WO1997015661A1 - Composition microporeuse tuant les pathogenes - Google Patents

Composition microporeuse tuant les pathogenes Download PDF

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Publication number
WO1997015661A1
WO1997015661A1 PCT/US1996/017136 US9617136W WO9715661A1 WO 1997015661 A1 WO1997015661 A1 WO 1997015661A1 US 9617136 W US9617136 W US 9617136W WO 9715661 A1 WO9715661 A1 WO 9715661A1
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composition
bound
oxidase
microporous
blood
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PCT/US1996/017136
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English (en)
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Robert C. Allen
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Eoe, Inc.
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Priority to AU75217/96A priority Critical patent/AU703034B2/en
Priority to KR1019980703010A priority patent/KR19990067070A/ko
Priority to JP9516810A priority patent/JP2000500741A/ja
Priority to EP96937748A priority patent/EP0857207A4/fr
Priority to CA 2235832 priority patent/CA2235832A1/fr
Publication of WO1997015661A1 publication Critical patent/WO1997015661A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N61/00—Biocides, pest repellants or attractants, or plant growth regulators containing substances of unknown or undetermined composition, e.g. substances characterised only by the mode of action
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
    • A61L2/08—Radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds

Definitions

  • the present invention provides a microporous substance for sequestering viruses and other pathogenic organisms in lethal proximity to a singlet oxygen generating system bound to the microporous substance.
  • Photodynamic Oxygenating Activity The microbicidal effects of photodynamic action have been known for a century (Raab, 1900). Recently, photochemical-based methodologies have been developed to inactivate viruses in blood (Matthews et al., 1988; Sieber et al., 1989; Neyndorffet al., 1990; O'Brien et al., 1990; Horowitz et al., 1991) and blood products (Lin et al., 1989; Prodouz et al., 1991; Dodd et al., 1991: Margolis-Nunno et al., 1992).
  • a photosensitizing molecule and light (hv) at an appropriate wavelength (color) can generate molecular oxygen (O 2 ) that has potent oxygenating activity capable of destroying pathogenic microbes (see Mohr et al., 1995, for general review), although few have succeeded in devising practical applications for this killing method.
  • O 2 molecular oxygen
  • psoralen activated by ultraviolet A hght is reported to inactivate vesicular stomatitis virus (Margolis-Nunno et al., 1992), and hypericin activated by fluorescent light is reported to inactivate HIV-l (Lavie et al., 1995).
  • Photosensitizers can use either a type I or a type II mechanism (Schenck and Koch, 1960). The equations below explain the two types of mechanisms, and use the following terminology.
  • the "spin multiplicity" of an atom or molecule is the spectroscopic expression of its total spin quantum number (_S) as defined by the expression 12S
  • the spin multiplicities ofthe reactants, i.e., photosensitizer and O 2 are described by an antecedent superscript; and an asterisk is used to indicate electronically excited molecules.
  • a photosensitizer in the S 0 state, has a singlet multiplicity; 12(0)
  • + 1 1
  • the initial event is the same.
  • the ground state singlet multiplicity dye ⁇ Photosensitizer absorbs a photon of light and is transformed to its electronically excited singlet state ⁇ Photosensitizer*):
  • the radicals generated can react with other radicals in annihilation reactions or can react with higher multiplicity molecules in radical propagation reactions.
  • the photosensitizer radical can be harnessed via a type II reaction to the production of oxygen radicals that are capable of destroying pathogenic organisms.
  • the reaction of oxygen with biomolecules is highly exergonic, but such reactions do not occur spontaneously.
  • Biomolecules are typically of singlet multiplicity, but the oxygen we breathe is a paramagnetic molecule of triplet multiplicity. In accordance with the Wigner spin conversation rules, reactions of this form of oxygen with biomolecules are of low probability (Alien, 1994).
  • ground state triplet oxygen ( 3 O 2 ) can be converted to singlet oxygen in a type ⁇ reaction with the triplet electronically excited photosensitizer molecule:
  • This triplet-triplet annihilation proceeds through a singlet reaction surface, and yields the ground state singlet photosensitizer molecule and singlet molecular oxygen
  • Photosensitizer-mediated inactivation of viruses in blood occurs principally through such a type ⁇ singlet oxygen mechanism, whereas both type I and II mechanisms contribute to erythrocyte cytotoxicity (Rywkin et al., 1992).
  • Singlet electronically excited molecules have relatively short lifetimes, e.g., IO" 8 second. Their ephemeral nature limits their participation in chemical reactions. Their fate is to return to ground state by photon emission. Fluorescence is the energy product of singlet excited molecules relaxing to their singlet ground state. Triplet electronically excited molecules have relatively long lifetimes, e.g., IO' 2 to IO 2 seconds. Phosphorescence is the energy product of triplet excited molecules relaxing to their singlet ground state. The Equation 2 transition of an excited singlet state to an excited triplet state by intersystem crossing is a low probability event. Therefore, the excited triplet is in a meta-stable state and can participate in chemical reactions.
  • the phosphorescence of triplet* ->singlet transitions is highly susceptible to chemical quenching, especially to O 2 quenching.
  • the relatively longer lifetime of the triplet excited state is a consequence ofthe low probability ofthe spin transition.
  • Oxygen is unusual in that its ground state is triplet. Relaxation from its singlet excited state to ground state triplet also requires intersystem crossing but in the opposite direction. The low probability of this transition is responsible for the relative meta-stability of 1 O 2 *.
  • the lifetime of *O * in aqueous solution is reported to be in the microsecond range (Merkel and Kearns, 1972). As such, O 2 * can serve as a chemical reactant, but because of its brief lifespan, its radius of reactivity is confined to within 0.1 to 0.2 micrometer of its point of generation (Lindig and Rodgers, 1981).
  • Haloperoxidases such as myeloperoxidase (MPO) and eosinophil peroxidase (EPO), can catalyze the hydrogen peroxide oxidization of chloride or bromide to hypochlorous or hypobromous acid (Allen, 1975a,b):
  • haloperoxidases provide an enzymatic system for generating ⁇ 2 * , the same reactive product that can be generated by photosensitizers.
  • Carbohydrate-Lectin Mechanisms in Microbial Infection and Defense The mechanisms by which viruses infect cells generally employ lectins, which are a family of proteins that bind tightly to specific sugars on glycoproteins or glycolipids.
  • lectins which are a family of proteins that bind tightly to specific sugars on glycoproteins or glycolipids.
  • the example of influenza virus illustrates how a viral lectin participates in the infection process.
  • the hemagglutinin protein on the surface of an influenza virus particle will first attach to the cell by binding to sialic acid on the cell's membrane (Weis et al., 1988).
  • influenza hemagglutinin is a lectin that binds specifically to sialic acid.
  • HTV-1 and HTV-2 also possess lectin-like activity that may be required for cell binding and infection.
  • Two envelope glycoproteins of the human immunodeficiency virus (HIV-l) appear to have lectin-like activity.
  • the HIV-l outer -membrane protein, gpl20, and the transmembrane protein, gp41 are both derived from cleavage of a gpl60 precursor, and both behave as mannosyl and N-acetylglucosaminyl (GlcNAc) binding lectins (Haihar et al., 1992a and b).
  • the ⁇ -D-mannosyl and ⁇ -D-glucosyl residues of glycoproteins on host cell membranes may serve as anchoring sites for these HTV-1 proteins, thus facilitating viral entry into the cell.
  • This mechanism may account for the infection of non-CD4 cells and/or may assist in the CD4-dependent infectious mechanism.
  • MBP mannosyl- specific serum lectin, mannan-binding protein
  • Concanavalin A (ConA), a plant lectin with affinity for ⁇ -D-mannosyl and ⁇ -D-glucosyl residues, also attaches to HTV-1, although it does not interfere with viral gpl20 envelope glycoprotein binding to CD4 (Gattegno et al., 1992).
  • Con-A bound HTV for lymphoid cells is reduced in a carbohydrate specific, dose-dependent manner (Gattegno et al., 1992).
  • Many bacteria are known to bind and agglutinate erythrocytes and this agglutination is inhibited in some cases by specific simple sugars, indicating that bacterial lectins mediate this binding.
  • Mannose-specific surface lectins are present on Escherichia coli, Klebsiella pneumonia, Salmonella spp., Serratia marcescens, Shigella spp., Enterobacter spp., and Erwinia spp. (Duguid and Old, 1980; Speert et al., 1984).
  • An E coli lectin has also been reported with binding specificity for N-acetylglucosamine (Vaisanen-Rhen et al., 1983).
  • fimbriae or pili filamentous bacterial appendages
  • the relative specificity of E. coli Type 1 fimbriae lectin binding to various mannose-derivatives and complex mannans has been reported (Firon et al., 1983). Lectin binding was directed to short oligomannose chains of N-linked glycoproteins. Such structures are common to mucosal cell surfaces (Sharon and Lis, 1982). The same pattern of lectin specificity was observed from several members of the same bacterial genus (Firon et al., 1984), suggesting that mannose may play a major role in infection by a wide variety of bacteria.
  • Haloperoxidases The leukocyte haloperoxidases myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are cationic glycoproteins that selectively bind to and kill bacteria (Allen, 1992, U.S. patent application Serial No. 07/660,994; Allen, 1995, U.S. No. 5,389,369). MPO and EPO binding and killing specificity for certain gram negative microbes involves a lectin-carbohydrate binding mechanism.
  • MPO myeloperoxidase
  • EPO eosinophil peroxidase
  • MPO and EPO are rich in mannose and N-acetylglocosamine (Yamada et al., 1981; Miyasaki et al., 1986; Olsen et al., 1985; Allen, unpublished observations of lectin binding specificities.
  • MPO and EPO can be bound by the mannose-specific pili lectins of many gram-negative microbes including Escherichia coli, Kelbsiella pneumonia, Salmonella spp., Serratia marcescens, Shigella spp., Enterobacter spp., and Erwinia spp. (Duguid and Old, 1980; Speert et al., 1984).
  • MPO and EPO are known to inactivate viruses, including HTV-1 (Klebanoff and Belding, 1974; Klebanoff and Coombs, 1991). Furthermore, NLH-sponsored contract studies using ExOxEmis prepared MPO and EPO demonstrate high leval inactivation of AZT-resistant HIV-l.
  • Microporous substances exist that are interlaced with regularly spaced channels of subcellular dimensions.
  • the network of pore channels can comprise a substantial proportion ofthe total volume of the microporous substance.
  • Porous substances include crystalline aluminosilicates or aluminophosphates, and gels composed of cross-linked dextran.
  • Porath and Flodin (1959) introduced the concept of using cross-linked dextran beads, i.e., Sephadex (Pharmacia), as a chromatography method for rapid separation of macromolecules based on size.
  • Sephadex Pharmacia
  • gel filtration the procedure does not require the use of gel and is not a true filtration.
  • the three ⁇ dimensional molecular network of the bead consists of many open pores of a certain size range. If the pore size is too small to allow entry of a large molecule, the molecule will be excluded, whereas molecules smaller than the pore size can enter the pore matrix.
  • Molecules too large to enter the pore matrix are thus excluded from this compartment of the gel, and will travel flow through the gel at the same speed as an aqueous solution. Much larger amounts of solution are required to elute the smaller molecules that have accessed the labyrinth within the pores.
  • the smaller pore- penetrating molecules will be retained by an equilibrium of molecules entering and leaving the pores.
  • the size and shape ofthe small molecules and the size and shape of the pores will determine the equilibrium condition, and as such, the degree of physical association determines the delay in elution time.
  • the gel filtration technique has been used to isolate and purify viruses.
  • bovine papilloma virus has been purified from crude extracts of bovine warts by gel filtration using Sephacryl® S-l 000 Superfine (Pharmacia Fine Chemicals, Uppsala, Sweden). Hjorth and Moreno-Lopez, J. Virol. Methods, 5:151-158 (1982).
  • barley yellow dwarf virus has been purified from infected oats using gel filtration on Sephacryl® S-l 000 Superfine. Hewish and Shukula, J. Virol. Methods, 7:223-228 (1983).
  • a variety of molecules can be covalently bound to sieving materials that have been chemically activated.
  • a number of well-known techniques are available for these purposes, e.g., as provided in Affinity Chromatography (Pharmacia Fine Chemicals), which is hereby inco ⁇ orated by reference.
  • the invention provides a microporous substance having pores sized to permit entry of pathogenic particles but exclude blood cells, wherein a singlet oxygen generating system is bound to the microporous substance.
  • Representative microporous substances for this purpose include controlled pore glass and carbohydrate polymers.
  • the pores are sized in the range of from about 0.1 ⁇ m to about 1.0 ⁇ m to permit entry of virus particles.
  • the pores may be sized up to about 3 ⁇ m to permit entry of bacteria.
  • the singlet oxygen generating system may be a light-activated photosensitizer or an enzymatic system.
  • Suitable photosensitizers include phtholocyanines, porphyrins, methylene blue, hypericin, fluorescein derivatives, and psoralen.
  • a preferred enzymatic system for generating singlet oxygen includes an oxidase and a haloperoxidase.
  • the oxidase is preferably capable of oxidizing a substrate present in blood.
  • Suitable oxidases for this purpose include lactate oxidase, oxalate oxidase, glucose oxidase, and cholesterol oxidase.
  • the haloperoxidase may be selected from among myeloperoxidase, eosinophil peroxidase, and fungal chloroperoxidase.
  • the singlet oxygen generating system may be bound to the microporous substance by covalent bonding or non-covalent binding.
  • the microporous substance may constitute or be configured into a device such as a bead, wafer, gel filtration matrix, filter, bag, or tubing.
  • a ligand that binds to a pathogenic particle is also bound to the microporous substance.
  • the ligand may be selected from among high mannose glycans, N-glucosamine, the N-acetylglucosaminyl core of oligosaccharides, the monosyl core of complex-type N-linked glycans, mannan, ⁇ - methylmannoside, haloperoxidases, sulfated polysaccharides, low molecular weight dextran sulfate, and lectins.
  • Preferred ligands for this purpose include the haloperoxidase myeloperoxidase and the lectin conconavalin A.
  • the microporous substance takes the form of a carbohydrate polymer bearing conconavalin A to which lactate oxidase and myeloperoxidase are respectively bound.
  • FIGURE 1 depicts a microporous substance in contact with blood, and illustrates how a pore diameter of 1 ⁇ m effectively excludes red blood cells (RBC) but permits viral particles to enter the pores.
  • FIGURE 2 illustrates a pore channel (lumen) of a microporous substance
  • FIGURE 3 illustrates the effective radius (overlapping red circles) within which the virus-killing activity of singlet oxygen is confined due to its brief lifetime.
  • the lifetime of singlet oxygen in aqueous solution is reportedly in the microsecond range, hence its radius of reactivity is limited to within 0.1 to 0.2 ⁇ m of its generation.
  • FIGURE 4 illustrates sieve material bearing conconavalin A to which MPO is non-covalently bound.
  • An oxidase is bound to the sieve material.
  • Virus are selectively retained within the pores by ligand-receptor interaction with the conconavalin A, the MPO, or both. Singlet oxygen generated by the immobilized enzymes kill or inactivate the virus.
  • This invention provides a microporous substance for purifying a biological fluid such as blood of pathogenic particles like virus and bacteria.
  • the microporous substance has pores that are sized to permit the entry of pathogenic particles but exclude desirable cells in the biological fluid, such as the cellular elements of blood.
  • a singlet oxygen generating system is bound to the microporous substance.
  • the microporous substance forms a sieving matrix in which pathogens (e.g., viruses, bacteria, prions, or other similarly-sized pathogenic particles) are removed and inactivated from blood and other biological fluids.
  • the enzyme components of a singlet oxygen generative system are bound to the channels of a sieving gel.
  • Pathogen-binding ligand molecules may also be bound to the matrix channels.
  • gel particles are polymeric materials including natural and synthetic polymers.
  • a polymer is a high molecular weight compound consisting of smaller subunits that are covalently linked together to form long chains that are straight or branched and that also often have interchain linkages among the subunits.
  • Polymeric materials used in gel filtration include natural polysaccharides such as agarose, dextran, and cellulose, and synthetic polymers such as polyacrylamide, polytrisacrylamide, and hydroxylated vinyl polymers. These polymers may be formed into particles (or beads) that have varying pore size.
  • the pore size ofthe matrix may be controlled by the extent of cross-linking between the polymer chains of the gel particle.
  • highly cross-linked polymers have smaller pore sizes than those polymers that are lightly cross-linked.
  • the porous sieving matrix can consist of cross-linked carbohydrate, e.g., polydextran, controlled pore glass, or other appropriate material.
  • cross-linked carbohydrate e.g., polydextran, controlled pore glass, or other appropriate material.
  • these materials are commercially available, including cross-linked agarose and bisacrylamide cross-linked allyl dextran (Sepharose® and Sephacryl® gels, respectively; Pharmacia Fine Chemicals, Uppsala, Sweden).
  • the polymeric material is a polysaccharide cross-linked to varying degrees with bisacrylamide.
  • a variety of devices can be readily constructed from the sieving material, for example, beads, tubes, flat porous surfaces, lining plastic blood bags, filters, hollow cellulose tube filters, and gel filtration matrices.
  • the sieving material will contain pores, a pore being a channel or void or intersticial space within a solid material that communicates with the outside liquid and permits the passage of liquids or gases through the material.
  • the diameter ofthe pore channel will be large enough to allow the entry of pathogenic viruses, bacteria, or other similarly-sized pathogens. As illustrated in FIGURE 1, the pore diameter will be small enough to exclude desirable cells, particularly the cellular components of blood.
  • virus particles that contaminate blood range in diameter from 20 (parvoviruses) to 150 nm (0.02 to 0.15 ⁇ m). Platelets, the smallest cellular elements of blood have a free-floating diameter of approximately 3,000 nm (3 ⁇ m). Erythrocytes are next in size with a diameter of 7.2 to 7.9 ⁇ m. As such, any material with pore sizes in the range of 155 nm (about 0.15 ⁇ m) to about 2,500 nm (or less than 3 ⁇ m) will physically accommodate virus particles while excluding platelets and other cellular elements of blood.
  • Pore sizes of about 2.5 ⁇ m are particularly useful for sequestering non-viral pathogens having diameters less than the diameter of platelets.
  • Bacteria are typically less than 2 ⁇ m in diameter, and most or all bacteria will enter pore channels having diameters of about 2-3 ⁇ m. Thus, pore sizes just slightly smaller than the 3 ⁇ m diameter of blood platelets will accommodate most pathogens that are expected to contaminate blood supplies.
  • a photosensitizer such as a fluorescein derivative or a phthalocyanine derivative or other appropriate molecule, is bound covalently or non-covalently to the surfaces of the channels present in the pores of the sieving material.
  • a “photosensitizer,” sometimes known also as a “photodynamic dye,” is a compound or a molecule that is capable of transferring its excited state energy to another compound or molecule.
  • a photosensitizer becomes electronically excited after absorbing a photon of light, and subsequently transfers its excited state energy to another compound or molecule.
  • a photosensitizer that transfers its excited energy to an oxygen molecule (i.e., ground state oxygen, ⁇ 02) thereby generating an excited oxygen molecule (i.e., excited state oxygen, IO2) a is a singlet oxygen photosensitizer.
  • Photosensitizers are characterized by the property of being converted to the triplet excited state when illuminated with light of an appropriate wavelength.
  • the excited triplet photosensitizer molecule then participates in a type II reaction with ground state triplet oxygen present in the blood to generate highly reactive singlet oxygen, 1 O 2 * , which is capable of killing pathogenic organisms.
  • Photosensitizers contemplated by the subject invention are those that absorb visible light (from about 350 nm to about 700 nm) and near-infrared light (from about 700 nm to about 1000 nm).
  • Photosensitizers preferred for purifying blood include compounds that absorb visible light in the red region (from about 600 nm to about 700 nm) and in the near-infrared region, as the light required to activate these photosensitizers will not be absorbed by the red blood cells.
  • Exemplary families of photosensitizers suitable for the subject invention include acridines, fluoresceins, xanthines, rhodamines, porphyrins, cyanines, and phthalocyanines.
  • a virus or bacteria-binding composition is also bound covalently or non-covalently to the surface ofthe pore channels to aid in the capture and retention of pathogen particles.
  • the enhanced capture of viruses can be accomplished by linking substances that can serve as ligands, such as concanavalin A or other lectins, to the surfaces of the pore channels.
  • the capture of additional pathogens can be accomplished by coating the pore channels with substances such as sialic acid, mannose, N-acetylglucosamine, haloperoxidases such as myeloperoxidase or eosinophil peroxidase, or any other substance known to have the capacity of binding with molecules present on the surfaces of viruses and bacteria.
  • substances such as sialic acid, mannose, N-acetylglucosamine, haloperoxidases such as myeloperoxidase or eosinophil peroxidase, or any other substance known to have the capacity of binding with molecules present on the surfaces of viruses and bacteria.
  • a significant advantage of this embodiment is that the ligand-mediated binding of pathogens in and of itself partially serves the purpose of removing pathogenic particles from the blood.
  • the sieve material is bound covalently or non-covalently with an enzymatic system capable of chemically generating HOC1 and *O .
  • an enzymatic system capable of chemically generating HOC1 and *O .
  • One such system is comprised ofthe previously described haloperoxidases, which require halide and H2O2 as substrates generating singlet oxygen.
  • Haloperoxidases catalyze the hydrogen peroxidase oxidation of chloride or bromide to hypochlorous or hypobromous acid.
  • the amount of chloride normally present in blood provides a sufficient amount of halide for reactions catalyzed by myeloperoxidase (Allen, U.S. Patent No. 5,389,369).
  • hypohalous acid thus produced reacts with an additional hydrogen peroxide molecule to yield singlet molecular oxygen.
  • Virus or bacteria that are in the immediate vicinity of the reactive singlet oxygen molecules will be inactivated or killed. Blood components that are too large to enter the pores will not be significantly exposed to either H2O2 or singlet oxygen.
  • Haloperoxidases suitable for the subject invention include myeloperoxidase, eosinophil peroxidase, and fungal chloroperoxidase. Oxidases capable of acting on substrates normally present in the blood can be conveniently employed to provide the hydrogen peroxide substrate required by the haloperoxidase.
  • Such oxidases include lactate oxidase, oxalate oxidase, cholesterol oxidase, glucose oxidase, or other oxidases capable of using substrates in the blood. If insufficient amounts of, e.g., lactate or glucose are present, these substrates could be added to the blood or to the blood-collecting bags to ensure sufficient hydrogen peroxide generation by the chosen oxidase. Lactate oxidase produces H 2 O 2 in the following reaction:
  • Lactate is an excellent substrate in that it is typically present in a relatively high concentration in blood.
  • the venous blood of normal healthy adults has a lactate concentration of 1 mm (9 mg dL).
  • Lactate is the end product of glycolytic metabolism and is the major product of erythrocyte metabohsm of glucose. Every molecule of glucose metabolized yields two molecules of lactate. Lactate continues to be generated by erythrocytes during blood bank storage. The peroxide generated by lactate oxidase thus provides a substrate for the sieve-bound haloperoxidase.
  • Suitable substrates for other oxidases are also present in the blood, e.g., oxalate oxidase (oxalate:O 2 oxidoreductase, EC 1.2.3.4), cholesterol oxidase (cholesterol :O 2 oxidoreductase, EC 1.1.3.6), glucose oxidase, etc., can be employed in a similar manner.
  • oxalate oxidase oxalate:O 2 oxidoreductase, EC 1.2.3.4
  • cholesterol oxidase cholesterol oxidase
  • glucose oxidase etc.
  • These and other oxidases are well-known and several of them are described in some detail in U.S. Patent No. 5,389,369.
  • FIGURE 2 schematically depicts a section through a virus-entrapping pore with surface bound lactate oxidase and myeloperoxidase.
  • This pore is approximately 0.2 to 0.3 ⁇ m in diameter and contains two viral particles the size of HTV, i.e., approximately 0.1 ⁇ m in diameter. Larger pores can be used to entrap bacteria and even cellular pathogens. Aside from the advantage of capturing pathogen particles, the isolated milieu of the pore also serves to concentrate and link the substrates and products required for effective enzymatic interaction.
  • FIGURE 2 further illustrates how the oxidation of lactate to pyruvate drives the generation of ⁇ 2 * in close proximity to the captured virus particles.
  • FIGURE 3 depicts the range (overlapping circles) within which the activity of ⁇ 2 * is limited because of its brief lifespan.
  • conconavalin A can be used to non ⁇ covalently bind the haloperoxidase to the microporous substratum.
  • Conconavalin A is a protein lectin derived from the jack bean.
  • the conconavalin A molecule has four sites capable of binding non-covalently to carbohydrates, and thus has the capacity to agglutinate a variety of animal cells.
  • the tetravalent molecule concanavalin A can serve as a "platform" for bringing several components of the composition into proximity within the pores of the microporous matrix.
  • one of the reactive sites of the conconavalin A is first covalently bound to the microporous substrate itself, and then the lectin's reactive sites are allowed to bind with haloperoxidase and with mannosylated oxidase. Under empirically adjusted stoichiometric conditions, the lectin molecules will still have reactive sites available to bind with a receptor present on a virus or bacterium.
  • FIGURE 4 depicts covalently bound conconavalin A to which MPO is non-covalently bound.
  • the oxidase is bound directly to the sieve material, but in other applications it can be mannosylated and bound non-covalently to the conconavalin A.
  • H 2 O 2 generation to within the pore environment has multiple advantages. It maximizes contact between pathogen particles and pore-bound myeloperoxidase, limits H 2 O 2 dilution, and insures that H 2 O will not contact with erythrocytes (and thus avoids the conversion of H 2 O 2 by erythrocyte catalase). Blood cellular components are also protected by limiting the destructive effect of H 2 O 2 to the pore environment. Most importantly, this confines peroxide generation by the oxidase to the location of MPO. Confinement to the pore space also insures that HOCl, the product of H 2 O -dependent MPO-catalyzed chloride oxidation, will react with an additional H 2 O 2 to yield 2 *.
  • Enzymatic generation of ⁇ 2 * within the pore space ensures a high level of focused anti-pathogen activity without requiring an exogenous source of energy. Unlike systems reliant on photosensitizers, the enzymatic generation of *O 2 * has the advantage of not requiring an external light source. Spatial confinement optimizes the reactive potential of l 0 2 * with its limited lifetime by localizing the pathogen to within the less than 0.2 ⁇ m radius of reactivity required for maximum viricidal or bacteriocidal action.
  • the invention thereby provides compositions useful for removing pathogenic particles, particularly viruses, bacteria, and prions, from blood or other fluids.
  • the subject microporous compositions can be used to remove viruses from pharmaceutical preparations that are produced by cultured eucaryotic or procaryotic cells.
  • the compositions moreover can be applied to remove pathogens from virtually any fluid whose purity is desired.
  • the subject compositions are composed in whole or part of a microporous substance configured so that its pores open onto a surface that interfaces with the liquid to be purified.
  • Representative microporous substances include controlled pore glass and polymers such as cross-linked carbohydrates. These pores are of appropriate size to permit the entry of pathogenic particles such as viruses and bacteria. Methods for creating pores of the desired diameter are known in the art. For example, the size of pores in cross-linked polymers can be controlled by manipulating the amount of cross-linkers, while the pores in glass beads can be established by controlled etching. Particularly useful are pores ranging in size from about 0.1 ⁇ m to about 1.0 ⁇ m to permit entry of virus particles, and pores sized up to about 2.5 ⁇ m to permit entry of bacteria. Pores of less than about 2.5 ⁇ m in diameter provide the advantage of excluding cellular components ofthe fluid, e.g., blood, from the interstices ofthe porous substance.
  • compositions may also employ one or more ligand-receptor pairs.
  • a ligand is a molecule or group of molecules that binds to a receptor molecule or site on the surface of a cell or virus.
  • Such a pair consists of two molecules with a sufficient affinity for one another such that if one member ofthe pair is bound to the microporous substance and the other member is present on the surface of a microbe, i.e., a virus or bacterium, the microbe will become at least temporarily attached to the microporous substance by virtue ofthe non-covalent binding between the two members ofthe pair.
  • the member ofthe ligand- receptor pair that is bound to the microporous surface is referred to as the "ligand", and the Ugand-binding molecules on the surface of a virus or bacterium is the "receptor".
  • Conconavalin A is an example of a ligand capable of binding mannan oligosaccharide receptor(s) on viruses such as HTV-l.
  • Suitable ligands include high mannose glycans, mannosylated surfaces, the manosyl core of complex- type N-linked glycans, N-acetylglucosamine and its derivatives, mannan, sulfated polysaccharides such as heparin or low molecular weight (MW) dextran sulfate (DS), and lectins such as conconavalin A and other lectins that share conconavalin A's affinity for ⁇ -D-mannosyl, ⁇ -D-glucosyl residues, and N-acetylglucosamine.
  • Conconavalin A attaches to HTV-l (Gattegno et al., 1992), and can also bind to EPO, MPO, and mannosylated oxidase.
  • the subject compositions require that the ligands be bound to the microporous substance, especially to the pores that permeate this substance. Methods are widely available for covalently linking these types of molecules to polymeric surfaces.
  • the subject microporous substances can be configured into a variety of devices. Such devices include beads, gel filtration matrices, filters, bags, tubing, and filters composed of hollow cellulose tubules. For example, to remove microbes from blood, the blood is passed ex vivo through a filter or a column containing the microporous substance. The blood can be filtered prior to being placed inside storage bags, or filtered upon removal from the bags, or both.
  • the inner surface of bags in which blood is stored can be coated with a thin layer ofthe microporous substance, or tubing lined with the substance or beads or wafers composed ofthe substance can be simply placed inside the bag to inactivate particulate viruses and/or bacteria while the blood is stored in the bag.
  • any composition having pores sized to the required dimensions can be employed as the microporous substance, i.e., the sieve material.
  • the sieve material i.e., the sieve material.
  • Sephacryl S-l 000 (Pharmacia, Sigma S-l 000) is a cross-linked co-polymer of allyl dextran and N,N'- methylenebisacrylamide with a bead diameter of 40-105 ⁇ m and a pore-size exclusion limit of 1 x IO 9 daltons.
  • Sepharose CL 2B (Pharmacia, Sigma CL-2B-300), a cross ⁇ linked agarose with a bead diameter of 60-200 ⁇ m and a pore-size exclusion of 0.2 x IO 9 daltons, may also be used with small viruses.
  • Controlled pore glass is prepared by heating treating borosilicate glass and leaching out the boric oxide with acid.
  • Controlled pore Glyceryl Glass (Sigma GG3000-200) is available in 120-200 mesh (75-125 ⁇ m in diameter) with a pore diameter of 0.3 ⁇ m (300 nm).
  • the sieve materials were brought up to 50 ml with distilled H 2 O, and placed on a rocker for 30-60 min. Next, the material was allowed to settle, and the aqueous supernatant was decanted. The material was washed as before with a second 50 ml of distilled water. After the second water wash had been decanted, 2 M phosphate buffer (PB), pH 11.4, (536g Na 2 HPO 4 -7H 2 O/L H 2 O and adjusted with NaOH to pH 11.4) was added to a final volume of 50 ml. The solution was placed under vacuum for 1 hour to degas, then placed into an ice bath to cool. 3. Activating the Sieve Material with Cyanogen Bomide (CNBr, (Sigma C-6388V
  • MPO-Lactate Oxidase MPO-LOx
  • EPO-LOx EPO-LOx
  • concanavalin A concanavalin A
  • MPO-LOx and lactate oxidase i: Five ml of myeloperoxidase (10 mg, porcine, ExOxEmis, Lot 1899201, 70 nm total) was combined with 3.2mg lactate oxidase, Pediococcus species (3.2mg, 108 units, Sigma L-06380).
  • EPO-LOx and lactate oxidasei: Five ml of eosinophil peroxidase (10 mg, porcine, ExOxEmis, Lot 1929201, 150 nmol total) was combined with 3.2mg lactate oxidase (Pediococcus species, 3.2mg, 108 units, Sigma L-06380).
  • Con- A Ten mg of concanavalin A, Type TV Canavalin ensiformis (Sigma C-2010) was dissolved in 5ml distilled H O.
  • an oxidase capable of using substrates present in blood could be concomitantly prepared and permitted to bind to the activated beads.
  • Beads activated as described above, were resuspended in 0.25 M bicarbonate buffer (BB), pH 9.0 (21g NaHCO 3 /L H 2 O adjusted to pH with NaOH) to a final volume of 30ml.
  • BB bicarbonate buffer
  • pH 9.0 21g NaHCO 3 /L H 2 O adjusted to pH with NaOH
  • Each 30ml bead suspension was divided equally into 3 tubes, for a total of nine tubes, including three each of Sepharose 2B, Sephacryl S-l 000, and Glyceryl-Glass as described above.
  • To each tube of bead preparations was added: 1.75 ml of MPO-LOx, 1.75 ml of EPO-LOx, and 1.75 ml of Con-A.
  • the tubes were then placed on a rocker table for gentle mixing and allowed to react overnight (12 hr) at 22°C.
  • a 1 M glycine solution was prepared (75 g glycine/1) and added to each bead suspension to a final volume of 50ml.
  • the glycine is provided to react with any unreacted iminocarbonates remaining on the beads. It was apparent from the color of the derivatized beads that the haloperoxidases had successfully bound to the beads.
  • the bead preparations once they had settled, displayed the pale green color characteristic of MPO and the orange- brown color of EPO. UV- Visible spectroscopy was performed to quantify the protein remaining in the supernatant after the beads had settled.
  • the amount of EPO bound to the beads was quantified as the reduced (dithionite)-oxidized difference spectrum at 449 nm using an extinction coefficient of 125 mM ⁇ cm- 1 :
  • bacteria are susceptible to virus infection and lysis. This type of virus is called a bacteriophage or phage. Bacteriophages are comparable to animal viruses in size, and their presence can be measured by their ability to form plaques, i.e., zones of lysis or clearing, produced on a "lawn" of susceptible bacteria. The number of bacteriophage can be measured as the number of plaque forming units per volume of sample.
  • Bacteriophage are not enveloped, and present a different surface than the HTV and hepatitis viruses of interest with regard to blood infectivity. However, their size and ease of detection allow testing of the size specific inactivating quality of the subject Pore:Oxidase-Haloperoxidase preparations.
  • Bacteriophage suspensions are contacted with the MPO- and EPO-bound beads prepared above. Bacteriophage enter the pores ofthe beads and interact non ⁇ covalently with the conconavalin A and/or the EPO or MPO present in the pore channels. After the beads are permitted to settle, the supernatant is collected and the titer of bacteriophage therein is determined using standard bacteriological techniques. As controls, bacteriophage suspensions are contacted with beads that have been subjected to all of the above preparatory steps except that no MPO or EPO was added during the binding step. The effectiveness of phage killing by the MPO and EPO enzyme system bound to the beads is determined by comparing the resulting control and experimental titers. 6. A Bacterial Model of Pore:Lactate-Oxidase-Haloperoxidase Inactivation.
  • the functional capacities of untreated and MPO-LOx bound sieve material was tested using Escherichia coli, a short gram-negative bacillus with a cell width of approximately 0.5 ⁇ m.
  • the E. coli were cultured in trypticase soy broth for approximately 16 hours at 37°C.
  • the bacteria were concentrated by centrifugation, and the final concentration adjusted to IO 6 to IO 7 bacteria per milliliter by turbidimetric measurement (titration).
  • E. coli CFU The small decrease in E. coli CFU observed with untreated sieve materials is thought to reflect binding and capture of the bacteria without killing.
  • the removal and/or killing of E. coli by the different MPO-LOx bound sieve materials was proportional to the pore size.
  • Three different MPO-LOx-bound sieve materials were tested. Of these, the pore size of the glyceryl-glass was greater than that of the Sephacryl SI 000, while the smallest pores of the group were those of Sephorose CL-2B. Accordingly, MPO-LOx-bound sephorose CL-2B was the least effective of the three sieve materials tested. This latter material also bound the least amount of MPO-LOx.

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Abstract

L'invention concerne une substance microporeuse dont les pores sont dimensionnés de sorte que les particules pathogènes puissent y pénétrer et que les globules sanguins en soient exclus, un système générant de l'oxygène singulet étant lié à ladite substance microporeuse.
PCT/US1996/017136 1995-10-25 1996-10-25 Composition microporeuse tuant les pathogenes WO1997015661A1 (fr)

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AU75217/96A AU703034B2 (en) 1995-10-25 1996-10-25 Microporous pathogen-killing composition
KR1019980703010A KR19990067070A (ko) 1995-10-25 1996-10-25 미공성 병원균-사멸 조성물
JP9516810A JP2000500741A (ja) 1995-10-25 1996-10-25 微小孔性病原体殺傷組成物
EP96937748A EP0857207A4 (fr) 1995-10-25 1996-10-25 Composition microporeuse tuant les pathogenes
CA 2235832 CA2235832A1 (fr) 1995-10-25 1996-10-25 Composition microporeuse tuant les pathogenes

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NL1009472C2 (nl) * 1998-06-23 1999-12-27 Univ Leiden Werkwijze voor het inactiveren van virussen.
WO2003077873A2 (fr) * 2002-03-15 2003-09-25 Ceramoptec Industries, Inc. Photoamorceurs pour therapie photodynamique relative aux infections microbiennes
US6630128B1 (en) 1998-08-28 2003-10-07 Destiny Pharma Limited Porphyrin derivatives their use in photodynamic therapy and medical devices containing them
EP1679117A2 (fr) * 2005-01-10 2006-07-12 HaemoSys GmbH Sytème d'adsorption pour éliminer les virus et les composés viraux de fluides, en particulier du sang et du plasma sanguin
WO2008039136A1 (fr) * 2006-09-29 2008-04-03 Ge Healthcare Bio-Sciences Ab Matrice de séparation pour une purification virale
WO2012107914A1 (fr) 2011-02-11 2012-08-16 Marc Bonneau Dispositif et procédé de décontamination et de stérilisation, notamment pour des produits alimentaires ou agricoles, des fluides ou des matériels industriels
US11628381B2 (en) 2012-09-17 2023-04-18 W.R. Grace & Co. Conn. Chromatography media and devices

Families Citing this family (1)

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CN110743035A (zh) * 2019-11-06 2020-02-04 广西大学 一种智能抗菌的水凝胶的制备方法及其应用

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US4869826A (en) * 1987-09-01 1989-09-26 Process Biotechnology, Inc. Cellular adsorbents for removal of viral contaminants from blood and complex protein solutions
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL1009472C2 (nl) * 1998-06-23 1999-12-27 Univ Leiden Werkwijze voor het inactiveren van virussen.
WO1999066962A1 (fr) * 1998-06-23 1999-12-29 Boston Clinics Pdt B.V. Methode d'inactivation de virus
US6630128B1 (en) 1998-08-28 2003-10-07 Destiny Pharma Limited Porphyrin derivatives their use in photodynamic therapy and medical devices containing them
WO2003077873A2 (fr) * 2002-03-15 2003-09-25 Ceramoptec Industries, Inc. Photoamorceurs pour therapie photodynamique relative aux infections microbiennes
WO2003077873A3 (fr) * 2002-03-15 2004-07-29 Ceramoptec Ind Inc Photoamorceurs pour therapie photodynamique relative aux infections microbiennes
EP1679117A2 (fr) * 2005-01-10 2006-07-12 HaemoSys GmbH Sytème d'adsorption pour éliminer les virus et les composés viraux de fluides, en particulier du sang et du plasma sanguin
EP1679117A3 (fr) * 2005-01-10 2007-12-26 HaemoSys GmbH Sytème d'adsorption pour éliminer les virus et les composés viraux de fluides, en particulier du sang et du plasma sanguin
WO2008039136A1 (fr) * 2006-09-29 2008-04-03 Ge Healthcare Bio-Sciences Ab Matrice de séparation pour une purification virale
US8481298B2 (en) 2006-09-29 2013-07-09 Ge Healthcare Bio-Sciences Ab Separation matrix for viral purification
WO2012107914A1 (fr) 2011-02-11 2012-08-16 Marc Bonneau Dispositif et procédé de décontamination et de stérilisation, notamment pour des produits alimentaires ou agricoles, des fluides ou des matériels industriels
US11628381B2 (en) 2012-09-17 2023-04-18 W.R. Grace & Co. Conn. Chromatography media and devices

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JP2000500741A (ja) 2000-01-25
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AU703034B2 (en) 1999-03-11
EP0857207A4 (fr) 2002-10-02
KR19990067070A (ko) 1999-08-16

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