include dithiothreitol (DTT), mercaptoethanol (ME), 2-mercaptoethylamine hydrochloride (2-MEA.HC1) all of which are typically utilized around neutral pH. These three reagents all include free thiol groups and can be removed from the protein by filtration prior to reacting the protein with the nanofibers to reduce competitive reactions during the conjugation of the protein. Cysteine- Cysteine disulfide bonds are frequently found in proteins as a method of maintaining the protein structure such that cleavage of these disulfide bonds can result in denaturation of the protein. Other structural features such as P -sheets, helix bundles and hairpin structures can maintain a protein’s conformation without the use of disulfide linkages.
- proteins useful for affinity binding of other proteins or useful biotherapeutic molecules are produced recombinantly or isolated from natural sources.
- the protein is one which has been produced recombinantly so as to include a disulfide linkage that is not involved in the configurational stability of the resulting protein; preferred proteins of this type include Protein A, Protein A/G and Protein G.
- Other preferred proteins include antibodies, monoclonal and polyclonal, the recognition fragments F(ab’) and F(ab’)2 which can be produced recombinantly or following an enzymatic treatment to cleave the fragments from the intact antibody (Rosenstein et.al., Curr Protoc Mol Biol.
- disulfide bonds which are reduced in the F(ab’)2 are distal to the binding site of the protein and can be cleaved to form the individual fragments without loss of the ability to bind to other proteins, most likely because disulfide linkages that help to maintain the protein configuration occur within a P- secondary structure such as a P-sheet which maintains the protein configuration and protects these disulfides from reduction.
- the protein is in an aqueous solution at a concentration from 5 to 50 mM; preferably the pH of the solution is from 8 to 9.5, preferably from 8.5 to 9.3, preferably from 8.7 to 9.1 and the molecular weight of the protein is no greater than 150,000 Daltons, preferably no greater than 100,000 Daltons and preferably no greater than 75,000 Daltons.
- a dielectric barrier discharge atmospheric pressure plasma process is used to attach the multifunctional monomer injected as liquid aerosol to the nanofiber substrate.
- the dielectric barrier discharge plasma process preferably is a homogenous glow discharge process.
- Homogeneous glow discharge plasma processes are known in the art to produce spatially uniform low temperature electrons from injected gases at atmospheric pressures. Ions collide with the injected monomers producing ionized species that may self-polymerize in aerosol prior to substrate deposition.
- Preferred gases suitable for plasma generation include carbon dioxide, nitrogen, argon, and/or helium. The flow of gases in plasma form and the injected monomer aerosol passes through a nozzle with a defined cross-sectional area.
- the nozzle is rectangular in geometry with an interelectrode gap and a width larger than the width of the nonwoven.
- the inter-electrode gap is preferably 0.5-10 mm, preferably, 0.8-2mm.
- the cross-sectional area of the nozzle used in the examples below is 5 cm 2 with a nozzle width of 40cm.
- gas flow rates range from 2-150 slm/cm 2 , where slm is in units of standard liters per minute with standard conditions of a gas volume are at temperature of 0°C and pressure of 1 atm (lOlkPa), preferably from 60 to 100 slm/cm 2 .
- Gas flow rates through the plasma head will depend on plasma head size and substrate width to be coated with monomer as well as on environmental temperature and pressure of the gas. Injected monomer flow rates depend on the amount of monomer to be attached per area of substrate. High injection rates of monomer may lead to self-polymerization prior to deposition on the substrate. Preferably, injection rates of aerosolized monomer range from 0.2 slm/cm 2 to 5 slm/cm 2 . Dilutions of monomers may be achieved by one or both means of liquid solution mixing, by dissolution or suspension of a solid monomer in water or an appropriate solvent, and by introduction of greater gas flow into the liquid monomer pure compound, suspension, or solvent mixture.
- Preferred dilution ratios range from of 2: 1 to 50: 1, preferably from 3: 1 to 20: 1.
- alternating current powered covered electrodes generate the plasma through a narrow gap at atmospheric pressures from 0.9-1.1 atm (91-111 kPa).
- plasma source voltage ranges from 1-100 kV with preferred range of 5-30 kV.
- supplied power for initial activation and reaction with monomer is in the range of 2 to 300W/cm 2 , preferably 20 to 200 W/cm 2 , preferably 30 to 160 W/cm 2 .
- supplied power in the presence of protein is in the range of 1 to 100 W/cm 2 , preferably 2 to 40 W/cm 2 , preferably 3 to 20 W/cm 2 .
- the distance between the substrate and the plasma head is 1-10 mm, preferably 2-5 mm.
- the substrate is pretreated using carbon dioxide, nitrogen, argon, and/or helium in an atmospheric plasma process without monomer.
- Nonwoven webs from different non carbohydrate based polymers were prepared by either an electrospinning process similarly to the one described in https://pubs.acs.org/doi/10.1021/acs.chemrev.8b00593, or an electroblowing process as described in U.S. Patent number 7,618,579.
- the nonwoven webs were then optionally consolidated as described in U.S. Patent number US8697587B2 (Sample 3).
- the spinning and consolidation processes were tuned such that porosity of the resulting webs could range between 35% and 95%, and the mean flow pore size between 0.1 pm and 5 pm.
- the various nonwoven substrates were pretreated through a plasma, and then processed through the plasma coating process (see, e.g., US 8,663,751, US 9,890,260 and US 9,938,388) using the functionalizing agent (“agent”) and process conditions resulting in various amounts of agent being injected into the plasma and applied to the substrates.
- agent functionalizing agent
- Each nanofiber nonwoven web is subjected to an activation pass through the plasma treatment device, in which the top and bottom surfaces of the web are exposed to a plasma of CO2 and N2 at a flow rate of 80 slm/cm 2 and 8 slm/cm 2 power level of 300 W/m 2 .
- the activated nanofiber web passes through a N2 plasma at flow rate of 80 slm/cm 2 .
- the distance between the electrode and substrate is about 2 mm.
- a measure of amount of agent injected per mass of substrate is determined by gravimetric means of weighing the supply solution at the start and finish of the plasma process taking the difference between start and finish and dividing the difference by the mass of substrate processed. Table II
- the epoxy functionalized nanofiber mats are conjugated with a reactive cysteine on recombinant Protein A.
- This reaction involves first reducing disulfide bonds in the Protein A dimer using TCEP-HC1 (Tris(2-carboxyethyl)phosphine hydrochloride) [See: Thermo Fisher Application Notes https://www.thermofisher.com/us/en/home/references/molecular-probes-the- handbook/thiol-reactive-probes/introduction-to-thiol-modification-and-detection.html] in aqueous solution at 5 - 50 mM concentration and pH 6-8.
- the Protein A with a free reactive thiol is then allowed to react with the epoxy-activated nanofiber mat in the presence of a salting out buffer e.g. sodium sulfate to increase the coating of the Protein A on the surface of the fibers and as a result increasing the local concentration of Protein A at the surface of the fiber which increases the amount of Protein A conjugated onto the nanofiber mat [See Thermo Fisher Application Notes: https://www.thermofisher.com]
- the saturated binding capacity, SBC can be measured by placing a 2.5 cm diameter disc of the membrane into a Swinnex filter holder and using an AKTA PureTM, elute a 10 mL solution of human IgG (0.2 mg/mL) in PBS buffer, pH 7.4 at 0.5 ml/min through the membrane.
- the filter is then washed with about ten bed volumes of the PBS buffer and then the human IgG is eluted from the membrane with 5 bed volumes of buffer (e.g. 0.1 M glycine at pH 3), quickly neutralized to about pH 7 to avoid denaturation and the concentration measured spectrophotometrically, using the formula below or by integration of the elution curve and comparing with a calibration curve (e.g. Hahn et. al. Journal of Chromatography A. 2005, 1093, 98):
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Abstract
A method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 µm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb is described. The method comprises steps of: a) exposing the nanoweb and an epoxide material to an atmospheric plasma to produce a coated nanoweb; and b) exposing the coated nanoweb to a molecule capable of forming covalent bonds.
Description
PROCESS FOR PREPARATION OF FUNCTIONALIZED FIBER
Background
This invention relates generally to a method for producing a functionalized nanoweb useful in chromatography.
During the production of biopharmaceuticals, in particular antibody derived therapeutics, e.g., monoclonal antibodies, an affinity capture and elute purification step is frequently employed. This step most typically involves the binding of the desired therapeutic by a protein ligand conjugated to a chromatography support. Some support materials require surface functionalization prior to attachment of the protein ligand to the functionalized support surface. Fibers have been used as support materials, e.g., in Z. Ma et al., J. Chromatogr. B, 877 (2009) 3686-3694, fibers are exposed to an air plasma in the presence of methacrylic acid to produce a functionalized fiber. In a separate step, in a liquid-phase reaction, a protein is bound to the functionalized fiber via amino groups on the protein.
Statement of Invention
The present invention is directed to a method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb; said method comprising steps of: a) exposing said nanoweb and a polyepoxide material to an atmospheric plasma to produce a coated nanoweb; and b) exposing the coated nanoweb to a molecule capable of forming covalent bonds, preferably a protein.
The present invention is further directed to a functionalized nanoweb comprising a polymer and a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume %; wherein the polymer is an ether or ester of an epoxy alcohol.
Detailed Description
All percentages are weight percentages (wt%), and all temperatures are in °C, unless otherwise indicated. Averages are arithmetic averages unless indicated otherwise. All operations are performed at room temperature (from 18 to 25 °C) unless specified otherwise. The terms “(meth)acrylate” , “(meth)acrylic”, and “(meth)acrylamide” mean acrylate or methacrylate, acrylic or methacrylic, and acrylamide or methacrylamide, respectively (collectively “acrylic monomers”). An acrylic polymer is a polymer comprising at least 50 wt% polymerized units of (meth)acrylic acid, alkyl, glycidyl or hydroxyalkyl (meth)acrylates, alkyl, glycidyl and/or hydroxyalkyl (meth)acrylamides, or a combination thereof. A carbohydrate polymer is a polymer comprising polymerized units of sugar molecules, i.e., a polysaccharide.
A web of randomly distributed fibers is commonly referred to as a “nonwoven.” The fibers can be bonded to each other or unbonded, preferably unbonded. A “nanoweb” is a nonwoven web comprising at least one nanofiber. A nanoweb may also be referred to as a “nanofiber mat.” Preferably, the nanofibers in the nanoweb are “continuous,” i.e., having been laid down in one continuous stream to form the web. Fiber diameters may be determined by SEM picture examination. Preferably the fiber has a diameter from 0.1 to 1 pm. Preferably, at least 95% of the fiber has a diameter in the stated range, based on length of the fiber. Preferably, if less than 95% of the fiber has a diameter in the stated range, then fiber diameter is the arithmetic average of at least 50 measurements, preferably at least 100 measurements.
Preferably, the nanoweb has a mean flow pore size of at least 0.15 pm, preferably at least 0.2 pm, preferably at least 0.25 pm; preferably no more than 2 pm, preferably no more than 1 pm, preferably no more than 0.5 pm. Mean flow pore size is a calculated quantity from material porometry measurements, where the dry sample is subjected to airflow at various flow rates, then wetted with a fluid of known surface tension and air flows are returned at steadily increasing flow rate until the last wetted pore of the material is evacuated with air. A mean flow pore is determined from a /i slope of the dry air flow curve intersecting the wet flow curve. Relationships between fiber diameter and mean flow pore size have been determined. Simmonds et al proposed the following relationship to determine mean flow pore size, Dmean,pore from the mean fiber diameter of the nonwoven, Daber (see Simmonds GE, Bomberger JD, Bryner MA. Designing nonwovens to meet pore size specifications. J Eng Fibers Fabrics. 2007;2(l), 1 - 15.) Dmean,pore — 0.39267 *Dfiber/(l -porosity)
Preferably, the nanoweb has a porosity from 40 to 90 volume %, preferably at least 50 volume%, preferably at least 60 volume %, preferably at least 65 volume%; preferably no more than 85 volume%, preferably no more than 80 volume%, preferably no more than 75 volume%. It is believed that fluid flow through the nanofiber mat is facilitated by high porosity, and binding capacity of a substrate is improved when the pore size is small. Preferably when pore size is 0.1 to 1 micron, porosity is 50 to 90 volume%; preferably when pore size is 0.1 to 0.5 micron, porosity is from 65 to 85 volume%. Porosity is calculated from the following equation: Porosity = 1- (mass of fiber in g/cm2/(Thickness of nanofiber mat in cm*Polymer Density in g/cm3))
Preferably, the fiber comprises a synthetic polymer; preferably poly(vinylidene fluoride) (PVDF), copolymers of PVDF such as poly(vinylidene fluoride-co-trifluoroethylene), polyamide, polyethersulfone (PES), polyethylene, polypropylene, polyester, polyimide or a combination thereof; preferably the fiber comprises PVDF, polyethersulfone, nylon or a combination thereof. Preferably, a PVDF polymer has a number average molecular weight is from 100,000 to 2,000,000 daltons, preferably from 200,000 to 500,000. Preferably, a polyamide has a number average molecular weight from 5,000 to 40,000, preferably from 10,000 to 20,000. Preferably, a polyethersulfone has a number average molecular weight from 20,000 to 80,000, preferably from 40,000 to 60,000. In a preferred embodiment of the invention the fiber comprises PVDF; preferably the fiber comprises at least 50 wt% PVDF, preferably at least 80 wt%, preferably at least 90 wt%, preferably at least 95 wt%.
The polyepoxide material comprises at least two epoxy groups; preferably at least three. Preferably, the polyepoxide material is an ether or ester of an epoxy alcohol, preferably an ester of glycidyl alcohol. Preferably, the epoxide material is a polymeric poly epoxide which is an ester of a polymer comprising carboxylic acid groups, preferably poly((meth)acrylic acid) or an ether of a polymer comprising hydroxyl groups. Preferably, the polymer is an acrylic polymer (preferably one comprising at least 75 wt% acrylic polymers, preferably at least 90 wt%) or an ether of a non-acrylic vinyl polymer (e.g., poly(allyl glycidyl ether, poly(vinylglycidyl ether). Preferred examples of polymeric polyepoxide materials include, but are not limited to: poly(glycidyl (meth)acrylate), poly(allyl glycidyl ether), poly(vinyl glycidyl ether), poly(glycidyloxyalkyl (meth)acrylate) and poly(glycidyl (meth)acrylate)-co-poly(hydroxy ethyl (meth)acrylate) (preferably having from 24 to 75% poly(glycidyl (meth)acrylate); preferably poly(glycidyl methacrylate) (polyGMA). Preferably, the polymeric polyepoxide has a number-
average molecular weight from 5,000 to 50,000, preferably 5,000 to 40,000, preferably 8,000 to 30,000, preferably 8,000 to 25,000. Preferably, in the polymeric polyepoxide at least 50% of the polymerized monomer units have an epoxy group, preferably at least 75%, preferably at least 85%.
In a preferred embodiment, the polyepoxide material is an aliphatic ether or ester compound having from two to four glycidyl groups, preferably two to four. Preferably, the glycidyl groups are attached via ether or ester linkages. Preferably, the aliphatic ether or ester compound has a molecular weight no greater than 1000 Daltons, preferably no greater than 700 Daltons, preferably no greater than 600 Daltons, preferably no greater than 500 Daltons. Preferred examples of aliphatic ethers having from one to four glycidyl groups include, but are not limited to: pentaerythritol tetraglycidyl ether, trimethylolpropane triglycidyl ether (TMPTE), glycerol triglycidyl ether and 1,4-butanediol di glycidyl ether. Preferred examples of aliphatic esters having from two to four glycidyl groups include, but are not limited to: l,2,4-tris(2- oxiranylmethyl) 1,2,4-butanetricarboxylate (CAS# 93164-66-0), 3-butene-l,2,3-tricarboxylic acid, l,2,3-tris(2-oxiranylmethyl)ester (CAS# 2639793-39-6), 2,2’-[2-ethyl-2-[[(2- oxiranylcarbonyl)oxy]methyl]-l,3-propanediyl] bis(2-oxiranecarboxylate) (CAS# 98306-86-6), l,4-bis(2-oxiranylmethyl)-2-(2-oxiranylmethoxy)butanedioate (CAS# 100620-09-5), 1 ,2,3-tris(2- oxiranylmethyl)-2-hydroxy-l,2,3-propanetricarboxylate (CAS# 1100856-07-2), 2, 2’, 2” -(1,2,3 - propanetriyl)tris(2-oxiranepropanoate) (CAS# 502516-01-0), 2-oxiranemethanol, a-[(ethoxy-2- oxiranylmethoxy)-2-oxiranylmethoxy]-, 2-acetate (CAS# 96116-54-0), 2,4-bis(2- oxiranylmethyl)-l-(2-propen-l-yl)- 1,2,4-butanetricarboxylate (CAS# 94431-00-2), hexanedioic acid, 2,2,4(or 2,4,4)-trimethyl-, bis(oxiranylmethyl) ester (CAS# 53445-36-6), hexanedioic acid, 2,3,4(or 2,3,5)-trimethyl-, bis(oxiranylmethyl) ester (CAS# 27043-02-3), 1,3-propanediol, 2,2- bis[(2-oxiranylmethoxy)methyl]-, 1,3-diacetate (CAS# 172950-03-7), hexanedioic acid, 2,5- dimethyl-, l,6-bis(2-oxiranylmethyl) ester (CAS# 25677-86-5), l,5-bis(2-oxiranylmethyl)-2,4- dimethylpentanedioate (CAS# 7446-86-8), 2-oxiranemethanol, a-[2-oxiranyl(2- oxiranylmethoxy)methoxy]-, 2-acetate (CAS# 94972-23-3), glycidic acid, tetraester with pentaerythritol (CAS# 98346-14-6), oxiraneacetic acid, 2,2-bis[[(oxiranylacetyl)oxy]methyl-l,3- propanediyl ester (CAS# 251921-39-8), 1,2,3,4-butanetetracarboxylic acid, l,2,3,4-tetrakis[2-(2- oxiranyl)ethyl] ester (CAS# 1376872-02-4), l,2,3-tris(2-oxiranylmethyl) 2-(2-oxiranylmethoxy)- 1,2,3-propanetricarboxylate (CAS# 111416-32-1), l,4-bis(2-oxiranylmethyl) (2Z)-2-butenedioate (CAS# 21767-55-5), l,4-bis(2-oxiranylmethyl) (2E)-2 -butenedioate (CAS# 43136-00-1), 2-(2-
oxiranylmethoxy)ethyl 2-methyl-2-propenoate (glycidyl ether of HEMA, CAS# 30491-79-3). In a preferred embodiment, the polymer is a polymer of an aliphatic ether having from two to four glycidyl groups.
The molecule capable of forming covalent bonds can include, but are not limited to, molecules that include nucleophilic groups. These nucleophilic groups can include, but are not limited to, hydroxyl, amine or thiol groups. These groups can be found in amino acids, peptides, proteins, nucleic acids, polynucleic acids, viruses and cells among others. Preferably the molecule capable of forming a covalent bond is a protein.
In a preferred embodiment of the invention, the coated nanoweb is contacted with the protein in an aqueous medium. Preferably, the protein is capable of binding other proteins to an active site and comprises a free thiol group. Preferably, the aqueous medium comprising the protein also comprises inorganic salts in a concentration from 1 to 2.25M, preferably from 1.25 to 2.25M. Preferred salts are those recognized in the literature as having antichaotropic activity. Examples of antichaotropic salts include sodium sulfate, ammonium sulfate, and magnesium or zinc salts, including chloride, phosphate and carbonate.
The present invention is further directed to a functionalized nanoweb comprising a polymer and a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume %; wherein the polymer is an ether or ester of an epoxy alcohol; preferably an ether or ester of glycidyl alcohol; preferably an ester of a polymer comprising carboxylic acid groups, preferably poly((meth)acrylic acid). Preferences for mean flow pore size and porosity are the same as those stated previously in the description of the method. Preferably, the fiber comprises PVDF, preferably the fiber comprises at least 50 wt% PVDF, preferably at least 80 wt%, preferably at least 90 wt%, preferably at least 95 wt%. Preferably, the polymer forms a continuous coating covering at least 25% of the fiber surface, preferably at least 50%, preferably at least 75%.
Preferably, the protein comprises a free cysteine thiol group, i.e., a thiol group (-SH) which is not part of a disulfide linkage, and said protein is active with respect to binding other proteins to an active site. Preferably, the protein comprising a free cysteine thiol group is obtained by cleaving a disulfide bond in the protein which is not required for maintaining the integrity of the protein binding site, i.e., the protein’s activity for binding other proteins. Preferably, the disulfide is reduced using Tris(2-carboxyethylphosphine) hydrochloride (TCEP.HC1) which can be used at a pH between 1.5 and 9.0 depending upon the stability of the
protein being reduced at that pH. A typical pH for the reduction is pH 3 to pH 8, using nonphosphate buffers e.g. TRIS, HEPES, Borate. One advantage of TCEP is that it does not contain a thiol group and therefore does not require removal before reacting the free sulfhydryl of the protein with the epoxide groups on the nanofiber. Additional reducing agents include dithiothreitol (DTT), mercaptoethanol (ME), 2-mercaptoethylamine hydrochloride (2-MEA.HC1) all of which are typically utilized around neutral pH. These three reagents all include free thiol groups and can be removed from the protein by filtration prior to reacting the protein with the nanofibers to reduce competitive reactions during the conjugation of the protein. Cysteine- Cysteine disulfide bonds are frequently found in proteins as a method of maintaining the protein structure such that cleavage of these disulfide bonds can result in denaturation of the protein. Other structural features such as P -sheets, helix bundles and hairpin structures can maintain a protein’s conformation without the use of disulfide linkages. Preferably, proteins useful for affinity binding of other proteins or useful biotherapeutic molecules are produced recombinantly or isolated from natural sources. Preferably, the protein is one which has been produced recombinantly so as to include a disulfide linkage that is not involved in the configurational stability of the resulting protein; preferred proteins of this type include Protein A, Protein A/G and Protein G. Other preferred proteins include antibodies, monoclonal and polyclonal, the recognition fragments F(ab’) and F(ab’)2 which can be produced recombinantly or following an enzymatic treatment to cleave the fragments from the intact antibody (Rosenstein et.al., Curr Protoc Mol Biol. 2020 Jun;131(l):el l9.doi: 10.1002/cpmb. l l9). Preferably, disulfide bonds which are reduced in the F(ab’)2 are distal to the binding site of the protein and can be cleaved to form the individual fragments without loss of the ability to bind to other proteins, most likely because disulfide linkages that help to maintain the protein configuration occur within a P- secondary structure such as a P-sheet which maintains the protein configuration and protects these disulfides from reduction. Preferably, the protein is in an aqueous solution at a concentration from 5 to 50 mM; preferably the pH of the solution is from 8 to 9.5, preferably from 8.5 to 9.3, preferably from 8.7 to 9.1 and the molecular weight of the protein is no greater than 150,000 Daltons, preferably no greater than 100,000 Daltons and preferably no greater than 75,000 Daltons.
Preferably, a dielectric barrier discharge atmospheric pressure plasma process is used to attach the multifunctional monomer injected as liquid aerosol to the nanofiber substrate. The dielectric barrier discharge plasma process preferably is a homogenous glow discharge process.
Homogeneous glow discharge plasma processes are known in the art to produce spatially uniform low temperature electrons from injected gases at atmospheric pressures. Ions collide with the injected monomers producing ionized species that may self-polymerize in aerosol prior to substrate deposition. Preferred gases suitable for plasma generation include carbon dioxide, nitrogen, argon, and/or helium. The flow of gases in plasma form and the injected monomer aerosol passes through a nozzle with a defined cross-sectional area. Preferably, for treating nonwoven roll goods, the nozzle is rectangular in geometry with an interelectrode gap and a width larger than the width of the nonwoven. The inter-electrode gap is preferably 0.5-10 mm, preferably, 0.8-2mm. The cross-sectional area of the nozzle used in the examples below is 5 cm2 with a nozzle width of 40cm. Preferably, gas flow rates range from 2-150 slm/cm2, where slm is in units of standard liters per minute with standard conditions of a gas volume are at temperature of 0°C and pressure of 1 atm (lOlkPa), preferably from 60 to 100 slm/cm2. Gas flow rates through the plasma head will depend on plasma head size and substrate width to be coated with monomer as well as on environmental temperature and pressure of the gas. Injected monomer flow rates depend on the amount of monomer to be attached per area of substrate. High injection rates of monomer may lead to self-polymerization prior to deposition on the substrate. Preferably, injection rates of aerosolized monomer range from 0.2 slm/cm2 to 5 slm/cm2. Dilutions of monomers may be achieved by one or both means of liquid solution mixing, by dissolution or suspension of a solid monomer in water or an appropriate solvent, and by introduction of greater gas flow into the liquid monomer pure compound, suspension, or solvent mixture. Preferred dilution ratios (solvent or carrier to monomer) range from of 2: 1 to 50: 1, preferably from 3: 1 to 20: 1. Preferably, alternating current powered covered electrodes generate the plasma through a narrow gap at atmospheric pressures from 0.9-1.1 atm (91-111 kPa). Preferably, plasma source voltage ranges from 1-100 kV with preferred range of 5-30 kV.
Preferably supplied power for initial activation and reaction with monomer is in the range of 2 to 300W/cm2, preferably 20 to 200 W/cm2, preferably 30 to 160 W/cm2. Preferably supplied power in the presence of protein is in the range of 1 to 100 W/cm2, preferably 2 to 40 W/cm2, preferably 3 to 20 W/cm2. Preferably, the distance between the substrate and the plasma head is 1-10 mm, preferably 2-5 mm.
Preferably, the substrate is pretreated using carbon dioxide, nitrogen, argon, and/or helium in an atmospheric plasma process without monomer.
EXAMPLES
Nonwoven webs from different non carbohydrate based polymers, used as examples and shown in Table I, were prepared by either an electrospinning process similarly to the one described in https://pubs.acs.org/doi/10.1021/acs.chemrev.8b00593, or an electroblowing process as described in U.S. Patent number 7,618,579. The nonwoven webs were then optionally consolidated as described in U.S. Patent number US8697587B2 (Sample 3). The spinning and consolidation processes were tuned such that porosity of the resulting webs could range between 35% and 95%, and the mean flow pore size between 0.1 pm and 5 pm.
The various nonwoven substrates were pretreated through a plasma, and then processed through the plasma coating process (see, e.g., US 8,663,751, US 9,890,260 and US 9,938,388) using the functionalizing agent (“agent”) and process conditions resulting in various amounts of agent being injected into the plasma and applied to the substrates. PolyGMA was CAS#25067-05-4 (Mn=10, 000-20, 000, 4 wt% in DMSO). Plasma process conditions for epoxide deposition on example nonwovens are shown in Table II. Each nanofiber nonwoven web is subjected to an activation pass through the plasma treatment device, in which the top and bottom surfaces of the web are exposed to a plasma of CO2 and N2 at a flow rate of 80 slm/cm2 and 8 slm/cm2 power level of 300 W/m2. The activated nanofiber web passes through a N2 plasma at flow rate of 80 slm/cm2. The distance between the electrode and substrate is about 2 mm. A measure of amount of agent injected per mass of substrate is determined by gravimetric means of weighing the supply solution at the start and finish of the plasma process taking the difference between start and finish and dividing the difference by the mass of substrate processed.
Table II
The epoxy functionalized nanofiber mats are conjugated with a reactive cysteine on recombinant Protein A. This reaction involves first reducing disulfide bonds in the Protein A dimer using TCEP-HC1 (Tris(2-carboxyethyl)phosphine hydrochloride) [See: Thermo Fisher Application Notes https://www.thermofisher.com/us/en/home/references/molecular-probes-the- handbook/thiol-reactive-probes/introduction-to-thiol-modification-and-detection.html] in aqueous solution at 5 - 50 mM concentration and pH 6-8. The Protein A with a free reactive thiol is then allowed to react with the epoxy-activated nanofiber mat in the presence of a salting out buffer e.g. sodium sulfate to increase the coating of the Protein A on the surface of the fibers and as a result increasing the local concentration of Protein A at the surface of the fiber which increases the amount of Protein A conjugated onto the nanofiber mat [See Thermo Fisher Application Notes: https://www.thermofisher.com]
The saturated binding capacity, SBC, can be measured by placing a 2.5 cm diameter disc of the membrane into a Swinnex filter holder and using an AKTA Pure™, elute a 10 mL solution of human IgG (0.2 mg/mL) in PBS buffer, pH 7.4 at 0.5 ml/min through the membrane. The filter is then washed with about ten bed volumes of the PBS buffer and then the human IgG is eluted from the membrane with 5 bed volumes of buffer (e.g. 0.1 M glycine at pH 3), quickly neutralized to about pH 7 to avoid denaturation and the concentration measured spectrophotometrically, using the formula below or by integration of the elution curve and
comparing with a calibration curve (e.g. Hahn et. al. Journal of Chromatography A. 2005, 1093, 98):
Protein concentration
Protein concentration (mg/ml): Absorbance xdilution x (Molecular weight / Molar extinction coefficient)
Results of saturated binding capacity measurements on conjugated epoxy treated nanofiber surfaces are shown in table III indicating the performance increase of capability of the total structure to bind with increasing deposition of epoxide via plasma.
In addition, an epoxy-functionalized nanoweb as described above was conjugated with Protein A supplied by the Repligen Corporation of Waltham, MA. This type of Protein A binds to the epoxy-functionalized nanoweb via an amine group. The successful conjugation was demonstrated by the binding of IgG to the Protein A-functionalized nanoweb, as demonstrated by the results in Table IV. The SBC testing was performed as described above.
Table IV
Claims
1. A method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb; said method comprising steps of: a) exposing said nanoweb and a polyepoxide material to an atmospheric plasma to produce a coated nanoweb; and b) exposing the coated nanoweb to a molecule capable of forming covalent bonds.
2. The method of claim 1 in which the nanoweb is not a carbohydrate polymer.
3. The method of claim 1 or claim 2 in which the nanoweb comprises PVDF, polyamide, polyethersulfone, polyethylene, polypropylene, polyester or polyimide.
4. The method of any preceding claim in which the molecule capable of forming covalent bonds comprises molecules that include at least one of hydroxyl, amine or thiol groups.
5. The method of any preceding claim in which the nanoweb is exposed to a polymerizable vinyl monomer in the presence of an atmospheric plasma prior to or concurrently with the poly epoxide material.
6. The method of any preceding claim in which the polyepoxide material is a glycidyl ester of a polymeric acid.
7. The method of any preceding claim in which the polyepoxide material is a glycidyl ether of a polymeric alcohol.
8. The method of any preceding claim in which the polyepoxide material is an ether comprising at least two glycidyl groups.
9. The method of claim 8 in which the ether is an aliphatic ether comprising from two to four glycidyl groups.
10. The method of any preceding claim in which said molecule comprises at least one protein.
11. The method of claim 10 in which the protein is capable of binding other proteins to an active site.
12. The method of any preceding claim in which the polyepoxide material is a glycidyl ester or glycidyl ether.
13. The method of any of claims 5 to 12 in which the polymerizable vinyl monomer is a glycidyl ether, a glycidyl ester or a carboxylic acid monomer.
14. A functionalized nanoweb comprising a polymer and a nanoweb; said nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume %; wherein the polymer is an ether or ester of an epoxy alcohol.
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