JP7265586B2 - Hybrid felt of electrospun nanofibers - Google Patents

Description

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

Microfiber and nanofiber membranes or "felts" are used in a variety of both biological and industrial applications. For example, felts are used for reinforcement with textiles, protective clothing, catalytic media, agricultural applications, sensors for environmental, medical and military surveillance, biomedical applications (e.g. bioseparations, tissue engineering and wound dressings), electronic applications (e.g. capacitors, transistors and diodes), as well as space applications (eg, support structures for solar sails and space mirrors). Microfiber and nanofiber felts are particularly convenient for purifying biological materials such as proteins, nucleic acids, carbohydrates, bacteria, viruses, cells and the like. They are useful in all fluid applications, both liquid and gaseous.

The biopharmaceutical therapeutics industry is expanding as more and more biopharmaceuticals are approved for sale. In addition, extensive use of biologically-based diagnostic tools provides high-throughput, sensitive diagnostic testing for a variety of disease states. Biological materials (eg, recombinant proteins, monoclonal antibodies, viral vaccines and nucleic acids) must be efficiently produced, purified, and used for both therapeutic and diagnostic agents.

Conventional purification methods, for example, use packed microbeads for adsorption/chromatography, ultrafiltration, and precipitation/crystallization to separate the desired biological material from by-products and other contaminants. A method of separation can be mentioned. Although these conventional separation methods give results suitable for biological applications, they are limited in terms of yield, processing time and purity. These limitations are primarily due to the slow diffusion rate of relatively large biomolecules, thereby limiting the ability of the material to be purified (i.e., the "target material") to reach available binding sites deep within the separation matrix. is limited. In addition, these systems can only be used for a limited number of cycles, and some can only be used once.

Ion exchange (IE) and hydrophobic interaction (HI) adsorption/chromatography are two examples of the more robust conventional separation techniques that are widely used for the separation of biological materials. These are generally less efficient overall than specific affinity-based separation techniques, e.g. It is also useful for purifying from products and impurities.

Affinity-based adsorption/chromatography can be more efficient than IE and HI, but is generally difficult to manufacture due to the complexity of generating and purifying biological ligands such as monoclonal antibodies and nucleic acids. and expensive. Such ligands are often very sensitive to environmental conditions (e.g. temperature, pH, ionic strength, etc.) and can be easily degraded, thus disrupting the affinity interactions required for adsorption. be done. Moreover, disruption of binding interactions can be difficult without using harsh conditions that can reduce biological activity and thus the utility of the target substance and/or the reusability of the purification media.

Membranes have been described that are useful for purifying biological materials (see, eg, Bioprocessing for Value-Added Products from Renewable Resources, Shang-Tian Yang, Ed., Chapter 7). Recently, membrane adsorption/chromatography using nanometer-diameter fibers constructed into mats with controlled thickness (i.e., "nanofiber felts") has shown 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 pore size, affinity characteristics as well as other performance criteria can be tightly controlled.

Single-component nanofiber felts previously described have provided promising results, but are often less efficient than would be desirable in terms of felt stability and material and time requirements. This is especially true when the target substance is present in the starting material to be purified in only low concentrations 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. That need is met by the embodiments disclosed below.

The following simplified summary provides a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview and is intended to neither identify key/critical elements nor 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. Composite nanofibers can be electrospun from a mixture of derivatized cellulose and a first non-cellulosic polymer, and single-component nanofibers can be the same or different from the first non-cellulosic polymer and a second non-cellulose polymer. It can be electrospun from the system polymer. Generally, the first and second non-cellulosic polymers are differentially removable from the nanofiber felt. This is because one of the non-cellulosic polymers is removed (using solvent or heat or a combination of solvent and heat) to a greater extent than the other (e.g., a difference in removability of 10% or more, e.g., 20% or 50%). use) means that a condition exists.

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

When the derivatized cellulose is an inorganic ester, it can be cellulose nitrate and cellulose sulfate, and when it is an alkyl cellulose, it can be hydroxyethyl cellulose or carboxymethyl 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 that forms the single component nanofibers is a synthetic polymer such as a vinyl polymer, polyamide, polyimide, polyester or copolymers thereof.

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

The as-formed nanofiber felt can be further processed by regenerating (ie, converting back to cellulose) the derivatized cellulose in the composite nanofibers. Methods of making nanofiber felts may also 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 binding affinity ligands with specific affinities to specific target molecules purified from the fluid.

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

Other aspects of the invention are found elsewhere in this specification.

1 is a schematic diagram showing an electrospinning method; FIG.

The present invention relates generally to hybrid felts constructed from electrospun nanofibers, eg, for use in chemical and biological separations. Hybrid nanofiber felts have high resolution and provide 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, are "hybrid" felts).

The felts of the present invention are composed of more than one type of polymer (ie, are "hybrid" felts). This includes hybrid felts made from a combination of single-component nanofibers and "composite" nanofibers (e.g., nanofibers made from a mixture of two or more materials) into a "hybrid" felt. . 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 be able to. In some embodiments, removal of the first non-cellulosic polymer simultaneously converts the derivatized cellulose back to cellulose, ie, the cellulose is "regenerated."

The nanofibers in the felt of the present invention are produced using an electrospinning technique. It refers to the production of fibers based on stretching an extruded polymer 'jet' 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 the exemplary scope to which such terms are entitled.

The terms "one," "a," or "an," as used in this disclosure, unless otherwise indicated, mean "at least one." means no less than

The terms "invention" or "the present invention" as used herein are not intended to be limiting, and are not intended to refer to any single embodiment of the particular invention, although both the specification and patents It encompasses 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 pressure drop per unit thickness of the felt. Permeability above 500 L/(min·m ² ·10 ⁵ Pa) is considered "high".

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

As used herein, the term "capacity" refers to the amount of bound product per unit adsorbent. Protein adsorption capacity is considered "high" above 100 mg protein/g adsorbent.

The terms "membrane", "felt" and "mat" are used interchangeably herein to refer to a nonwoven or randomly laid assembly of fibers.

As used herein, the term "nanofiber felt" refers to an assembly of nanofibers arranged in a substantially planar fashion, but includes microfibers added for strength, flux enhancement, etc. Sometimes.

As used herein, the term "microfiber" refers to fibers having a diameter greater than 1.0 micrometer, generally greater than or equal to 1.0 micrometer and less than or equal to 1.0 millimeter.

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

As used herein, the term "hybrid nanofiber felt" refers to at least two types of polymers in combination of single component fibers or composite fibers and at least one other single component fiber or at least one other composite fiber. refers to a nonwoven or randomly laid assembly of fibers.

As used herein, the term "single-component nanofibers" refers to nanofibers produced 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 superimposed assembly of fibers.

As used herein, the term "composite nanofibers" are nanofibers made from at least two different polymers.

As used herein, the term "moderately elevated temperature" refers to a temperature of 24°C or higher and 110°C or lower.

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

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

The term "spin dope" as used herein refers to the polymer solution used in the electrospinning process.

The term "electrospinning" as used herein refers to applying electric forces to a spin dope to form nanofibers.

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

The term "chemically stable" as used herein means that the polymer is not soluble in solvents such as water or common 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. Cellulose is therefore the most abundant organic compound on earth. Cellulose is derived from D-glucose units linked to linear polymers through β(1-4) glycosidic linkages. In biological and industrial applications, cellulose is purified from plants, wood pulp or cotton and converted into many useful materials 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.

Cellulosic separation media have many advantages, but unfortunately they are chemically unstable (ie, degrade) in strong acids and bases. Furthermore, dissolution of cellulose requires the use of special solvent mixtures such as N-methylmorpholine-N-oxide (NMMO) and water or 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 stringent cleanliness regulations required by the FDA.

The cellulose fibers are conventionally produced by wet-spinning and previously contain derivatives of cellulose. This is because it is very difficult to electrospun cellulose directly from solution or melt. Various research efforts have been devoted to electrospinning cellulose derivatives such as cellulose acetate to prepare cellulose nanofibers. Unlike cellulose, cellulose acetate is soluble in many common solvents such as acetone. Cellulose acetate can be electrospun into nanofibers, and the nanofibers can be subjected to post-spinning treatments of hydrolysis/deacetylation to produce regenerated cellulose nanofibers.

Therefore, in the practice of the invention, one of the polymers in the hybrid nanofiber felt is derivatized cellulose. Cellulose can be readily derivatized using well-known methods to convert the -OH groups of individual glucose units into other moieties with higher or lower reactivity, varying charge, and the like. Such derivatized cellulose species exhibit enhanced stability when exposed to solvents and exhibit other desirable physical properties. Many cellulose derivatives are readily available commercially. 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 celluloses (hydroxy ethyl cellulose, carboxymethyl cellulose).

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

Although derivatized cellulose is the majority by mass in the non-cellulosic polymer hybrid nanofiber felt, incorporation of additional types of fibers into the felt imparts the functionality required for felt applications. . Thus, having additional fibers within the felt imparts increased mechanical strength to the felt and allows multiple functionalities to be incorporated into the felt, providing stability and stability as discussed elsewhere herein. It is desirable because it can provide other aspects to the manufacturing method. Indeed, unexpectedly, the inclusion of even a small fraction of non-cellulosic polymers in the hybrid nanofiber felt improves the electrospinning process, and in particular the hybrid nanofiber felt can be combined with composite nanofibers. It has also been discovered by the inventors that when composed of both single-component nanofibers, the finished product can be adapted for a variety of biological and industrial applications.

Synthetic polymeric nanofibers (eg, those made from vinyl and acrylic polymers) offer a wide range of chemical functionality for bioseparations and other applications. By combining different polymer units, the surface chemistry of the resulting fiber can be controlled as part of the electrospinning process, directly imparting functionality to the produced nanofibers. Alternatively, and similar to conventional micrometer-scale fibers, the surface functionality of polymeric nanofibers can be adjusted by applying an electric field to match the specific functionality requirements for various bioseparation applications (discussed below). It can be chemically modified after spinning. The chemistry of functionalization is well known in polymer technology. They also withstand the rigorous cleaning regimes typically associated with bioprocessing. Exemplary functionalization chemistries are also described in further detail elsewhere herein.

Synthetic carbon-based adsorption media and filtration membranes are often much more chemically robust than cellulosic media, thus requiring strong acids and bases to clean the separation media between uses. can be used when In addition, hybrid nanofibers containing both cellulosic and non-cellulosic polymers (e.g., polyacrylonitrile and polyvinyl alcohol) have much larger specific surface areas and high Indicates mechanical strength. Thus, there is an observable synergy when composite nanofibers contain both cellulosic and non-cellulosic polymers.

(1) thermoplastic homopolymers such as vinyl polymers, acrylic polymers, polyamides, polyesters, polyethers and polycarbonates; (2) thermal polymers such as vinyl-co-vinyl polymers, acrylic-co-acrylic copolymers and vinyl-co-acrylic polymers; (3) elastic polymers such as triblock copolymer elastomers, polyurethane elastomers and ethylene-propylene-diene-elastomers; (4) high performance polymers such as polyimides and aromatic polyamides; (5) poly(p-phenylene terephthalamide). (6) textile polymers such as polyethylene terephthalate and polyacrylonitrile; (7) conductive polymers such as polyaniline; and (8) biocompatible polymers such as polycaprolactone, polylactide, chitosan and polyglycolide. Many polymers have been successfully electrospun into nanofibers, including organic polymers (or “biopolymers”). As noted above, the polymer can also be a copolymer of two or more of the polymer types listed above.

Examples of additional polymers that can be added into the hybrid nanofiber felt are polyacrylonitrile (PAN), polyimides, polyamides (nylon 6, nylon 6,6, nylon 6,10, etc.), polyesters (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 a single component derivatized cellulose solution is unstable, resulting in low yields, low efficiency (long hours and many interruptions), and only 1 This is partly due to the possibility of producing low-quality nanofibers (broad size distribution, fragility, etc.) with only one chemical functionality. Therefore, it may be necessary to combine cellulose derivatives with non-cellulosic polymers that stabilize the electrospinning process in order to efficiently make large quantities of high-quality nanofiber felts with multiple functionalities.

The non-cellulosic polymer of the composite nanofibers of the present invention may consist of synthetic carbon-based polymers removable from the nanofiber felt by exposure to elevated temperatures and/or solvents. Exposure of the nanofiber felt to a mixture of solvents or a combination of elevated temperatures and solvents can be done 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 (eg, those made from vinyl and acrylic polymers) offer a wide range of chemical functionalities for bioseparation applications. By combining different polymer units, the surface chemistry of the resulting fiber can be controlled as part of the electrospinning process, directly imparting functionality to the produced nanofibers. Alternatively, and similar to conventional micrometer-scale fibers, the surface functionality of polymeric nanofibers can be adjusted by applying an electric field to match the 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 to lend themselves to a wide variety of uses. Such functionalization chemistries are well known in the polymer art. They also withstand the rigorous cleaning regimes typically associated with bioprocessing.

(1) thermoplastic homopolymers such as vinyl polymers, acrylic polymers, polyamides, polyesters, polyethers and polycarbonates; (2) thermal polymers such as vinyl-co-vinyl polymers, acrylic-co-acrylic copolymers and vinyl-co-acrylic polymers; (3) elastic polymers such as triblock copolymer elastomers, polyurethane elastomers and ethylene-propylene-diene-elastomers; (4) high performance polymers such as polyimides and aromatic polyamides; (5) poly-p-phenylene terephthalamide. (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. We have successfully electrospun many polymers into nanofibers, including

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

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

Electrospinning Electrospinning is a technique in which only electric forces are used to drive the spinning process to produce polymer fibers from solution or melt. Electrospinning produces fibers with diameters in the nanometer range, unlike conventional spinning techniques (e.g., solution spinning and melt spinning), which can produce fibers with diameters in the micrometer range (approximately 5-25 μm). can do. Electrospun polymeric nanofibers have many surprising properties, including small fiber diameter and concomitant large specific surface area, high degree of polymer orientation and resulting excellent mechanical properties. Additionally, felts made with electrospun polymeric 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 "nanofabrication methods", thereby making them relatively easy to assemble and process into applications, at low cost. become nanofibers.

In general, nanofiber formation is a delicate and complex balance of the three main forces involved in the electrospinning process, including electrical, surface tension and viscoelastic forces. Of these three forces, the electric force always favors the formation of products with the greatest surface area. Surface tension always favors the formation of products with minimal surface area. The viscoelastic force is a force that varies significantly with solvent evaporation and is the main reason for preventing the electrospinning jet/filament from breaking into droplets. The viscoelastic force opposes the electric force when the electric force predominates. When surface tension prevails, viscoelastic forces oppose surface tension.

Theoretically, the smallest nanofibers can be formed under two conditions. If (1) the excess charge density carried by the electrospinning jet is high, and (2) the time is long enough to prevent capillary breakage of the jet/filament, the viscoelastic forces will prevent capillary breakage of the jet/filament. If the electric force is high enough to allow the jet to be stretched effectively, but low enough to allow the jet to be effectively stretched. In condition (1), adding a soluble electrolyte to the spin dope (e.g., adding a strong electrolyte such as NaCl to the aqueous polyethylene oxide solution) greatly increases the excess charge density carried by the jet, resulting in more It has been found that small diameter nanofibers can be formed. However, this method also produces negative effects such as (a) reduced flow rate and consequent reduced productivity of the nanofibers and (b) electrolyte contamination of the prepared nanofibers. It can be difficult to remove the electrolyte without sacrificing the properties of the nanofibers.

Condition (2) requires further understanding of jet solidification. In general, jet solidification is closely related to solvent volatility. If the solvent is too volatile, the time to effectively draw the electrospinning jet/filament is short. Thus, fibers of relatively large diameter are obtained. If the solvent volatility is too low, the electrospinning jet/filament can break into droplets upon stretching. Beads and/or beaded fibers are thus obtained.

The electrospinning process generally includes three steps. (1) the initiation of the electrospinning jet/filament and elongation along the straight trajectory of the jet; (2) the development of bending instability and further elongation of the jet (causing the jet to follow looped and spiral paths to a very high degree); and (3) solidification of the jet by solvent evaporation or cooling, thereby forming 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 generally described as follows.

Step 1: As shown in Figure 1, a 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 an electrode (e.g., copper wire) (3) to the solution. An electrically grounded collector (4) is placed at a fixed 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 forces overcome 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 helical loop. This phenomenon is called "bending (or whipping) instability". Bending instability typically causes the length of the jet to grow over 10,000 times in a very short period of time (less than 50 ms). Therefore, the elongation rate during bending instability is extremely high (up to 1,000,000 s ^-1 ). This extremely fast elongation rate allows the chains of the polymer to be effectively stretched and closely aligned along the nanofiber axis.

Step 3: The jet solidifies by solvent evaporation 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 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.

a. Removal of First Non-Cellulosic Polymer Optionally, one or more of the polymers present in the composite nanofiber, particularly the non-cellulosic polymer, is removed using high heat and/or solvent(s). can be removed. Removing 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 is left with size-controlled "pores" where the non-cellulosic polymer previously occupied space. . This additional "void space" provides a greater surface area to the resulting nanofiber felt, e.g., increases adsorptive binding capacity for separations, improves selectivity of separations based on size, and provides additional porosity. can improve throughput from Removal of the non-cellulosic polymer eliminates the opportunity for multiple functionalities (that were present in the composite nanofibers) to reside directly on the remaining cellulosic polymer nanofibers.

b. Cellulose Regeneration After preparing the “as-electrospun” nanofibers, the derivatized cellulose can be converted to cellulose by a regeneration process. Regenerated cellulose has the same properties as the previously described pure natural cellulose. The regeneration process is completed by contacting the nanofibers containing the 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 ion exchange groups such as weak or strong acids and bases (e.g., carboxylic acids and amines), hydrophobic groups such as phenolic compounds, and affinity ligands such as antibodies or enzyme substrates. additions.

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

Furthermore, nanofiber felts for use in bioseparation should exhibit several qualities. (1) small-diameter fibers that allow for maximum amount of specific surface area (this criterion is most important for adsorption processes and less important for strictly size-based separations discussed below), (2) flow rate in adsorption applications. (3) the fibers are exposed to potentially high operating pressures and stresses; (4) the fibers should have a well-defined and spatially consistent size and chemical composition;

In adsorption processes where macromolecular products such as proteins, nucleic acids and viruses are the primary targets, the extremely large specific surface area associated with nanofiber felts provides a vast number of potential binding sites for bioadsorptive separations. Nanofibers can be modified to contain a staggering number of binding sites, adsorption occurs almost exclusively on the surface of the fiber, and thus binding sites are readily available and can be used to target relatively large target molecules. does not need to be diffused internally. When using traditional porous resin beads, interdiffusion can often limit the capacity of many adsorption processes for bioproducts. Furthermore, because nanofiber membranes can be made with many different chemistries, adsorption ligands can be tailored to meet specific separation needs (eg, ionicity, hydrophobicity, and affinity). In some cases, ligands can be incorporated into the nanofibers from the source material during electrospinning, or the surface can be chemically modified to provide the desired adsorption agents after the nanofibers are produced.

Two of the most important characteristics of the separation operation are (1) the flow through the micro- and macropores of the felt (as opposed to densely packed resin beads), and (2) its Adsorption takes place on the surface of the fiber and no internal diffusion is required. These factors reduce the concern of high pressure drop at high flow rates and eliminate the slow intraparticle diffusion required for adsorption within the resin beads. It was found that the binding capacity of biomolecules to currently available absorbent felts is similar to that of resin beads in terms of size, but can be operated at a processing flow rate more than 10 times faster than that of packed beds. These factors lead to 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 between 20-300 nm). This is because large biomolecules are extremely difficult to purify using packed beds due to severe mass transfer limitations 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 produced using simple chemical modifications such as sulfonation of polystyrene fibers with sulfuric acid. Using grafting reactions, atom transfer radical polymerization (ATRP), and plasma treatment, ion-exchange surface functional groups and three-dimensional tethers can be isolated from various polymer substrates, including polypropylene, polyvinylidene difluoride, polysulfone, etc. was created. Phenyl and butyl groups can also be introduced as hydrophobic interaction ligands. Often the surface of the polymer membrane must be further modified towards increased hydrophilicity to prevent non-specific binding. This was accomplished by incorporating poly(ethylene glycol) and other polyols into the surface.

The ion exchange capacity of hybrid nanofiber felts can also be enhanced, for example, by introducing diethylaminoethyl (DEAE) groups as weak anion exchange ligands or carboxylic acids as weak cation exchange ligands.

d. Surface Functionalization with Antimicrobials In one embodiment of the invention, the non-cellulosic polymer is polyacrylonitrile (PAN). Fiber membranes of PAN have been widely adopted in filtration because of their thermal stability, high mechanical properties, and chemical resistance. Electrospun PAN nanofiber felts have been of particular interest due to properties such as small fiber diameter and associated high specific surface area, as well as the ability to control pore size between nanofibers and incorporate antimicrobial agents at the nanoscale. Felts composed of nanofibers with antimicrobial functionality have received increasing attention due to concerns over purified water and/or filtered air quality and processing costs. Water and air filters (especially those operating in dark, damp conditions) are constantly subject to attack from environmental microorganisms. Microorganisms (such as bacteria) that can be easily captured by filters grow rapidly to form biofilms. The quality of purified water and/or filtered air is compromised when microorganisms build up on the filter surface. Moreover, their enhancement also has an unfavorable effect on water and/or air flow.

In addition, biofilm-contaminated filters are difficult to clean. High pressures are normally required during operation. This increases costs. In the reported method, antimicrobial agents (such as N-halamine and silver ions/nanoparticles) are directly incorporated into the spin dope, thus the antimicrobial agent molecules/particles are distributed throughout the nanofibers (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, mainly because high antimicrobial content can seriously affect the electrospinning process and/or compromise the properties of the resulting nanofibers. A potential solution to these problems is to introduce antibacterial functionality to the nanofiber surface after the nanofibers are produced (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 nitrile (-C[identical to]N) groups in PAN can be chemically converted to amidoxime (-C( _NH2 )=NOH) groups. Amidoxime groups can coordinate with a wide range of metal ions, including silver ions, and can reduce the coordinated silver ions to silver nanoparticles. Both silver ions and silver nanoparticles are antibacterial agents with high antibacterial effects.

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

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

Unlike modifying the nanofiber surface for ion-exchange and hydrophobic interaction functionalities, incorporation of affinity ligands onto nanofibers can be more difficult. The methods often involve first modifying the surface to create coupling sites for immobilization of the ligand, followed by binding of the ligand to the active site. Importantly, both the initial surface modification and ligand coupling should be robust so as not to leach out during processing.

In some cases, simple carboxyl groups of methacrylic acid grafted to the surface serve as active coupling sites by creating covalent amide bonds between functionalized carboxyl groups and exposed amine groups on protein ligands. be able to. Similarly, oxidation of cellulose (when properly controlled) can form covalent bonds to primary amines of proteins (including protein A and protein G), particularly through the lysines of amino acids. can be provided on the fiber surface. In another case, surface functionalization with common affinity dyes (eg Cibacron Blue, which can bind some proteins) can be directly coupled to cellulose nanofibers.

More sophisticated, 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) as a block copolymer with poly-D,L-lactide (PDLLA). After electrospinning, glycol can be coupled with biocytin (which is capable of affinity interaction with streptavidin fusion proteins) 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. b-PEG-NH2 (PLGA-b-PEF-NH2) diblock copolymers can be created. Finally, in some cases it is possible to use the unique active sites associated with some nanofiber matrices. For example, Concanavalin A (an affinity tag for lectins associated with glycol-proteins and/or other glycol conjugates) has been successfully coupled 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 As an orthogonal purification mechanism to adsorption, size-based separation is also routinely used in downstream bioprocessing. Depth filtration and microfiltration are common operations used for clarification of fermentation broths to remove cells (approximately 1-20 μm) and cell debris (0.1-1 μm) from bioreactor slurries. Membrane-based nanofiltration is used for virus exclusion and/or purification of 20-200 nm virus particles, and ultrafiltration is commonly used for protein concentration and purification. In any case, some properties of the separation medium are desirable. First, well-defined size cutoffs are desirable to obtain closely controlled separations. Second, highly porous materials are needed for high throughput processing without excessive pressure requirements to minimize operating time and/or membrane area requirements. And third, chemical and physical robustness is desirable for harsh cleaning conditions and operations under moderate pressure. Nanofiber felts can be produced inexpensively in large quantities from mechanically and chemically tough fibers, and have well-controlled pore sizes in the fibers (or as hollow fibers), so advanced size-based It presents surprisingly good opportunities as a separation medium. Polymer nanofibers generally exhibit minimal amounts of non-specific binding, but can 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/biological particles with attendant fouling, but can withstand harsh regeneration conditions.

To date, nanofiber meshes for size-based separation have been primarily applied to the isolation of nanometer- and micrometer-scale bioparticles (or surrogates) by depth filtration mechanisms. The increased specific surface area of the nanofibers within the filtration mat provides a more tortuous path, increasing the chances of capturing desired particles from solution while maintaining high porosity. 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 could remove polystyrene particles between 0.5 and 10 μm. Ceramic nanofiber meshes were probably the most widely used. For one example, the combination of large titanaate nanofibers and smaller boehmite nanofibers is capable of very high fluxes (1000 L/m2.h) at relatively low pressure driving forces (20 kPa) and particles larger than 60 nm. was substantially all removed from the solution. Note that many applications of micro- and nano-depth filtration also rely on the chemisorption of particles to surfaces, the nanofibers of which can be readily manufactured to specifically adsorb the desired impurities.

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

Additionally, the pore sizes of the fibers in the felt typically have a tight pore size distribution (greater than 90% of the nanofibers fall between 100 nm and 500 nm) to prevent channeling and exceed the desired size cutoff for filtration operations. Keep only seeds. Finally, nanofiber felts are typically sufficiently mechanical to operate under conditions of potentially large pressure drops (up to 100 psi) and high flow rates (flux values greater than 30 L/(min·m ² )). and chemically robust enough to withstand potentially harsh cleaning regimes (often involving 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 noted above, the hybrid nanofelts of the present invention can be formed from various combinations of polymers and nanofibers. Examples of these include, for example:
• A composite nanofiber felt wherein all of the nanofibers in the felt consist 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 monocomponent nanofibers. • A nanofiber felt consisting of at least one monocomponent nanofiber and at least one composite nanofiber.

In addition to the nanofelt configurations described above, the nanofelt of the present invention may also contain microfibers to add stability, strength, and other physical properties of the felt for use in specific applications. 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 Prior Art Cellulose Acetate Single-Component Nanofiber Felts Cellulose acetate single-component nanofiber felts are described in 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 (approximately 30,000 g/mol average molecular weight), NaOH, NaCl, acetone, N,N-dimethylacetamide (DMAc) and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich Co. (Milwaukee, WI). bottom. 2-(Diethylamino)ethyl chloride hydrochloride (DAECH) of 98% purity was purchased from Alfa Aesar Co. (Ward Hill, Mass.).

A solution of 15% (mass fraction) cellulose acetate in acetone/DMAc (2/1 mass ratio) was prepared at room temperature. The solution was added to the syringe. The electrospinning setup included a high voltage power supply and laboratory produced rollers. 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 randomly overlaid felts on an electrically grounded aluminum foil covering the roller. A heat lamp was used to dry the nanofiber felt during electrospinning, and the felt was further dried in a vacuum oven after electrospinning. Overall, the electrospinning process was relatively unstable and interrupted frequently 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 .

As-electrospun cellulose acetate nanofiber felts were first hydrolyzed/deacetylated by soaking in 0.05 M NaOH aqueous solution for 24 h. The product, called regenerated cellulose nanofiber felt, was then rinsed three times in distilled water and dried in a vacuum oven at 60°C. The samples were immersed in a 15% (mass fraction) DAECH aqueous solution for 10 minutes and then dried at 60°C. The sample was then immersed in a 0.5M NaOH aqueous solution at 90°C for 10 minutes. The samples were rinsed three times in distilled water and dried at 60°C to obtain DEAE anion-exchanged cellulose nanofiber felts.

[Example 2]
Preparation of Hybrid Nanofiber Felts of CA/PEO Composite Nanofibers and PAN _Single Component Nanofibers and diethylaminoethyl chloride were purchased from Sigma-Aldrich Co. (Milwaukee, WI).

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

During the electrospinning process, two syringes filled with spin dopes of PAN or CA+PEO were placed on opposite sides of a lab-produced roller. Overall, the electrospinning process is very stable, sustainable for a long time (>48 h), and consists of CA+PEO composite nanofibers and PAN nanofibers (self-supporting ) or supported on medical cotton gauze) electrospun hybrid nanofibrous mats were collected on an electrically grounded aluminum foil covering the roller.

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

[Example 3]
Preparation of Hybrid Nanofiber Felts of CA/PVP Composite Nanofibers and PAN _Single Component Nanofibers and diethylaminoethyl chloride were purchased from Sigma-Aldrich Co. (Milwaukee, WI).

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

During the electrospinning process, two syringes loaded with PAN or CA+PVP spin dopes were placed on opposite sides of the laboratory-produced roller. Overall, the electrospinning process is very stable and sustainable for a long time (>48 hours), consisting of CA+PVP composite nanofibers and PAN nanofibers (self-supporting or medical cotton The electrospun hybrid nanofibrous mat (supported by gauze) was collected on an electrically grounded aluminum foil covering the roller.

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

[Example 4]
Preparation of Hybrid Nanofiber Felts of CA/PEO Composite Nanofibers and Nylon 6 Single Component Nanofibers Cellulose Acetate (CA), Polyethylene Oxide (PEO), Chloroform ( _CHCl3 ), Hexafluoroisopropanol (HFIP), Nylon 6, and Nylon 6 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 dopes, the polymer was dissolved in HFIP. For CA+PEO spin dopes, 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 dopes were placed on opposite sides of the lab-produced roller. Overall, the electrospinning process is very stable and sustainable for a long time (>48 hours), consisting of CA+PEO composite nanofibers and nylon 6 nanofibers (free-standing or medical cotton The electrospun hybrid nanofibrous mat (supported by gauze) was collected on an electrically grounded aluminum foil covering the roller.

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. The mat was then hydrolyzed/deacetylated by soaking in aqueous NaOH for 24 hours. This process also dissolves/removes the PEO from the mat. The resulting hybrid nanofibrous mat of regenerated cellulose nanofibers and nylon 6 nanofibers was rinsed with distilled water and dried.

[Example 5]
Preparation of Hybrid Nanofiber Felts of CA/PVP Composite Nanofibers and Nylon 6 Monocomponent Nanofibers Cellulose Acetate (CA), Polyvinylpyrrolidone (PVP), Chloroform ( _CHCl3 ), Hexafluoroisopropanol (HFIP), Nylon 6, and Nylon 6 Diethylaminoethyl chloride was purchased from Sigma-Aldrich Co. (Milwaukee, WI).

Nylon 6 and CA+PVP spin dopes were prepared separately. Briefly, for nylon 6 spin dope preparation, nylon 6 was dissolved in HFIP to form a solution. For CA+PEO spin dopes, 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 dopes were placed on opposite sides of the lab-produced roller. Overall, the electrospinning process is very stable, sustainable for a long time (>48 hours), and consists of CA+PVP composite nanofibers and nylon 6 nanofibers (self-supporting or medical The electrospun hybrid nanofibrous mat (supported on cotton gauze) was collected on an electrically grounded aluminum foil covering the roller.

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. The mat was then hydrolyzed/deacetylated by soaking in aqueous NaOH for 24 hours. This process also dissolves/removes the PVP from the mat. The resulting hybrid nanofibrous mat 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) were used for batch adsorption, dynamic adsorption, flow dispersion and permeability. The previous studies were compared with commercially available regenerated cellulose adsorption membranes and cotton balls. Commercially available cellulose membranes and cotton balls underwent the same electrospinning post-treatments as the single component nanofiber felts and hybrid nanofiber felts prior to testing.

A batch adsorption experiment was completed to determine the Langmuir equilibrium adsorption isotherm. For batch analysis, single component felt, hybrid felt, commercial cellulose and cotton balls were rinsed with buffer, cut into individual pieces of approximately 1 cm ² and weighed. For each medium, ten individual pieces (approximately 100 mg) were then placed in 15 milliliter (mL) centrifuge tubes. A stock solution of target protein was prepared at 2.0 mg/mL by mixing a known mass of lyophilized protein with buffer. Appropriate combinations of stock solution and buffer were added to each tube containing cut felt strips, commercially available cellulose membranes, or cotton balls to bring the final volume in each tube to 14 mL and the initial protein concentration. Concentrations were between 0.0 mg/mL and 2.0 mg/mL protein. A 1.0 mL liquid sample was immediately taken from each of the different initial protein concentrations and UV absorbance measured at 280 nm. The samples were then placed in an end-over-end mixer rotating at about 40 revolutions per minute (rpm). After mixing for a minimum of 24 hours, the liquid of each sample was removed and protein concentration was determined by UV-280 nm absorbance using a Genesys 10 UV spectrophotometer purchased from Thermo Electron Corporation (Madison, Wis.). The difference allowed to calculate the protein adsorbed to the felt. Test tubes with 2.0 mg protein/mL and no felt were also prepared to assess the potential for proteins to adsorb to the tube surface. Adsorption to the test tube surface was not observed. Similarly, controls were monitored to detect possible leached chemicals that could contribute to the UV-280 nm absorbance and non-specific binding of proteins to underivatized membranes (after renaturation with NaOH). evaluated. No leaching or non-specific binding was observed for any of the samples. A Langmuir adsorption isotherm was then generated and the modeling constants (Q _max and K _d ) were determined by a least-squares regression fit to the following equations:

式中、Qはタンパク質の膜への吸着平衡濃度(mg/g)であり、Q_maxは漸近最大飽和容量であり、Cはタンパク質の液相平衡濃度(mg/mL)であり、K_dは脱着定数(mg/mL)である。Qの高値及びK_dの低値は、効率的な吸着プロセスを示す。吸着分析に続いて、液体をデカンテーションし、フェルトを14mLのバッファーで1時間混合すると共に洗浄した。洗浄液を除去した後、1M NaClを含むバッファーを試験管に添加し、1時間混合し、UV-280nm吸光度によるタンパク質濃度用の液体をサンプリングした。溶離百分率を、研究の吸着時期に見られた結合量に基づいて算出した。

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 values of Q and low values of K _d indicate efficient adsorption processes. Following adsorption analysis, the liquid was decanted and the felt was mixed and washed with 14 mL of buffer for 1 hour. After removing the wash solution, buffer containing 1M NaCl was added to the tube, mixed for 1 hour, and the liquid was sampled for protein concentration by UV-280 nm absorbance. Elution percentages were calculated based on the amount of binding seen during the adsorption phase of the study.

The results, shown in Table 2 below, indicated that the electrospun hybrid nanofiber felt had the highest capacity followed by the single component cellulose nanofiber felt. Both nanofiber-based adsorbent materials had significantly higher saturation capacities than any of the commercial 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 results from increased binding capacity. In all cases, quantitative elution of proteins can be performed.

The permeabilities of buffers through single-component and hybrid nanofiber felts and commercial regenerated cellulose adsorbent membranes were measured. Higher permeability values indicate the ability to operate at higher throughput (faster processing times) and/or lower pressures. Both of these provide significant benefits to the manufacturing process. A small-scale "coin" membrane adsorption holder was utilized in all experiments. The unit allows an effective filtration area of approximately 1.5 cm ² and is sealed with an O-ring to prevent leakage. First, the pressure drop of only the system with the membrane holder in place but no membrane present was evaluated at flow rates ranging from 2.0 mL/min to 30.0 mL/min. Layers of each nanofiber felt or commercial membrane were then sequentially added 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 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 significantly higher than that of the corresponding commercial regenerated cellulose samples. 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), functionalized commercial membranes. similar to, but much lower than nanofelt.

Systems analysis of variance was performed on hybrid and single component nanofiber felts and commercial regenerated cellulose adsorbent membranes to determine the degree of axial mixing with different numbers of layers in place. Lower axial mixing (better flow distribution) is desirable to minimize channeling and premature breakthrough during the adsorption process. The same configuration used for the 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 curve was analyzed to calculate the Peclet (Pe) number by a least squares fit of the following equation.

式中、C_outは溶出液280nm吸光度であり、C_inは入口280nm吸光度であり、Vは添加されたアセトン溶液の体積であり、V₅₀はC_out/C_in=0.50の場合の体積である。より大きいPe値を使用して、プラグフロー(より低い軸混合及びよりよいフロー分散)により近似しているという望ましい性質を示した。

where C _out is the eluent 280 nm absorbance, C _in is the inlet 280 nm absorbance, V is the volume of the added acetone solution, and V ₅₀ is the volume when C _out /C _in =0.50. . A higher Pe value was used to indicate the desirable properties of being more closely approximated by plug flow (lower axial mixing and better flow dispersion).

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

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

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

Table 5 below shows the dynamic binding capacities of proteins at 10% breakthrough on nanofiber felts and commercial regenerated cellulose adsorbent membranes. The hybrid nanofiber mats had substantially higher dynamic capacities compared to any other adsorbent media evaluated. Furthermore, the elution results indicated that, within experimental uncertainty, protein elution was complete for each adsorption system and no loss in overall mass balance was observed.

The foregoing examples are set forth to provide those skilled in the art with a complete disclosure and description of the making and use of the composition embodiments and are intended to limit the scope of what the inventors regard as their invention. not something to do. Modifications of the above-described modes of carrying out the invention (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 hereby incorporated by reference as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. As such, incorporated herein by reference.

The present disclosure also discloses the following embodiments.
[Embodiment 1]
An electrospun hybrid nanofiber felt comprising composite nanofibers and single component nanofibers, the composite nanofibers comprising a mixture of derivatized cellulose and a first non-cellulosic polymer, and the single component nanofibers 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]
2. The nanofiber felt of embodiment 1, wherein the derivatized cellulose is selected from the group consisting of organic esters of cellulose, inorganic esters of cellulose and alkylcelluloses.
[Embodiment 3]
3. 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]
3. The nanofiber felt of embodiment 2, wherein the derivatized cellulose comprises inorganic esters of cellulose selected from the group consisting of cellulose nitrate and cellulose sulfate.
[Embodiment 5]
3. The nanofiber felt of embodiment 2, wherein the derivatized cellulose comprises an alkylcellulose selected from the group consisting of hydroxyethylcellulose and carboxymethylcellulose.
[Embodiment 6]
3. The nanofiber felt of embodiment 1, wherein the first non-cellulosic polymer comprises a synthetic polymer.
[Embodiment 7]
7. The nanofiber felt of embodiment 6, wherein the synthetic polymer is selected from the group consisting of vinyl polymers, polyethers, acrylic polymers, polyesters, polycarbonates and polyurethanes.
[Embodiment 8]
3. The nanofiber felt of embodiment 1, wherein the first non-cellulosic polymer comprises a natural polymer.
[Embodiment 9]
9. A nanofiber felt according to embodiment 8, wherein the natural polymer is selected from the group consisting of polysaccharides, polyamides and polylactides.
[Embodiment 10]
10. A nanofiber felt according to embodiment 9, wherein the natural polymer is a polysaccharide selected from the group consisting of starch and chitin.
[Embodiment 11]
10. A nanofiber felt according to embodiment 9, wherein the natural polymer is a polyamide selected from the group consisting of protein and gelatin.
[Embodiment 12]
3. The nanofiber felt of embodiment 1, wherein the second non-cellulosic polymer comprises a synthetic polymer.
[Embodiment 13]
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]
7. 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 making the nanofiber felt of embodiment 1, comprising:
a) separately preparing a composite polymer spin dope and a single component polymer spin dope;
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 collecting the solidified nanofibers as a randomly superimposed or partially aligned nanofiber felt.
[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]
18. The method of embodiment 17, wherein surface functionalization comprises attachment of an affinity ligand.
[Embodiment 21]
A method of purifying a biomolecule from a fluid, comprising:
a) preparing a nanofiber felt according to embodiment 14;
b) flowing the fluid through the nanofiber felt; and
c) A method comprising recovering biomolecules from the nanofiber felt.

Claims (36)

An electrospun hybrid nanofiber felt comprising composite nanofibers and single component nanofibers for use in chemical or biological separations, wherein the composite nanofibers are composed of derivatized cellulose and a first non-cellulosic polymer. nanofibers comprising a mixture, wherein the single component nanofibers comprise a second non-cellulosic polymer, wherein at least a portion of the first or second non-cellulosic polymer is removed from the nanofiber felt felt. 2. The nanofiber felt of claim 1, wherein the derivatized cellulose is selected from the group consisting of organic esters of cellulose, inorganic esters of cellulose and alkylcelluloses. 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 of claim 2, wherein the derivatized cellulose comprises inorganic esters of cellulose selected from the group consisting of cellulose nitrate and cellulose sulfate. 3. The nanofiber felt of claim 2, wherein the derivatized cellulose comprises an alkylcellulose selected from the group consisting of hydroxyethylcellulose and carboxymethylcellulose. 2. The nanofiber felt of claim 1, wherein the first non-cellulosic polymer comprises a synthetic polymer. 7. 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. 2. The nanofiber felt of claim 1, wherein the first non-cellulosic polymer comprises a natural polymer. 9. Nanofiber felt according to claim 8, wherein the natural polymer is selected from the group consisting of polysaccharides, polyamides and polylactides. 10. A nanofiber felt according to claim 9, wherein the natural polymer is a polysaccharide selected from the group consisting of starch and chitin. 10. Nanofiber felt according to claim 9, wherein the natural polymer is a polyamide selected from the group consisting of protein and gelatin. 2. The nanofiber felt of claim 1, wherein the second non-cellulosic polymer comprises a synthetic polymer. 13. The nanofiber felt of 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 or second non-cellulosic polymer is a copolymer. 2. The nanofiber felt of claim 1, wherein the derivatized cellulose of the composite nanofibers is regenerated to cellulose. 2. The nanofiber felt of claim 1, wherein the single component nanofibers contain surface functional groups . 17. The nanofiber felt of claim 16, wherein the surface functional groups comprise affinity ligands. 18. Nanofiber felt according to claim 17, wherein the affinity ligand comprises protein A or protein G. 17. The nanofiber felt of Claim 16, wherein the surface functional groups comprise ion exchange groups. 20. The nanofiber felt of Claim 19, wherein the ion exchange groups comprise strong acids, weak acids, or bases. 20. The nanofiber felt of Claim 19, wherein the ion exchange groups comprise diethylaminoethyl or carboxylic acid. 17. The nanofiber felt of Claim 16, wherein the surface functional groups comprise hydrophobic groups . 23. The nanofiber felt of claim 22, wherein the hydrophobic groups comprise phenyl groups, butyl groups, or both phenyl and butyl groups. An electrospinning method for making a nanofiber felt for use in chemical or biological separations comprising:
a) separately preparing a composite polymer spin dope and a single component polymer spin dope, the composite polymer spin dope comprising a mixture of a derivatized cellulose and a first non-cellulosic polymer; comprises a second non-cellulosic polymer, step
b) entering 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;
e) collecting the solidified nanofibers as a randomly superimposed or partially aligned nanofiber felt; and
f) differentially removing at least a portion of at least one of the first or second non-cellulosic polymers from the nanofiber felt;
method including. 25. The method of claim 24, further comprising converting the derivatized cellulose to cellulose. 25. The method of claim 24, further comprising surface functionalizing one or more of the nanofibers in the nanofiber felt. 27. The method of claim 26 , wherein surface functionalization comprises attachment of affinity ligands. 28. The method of claim 27, wherein the affinity ligand comprises protein A or protein G. 27. The method of claim 26 , wherein the surface functionalization comprises attachment of ion exchange groups. 30. The method of claim 29, wherein the ion exchange groups comprise strong acids, weak acids, or bases. 30. The method of claim 29, wherein the ion exchange group comprises diethylaminoethyl or carboxylic acid. 27. The method of claim 26 , wherein the surface functionalization comprises attachment of hydrophobic groups . 33. The method of claim 32, wherein the hydrophobic group comprises a phenyl group, a butyl group, or both phenyl and butyl groups. A method of purifying a biomolecule from a fluid, comprising:
a) providing an electrospun hybrid nanofiber felt comprising composite nanofibers and single component nanofibers, the composite nanofibers comprising a mixture of derivatized cellulose and a first non-cellulosic polymer, wherein the single component wherein the nanofibers comprise a second non-cellulosic polymer and at least one of the first or second non-cellulosic polymers is differentially removed from the nanofiber felt;
b) flowing the fluid through the nanofiber felt; and
c) A method comprising recovering biomolecules from the nanofiber felt. 35. The method of claim 34, wherein the biomolecule comprises one or more of proteins, nucleic acids or viruses. 35. The method of claim 34, wherein one or more of the nanofibers are surface functionalized with affinity ligands, ion exchange groups, or hydrophobic groups .

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