WO2012068442A1 - Fibre de grande aire de surface et membranes non tissées destinées à être utilisées dans des bioséparations - Google Patents

Fibre de grande aire de surface et membranes non tissées destinées à être utilisées dans des bioséparations Download PDF

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Publication number
WO2012068442A1
WO2012068442A1 PCT/US2011/061353 US2011061353W WO2012068442A1 WO 2012068442 A1 WO2012068442 A1 WO 2012068442A1 US 2011061353 W US2011061353 W US 2011061353W WO 2012068442 A1 WO2012068442 A1 WO 2012068442A1
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Prior art keywords
fibers
surface area
sample
target agent
gma
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PCT/US2011/061353
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English (en)
Inventor
Patrick Vasconcelos Gurgel
Yong Zheng
Steven James Burton
Ruben G. Carbonell
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Pathogen Removal And Diagnostic Technologies Inc.
North Carolina State University
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Application filed by Pathogen Removal And Diagnostic Technologies Inc., North Carolina State University filed Critical Pathogen Removal And Diagnostic Technologies Inc.
Publication of WO2012068442A1 publication Critical patent/WO2012068442A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0002—Organic membrane manufacture
    • B01D67/002—Organic membrane manufacture from melts
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/39—Electrospinning

Definitions

  • the present invention relates to the use of high-surface area fibers for the purpose of bioseparation of biologic and biotherapeutic compounds for their removal, separation, extraction or purification from a sample such as blood, plasma, serum, urine, tissue homogenates, cell culture media, fermentation broth or manufactured preparation.
  • the present invention also relates to the use of nonwoven material containing of high-surface area fibers for the purpose of said bioseparation; and the related methods for bioseparation using nonwoven material containing of high-surface area fibers.
  • the present invention further concerns devices comprising nonwoven material containing high-surface area fibers for performing such bioseparation.
  • Biotherapeutic compounds such as recombinant proteins, monoclonal antibodies, attenuated viruses, and nucleic acid segments, are a class of therapeutics that have become increasingly important in the past few years.
  • the bioseparations industry is under pressure to develop improved methodologies for the cost-effective recovery and purification of these compounds from complex media, as well as to develop processes to remove potential pathogens from said feed-streams.
  • the industry is currently being driven by a need to replace the conventional downstream separation processes with disposable systems, which have the potential to decrease labor and operational expenses associated with repetitive cleaning-in-place (CIP) and sterilization procedures.
  • CIP cleaning-in-place
  • Packed bed chromatography is commonly used in the bioseparation industry for capture of target proteins and other compounds.
  • Packed bed chromatography is characterized by the use of resins, gels, beads or other particles that are packed in a column for capturing and eluting a liquid sample through such column.
  • the technique has, however, widely known drawbacks such as slow intra- particle diffusion, high pressure drops across the column bed, relatively slow throughput, and high cost of chromatography media.
  • the costs of conventional bead-packed bed columns are typically high, which makes the development of disposable column systems a challenge.
  • a method for separating a target agent from a sample comprises the steps of: (a) providing the sample containing the target agent;
  • the method for separating a target agent from a sample further comprises the step of (d) collecting the sample resulting from step (c).
  • the method for separating a target agent from a sample further comprises the steps of (d) retrieving the sample resulting from step (c), and (e) collecting the target agent bound to the nonwoven material by eluting through the nonwoven material an elution solution interfering with the binding between the target agent and the fibers so as detach the target agent from the fibers.
  • the interest of a user of the present invention could be in obtaining a sample depleted from the target agent and therefore, the sample resulting from step (c) is collected for further uses and the nonwoven material with the bound target agent can be disposed or cleaned for being reused.
  • the interest of a user of the present invention could be in obtaining the target agent purified or separated from the sample and therefore, the sample resulting from step (c) is retrieved from the nonwoven material and possibly discarded or disposed, and the target agent that has bound to the nonwoven material is collected by eluting through the nonwoven material an elution solution interfering with the binding between the target agent and the fibers so as detach the target agent from the fibers.
  • the interest of such a user is in the resulting elution solution containing the unbound target agent that can be further used.
  • a device for separating a target agent from a sample comprising: (a) an inlet for receiving the sample;
  • the device can be disposable.
  • a method for separating a target agent from a sample comprising the steps of:
  • the method for separating a target agent from a sample may further comprise the step of:
  • the sample may be continuously introduced to the device via the inlet, and continuously collected via the outlet. Collecting and retrieving are used interchangeably in the present application.
  • the method for separating a target agent from a sample further comprises the step of:
  • a fourth aspect of the invention concerns a method for regenerating a device after its first use for the purpose of performing a second use, said method comprises the steps of:
  • the step (a) may not be performed.
  • the elution solution and the cleaning solution include, without limitation, acidic or basic buffers and solutions, salt solutions, guanidine, ammonium sulphate, chaotropic agents, detergents and co-factors in concentrated or diluted form.
  • the cleaning solution is the same that the elution solution but in a more concentrated form.
  • Many regeneration solutions can be used in accordance with this aspect of the invention, including, without limitation, acidic, neutral or basic buffers, salt solutions, and co-factor solutions.
  • the regeneration solution is a solution similar to next sample to be applied to the nonwoven material.
  • the regeneration solution can be an isotonic saline solution.
  • the regeneration solution is this specific buffer.
  • the nonwoven material is a membrane.
  • the nonwoven material may also consist of stacked membranes.
  • the high-surface area fibers of the present invention are preferably winged fibers or nanofibers.
  • the high- surface area fibers are winged fibers.
  • the high-surface area fibers have a surface area of at least 20,000 cm 2 /g, more preferably a surface area of at least 100,000 cm 2 /g, further preferably a surface area of at least 140,000 cm 2 /g, further more preferably a surface area of at least 200,000 cm 2 /g, also more preferably a surface area of at least 250,000 cm 2 /g, and also further preferably a surface area of at least 300,000 cm 2 /g.
  • the high-surface area fibers are nanofibers.
  • the nanofibers have preferably a diameter less than 1 pm, more preferably a diameter less than 0 5 pm, and also preferably a diameter between 50 nm and 500 nm.
  • the high-surface area fibers are made of cellulose, polyamide, polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polyethylene (PE), polypropylene (PP), polyolefin, polyethylene oxide (PEOX), polyphenol formaldehyde) (PPF), polyvinylalcohol (PVOH), polyvinylchloride (PVC), aromatic polyamide, polyacrylonitrile (PAN), polyurethane (PU), polyacetal, liquid-crystalline polyester, polysaccharide or a combination thereof.
  • the fibers are made of polybutylene terephthalate (PBT).
  • the fibers are made of polyethylene terephthalate (PET). According to a further embodiment, the fibers are made of polypropylene (PP). According to another further embodiment, the fibers are winged fibers made of polybutylene terephthalate (PBT), polyethylene terephthalate (PET) or polypropylene (PP) and have a surface area of at least 140,000 cm 2 /g.
  • PET polyethylene terephthalate
  • PP polypropylene
  • the fibers are coated with acrylate monomers.
  • the acrylate monomers are preferably 2-hydroxylethyl methacrylate (HEMA), acrylamide, acrylic acid, acrylonitrile, methyl rmethacrylate, glycidyl methacrylate (GMA) or a combination thereof.
  • Preferred acrylate monomers for coating are glycidyl methacrylate (GMA).
  • Other preferred acrylate monomers for coating are a combination of 2-hydroxylethyl methacrylate (HE A) and glycidyi methacrylate (GMA).
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram illustrating an exemplary embodiment of the present invention.
  • FIG. 1 A block diagram
  • the sample can be, without limitation, blood, plasma, serum, urine, tissue homogenates, cell culture media, fermentation broth or manufactured preparation.
  • the target agent can be, without limitation, a protein, a peptide, a lipid, a DNA molecule, a RNA molecule, an organic molecule, an inorganic molecule, a cell, a virus, a bacterium, a toxin or a prion.
  • the method of bioseparation of the present invention uses a nonwoven material containing or made of high-surface area fibers in absence of chromatographic media.
  • chromatographic media it is intended porous resins, beads of agarose, cellulose, glass, silica, dextran, acrylates, acrylamide, or other polymeric materials, or packed beds as described in the background, or the like. It has been surprisingly discovered that high-surface area fibers provide a so high binding capacity that it is no more desirable the use of chromatographic media.
  • using a nonwoven material made of high-surface area fibers without beads for chromatography is a cheaper way to manufacture a separation device. Using beads for chromatography is expensive and using only nonwoven materials made of high-surface area fibers has a real economic advantage.
  • Figure 1 shows the infrared spectra of a polybutyleneterephthalate (PBT) winged nonwoven fibers before and after grafting with glycidyi methacrylate (G A) (PBT and PBT-GMA, respectively). Arrows indicate the characteristic peaks for GMA and are showing that the PBT winged fabrics have been grafted with GMA.
  • PBT polybutyleneterephthalate
  • G A glycidyi methacrylate
  • Figures 2A-B show scanning electron microscope (SEM) images of a PBT winged nonwoven fibers before (2A) and after (2B) grafting with GMA, providing evidence that the structures conferring high-surface area fibers remain open and accessible after grafting.
  • SEM scanning electron microscope
  • Figure 3 shows the infrared spectra of polyethyleneterephthalate (PET) winged nonwoven fibers before and after grafting with GMA (PET and PET-GMA, respectively). Arrows indicate the characteristic peaks for GMA.
  • Figures 4A-B show SEM images of PET winged nonwoven fibers before (4A) and after (4B) grafting with GMA, providing evidence that the structures conferring high-surface area fibers remain open and accessible after grafting.
  • Figure 5 shows the infrared spectra of a polypropylene (PP) winged nonwoven fibers before and after grafting with GMA (PP and PP-PGMA, respectively). Arrows indicate the characteristic peaks of GMA.
  • PP polypropylene
  • Figures 6A-B show SEM images of PP winged fiber nonwoven fibers before (6A) and after (6B) grafting with GMA, providing evidence that the structures conferring high-surface area fibers remain open and accessible after grafting.
  • Figure 7 shows the infrared spectra of a PBT winged nonwoven fibers before and after grafting with poly(HEMA-co-GMA)-DEA (PBT winged and PBT winged-g-poly(HEMA-co-GMA)- DEA, respectively).
  • Figures 8A-B show SEM images of a PBT winged nonwoven fibers before (8A) and after (8B) modification with poly(HEMA-co-GMA)-DEA, providing evidence that the structures conferring high-surface area fibers remain open and accessible after grafting and modification.
  • Figure 9 shows the infrared spectra of a PET winged nonwoven fibers before and after modification with poly(HEMA-co-GMA)-DEA (PET winged and PET winged-g-poly(HEMA-co-GMA)-DEA, respectively).
  • Figures 10A-B show SEM images of a PET winged nonwoven fibers before ( 0A) and after (10B) modification with poly(HEMA-co-GMA)-DEA. providing evidence that the structures conferring high-surface area fibers remain open and accessible after grafting and modification.
  • Figure 1 1 shows the infrared spectra of a PP winged nonwoven fibers before and after modification with poly(HEMA-co-GMA)-DEA (PP winged and PP winged-g-poly(HEMA-co-GMA)-DEA, respectively).
  • Figures 12A-B show SEM images of PP winged nonwoven fibers before (12A) and after (12B) modification with poly(HEMA-co-GMA)-DEA, providing evidence that the structures conferring high-surface area fibers remain open and accessible after grafting and modification.
  • Figure 13 shows the infrared spectra of a standard PP nonwoven fibers before and after modification with poly(HEMA-co-GMA)-DEA (PP and PP-g- poly(HEMA-co-GMA)-DEA, respectively).
  • Figures 14A-B show SEM images of a standard PP nonwoven fibers before (14A) and after (14B) modification with poly(HEMA-co-GMA)-DEA, providing evidence that the structures conferring high-surface area fibers remain open and accessible after grafting and modification.
  • Nonwoven materials possess many unique characteristics that make them a desirable medium for bioseparations.
  • the materials have an engineered interconnected porous structure, allowing for convective mass transfer, while possessing negligible diffusion limitations.
  • Nonwoven materials also have a relatively large surface-to-volume ratio, and a controllable wide-range mesh pore size, which potentially allows for the handling of complex mixtures containing particulate materials, such as cells and cell debris, and the capture of large macromolecules, such as protein complexes, viruses, and even bacterial cells.
  • the porosity of nonwoven materials is controllable, and allows for the use of the materials at relatively high flow rates with a small pressure drop through the material.
  • another advantage of using nonwoven materials is that they can be produced in a variety of shapes and fiber diameters at low cost, and high production throughput (hundreds of square meters of material per minute).
  • nonwoven membranes offer a good opportunity to develop disposable bioseparation devices, for example.
  • one of the limitations of membranes has always been the relatively low capacity of such materials, especially compared to porous resins, which possess a very high surface area to weight ratio.
  • the inventors have found that high-surface area fibers can be used effectively to bind target agents, and by using nonwoven membranes made of high-surface area fibers, the inventors have produced devices with improved binding capacities.
  • Textiles made from the winged fibers are highly absorbent, because liquid can be drawn into the channels between the projections around the core of the fiber.
  • the shape of the fiber results in there being a greater area between the fibers for liquid absorption, and faster wicking compared to standard fibers having a round cross-sectional shape.
  • the textiles are also useful as filters, for example for filtering air, water and other liquids.
  • high-surface area fibers may be used for separating or purifying a target agent from a sample, by binding said target agent to the surface of the fibers.
  • the high-surface area fibers are utilized in separation devices in the form of a nonwoven material, most preferably in the form of one or more nonwoven membranes.
  • nonwoven materials made from high-surface area fibers enables a higher binding capacity to be achieved compared to nonwoven materials made of traditional fibers, and results in making the bioseparation/purification process more efficient and more economical.
  • bioseparation used herein is intended to include the purification, separation, removal or extraction of biological compounds or therapeutic compounds from a liquid or gaseous sample, by binding such compounds to the nonwoven material of the present invention.
  • Such bioseparation, purification, separation, removal or extraction is effective to remove all the compounds present in the sample, preferably at least 99.9% of the compounds present in the sample, more preferably at least 99.5%, further preferably at least 99.0%, further more preferably at least 95.0%, also preferably at least 90.0%, and also further preferably at least 80.0%.
  • the high-surface area fibers according to the present invention are intended to mean fibers possessing a high surface-to-volume ratio.
  • such high-surface area fibers by virtue of their shape, possess a high surface-to-volume ratio compared to a standard fiber having an approximately circular cross-sectional shape.
  • the high-surface area fibers according to the present invention possess a high-surface area to weight ratio.
  • the high-surface area fibers used in accordance with the present invention have a high-surface area, preferably a surface area of at least 20,000 cm 2 /g, more preferably a surface area of at least 100,000 cm 2 /g, further preferably a surface area of at least 140,000 cm 2 /g, further more preferably a surface area of at least 200,000 cm 2 /g, also more preferably a surface area of at least 250,000 cm 2 /g, and also further preferably a surface area of at least 300,000 cm 2 /g.
  • a wide variety of high-surface area fibers can be used to produce materials of the invention.
  • a preferred embodiment of the high-surface area fibers suitable for use in the present invention is a so-called "winged" fiber, as described in US patent application 1 1 /592,370, Pourdeyhimi and Chappas.
  • Winged fibers and textiles made from winged fibers possess a high surface area of 140,000 cm 2 /g or higher due to their structure, which comprises a middle core with several projections arranged radially along its length.
  • Another preferred embodiment of the high-surface area fibers according to the present invention includes fibers of small diameter.
  • the fibers of small diameter that can be used in the present invention have a diameter of 1 pm or smaller, preferably of 0.5 pm or smaller, more preferably of 0.25 pm or smaller.
  • Other preferred examples of high-surface area fibers suitable for use in the present invention are thin fibers having a diameter of less than 1 pm, which may be referred to as "nanofibers".
  • Nanofibers suitable for use in the present invention preferably have a diameter of at least 50 nm. The diameter is preferably no more than 500 nm, and therefore the preferred range is 50 nm to 500 nm (between 0.050 pm and 0.500 pm).
  • Nanofibers can be prepared by known techniques, such as electrospinning, melt spinning, dry spinning, wet spinning , melt blowing, and extrusion methodologies, including the technique known as "islands in the sea”.
  • electrospinning melt spinning
  • dry spinning wet spinning
  • melt blowing melt blowing
  • extrusion methodologies including the technique known as "islands in the sea”.
  • the thicker fibers provide strength to the fabric, while the small diameter fibers are made of polymers capable of capturing the target material in a complex mixture (such as prions from blood or plasma products, or a specific protein from a cell culture medium, fermentation broth, or tissue homogenate).
  • High-surface area fibers can be produced using a variety of polymers.
  • Preferred examples are cellulose (cotton), polyamide (nylon), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polyethylene (PE), polypropylene (PP) and other polyolefins, polyethylene oxide (PEOX), polyphenol formaldehyde) (PPF), polyvinylalcohol (PVOH), polyvinylchloride (PVC), aromatic polyamide (Twaron®, Kevlar®, or Nomex®), polyacrylonitrile (PAN), polyurethane (PU), polyacetals, liquid-crystalline polyesters, polysaccharides or combinations thereof.
  • the fibers and nonwovens produced therefrom can be subjected to activation processes such as self-assembled monolayers (SAMs), physical or chemical vapor deposition (PVD or CVD), UV or plasma surface treatment, surface coating and grafting, among others.
  • activation processes such as self-assembled monolayers (SAMs), physical or chemical vapor deposition (PVD or CVD), UV or plasma surface treatment, surface coating and grafting, among others.
  • One or more of the activation processes can be used to provide conformal layers of functional molecules coating the base polymer, effectively changing the chemical and physical characteristics of the fiber, allowing for different surface derivatizations, introduction of functional groups, attachment of ligands, or change in hydrophobic character.
  • Such materials can be used for the purification of biological compounds by solid-phase separation processes including but not limited to ion-exchange chromatography, hydrophobic interaction chromatography and affinity chromatography.
  • winged fibers and nonwoven material comprising winged fibers may be grafted with a conformal polymer coating.
  • conformal coating the inventors refer to a coating that conforms to the surface of the fibers, thus achieving full coverage of the fibers by an approximately uniform thickness of the grafted polymer. Conformal coatings are required for nonwoven system applications that necessitate complete control of surface properties, such as diagnostics, separations and other applications where the mats are to be exposed to complex mixtures.
  • winged fibers, or nonwoven membrane comprising winged fibers, having a conformal polymer coating.
  • Grafting may be effected using known technologies.
  • the method of coating described in the International Application WO 2009/ 51 593 has been found by the present inventors to be particularly effective.
  • nonwoven membranes made of winged fibers may be given a conformal coating of poly(glycidyl methacrylate) (pGMA) by grafting glycidyl methacrylate (GMA) monomers onto the surface and curing, in accordance with the method described in the I nternational Application WO 2009/151 593.
  • pGMA poly(glycidyl methacrylate)
  • GMA glycidyl methacrylate
  • the process starts with exposing the high-surface area fibers of the present invention or the nonwoven material made of such fibers to UV irradiation in the range between 1 50 to 300 nm in air. During the exposure, ozone is simultaneously generated as a result of 0 2 exposure to UV light.
  • the objective behind the use of UV irradiation plus ozone treatment in this invention is not to generate radicals or peroxides on the fiber surface. Instead, the goal is to etch the surface to increase its roughness, and simultaneously to increase the concentration of hydroxyl and other oxygen-containing compounds. The combined effect significantly increases the adsorption of initiators in the subsequent grafting step.
  • Polymer fibers may have a smooth or glazed surface, which is the consequence of the fiber production conditions, as the polymer melts or solution passes through a fine nozzle at very high speed.
  • a glazed surface prevents other molecules from attaching to the surface.
  • a rough surface can increase the adsorption of other molecules, such as initiators, to the surface.
  • Initiators are molecules that can produce free radicals under mild conditions and initialize radical polymerization reactions. The interactions between polar groups such as hydroxyl and other oxygen containing compounds, and initiators, can further help stabilizing the adsorption.
  • UV irradiation plus ozone is very effective in etching only a very thin layer of the fiber surface to increase its roughness and simultaneously generating hydroxyl and carbonyl groups.
  • Other approaches such as plasma treatment, peroxide oxidation, base and acid or any method which can increase surface roughness and render oxidization, can also be used for this purpose.
  • Some polymers are made from monomers which already containing polar groups, such as amines, carbonyls and hydroxyls etc. Initiators may adsorb to these surfaces to such an extent that a conformal coating can be obtained even without pre-treatment.
  • pre-treatment is indispensable for a conformal coating.
  • the functional monomers can be grafted to the surface by free radical polymerization.
  • This process can use UV-initialized radical polymerization or thermally-initialized radical polymerization.
  • Photosensitizers and thermally decomposable initiators should be used in the respective processes.
  • Photosensitizers include benzophenone, anthraquinone, naphthoquinone or any compound involving hydrogen abstraction for initialization.
  • Thermally decomposable initiators include azo compounds or peroxide compounds.
  • the monomer concentration is in the range of 1 to 20%.
  • the initiator concentration is in the range of 0.5 to 7%. Alcohols and hydrocarbons can be used as solvents.
  • the grafting is carried out between approximately 1 and 120 minutes.
  • acrylate monomers can be selected for grafting, for example, 2-hydroxylethyl methacrylate, acrylamide, acrylic acid, acrylonitrile, methyl methacrylate, glycidyl methacrylate and similar acrylate derivatives.
  • any monomer which can be polymerized by radical polymerization can be used for grafting.
  • a continuous UV irradiation of 300-450 nm is required for UV-initialized grafting.
  • a pre-treated substrate pre-soaked with the solution of monomer and photosensitizer is inserted between two thin glass plates (or a confined geometry) and exposed to UV for a determined amount of time.
  • Confined geometry forming a saturated vapor phase near the surface of the substrate, has the advantage of preventing fast loss of solvent.
  • the confined geometry also minimizes the volume of the grafting solution and allows for the absence of degassing and inert gas protection.
  • the glass plates may be pre-treated with mold release agents, for example Frekote®.
  • the grafting can be performed at room temperature or at an elevated temperature, but far below the boiling temperature of monomer solution. Cooling is necessary when solvent evaporates too fast.
  • An elevated temperature is required for thermally-initialized grafting, where initiators can decompose efficiently. Same confined geometries can also be used.
  • the substrates are washed with appropriate solvents to extract unreacted monomers and unattached homopolymers.
  • Water is a good solvent for monomers and homopolymers which are aqueous soluble. Otherwise, extraction can be done by alcohols, hydrocarbons, or with any other suitable solvent.
  • PolyG A may be used to bind the target agents directly.
  • pG A also provides means of attaching ligands to the surface of the fibers.
  • pGMA and polyGMA are used interchangeably.
  • pGMA may be modified by the attachment of further functional groups such as, without limitation to, amino groups, carboxyl groups, hydroxyl groups and sulfhydryl groups, so as to capture and purify proteins by electrostatic interactions
  • the material can be aminated, for example, by immersing the membrane in a 30% dimethyalamine solution in water at temperatures ranging from 22°C to 45°C for 1 hour to 16 hours, following rinsing and drying.
  • pGMA may be modified by the attachment of further functional groups such as amino groups, epoxy groups, carbonyl groups, and hydroxyl groups, among others, which may be used to facilitate the attachment of ligands.
  • Suitable ligands include, but are not limited to, peptides and synthetic ligands, but many different types of ligands are contemplated by the inventors, including larger compounds such as proteins, antibodies and bacteria.
  • the sample that is used in the present invention may be a gaseous or liquid sample.
  • the invention is used to separate or purify a target agent from a liquid sample.
  • the sample is a biological sample such as whole blood, blood-derived compositions or components, plasma, plasma derivatives, serum, urine, tissue homogenates, cell culture media, fermentation broths, before or after clarification, process intermediate solutions derived from processing of such samples, and manufactured preparation.
  • a manufactured preparation is a preparation obtained at the final step or at an intermediate step of the manufacture of a compound, a peptide, a protein or the like.
  • High-surface area fibers can be used to separate or purify a wide range of target agents from a sample.
  • the target agents are preferably biological compounds or therapeutic compounds.
  • Preferred target agents include, without limitation, proteins, peptides, lipids, DNA, RNA, SiRNA, simple and complex carbohydrates, whole cells, viruses, attenuated viruses, cell structures, toxins, prions, peptides, proteins, RNA molecules, DNA molecules, monoclonal antibodies, antigens, microorganisms such as bacteria and viruses, microorganisms material such as bacteria cell membranes or virus capsule molecules, antibodies, cells such as white blood cells, organic compounds or inorganic compounds.
  • the present invention is particularly useful for the separation or purification of biological compounds that are difficult to capture using current techniques, eg chromatography columns.
  • biological materials include biotherapeutics, for example, peptides, proteins, polynucleic acids, SiRNA, RNA, monoclonal antibodies, attenuated and non-attenuated viruses, antigens, toxins, prion, protein, and bacterial cells.
  • the nonwoven materials of the present invention are typically used in the form of a membrane, i.e. a flat, sheet structure with a thickness that is considerably less than its other dimensions. It will be appreciated, however, that alternative shapes may be used.
  • the nonwoven material may be formed into a three-dimensional article by forming a cylinder from a sheet of nonwoven, or forming a stack of individual nonwoven membranes using pre-cut disks of the material.
  • a device for bioseparation comprises nonwoven material composed of high- surface area fibers.
  • the high-surface area fibers are coated as above-described.
  • the coated fibers are modified so as to attach thereto functional groups such as amino groups or to attach ligands.
  • the ligands can be specific to toxic compounds in order to detoxify biological sample such as blood or serum.
  • the ligands are specific to therapeutic compounds and the device can be used for purifying a therapeutic compound from a crude solution in a final step of manufacturing.
  • a device for separating or purifying a target agent from a sample which device comprises an inlet for introducing the sample into the device; and nonwoven material comprising high-surface area fibers.
  • the nonwoven material is located in a binding chamber for contacting the sample with the nonwoven material and allowing the target agent to bind the high-surface area fibers.
  • the device further comprises an outlet for collecting, retrieving or recovering the processed sample.
  • the nonwoven material is in the form of a nonwoven membrane, or stacked layers of two or more nonwoven membranes, which are used in accordance with known membrane separation methods.
  • the sample is introduced onto the top membrane and diffuses through the nonwoven material, and the processed sample is collected.
  • the processed sample can remain in the separation device, rather than being removed via an outlet. This is particularly useful for performing the bioseparation of target agents from small volumes of sample.
  • the sample is introduced in the separation device and collected from the separation device in a continuous manner. To do so, the sample is continuously introduced into the device via an inlet and the processed sample continuously collected via an outlet.
  • the nonwoven material of the device is reusable performing another bioseparation.
  • the following solutions are applied to the nonwoven material:
  • an elution solution is eluted through the nonwoven material, wherein said elution solution interferes with the binding between the target agent and the fibers so as to detach the target agent from the fibers;
  • the step (a) may not be performed.
  • the elution solution and the cleaning solution include, without limitation, acidic or basic buffers and solutions, salt solutions, guanidine, ammonium sulphate, chaotropic agents, detergents and co-factors in concentrated or diluted form.
  • the cleaning solution is in a more concentrated form than the elution solution.
  • Many regenerating solutions can be used in accordance with this embodiment of the invention, including, without limitation, acidic, neutral or basic buffers, salt solutions, and co-factor solutions.
  • the regeneration solution is a solution similar to next sample to be applied to the nonwoven material.
  • the regeneration solution can be an isotonic saline solution.
  • the regeneration solution is this specific buffer.
  • Example 1 Grafting polybutyleneterephthalate (PBT) winged fibers with glycidyl methacrylate (GMA) monomer
  • winged fibers made of polybutyleneterephthalate (PBT) were grafted with a glycidyl methacrylate (GMA) monomer.
  • the used coating method ensures the production of a conformal coating of GMA on top of the winged fibers, and makes the resulting material homogeneous in terms of surface characteristics.
  • the monomers of GMA polymerized together on the surface of the fibers and are then called polyGMA or pGMA.
  • Nonwoven membranes (containing PBT fibers) of dimension 2 * 4 cm were prepared. All the samples were pre-treated by exposure to UV light at 254 nm for 10 minutes for each side of the nonwoven material.
  • Membranes were then soaked with 15% GMA monomer solution with initiator to monomer ratio of 1 :5 and sandwiched between two glass slides. The glass-nonwoven sandwiches were then exposed a second time to UV light, this time at 365 nm for 15 minutes. After grafting, all samples were washed with THF and methanol.
  • FTIR and SEM were used to characterize the nonwoven membranes before and after grafting.
  • the FTIR spectra of the PBT nonwoven membranes before and after grafting are shown in Figure 1.
  • the images of the original PBT nonwoven membrane (Fig. 2A), taken at different magnifications, show that the nonwoven has a radial, fin-like structure (wings).
  • Fig. 2B After grafting (Fig. 2B), the inter-fiber pores were left virtually unchanged by the process.
  • the grafted fiber surface showed a different topology from the original, indicating the presence of a pGMA layer.
  • the wing/fin structure of the fiber was retained and not blocked by the grafting layers.
  • Example 2 Grafting polyethyleneterephthalate (PET) winged fibers with glycidyl methacrylate (GMA) monomer
  • PET winged fibers were grafted with glycidyl methacrylate (GMA) monomer using the methodology described in Example 1.
  • GMA glycidyl methacrylate
  • Figure 3 the infrared (IR) spectra of the PET fibers before and after grafting are shown. Similar to PBT and GMA, PET also has carbonyl groups. Therefore, the peak at 1720 cm “1 is not a good indicator for pGMA grafting. However, the appearance of new peaks at 1 150 cm "1 and 908 cm "1 for the grafted PET indicate pGMA grafting.
  • FIG. 4 Images of the PET fibers before and after grafting are shown in Figure 4.
  • the original PET fibers have a structure similar to the one observed for the polypropylene (PP) fibers (Fig. 4A).
  • Fig. 4B After grafting (Fig. 4B), the inter-fiber pores remain intact from the grafting process.
  • the fiber surface shows grafted pGMA.
  • the space between fiber fins was not blocked or clogged by the grafting.
  • the grafted layer extended to the bottom of the fiber through the space between wings, and the side walls of the wings also seem to be grafted with a smooth layer of pGMA.
  • Example 3 Grafting polypropylene (PP) winged fibers with glycidyl
  • GMA methacrylate
  • PP winged fibers were grafted with glycidyl methacrylate (GMA) monomer using the methodology described in Example 1.
  • Example 4 Comparing the binding capacity of the grafted winged fibers with the binding capacity of grafted standard fibers U 2011/061353
  • the materials tested are in the form of membranes and were grafted with 2-hydroxyl ethyl methacrylate (HEMA) and glycidyl methacrylate (GMA), to produce a coating of poly(2-hydroxyl ethyl methacrylate-co- glycidyl methacrylate) (poly(HEMA-co-GMA)).
  • the materials were further modified with diethylamine, to introduce a reactive amino group, which confers positive charges to the material, forming an ion-exchange surface.
  • the final modified coating is poly(2-hydroxy-ethyl-methacrylate-co-2-diethylamino-2-hydroxy-ethyl- methacrylate) (poly(HE A-co-GMA)-DEA).
  • This copolymer at the surface of the fibers increases the hydrophilic character of the surface, and allows for the control of the density of the amino groups, and the production of membranes with a variety of charge levels.
  • PBT, PET and PP winged fiber-based materials were provided by Allasso Industries®, Inc. (Raleigh, NC, USA). They have basis weights of 58 gsm (g/m 2 ), 62 gsm and 76 gsm, respectively.
  • the PP standard fibers have a round cross-section shape and a smooth surface, have, a basis weight of 40 gsm, and were used as control in the experiment.
  • the monomer solution used for grafting consists of HEMA and GMA in a ratio of 9: 1. Grafting process was performed as described above in Example 1 , with the exception that the grafting time was extended to 90 minutes. FTIR and SEM were taken on the samples before and after modification as discussed below in Examples 5, 6, 7 and 8.
  • the binding capacity of the grafted PP standard fiber-based material was determined to be 13 mg of protein per gram of membrane (mg/g), while the binding capacity of the grafted PP winged fiber-based material is 21 mg/g, showing an increase of about 60% in binding capacity.
  • the grafted PBT winged fiber-based material shows a BSA binding capacity of about 23 mg/g, while the grafted PET winged fiber-based material have a binding capacity of about 13 mg/g.
  • Example 5 Polybutyleneterephthalate (PBT) winged fibers grafted with a combination of HEMA and GMA monomers and aminated with DEA [0101]
  • Figure 7 shows the IR spectra of the poly(HEMA-co-GMA)-DEA) PBT winged fibers before and after grafting/modification process as described in Example 4. Arrows in the figure indicate the characteristic peaks for OH at 3500 cm "1 and C-0 at 1 150 cm "1 . The appearance of a peak at the first position is a clear indication of HEMA graft. The shoulder shown at the second position can be attributed to both HEMA and GMA. Since tertiary amines generally do not absorb in the IR range, there is no observable peak for tertiary amine in the spectra.
  • Figures 8A-B show images of the poly(HEMA-co-GMA)-DEA) PBT fiber nonwoven material before (Fig. 8A) and after (Fig. 8B) grafting/modification process. Those images illustrate the typical state of the winged material before grafting/amination, and suggest, by visual examination, that the surface modification with HEMA-co-GMA followed by aminatlon with diethyl amine did not change significantly the integrity of the PBT winged fiber nonwoven material.
  • Example 6 Polyethyleneterephthalate (PET) winged fibers grafted with a combination of HEMA and GMA monomers and aminated with DEA
  • Figure 9 shows the IR spectra of the PET winged fiber nonwoven material before and after grafting/modification process as described in Example 4. Similar to what was observed for the PBT sample, the new peak and shoulder, respectively at 3500 and 1 150 cm ' 1 represent the results of poly(HEMA-co-GMA) graft.
  • Example 7 Polypropylene (PP) winged fibers grafted with a combination of
  • FIG. 1 1 the IR spectra of the PP winged fiber nonwoven before and after modification are shown. New peaks at 3500 cm “1 , 1720 cm “1 and 1 150 cm “1 clearly indicate the grafting of poly(HEMA-co-GMA).
  • the PP sample shows a much larger hydroxyl peak than PET and PBT. This indicates more HEMA was grafted to the PP surface than the PET and PBT surface, even under the same grafting conditions. This implies that the chemical property of the surface may play a role in determining the composition of the copolymer graft.
  • Example 8 Polypropylene (PP) standard fibers grafted with a combination of
  • Figure 13 shows the IR spectra of the PP standard fiber nonwoven material before and after grafting/modification as described in Example 4. New peaks at 3500 cm “1 , 1720 cm “1 and 1150 cm 1 clearly indicate the grafting of poly(HEMA-co-PGMA). A high level of grafting of HE A was obtained when using the PP standard fiber nonwoven material.
  • Example 9 Modification of G A grafted PBT membranes by attachment of a ligand
  • GMA grafted PBT membranes made of standard fibers and high- surface area fibers
  • These GMA grafted PBT membranes were then modified by attachment of Mimetic Blue SA ligand using the available epoxy groups.
  • This ligand has affinity to albumin, a blood protein.
  • the membranes were put in contact with the Blue SA ligand dissolved in water, and the pH was adjusted to 12 using 10 M NaOH. The membranes were incubated with the solution overnight at 50°C. After treatment the membranes were removed from the ligand solution and washed with pure water. The presence of blue color in the membrane was an indication of the successful attachment of the ligand.
  • Example 10 Use of Mimetic Blue SA ligand modified GMA grafted PBT membranes for separating albumin in a sample
  • the membranes from Example 9 were used for binding albumin by affinity using the Mimetic Blue SA ligand.
  • a non-modified grafted PBT membrane made of standard fibers was used as a control.
  • the membranes were cut into 12 discs (10 mm diameter, total area 9.4 cm 2 ), stacked and placed into a column, with adaptors holding the membranes in place.
  • the membranes were equilibrated using 50 mM sodium phosphate at pH 6.0, followed by loading a solution of human serum albumin at 1 .8 mg/mL at a flow rate of 1 mL/min.
  • the membranes were then washed with equilibration buffer, and the bound albumin was eluted from the membranes using a solution of 50 mM sodium phosphate and 50 mM sodium caprylate in water at pH 6.0.
  • the results obtained show that the control membrane (i.e. non-modified GMA grafted PBT membrane made of standard fibers) has captured no albumin.
  • the control membrane i.e. non-modified GMA grafted PBT membrane made of standard fibers
  • the membrane with high-surface area fibers had a capacity 4 times higher than the capacity displayed by the membrane with standard fibers after ligand-modification.
  • Nonwoven materials made of winged fibers have higher binding capacity than the standard nonwoven materials consisting only of circular cross-section fibers.
  • Copolymer can be grafted on the nonwoven membrane surface without causing significant changes to the porous structure of the membrane or to the corrugations on the surface of the fiber.
  • Nonwoven materials formed from grafted high-surface area fibers offer exceptional properties for the capture and purification of biological compounds.

Abstract

La présente invention concerne l'utilisation d'un matériau non tissé contenant des fibres de grande aire de surface pour la bioséparation de composés biologiques, thérapeutiques ou toxiques d'échantillons biologiques afin de séparer les composés des échantillons, de purifier un composé ou de détoxiquer un échantillon biologique. La présente invention concerne également un dispositif de bioséparation qui présente une capacité de liaison accrue et qui est plus économique à fabriquer.
PCT/US2011/061353 2010-11-19 2011-11-18 Fibre de grande aire de surface et membranes non tissées destinées à être utilisées dans des bioséparations WO2012068442A1 (fr)

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US9029517B2 (en) 2010-07-30 2015-05-12 Emd Millipore Corporation Chromatography media and method
WO2018015871A1 (fr) * 2016-07-18 2018-01-25 North Carolina State University Greffage de non-tissés induit par la chaleur pour une séparation d'échange d'ions de grande capacité
CN108025266A (zh) * 2015-07-30 2018-05-11 北卡罗莱纳州立大学 用于高容量离子交换生物分离的接枝海岛型非织造物
US10449517B2 (en) 2014-09-02 2019-10-22 Emd Millipore Corporation High surface area fiber media with nano-fibrillated surface features
US11236125B2 (en) 2014-12-08 2022-02-01 Emd Millipore Corporation Mixed bed ion exchange adsorber

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US20040241436A1 (en) * 2002-11-12 2004-12-02 The Regents Of The University Of California Nano-porous fibers and protein membranes
US20060234210A1 (en) * 2004-04-14 2006-10-19 Affinergy, Inc. Filtration device and method for removing selected materials from biological fluids

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US20040241436A1 (en) * 2002-11-12 2004-12-02 The Regents Of The University Of California Nano-porous fibers and protein membranes
US20060234210A1 (en) * 2004-04-14 2006-10-19 Affinergy, Inc. Filtration device and method for removing selected materials from biological fluids

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9029517B2 (en) 2010-07-30 2015-05-12 Emd Millipore Corporation Chromatography media and method
US9815050B2 (en) 2010-07-30 2017-11-14 Emd Millipore Corporation Chromatography media and method
US11305271B2 (en) 2010-07-30 2022-04-19 Emd Millipore Corporation Chromatography media and method
US10449517B2 (en) 2014-09-02 2019-10-22 Emd Millipore Corporation High surface area fiber media with nano-fibrillated surface features
US11236125B2 (en) 2014-12-08 2022-02-01 Emd Millipore Corporation Mixed bed ion exchange adsorber
EP3328527A4 (fr) * 2015-07-30 2019-04-10 North Carolina State University Non-tissés à îlots dans la mer greffés pour la bioséparation d'échange d'ions à haute capacité
US20190001281A1 (en) * 2015-07-30 2019-01-03 North Carolina State University Grafted Islands-In-The-Sea Nonwoven For High Capacity Ion Exchange Bioseparation
US11027243B2 (en) 2015-07-30 2021-06-08 North Carolina State University Grafted islands-in-the-sea nonwoven for high capacity ion exchange bioseparation
CN108025266A (zh) * 2015-07-30 2018-05-11 北卡罗莱纳州立大学 用于高容量离子交换生物分离的接枝海岛型非织造物
CN109476861A (zh) * 2016-07-18 2019-03-15 北卡罗莱纳州立大学 用于高容量离子交换分离的非织造物的热诱导接枝
EP3484947A4 (fr) * 2016-07-18 2020-02-12 North Carolina State University Greffage de non-tissés induit par la chaleur pour une séparation d'échange d'ions de grande capacité
RU2715660C1 (ru) * 2016-07-18 2020-03-02 Норт Каролина Стейт Юниверсити Термически индуцированная прививка нетканых материалов для высокоэффективного ионообменного разделения
WO2018015871A1 (fr) * 2016-07-18 2018-01-25 North Carolina State University Greffage de non-tissés induit par la chaleur pour une séparation d'échange d'ions de grande capacité

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