JP2020079475A - Hybrid felt of electro-spun nanofibers - Google Patents

Abstract

To provide a composition used for biological and chemical separation and used in other applications as a whole, and more specifically, to provide a hybrid felt made of electro-spun nanofibers having high transmittance and high capacity.MEANS FOR SOLVING THE PROBLEM: Such a hybrid felt uses a derivatized cellulose and at least one non-cellulosic polymer that can be removed from the felt by being subjected to a moderately elevated temperature and/or a solvent capable of dissolving the non-cellulosic polymer and leaves a porous nanofiber felt having a uniform pore diameter and other improved properties as compared to a single-component nanofiber felt.SELECTED DRAWING: Figure 1

Description

  The present invention relates generally to compositions for use in biological and chemical separations and other applications. More specifically, the present invention relates to hybrid felts made from electrospun nanofibers having high permeability and high capacity.

  Microfiber and nanofiber membranes or "felts" have various uses in both biological and industrial applications. For example, felts are textile reinforcements, protective clothing, catalytic media, agricultural applications, environmental, sensors for medical and military surveillance, biomedical applications (e.g. bioseparation, tissue engineering and wound dressings), electronic applications (e.g. Capacitors, transistors and diodes), and space applications (eg, support structures for solar sails and space mirrors). Microfibers and nanofiber felts are particularly convenient for purifying biological materials such as proteins, nucleic acids, carbohydrates, bacteria, viruses and cells. They are useful in all liquid and gaseous fluid applications.

  The biopharmaceutical therapeutics industry is expanding as more and more biopharmaceuticals are approved for sale. Furthermore, a wide range of biologically-based diagnostic tools are used to perform high-throughput, highly sensitive diagnostic tests for a variety of pathologies. For both therapeutic and diagnostic agents, biological materials (eg, recombinant proteins, monoclonal antibodies, viral vaccines and nucleic acids) must be efficiently produced and purified and used.

  Conventional purification methods include, for example, packed microbeads for adsorption/chromatography, ultrafiltration, and precipitation/crystallization to extract the desired biological material from by-products and other contaminants. The method of separating is mentioned. Although these conventional separation methods provide results suitable for biological applications, they are limited in terms of yield, processing time and purity. These limits are primarily due to the slow diffusion rate of relatively large biomolecules, which allows the substance to be purified (ie, the “target substance”) to reach available binding sites deep within the separation matrix. Is limited. Furthermore, these systems can only be used for a limited number of cycles, some of them only once.

  Ion exchange (IE) and hydrophobic interaction (HI) adsorption/chromatography are two examples of more robust conventional separation techniques widely used for the separation of biological materials. These are generally less efficient than specific affinity-based separation techniques, such as antibody-based separations, but when the separation conditions are carefully selected, many target substances are undesired side-effects. It is also useful for purifying products and impurities.

  Affinity-based adsorption/chromatography may be more efficient than IE and HI, but is generally difficult to manufacture due to the complex production and purification of biological ligands such as monoclonal antibodies and nucleic acids It is expensive. Such ligands are often very sensitive to environmental conditions (eg, temperature, pH, ionic strength, etc.) and can easily degrade, thus destroying the affinity interactions required for adsorption. To be done. Furthermore, disrupting binding interactions can be difficult without harsh conditions that can reduce biological activity and thus the usefulness of the target substance and/or reusability of the purification medium.

Membranes that are useful for purifying biological materials have been described (see, eg, Bioprocessing for Value-Added Products from Renewable Resources, Shang-Tian Yang, Ed., Chapter 7). Recently, membrane adsorption/chromatography using nanometer-sized fibers built into mats with controlled thickness (i.e., ``nanofiber felts'') holds considerable promise for use in bioseparations (Todd J. Menkhaus, et al., "Chapter 3: Applications of Electrospun Nanofiber Membranes for Bioseparations ^" , in Handbook of Membrane Research, Stephan V. Gorley, Ed.). Such nanofiber felts are superior to microfiber felts because they allow tighter control of pore size, affinity properties, and other performance criteria.

  While the single-component nanofiber felts described above have provided promising results, they are often less efficient than would be desirable in terms of felt stability and material and time requirements. This is especially true if the target substance is only present in low concentrations in the starting material to be purified and is rich in contaminants and/or synthetic by-products. Therefore, there is a need to improve felt stability and purification efficiency of biological products. The needs disclosed are met by the embodiments disclosed below.

  The following brief summary provides a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview and is not intended to identify key/critical elements or delineate the scope of the claimed subject matter. Its purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented below.

  In one embodiment, the present invention is an electrospun hybrid nanofiber felt formed from composite nanofibers and single component nanofibers. The composite nanofibers can be electrospun from a mixture of derivatized cellulose and a first non-cellulosic polymer, and the single component nanofibers can be the same as or different from the first non-cellulosic polymer. It can be electrospun from a polymer. Generally, the first and second non-cellulosic polymers are differentially removable from the nanofiber felt. This means that one of the non-cellulosic polymers is significantly (eg a difference in removability of 10% or more, eg 20% or 50%) removed from the other (solvent or heat or combination of solvent and heat (To be used) means that the condition exists.

  The derivatized cellulose in the composite nanofibers can be an organic ester of cellulose, an inorganic ester of cellulose or an alkyl cellulose. The organic ester of cellulose can be cellulose acetate, cellulose triacetate or cellulose proprionate.

  The derivatized cellulose can be cellulose nitrate and cellulose sulfate when it is an inorganic ester, and can be hydroxyethyl cellulose or carboxymethyl cellulose when it is an alkyl cellulose.

  The first non-cellulosic polymer is a vinyl polymer, polyether, acrylic polymer, polyester, polycarbonate, polyurethane, polysaccharide (e.g. starch or chitin), polyamide (e.g. protein or gelatin), polylactide, polyglycolide or their. It can be a synthetic or natural polymer such as a copolymer.

  In one embodiment, the second non-cellulosic polymer forming the single component nanofiber is a synthetic polymer such as vinyl polymer, polyamide, polyimide, polyester or copolymers thereof.

  In another embodiment, the invention is an electrospinning method of making the above nanofiber felt, which method can be described in the following steps. a) preparing the composite polymer spin dope and the single-component polymer spin dope separately, b) putting the spin dope into two different spinnerets, c) applying a voltage to each spin dope with an electrode, d) spinning Separately electrospinning composite and single component nanofibers from a spinneret, and e) collecting the solidified nanofibers as randomly superposed or partially aligned nanofiber felts.

  The as-formed nanofiber felt can be further processed by regenerating (ie, converting and converting back to cellulose) the derivatized cellulose in the composite nanofibers. The method of making the nanofiber felt may include the additional step of removing some or all of the non-cellulosic first polymer from the composite nanofibers. Alternatively or in addition to these steps, the method of making the nanofiber felt may include surface functionalizing one or more of the polymeric nanofibers in the nanofiber felt. Such surface functionalization may involve the binding of an affinity ligand with a specific affinity for a particular target molecule that is purified from the fluid.

  In another embodiment, the invention is a method of purifying a biomolecule from a fluid, which method can be described in the following steps. Preparing a nanofiber felt according to the method described above, b) flowing the fluid through the nanofiber felt, and recovering biomolecules from the nanofiber felt.

  Other aspects of the invention can be found elsewhere in the specification.

It is a schematic diagram showing an electrospinning method.

  The present invention relates generally to hybrid felts composed of electrospun nanofibers used, for example, in chemical and biological separations. The hybrid nanofiber felt has high resolution and gives reproducibility under high flow and pressure over multiple cycles. Such nanofiber felts exhibit a complex, interconnected three-dimensional porous structure and a relatively large surface area.

  In particular, hybrid nanofiber felts are composed of more than one polymer type (ie, “hybrid” felts).

  The felts of the present invention are composed of more than one polymer type (ie, "hybrid" felts). This includes hybrid felts made into "hybrid" felts from a combination of single component nanofibers and "composite" nanofibers (eg nanofibers made from a mixture of two or more materials). .. For "composite" nanofibers, the "backbone polymer" is the derivatized cellulose and the first non-cellulosic polymer is removed from the fiber/felt by exposure to high temperature or chemical solvent or both high temperature and chemical solvent. be able to. In some embodiments, removing the first non-cellulosic polymer simultaneously converts the derivatized cellulose back to cellulose, ie, "regenerates" the cellulose.

  The nanofibers in the felt of the present invention are manufactured using electrospinning techniques. This refers to the production of fibers based on the extruded polymer "jet" being stretched into nanofibers by exposing the extruded polymer "spin dope" to an electrostatic field.

  These and other exemplary aspects of the invention are described in further detail below.

Definitions In the following description, several terms are used generically. The following non-limiting definitions provide a clear and consistent understanding of the specification and claims, including exemplary ranges where such terms are provided.

  The terms "one," "a," or "an" as used in this disclosure, unless otherwise indicated, are "at least one" or "one." Means more than.

  As used herein, the term "invention" or "invention" is not intended to be limiting and is not intended to refer to any single embodiment of a particular invention, although the specification and patents Includes all possible embodiments recited in the claims.

As used herein, the term "permeance" refers to the flux of fluid through a nanofiber felt per unit thickness of felt and per unit pressure drop. It is considered to be “high” when the transparency exceeds 500 L/(min·m ² ·10 ⁵ Pa).

  The term "flux" refers to the flow rate of fluid through a nanofiber felt per unit time per unit area of surface exposed to the flow.

  As used herein, the term "capacity" refers to the amount of bound product per unit adsorbent. The protein adsorption capacity is considered to be “high” when the protein exceeds 100 mg/adsorbent 1 g.

  The terms "membrane", "felt" and "matt" are used interchangeably herein and refer to a non-woven or randomly superposed assembly of fibers.

  As used herein, the term "nanofiber felt" refers to an aggregate of nanofibers arranged in a substantially planar manner, including microfibers added to improve strength, flux, etc. Sometimes.

  As used herein, the term "microfiber" refers to a fiber having a diameter of greater than 1.0 micrometer, generally 1.0 micrometer and greater than 1.0 millimeter.

  As used herein, the term "nanofiber" refers to a fiber having a diameter of less than 1.0 micrometer, generally greater than or equal to 10 nanometers and less than or equal to 1.0 micrometer, such as 200 nm to 600 nm.

  As used herein, the term "hybrid nanofiber felt" comprises at least two types of polymers that combine a single component fiber or composite fiber with at least one other single component fiber or at least one other composite fiber. Refers to a non-woven or randomly superposed assembly of fibers.

  As used herein, the term “single component nanofiber” refers to a nanofiber made from a single polymer.

  As used herein, the term "single-component nanofiber felt" refers to an assembly of many single-component nanofibers into a non-woven or randomly superposed assembly of fibers.

  As used herein, the term "composite nanofiber" is a nanofiber made from at least two different polymers.

  As used herein, the term "moderately elevated temperature" refers to temperatures above 24°C and below 110°C.

  As used herein, the term "differentially removable" means that when a hybrid nanofiber felt is composed of at least two non-cellulosic polymers, one non-cellulosic polymer is the other non-cellulosic polymer. This means that the conditions (high temperature and/or solvent exposure) can be chosen to be removed significantly (at least 10% difference, up to 100% vs. 0%) over the polymer.

  As used herein, the term "solvent" refers to any single component liquid or mixture of liquids that is capable of dissolving one or more components of the nanofiber felt.

  As used herein, the term "spin dope" refers to polymer solutions used in electrospinning processes.

  As used herein, the term "electrospinning" refers to the application of electric forces to spin dopes to form nanofibers.

  As used herein, the term "thermally stable" means that the polymer does not collapse in the temperature range of 50-110°C.

  As used herein, the term "chemically stable" means that the polymer is not soluble in solvents such as water or commonly used organic solvents (eg alcohols and hydrocarbons) and mixtures thereof.

Derivatized Cellulose Cellulose is a structural component found in the cell walls of plants and algae. It is also secreted by some bacteria. Therefore, cellulose is the most abundant organic compound on earth. Cellulose is derived from D-glucose units linked to a linear polymer via β(1-4) glycosidic bonds. In biological and industrial applications, cellulose is refined from plants, wood pulp or cotton and converted into many useful substances such as paper, cellophane, rayon, biofuels and the like. The usefulness of cellulose can be largely attributed to its physical properties. Cellulose is odorless, hydrophilic, relatively insoluble, has very low non-specific binding and is biodegradable.

  Although cellulosic separation media have many advantages, unfortunately they are chemically unstable (ie, decompose) in strong acids and bases. Furthermore, dissolution of cellulose requires the use of N-methylmorpholine-N-oxide (NMMO) and water or a special solvent mixture such as lithium chloride and N,N-dimethylacetamide. This limits the use of cellulosic media to operations that do not require the harsh regeneration conditions often required in the biopharmaceutical industry to meet the stringent cleaning regulations required by the FDA.

  Cellulose fibers are conventionally produced by wet spinning and pre-comprised a derivative of cellulose. This is because it is very difficult to electrospin cellulose directly from solution or melt. Various research efforts have been devoted to electrospin cellulose derivatives such as cellulose acetate to prepare cellulose nanofibers. Unlike cellulose, cellulose acetate is soluble in many commonly used solvents such as acetone. Cellulose acetate can be electrospun into nanofibers and regenerated cellulose nanofibers can be produced by subjecting the nanofibers to a hydrolytic/deacetylation post-spin treatment.

  Therefore, in the practice of the present invention, one of the polymers in the hybrid nanofiber felt is derivatized cellulose. Cellulose can be readily derivatized using well known methods that convert the -OH groups of individual glucose units to other moieties with higher or lower reactivity, different charges, etc. Such derivatized cellulosic species exhibit improved stability when exposed to solvents and also exhibit other desirable physical properties. Many cellulose derivatives are easily available as commercial products. Exemplary derivatized cellulose species include, for example, organic esters (cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate, cellulose acetate butyrate), inorganic esters (cellulose nitrate, cellulose sulfate), and alkyl cellulose (hydroxyl. Ethyl cellulose, carboxymethyl cellulose).

  The hybrid nanofiber felts of the present invention will typically have a majority (ie, 51% or more) by mass of derivatized cellulose, eg, greater than 60% or greater than 70%.

Non-cellulosic polymers Hybrid nanofibers Although the majority of the mass in derivatized cellulose is derivatized cellulose, the incorporation of additional types of fibers into the felt imparts the functionality required for the felt application. . Thus, having an additional fiber in the felt imparts increased mechanical strength to the felt, allows multiple functionalities to be incorporated into the felt, stability and described elsewhere in this specification. It is desirable because the manufacturing method can be provided with another embodiment. In fact, unexpectedly, the inclusion of even a small fraction of non-cellulosic polymers in the hybrid nanofiber felts improved the electrospinning process, and in particular, the hybrid nanofiber felts could be used in combination with composite nanofibers. It has also been discovered by the inventors that the finished product can be adapted to various biological and industrial applications if it consists of both one-component nanofibers.

  Synthetic polymer nanofibers, such as those made from vinyl and acrylic polymers, exhibit a wide range of chemical functionality for bioseparation and other applications. By combining different polymer units, the interfacial chemistry of the resulting fiber can be controlled as part of the electrospinning process, directly imparting functionality to the nanofibers produced. Alternatively, and like conventional micrometer-scale fibers, the electric field can be tailored to tailor the surface functionality of polymer nanofibers to specific functionality requirements for various bioseparation applications (discussed below). It can be chemically modified after spinning. Functionalization chemistries are well known in the polymer arts. They also withstand the rigorous cleanliness typically associated with bioprocesses. Exemplary functionalization chemistries are also described in further detail elsewhere in this specification.

  Synthetic carbon-based adsorption media and filtration membranes are often much more chemically robust than cellulosic media and therefore require strong acids and bases to clean the separation media between uses. It can be used when In addition, hybrid nanofibers containing both cellulosic and non-cellulosic polymers (e.g., polyacrylonitrile and polyvinyl alcohol) have much higher specific surface area and higher surface area compared to single-component cellulose or single-component synthetic polymer nanofibers. Indicates mechanical strength. Therefore, there is an observable synergistic effect when the composite nanofibers include both cellulose and non-cellulosic polymers.

  (1) Thermoplastic homopolymers such as vinyl polymers, acrylic polymers, polyamides, polyesters, polyethers and polycarbonates, (2) heats such as vinyl-co-vinyl polymers, acrylic-co-acrylic copolymers and vinyl-coacrylic polymers. Plastic copolymers, (3) triblock copolymer elastomers, polyurethane elastomers and elastic polymers such as ethylene-propylene-diene-elastomer, (4) high-performance polymers such as polyimide and aromatic polyamide, (5) poly(p-phenylene terephthalamide) ) Or polyaramid or other liquid crystal polymer, (6) polyethylene terephthalate or polyacrylonitrile or other textile polymer, (7) polyaniline or other conductive polymer, and (8) polycaprolactone, polylactide, chitosan or polyglycolide biocompatible Many polymers have been successfully electrospun into nanofibers, including reactive polymers (or "biopolymers"). As noted above, the polymer can also be a copolymer of two or more of the polymer species listed above.

  Examples of additional polymers that can be added to the hybrid nanofiber felt are polyacrylonitrile (PAN), polyimide, polyamide (nylon 6, nylon 6,6, nylon 6,10, etc.), polyester (polyethylene terephthalate, etc.). , As well as electrospun as single component nanofibers from their copolymers.

Composite Nanofibers In one embodiment of the invention, the hybrid nanofiber felt comprises composite nanofibers. This is because the electrospinning process used to make nanofibers from single-component derivatized cellulose solutions is unstable, resulting in low yield, low efficiency (long time and many interruptions), and only 1 Partly due to the possibility of producing low-quality nanofibers with only one chemical functionality (wide size distribution, brittleness, etc.). Therefore, it may be necessary to combine a cellulose derivative with a non-cellulosic polymer that stabilizes the electrospinning process in order to efficiently make large quantities of high quality nanofiber felt with multiple functionalities.

  The non-cellulosic polymer of the composite nanofibers of the present invention may consist of a synthetic carbon-based polymer that is removable from the nanofiber felt by exposure to elevated temperatures and/or solvents. The exposure of the nanofiber felt to the solvent mixture or the combination of elevated temperature and solvent can occur simultaneously or sequentially. The presence of non-cellulosic polymers in the electrospinning process also enhances nanofiber stability and other aspects of the method described elsewhere herein.

  Synthetic polymeric nanofibers, such as those made from vinyl and acrylic polymers, exhibit a wide range of chemical functionalities for bioseparation applications. By combining different polymer units, the interfacial chemistry of the resulting fiber can be controlled as part of the electrospinning process, directly imparting functionality to the nanofibers produced. Alternatively, and like conventional micrometer-scale fibers, the electric field can be tailored to tailor the surface functionality of polymer nanofibers to specific functionality requirements for various bioseparation applications (discussed below). It can be chemically modified after spinning. Synthetic polymer nanofibers offer a tremendous range of potential functionalization chemistries that lend themselves to a wide variety of uses. The chemistries of such functionalization are well known in the polymer arts. They also withstand the rigorous cleanliness typically associated with bioprocesses.

  (1) Thermoplastic homopolymers such as vinyl polymers, acrylic polymers, polyamides, polyesters, polyethers and polycarbonates, (2) heats such as vinyl-co-vinyl polymers, acrylic-co-acrylic copolymers and vinyl-coacrylic polymers. Plastic copolymers, (3) triblock copolymer elastomers, polyurethane elastomers and elastic polymers such as ethylene-propylene-diene-elastomer, (4) high-performance polymers such as polyimide and aromatic polyamide, (5) poly-p-phenylene terephthalamide Liquid crystal polymers such as and polyaramid, (6) textile polymers such as polyethylene terephthalate and polyacrylonitrile, (7) conductive polymers such as polyaniline, and (8) biocompatible polymers such as polycaprolactone, polylactide and polyglycolide. Including, we succeeded in electrospinning many polymers into nanofibers.

  Exemplary non-cellulosic polymers for making composite nanofibers include, for example, polyethylene oxide, poly(vinylpyrrolidone), poly(vinyl acetate), poly(vinyl alcohol), polysaccharides (chitin, starch, etc.), polystyrene, And poly(methyl methacrylate).

  The non-cellulosic polymer is usually present in the composite nanofibers in an amount of 49% by mass or less, such as 30% by mass or 25% by mass.

Electrospinning Electrospinning is a technique that utilizes only electric forces to drive the spinning process and produce polymer fibers from a solution or melt. Unlike conventional spinning techniques (e.g., solution spinning and melt spinning) that can produce fibers in the micrometer diameter range (about 5-25 μm), electrospinning produces fibers in the nanometer diameter range. can do. Electrospun polymer nanofibers have many surprising properties, including small fiber diameter and associated large specific surface area, high degree of polymeric orientation and resulting excellent mechanical properties. Furthermore, felts made with electrospun polymer nanofibers exhibit controlled pore size when compared to nanofibers made using other fabrication techniques. Unlike nanorods, nanotubes and nanowires, which are mostly produced by synthetic methods, electrospun nanofibers are produced by the “nano-manufacturing method”, which is a low cost that is relatively easy to assemble and process into applications. Become a nanofiber.

  In general, the formation of nanofibers is a delicate and intricate balance of the three main forces involved in the electrospinning process, including electrical, surface tension and viscoelastic forces. Of these three forces, electric forces always promote the formation of products with the greatest surface area. Surface tension always promotes the formation of products with minimal surface area. Viscoelastic force is a force that varies significantly with solvent evaporation and is the main reason to prevent electrospinning jets/filaments from breaking into droplets. When the electric force is dominant, the viscoelastic force acts in opposition to the electric force. When the surface tension is dominant, viscoelastic forces counteract the surface tension.

  Theoretically, the smallest nanofibers can be formed under two conditions. (1) when the excess charge density carried by the electrospinning jet is high, and (2) the time is long enough to prevent jet/filament capillary rupture, and the viscoelastic force prevents jet/filament capillary rupture. High enough to do so, but the electric force is low enough to effectively stretch the jet. In condition (1), adding a soluble electrolyte to the spin dope (for example, adding a strong electrolyte such as NaCl to an aqueous solution of polyethylene oxide) significantly increases the excess charge density carried by the jet, and It has been found that small diameter nanofibers can be formed. However, this method also produces negative effects such as (a) reduction of flow rate and consequent reduction of productivity of nanofibers, and (b) contamination of prepared nanofibers with electrolyte. Removal of the electrolyte can be difficult without sacrificing the properties of the nanofibers.

  Condition (2) requires a further understanding of jet solidification. Generally, jet solidification is closely related to solvent volatility. If the solvent volatility is too high, the time to effectively draw the electrospun jet/filament is short. Thus, a relatively large diameter fiber is obtained. If the solvent volatility is too low, the electrospun jet/filament may break into droplets upon stretching. Therefore, beads and/or beaded fibers are obtained.

  The electrospinning process generally involves three steps. (1) electrospinning jet/filament initiation and extension along the jet's linear trajectory; (2) growth and further extension of the jet's bending instability, which causes the jet to follow loop and spiral paths (Can be elongated); and (3) solidification of the jet by solvent evaporation or cooling, which forms nanofibers. Figure 1 schematically shows the process of electrospinning (Hao Fong, In Polymeric Nanostructures and Their Applications, Volume 2: Applications: Chapter 11, Electrospun Polymer, Ceramic, Carbon/Graphite Nanofibers and Their Applications, Hari S. Nalwa Editor, American Scientific Publishers, Los Angeles, CA (ISBN: 1-58883-070-5), 2007, pp. 451 -474.

  An exemplary electrospinning process can be described generally as follows.

  Step 1: As shown in FIG. 1, spin dope (e.g., polymer solution) is placed in a container equipped with a spinneret (1) and a high voltage DC (2), typically in the range of 5-40 kilovolts, is applied to the electrode (e.g., Apply to the solution through the copper wire (3). An electrically grounded collector (4) is placed at a certain distance (known as the gap distance) (5) from the spinneret. Gap distances can range from a few centimeters to a meter. When the electrostatic field reaches a critical value and the electrical force overwhelms the surface tension and viscoelastic forces, the jet/filament is ejected and travels a straight distance (known as the jet length).

Step 2: The jet then begins to bend, forming a spiral loop. This phenomenon is called "bending (or whipping) instability". Bend instability typically extends the jet length by more than 10,000 times in a very short time (less than 50 ms). Therefore, the elongation rate during bending instability is extremely high (up to 1,000,000 s ^-1 ). This extremely high elongation rate allows the polymer chains to be effectively stretched and closely aligned along the nanofiber axis.

  Step 3: The jet solidifies by evaporation of the solvent or when the melt cools below the solid-liquid transition temperature. The longer the solidification time, the longer the jet can be. Solidification time is related to many factors such as solvent vapor pressure, solvent diffusivity, volumetric charge density carried by the jet, and the strength of the applied electrostatic field.

Optional Electrospinning Post-Treatment After solidifying the collected nanofibers, there are several steps that can be taken to "customize" the nanofibers for a particular use. Exemplary additional steps are described below.

Removal of the first non-cellulosic polymer optionally using one or more of the polymers present in the composite nanofibers, especially the non-cellulosic polymer, using high heat and/or solvent(s). It can be removed. Removal of the first non-cellulosic polymer adds surface area and improves the porosity of the remaining cellulosic polymer. This is because after removal of the non-cellulosic polymer, the cellulosic polymer retains size-controlled "pores" where the non-cellulosic polymer previously occupied the space. .. This additional "void space" provides a larger surface area to the resulting nanofiber felt, for example to increase adsorptive binding capacity for separations, improve selectivity of size-based separations, and additional porosity. Therefore, the throughput can be improved. Removal of the non-cellulosic polymer eliminates the opportunity for multiple functionalities (which were present in the composite nanofibers) to be present directly on the remaining cellulosic polymeric nanofibers.

b. Cellulose Regeneration After the "as electrospun" nanofibers have been prepared, the derivatized cellulose can be converted to cellulose by a regeneration process. Regenerated cellulose has the same properties as the pure natural cellulose previously described. The regeneration process is completed by contacting the nanofibers containing derivatized cellulose with, for example, a strong base (eg, sodium hydroxide) or other solvent. After the regeneration reaction to convert to cellulose, the nanofibers can be washed to remove excess solvent used during the process.

c. Surface functionalization
After preparing the hybrid nanofiber felt, the fiber surface can be functionalized. Non-limiting examples of functionalization include weak or strong acids, and ion exchange groups such as bases (e.g., carboxylic acids and amines), hydrophobic groups such as phenolic compounds, and affinity ligands such as antibodies or enzyme substrates. Addition.

  For use in bioseparations, the hybrid nanofiber felts of the invention are ideally biologically inert, including non-specific binding of insoluble solids such as cells and cell debris, as well as proteins, sugars, It means that undesired interactions with nucleic acids, viruses and other soluble components present in many biologically produced systems should be blocked.

  Furthermore, nanofiber felts for use in bioseparation should exhibit several qualities. (1) Small diameter fibers that allow the maximum amount of specific surface area (this criterion is most important for the adsorption process and less important for the strictly size-based separations described below), (2) flow rate in adsorption applications. Well-controlled narrow pore size distribution between fibers, enabling even distribution and tight size cutoffs for size-based separations, (3) fibers are potentially high operating pressures and harsh It should have good mechanical and chemical stability to withstand various cleaning conditions, and (4) the fiber should have a well-defined, spatially consistent size and chemical composition.

  In adsorption processes where macromolecular products such as proteins, nucleic acids and viruses are the main targets, the extremely large specific surface area associated with nanofiber felts provides a vast number of potential binding sites for bioadsorption separations. Nanofibers can be modified to contain a surprising number of binding sites and adsorption occurs almost exclusively on the surface of the fiber, thus making the binding sites readily available and relatively large target molecules. Need not be diffused inside. When using traditional porous resin beads, internal diffusion can often limit the capacity of many adsorption processes for bioproducts. Furthermore, because nanofiber membranes can be made with a variety of different chemistries, adsorbent ligands can be tailored to meet specific separation needs (eg, ionic, hydrophobic, and affinity). In some cases, the ligand can be incorporated into the nanofibers from the material during electrospinning, or the surface can be chemically modified to provide the desired adsorptive agent after the nanofibers have been produced.

  Two of the most important properties of the separation operation are (1) the flow (as opposed to densely packed resin beads) passing through the felt micropores and macropores, and (2) that The adsorption is done on the surface of the fiber and no internal diffusion is required. These factors reduce the concern of high pressure loss at high flow rates and eliminate the slow intra-particle diffusion required for adsorption within resin beads. Although the binding ability of biomolecules to currently existing adsorption felts is similar to that of resin beads in terms of size, it has been revealed that they can be operated at a processing flow rate that is more than 10 times faster than a packed bed. These factors result in much faster processing times and potentially higher binding levels for purifying valuable biological products. This is highly desirable, especially for large biomolecules (molecular weight greater than 250 kDa and/or hydrodynamic diameter of 20-300 nm). This is because large biomolecules are extremely difficult to purify using packed beds due to the strict mass transfer limits within the pores of resin beads.

  The surface of the nanofiber felts of the present invention can be modified to provide ion exchange and hydrophobic interaction chemistry. Cation exchange media were generated using simple chemical modifications such as sulfonation of polystyrene fibers with sulfuric acid. Ion exchange surface functional groups and three-dimensional tethers from various polymer substrates including polypropylene, polyvinylidene difluoride, polysulfone, etc. using grafting reactions, atom transfer radical polymerization (ATRP), and plasma treatment. Was created. Phenyl and butyl groups can also be introduced as hydrophobic interacting ligands. Often, the surface of the polymer membrane must be further modified towards increased hydrophilicity to prevent non-specific binding. This was accomplished by introducing poly(ethylene glycol) and other polyols onto the surface.

  The ion exchange capacity of the hybrid nanofiber felt can also be improved, for example by introducing a diethylaminoethyl (DEAE) group as the weak anion exchange ligand or a carboxylic acid as the weak cation exchange ligand.

d. Surface Functionalization with Antimicrobial In one embodiment of the invention, the non-cellulosic polymer is polyacrylonitrile (PAN). PAN fiber membranes have been widely adopted in filtration due to their thermal stability, high mechanical properties, and chemical resistance. Electrospun PAN nanofiber felts have been of particular importance due to their properties such as small fiber diameter and associated large specific surface area, as well as controlled pore size between nanofibers and the capacity to incorporate antimicrobial agents at the nanoscale. Felts composed of nanofibers with antibacterial functionality have attracted increasing attention due to concerns over the quality of purified water and/or filtered air and processing costs. Water and air filters, especially those operating in dark and humid conditions, are always susceptible to attack by environmental microbes. Microorganisms (such as bacteria) that can be easily captured by the filter rapidly grow to form biofilms. When microorganisms intensify on the filter surface, the quality of purified water and/or filtered air is impaired. Moreover, their enhancement also adversely affects the flow of water and/or air.

  In addition, filters contaminated with biofilm are difficult to clean. High pressures are usually required during operation. This increases costs. In the reported method, antibacterial agents (such as N-halamine and silver ions/nanoparticles) were directly incorporated into the spin dope, and thus the antimicrobial agent molecules/particles were distributed throughout the nanofiber (Xinbo Sun, Lifeng Zhang, Zhengbing Cao, Ying Deng, Li Liu, Hao Fong, and Yuyu Sun. "Electrospun Composite Nanofiber Fabrics Containing Uniformly Dispersed Antimicrobial Agents as an Innovative Type of Polymeric Materials with Superior Anti-Infective Efficacy". ACS Applied Materials and Interfaces, 2(4 ), 952-956, 2010).

  However, this often leads to process problems, as a high content of antimicrobial agents can seriously affect the electrospinning process and/or impair the properties of the resulting nanofibers. A potential solution to these problems is to introduce antibacterial functionality to the nanofiber surface after the nanofibers have been formed (Lifeng Zhang, Jie Luo, Todd J. Menkhaus, Hemanthram Varadaraju, Yuyu Sun, and Hao Fong. "Antimicrobial Nano-fibrous Membranes Developed from Electrospun Polyacrylonitrile Nanofibers". Journal of Membrane Science, 369, 499-505, 2011).

It is known that the nitrile (-C≡N) group in PAN can be chemically converted to an amidoxime (-C(NH ₂ )=NOH) group. The amidoxime group can coordinate with a wide range of metal ions including silver ions, and can reduce the coordinated silver ions into silver nanoparticles. Both silver ions and silver nanoparticles are antibacterial agents having a high antibacterial effect.

e. Other Examples A promising alternative to packed bed chromatography and other separation techniques is the use of the hybrid nanofiber felts of the invention as selective adsorption membranes. This style of adsorption utilizes nanofiber felts as supports for the ligands used in the selective adsorption process.

  Selective adsorption involves the "active" surface functionalization of hybrid nanofiber felts, allowing direct capture (adsorption) of target substances. Such modifications are simplified when the hybrid nanofiber felts include chemical moieties that are relatively easy to chemically modify to provide adsorption sites on their surface.

  Unlike modifying the nanofiber surface for ion exchange and hydrophobic interaction functionalities, the incorporation of affinity ligands onto the nanofibers can be more difficult. The method often requires first modifying the surface to create a coupling site for immobilization of the ligand, followed by attachment of the ligand to the active site. Importantly, both the initial surface modification and the coupling of the ligand should be robust so that they do not leach during processing.

  In some cases, the simple carboxyl group of methacrylic acid grafted to the surface acts as an active coupling site by creating a covalent amide bond between the functionalized carboxyl group and the exposed amine group on the protein ligand. be able to. Similarly, the oxidation of cellulose (when properly controlled) is capable of forming covalent bonds to primary amines of proteins (including protein A and protein G), especially through the amino acid lysine. Can be provided on the fiber surface. In other cases, surface functionalization with common affinity dyes (eg, Cibacron Blue, which can bind some proteins) can be coupled directly to cellulose nanofibers.

  More elaborately, bioactive sites for protein ligand immobilization can be incorporated into the nanofiber backbone during nanofelt construction. An example of this is the use of polyethylene glycol (PEG) with poly D,L lactide (PDLLA) as a block copolymer. After electrospinning, glycol can be coupled with biocytin (which can have an affinity interaction with streptavidin fusion protein) to create affinity nanofibers. Similarly, polycaprolactone (PCL) and poly(D,L-lactic-co-glycolic acid)-containing surface aminated nanofibers for coupling to proteins using homobifunctional coupling agents. A b-PEG-NH2 (PLGA-b-PEF-NH2) diblock copolymer can be created. Finally, it is possible in some cases to use the intrinsic active site associated with some nanofiber matrices. For example, successful coupling of Concanavalin A (an affinity tag for lectins associated with glycol-proteins and/or other glycol conjugates) to chitosan-based nanofibers.

  Other techniques for attaching specific ligands to cellulosic compounds and/or synthetic polymers are known in the chemical arts.

Size-Based Separation Size-based separations are also routinely used in downstream bioprocessing as an orthogonal purification mechanism for adsorption. Deep filtration and microfiltration are common procedures used for clarification of fermentation broth, where cells (about 1-20 μm) and cell debris (0.1-1 μm) are removed from the bioreactor slurry. Nanofiltration with a membrane is used for virus exclusion and/or purification of 20-200 nm virus particles, and ultrafiltration is usually used for protein concentration and purification. In all cases, some properties of the separation medium are desirable. First, a well-defined size cutoff is desirable to obtain a tightly controlled separation. Second, a high porosity material is required to perform high process processing without excessive pressure requirements to minimize operating time and/or membrane area requirements. And third, chemical and physical robustness are desirable for harsh cleaning conditions and operations under moderate pressure. Nanofiber felts can be produced in large quantities at low cost from mechanically and chemically tough fibers and have well-controlled pore size of the fibers (or as hollow fibers), so they are based on advanced size. It offers a surprisingly good opportunity as a separation medium. Polymer nanofibers generally exhibit the least amount of non-specific binding, but may suffer from lower chemical robustness than carbon and ceramic fibers. Ceramic fibers suffer from brittleness and the potential for large amounts of non-specific adsorption of biomass/bioparticles with concomitant fouling, but can withstand harsh regeneration conditions.

  To date, nanofiber meshes for size-based separations have primarily been applied to the isolation of biometric particles (or surrogates) on the nanometer and micrometer scale by a depth filtration mechanism. The increased specific surface area of the nanofibers in the filtration mat provides a more tortuous path, increasing the chance of capturing the desired particles from solution while maintaining high porosity. The polymer, carbon, and ceramic nanofibers were all evaluated and all were able to separate the desired particle size from the mixture while maintaining high flux. Specifically, electrospun nanofibers made from polyvinylidene fluoride (PVDF) and nylon 6 were able to remove polystyrene particles between 0.5 and 10 μm. Ceramic nanofiber mesh is probably the most widely used. An example is the combination of large titanaate nanofibers and smaller boehmite nanofibers, which enables very high flux (1000L/m2.h) with relatively low driving force (20kPa) and particles larger than 60nm. Indicates that substantially all of the can be removed from the solution. It should be noted that many applications of micro and nano depth filtration also rely on chemisorption of particles on surfaces, the nanofibers of which can be easily manufactured and specifically adsorb desired impurities.

Nanofelt construction/construction Utilization within a given bed volume for potential binding by utilizing fibers in the submicron to nanometer diameter range (1-1000 nm, called "nanofiber" felts) The possible surface area would increase by as much as two orders of magnitude. By controlling the pore size of the nanofiber felt, the pressure drop and hydrodynamic flow properties can also be controlled, making it as efficient as the microfiber felt.

In addition, the pore size of the fibers in the felt usually has a close pore size distribution (more than 90% of the nanofibers are in the 100 nm to 500 nm range) to prevent channeling and exceed the desired size cutoff for filtration operations. Keep only seeds. Finally, nanofiber felts are usually sufficiently mechanical to operate under potentially high pressure drops (up to 100 psi) and high flow rates (flux values above 30 L/(min·m ² )). Tough, and chemically robust enough to withstand potentially harsh cleaning controls (often containing strong acids, bases, and organic solvents) without breaking.

In one embodiment, the nanofiber felt consists of composite nanofibers (one derivatized cellulosic polymer plus one non-cellulosic polymer) and single component nanofibers (non-cellulosic polymer). However, as mentioned above, the hybrid nanofelts of the present invention can be formed from various combinations of polymers and nanofibers. Examples of these include the following.
A composite nanofiber felt, where the nanofibers in the felt are all composed of a single type of composite nanofiber made from a coextruded mixture of a backbone polymer and a first non-cellulosic polymer.
A nanofiber felt consisting of at least two different single-component nanofibers. A nanofiber felt consisting of at least one single-component nanofiber and at least one composite nanofiber.

  In addition to the nanofelt constructions described above, the nanofelts of the present invention may also include microfibers to add stability, strength and other physical properties of the felt to suit its use in a particular application. When compared to single component nanofiber felts, the hybrid nanofiber felts of the present invention exhibit the following exemplary improved properties.

[Example]
[Example 1]
Preparation of Cellulose Acetate Single-Component Nanofiber Felt Handbook of Membrane Research, Chapter 3, Applications of Electrospun Nanofiber Membranes for Bioseparations, Todd J. Menkhaus, et al, Nova Science Publishers, Inc ., edited by Stephan V. Gorley. Cellulose acetate (average molecular weight about 30,000 g/mol), NaOH, NaCl, acetone, N,N-dimethylacetamide (DMAc) and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich Co. (Milwaukee, WI). did. 98% pure 2-(diethylamino)ethyl chloride hydrochloride (DAECH) was purchased from Alfa Aesar Co. (Ward Hill, MA).

A solution of 15% (mass fraction) cellulose acetate in acetone/DMAc (mass ratio 2/1) was prepared at room temperature. The solution was added to the syringe. The electrospinning setup included a high voltage power supply and a laboratory produced roller. During electrospinning, a positive high voltage of 15 kV was applied to the needle and a syringe pump was used to maintain a flow rate of 1.0 mL/hr. Cellulose acetate nanofibers were collected as felts randomly laminated onto an electrically grounded aluminum foil covering the rollers. The nanofiber felt was dried during electrospinning using a heating lamp and after electrospinning the felt was further dried in a vacuum oven. Overall, the electrospinning process was relatively unstable, with frequent interruptions at approximately 2 hour intervals. The collected cellulose acetate nanofiber felt had a thickness of about 225 μm and a mass per unit area of about 60 g/m ² .

  The as-electrospun cellulose acetate nanofiber felt was first hydrolyzed/deacetylated by immersion in 0.05M aqueous NaOH solution for 24 hours. The product called regenerated cellulose nanofiber felt was then rinsed 3 times in distilled water and dried in a vacuum oven at 60°C. The sample was immersed in a 15% (mass fraction) DAECH aqueous solution for 10 minutes and subsequently dried at 60°C. Then, the sample was immersed in a 0.5 M NaOH aqueous solution at 90° C. for 10 minutes. The sample was rinsed 3 times in distilled water and dried at 60° C. to obtain DEAE anion exchange cellulose nanofiber felt.

[Example 2]
Preparation of hybrid nanofiber felt of CA/PEO composite nanofiber and PAN single component nanofiber Cellulose acetate (CA), polyethylene oxide (PEO), chloroform (CHCl ₃ ), dimethylformamide (DMF), polyacrylonitrile (PAN), And diethylaminoethyl chloride were purchased from Sigma-Aldrich Co. (Milwaukee, WI).

Spin dope of PAN and CA+PEO were prepared separately. Briefly, for the preparation of PAN spin dope, PAN was dissolved in DMF to make a solution. For CA+PEO spin dope, CA plus PEO in CHCl ₃ /DMF with diethylaminoethyl chloride was prepared.

  During the electrospinning process, two syringes filled with a spin dope of PAN or CA+PEO were placed on opposite sides of the laboratory produced roller. Overall, the electrospinning process is very stable, long-lasting (>48 hours) and composed of CA+PEO composite nanofibers and PAN nanofibers (self-supporting Electrospun hybrid nanofibrous mats) or supported on medical cotton gauze) were collected on an electrically grounded aluminum foil covering the rollers.

  The as-electrospun CA+PEO+PAN hybrid nanofibrous mat was then annealed for 24 hours to complete the phase dispersion of CA and PEO. Then, the mat was hydrolyzed/deacetylated by immersing the mat in an aqueous NaOH solution for 24 hours. The resulting hybrid nanofibrous mat consisting of regenerated cellulose nanofibers and PAN nanofibers was rinsed with distilled water and dried.

[Example 3]
Preparation of hybrid nanofiber felt of CA/PVP composite nanofiber and PAN single component nanofiber Cellulose acetate (CA), polyvinylpyrrolidone (PVP), chloroform (CHCl ₃ ), dimethylformamide (DMF), polyacrylonitrile (PAN), And diethylaminoethyl chloride were purchased from Sigma-Aldrich Co. (Milwaukee, WI).

Spin dope of PAN and CA+PVP were prepared separately. Briefly, for the preparation of PAN spin dope, PAN was dissolved in DMF to make a solution. For CA+PEO spin dope, CA plus PVP in CHCl ₃ /DMF with diethylaminoethyl chloride was prepared.

  During the electrospinning process, two syringes loaded with a spin dope of PAN or CA+PVP were placed on opposite sides of the lab-produced roller. Overall, the electrospinning process is very stable, long-lasting (>48 hours) and composed of CA+PVP composite nanofibers and PAN nanofibers (self-supporting or medical cotton). Electrospun hybrid nanofibrous mats (supported by gauze) were collected on an electrically grounded aluminum foil covering the rollers.

  The as-electrospun CA+PVP+PAN hybrid nanofibrous mat was then annealed for 24 hours to complete the phase dispersion of CA and PVP. Then, the mat was hydrolyzed/deacetylated by immersing the mat in an aqueous NaOH solution for 24 hours. This process also dissolves/removes PVP from the mat. The resulting hybrid nanofibrous mat consisting of regenerated cellulose nanofibers and PAN nanofibers was rinsed with distilled water and dried.

[Example 4]
Preparation of hybrid nanofiber felt of CA/PEO composite nanofiber and nylon 6 single component nanofiber Cellulose acetate (CA), polyethylene oxide (PEO), chloroform (CHCl ₃ ), hexafluoroisopropanol (HFIP), nylon 6, and Diethylaminoethyl chloride was purchased from Sigma-Aldrich Co. (Milwaukee, WI).

Nylon 6 and CA+PEO spin dopes were prepared separately. Briefly, for the preparation of nylon 6 spin dope, the polymer was dissolved in HFIP. For CA+PEO spin dope, CA plus PEO in CHCl ₃ /DMF with diethylaminoethyl chloride was prepared.

  During the electrospinning process, two syringes loaded with nylon 6 or CA+PEO spin dope were placed on opposite sides of the lab-produced roller. Overall, the electrospinning process is very stable, can last for a long time (>48 hours) and consists of CA+PEO composite nanofibers and nylon 6 nanofibers (freestanding or medical cotton). Electrospun hybrid nanofibrous mats (supported by gauze) were collected on an electrically grounded aluminum foil covering the rollers.

  The as-electrospun CA+PEO+nylon 6 hybrid nanofibrous mat was then annealed for 24 hours to complete the phase dispersion of CA and PEO. Then, the mat was hydrolyzed/deacetylated by immersing the mat in an aqueous NaOH solution for 24 hours. This process also dissolves/removes PEO from the mat. The resulting hybrid nanofibrous mat consisting of regenerated cellulose nanofibers and nylon 6 nanofibers was rinsed with distilled water and dried.

[Example 5]
Preparation of hybrid nanofiber felt of CA/PVP composite nanofiber and nylon 6 single component nanofiber Cellulose acetate (CA), polyvinylpyrrolidone (PVP), chloroform (CHCl ₃ ), hexafluoroisopropanol (HFIP), nylon 6, and Diethylaminoethyl chloride was purchased from Sigma-Aldrich Co. (Milwaukee, WI).

Nylon 6 and CA+PVP spin dopes were prepared separately. Briefly, for the preparation of nylon 6 spin dope, nylon 6 was dissolved in HFIP to make a solution. For CA+PEO spin dope, CA plus PVP in CHCl ₃ /DMF with diethylaminoethyl chloride was prepared.

  During the electrospinning process, two syringes loaded with nylon 6 or CA+PVP spin dope were placed on opposite sides of the lab-produced roller. Overall, the electrospinning process is very stable, long-lasting (>48 hours) and composed of CA+PVP composite nanofibers and nylon 6 nanofibers (self-supporting or medical Electrospun hybrid nanofibrous mats (supported by cotton gauze) were collected on an electrically grounded aluminum foil covering the rollers.

  The as-electrospun CA+PVP+nylon 6 hybrid nanofibrous mat was then annealed for 24 hours to complete the phase dispersion of CA and PVP. Then, the mat was hydrolyzed/deacetylated by immersing the mat in an aqueous NaOH solution for 24 hours. This process also dissolves/removes PVP from the mat. The resulting hybrid nanofibrous mat consisting of regenerated cellulose nanofibers and nylon 6 nanofibers was rinsed with distilled water and dried.

[Example 6]
Performance Evaluation of Single-Component and Hybrid Nanofiber Felts Single-Component Nanofiber Mats (Example 1) and Hybrid Nanofiber Mats (Examples 2-6) for Batch Adsorption, Dynamic Adsorption, Flow Dispersion and Permeability The studies were compared with commercial regenerated cellulose adsorbent membranes and cotton balls. Commercially available cellulose membranes and cotton balls underwent the same electrospinning post-treatments as single component nanofiber felts and hybrid nanofiber felts before testing.

The batch adsorption experiment was completed and the Langmuir equilibrium adsorption isotherm was determined. For batch analysis, single component felts, hybrid felts, commercially available cellulose and cotton balls were rinsed with buffer, cut into individual pieces of approximately 1 cm ² and weighed. For each medium, 10 individual pieces (about 100 mg) were then placed in a 15 milliliter (mL) centrifuge tube. A stock solution of target protein was prepared at 2.0 mg/mL by mixing a known mass of lyophilized protein with buffer. Appropriate combination of stock solution and buffer was added to each test tube containing chopped felt pieces, commercial cellulose membranes or cotton balls to give a final volume of 14 mL of each tube and initial protein. The concentration was between 0.0 mg/mL and 2.0 mg/mL of protein. A 1.0 mL liquid sample was taken immediately from each of the different initial protein concentrations and UV absorbance was measured at 280 nm. The sample was then placed in an inversion mixer rotating at about 40 revolutions per minute (rpm). After mixing for a minimum of 24 hours, the liquid for each sample was removed and the protein concentration was determined by UV-280 nm absorbance using a Genesys 10 UV spectrophotometer purchased from Thermo Electron Corporation (Madison, WI). The protein adsorbed on the felt could be calculated from the difference. Test tubes without 2.0 mg protein/mL and felt were also prepared to evaluate the possibility of proteins adsorbing on the test tube surface. No adsorption was observed on the surface of the test tube. Similarly, controls were monitored for potential leached chemicals that may contribute to UV-280 nm absorbance, and non-specific binding of protein to underivatized membranes (after regeneration with NaOH). evaluated. No leaching or non-specific binding was observed for any of the samples. Langmuir adsorption isotherms were then generated and modeling constants (Q _max and K _d ) were determined by least squares regression fit to

Where Q is the adsorption equilibrium concentration of the protein on the membrane (mg/g), Q _max is the asymptotic maximum saturation capacity, C is the liquid phase equilibrium concentration of the protein (mg/mL), and K _d is Desorption constant (mg/mL). High Q and low K _d indicate an efficient adsorption process. Following adsorption analysis, the liquid was decanted and the felt mixed and washed with 14 mL of buffer for 1 hour. After removing the washing solution, a buffer containing 1 M NaCl was added to the test tube and mixed for 1 hour, and a liquid for protein concentration by UV-280 nm absorbance was sampled. Elution percentage was calculated based on the amount of binding found at the time of adsorption of the study.

  The results, shown in Table 2 below, showed that electrospun hybrid nanofiber felts had the highest capacity, followed by single-component cellulose nanofiber felts. Both nanofiber based adsorbent materials had significantly higher saturation capacities than any of the commercially available cellulosic adsorbent media at all equilibrium liquid phase concentrations. Regenerated cellulose microfiber felt and cotton balls showed the lowest binding capacity. The high specific surface area combined with the unique nanofiber felt morphology is due to the increased binding capacity. In all cases, quantitative elution of proteins can be performed.

The permeability of the buffer through single component and hybrid nanofiber felts and commercially available regenerated cellulose adsorption membranes was measured. Higher permeability values indicate higher throughput (faster treatment time) and/or the ability to operate at lower pressure. Both of these bring significant benefits to the manufacturing process. A small scale "coin" membrane adsorption holder was used in all experiments. The unit allows an effective filtration area of about 1.5 cm ² and is sealed with an O-ring to prevent leaks. First, the pressure drop of only the system with the membrane holder in place but without the membrane was evaluated at flow rates ranging from 2.0 mL/min to 30.0 mL/min. The layers of nanofiber felt or commercial membrane, respectively, were then added sequentially to the unit while measuring the pressure drop at various flow rates. For each felt/membrane, 1, 3, 5, 7, and 9 layers were evaluated. The pressure drop of the system was subtracted from the pressure drop measured with the felt/membrane in place to calculate the permeability of the felt/membrane at each flow rate. A minimum of 5 flow rates and corresponding pressure readings were taken for each different number of layers.

As shown in Table 3 below, the permeability of the buffer through the stack of hybrid nanofiber felts is significantly higher than that of the single component felts, and the permeability of the single component nanofiber felts is comparable to that of the corresponding commercially available regenerated cellulose sample. At least 5 times higher than permeability. Also, for comparison, a 15 cm packed bed of Sepharose Fast Flow was reported by the manufacturer to have a permeability of about 7 L/(min·m ² ·10 ⁵ Pa), a functionalized commercial membrane. But much lower than nanofelt.

  System ANOVA was performed on hybrid and single component nanofiber felts and commercially available regenerated cellulose adsorbent membranes to determine the degree of axial mixing using different numbers of layers in place. Lower axial mixing (better flow distribution) is desirable to minimize channeling and premature leakage during the adsorption process. The same configuration used for permeability analysis was used for the system dispersion test except that the flow rate was maintained at 1.0 mL/min throughout the process. After equilibrating the felt/membrane stack with buffer, a solution of 1% (volume content) acetone in buffer was added to the system. The online absorbance at UV-280 nm was monitored and the resulting curves were analyzed to calculate the Peclet (Pe) number by least squares fit of

Where C _out is the eluate 280 nm absorbance, C _in is the inlet 280 nm absorbance, V is the volume of added acetone solution and V ₅₀ is the volume for C _out /C _in =0.50. . Larger Pe values were used to show the desirable property of being more similar to plug flow (lower axial mixing and better flow distribution).

  Table 4 below summarizes the Pe number results determined for various numbers of layers of hybrid and single component nanofiber mats and commercially available regenerated cellulose felt/membrane layers. The results show that the nanofiber felts produced in this study have similar hydrodynamics.

  Dynamic breakthrough analyses were completed to evaluate adsorption efficiency when operated under flow conditions. It is shown that the higher the volume at low leakage%, the more efficient the adsorbent material. The dynamic leak experiment was completed using a Pall Mustang coin holder according to the manufacturer's recommendations. Nine layers of either nanofiber felt or commercial membrane were used in the analysis. All experiments were run on an AKTA Purifier (GE Healthcare, Piscataway, NJ), UV-280nm absorbance, pH, and conductivity were measured online and controlled with Unicorn software version 5.01. Fractions were automatically collected by the system in 0.60 mL aliquots (approximately 2 bed volumes). A minimum of 10 bed volumes was used for equilibration. Step elution to 100% buffer B (equilibration buffer with 1.0 M NaCl) was used for each experiment. The flow rate was maintained at a value of 1.0 mL/min for all dynamic leak tests. Protein stock solution prepared at 1.5 mg/mL in buffer was dosed until 100% leakage was achieved. The felt was then washed with a minimum of 10 bed volumes of buffer before desorption. All eluents (flow-through during loading, washing, and elution) were collected, weighed, volume determined, and protein concentration analyzed by UV-280 nm absorbance. The protein mass balance was then calculated based on the volume input and all fractions collected during the process.

  The final performance evaluation of any adsorption system is a dynamic leak analysis, a combination of equilibrium binding capacity, adsorption kinetics, and system dispersion. It is also a direct application of volume for flow-through mode operation in which bound molecules do not need to be selectively eluted from other impurities.

  Table 5 below shows the dynamic binding capacity of proteins at 10% leak with nanofiber felt and commercial regenerated cellulose adsorption membranes. The hybrid nanofiber mats were substantially higher in dynamic capacity compared to any other adsorbent media evaluated. Furthermore, the elution results showed that within the uncertainty of the experiment, the protein elution was complete for each adsorption system and no loss was observed in the overall mass balance.

  The examples set forth above are provided to provide those of ordinary skill in the art with a complete disclosure and description of making and using the embodiments of the composition and limit the scope of what the inventors regard as the invention. Not something to do. Modifications of the above manner (implementing the invention, which are obvious to those skilled in the art) are intended to be within the scope of the following claims. All publications, patents and patent applications cited in this specification are each individual publication, patent or patent application being specifically and individually indicated to be incorporated by reference. As such, are hereby incorporated by reference.

The examples set forth above are provided to provide those of ordinary skill in the art with a complete disclosure and description of making and using the embodiments of the composition and limit the scope of what the inventors regard as the invention. Not something to do. Modifications of the above manner (implementing the invention, which are obvious to those skilled in the art) are intended to be within the scope of the following claims. All publications, patents and patent applications cited in this specification are each individual publication, patent or patent application being specifically and individually indicated to be incorporated by reference. As such, are hereby incorporated by reference.
The following embodiments are also disclosed in the present disclosure.
[Embodiment 1]
An electrospun hybrid nanofiber felt comprising a composite nanofiber and a single-component nanofiber, the composite nanofiber comprising a mixture of derivatized cellulose and a first non-cellulosic polymer, the single-component nanofiber comprising a second A nanofiber felt comprising a non-cellulosic polymer, wherein the first and second non-cellulosic polymers are differentially removable from the nanofiber felt.
[Embodiment 2]
The nanofiber felt according to embodiment 1, wherein the derivatized cellulose is selected from the group consisting of organic esters of cellulose, inorganic esters of cellulose and alkyl celluloses.
[Third Embodiment]
The nanofiber felt of embodiment 2, wherein the derivatized cellulose comprises an organic ester of cellulose selected from the group consisting of cellulose acetate, cellulose triacetate and cellulose propionate.
[Embodiment 4]
The nanofiber felt of embodiment 2, wherein the derivatized cellulose comprises an inorganic ester of cellulose selected from the group consisting of cellulose nitrate and cellulose sulfate.
[Fifth Embodiment]
The nanofiber felt according to embodiment 2, wherein the derivatized cellulose comprises an alkyl cellulose selected from the group consisting of hydroxyethyl cellulose and carboxymethyl cellulose.
[Sixth Embodiment]
The nanofiber felt of embodiment 1, wherein the first non-cellulosic polymer comprises a synthetic polymer.
[Embodiment 7]
7. The nanofiber felt according to embodiment 6, wherein the synthetic polymer is selected from the group consisting of vinyl polymers, polyethers, acrylic polymers, polyesters, polycarbonates and polyurethanes.
[Embodiment 8]
The nanofiber felt of embodiment 1, wherein the first non-cellulosic polymer comprises a natural polymer.
[Embodiment 9]
The nanofiber felt according to embodiment 8, wherein the natural polymer is selected from the group consisting of polysaccharides, polyamides and polylactides.
[Embodiment 10]
Nanofiber felt according to embodiment 9, wherein the natural polymer is a polysaccharide selected from the group consisting of starch and chitin.
[Embodiment 11]
The nanofiber felt according to embodiment 9, wherein the natural polymer is a polyamide selected from the group consisting of proteins and gelatin.
[Embodiment 12]
The nanofiber felt of embodiment 1, wherein the second non-cellulosic polymer comprises a synthetic polymer.
[Embodiment 13]
The nanofiber felt according to embodiment 12, wherein the synthetic polymer is selected from the group consisting of vinyl polymers, polyamides, polyimides and polyesters.
[Embodiment 14]
The nanofiber felt of embodiment 6, wherein the first non-cellulosic polymer is a copolymer.
[Embodiment 15]
13. The nanofiber felt of embodiment 12, wherein the second non-cellulosic polymer is a copolymer.
[Embodiment 16]
An electrospinning method for producing the nanofiber felt according to Embodiment 1,
a) preparing the composite polymer spin dope and the single-component polymer spin dope separately,
b) putting the spin dope into two different spinnerets,
c) applying a voltage to each spin dope using an electrode,
d) separately electrospinning the composite and single component nanofibers from the spinneret, and
e) collecting the solidified nanofibers as randomly superposed or partially aligned nanofiber felts
Including the method.
[Embodiment 17]
15. The method of embodiment 14, further comprising converting the derivatized cellulose to cellulose.
[Embodiment 18]
15. The method of embodiment 14, further comprising removing at least a portion of the non-cellulosic first polymer from the composite nanofibers in the nanofiber felt.
[Embodiment 19]
15. The method of embodiment 14, further comprising surface functionalizing one or more of the nanofibers in the nanofiber felt.
[Embodiment 20]
The method of embodiment 17, wherein the surface functionalization comprises binding of an affinity ligand.
[Embodiment 21]
A method for purifying a biomolecule from a fluid, comprising:
a) preparing the nanofiber felt according to embodiment 14,
b) flowing a fluid through the nanofiber felt, and
c) Step of recovering biomolecule from nanofiber felt
Including the method.

Claims (21)

  An electrospun hybrid nanofiber felt comprising a composite nanofiber and a single-component nanofiber, the composite nanofiber comprising a mixture of derivatized cellulose and a first non-cellulosic polymer, the single-component nanofiber comprising a second A nanofiber felt comprising a non-cellulosic polymer, wherein the first and second non-cellulosic polymers are differentially removable from the nanofiber felt.   The nanofiber felt according to claim 1, wherein the derivatized cellulose is selected from the group consisting of organic esters of cellulose, inorganic esters of cellulose and alkyl celluloses.   3. The nanofiber felt of claim 2, wherein the derivatized cellulose comprises an organic ester of cellulose selected from the group consisting of cellulose acetate, cellulose triacetate and cellulose propionate.   3. The nanofiber felt according to claim 2, wherein the derivatized cellulose comprises an inorganic ester of cellulose selected from the group consisting of cellulose nitrate and cellulose sulfate.   The nanofiber felt according to claim 2, wherein the derivatized cellulose comprises an alkyl cellulose selected from the group consisting of hydroxyethyl cellulose and carboxymethyl cellulose.   The nanofiber felt of claim 1, wherein the first non-cellulosic polymer comprises a synthetic polymer.   7. The nanofiber felt according to claim 6, wherein the synthetic polymer is selected from the group consisting of vinyl polymers, polyethers, acrylic polymers, polyesters, polycarbonates and polyurethanes.   The nanofiber felt of claim 1, wherein the first non-cellulosic polymer comprises a natural polymer.   9. The nanofiber felt according to claim 8, wherein the natural polymer is selected from the group consisting of polysaccharides, polyamides and polylactides.   10. The nanofiber felt according to claim 9, wherein the natural polymer is a polysaccharide selected from the group consisting of starch and chitin.   10. The nanofiber felt according to claim 9, wherein the natural polymer is a polyamide selected from the group consisting of proteins and gelatin.   The nanofiber felt of claim 1, wherein the second non-cellulosic polymer comprises a synthetic polymer.   13. The nanofiber felt according to claim 12, wherein the synthetic polymer is selected from the group consisting of vinyl polymers, polyamides, polyimides and polyesters.   7. The nanofiber felt of claim 6, wherein the first non-cellulosic polymer is a copolymer.   13. The nanofiber felt of claim 12, wherein the second non-cellulosic polymer is a copolymer. An electrospinning method for producing the nanofiber felt according to claim 1,
a) preparing the composite polymer spin dope and the single-component polymer spin dope separately,
b) putting the spin dope into two different spinnerets,
c) applying a voltage to each spin dope using an electrode,
d) separately electrospinning the composite and single component nanofibers from the spinneret, and
e) A method comprising the step of collecting the solidified nanofibers as randomly superposed or partially aligned nanofiber felts.   15. The method of claim 14, further comprising converting the derivatized cellulose to cellulose.   15. The method of claim 14, further comprising removing at least a portion of the non-cellulosic first polymer from the composite nanofibers in the nanofiber felt.   15. The method of claim 14, further comprising surface functionalizing one or more of the nanofibers in the nanofiber felt.   18. The method of claim 17, wherein the surface functionalization comprises binding an affinity ligand. A method for purifying a biomolecule from a fluid, comprising:
a) preparing the nanofiber felt of claim 14,
b) flowing a fluid through the nanofiber felt, and
c) A method comprising recovering biomolecules from nanofiber felt.