TITLE: ELECTROSPUN COMPOSITE FIBERS AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of Great Britain Application Ser. No.
2314188.0 filed on 18 September 2023 titled “ELECTROSPUN COMPOSITE FIBERS AND USES THEREOF”, and Great Britain Application Ser. No. 2406482.6 filed on 9 May 2024 titled “ELECTROSPUN COMPOSITE FIBERS”, each of which is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present disclosure relates generally to electrospun composite fibers and their use. The composite fibers can be part of membranes, which can be used for applications in industries where there is a desire to separate and capture target components such as biomolecules, viruses, cells and other materials. The composite fibers and membranes comprised thereof are particularly useful for bioseparation methods.
BACKGROUND
[0003] The background description provided herein gives context for the present disclosure. Work of the presently named inventors, as well as aspects of the description that may not otherwise qualify as prior art at the time of filing, are neither expressly nor impliedly admitted as prior art.
[0004] Fiber-based membranes (including both microfiber and nanofiber technologies) have a variety of different uses for both biological and industrial separations. Microfiber and nanofiber felts are particularly well suited for purifying biological substances, such as proteins, nucleic acids, carbohydrates, bacteria, viruses, cells, and the like. They are useful in all fluid applications, both liquid and gaseous. The manufacturing of biotherapeutics, vaccines, and other treatments are increasingly reliant on the separation of particular target components from a number of other non-target components. Target components can vary in size and properties, including even fragments of molecules, viruses and nucleic acid materials. The ability to capture specific components efficiently requires separation devices and methods that can handle high volumes of fluid at increasingly fast flow rates. The media utilized in bioseparations can vary from membranes to beads (including resins) and other materials. In order to effectively capture targeted components, it is necessary to have separation devices that house the media and provide desirable fluid dynamics including fluid paths and the ability to withstand strong pressure. Additionally, the ability to increase the surface area per volume of the media capable of capturing targeted components is important to improving the efficiency of the bioseparation process.
[0005] Conventional purification methodologies, such as column chromatography separations and certain membrane-based technologies, are limited in terms of binding capacity and processing time. These limitations are primarily due to slow diffusion rates of relatively large biomolecules, which limits the ability of the target component to access available binding sites deep within the separation media. In addition, these systems can be extremely large and require excessive amounts of separation media.
[0006] Various media types and configurations have been utilized over the years including beads (see, e.g, W01996/009116) and membrane technologies in different configurations, including pleated and rolled (see, e.g, U.S. 2013/0092620) and layered (see, e.g., U.S. 9,120,037). However, separation devices with increased binding capacity, maximized flow rates of the mobile phase, and improved usage and reduced pressure across the entire separation media have lagged behind the corresponding improvements in ligand and binding chemistries. Thus, while various configurations have been achieved for different separation media (such as membranes and resins), the existing device configurations still fail to achieve the potential of the separation media themselves.
[0007] Fiber membranes incorporating both cellulose nanofibers and synthetic polymer reinforcement nanofibres have been developed to improve the surface area, porosity and mechanical properties of fiber membranes used in bioseparations. See, for example, US 9,604, 168B2 and US 10,293,289 B2. Use of composite fiber technology where separate polymers are incorporated into a single fiber was also described, but only as a mechanism to produce enhanced cellulose based membranes by removal of the one or more noncellulose components. Further, much of the electrospun fiber-based membrane production has focused around needle spinning. To prepare a composite fiber membrane via electrospinning, two separate spin dopes are typically required. This has limited composite fiber membrane preparation to needle-based spinning and also limited the nature of the needle-based methodology and resultant products. There is a need to improve the fiber-based membrane materials and expand the compatibility of the underlying chemistries for preparation by techniques inclusive of needle electrospinning and non-needle electrospinning.
[0008] Thus, there remains a need for improved fiber-based membranes and methods of electrospinning fibers for use in membranes.
BRIEF SUMMARY OF PREFERRED EMBODIMENTS
[0009] The following objects, features, advantages, aspects, and/or embodiments, are not exhaustive and do not limit the overall disclosure. No single embodiment need provide each and every object, feature, or advantage. Any of the objects, features, advantages, aspects, and/or embodiments disclosed herein can be integrated with one another, either in full or in part.
[0010] It is a primary object, feature, and/or advantage of the present disclosure to improve on or overcome the deficiencies in the art.
[0011] A preferred embodiment is a membrane comprising a nonwoven collection of composite fibers that are microfibers, nanofibers, or a mixture thereof; the composite fibers comprising i) cellulose or a cellulose derivative and ii) a non-cellulose polymer comprising collagen, chitosan, agarose, agarose acetate, a vinyl polymer, a vinyl copolymer, an acrylic polymer, aramid, an acrylic copolymer, a polyacrylic acid, a polymethacrylic acid, polyacrylonitrile, a polyethylene oxide, a polyimide, polyethyleneimine, a polyamide, a polyester, polystyrene, a polysulfone, poly caprolactone, or a mixture thereof; and pores and channels formed between and/or among the composite fibers.
[0012] A preferred embodiment is a method of preparing a collection of composite fibers, the method comprising electrospinning a polymer solution comprising a cellulose derivative, a non-cellulose polymer, and a solvent to form the collection of composite fibers; wa non-cellulose polymer comprising collagen, chitosan, agarose, agarose acetate, a vinyl polymer, a vinyl copolymer, an acrylic polymer, aramid, an acrylic copolymer, a polyacrylic acid, a polymethacrylic acid, polyacrylonitrile, a polyethylene oxide, a polyimide, polyethyleneimine, a polyamide, a polyester, polystyrene, a polysulfone, polycaprolactone, or a mixture thereof; and optionally, forming a membrane with the collection of composite fibers; the membrane including pores and/or channels.
[0013] A preferred embodiment is use of the composite fibers for the capture, separation, purification, filtration, concentration, characterization, quantitation or analysis of biological compounds. Preferably, the biological compounds comprise one or more of an amino acid, a peptide, an affimer, a biomolecule and/or fragment thereof, a protein, an enzyme, a glycoprotein, a lipopolysaccharide, an antibody and/or a fragment thereof, an LNP, a nucleic acid, an organic polymer, a virus, a VLP, an extracellular vesicle, an exosome, a bacterium, a cell, a cell-related structure, or a mixture thereof.
[0014] These and/or other objects, features, advantages, aspects, and/or embodiments will become apparent to those skilled in the art after reviewing the following brief and detailed descriptions of the drawings. The present disclosure encompasses (a) combinations of disclosed aspects and/or embodiments and/or (b) reasonable modifications not shown or described.
BRIEF DESCRIPTION OF THE FIGURE
[0015] Several embodiments in which the present disclosure can be practiced are illustrated and described in detail, wherein like reference characters represent like components throughout the several views. The figures are presented for exemplary purposes and may not be to scale unless otherwise indicated.
[0016] FIG. 1 is a scanning electron microscope (SEM) image of a collection of electrospun composite fibers comprised of a cellulose derivative, a non-cellulose polymer, and a sacrificial polymer.
[0017] FIG. 2 is a table summarizing observed properties of various polymer solutions prepared with varying polymer and solvent concentrations. The polymers employed for this testing were cellulose acetate (CA), polyacrylonitrile (PAN), and polyethylene oxide (PEO); the solvents employed were dimethylformamide (DMF) and dimethylacetamide (DMAc). FIG. 2 corresponds to the testing described in Example 6.
[0018] FIGS. 3A-3O are scanning electron microscope (SEM) images of polymer depositions following electrospinning of the solutions summarized in FIG. 2.
[0019] FIG. 4 is a table summarizing observed properties of various polymer solutions prepared with varying polymer and solvent concentrations. The polymers employed for this testing were cellulose acetate (CA), polyacrylonitrile (PAN), and polyethylene oxide (PEO); the solvents employed were dimethylformamide (DMF) and dimethylacetamide (DMAc). FIG. 4 corresponds to the testing described in Example 6.
[0020] FIGS. 5A-5G are SEM images of fibers formed via electrospinning of the polymer solutions summarized in FIG. 4. [0021] FIGS. 6A-6F are SEM images of membranes prepared via electrospinning of Test Solution A.
[0022] FIGS. 7A-7D are SEM images of membranes prepared via electrospinning of Test Solution B.
[0023] FIGS. 8A-8D are SEM images of membranes prepared via electrospinning of Test Solution C.
[0024] An artisan of ordinary skill in the art need not view, within isolated figure(s), the near infinite distinct combinations of features described in the following detailed description to facilitate an understanding of the present disclosure.
DETAILED DESCRIPTION
[0025] The present disclosure relates to composite fibers for use in separation membranes, particularly, bioseparations. The composite fibers comprise cellulose or a cellulose derivative and a non-cellulose polymer. Beneficially, the composite fibers and membranes comprised of the composite fibers have increased robustness and mechanical strength. These improvements increase the ease of handling and use in combination with higher flow-rates and back-pressures
[0026] It is further to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Further, all units, prefixes, and symbols may be denoted in its SI accepted form.
[0027] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 1!4, and 4%. This applies regardless of the breadth of the range. [0028] Definitions
[0029] So that the present invention may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.
[0030] The term “about,” as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, angle, capacity, concentration, conductivity, density, diameter, distance, flowrate, flux, mass, pH, pressure, time, slope, temperature, viscosity, and volume. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations. Whether or not modified by the term “about,” the claims include equivalents to the quantities.
[0031] The methods and compositions of the present invention may comprise, consist essentially of, or consist of the components and ingredients of the present invention as well as other ingredients described herein. As used herein, “consisting essentially of’ means that the methods, systems, apparatuses and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed methods, systems, apparatuses, and compositions.
[0032] The term "actives" or "percent actives" or "percent by weight actives" or "actives concentration" are used interchangeably herein and refers to the concentration of those ingredients involved in cleaning expressed as a percentage minus inert ingredients such as water or salts. It is also sometimes indicated by a percentage in parentheses, for example, “chemical (10%).”
[0033] As used herein, the term “alkyl” or “alkyl groups” refers to saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (or “cycloalkyl” or “alicyclic” or “carbocyclic” groups) (e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups). [0034] Unless otherwise specified, the term “alkyl” includes both “unsubstituted alkyls” and “substituted alkyls.” As used herein, the term “substituted alkyls” refers to alkyl groups having substituents replacing one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxy carbonyloxy, aryloxy, aryloxy carbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxy carbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonates, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups.
[0035] In some embodiments, substituted alkyls can include a heterocyclic group. As used herein, the term “heterocyclic group” includes closed ring structures analogous to carbocyclic groups in which one or more of the carbon atoms in the ring is an element other than carbon, for example, nitrogen, sulfur or oxygen. Heterocyclic groups may be saturated or unsaturated. Exemplary heterocyclic groups include, but are not limited to, aziridine, ethylene oxide (epoxides, oxiranes), thiirane (episulfides), dioxirane, azetidine, oxetane, thietane, dioxetane, dithietane, dithiete, azolidine, pyrrolidine, pyrroline, oxolane, dihydrofuran, and furan.
[0036] The term “and/or” means any one of the items, any combination of the items, or all of the items with which this term is associated. [0037] The terms “capacity” and “binding capacity” as used herein refer to the amount of product bound per unit of adsorbent.
[0038] The term “chemically stable” as used herein means that a material (including, but not limited to, a membrane and composite fiber) is not soluble in solvents such as water or common organic solvents (e.g., alcohols and hydrocarbons), and their mixtures; further it includes resistance to a range of pH conditions including at least from about 3 to about 11, and resistance to buffer additives.
[0039] The term “temperature stable” as used herein means that a material (including, but not limited to, a membrane and composite fiber) is stable in temperature conditions in the range of from about 0 °C to about 100 °C.
[0040] The term “electrospinning” as used herein refers to the application of electric forces to the spin dope to form the nanofibers.
[0041] The term “exemplary” refers to an example, an instance, or an illustration, and does not indicate a most preferred embodiment unless otherwise stated.
[0042] The term “extracellular vesicles” includes vesicles of any size, including exosomes and larger extracellular vesicles. As used herein, “exosomes” as used herein refers to small, secreted vesicles (typically about 30-150 nm) which may contain, or have present in their membrane, nucleic acid, protein, or other biomolecules and may serve as carriers of this cargo between diverse locations in a body or biological system. [0043] The term “flowrate” as used herein refers to the volume of liquid expressed in dry media volumes (MVs) flowed through membrane per time expressed in minutes. Flowrate is considered high if it is above 20 MV/min.
[0044] The term “flux” refers to the flow rate of fluid passing through the separation media per unit time, per unit of facial area exposed to the flow.
[0045] The term “membrane” as used herein refers to a non-woven or randomly overlaid collection of electrospun composite fibers comprised of at least two types of polymers in a single fibers.
[0046] The term “ligand” as used herein refers to molecules or compounds capable of interaction with target compounds, such as antibodies, nucleic acids, proteins, and viruses, or particles or fragments thereof.
[0047] The term “lipid nanoparticle” or “LNP” as used herein is a general term to describe lipid-based particles in the submicron range. LNPs can have structural characteristics of liposomes and/or have alternative non-bilayer types of structures. LNPs constitute an alternative to other particulate systems, such as emulsions, liposomes, micelles, microparticles and/or polymeric nanoparticles, for the delivery of active ingredients, such as oligonucleotides and small molecule pharmaceuticals.
[0048] The term “microfibers” as used herein refers to fibers with diameters larger than 1.0 micrometer, and generally between 1.0 micrometer and 1.0 millimeter; as measured by scanning electron microscope (SEM).
[0049] The term “nanofibers” as used herein refers to fibers with diameters smaller than of 1.0 micrometer, and generally between 10 nanometers and 1.0 micrometer, such as between 200 nm and 600 nm; as measured by scanning electron microscope (SEM). [0050] The term “nanofiber felt” as used herein refers to a collection of nanofibers in a substantially planar array, which may also include randomly overlaid fibers, woven fibers, non-woven fibers, electrospun nanofibrous arrays, or non-electrospun nanofibrous arrays.
[0051] As used herein, the term “nucleic acid” refers to nucleic acids in the form of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or peptide nucleic acid (PNA), as well as analogs, derivatives, or any combination thereof. Such a derivative could contain, for example, a nucleotide analog or a “backbone” bond other than a phosphodiester bond, for example, a phosphotriester bond, a phosphoramidate bond, a phosphorothioate bond, or a thioester bond. Naturally-occurring RNA molecules include, but are not limited to, transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), or genomic RNA, such as that from influenza or hepatitis C viruses. Other forms of RNA include, but are not limited to, small interfering RNA (siRNA) and microRNA (miRNA). The nucleic acid molecules can be single-stranded (ss; and which can be sense or antisense), double-stranded (ds) or a combination of the two, and can be linear or circular, the latter of which can be open-circular or closed- circular. The nucleic acid molecule can be a vector. The vector can be a viral vector, preferably a lentivirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, a vesicular stomatitis virus (VSV) vector, a herpes simplex virus (HSV) vector, a vaccinia virus vector, a reo virus vector, a pox virus vector, an influenza virus vector, a respiratory syncytial virus vector, a parainfluenza virus vector, a foamy virus vector, a measles virus vector or a retrovirus vector. [0052] As used herein the term “polymer” refers to a molecular complex comprised of more than ten monomeric units and generally includes, but is not limited to, homopolymers, copolymers, such as for example, block, graft, random and alternating copolymers, terpolymers, and higher "x"mers, further including their analogs, derivatives, combinations, and blends thereof. Furthermore, unless otherwise specifically limited, the term “polymer” shall include all possible isomeric configurations of the molecule, including, but are not limited to isotactic, syndiotactic and random symmetries, and combinations thereof. Furthermore, unless otherwise specifically limited, the term “polymer” shall include all possible geometrical configurations of the molecule.
[0053] The terms “protein”, “polypeptide”, or “peptide” can be used interchangeably and refer to any natural or recombinant molecule comprising amino acids joined together by peptide bonds between adjacent amino acid residues. A “peptide bond”, “peptide link”, or “amide bond” is a covalent bond formed between two amino acids when the carboxyl group of one amino acid reacts with the amino group of the other amino acid. The terms “amino acid”, “amino acid residue”, and “residue” may be used interchangeably herein.
[0054] The terms “purifying,” “separating,” or “isolating,” as used interchangeably herein, refer to increasing the degree of purity of a molecule of interest or a target molecule from a composition or sample comprising the molecule and one or more impurities.
[0055] As used herein, the term “sample” refers to any liquid or solid material to be used in the processes described herein. For example, a sample can be a solution containing eukaryotic or prokaryotic cells or cellular material, or virus or viral material, or bacteria or bacterial material, or microorganisms or pathogens. A sample can be essentially water, or a buffered solution or be composed of any artificially introduced chemicals and may or may not contain nucleic acids or proteins.
[0056] As used herein, “biological sample” refers to any sample obtained from a living, recently living or viral source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid or protein or other macromolecule can be obtained. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample. Biological samples can include biological solid material or biological fluid or a biological tissue. Examples of biological solid materials include tumors, cell pellets, or biopsies. Examples of biological fluids include cell cultures, cell homogenates, suspension of cells in a medium, urine, blood, plasma, serum, sweat, saliva, semen, stool, sputum, cerebral spinal fluid, mouth wash, tears, mucus, sperm, amniotic fluid, or the like. Biological tissues are aggregates of cells, usually of a particular kind, together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries, and individual cell(s). Also included the definition of biological sample are soil, water and other environmental samples including industrial waste and natural bodies of water (lakes, streams, rivers, oceans) that can contain viruses, bacteria, fungi, algae, protozoa, and components thereof.
[0057] The term “spin dope” as used herein refers to the polymer solution that is used in the electrospinning process.
[0058] The term “target component” includes, but is not limited to, any particular component which is desired to be separated and captured. Preferably, “target components” are soluble components, including but not limited to, biological entities (including, but not limited to, cells, viruses, proteins, extracellular vesicles, nucleic acids, peptides, polypeptides, etc.), biocomponents, and chemical components (including, but not limited to, synthetic organics, metal ions, small molecules, etc.). In some embodiments, the target component can be found in and/or isolated from a biological sample.
[0059] The term “thermally stable” as used herein means that a material (including, but not limited to, a separation media) does not disintegrate in the temperature range from 50-110°C.
[0060] The term “substantially” refers to a great or significant extent. “Substantially” can thus refer to a plurality, majority, and/or a supermajority of said quantifiable variables, given proper context.
[0061] A “vector” is a composition of matter which comprises an isolated nucleic acid and is a transportation and delivery vehicle for a nucleic acid to the interior of a cell. Examples of vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, exosomes and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. [0062] A “virus-like particle” or “VLP” as used herein refers to at least one virus particle, which does not contain any nucleic acid. VLPs can thus be used for vaccination or for inducing an immunogenic reaction in a subject. However, due to the absence of nucleic acids, VLPs will not be able to replicate in a host cell and are thus non- replicative.
[0063] The term “weight percent,” “wt.%,” “percent by weight,” “% by weight,” and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent,” “%,” and the like are intended to be synonymous with “weight percent,” “wt.%,” etc.
[0064] The present disclosure is not to be limited to that described herein. Mechanical, electrical, chemical, procedural, and/or other changes can be made without departing from the spirit and scope of the present disclosure. No features shown or described are essential to permit basic operation of the present disclosure unless otherwise indicated. [0065] Composite Fibers
[0066] Disclosed herein are composite fibers comprised of a cellulose and a noncellulose polymer, where each fiber is composed of both the cellulose and non-cellulose polymer. The composite fibers can further comprise one or more sacrificial polymers, which are included during the electrospinning process and subsequently removed prior to the intended end-use (i.e. , separation process). The composite fibers can comprise mixtures of cellulose polymers and/or mixtures of non-cellulose polymers.
[0067] Preferably, the composite fibers have an average diameter of from about 10 nm to about 10 pm, more preferably of from about 10 nm to about 5 pm, most preferably from about 20 nm to about 1 pm; as measured by scanning electron microscope (SEM). [0068] Preferably, the cellulose or cellulose derivative and the non-cellulose polymer are in a ratio by weight of from about 2: 1 to about 4.5:1, more preferably from about 1.5: 1 to about 4.5: 1, more preferably about 2: 1 to about 3:1, more preferably about 2.5:1 (cellulose or cellulose derivative to non-cellulose polymer).
[0069] Beneficially, the composite fibers and membranes comprised of the composite fibers can be chemically stable, temperature stable, and have robust mechanical properties. Thus, the composite fibers and membranes comprised of the composite fibers can be used in a wide variety of conditions, including, but not limited to, variations of pH, buffer additives, pressures, flow rates, and temperatures.
[0070] Cellulose
[0071] The composite fibers comprise cellulose or a celluloses derivative. Cellulose is not amenable to electrospinning on its own and needs to be prepared via a cellulose derivative. Cellulose can easily be derivatized using well known methods by converting the -OH group of the individual glucose units into other moieties. Subsequent to electrospinning, the cellulose derivative can be regenerated into cellulose, retained as a cellulose derivative, or further derivatized to a different cellulose derivative.
[0072] Such derivatized cellulose species exhibit enhanced stability when exposed to solvents and other desirable physical properties. Many cellulose derivatives are readily commercially available. Exemplary derivatized cellulose species include, for example: organic esters (cellulose acetate, triacetate, propionate, acetate propionate, acetate butyrate); inorganic esters (cellulose nitrate, cellulose sulfate); an organic cellulose ether, and alkyl cellulose (ethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose), and mixtures thereof. Most preferred cellulose derivatives include cellulose acetate, ethyl cellulose, or a mixture thereof.
[0073] Non-Cellulose Polymer
[0074] The composite nanofiber further comprises a non-cellulose polymer. The non- cellulose polymer can be a natural polymer and/or a synthetic polymer. Preferred natural polymers include, but are not limited to, collagen, chitosan, agarose, agarose acetate, and combinations thereof. Preferred synthetic polymers include, but are not limited to, those comprised of vinyl polymers, acrylic polymers, and copolymers thereof. More preferred synthetic polymers include, but are not limited to, polyacrylic acid, polymethacrylic acid, polyacrylonitrile (PAN), polyethylene oxides, polyimides, polyethyleneimine (PEI), polyamides (nylon 6, nylon 6,6, nylon 6,10, etc.), polyesters (polyethylene terephthalate, etc.), polystyrene, polysulfones, poly caprolactone, and copolymers thereof.
[0075] Preferred polymers can include, but are not limited to, thermoplastic homopolymers such as vinyl polymers, acrylic polymers, polyamides, polyesters, polyethers, and polycarbonates including, but not limited to, polyacrylic acid, polymethacrylic acid, nylon, poly ethersulfone, and polyacrylonitrile, (2) thermoplastic copolymers such as vinyl-co-vinyl polymers, acrylic-co-acrylic copolymers and vinyl- coacrylic polymers, (3) elastomeric polymers such as triblock copolymer elastomers, polyurethane elastomers, and ethylene-propylene-diene-elastomers, (4) high performance polymers such as polyimides and aromatic polyamides, (5) liquid crystalline polymers such as poly(p-phenylene terephthalamide) and polyaramid, (6) textile polymers such as polyethylene terephthalate and polyacrylonitrile, (7) electrically conductive polymers such as polyaniline, as well as (8) biocompatible polymers (i.e. “biopolymers”) like polycaprolactone, polylactide, chitosan and polyglycolide; and mixtures thereof. As described, the polymer may also be a copolymer of two or more of the above-named polymer species.
[0076] The non-cellulose polymer can act to increase the structural integrity of the membrane. For example, the inclusion of a non-cellulose polymer in the composite fiber can reinforce the fiber and the membrane thereby providing a significant increase in robustness and mechanical strength. These improvements increase the ease of handling and use in combination with higher flow-rates and back-pressures. Additionally, we have found that the average fiber diameter can be more homogeneous when prepared as a composite fiber. For example in a preferred embodiment, the average fiber diameter of the composite fibers is within about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6%, or about 5%; according to measurements taken by SEM.
[0077] Previously, it was thought that separate non-cellulose fibers would have to be prepared to increase the mechanical robustness and properties of the resultant membrane; however, we have found that there is a way to prepare composite fibers so that the fibers themselves have the improved mechanical properties, and not just the membrane. This is important because if only some fibers have the mechanical improvements and others do not, the membrane can have inconsistent mechanical properties and some fibers can be damaged by the intended high pressure and flow rates. By use of composite fibers, all of the fibers forming the membrane have consistent and improved mechanical properties.
[0078] Sacrificial Polymer
[0079] Optionally, the composite nanofiber can comprise a sacrificial polymer, which is removed from the membrane. By removing the sacrificial polymer, the surface area of the fibers is increased. Further, removal can act to provide pores and channels in the membrane of homogenous or substantially homogenous diameter according to measurements taken via capillary flow porometry. Preferably, the pores and channels have an average diameter of from about 1 nm to about 10 micrometers according to measurements taken via capillary flow porometry. A preferred instrument for capillary flow porometry is a Porolux 200 available from Aptco.
[0080] The sacrificial polymer is differentially removable from the cellulose and noncellulose polymer such that the cellulose and non-cellulose polymer remain after the sacrificial polymer has been removed. The sacrificial polymer can be differentially removable via thermal degradation or dissolving. Preferred sacrificial polymers include, but are not limited to, polyethylene oxide (PEO), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), gelatin, polyethylene glycol (PEG), xanthan gum, pectin, alginates, dextran, carboxymethyl cellulose (CMC), sodium acrylate, and combinations thereof. Most preferred sacrificial polymers include polyethylene oxide (PEO), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and combinations thereof.
[0081] Preferred differentially removable polymers are those that are water soluble. However, electrospinning with polymers dissolved in an aqueous solution can be challenging, particularly, when a more substantial amount of water is required for dissolving the polymers. Water has a very high surface tension and dielectric constant which makes it difficult to spin such polymers on their own. Thus, with water soluble polymers it can be preferred to include a water miscible solvent and/or a ‘facilitator’ polymer, which helps facilitate the electrospinning of the water soluble polymer. The facilitator is then preferably removed post-electrospinning via sacrificial removal or leaching. [0082] Preferably, the sacrificial polymer has a melting point below the glass transition temperature of the cellulose and non-cellulose polymer that make up the composite fiber. Thus, the melting point of the sacrificial polymer is preferably below about 230 °C, below about 220 °C, below about 210 °C, below about 200 °C, below about 190 °C, below about 180 °C, below about 170 °C, below about 160 °C, below about 150 °C, below about 140 °C, below about 130 °C, below about 120 °C, below about 110 °C, below about 100 °C, below about 95 °C, below about 90 °C, below about 85 °C, below about 80 °C, below about 75 °C, below about 70 °C, below about 65 °C, or below about 60 °C.
[0083] Membrane
[0084] A membrane can be formed from a collection of electrospun composite fibers of the present invention. Specifically, a membrane can comprise the collection of electrospun composite fibers which are randomly overlaid. As disclosed herein, the electrospun composite fibers can be microfibers, nanofibers, of a diameter larger than microfibers, or a combination thereof. Preferably, the membrane comprises channels and pores formed by the composite fibers. The channels and/or pores can be substantially uniform in diameter or be of varying diameters. Most preferably, the membrane is suitable for use in bioseparations.
[0085] The collection of electrospun composite fibers can be shaped as disclosed below and/or the membrane can be molded, folded, bent, pleated or otherwise shaped through mechanical manipulation. The membrane can comprise layers, including multiple collections of electrospun composite fibers and/or inert porous materials.
[0086] Electrospinning
[0087] The composite fibers and membranes formed therefrom can be prepared by electrospinning. Any suitable electrospinning technique can be used, including, but not limited to, needle electrospinning, open-surface electrospinning, and wire-based electrospinning.
[0088] Electrospinning is a technique that utilizes electric forces alone to drive the spinning process and to produce polymer fibers from solutions or melts. Unlike conventional spinning techniques (e.g. solution- and melt-spinning), which are capable of producing fibers with diameters in the micrometer range (approximately 5~25 pm), electrospinning is capable of producing fibers with diameters across a much broader range as it can include the nanometer range in addition to the micrometer range.
[0089] Electrospun polymer nanofibers possess many extraordinary properties including the small fiber diameter and the concomitant large specific surface area, the high degree of macromolecular orientation and the resultant superior mechanical properties.
[0090] Additionally, membranes made of electrospun nanofibers exhibit controlled pore sizes when compared to nanofibers that are made using other fabrication techniques. Unlike nanorods, nanotubes and nanowires that are produced mostly by synthetic methods, electrospun nanofibers are produced through a “nano-manufacturing process”, which results in low-cost nanofibers that are also relatively easy to assemble and process into applications.
[0091] In general, the formation of electrospun fibers is a delicate and complicated balance of three major forces involved in the electrospinning process, including the electrical force, the surface tension, and the viscoelastic force. Among these three forces, the electrical force always favors the formation of the product with the highest surface areas. The surface tension always favors the formation of the product with the smallest surface areas. The viscoelastic force is a force which varies significantly with the evaporation of the solvent and is the main reason preventing the breakup of the electrospinning jet/filament into droplets. When the electrical force is dominant, viscoelastic force works against the electrical force. When surface tension is dominant, viscoelastic force works against surface tension.
[0092] Theoretically, the smallest fibers, on the nanofiber scale, are capable of being formed under two conditions: (1) when the excess charge density carried by the electrospinning jet is high, and (2) when the time period is long enough and the viscoelastic force is high enough to prevent the capillary breakup of the jet/filament but low enough to allow the electrical force to effectively stretch the jet.
[0093] For condition (1), it has been found that the addition of soluble electrolytes to the spin dope (e.g., addition of strong electrolytes such as NaCl to the polymer solution to be electrospun) can significantly increase the excess charge density carried by the jet and cause the formation of smaller diameter nanofibers. This method, however, also creates negative effects such as (a) a smaller flow rate and the resulting decrease in nanofibers productivity, and (b) the contamination of the prepared nanofibers by the electrolytes. The removal of the electrolytes without sacrificing the properties of nanofibers may be difficult.
[0094] For condition (2), further understanding of jet solidification is required. In general, the jet solidification is closely related to the volatility of solvent. If the solvent volatility is too high, the time period for effectively stretching the electrospinning jet/filament is short. Consequently, fibers with relatively large diameters will be obtained. If the solvent volatility is too low, the electrospinning jet/filament is likely to break up into droplets as a result of the stretching. Consequently, beads and/or beaded fibers will be obtained.
[0095] The electrospinning process generally includes three steps: (1) initiation of the electrospinning jet/filament and the extension of the jet along a straight trajectory; (2) growth of the bending instability and the further elongation of the jet, which allows the jet to become very long and thin while following a looping and spiraling path; and (3) the solidification of the jet through solvent evaporation or cooling, which leads to the formation of electrospun fibers.
[0096] In the case of an open surface electrospinning system, a carriage carrying the polymer solution mixture drops small droplets of the solution onto an open-surface rail as it moves along backwards and forwards on top of the rail. When a potential difference (voltage) is created between the open-surface rail and the top collector substrate, the small droplets form into jets which then produce fibers that are projected upwards towards the collector. Two or three separate but parallel open-surface rails can be used at the same time to increase the volume of fibers produced.
[0097] In the case of a wire-based electrode electrospinning system, there is a wire electrode that is constantly wetted by a carriage carrying the polymer solution along the wire. The carriage is in direct contact with the wire as it moves backwards and forwards. The jets emerge directly from the wire and upward towards a moving substrate on to which fibers are collected. A wire-based electrode system can use one, two, three, or four wire electrodes in parallel configuration at the same time to increase fiber yield.
[0098] Both of these electrospinning systems (open surface and wire-based electrode) are designed to work with a single polymer solution at a time as the source of applied voltage is a single power supply that can generate only one potential difference in the entire electrospinning chamber.
[0099] Preparation of the Spin Dope (Polymer Solution)
[0100] A spin dope can be acquired with the desired polymer(s) dissolved in solvent, or it can be prepared by dissolving polymers in a solvent. The spin dope comprises a cellulose derivative and a non-cellulose polymer (such as polyacrylic acid, polyacrylonitrile, aramid, polyamide, polystyrene, chitosan, polyethyleneimine, or a mixture thereof) dissolved in solvent. The cellulose derivative and non-cellulose polymer can be separately dissolved and combined, stepwise dissolved in a single solvent mixture, or simultaneously dissolved in solvent mixture. In a further embodiment, a sacrificial polymer can be dissolved in a solvent and combined with the cellulose derivative and non-cellulose polymer.
[0101] Traditionally, chloroform is used to dissolve the cellulose derivative. However, we found chloroform to be incompatible with a preferred non-cellulose polymer, PAN. Further, while some solvents could dissolve a non-cellulose polymer well, they do not effectively dissolve cellulose derivative polymers. As such, it was necessary to find solvents that could be mixed together and remain in solution and which could effectively dissolve cellulose derivative polymers and PAN.
[0102] Solvents that met these criteria were found to include, but are not limited to, acetone, dimethylformamide (DMF), dimethylacetamide (DMAc), dimethyl carbonate (DMC), cyclopentanone (CPO), dimethylsulfoxide (DMSO), ethanol, methanol, water, or a mixture thereof.
[0103] The polymer solution preferably has a viscosity of from about 300 centipoise to about 3500 centipoise, more preferably from about 300 centipoise to about 3000 centipoise, at about 20 °C as measured by a viscometer. We found that if the viscosity is too high (e.g., -6500 cp) the electrospinning process is more difficult and the fiber diameter is too large for most applications. To achieve a desired viscosity, the amounts of polymer or solvent can be adjusted. Further, use of a sacrificial polymer can also be added to modify viscosity.
[0104] The polymer solution preferably has a conductivity of from about 10 ps/cm to about 100 ps/cm, more preferably from about 15 ps/cm to about 95 ps/cm, at about 20 °C, most preferably from about 20 ps/cm to about 90 ps/cm, at about 20 °C as measured by an electrical conductivity meter.
[0105] Following obtaining or preparing a spin dope, the fibers can be prepared by electrospinning. An example, nonlimiting electrospinning process can generally be described as follows:
[0106] Step 1: A spin dope (e.g., a polymer solution) is placed in one or more containers in fluid communication with one or more spinnerets, and DC high voltage, usually in the range from 5-40 kilovolts, is applied to the solution through an electrode (e.g, a copper wire). An electrically grounded collector is placed at a certain distance (known as the gap distance) away from the spinneret. The gap distance may range from a few centimeters up to one meter. When the electrostatic field reaches a critical value, and the electric force overcomes surface tension and viscoelastic forces, a jet/filament is ejected and travels straight for a certain distance (known as the jet length).
[0107] Step 2: The jet then starts to bend, forming helical loops. This phenomenon is termed “bending (or whipping) instability.” Typically, the bending instability causes the length of a jet to elongate by more than 10,000 times in a very short time period (50 ms or less). Thus, the elongational rate during the bending instability is extremely high (up to 1,000,000 s'1). This extremely fast elongational rate can effectively stretch the chain of macromolecules and closely align them along the nanofiber axis.
[0108] Step 3: The jet solidifies, either though evaporation of the solvent or when the melt cools below the solid-liquid transition temperature. The longer the solidification time, the more the jet can be lengthened. The solidification time is related to many factors such as solvent vapor pressure, solvent diffusivity, volumetric charge density carried by the jet, and strength of the applied electrostatic field.
[0109] These steps can be performed by an electrospinning system with one spinneret or multiple spinnerets. Preferably the electrospinning system has between 1 and 6 spinnerets; although systems with more spinnerets can be used. Preferred electrospinning systems include needle-based systems, open-surface systems, and wirebased electrode systems. The methods disclosed herein would be suitable on other types of electrospinning systems, such that the manufacture of the composite fibers disclosed herein is not limited by the electrospinning system. [0110] Optional Post-Electrospinning Processing
[oni] After solidification of the collected electrospun fibers, there are certain additional steps that can be performed in order to “customize” the fibers for particular uses. Exemplary additional steps are discussed below:
[0112] Cellulose Regeneration
[0113] Following preparation of the electrospun fibers, the derivatized cellulose can be converted into cellulose through the process of regeneration. The regenerated cellulose will have the same properties as pure native cellulose described. The regeneration process is completed by contacting nanofibers containing derivatized cellulose with, for example, a strong base (e.g., sodium hydroxide), or other reagent. Following the regeneration reaction for conversion to cellulose, the nanofibers can be washed to remove any excess solvent used during the process.
[0114] Cellulose Crosslinking
[0115] The regenerated cellulose can be crosslinked by combining the regenerated cellulose with a crosslinking system comprised of a crosslinking agent and a catalyst in solution (the solution can be a solvent or water) (one example of such crosslinking is described in WO2017/189977); permitting the regenerated cellulose to be in contact with the crosslinking system for a sufficient time (such as from 1 minute to 1 hour); and cured. The curing can be thermal (i.e., heating), light (e.g., subjecting the regenerated cellulose and crosslinking system to light waves), electromagnetic radiation, or any other method of curing appropriate for the crosslinking system and fiber materials; the type of curing will be based on the crosslinking system and catalyst chosen.
[0116] Preferred crosslinking agents can include, but are not limited to, aldehydes, organochlorides, ethers, multi-functional carboxylic acids, urea derivatives, glycidyl ethers, and mixtures thereof. More preferably, the crosslinking agents can include, but are not limited to, citric acid, malic acid, maleic acid, itaconic acid-maleic acid, 1,2, 3, 4 buthanetetracarboxylic acid (BTCA), sodium hypophosphite, ammonium persulfate, sodium hydroxide, aluminum sulfate, glyoxal, glycerol, epichlorohydrin, epibromohydrin, 1,4 butanediol diglycidyl ether, and mixtures thereof.
[0117] The catalyst used can depend on the crosslinking agent. Preferred catalysts include, but are not limited to, cyanamide, boric acid, aluminum sulfate, ammonium persulfate, sodium hypophosphite, magnesium chloride, phosphate-containing compounds, and mixtures thereof.
[0118] Removal of Sacrificial Polymer
[0119] If a sacrificial polymer is included in the spin dope, it can be removed from the resultant composite fibers and membrane. The sacrificial polymer can be removed based on a differential removal technique specific to the sacrificial polymer species. For example, if the sacrificial polymer is dissolvable in a solvent that the cellulose derivative and non-cellulose polymer fibers are not soluble in, then the sacrificial polymer can be removed by dissolving. If the sacrificial polymer has a lower melting point than the cellulose derivative and non-cellulose polymer fibers, then the sacrificial polymer can be removed by melting. It is possible the sacrificial polymer can be removed by a combination of heating and dissolving.
[0120] Shaping
[0121] The collection of electrospun composite fibers can be shaped in a desired configuration. Preferred shapes include, but are not limited to, a wafer, a rectangle, a square, a cylinder, a star, a sheet, or another desired shape.
[0122] Layering
[0123] The collection of electrospun composite fibers can be layered with another collection of electrospun fibers or with an inert porous material (including, but not limited to, a sheet, tube, or cylinder).
[0124] Surface Functionalization
[0125] The electrospun composite fibers can also be surface functionalized. For use in bioseparation, the composite fibers are ideally biologically inert, meaning it generally resists non-specific binding of insoluble solids such as cells and cellular debris, as well as unwanted interactions with proteins, sugars, nucleic acids, viruses, and other soluble components present in many biologically produced systems.
[0126] Thus, to capture target molecules, the electrospun composite fibers are modified so as to comprise one or more surface functionalizations. The surface functionalizations can be part of the electrospun composite fibers or attached to the electrospun composite fibers. Any suitable surface functionality for a particular separation technique can be utilized with the electrospun composite fibers. Preferred surface functionalities include, but are not limited to, ion exchange groups, hydrophobic groups, chelating groups, affinity ligands, mixed mode ligands (also referred to as multi-modal ligands).
[0127] Target “biocomponents” include, but are not limited to, biomolecules and fragments thereof. Preferred target biocomponents, include, but are not limited to, an amino acid, a peptide, an affimer, a biomolecule and/or fragment thereof, a protein, an enzyme, a glycoprotein, a lipopolysaccharide, an antibody and/or a fragment thereof, an LNP, a nucleic acid, an organic polymer, a virus, a VLP, an extracellular vesicle, an exosome, a bacterium, a cell, and a cell-related structure. Preferred antibodies include but are not limited to, monoclonal antibodies, single-chain antibodies, bi-specific antibodies, multi-specific antibodies, antibody conjugates, antibody fusion proteins, drug-antibody conjugates, antibody fragments, Fab fragments, Fv fragments, gamma globulins (IgG), IVIG, IgM, IgA, IgE, hyperimmune gamma globulins and isoagglutinins. Preferred viruses, include, but are not limited to, adeno-associated virus (AAV), measles virus and lentivirus. Preferred cells and cell-related structures are derived from plants or animals; most preferably cells and cell-related structures are animal derived.
[0128] The surface functionalizations can be directly attached to the composite fibers or attached by a linker. If by a linker, the linker preferably has a chain length of from about 1 carbon to about 20 carbons, more preferably from about 2 carbons to about 19 carbons, still more preferably from about 6 carbons to about 18 carbons.
[0129] The surface functionalizations can be present on the collection of electrospun composite fibers at density of from about 0.001 mg/g to about 999 mg/g. Embodiments provide the ligand can be present at density of from about 0.001 mg/g to about 500 mg/g, from about 0.01 mg/g to about 400 mg/g, from about 0.1 mg/g to about 300 mg/g, from about 0.5 mg/g to about 200 mg/g, from about 1 mg/g to about 100 mg/g, about 10 mg/g to about 100 mg/g, from about 10 mg/g to about 200 mg/g, from about 10 mg/g to about 250 mg/g, from about 10 mg/g to about 300 mg/g, from about 10 mg/g to about 400 mg/g, from about 10 mg/g to about 500 mg/g, from about 10 mg/g to about 600 mg/g, from about 10 mg/g to about 700 mg/g, from about 10 mg/g to about 750 mg/g, from about 10 mg/g to about 800 mg/g, from about 10 mg/g to about 900 mg/g, from about 50 mg/g to about 100 mg/g, from about 50 mg/g to about 200 mg/g, from about 50 mg/g to about 250 mg/g, from about 50 mg/g to about 300 mg/g, from about 50 mg/g to about 400 mg/g, from about 50 mg/g to about 500 mg/g, from about 50 mg/g to about 600 mg/g, from about 50 mg/g to about 700 mg/g, from about 50 mg/g to about 750 mg/g, from about 50 mg/g to about 800 mg/g, from about 50 mg/g to about 900 mg/g, from about 50 mg/g to about 1000 mg/g, from about 100 mg/g to about 200 mg/g, from about 100 mg/g to about 250 mg/g, from about 100 mg/g to about 300 mg/g, from about 100 mg/g to about 400 mg/g, from about 100 mg/g to about 500 mg/g, from about 100 mg/g to about 600 mg/g, from about 100 mg/g to about 700 mg/g, from about 100 mg/g to about 750 mg/g, from about 100 mg/g to about 800 mg/g, from about 100 mg/g to about 900 mg/g, from about 100 mg/g to about 1000 mg/g, from about 200 mg/g to about 250 mg/g, from about 200 mg/g to about 300 mg/g, from about 200 mg/g to about 400 mg/g, from about 200 mg/g to about 500 mg/g, from about 200 mg/g to about 600 mg/g, from about 200 mg/g to about 700 mg/g, from about 200 mg/g to about 750 mg/g, from about 200 mg/g to about 800 mg/g, from about 200 mg/g to about 900 mg/g, from about 200 mg/g to about 1000 mg/g, from about 250 mg/g to about 300 mg/g, from about 250 mg/g to about 400 mg/g, from about 250 mg/g to about 500 mg/g, from about 250 mg/g to about 600 mg/g, from about 250 mg/g to about 700 mg/g, from about 250 mg/g to about 750 mg/g, from about 250 mg/g to about 800 mg/g, from about 250 mg/g to about 900 mg/g, from about 250 mg/g to about 1000 mg/g, from about 300 mg/g to about 400 mg/g, from about 300 mg/g to about 500 mg/g, from about 300 mg/g to about 600 mg/g, from about 300 mg/g to about 700 mg/g, from about 300 mg/g to about 750 mg/g, from about 300 mg/g to about 800 mg/g, from about 300 mg/g to about 900 mg/g, from about 300 mg/g to about 1000 mg/g, from about 400 mg/g to about 500 mg/g, from about 400 mg/g to about 600 mg/g, from about 400 mg/g to about 700 mg/g, from about 400 mg/g to about 750 mg/g, from about 400 mg/g to about 800 mg/g, from about 400 mg/g to about 900 mg/g, from about 400 mg/g to about 1000 mg/g, from about 500 mg/g to about 600 mg/g, from about 500 mg/g to about 700 mg/g, from about 500 mg/g to about 750 mg/g, from about 500 mg/g to about 800 mg/g, from about 500 mg/g to about 900 mg/g, from about 500 mg/g to about 1000 mg/g, from about 600 mg/g to about 700 mg/g, from about 600 mg/g to about 750 mg/g, from about 600 mg/g to about 800 mg/g, from about 600 mg/g to about 900 mg/g, from about 600 mg/g to about 1000 mg/g, from about 700 mg/g to about 750 mg/g, from about 700 mg/g to about 800 mg/g, from about 700 mg/g to about 900 mg/g, from about 700 mg/g to about 1000 mg/g, from about 750 mg/g to about 800 mg/g, from about 750 mg/g to about 900 mg/g, from about 750 mg/g to about 1000 mg/g, from about 800 mg/g to about 900 mg/g, from about 800 mg/g to about 1000 mg/g, or from about 900 mg/g to about 1000 mg/g. Embodiments provide the ligand may be present at density of from about 10 mg/g, about 50 mg/g, about 100 mg/g, about 200 mg/g, about 250 mg/g, about 300 mg/g, about 400 mg/g, about 500 mg/g, about 600 mg/g, about 700 mg/g, about 750 mg/g, about 800 mg/g, about 900 mg/g, or about 1000 mg/g.
[0130] Preferably, the surface functionalized electrospun composite fibers have a dynamic binding capacity on a volume basis between about 1 pg/mL and about 400 mg/ml of the electrospun composite fibers; more preferably between about 5 pg/mL and about 400 mg/mL of the electrospun composite fibers. It should be understood that the binding capacity can vary depending the target molecule. For example, if the target molecule is a small molecule or microscopic material (e.g., virus) the amount captured compared to the mass of electrospun composite fibers will be small. However, in some applications the binding capacity can be greater due to capture of a larger molecule. Accordingly, in some embodiments, the electrospun composite fibers preferably have a binding capacity on a volume basis of at least 1 pg/mL of the electrospun composite fibers, at least about 5 pg/mL of the electrospun composite fibers, at least about 10 pg/mL of the electrospun composite fibers, at least about 15 pg/mL of the electrospun composite fibers, at least about 20 pg/mL of the electrospun composite fibers, at least about 25 pg/mL of the electrospun composite fibers, at least about 30 pg/mL of the electrospun composite fibers, at least about 35 pg/mL of the electrospun composite fibers, at least about 40 pg/mL of the electrospun composite fibers, at least about 45 pg/mL of the electrospun composite fibers, at least about 50 pg/mL of the electrospun composite fibers, at least about 55 pg/mL of the electrospun composite fibers, at least about 60 pg/mL of the electrospun composite fibers, at least about 65 pg/mL of the electrospun composite fibers, at least about 70 pg/mL of the electrospun composite fibers, at least about 75 pg/mL of the electrospun composite fibers, at least about 80 pg/mL of the electrospun composite fibers, at least about 85 pg/mL of the electrospun composite fibers, at least about 90 pg/mL of the electrospun composite fibers, at least about 95 pg/mL of the electrospun composite fibers, at least about, 100 pg/mL of the electrospun composite fibers, at least about 110 pg/mL of the electrospun composite fibers, at least about 120 pg/mL of the electrospun composite fibers, at least about 130 pg/mL of the electrospun composite fibers, at least about 135 pg/mL of the electrospun composite fibers, at least about 140 pg/mL of the electrospun composite fibers, at least about 150 pg/mL of the electrospun composite fibers, at least about 160 pg/mL of the electrospun composite fibers, at least about 170 pg/mL of the electrospun composite fibers, at least about 180 pg/mL of the electrospun composite fibers, at least about 190 pg/mL of the electrospun composite fibers, at least about 200 pg/mL of the electrospun composite fibers, at least about 250 pg/mL of the electrospun composite fibers, at least about 300 pg/mL of the electrospun composite fibers, at least about 350 pg/mL of the electrospun composite fibers, at least about 400 pg/mL of the electrospun composite fibers, at least about 450 pg/mL of the electrospun composite fibers, at least about 500 pg/mL of the electrospun composite fibers, at least about 550 pg/mL of the electrospun composite fibers, at least about 600 pg/mL of the electrospun composite fibers, at least about 650 pg/mL of the electrospun composite fibers, at least about 700 pg/mL of the electrospun composite fibers, at least about 750 pg/mL of the electrospun composite fibers, at least about 800 pg/mL of the electrospun composite fibers, at least about 850 pg/mL of the electrospun composite fibers, at least about 900 pg/mL of the electrospun composite fibers, at least about 950 pg/mL of the electrospun composite fibers, at least about 1 mg/mL of the electrospun composite fibers, at least about 2 mg/mL of the electrospun composite fibers, at least about 3 mg/mL of the electrospun composite fibers, at least about 4 mg/mL of the electrospun composite fibers, at least about 5 mg/mL of the electrospun composite fibers, at least about 6 mg/mL of the electrospun composite fibers, at least about 7 mg/mL of the electrospun composite fibers, at least about 8 mg/mL of the electrospun composite fibers, at least about 9 mg/mL of the electrospun composite fibers, at least about 10 mg/mL of the electrospun composite fibers, at least about 15 mg/mL of the electrospun composite fibers, at least about 20 mg/mL of the electrospun composite fibers, at least about 25 mg/mL of the electrospun composite fibers, at least about 30 mg/mL of the electrospun composite fibers, at least about 35 mg/mL of the electrospun composite fibers, at least about 40 mg/mL of the electrospun composite fibers, at least about 45 mg/mL of the electrospun composite fibers, at least about 50 mg/ml of the electrospun composite fibers, at least about 55 mg/ml of the electrospun composite fibers, at least about 65 mg/ml of the electrospun composite fibers, at least about 70 mg/ml of the electrospun composite fibers, at least about 75 mg/ml of the electrospun composite fibers, at least about 80 mg/ml of the electrospun composite fibers, at least about 85 mg/ml of the electrospun composite fibers, at least about 90 mg/ml of the electrospun composite fibers, at least about 95 mg/ml of the electrospun composite fibers, at least about 100 mg/ml of the electrospun composite fibers, at least about 110 mg/ml of the electrospun composite fibers, at least about 120 mg/ml of the electrospun composite fibers, at least about 130 mg/ml of the electrospun composite fibers, at least about 140 mg/ml of the electrospun composite fibers at least about 150 mg/ml of the electrospun composite fibers.
[0131] Preferably, the surface functionalized collection of electrospun composite fibers has a dynamic binding capacity for proteinaceous target components on a mass basis of at least about 10 mg/g of the electrospun composite fibers in hydrated form, at least about 95 mg/g of the electrospun composite fibers, at least about 100 mg/g of the electrospun composite fibers, at least about 105 mg/g of the electrospun composite fibers, at least about 110 mg/g of the electrospun composite fibers, at least about 115 mg/g of the electrospun composite fibers, at least about 120 mg/g of the electrospun composite fibers, at least about 125 mg/g of the electrospun composite fibers, at least about 130 mg/g of the electrospun composite fibers, at least about 125 mg/g of the electrospun composite fibers, at least about 130 mg/g of the electrospun composite fibers, at least about 135 mg/g of the electrospun composite fibers, at least about 140 mg/g of the electrospun composite fibers, at least about 145 mg/g of the electrospun composite fibers, at least about 150 mg/g of the electrospun composite fibers, at least about 155 mg/g of the electrospun composite fibers, at least about 160 mg/g of the electrospun composite fibers, at least about 165 mg/g of the electrospun composite fibers, at least about 170 mg/g of the electrospun composite fibers, and at least about 175 mg/g of the electrospun composite fibers.
[0132] Preferably, the surface functionalized collection of electrospun composite fibers has a dynamic binding capacity for nucleic acid components on a mass basis of at least about 2 mg/mL of electrospun composite fibers, at least about 5mg/mL of electrospun composite fibers, at least about lOmg/mL of electrospun composite fibers, at least about 15mg/mL of electrospun composite fibers and at least about 20mg/mL of electrospun composite fibers. [0133] In the case of virus particles or VLPs, binding capacity can be measured based on the number of particles bound per mL of electrospun composite fibers. Accordingly, binding capacity for virus particles and VLPs is between about 1 x IO10 particles per mL and about 1 x 1011 particles per mL, more preferably between about 1 x IO10 particles per mL and about 1 x 1012 particles per mL, still more preferably between about 1 x IO10 particles per mL and about 1 x 1013 particles per mL, between about 1 x 1010 particles per mL and about 1 x 1014 particles per ml and most preferably between about 1 x 1012 particles per mL and about 1 x 1014 particles per mL.
[0134] Ion Exchange Surface Functionalizations
[0135] Suitable ion exchange surface functionalizations include, but are not limited to, anion exchange groups, cation exchange groups, and mixtures thereof.
[0136] Anion exchange groups contain one or more moi eties which are positively charged and attract anionic molecules. Preferred anion exchange groups include, but are not limited to, tertiary and quaternary amines. Preferred anion exchange groups include, but are not limited to, amine-based moieties, including but not limited to quaternary ammonium groups such as alkyltrimethyl ammonium groups (sometimes referred to as Q groups) diethyl-(2-hydroxy-propyl) aminoethyl groups (sometimes referred to as a QAE exchange group), trimethylaminoethyl groups (sometimes referred to as TMAE groups), tertiary amine groups such as diethylaminoethyl (DEAE), and mixture thereof. Preferred amine based moieties include, but are not limited to — N+(CHs)3, — N+(C2H5)2H, — CH2CH2N+(CH3)3, — O— CH2CH2— N+(CH3)3, — CH2CH2N+(C2H5)2H, —
CH2CH2N+(C2H5)2(CH2CH(OH)CH3), and — CH2CH2N+(CH3)2H moieties.
[0137] Cation exchange groups contain one or more moieties which are negatively charged and attract cationic molecules. Preferred cation exchange groups include, but are not limited to, one or more carboxylate ( — COO ), sulphonate ( — SO3 ), phosphonate ( — P(OH)2O ) groups, or mixtures thereof. Preferred weak cation exchange groups include one or more of — CH2COO , — O-CH2COO , — CH2OCH2COO (sometimes referred to as a CM ion-exchange group). Preferred strong cation exchange groups include one or more of — SO3 (sometimes referred to as an S ion-exchange group), — CH2CH2CH2SO3 .(sometimes referred to as an SP ion-exchange group) — CH2CH2SO3 . — P(OH)2O , and mixtures thereof. [0138] Hydrophobic Surface Functionalizations
[0139] Hydrophobic surface functionalizations are used for separating molecules based on their relative hydrophobicity. Surface functional groups suitable for this include one or more hydrophobic moieties. Preferred hydrophobic moieties include, but are not limited to, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, octyl, decyl or phenyl groups, or mixtures thereof.
[0140] Affinity Ligand Surface Functionalizations
[0141] Affinity ligand surface functionalizations separate molecules based on their affinity to particular ligands. In a preferred embodiment, an affinity ligand is a chemical or biological ligand, which for example, can rely on a multiplicity of attractive forces including electrostatic, hydrophobic, hydrogen bonding and Van der Waal’s forces in combination with an advantageous spatial orientation of binding groups as exists, for example, in the case of the binding of antibodies to antigens, protein A to IgG or substrates and inhibitors to enzymes.
[0142] Suitable affinity ligands include, but are not limited to, chemical ligands, dyes, small molecule biomimetic ligands, immobilized metal ions, affimers, aptamers, affibodies, peptides, polypeptides, antibodies, enzyme inhibitors, lectins and bacterial immunoglobulin binding proteins.
[0143] Preferred affinity ligands include, but are not limited to, monoclonal antibodies, polyclonal antibodies, antibody fragments, dyes, histidine, a bacterial immunoglobulin binding protein, an immobilized metal ion, nucleic acids, oligonucleotides, nucleotides, lectins, proteins, peptides, polypeptides, oligopeptides, polysaccharides, oligosaccharides, sugars, antigens, aptamers, affimers, affibodies, small molecule biomimetic ligands, small organic compounds, chemical ligands, synthetic affinity ligands (typically with molecular weights <2,000 including, but not limited to, triazine ligands, aminophenyl boronate, and aminobenzamidine), enzyme inhibitors, drugs and other ligands. Examples of suitable affinity ligands are available in the published literature and are well known.
[0144] In a preferred embodiment the ligand can bind an immunoglobulin. Nonlimiting, examples of such ligands include Protein A, Protein G, and Protein L. As used herein, “Protein A” refers to recombinant Protein A (which may have an altered sequence compared to Protein A found in Staphylococcus aureus) and tagged Protein A (as described in EP-B-0873353 and U.S. Pat. No. 6,399,750, the detailed disclosures of which are incorporated herein by reference). Protein A may be a modified variant of Protein A, for instance cysteine modified variants of Protein A.
[0145] Small molecule biomimetic ligands are small molecule chemical ligands with a molecular mass < 2,000 D that mimic the binding of interactions of naturally occurring ligands.
[0146] A preferred antibody and antibody fragment includes, but is not limited to, IgG, IgM, monoclonal antibodies, camelid antibodies, antibody fragments, Fab, Fv, and combinations thereof.
[0147] Certain dyes can be employed as affinity ligands. Preferred dyes include, but are not limited to, Procion Blue HB, Procion Yellow HE-4R, Procion Red HE-3B, and Cibacron Blue F3G.
[0148] Another category of compounds useful as affinity ligands includes groups containing a metal ion, i.e., an immobilized metal ion. Preferred groups containing metal cations are chelating groups which contain and immobilize one or more metal cations. Preferably the metal cation contained is one or more of copper, nickel, zinc, iron and cobalt; most preferably Cu2+, Ni2+, Zn2+, Fe3+, Co2+ or a combination thereof. [0149] Mixed Mode Ligand Surface Functionalizations
[0150] Mixed mode ligands (sometimes referred to as multimodal ligands) utilize two or more types of binding interaction via a single ligand, i.e., a single ligand possessing at least two of an anion exchange group, a cation exchange group, a polar group, a hydrophobic group or a hydrogen bonding group, which separates molecules based on two or more characteristics of the target molecule. In a preferred embodiment, a mixed mode ligand comprises an ion exchange group in addition to a hydrophobic group or a hydrogen bonding group. In a preferred embodiment, a mixed mode ligand comprises a hydrophobic group in addition to a hydrogen bonding group, a polar group, or ion exchange group. In a preferred embodiment, a mixed mode ligand comprises a hydrogen bonding group in addition to an ion exchange group or hydrophobic group. Preferred mixed mode ligands include, but are not limited to, those comprising an N- benzyl methyl ethanolamine group, an N-benzoyl-homocysteine group, or combinations thereof. [0151] A surface functionalized collection of electrospun composite fibers for use in such methods may also contain one or more hydrophobic groups which are ionizable, for use in so-called Hydrophobic Charge Induction Chromatography (HCIC). Thus, in one embodiment, a mixed mode ligand comprises a HCIC compatible group. Preferred mixed mode ligands suitable for suitable for HCIC include, but are not limited to, 4- mercapto-ethyl-pyridine (MEP) groups and octylamine groups.
Membranes
[0152] The collection of composite fibers can be formed into a membrane for use in separation methods. Beneficially, the composite nanofiber membranes have complex interconnected, three-dimensional porous structures, relatively large surface areas, and increased mechanical properties.
[0153] The raw electrospun cellulose acetate fiber utilized can be converted back to cellulose, i.e., the cellulose is “regenerated,” after electrospinning.
[0154] The composite nanofibers are manufactured using an electrospinning technique. This refers to the manufacture of fibers based on exposure of an extruded polymer “spin dope” to an electrostatic field which results in elongation of the extruded polymer “jet” into a nanofiber.
[0155] In a preferred embodiment, a separation media comprises a fibrous membrane and is particularly suitable for use in bioseparations and exhibits one or more of the following technical features: (1) small diameter fibers to allow for the largest amount of specific area; (2) well-controlled and narrow pore size distribution between fibers to allow for even flow distribution during adsorptive applications; (3) fibers having excellent mechanical and chemical stability to withstand potentially high operating pressures and harsh cleaning conditions; and (4) fibers having a well-defined and spatially consistent size and chemical composition. In a more preferred embodiment, the membrane support comprises crosslinked fibers. In a most preferred embodiment, the membrane support comprises crosslinked cellulose nanofibers such as the crosslinked nanofibers described in WO2017/189977, which is incorporated by reference in its entirety.
[0156] Separation Methods
[0157] A crude feed is passed through a membrane comprised of a collection of electrospun composite fibers; preferably, the composite fibers are surface functionalized. The crude feed can be a gas or a liquid. Further, the crude feed comprises one or more target components. The crude feed passes through the membrane, target molecules are separated from the crude feed, and an effluent is discharged from the membrane. Optionally, the effluent travels to a downstream application. Downstream applications can include, but are not limited to, an instrument and/or sensor. In a preferred embodiment, the effluent is collected in a fraction collector.
[0158] One or more instruments can be utilized for identifying and/or quantifying the presence or absence of particular materials in the effluent (including, but not limited to, the target component). Preferably, any instruments are in fluid communication with the separation device(s) such an instrument is present before and/or after the separation device. Instruments can include, but are not limited to, a chromatography work station, a fast protein liquid chromatography system (“FPLC”), an Akta system, a process chromatography skid, a membrane chromatography skid, a high performance liquid chromatography instrument (“HPLC”), an ultra-performance liquid chromatography instrument (“UPLC”), a next generation chromatograph (“NGC”), a mass chromatograph (“MS”), a gas chromatograph (“GC”), a colorimeter, a mass spectrometer, a fluorometer, a photometer, a spectrometer, a spectrophotometer, a X-ray photoelectron spectrometer (“XPS”), a sequencing instrument, and a next generation sequencing (“NGS”) sequencing instrument. Preferred sensors can include a pH sensor, a conductivity sensor, a UV detector, a visible light detector, or combination thereof. Preferred instruments and sensors are non-destructive.
[0159] The instruments and/or sensors can be in communication with a system control that detects a particular change state (e.g., presence or absence of a material in the effluent, change in pH, change in conductivity, etc.), such that the system control can stop the flow of crude feed, start a buffer cycle and/or a clean cycle (such as a clean-in- place).
[0160] Any number of membranes can be arranged in a system. Preferably a system comprises at least 2 membranes, at least 3 membranes, at least 4 membranes, or at least 5 membranes; where the membranes are in operation in parallel, series or a combination thereof. In an embodiment, a system comprises between 1 and 100 membranes in series, parallel or a combination thereof. [0161] In a system, the membranes can be arranged in series and/or in parallel. If arranged in parallel, the system has the ability to perform a separation on significantly more fluid over a period of time due to the increased volume achieved by use of separation devices. If arranged in a series, the system has the ability to employ different separation techniques and target different biological or non-biological substances as fluid flows through each membrane. In a preferred embodiment, the system comprising one or more membranes can be arranged so that flow can be redirected from one membrane to another membrane without a substantial break, or any break, in the flow while a membrane is repaired, replaced, inspected, or otherwise removed from the fluid flow. The arrangement of the membranes in a system can be varied to suit the particular arrangement desired. The system preferably further comprises one or more of a pump, a process controller, a fraction collector, a feed line, a discharge line, and optionally one or more instruments and/or sensors.
[0162] Preferred pumps include, but are not limited to, a peristaltic pump, a diaphragm pump, a syringe pump, a positive displacement pump, a quattroflow pump, a centrifugal pump and a lobe pump connected in series and in fluid communication with a separation device and one or more instruments or sensors including, but not limited to, a UV detector, a visible light detector, a conductivity sensor, a pH sensor, a pressure sensor, a temperature sensor, a liquid flow sensor, a refractive index detector or a diode array detector, or any combination thereof, any or all of which may optionally be connected to a recording device and/or a controller device. Thus, the system need not be limited to a chromatography work station, an FPLC, an Akta system, a process chromatography skid, a membrane chromatography skid, or other traditional chromatography systems. [0163] A preferred method of use comprises a rapid cycling mode, where the membrane is optionally prepared for a separation via a buffer cycle (where an equilibrium buffer is passed through the membrane); then crude feed is passed through the membrane. After passing through the membrane the crude feed is considered an effluent. The effluent is then preferably provided to an instrument and/or sensor via a discharge feed and then to a fraction collector. When a particular state is reached (whether by time or sensing of the presence or absence of a condition), the crude feed is no longer fed through the membrane; optionally a purge cycle can be performed to remove any impurities and contaminants from the membrane; whether a purge cycle is performed or not, the target compound is then released from the membrane via an elution cycle; preferably another buffer and/or clean cycle is performed to release any residual target compound; the same or other buffer cycles can be performed to regenerate, sanitise or re-equilibrate (or combinations thereol) the membrane so that it is prepared for another separation cycle for capturing target components. These methods can be performed manually with a person overseeing the various steps, via a programmed method, or an automated system. [0164] Accordingly, a system for separating one or more target molecules can comprise a feed line, a membrane comprising a collection of electrospun composite fibers, a discharge line, optionally one or more instruments and/or sensors, and a fraction collector. The collection of electrospun fibers comprises one or more surface functionalizations. The feed line is in fluid communication with the membrane and provides the crude feed. The discharge line is in fluid communication with membrane and is what the effluent travels through to the fraction collector. The optional one or more instruments and/or sensors can be in fluid communication with the discharge line and/or fraction collector. Related methods of controlling fluid through the system and methods of separating a one or more target components can be performed with this system as disclosed herein.
[0165] It should be understood that while operating at a higher flow rate can be preferable, the flow rate can be determined by the nature of the system providing fluid pressure, which can be limiting on the flow rate achieved. The membrane disclosed herein can withstand high flow rates without mechanical compromise.
[0166] The composite fibers are capable of sustaining a flow rate of a fluid at a pressure of up to about 1.0 bar, up to about 1.5 bar, up to about 2.0 bar, up to about 2.5 bar, up to about 3.0 bar, up to about 3.5 bar, up to about 4.0 bar, up to about 4.5 bar, up to about 5.0 bar, up to about 5.5 bar, up to about 6.0 bar, up to about 6.5 bar, up to about 7.0 bar, up to about 7.5 bar, up to about 8.0 bar, up to about 8.5 bar, up to about 9.0 bar, up to about 9.5 bar, up to about 10 bar.
Example Embodiments
[0167] The inventions are defined in the claims. However, below is anon-exhaustive list of non-limiting embodiments in numbered format. Any one or more of the features of these embodiments may be combined with any one or more features of another example, embodiment, or aspect described herein. Accordingly, the following numbered clauses form part of the present disclosure but do not form part of the claims: [0168] 1. A membrane comprising: a nonwoven collection of composite fibers that are microfibers, nanofibers, or a mixture thereof; the composite fibers comprising i) cellulose, a cellulose derivative, or a mixture thereof and ii) a non-cellulose polymer comprising collagen, chitosan, agarose, agarose acetate, a vinyl polymer, a vinyl copolymer, an acrylic polymer, aramid, an acrylic copolymer, a polyacrylic acid, a polymethacrylic acid, polyacrylonitrile, a polyethylene oxide, a polyimide, polyethyleneimine, a polyamide, a polyester, polystyrene, a polysulfone, poly caprolactone, or a mixture thereof; and pores and channels formed between and/or among the composite fibers.
[0169] 2. The membrane of clause 1, wherein the pores and channels have an average diameter of from about 1 nm to about 10 micrometers as measured by a capillary flow porometry instrument.
[0170] 3. The membrane of any one of clauses 1-2, wherein the composite fibers have an average diameter of from about 10 nm to about 10 pm as measured by a capillary flow porometry instrument.
[0171] 4. The membrane of any one of clauses 1-3, wherein the non-cellulose polymer comprises polyacrylonitrile.
[0172] 5. The membrane of any one of clauses 1-4, wherein the non-cellulose polymer comprises polyacrylic acid.
[0173] 6. The membrane of any one of clauses 1-5, wherein the composite fibers comprise one or more surface functionalizations.
[0174] 7. The membrane of clause 6, wherein the one or more surface functionalizations comprise an ion exchange group, a hydrophobic group, an affinity ligand, a mixed mode ligand, or a combination thereof.
[0175] 8. The membrane of clause 7, wherein the one or more surface functionalizations comprise an anion exchange group, a cation exchange group, or a mixture thereof.
[0176] 9. The membrane of clause 8, wherein the ion exchange group comprises one or more of a tertiary amine, a quaternary amine, a quaternary ammonium, 6- chloranyl-3-[(2-pentyl-2,3-dihydro-l,3-thiazol-4-yl)methyl]quinazolin-4-one, diethyl- (2 -hydroxy-propyl) aminoethyl, diethylaminoethyl, trimethylaminoethyl — N+(CH3)3, — N+(C2H5)2H, — CH2CH2N+(CH3)3, — O— CH2CH2— N+(CH3)3, — CH2CH2N+(C2H5)2H, — CH2CH2N+(C2H5)2(CH2CH(OH)CH3), — CH2CH2N+(CH3)2H, a carboxylate, a sulphonate, a phosphonate, — CH2COO , — O-CH2COO , — CH2OCH2COO , — SO3 , — CH2CH2CH2SO3 , — CH2CH2SO3 , — P(OH)2O , or a mixture thereof.
[0177] 10. The membrane of clause 7, wherein the one or more surface functionalizations comprise a hydrophobic group.
[0178] 11. The membrane of clause 10, wherein the hydrophobic group comprises a propyl group, an isopropyl group, a butyl group, an isobutyl group, a pentyl group, a hexyl group, an octyl group, a decyl group, a phenyl group, or a mixture thereof.
[0179] 12. The membrane of clause 7, wherein the one or more surface functionalizations comprise an affinity ligand.
[0180] 13. The membrane of clause 12, wherein the affinity ligand comprises a monoclonal antibody, a polyclonal antibody, an antibody fragment, a bacterial immunoglobulin binding protein, a chemical ligand, a dye, an enzyme inhibitor, histidine, an immobilized metal ion, a nucleic acid, an oligonucleotide, a nucleotide, a lectin, a protein, an oligopeptide, a polysaccharide, an oligosaccharide, a sugar, a peptide, a polypeptide, an antigen, an aptamer, an affimer, an affibody, small molecule biomimetic ligand, a small organic compound, a synthetic affinity ligand, or a mixture thereof.
[0181] 14. The membrane of clause 13, wherein affinity ligand is Protein A, Protein G, or Protein L.
[0182] 15. The membrane of clause 13, wherein the affinity ligand comprises IgG, IgM, a monoclonal antibody, a camelid antibody, an antibody fragment, Fab, Fv, or a mixture thereof.
[0183] 16. The membrane of clause 7, wherein the one or more surface functionalizations comprise a mixed mode ligand.
[0184] 17. The membrane of clause 16, wherein the mixed mode ligand comprises at least two of an anion exchange group, a cation exchange group, a polar group, a hydrophobic group, and a hydrogen bonding group.
[0185] 18. The membrane of clause 16 or 17, wherein the mixed mode ligand comprises an N-benzyl methyl ethanolamine group, an N-benzoyl-homocysteine group or mixture thereof. [0186] 19. The membrane of any one of clauses 6-18, wherein the one or more surface functionalizations are atached to the composite fibers by a linker.
[0187] 20. The membrane of any one of clauses 1-19, wherein the cellulose comprises cellulose acetate.
[0188] 21. The membrane of any one of clauses 1-19, wherein the cellulose comprises ethyl cellulose.
[0189] 22. The membrane of any one of clauses 1-21, wherein the cellulose is regenerated cellulose.
[0190] 23. The membrane of any one of clauses 1-22, wherein the ratio of cellulose to non-cellulose polymer is from about 4.5 : 1 to about 1:2, preferably about 2: 1 to about 4.5: 1 or about 1.5: 1 to about 1:2.
[0191] 24. The membrane of any one of clauses 1-23, wherein the composite fibers have a homogeneous average fiber diameter as measured by SEM.
[0192] 25. A method of preparing a collection of composite fibers comprising: electrospinning a polymer solution comprising a cellulose derivative, a non-cellulose polymer, and a solvent to form the collection of composite fibers; wherein the non- cellulose polymer comprises polyacrylic acid, polyacrylonitrile, aramid, polyamide, polystyrene, chitosan, or a mixture thereof; and optionally, forming a membrane with the collection of composite fibers; the membrane including pores and/or channels.
[0193] 26. The method of clause 25, wherein the polymer solution further comprises a sacrificial polymer.
[0194] 27. The method of clause 26, wherein the sacrificial polymer has a melting point of below about 100 °C, preferably below about 65 °C.
[0195] 28. The method of clause 27, further comprising, after the electrospinning step, heating the membrane to a temperature sufficient to melt the sacrificial polymer and remove it from the membrane.
[0196] 29. The method of clause 27 or 28, wherein the heating is performed at a temperature up to about 65°C.
[0197] 30. The method of clause 26, wherein the sacrificial polymer is dissolvable.
[0198] 31. The method of clause 30, further comprising, after the electrospinning step, dissolving the sacrificial polymer to remove it from the membrane. [0199] 32. The method of any one of clauses 25-31, further comprising a step, before the electrospinning step, of forming the polymer solution by mixing the cellulose derivative and polyacrylonitrile with the solvent.
[0200] 33. The method of any one of clauses 25-32, wherein the solvent comprises acetone, dimethylformamide (DMF), dimethylacetamide (DMAc), dimethyl carbonate (DMC), cyclopentanone (CPO), dimethylsulfoxide (DMSO), ethanol, methanol, water, or a mixture thereof.
[0201] 34. The method of any one of clauses 25-33, wherein the cellulose derivative and non-cellulose polymer are added in a ratio of from about 4.5 : 1 to about 1 :2, preferably about 2: 1 to about 4.5:1 or about 1.5: 1 to about 1:2, cellulose derivative to noncellulose polymer.
[0202] 35. The method of any one of clauses 25-34, wherein the polymer solution has a viscosity of from about 300 cP to about 3500 cP at about 20 °C as measured by a viscometer.
[0203] 36. The method of any one of claims 25-33, wherein the polymer solution has a conductivity of from about 10 ps/cm to about 100 ps/cm at about 20 °C as measured by an electrical conductivity meter.
[0204] 37. The method of any one of clauses 25-36, wherein the cellulose derivative is a cellulose ester, an alkyl cellulose, an organic cellulose ether, or a mixture thereof.
[0205] 38. The method of any one of clauses 25-37, wherein the cellulose derivative is cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate, cellulose acetate butyrate, cellulose nitrate, cellulose sulfate, hydroxyethyl cellulose, carboxymethyl cellulose, ethyl cellulose, or a mixture thereof.
[0206] 39. The method of any one of clauses 25-38, wherein the cellulose derivative comprises cellulose acetate.
[0207] 40. The method of any one of clauses 25-39, wherein the cellulose derivative comprises ethyl cellulose.
[0208] 41. The method of any one of clauses 25-40, further comprising, after the electrospinning, regenerating the cellulose derivative into cellulose.
[0209] 42. The method of any one of clauses 25-41, further comprising surface functionalizing the cellulose derivative or cellulose. [0210] 43. The method of any one of clauses 25-42, wherein the electrospinning is performed by a non-needle based electrospinning system.
[0211] 44. The method of clause 43, wherein the electrospinning system is an open surface system or a wire-based electrode system.
[0212] 45. The method of any one of clauses 25-42, wherein the electrospinning is performed by a needle-based electrospinning system.
[0213] 46. Use of the fibers of any one of clauses 1-45 for the capture, separation, purification, filtration, concentration, characterization, quantitation or analysis of biological compounds.
[0214] 47. The use of clause 46, wherein the biological compounds comprise one or more of an amino acid, a peptide, an affimer, a biomolecule and/or fragment thereof, a protein, an enzyme, a glycoprotein, a lipopolysaccharide, an antibody and/or a fragment thereof, an LNP, a nucleic acid, an organic polymer, a virus, a VLP, an extracellular vesicle, an exosome, a bacterium, a cell, a cell-related structure, or a mixture thereof.
[0215] 48. The use of clause 47, wherein the antibody and/or fragment thereof comprises a monoclonal antibody, a single-chain antibody, a bi-specific antibody, a multi-specific antibody, an antibody conjugate, an antibody fusion protein, a drugantibody conjugate, an antibody fragment, a Fab fragment, a Fv fragment, a gamma globulin (IgG), an IVIG, an IgM, an IgA, an IgE, a hyperimmune gamma globulin, an isoagglutinin, or a mixture thereof.
[0216] 49. The use of clause 47, wherein the virus comprises a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vesicular stomatitis virus (VSV), a herpes simplex virus (HSV), a vaccinia virus, a reo virus, a pox virus, an influenza virus, a respiratory syncytial virus, a parainfluenza virus, a foamy virus, a measles virus or a retrovirus, or a mixture thereof.
EXAMPLES
[0217] Preferred embodiments of the present disclosure are further exemplified in the following non-limiting Examples. It should be understood that these Examples, while indicating certain preferred embodiments, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the inventions, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
[0218] Example 1
[0219] Composite fibers were prepared by electrospinning different polymer solutions. The different polymer solutions were prepared to assess the solubility of a cellulose derivative and PAN, the spinnability of the solutions, the viscosities of the solutions, the conductivity of the solutions, and the properties of the resultant fibers.
[0220] Two polymer solutions of 7.5 wt.% cellulose acetate and 3 wt.% PAN were formed with a solvent mixture of DMC, CPO, and DMSO.
[0221] The first polymer solution was prepared by dissolving cellulose acetate in DMC and CPO, and dissolving PAN in DMSO. The resultant solutions were mixed to form a polymer solution, which was analyzed. The polymer solution had a viscosity of 1336 ± 13.64 cp, 98.0%, 22 rpm, 19.2 °C, and a conductivity of 49.6 pS/cm at 19.0 °C. The viscosity was measured by a Brookfield DV2T viscometer with an LV62 spindle from Ametek for a duration of 2 minutes with each measurement being recorded every 10 seconds for the 2 minute duration. The conductivity was measured by a Jenway 4510 electrical conductivity meter with a glass probe; the probe was immersed in a 50 mL polymer solution until the meter’s monitor confirms stability of the sample measurement and a readout of the conductivity and temperature.
[0222] The second polymer solution was prepared by directly dissolving cellulose acetate and PAN in a solvent mixture of DMC, CPO, and DMSO. The polymer solution had a viscosity of 1466 ± 15.00 cp, 97.7%, 20 rpm, 19.6 °C, and a conductivity of 48.3 pS/cm at 19.5 °C. The viscosity and conductivity were measured as described above.
[0223] A third polymer solution was prepared, this time with 10 wt.% cellulose acetate and 4 wt.% PAN were formed with a solvent mixture of DMC, CPO, and DMSO. This polymer solution was prepared by separately dissolving the cellulose acetate in DMC and CPO, and dissolving the PAN in DMSO, then combining the two solutions. The polymer solution had a viscosity of 6346±65.2, 97.3%, 4.6 rpm, at 21.2 °C, and a conductivity of 21.3 ps/cm, 20.4 °C. The viscosity and conductivity were measured as described above.
[0224] These three polymer solutions were electrospun with an open surface system to form composite fibers.
[0225] Example 2
[0226] Composite fibers were prepared by electrospinning different polymer solutions. The different polymer solutions were prepared to assess the solubility of a cellulose derivative and PAN, the ability to include a sacrificial polymer, the spinnability of the solutions, the viscosities of the solutions, the conductivity of the solutions, and the properties of the resultant fibers.
[0227] Cellulose acetate and PAN were again dissolved maintaining a ratio of 2.5: 1 (CATAN) for purposes of consistency with the prior test data. For this polymer solution, the solvent mixture was DMF and DMAc. Small amounts (0.25-1 wt.%) of polyethylene oxide (PEO) were added to assess the ability to include a sacrificial polymer. The PEO was found to significantly affect solution viscosity but not the electrical conductivity.
[0228] It was observed that the polymer solution maintained good stability for multiple days, except with the highest tested concentration of PEO (1.0 wt.%).
[0229] Each of the solutions were found to have excellent spinnability. The as-spun fibers were smooth (bead-free) and had mean diameters of between about 1 pm and about 1.5 pm. Fiber diameter was measured via a Phenom G6 ProX SEM. The electrospun membrane was gold sputter-coated under vacuum until a thickness of about 5 nms was achived. The sample was then placed in the SEM and five images were taken at a magnification of 5000x at an accelerating voltage of 5kV; Fibrometeric software was then used to calculate the mean fiber diameter based on the SEM images. Production of smaller diameter fibers (including nanofibers) is achievable by adjustments to the composition of the co-solvent and/or process parameters for the electrospinning; however, these were kept consistent in this testing for comparative purposes.
[0230] The fibers were characterized via EDX, which verified the presence of both cellulose acetate and PAN in the individual fibers confirming that composite fibers were formed.
[0231] A second set of polymer solutions was prepared where cellulose acetate and PAN were again dissolved maintaining a ratio of 2.5:1 (CATAN) for purposes of consistency with the other test data. For this polymer solution, the solvent mixture was DMF, DMC, and CPO. Small amounts (0.25-1 wt.%) of polyethylene oxide (PEO) were also added to assess the ability to include a sacrificial polymer. Again, the PEO was found to significantly affect solution viscosity but not the electrical conductivity.
[0232] Again, the polymer solutions were observed to maintain good stability for multiple days.
[0233] Each of the solutions were found to have excellent spinnability . The as-spun fibers were smooth (bead-free) and had mean diameters of between about 1 pm and about 6 pm; calculated as described above. Again, we note that production of smaller diameter fibers (including nanofibers) is achievable by adjustments to the composition of the co-solvent and/or process parameters for the electrospinning; however, these were kept consistent in this testing for comparative purposes.
[0234] These fibers were also characterized via EDX, which verified the presence of both cellulose acetate and PAN in the individual fibers confirming that composite fibers were formed.
[0235] A third set of polymer solutions was prepared where cellulose acetate and PAN were again dissolved maintaining a ratio of 2.5: 1 (CATAN) for purposes of consistency with the other test data. For this polymer solution, the solvent mixture was DMSO, DMC, and CPO. Small amounts (0.25-1 wt.%) of polyethylene oxide (PEO) were also added to assess the ability to include a sacrificial polymer. Again, the PEO was found to significantly affect solution viscosity but not the electrical conductivity.
[0236] Again, the polymer solutions were observed to maintain good stability for multiple days.
[0237] Each of the solutions were found to have excellent spinnability. The as-spun fibers were smooth (bead-free) and had mean diameters of about 1 pm (calculated as described above); this was particularly found for the solutions that included PEO versus those without PEO, some of which beaded. Again, we note that production of smaller diameter fibers (including nanofibers) is achievable by adjustments to the composition of the cosolvent and/or process parameters for the electrospinning; however, these were kept consistent in this testing for comparative purposes.
[0238] Example 3 [0239] A polymer solution was prepared where cellulose acetate and PAN were dissolved in a ratio of 3: 1 (CATAN). For this polymer solution, the solvent mixture was DMC, CPO, and DMF. PEO was also dissolved in an amount of 1 wt.% relative to the CA. The solution was electrospun via a non-needle-based system to form a collection of composite fibers. The collection of composite fibers was assessed via scanning electron microscope. FIG. 1 provides an SEM image of these fibers where it can be seen that the fibers are formed well and generally consistent such that there is a homogeneous fiber diameter which can be observed.
[0240] Example 4
[0241] Multiple polymer solutions were prepared with polyacrylic acid and ethyl cellulose mixed together in different solvent/co-solvent mixtures, including, DMF, DMSO, DMSO/EtOH, DMAc/EtOH, acetone/EtOH, DMC/EtOH. The co-solvent ratio was dependent on the solvent combinations, including for example, about 1 : 1 to about 7:3 (w/w) of DMSO/EtOH; about 3:7 to about 1: 1 (w/w) of DMAc/EtOH; about 3:7 to about 7:3 (w/w) of acetone/EtOH; about 3:7 to about 7:3 (w/w) of DMC/EtOH; about 3:7 to about 1 : 1 (w/w) of DMC/MeOH. In these example polymer solutions, the ratio of PAA:EC was varied from about 1:2 to about 2: 1. Water could also be added into the cosolvent (e.g., with acetone, ethanol, and/or methanol) to tune the process stability of electrospinning.
[0242] One example polymer solution was prepared to test the dissolvability of both polyacrylic acid and ethyl cellulose in a single polymer solution and to examine the stability of that polymer solution. For this test, polyacrylic acid and ethyl cellulose were added to a solution of DMSO and dissolved. The concentration of polyacrylic acid was about 6 wt.% and the concentration of ethyl cellulose was about 6 wt.%. The solution was stable for 24 hours and then electrospun. The solution provided good spinnability and asspun fibers were smooth and had mean diameters of about 1.3 to about 1.7 pm per SEM data (the SEM data was collected the same as described above). Based on the appearance and spinnability of the solution, it is expected the solution would have maintained stability longer than the initial 24 hours tested.
[0243] Next solvent/co-solvent systems were prepared and tested. The polymer concentration was retained at about 12 wt.% for each solution (including about 6 wt.% polyacrylic acid and about 6 wt.% ethyl cellulose). Solvent/co-solvent mixtures were tested inclusive of the following ranges and mixtures:
DMSO/EtOH: from about 5:7 to about 7:3 (w/w);
DMAc/EtOH: from about 3:7 to about 1:1 (w/w);
Acetone/EtOH: from about 3:7 to about 7:3 (w/w);
DMC/EtOH: from about 3:7 to about 7:3 (w/w);
DMC/MeOH: from about 3:7 to about 1:1 (w/w);
Again, the polymer solutions were stable for 24 hours and electrospun at that point. Based on the appearance and spinnability of the solutions, it is expected the solutions would have maintained stability longer than the initial 24 hours tested. The polymer solutions provided good spinnability and as spun fibers which were smooth, bead-free, and had diameters from about 0.9 to about 5 pm per SEM data (the SEM data was collected the same as described above). The as-spun fibers were tested for storage stability in both water and NaOH and were stable for at least 24 hours.
[0244] Fibers were prepared with different ratios of polyacrylic acid and ethyl cellulose to evaluate the impact of the polymer type on the fibers. Ratios prepared and tested were of polyacrylic acid to ethyl cellulose included, about 5:7, about 4:8, about 3:9, about 2:10, about 1:11, and about 0:25). It was found that the higher concentration of polyacrylic acid will provided increased stability of the fibers.
[0245] Example 5
[0246] Electrospun fibers formed with polyacrylic acid and ethyl cellulose were prepared, formed into prototype membranes, and lysozyme was adsorbed to evaluate protein binding capacity. The fibers were prepared with as follows:
About 5 wt.% polyacrylic acid and about 7 wt.% ethyl cellulose in a solvent/co- solvent mixture of DMC/EtOH (at a 1:1 (w/w) ratio).
About 4 wt.% polyacrylic acid and about 8 wt.% ethyl cellulose in a solvent/co- solvent mixture of DMC/EtOH (at a 1:1 (w/w) ratio).
About 3 wt.% polyacrylic acid and about 9 wt.% ethyl cellulose in a solvent/co- solvent mixture of DMC/EtOH (at a 1:1 (w/w) ratio).
About 2 wt.% polyacrylic acid and about 10 wt.% ethyl cellulose in a solvent/co- solvent mixture of DMC/EtOH (at a 1:1 (w/w) ratio). About 1 wt.% polyacrylic acid and about 11 wt.% ethyl cellulose in a solvent/co- solvent mixture of DMC/EtOH (at a 1:1 (w/w) ratio).
0 wt.% polyacrylic acid and about 25 wt.% ethyl cellulose in a solvent/co-solvent mixture of DMC/EtOH (at a 1:1 (w/w) ratio).
The highest concentration of polyacrylic acid tested (about 5 wt.% polyacrylic avid and about 7 wt.% ethyl cellulose) provided the highest binding capacity (between about 16 mg/ml and about 23 mg/ml lysozyme over the various membranes prepared and tested). The binding capacity steadily decreased thereafter with the 0 wt.% polyacrylic acid and 25 wt.% ethyl cellulose having 0 mg/ml lysozyme bound.
[0247] To further test increased lysozyme binding capacity, layered membranes were prepared from fibers electrospun from a polymer solution having about 5 wt.% polyacrylic acid and about 7 wt.% ethyl cellulose in a solvent/co-solvent mixture of DMC/EtOH (at a 1: 1 (w/w) ratio). Layered membranes were prepared with four layers, lysozyme was attached and the binding capacity was assessed. Consistently the binding capacities were in the range of about 18 mg/ml to about 55 mg/ml. In a number of embodiments, the binding capacities exceeded 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, and in some instances even 60 mg/ml and 70 mg/ml.
[0248] From the foregoing, it can be seen that the present disclosure accomplishes at least all of the stated objectives.
[0249] Example 6
[0250] Multiple solutions were prepared with CA, PAN, and PEO to test the solution properties, their spinnability, and the resultant fiber based on the variations in weight percentage of the different polymers and solvents. The solvent used was a mixture of DMF and DMAc. After preparation, the spinnability of the solutions, the viscosities of the solutions, the conductivity of the solutions, and the properties of the resultant fibers were evaluated. The viscosity was measured by a Brookfield DV2T viscometer with an LV62 spindle from Ametek for a duration of 2 minutes with each measurement being recorded every 10 seconds for the 2 minute duration. The conductivity was measured by a Jenway 4510 electrical conductivity meter with a glass probe; the probe was immersed in a 50 mL polymer solution until the meter’s monitor confirms stability of the sample measurement and a readout of the conductivity and temperature. The ingredient ratios and the evaluated properties are summarized in the table presented in FIG. 2. [0251] FIGS. 3A-3O are SEM images of the resultant fibers. As can be seen in these figures, solutions 1-1, 1-2, 1-3 and 1-6 had poor deposition resulting and did not form rounded fibers. Thus, these solutions were found to have poor spinnability. The remaining solutions provided good spinnability and resulted in good fiber formation. [0252] Further polymer solutions were prepared again utilizing CA, PAN, and PEO in a solvent mixture of DMF and DMAc. These solutions were also tested for their spinnability, viscosity, conductivity, and the resultant fiber based on the variations in weight percentage of the different polymers and solvents. The ingredient ratios and the evaluated properties are summarized in the table presented in FIG. 4.
[0253] FIGS. 5A-5G are SEM images of the resultant fibers. As can be seen in these figures, each of these solutions provided good spinnability and resulted in rounded fibers.
[0254] Example 7
[0255] Testing was performed to evaluate the ability to scale up the fiber formation from some of the preferred polymer solutions identified in Example 6, Specifically, solutions 1-14, 1-15, and 2-3 were prepared according to the same ingredient concentrations and ratios, but in larger quantity (600 g of solution rather than the previously tested 40 g).
[0256] The larger batches were prepared as follows:
Test Solution A: 600g solution: 72g CA (12 wt.%), 12g PAN (2 wt.%), 367.8g DMF, 147.12g DMAc, 1.08g PEO (1.5 wt.% of CA mass);
Test Solution B: 600g solution: 66g CA (11 wt.%), 18g PAN (3 wt.%), 367.8g DMF, 147.15g DMAc, 0.99g PEO (1.5 wt.% of CA mass); and
Test Solution C: 600g solution: 42g CA (7 wt.%), 42g PAN (7 wt.%), 368.4g DMF, 147.45g DMAc, 0.15g PEO (0.36 wt.% of CA mass)
[0257] Test Solution A corresponded to the ingredient ratios of solution 1-14, Test Solution B corresponded to the ingredient ratios of solution 1-15, and Test Solution C corresponded to the ingredient ratios of solution 2-3. Multiple membranes were prepared utilizing each of these test solutions.
[0258] The resultant membranes were analyzed with a scanning electron microscope and the images are provided in FIGS. 6A-6F (membrane prepared from Test Solution A), FIGS. 7A-7D (membrane prepared from Test Solution B), and FIGS. 8A-8D (membrane prepared from Test Solution C). Three membranes were prepared from Test Solution A and FIGS. 6A and 6B were taken of the same membrane, FIGS. 6C and 6D were taken of the same membrane, and FIGS. 6E and 6F were taken of the same membrane. Two membranes were prepared from Test Solution B and FIGS. 7A and 7B were taken of the same membrane and FIGS. 7C and 7D were taken of the same membrane. Two membranes were prepared from Test Solution C and FIGS. 8A and 8B were taken of the same membrane and FIGS. 8C and 8D were taken of the same membrane. As can be seen from FIGS. 6A-8D, each of these polymer solutions provided good spinnability and demonstrated scalability for manufacturing of membranes.
[0259] Example 8
[0260] Composite nanofiber membrane was prepared according to Examples 6 & 7 from a solution comprising: CA (10 wt.%), PAN (4 wt.%), DMF (61.4 wt.%), DMAc (24.575 wt%) and PEO (0.025%). The cellulose acetate component was deacetylated to form regenerated cellulose by incubating the electrospun membrane with 1.0M NaOH for 1 hour at 20 °C, followed by washing with water to neutral pH. This procedure also served to remove PEO from the electrospun fibers. The resulting membrane was dried in air at 100 °C for 30 minutes. The dried cellulose/PAN composite nanofiber membrane had a mean thickness of 197 pm and a mean GSM of 17.1 g.m'2. This was subsequently derivatized with DEAE groups by reacting with an aqueous solution of 6.7% (w/v) 2-Diethylaminoethyl Chloride in 0.67M NaOH for 1 hour at 40 °C following which any unreacted reagent was removed by washing with water at 20 °C to neutral pH and the DEAE nanofiber membrane dried in air at 40 °C. DEAE derivatized composite nanofiber membrane, 0.031 ml, was packaged into a suitably sized filter housing and 10ml of 1.1 mg/ml bovine serum albumin (BSA) solution in lOmM Tris buffer, pH 8.0 passed through over a period of 8 minutes at 4 °C. A BSA binding capacity of 66mg BSA/ml membrane volume was measured, confirming surface-functionalized composite nanofiber membrane is highly effective for the binding of biological compounds.
[0261] The above disclosure is not intended to refer to any single embodiment of the particular invention but encompass all possible embodiments as described in the specification and the claims. The “scope” of the present disclosure is defined by the appended claims, along with the full scope of equivalents to which such claims are entitled. The scope of the disclosure is further qualified as including any possible modification to any of the aspects and/or embodiments disclosed herein which would result in other embodiments, combinations, subcombinations, or the like that would be obvious to those skilled in the art. The above specification provides a description of the manufacture and use of the disclosed compositions and methods. Since many embodiments can be made without departing from the spirit and scope of the disclosure, the invention resides in the claims.