CN1284897A - Batch devices for reduction of compounds from biological compositions containing cells and methods of use - Google Patents

Batch devices for reduction of compounds from biological compositions containing cells and methods of use Download PDF

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Publication number
CN1284897A
CN1284897A CN98813840A CN98813840A CN1284897A CN 1284897 A CN1284897 A CN 1284897A CN 98813840 A CN98813840 A CN 98813840A CN 98813840 A CN98813840 A CN 98813840A CN 1284897 A CN1284897 A CN 1284897A
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Prior art keywords
platelet
adsorbent
equipment
resin
amino
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CN98813840A
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Chinese (zh)
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D·J·赫
T·A·番
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Cerus Corp
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Cerus Corp
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Priority claimed from PCT/US1998/000531 external-priority patent/WO1998030327A1/en
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Publication of CN1284897A publication Critical patent/CN1284897A/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28026—Particles within, immobilised, dispersed, entrapped in or on a matrix, e.g. a resin
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0278—Physical preservation processes
    • 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
    • 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
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/02—Blood transfusion apparatus
    • A61M1/0209—Multiple bag systems for separating or storing blood components
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/02—Blood transfusion apparatus
    • A61M1/0209—Multiple bag systems for separating or storing blood components
    • A61M1/0218—Multiple bag systems for separating or storing blood components with filters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
    • B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28004—Sorbent size or size distribution, e.g. particle size
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B01J20/28019—Spherical, ellipsoidal or cylindrical
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28028—Particles immobilised within fibres or filaments
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28057—Surface area, e.g. B.E.T specific surface area
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their surface properties or porosity
    • B01J20/28078—Pore diameter
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
    • C07D219/08—Nitrogen atoms
    • C07D219/10—Nitrogen atoms attached in position 9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/64—Acridine or hydrogenated acridine ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption

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Abstract

Methods and devices are provided for reducing the concentration of low molecular weight compounds in a biological composition containing cells while substantially maintaining a desired biological activity of the biological composition. The device comprises highly porous adsorbent particles, and the adsorbent particles are immobilized by an inert matrix.

Description

Reduce the batch device and the using method of chemical compound in the celliferous biological composition
Related application
The application is that the serial number of on January 6th, 1998 application is the part continuity of 09/003113 common pending application, and its full content is incorporated herein by reference.
Technical field
The application relates to the method and apparatus that reduces chemical compound in the biological composition.The molecular weight of these chemical compounds is the about 30000g/mol of about 100g/mol-.
Background technology
Big quantity research all concentrates on removes material from blood products.Great majority all cause leucocytes reduction in this research.Referring to, people's such as people's such as M.N.Boomgaard people's such as Transfusion34:311 (1994), F.Bertolini Vox Sang 62:82 (1992) and A.M.Joustra-Dijkhuis Vox Sang67:22 (1994) for example.Filter platelet and be and be used for reducing the leukocytic common methods of platelet concentrate.Referring to, people's such as M.Bock Transfusion 31:333 (1991) (Sepacell PL-5A for example, Asahi, Tokyo, Japan), people's such as J.D.Sweeney Transfusion 35:131 (1995) (Leukotrap PL, Miles Inc_Covina, CA) and people's such as M.van Marwijk Transfusion30:34 (1990) (Cellselect, NPBI, Emmer-Compascuum, TheNetherlands; Immugard Ig-500, Terumo, Tokyo, Japan).Yet these filter machinery and can not remove the relatively low chemical compound of molecular weight at present, other chemical compound commonly used when comprising for example psoralen, psoralen photoproducts and handling biofluid.
Adsorption method has been used for separation selectivity blood constitutent on the phospholipid polyalcohol.For example, several copolymers with different electric charges and the interaction of blood constitutent are estimated, comprised thrombocyte adhesiveness and protein adsorption.People's such as K.Ishihara J.Biomed.Mat.Res.28:1347 (1994).But these polymer design are not used to adsorb low molecular weight compound.
Various dialysis apparatuss can be removed low molecular weight compound from blood plasma and whole blood.For example, low-molecular-weight toxin and medical compounds can be successfully removed in dialysis.Therefore, dialysis can be used for, for example from blood products, remove psoralen and psoralen photoproducts.Unfortunately, dialysis procedure needs very complicated and expensive equipment at present.Therefore, it is just unrealistic dialysis machinery to be used for depolluting of a large amount of blood products.
Use polystyrene divinylbenzene, silica dioxide gel and acryloyl ester polymer absorption methylene blue had been described in the past.For example, PCT publication number WO91/03933 described free absorbent resin batch research (for example, Amberlite (Rohm and Haas (and Frankfurt, Germany) and Bio Beads (Bio-Rad Laboratories (Munich, Germany)).But, in being exposed to blood products, very carefully these absorbent resins are removed afterwards, so these methods have caused transferring the danger of resin particle.
In addition, equipment and the method for removing leucocyte-removing and virus inactivating agent (for example, psoralen, hypericin and for example dyestuff of methylene blue, toluidine blue and crystal violet) also disclosed.Specifically, PCT publication number WO95/18665 has described the filter of the fabric web that comprises laying, and this fabric web contains the polymeric matrices of mechanically stable.This net itself contains interlock fabric fiber that forms the substrate with gap and the fibril grain that distributes in this gap.But this equipment reduces the factor XI, plasma thromboplastin antecedent activity greatly, and this may make the product of handling its predetermined purposes that can not suit.
Therefore need simpler, safer and more economical device,, kept the biological activity of the biological composition of celliferous processing simultaneously basically to reduce the concentration of low molecular weight compound in the celliferous biological composition.
Invention is described
The invention provides the equipment that reduces compound concentration in the celliferous biological composition.This equipment comprises and containing by the fixed particulate adsorbing medium of inert base, and is the batch structure.Typically, in biological composition, use the molecular weight of the chemical compound that this equipment reduced to be the about 30000g/mol of about 100g/mol-.The contained cell of this biological composition comprises the cell that for example is suspended in Biomedia such as blood plasma or the tissue culture medium (TCM).This biological composition with kept its biological activity basically after these equipment contact.
The illustration chemical compound comprises pathogen inactivated chemical compound, dyestuff, mercaptan, plasticizer and complement activation.The equipment that is provided comprises by the fixed three-dimensional network absorbent particles of inert base.This immobilization has reduced the bleed danger of blood products of loose absorbent particles.And, simplified the operation that reduces the problem that accompanies with the loose absorbent particles of processing by inert base fixed absorbent granule.The fixed absorbent granule also can improve the ability that absorbent particles adsorbs chemical compound in the celliferous biological composition, and pair cell does not have mechanical damage.
The invention provides the concentration method that reduces biological respinse modifier in the celliferous biological composition, wherein this method has kept the required biological activity of biological composition basically.This method relates to this biological composition of a kind of device processes.
In one embodiment, this equipment comprises the inert base that contains a large amount of absorbent particles, and wherein the diameter range of absorbent particles is at the about 1500 μ m of about 100 μ m-, and wherein this equipment is used for batch machining.
In another embodiment, this biological respinse modifier is a complement activation.
In another embodiment, the absorbent particles of this equipment is for having good wettable polyaromatic absorbent particles.
In another embodiment, the absorbent particles of this equipment is the activated carbon granule from synthetic source.
In another embodiment, the inert base of this equipment is a synthetic polymeric fibers, and this synthetic polymeric fibers comprises the polymer core with high melting temperature of the sheath encirclement with low fusion temperature.
In another embodiment, the inert base of this equipment is a particle network, and this particle network comprises polyethylene particle.
In another embodiment, this biological composition comprises platelet.
In another embodiment, this biological composition comprises erythrocyte.
In another embodiment, this method also reduces the concentration of psoralen derivant in the biological composition or acridine derivatives.
In another embodiment, this method also reduces the concentration of dyestuff in the biological composition or quencher.
The accompanying drawing summary
Fig. 1 illustrates the perspective view of an embodiment of fiber, has shown its internal core and outer sheath, and they have formed the network of fibers of fixed absorbent medium.
Fig. 2 illustrates the part of an embodiment of fixed absorbent medium of the present invention.
Fig. 3 illustrates the cross-sectional view that the adsorbent pearl is fixed on an embodiment of the fixed absorbent medium that constitutes Fibrized resin in the fiber.
Fig. 4 illustrates in the fiber that the adsorbent pearl is fixed on the fixed absorbent medium and the cross-sectional view of an embodiment of the fixed absorbent medium of Fibrized resin sample is surrounded in heat-sealing.
Fig. 5 is for Dowex_XUS-43493 and the loose adsorbent pearl of Amberlite_XAD-16 HP and the fixed absorbent medium that the contains Amberlite_XAD-16 sketch map except that the comparison of the adsorption dynamics adsorption kinetics of the psoralen that deaminizes from platelet.
Fig. 6 is the sketch map of the comparison of the adsorption dynamics adsorption kinetics of removing the psoralen that deaminizes with the fixed absorbent medium that contains Amberlite_XAD-16 and the fixed absorbent medium of active carbon with two kinds of different loads from platelet.Fibrosis XAD-16 data are represented by circle, solid line; Fibrosis AQF-500-B data are represented by square, dash line; Fibrosis AQF-375-B is represented by triangle, dash line.
Fig. 7 is for p (HEMA)-coating and Dowex_XUS-43493 pearl uncoated the sketch map except that the comparison of the adsorption dynamics adsorption kinetics of the psoralen that deaminizes from platelet.
Fig. 8 is with Amberlite_XAD-16 and the sketch map of Dowex_XUS-43493 pretreatment to the comparison of the influence of the relative absorbability of amino psoralen with glycerinated solution.
Fig. 9 be Wetting Solution to Amberlite_XAD-16 in 100% blood plasma (below) and Dowex_XUS-43493 (above) dry adsorbent 4 '-(4-amino-2-oxa-) butyl-4,5 ', the comparison sketch map of the influence of 8-trimethylpsoralen absorbability, the sample of moistening is not designated as " No Tx " in alcoholic solution.Absorbability is reported as the percentage rate with respect to the optimal wet adsorbent power.
Figure 10 for the Amberlite_XAD-16 that uses moistening in several different solutions from blood plasma through adsorbing the comparison sketch map of amino psoralen in 3 hours.
Figure 11 is through adsorbing dynamic (dynamical) relatively sketch map of methylene blue in 2 hours from blood plasma.
Figure 12 illustrates acridine, acridine orange, 9-aminoacridine and 5-[(β-carboxyethyl) amino] chemical constitution of acridine.
Figure 13 is for drawing data (y-axle) and the 5-[(β-carboxyethyl of different resins to the adenine capacity) amino] sketch map of data (X-axle) of acridine capacity.
Figure 14 A and Figure 14 B are for removing 5-[(β-carboxyethyl with Dowex_XUS-43493 and Purolite_MN-200 and Amberlite_XAD-16 HP) amino] sketch map of comparison of adsorption dynamics adsorption kinetics of acridine.
Figure 15 is for removing the sketch map of comparison of the adsorption dynamics adsorption kinetics of 9-aminoacridine and acridine orange with Dowex_XUS-43493.
Figure 16 has described the fixedly batch structure of adsorption plant (IAD).
Figure 17 for the compare sketch map of comparison of different Ambersorb absorption isotherms of Purolite MN-200.
Figure 18 is for being exposed to fibrosis Pica G277 IAD (500g/m through continuous or termination 2) in after 24 hours at 4 ℃ of following 5-[(β-carboxyethyls in the unitary supernatant of 300mL PRBC in 4 weeks of storage) amino] sketch map of comparison of level of acridine and GSH.
Figure 19 is that closed material is to 5-[(β-carboxyethyl among the PRBC) amino] sketch map of influence of adsorption dynamics adsorption kinetics of acridine.
Figure 20 contains without fixing and through the sketch map of the comparison of the percentage of hemolysis of the adsorbent equipment of fixed absorbent particles Purolite MN-200.
Figure 21 is the sketch map without the comparison of the percentage of hemolysis of fixing and the fixed absorbent particles Pica G-277 active carbon of process.
Figure 22 for from platelet concentrate, remove 4 '-(4-amino-2-oxa-) butyl-4,5 ', dynamic (dynamical) sketch map of 8-trimethylpsoralen.
Figure 23 is the sketch map of the psoralen adsorption dynamics adsorption kinetics of fibre substrate IAD (AQF, square) and particle matrix IAD (Porex, triangle).
Implement best mode of the present invention
The invention provides the equipment that reduces compound concentration in the celliferous biological composition.This equipment comprises by the adsorbing medium of the fixed granulometric composition of inert base and is the batch structure.Typically, in biological composition, use chemical compound that this equipment reduced to have the molecular weight of the about 30000g/mol of about 100g/mol-.The contained cell of biological composition comprises the cell that for example is suspended in Biomedia such as blood plasma or the tissue culture medium (TCM).This biological composition with kept its biological activity basically after these equipment contact.
The illustration chemical compound comprises pathogen inactivated chemical compound, dyestuff, mercaptan, plasticizer and complement activation.The equipment that is provided comprises by the three-dimensional network of the fixed absorbent particles of inert base.Should fixedly reduce the bleed danger of blood products of loose absorbent particles.And, simplified the operation that reduces the problem that accompanies with the loose absorbent particles of processing by inert base fixed absorbent granule.The fixed absorbent granule also can improve the ability that absorbent particles adsorbs chemical compound in the celliferous biological composition, and pair cell does not have mechanical damage.
Definition
Term " acridine derivatives " is meant and contains tricyclic structure acridine (dibenzo [b, e] pyridine; The 10-naphthazine) chemical compound.These chemical compounds have affinity to nucleic acid, and can adhere on the nucleic acid by inserting non-covalently.Term " aminacrine " is meant that these acridine chemical compounds have one or more nitrogen-containing functional groups.The example of aminacrine comprises 9-aminoacridine and acridine orange (shown in Figure 12).
Term " absorbent particles " broadly is meant arbitrarily natural or synthetic particulate matter, it can with the interaction of molecules in the liquid, therefore this molecule is removed from liquid.The example of naturally occurring adsorbent includes but not limited to active carbon, silicon dioxide, kieselguhr and cellulose.The example of synthetic adsorbent includes but not limited to polystyrene, polyacrylic acid and carbon-bearing adsorbent.Absorbent particles is generally porous, has high surface usually, and can influence how interactional functional group (for example ionic, hydrophobic, tart, the alkaline) modification of this adsorbent and molecule with various.
Term " aromatic series ", " aromatic compound " etc. broadly are meant the chemical compound of the former subring with delocalized electron.This monocyclic compound benzene (C 6H 6) be common aromatic compound.But, electron delocalization can take place on more than one adjacent ring (for example, naphthalene (2 rings) and anthracene (3 rings)).Dissimilar aromatic compounds includes, but not limited to aromatic halide (aryl halide), heteroaromatic compounds, aromatic hydrocarbon (aromatic hydrocarbons) and aromatic nitro compound (aromatic nitro-compound).
Term " biocompatible bag quilt " broadly is meant with hydrophilic polymer covering surfaces (for example polystyrene bead surface), when this hydrophilic polymer contacts with blood products, can not cause being harmful to, poisonous or immunoreation, and by reducing cell adhesion forces, reducing protein adherence power or improve cell function and give this surface bigger biocompatibility.Suitable bag is biocompatible, even they have faint influence (if any) to the biomaterial that is in contact with it." faint " influence meaning is not see big difference biology compared with the control.In a preferred embodiment, biocompatible bag has been enhanced the surperficial blood compatibility of paradigmatic structure.For example, poly-(methacrylic acid 2-hydroxyl ethyl ester) (pHEMA) is often used in the bag quilt of armarium (for example blood filter).
Term " biocompatible container (housing) " is meant that broadly suitable dress biological material is as the container that contains platelet or erythrocytic compositions, bag, pipe, accepter etc.Suitable container is biocompatible, even they have faint influence (if any) to the biomaterial that comprises wherein." faint " influence meaning is to compare in the blood products function with described contrast herein erythrocyte, platelet and blood plasma are not seen big difference.Therefore, blood products can preserved in biocompatible container before the donee that transfuses blood.In a preferred embodiment, biocompatible container is the blood bag, comprises platelet storage container or erythrocyte storage container.
Term " container compatible with biological composition " is meant the suitable container of adorning celliferous biological composition, and said composition for example has cell culture compositions and contains platelet or erythrocytic compositions.These containers have faint influence to celliferous biological composition.The example of these containers includes, but not limited to Tissue Culture Plate, Tissue Culture Flask and blood bag.
Term " biological fluid " comprise synthetic medium, human body or the non-human body of cell culture medium, storage cell whole blood, blood plasma, platelet, erythrocyte, leukocyte, serum, lymph, saliva, breast, urine from or contain the product of above-mentioned arbitrarily single or mixture, have or do not have chemical addition agent solution.Preferably, this fluid is blood or the blood products that contains or do not contain chemical addition agent solution, more preferably blood plasma, platelet and erythrocyte, most preferably separation property blood transfusion blood plasma, erythrocyte and platelet.
Term " blood bag " is meant a class blood products container.
Term " blood products " is meant by (for example erythrocyte, leukocyte, platelet etc.) such as the liquid of systemic circulatory system and/or relevant cell compositions; Blood products includes, but not limited to blood cell, platelet mixture, serum and blood plasma.Term " platelet mixture " is meant a class blood products, and wherein this cell composition mainly or only has platelet.Platelet concentrate (PC) is a class platelet mixture, and wherein these platelet are attended by than the littler part of normal plasma part.In blood products, synthetic medium can remedy the shared volume of normal plasma; For example, platelet concentrate can limit platelet suspension in 35% blood plasma/65% synthetic medium.Often, this synthetic medium contains phosphate.
Term " blood separating mechanism " broadly is meant the equipment that blood separation can be become blood products (for example, platelet and blood plasma), machinery etc.The separation property blood transfusion system is a class blood separating mechanism.The separation property blood transfusion system generally includes the computerized device of blood separation equipment, complicated pipeline and filter network, collecting bag, anticoagulant and control all components.
Term " through crosslinked " broadly is meant to be connected to each other and forms the linear molecule of bidimensional or three-dimensional network.For example, divinylbenzene (DVB) is as the cross-linking agent that forms styrene diethylene benzene copoly mer.This term also comprises " super crosslinked ", wherein through super crosslinked network be with or solution in or for the linear polystyrene chain of solvent swelling state and difunctionality reagent is crosslinked makes.Various difunctionality reagent can be used for crosslinked (for example, referring to Davankov and Tsyurupa, Reactive Polymers 13:24-42 (1990); People such as Tsyurupa, ReactivePolymers 25:69-78 (1995)).
Term " cycle compound " is meant have one (that is monocyclic compound) or the chemical compound of (that is polycyclic compound) annular atoms more than.This term is not limited to contain the chemical compound of the ring of specific quantity atom.Although the contained ring of most of cycle compounds is 5 or 6 atoms, the ring of other quantity atom (for example 3 or 4 atoms) is also admitted by the present invention.Although atom mainly is a carbon atom in the ring, without limits to the concordance of these atoms.In general, the ring of polycyclic compound is adjacent one another are, and still, term " multi-ring " chemical compound comprises that those contain the chemical compound of a plurality of rings not adjacent to each other.
Term " dyestuff " broadly is meant the chemical compound of giving color.Dyestuff contains the chromophore that is connected on one or more cycle compounds and the group of auxochrome usually.Color is owing to chromophore, yet dyeing affinity is because auxochrome.Dyestuff has been divided into many classes, comprises azine dye (for example, dimethyl diaminophenazine chloride, safranine and azocazmine B); Azo dye; The azocazmine dyestuff; Take off the phenylmethane dyestuff; Fluorescent yellow dye; The ketimide dyestuff; Rosaniline dyes; Triphenhlmethane dye; Phthalocyanine dye; And hypericin.Should think can be with method and apparatus of the present invention through putting into practice and combining for the dyestuff of cycle compound arbitrarily.
Term " Fibrized resin " typically refers to fixed sorbent material, for example comprises, is embedded in the network of fibers or attached to the resin on the network of fibers.In one embodiment, this network of fibers is made up of polymer fiber.In another embodiment, these fibers are to be made up of the polymer core (for example, polyethylene terephthalate [PET]) of the higher melting temperature of being surrounded by the polymer sheath of relatively low melting temperature (for example, nylon or modified PET).Fibrized resin can heat this network of fibers down and make by the condition (temperature is enough melted sheath but do not melted core) that produces negative effect significantly at the absorbability that does not cause resin.Contain the place of pearl at this resin, heat in case the adsorbent pearl attached to producing " fibrosis pearl " on the outer polymer sheath.By producing the Fibrized resin that every definition area contains dose known amounts adsorbent pearl, can be by the Fibrized resin of cutting definition area, rather than the adsorbent pearl of weighing obtains (for example to be used to remove cycle compound, psoralen, particularly amino psoralen) and the Fibrized resin sample of other products.
Term " filter " broadly is meant and can makes equipment by being detained other component simultaneously of some component in the mixture, material etc.For example, filter can comprise that the aperture allows blood products (for example erythrocyte compositions) by keeping for example net of other component of resin particle simultaneously.Term " filter " is not limited to keep the device of some component.
Term " heterocyclic compound " is meant that broadly one of them or several ring contain the cycle compound of more than one atoms.Usually, carbon atom is main atom, and other atom for example comprises nitrogen, sulfur and oxygen atom simultaneously.The example of heterocyclic compound comprises furan, pyrroles, thiophene and pyridine.
Term " high-temperature activation process " is meant typically because the pyroprocess that raw material pyrolysis and/or oxidation cause the surface chemistry of surface area, porosity and treated material to change.
Term " fixed adsorbent equipment (IAD) " is meant and is embedded in the inert base or attached to the fixed absorbent material on the inert base.At inert base is the place of network of fibers, and term IAD can use with term Fibrized resin exchange ground.For example, fibrosis Ambersorb 572 and Ambersorb IAD (AQF) refer to same material.
Term " inert base " is meant synthetic arbitrarily or naturally occurring fiber or polymeric material, they can be used for fixing absorbent particles, and not influence the required biological activity of blood products basically.Although this substrate is to absorption or remove almost not contribution of process, it may be favourable to the concentration that reduces less organic compound.In addition, this inert base can interact with cell or protein component, makes cell remove (for example leukocyte eliminating) or removes deproteinize or other molecule.
Term " separation " is meant isolates a kind of material from the mixture that contains more than one components.For example, can from whole blood, isolate platelet.Isolating product needs not to be this product to be separated from other component fully.
Term " macropore " meaning usually is that the aperture is greater than about 500_.The term micropore is meant that the aperture is less than about 20_.The term mesopore be meant the aperture greater than about 20_ less than about 500_.
Term " macropore " is used to describe the pore structure of a large amount of apertures greater than about 500_.
Term " macroreticular " is a relative terms, and the meaning is to have degree of physical porous (that is, having big metering-orifice) structure, and the existing macropore of porous adsorbent structure has micropore again.
Term " net closure ", " mesh bag " etc. are meant and are processed into closure, bag, bag or the analog that contains a plurality of openings.For example, the present invention uses a kind of bag, and it is equipped with the fixed absorbent granule, and its hole dimension makes blood products can contact fixed absorbent particles, but fixed absorbent particles is retained in the bag.
Term " spacer " is meant and can or be separated into section or the equipment or the element of any type of part with an overall separation.For example, the present invention's blood bag of attempting to use a kind of spacer will suit to contain blood products is divided into two parts.Blood products accounts for the part of this bag before handling and in the processing procedure, and absorbent resin accounts for another part simultaneously.In one embodiment, after treatment of blood products, this spacer is removed (for example the integrity with this spacer changes), made that therefore the blood products of handling contacts with absorbent resin.This spacer can be positioned at the inside of bag, also can be in its outside.When using term " spacer ", the meaning " removed " in term is separately no longer existing of two parts of blood bag, needs not to be this spacer and links to each other with bag no longer in some way.
Term " photoproducts " be meant psoralen or other dyestuff (for example methylene blue, phthalocyanine dye) through the photochemical reaction that being exposed to ultraviolet radiation product extremely.
Term " polyvinyl aromatic compound " is meant the polymer that contains aromatic radical in main chain, and polyethylene terephthalate for example, or as the polymer of side group as polystyrene, or had not only contained aromatic radical but also as the polymer of side group in main chain.
Term " polystyrene network " broadly is meant and contains styrene (C 6H 5CH=CH 2) polymer of monomers; These polymer can be linear, are made up of single covalency alkane chain and phenyl substituent, or through crosslinked, common and-or right-phenylene residue or other difunctionality or super cross-linked structure are crosslinked, thereby formation two-dimensional polymer main chain or three-dimensional network.
Term " psoralen remover " is meant a kind of material or the equipment that can remove greater than about 80% psoralen from blood products for example; Preferably, greater than about 90%; More preferably greater than about 99%.The psoralen remover can also be removed other component in the blood products, for example psoralen photoproducts.
Phrase " reduction concentration " is meant removes a part of low molecular weight compound from biological composition.It is about 70% that the reduction of concentration simultaneously is preferably greater than, and more preferably from about 90%, most preferably from about 99%.
Phrase " is removed described freely chemical compound part (for example psoralen, psoralen derivant, isopsoralen, acridine, acridine derivatives, dyestuff, plasticizer or complement activation) in nearly all solution " and preferably is meant and removes greater than this chemical compound freely in about 80% the solution, more preferably remove greater than about 85%, be preferably greater than approximately 90% again, most preferably remove greater than about 99%.
Term " resin " is meant a kind of solid carrier (for example granule or pearl etc.), it can with solution or liquid (for example blood products) in comprise that the various little organic compound of psoralen interacts and absorption, has therefore reduced the concentration of these compositions in the solution.This process of removing is not limited in the mechanism of any specific.For example, can remove psoralen by hydrophobic or ionic interaction.Term " absorbent resin " broadly is meant natural organic matter and synthetic and composition thereof.
Term " alr mode " be meant can the mixed biologic compositions any means.The example of alr mode includes, but not limited to following mechanical agitator: back and forth, track, three-dimensional rotation device and rotary apparatus type agitator.
Term " electromagnetic shaker equipment " is meant the equipment of any type that can fully mix blood products sample of blood platelet concentrate.This equipment can have the timing mechanism that the mixing of making is limited in moment.
Term " sintered medium " is meant by applying the structure that heat and pressure form porous resin, comprises for example granule thermoplastic polymer.Porous resin can be made by following: the polymer beads that fusing point is low relatively mixes, and they is heated to this plastic particle partial melting, but still is penetrated in the porous resin with the fluid approach.The sintering adsorbent medium can mix with low-melting powder and heats and make similarly by carbon or other is high or not fused absorbent particles.The method of production porous plastic material is described among the US3975481,4110391,4460530,4880843 and 4925880, is incorporated herein by reference.This method makes the low melting point particle fusion form the porosu solid structure.By in sintering process, polymer beads being placed in the shaping jig and this sintered medium can being formed different shape.Before carrying out sintering process, mix with thermoplastic polymer particles and absorbent particles can be added in the sintered medium by absorbent particles.
Term " stabilizing agent " is meant the chemical compound or the compositions of the absorbability that can keep some adsorbent (for example Amberlite) under drying condition.In general, acceptable stabilizing agent should be able to water-soluble and ethanol (or other wetting agent), and is non-volatile with respect to water and ethanol, and can be safely with a small amount of conveying.The example of stabilizing agent includes, but not limited to glycerol and low-molecular-weight PEG.The difference of " moistening temperature agent " and " stabilizing agent " is that the former is considered to open once more the adsorbent hole without super crosslinked resin (for example Amberlite XAD-4, Amberlite XAD-16).Wetting agent can not prevent that generally the hole subsides under the drying condition, yet stabilizing agent can.The general discussion of moistening and wetting agent has statement in people's such as Pall US5501795, be incorporated herein by reference here.
Phrase " the required biological activity that has kept biological composition basically " is meant the performance (for example cell integrity) that has kept biological composition basically.In some embodiments, the cell integrity has reacted the potential performance of compositions in the treatment adjustment.For example, relating to erythrocytic place, if ATP level, extracellular potassium spill, percentage of hemolysis is retained in the erythrocyte of handling by methods described herein basically, activity in vivo is destroyed or do not have a big reduction so.The variation of the erythrocytic ATP level of for example, handling should be less than about 10%.The hemolysis levels of the erythrocyte of handling after storage should be less than about 1%, preferably less than about 0.8%.The variation that the erythrocytic extracellular potassium of handling spills should be less than about 15%.Relating to hematoblastic place, if for example platelet yield, pH, gather reaction, change of shape, GMP-140, form or hypotonic shock response and be retained in basically in the platelet of handling by methods described herein, activity in vivo is destroyed or do not have a big reduction so.For example, the platelet in the biological composition is lost preferably less than 15% after the storage; After storage 5 days more preferably 15%; Even after storage 5 days more preferably 10%.Should think that also phrase " keeps the every kind performance relevant with described blood products basically " and also can comprise the acceptable value of those skilled in the art described in the document, the document comprises for example Klein H.G_ed.Standards for Blood Banks andTransfusion Services, 17 ThEd_Bethesda, MD:AmericanAssociation of Blood Banks, 1996, be incorporated herein by reference.
Term " its coordinate " is meant function and the suitable equipment of biological activity that keeps biological composition when it is used for equipment of the present invention.For example, " on an equal basis " equipment or the substrate that contains absorbent particles is similarly kept cell viability or suitable thrombin level a kind of.
Term " low molecular weight compound " is meant the organic or biomolecule of molecular weight ranges at the about 30000g/mol of about 100g/mol-.Low molecular weight compound includes, but not limited to following chemical compound: little organic compound such as psoralen, acridine or dyestuff; Quencher is as glutathion; Plasticity extractable, for example plasticizer; Biological instrumentality as complement activation, has the molecular weight of the about 30000g/mol of about 100g/mol-; And polyamine derivatives.
Term " biological composition of suitable infusion " is meant that biological composition has kept its necessary biological property (for example platelet form), contain enough low-level any unwanted chemical compound (for example deactivation compounds, reaction instrumentality) simultaneously, so that infusion provides the function of wanting, but there is not harmful side effect.
Term " contrast " is meant the test of the relative effect research of carrying out different condition when it for example is used for the phrase of " relative comparison ".For example, in the place that biological composition is handled with a kind of equipment, " untreated contrast " should be meant treated under the same conditions biological composition, be this device processes of no use, perhaps (for example fixed granule and the loose granule) handled with the equipment of another form.
Term " 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen " also be called " S-59 ".
Term " N-(9-acridinyl)-Beta-alanine " also is called " 5-[(β-carboxyethyl) amino] acridine ".It is also called " S-300 ".
Term " XUS-43493 " is also called " Optipore 493 ".
Absorbent particles
In order to reduce the required biological activity that compound concentrations in the celliferous biological composition keeps biological composition simultaneously basically, be provided for the absorbent particles in a kind of equipment.Typically, these chemical compounds that reduced in biological composition have the molecular weight of the about 30000g/mol of about 100g/mol-.
Absorbent particles can be to make it join any regular in the inert base or irregularly shaped, but is preferably roughly spherical.These particulate diameters are greater than about 100 μ m and less than about 1500 μ m; Preferably, these particulate diameters are between the about 1300 μ m of about 200 μ m-; More preferably, these particulate diameters are between the about 900 μ m of about 300 μ m-.
Surface area is greatly these particulate features.Preferably, these granules have about 750m 2The about 3000m of/g- 2The surface area of/g.More preferably, these granules have about 1000m 2The about 3000m of/g- 2The surface area of/g.
The absorbent particles that is suitable for present device can be the material of any suitable, and to be this material be free from side effects basically to the biological activity of the biological composition of contact with it in its restriction.This absorbent particles can be for example, to be made by for example material of active carbon, hydrophobic resin or ion exchange resin.
In a preferred implementation, these absorbent resins are by active carbon natural or that synthetic source obtains.Preferably these active carbons derive from synthetic source.The non-limitative example of active carbon comprises: can from PICA USA Inc. (Columbus, OH) the Picatiff Medicinal_ of Huo Deing can be from Norit Americas, Inc. (Atlanta, the GA) Norit_ROX 0.8 of Huo Deing can be from Rohm ﹠amp; Haas (Philadelphia, PA) Ambersorb_572 of Huo Deing and can be from PICA (Columbus, OH) G-277_ of Huo Deing.
In another preferred embodiment, these granules can be hydrophobic resins.The non-limitative example of hydrophobic resin comprises following polyaromatic adsorbent: Amberlite_ adsorbent (for example, Amberlite_XAD-2, XAD-4 and XAD-16), and can be from Rohm ﹠amp; (Philadelphia PA) obtains Haas; Can be from Toso Haas (TosoHass, Montgomeryville, PA) the Amberchrom_ adsorbent of Huo Deing; Diaion_ //Sepabeads_ adsorbent (for example Diaion_HP20), can be from MitsubishiChemical America, (White Plains NY) obtains Inc.; Hypersol-Macronet_ absorbent resin (for example Hypersol-Macronet_ absorbent resin MN-200, MN-150 and MN-400) can (Bala Cynwyd PA) obtains from Purolite; With Dowex_ adsorbent (for example Dowex_XUS-40323, XUS-43493 and XUS-40285), can be from Dow Chemical Company (Midland, MI) acquisition.
Preferred granule is the hydrophobic resin that contains the polyaromatic adsorbent of super crosslinked polystyrene network, for example Dowex_XUS-43493 (being Optipore_L493 or V493 on the known commercial) and Purolite MN-200.
Super crosslinked polystyrene network as Dowex_XUS-43493 and Purolite MN-200 is nonionic macropore and macroreticular resin.Nonionic macroreticular and macropore Dowex_XUS-43493 to for example comprise 4 '-(4-amino-2-oxa-) butyl-4,5 ', the psoralen of 8-trimethylpsoralen has high affinity, and it has good wettability.Phrase " good wettability " meaning is that dried (for example substantially anhydrous) adsorbent needn't used wetting agent (for example ethanol) moistening for the concentration that reduces the medium and small organic compound of blood products effectively with before blood products contacts.
For example preferred its of the super crosslinked polystyrene network of Dowex_XUS-43493 and Purolite MN-200 is shaped as the spheroidal particle of diameter range at the about 1300 μ m of about 200 μ m-.Comprise that for example the absorbent particles of Dowex_XUS-43493 preferably has king-sized internal surface area and relative little hole (for example average diameter 46_).Particulate internal surface area can be at the about 1100m of about 300- 2/ g; The about 1100m of preferably about 900- 2/ g; Most preferably about 1100m 2/ g.Particulate most of hole can be greater than 25_ and less than 800_; The preferred about 150_ of about 25_-; The about 50_ of 25_-most preferably from about.When in the mechanism of not planning to limit the present invention to the reduction of carrying out little organic compound, the dominant mechanism that hydrophobic interaction is considered to adsorb.Its permeability can be by making micromolecule enter relative macromole (for example larger protein) thereby and the surface area of the more vast scale of cell optionally authorize adsorption process.Purolite_ has many characteristics similar to Dowex_XUS-43493, and for example to high affinity of psoralen and good wettability, and it also is best absorbent particles.
Can polystyrene be divided into ⅰ according to its synthesis mechanism and physics and functional characteristic) legacy network and ⅱ) super cross-linked network.Preferred adsorbent has big surface area, the hole that does not subside when dry, do not need moistening when being used to comprise erythrocyte or hematoblastic biological composition, and have few especially granule and foreign particle (for example dust, fiber, non-absorbent particles and unidentified granule).In addition, preferred adsorbent has a small amount of extractible residual monomer, cross-linking agent and other organic extractable.
This legacy network mainly is a styrene diethylene benzene copoly mer, and wherein divinylbenzene (DVB) is as cross-linking agent (that is the reagent that the linear polystyrene chain is linked together).These converging networks comprise " gel-type " polymer.This gel-type polymer is even, the atresia styrene-DVB copolymer that obtains by monomer copolymerizable.Macroporous adsorbent is represented the second quasi-tradition network.They are to obtain by copolymerization monomer in the presence of the diluent of the polystyrene chain that generates in precipitation.The polystyrene network that forms by this step has big relatively internal surface area (every gram polymer up to hundreds of square metre); Amberlite_XAD-4 is produced by this step.
Compare with above-mentioned legacy network, preferred adsorbent of the present invention (for example Dowex_XUS-43493) is super cross-linked network.These networks are by with the linear polystyrene chain or with solution or with solvent swelling state and difunctionality reagent is crosslinked makes; Preferred difunctionality reagent produces the cross-bridge of conformation constraint, it is believed that to have prevented that like this hole subsides when this adsorbent is in substantially anhydrous (i.e. " drying ") state.
Super cross-linked network it is believed that to have and other three key properties of legacy network phase region.At first, because these bridges make polystyrene chain keep separating, so the density of polymer chain is low.As a result, adsorbent has big relatively aperture surface area and aperture usually.Secondly, these networks can expand, that is, its volume increases when polymer phase contact organic molecule.It is " directly " when at last, this super cross linked polymer is in drying regime; That is, the rigidity of drying regime network has stoped the attraction of chain and chain.But, these chain relaxations when adsorbent is wetted, this has increased network expansible ability in liquid medium.Davankov and Tsyurupa, Reactive Polymers13:27-42 (1990); People Reactive Polymers 25:69-78 (1995) such as Tsyurupa, this paper is incorporated herein by reference.
Successfully utilize the bridge between several cross-linking agent production polystyrene chains, comprise dichloro-xylol (XDC), monochloro-dimethyl ether (MCDE), 1,4-two-chloromethyl biphenyl (CMDP), 4,4 '-two-(chloromethyl) biphenyl (CMB), dimethyl methyl acetal (DMF), p, p '-two-chloromethyl-1,4-diphenyl butane (DPB) and three-(chloromethyl)-mesitylene (CMM).Be reflected at these bridges of formation between the polystyrene chain by one of these cross-linking agent and styrene phenyl ring by Friedel-Crafts reaction.Therefore, the gained bridging is connected on the styrene phenol ring that occurs on two different polystyrene chains.Referring to for example US3729457, be incorporated herein by reference here.
Because these bridges have been eliminated the needs to " moistening " agent usually, so these bridges are particular importances.That is, these bridges have prevented that the hole subsides when adsorbent is in anhydrous basically (promptly " doing ") state, therefore adsorbent with needn't open once more with wetting agent before blood products contacts.In order to prevent that the hole from subsiding, should form the bridge of conformation constraint.The conformation that some difunctionality reagent limit as DPB does not cause; For example, DPB contains four continuous MU (methylene unit) of conformation being reset sensitivity.Therefore, DPB is used for preferred difunctionality reagent of the present invention.
The structure dependent characteristic of some of above-mentioned absorbent particles is listed in the Table A.
Table A
Figure 9881384000191
The processing absorbent particles
Can further process to remove subparticle, salt, potential extractable and endotoxin these absorbent particles.These can extract removing typically by using organic solvent, steam or treatment with supercritical fluid to realize of component.Preferably these granules are through sterilization.
The absorbent particles of the commercially available acquisition of " purification " (promptly finished) type is sold by present several company.Except these absorbent particles (for example resin) were processed, these adsorbents were tested by these companies, and final adsorbent guarantees aseptic (USP XXI), apyrogeneity (LAL) and do not have detectable extract (DVB and total organic matter).
Hot-working (for example steam) is a kind of effective ways of processing absorbent particles.F.Rodriguez，Principles?Of?Polymer?Systems，(HemispherePublishing?Corp.)，pp.449-53(3 rd.Ed_1989)。(Bellefonte PA) uses a kind of solvent-free, hot proprietary process to purify Dowex_XUS-43493 and Amberlite adsorbent in Supelco company.Use the main benefit of steam to be in adsorbent, not add any potential extractable.But a big defective is that this process can seize moisture from the hole of resin bead; The effective performance of some adsorbents needs the quilt moistening once more before the contact blood products of these pearls.
An advantage of purification/finished adsorbent is that low-level especially particulate diameter is less than 30 μ m.Preliminary test to the adsorbent (Dowex_XUS-43493 and Amberlite_XAD-16) of Supelco processing is to measure granule number.The result of these tests demonstrates does not have foreign particle (for example dust, fiber, non-absorbent particles and unidentified granule) and do not have subparticle (＜30 μ m) basically.
Wetting agent and stabilizing agent are used with absorbent resin
Can use certain methods to prevent the forfeiture of particle drying and absorbability, for example Amberlite_ loses some absorbabilitys down in some condition (for example dry).
In one approach, granule, material or equipment can be processed under airtight and the wetting state that can not become dry.This method is attended by several major defects.The level of extractable may increase in the material owing to go beyond the time limit, so the pot-life of these products may shorten.Because the moistening polymer does not typically pass through gamma-irradiation, therefore sterilization may be limited in steam course.The processing request assembly keeps the equipment of moisture state more difficult than dry processing equipment usually; For example, if time-lag is oversize between device assembles and the final sterilization, may consider biological load and endotoxin.
The second method that prevents the absorbability forfeiture is to use the adsorbent of being free from side effects through super-dry.As mentioned above, the macroreticular adsorbent (for example, Dowex_XUS-43493 and Purolite_MN-200) with highly cross-linked pore structure does not need wetting agent usually, and this is because this crosslinked hole that stoped subsides.Do not resemble Amberlite_XAD-16, these macroreticular adsorbents have kept very a high proportion of initial activity when it is dried.
In the third method, in the presence of non-volatile wetting agent by making Amberlite_XAD-16 and related adsorbent (for example, Amberlite_XAD-4) become hydrate can prevent forfeiture because of exsiccant absorbability.For example, when using Amberlite_XAD-16 as adsorbent, this adsorbent pearl can be partly dry in operation, sterilization and storage before using.When the moisture subcritical level of these adsorbents, the quick forfeiture (may because hole " subside ") of absorbability takes place; Therefore, in order to reach optimum efficiency, these holes have to " open " with wetting agent again before use.
When some absorbent resin is exposed to drying condition following time, stabilizing agent is effective near its maximum to keeping absorbability.It is believed that and use stabilizing agent to prevent that the adsorbent hole from subsiding.
Acceptable stabilizing agent should be water soluble and ethanol, and the second alcohol and water is non-volatile relatively, and to transfer safety on a small quantity.Glycerol and low molecular poly (for example PEG-200 and PEG-400) are the examples with stabilizing agent of these characteristics.Glycerol has positive blood compatibility history.In the chilled storage of erythrocyte goods, often it is added in the blood as antifreezing agent.Referring to people Transfusion 26:341-45 (1986) such as for example Chaplin; People Am.J.Vet.Res.42 (9) 1590-94 (1981) such as Valeri.The solution that will contain up to 1% glycerol transfers routinely, glycerite can be commercially available (Glyerolite 57 Solution for example, FenwalLaboratories, Deerfield, IL).The adsorbent pearl that resembles Amberlite_XAD-16 can be through stable in ethanol and glycerol.
The low molecular poly that is often used as the medicine base material also can be used as stabilizing agent.PEG is chemical general formula H (OCH 2CH 2) nThe liquid state of OH and solid polymer, wherein n is more than or equal to 4.The PEG goods are followed the quantity of corresponding its mean molecule quantity usually; For example, the molecular weight of PEG-200 is 200, and molecular weight ranges is 190-210.PEG is can many goods (for example Carbowax, Poly-G and Solbase) commercially available.
Be used for granulopectic inert base
By inert base fixed absorbent granule.This inert base can be by synthetic or natural polymer preparation.For example, this inert base can be synthetic or natural polymerization fibres, for example network of fibers.This inert base can be sintered polymer.Other assembly of this inert base and this equipment is preferably can be biocompatible, and can side effect not arranged because of the biological activity that contacts material basically.
Most preferably, these synthetic fibers are polyester fiber (Air QualityFiltration (AQF), Hoechst Celanese place (Charlotte, N.C.)).Other preferred example of synthetic fibers is polyethylene or Fypro.Other illustration synthetic fibers comprise polyolefin, polyvinyl alcohol and polysulfone fibre.
In a preferred implementation, this synthetic polymeric fibers comprises dystectic first polymer core that has that sheath with low fusion temperature surrounds.This polymer core can be polyester (polyethylene terephthalate).This sheath can be nylon or modified poly ester.Fiber can (Osaka be Japan) with Hoechst Trevira GmbH ﹠amp from Unitika; Co. (Augsberg, Germany) commercially available.
The natural polymer fiber of illustration comprises the cellulose fibre that derives from separate sources, and for example there are Corchorus olitorius L., kozu, kraft paper and abaca in these sources.To synthesize or the network of natural polymerization fibres is used to prepare the filter described in US4559145 and 5639376, this paper is incorporated herein by reference.
The synthetic polymer of suitable configurations sintered particles is high density polyethylene (HDPE), ultra-high molecular weight polyethylene, polypropylene, polyvinyl fluoride, politef, nylon 6.More preferably this sintered particles is a polyolefin, as polyethylene.
Aforesaid polymer fiber can be the absorbent resin that does not adhere to absorbent particles.These fibers can be formed on the network of fibers that network of fibers maybe can be fixed in sorbent fibers still less.The present invention comprises these fibers; These fibers preferably contain greatly, the surface area of porous, adsorptivity or be easy to reduce other adsorptivity instrument of low molecular weight compound concentration.
Particulate fixing
In one embodiment, absorbent particles is fixed by inert base, thereby makes the adsorbing medium that is used to reduce the medium and small organic compound substrate concentration of material.This inert base can be to contain three-dimensional network synthetic or natural fiber network and fixing absorbent particles wherein.
Preferably, this adsorbing medium contains the aperture absorbent particles with highly porous structure and very large internal surface area, as mentioned above, is fixed by this inert base.Preferably, when biomaterial was contacted with adsorbing medium, adsorbing medium was free from side effects basically to biological activity or other performance of material.
The adsorbent pearl is fixed on the technology that constitutes air filter on the network of fibers has description in US5486410 and US5605746, be incorporated herein by reference.As described in Figure 1, the polymer fiber 600 of network of fibers is made up of the polymer core with higher melt 602 (for example, polyethylene terephthalate (PET)) of polymer sheath 604 (for example, the nylon) encirclement with low relatively fusion temperature.Referring to people's such as Heagle US5190657, this paper is incorporated herein by reference.Fibrized resin by in network of fibers at first uniform distribution adsorbent pearl make.Next, with this network Fast Heating (for example, 180 ℃ * 1min.) make the polymer sheath fusion of fiber 600 and stick to adsorbent pearl 606 and other fiber on, thereby form crosslinked network of fibers, as shown in Figure 2.As (not to scale (NTS)) as described in Fig. 3 and Fig. 4, in general, this network of fibers contains three layers: two skin 607 and not too thick internal layers 609 that contain adsorbent pearl 606 and less fiber 600 that are wrapped in fiber 600 densely.In a preferred embodiment, the edge of Fibrized resin can be with polyurethane or other polymeric seal.Perhaps, as described in Fig. 3 and Fig. 4, can in the gained Fibrized resin, prepare heat seal 608 with predetermined space; For example, can in Fibrized resin, prepare heat seal with square pattern.Afterwards, can be by these heat seal with the cutting of this Fibrized resin, contain required adsorbent pearl quality (for example, preferably less than 5.0g and be more preferably less than 3.0g) and suitable size and be placed on resin sample in the blood products container thereby form.These heat seal are used to prevent that the Fibrized resin that cuts is worn and helps the fixed absorbent pearl.But, do not need to use these heat seal in order to put into practice the present invention.In a selectivity embodiment, as described in Figure 4, the adsorbent pearl is not fixed on these fibers originally on one's body, but still be fixed on fiber denser outer 607 between and have a heat seal 608; This embodiment also can be by making the fibrotic mediators sample contain a certain amount of adsorbent after these heat seal cuttings.
The present invention also attempts using adhesive agent (for example, binding agent) that absorbent resin is fixed on the fiber.And, although preferably the adsorbent pearl chemically is adhered on the network of fibers, also can physically be limited in these pearls in the network of fibers; This can realize so that make these pearls be in the appropriate location by for example surrounding these pearls with enough fibers.
Also attempted these absorbent particles to be fixed on other method in the network of fibers.Can use dried shop method to fix these granules, described in US5605746 and 5486410 (AQF patent), this paper is introduced into as a reference.Can use wet shop method to fix these granules, described in US4559145 and 4309247, this paper is introduced into as a reference.Can use the melt-blown method to fix these granules, described in US5616254, this paper is introduced into as a reference.When using wet shop method to make up substrate by the natural polymerization fibres, this inert base preferably includes bonding agent so that the adsorbent pearl is attached on the fiber.The non-limitative example of bonding agent comprises melamine, polyamine and polyamide.This substrate typically contains 1% or these bonding agent still less.
Using absorbent particles when agglomerating synthetic polymer particle makes up inert base, it is very important that absorbent particles has higher melting temperature than substrate.
In a preferred embodiment, absorbent particles is fixed in the fibre substrate that the heat bonding by the biological components network of fibers forms.Another embodiment relates to absorbent particles is fixed in the abiotic component fibre and working strength resin system, adhesive agent or other fusible fibers, thereby forms bonding between fiber and absorbent particles.The non-limitative example of useful fiber comprises polyester, nylon and polyolefin.(supplier who is used for the fiber of nonwoven industry lists in " AGuide to Fibers for Nonwovens; " Nonwovens Industry, June 1998, and 66-87.) example of wet-strength resins system comprises melamine/formaldehyde, chloropropylene oxide base resin, polyamine and polyamide.But use meldable fibre to be fixed in the fibre substrate granule open.Referring to for example US4160059.
Preferably, gained adsorbing medium unit are contains the adsorbent of known quantity.The adsorbent of unit are is about 100g/m 2-Yue 500g/m 2, preferably from about 250g/m 2-Yue 350g/m 2Like this, can measure the adsorbent (that is, need not claim Fibrized resin) of the appropriate amount of attempting to be used for specific purpose simply by the predetermined area of cutting fibre resin.
The preferred bio-compatible of this adsorbing medium (that is, not producing poisonous, harmful or immunoreation); Performance to the material of for example blood products (for example, platelet and thrombin) has minimum influence; And without poisonous extractable.Adsorbing medium fixed absorbent particles preferably have high mechanical stability (that is, do not have subparticle produce).The adsorbing medium that is used for batch device contains the adsorbent of about 25-85wt%, preferably with the about 100-500g/m of load 2The adsorbent that contains about 50-80% is more preferably with the about 250-350g/m of load 2The adsorbent that contains about 50-80%.
Bag is adsorbed the agent granule
The surperficial blood compatibility of granule, substrate or adsorbing medium can be improved by its surface by using the hydrophilic polymer bag.The hydrophilic polymer of illustration comprises poly-(methacrylic acid 2-hydroxyl ethyl ester) (pHEMA), it can be from for example Scientific Polymer Products, Inc. (Ontario, NY) obtain, and cellulose-based polymer, ethyl cellulose for example, it can (Midland MI) obtains from Dow Chemical.Referring to people such as for example Andrade, Trans.Amer.Soc.Artif.Int.Organs XVII:222-28 (1971).Other example of bag quilt comprises Polyethylene Glycol and polyethylene glycol oxide, also can be from Scientific Polymer Products, and Inc. obtains.This polymer coating can increase the blood compatibility and reduce because mechanical breakdown produces short grained danger.
This adsorbent surface also can be regulated with fixed heparin.In addition, can regulate reinforcing YIN-essence ion exchange polystyrene divinylbenzene adsorbent through heparin absorption.Heparin, a kind of polyanion will be adsorbed on the surface of the adsorbent with reinforcing YIN-essence ion exchange property very securely.The polystyrene divinylbenzene adsorbent that the various quaternary ammoniums of commercially available acquisition are regulated.
Can use a large amount of diverse ways to wrap quilt, comprise the radio frequency glow discharge polymerization described in US5455040, this paper is incorporated herein by reference with Wurster and wraps by method (by International Processing Corp. (Winchester KY) finishes).
In one embodiment, can be by absorbent particles (passing through air pressure usually) being suspended in the chamber so that for example the hydrophilic polymer of pHEMA can evenly be injected on the whole surface of absorbent particles and implements this Wurster bag by method.As described in embodiment 3, the Dowex_XUS-43493 that evenly sprays with pHEMA demonstrate that the platelet yield increases and with the bag of recruitment by change has produced unforeseeable effect to the platelet shape.Found that this Wurster bag is optionally wrapped the outer surface that is adsorbed the agent surface by method, to almost not influence of inner bore surface.
In a preferred embodiment, fixing adsorbing medium can be immersed in and wrap in the hydrophilic polymer by (referring to embodiment 3).This method is simpler and cost is lower than spray absorbent particles with hydrophilic polymer.
This method is not limited in the method for any this adsorbing medium of special time endoperidium.For example, in one embodiment, after making adsorbing medium but before this adsorbing medium of heat-sealing, carry out pHEMA bag quilt.In another embodiment, this adsorbing medium is at first sealed, carry out this pHEMA bag quilt then.Except wrapping, will use sintering process together with pHEMA and be used to remove discrete particles and fiber by this adsorbing medium.
Along with package amount increases, some little organic compound are arrived particle surface by bag will become more difficult, and this makes absorption power reduce.Therefore, along with package amount increases, must use usually the adsorbent of recruitment reach adsorbent with the bag quilt identical remove power.In one embodiment, the optimum level of pHEMA bag quilt is the minimum bag quilt (0.1-0.5%) that can observe the protective effect of platelet yield and external platelet function.
These bag quilts may be to the sensitivity of sterilizing.For example, the γ sterilization may cause bag to be crosslinked and/or split.Therefore, type of sterilization (E-beam and γ irradiation) and dosage may influence the performance of the adsorbent of the quilt that wraps.Usually, preferred E-beam.
Equipment
Provide equipment to be used for reducing the chemical compound of celliferous biological composition.This equipment is batch device.The example of batch device is shown in Figure 16.Batch device is known in the literature and be described in for example PCT application WO96/40857, is incorporated herein by reference.
Batch device of the present invention can comprise a kind of container of for example blood bag, comprises the adsorbent medium that contains immobilized particles.In one embodiment, blood products is added in the blood bag that contains adsorbent medium also with this bag stirring certain hour.
For example, in one embodiment, with for example fixedly the adsorbing medium of Dowex_XUS-43493 (for example be placed on the blood products container, PL 146 plastic containers (BaxterHealthcareCorp. (Deerfield, IL)) in, (HelmerLaboratories (Novesvill, IN)) or rotary apparatus (at room temperature kept on the HelmerLaboratories (Novesvill, IN)) about 24 hours and preserve under condition of different temperatures at the platelet shaking machine with this container.The size of blood products container can be the about 1200mL of about 600-.Reserve temperature can be about 4 ℃-Yue 22 ℃.
Can using method reduce the appearance of the diffusing absorbent particles that may from adsorbent medium, fluff.
The present invention relates to a kind of batch device, contain the fixed absorbent medium in the container that is retained in mesh bag/capsule for example.This net/capsule can be made of the system of knitting, nonwoven or membrane seal.In one embodiment, this braiding mesh bag can be made of medicinal polyester or nylon.It preferred embodiment is polyester.The film of commercially available acquisition includes, but not limited to Supor_200,800,1200 hydrophilic poly-sulfonic acid second diester (PES) film (Gelman Sciences (Ann Arbor, MI)); The polyvinylidene fluoride of Durapore_ hydrophilic modifying (PVDF) (Mantee AmericaCorp. (San Diego, CA)) and have the polysulfone membrane of the hydrophilic modifying of whole hydrophobic outlet, for example Gemini film (Millipore (Marborough, MA)); With (the Poretics (Livermore, CA)) of the film that contains Merlon with poly-vinylidene halide bag quilt.These containers can be sterilized after adding adsorbing base.
The preferred implementation that is used for celliferous biological composition
The invention provides the equipment that reduces the concentration of low molecular weight compound in the celliferous biological composition.These equipment comprise the adsorbing medium by the fixed granulometric composition of inert base.
Shown as the biological respinse modifier of milphosis toxin C 3a and terminal membrane attack complex SC5b-9 by whole blood and its component being handled (for example bleed filter leukocyte, singly adopt, recovery etc.) and storage production.In the rough sledding of operation and blood transfusion, these biological respinse modifiers have been involved.
In some embodiments, equipment of the present invention reduces or controls the concentration of complement activation in the celliferous biological composition.Compare with the compositions of this device processes of no use, when with this device processes said composition, wherein the concentration of complement activation is lowered or controls.In one embodiment, this adsorption plant comprises the fibrosis Ambersorb IAD that for example produces by AQF.In this embodiment, celliferous biological composition is exposed to makes the terminal component complex of C3a complement fragment and SC5b-9 reduce in this equipment compared with the control.In one embodiment, be exposed in this equipment and continue to make C3a complement fragment reduce about 10% at least compared with the control in 5 days.In another embodiment, be exposed in this equipment and continue to make C3a complement fragment reduce about 30% at least compared with the control in 5 days.In another embodiment, be exposed in this equipment and continue to make C3a complement fragment reduce about 50% at least compared with the control in 5 days.
In one embodiment, the invention provides a kind of equipment that contains hematoblastic biological composition compound concentrations that is used for reducing.Hematoblastic biological activity obtains keeping basically after with this device processes.The adsorbing medium of this embodiment comprises by the fixed absorbent particles of inert base.Preferred granule is a lot of holes and has greater than about 750m 2The surface area of/g.
The particularly preferred granule that is used for present embodiment is the polyaromatic adsorbent that contains super crosslinked polystyrene network, for example Dowex_XUS-43493 or Purolite MN-200.Preferred inert base comprises synthetic or natural polymerization fibres.This inert base comprises synthetic polymeric fibers in a preferred embodiment, and it comprises dystectic first polymer core that has that is had than the sheath encirclement of hanging down the fusion temperature.This polymer core can be the polyethylene terephthalate core.This sheath can be nylon sheath or modified poly ester sheath.Staple fibre can from Unitika (Osaka, Japan) and Hoechst Trevira commercially available.
By these equipment, material and the method reduction of present embodiment or the illustration chemical compound of controlling is psoralen, psoralen derivant, isopsoralen, psoralen photoproducts, acridine, acridine derivatives, methylene blue, plasticity extractable, biological respinse modifier, quencher and polyamine derivatives.
Typically will contain hematoblastic biological composition and be used for donations in 3 days, but can at room temperature preserve up to 7 days, therefore, it should be favourable that these platelet compositionss are kept contacting with adsorbing medium in whole storage.Preferably, this process should obtain acceptable platelet yield (for example, loss is lower than 10%).A kind of method that the present invention attempts makes storage prolong by the blood compatibility that improves adsorbent surface.
The adsorbing medium that use contains by the fixed absorbent particles of inert base allows to make under the situation that platelet count does not have to lose basically the concentration of low molecular weight compound to reduce.Phrase " basically not loss " is meant that being suitable for it wants purpose for example, the platelet product of suitable input human body, and can be for example in whole process, and more preferably at least 5 days platelet counts or loss function are lower than approximately 10%, preferably are lower than about 5%.And, there is not under the situation of loss the time that the time that platelet can contact with adsorbing medium can contact with absorbent particles separately greater than platelet basically at platelet count.Particulate fixing prolongation time of contact and the platelet loss number of making unexpectedly reduces.Do not have big loss for example to lose under about 20% the situation at platelet count, typically platelet can not with contact without fixed absorbent particles above about 20 hours.On the contrary, do not have basically at platelet count under the situation of loss, platelet can be contacted above 20 hours for example about 1-7 days with containing by the adsorbing medium of the fixed absorbent particles of inert base.
In addition, do not compare, exist the hematoblastic external platelet function of preserving under the situation of adsorbing medium (for example alteration of form, GMP-140, pH) to improve with there being the overtime storage platelet of adsorbing medium.Exist the platelet of preserving under the situation of adsorbing medium can have greater than about 6-less than about 7.5 pH.
In another embodiment, the invention provides and a kind ofly be used for reducing the concentration contain erythrocytic room temperature compositions low molecular weight compound (for example little organic compound) and kept the bioactive equipment of erythrocyte simultaneously basically.Typically, the chemical compound of removing by this equipment has the molecular weight of the about 30000g/mol of about 100g/mol-.This adsorbing medium contains by the fixed absorbent particles of inert base.The preferred particulates that is used for present embodiment has a lot of holes and has greater than about 750m 2The surface area of/g.
Preferably, the equipment that is used for the erythrocyte compositions is to have kept the bioactive equipment of erythrocyte basically after the concentration that reduces low molecular weight compound.In one embodiment, this erythrocyte equipment does not have negative effect because of contact to the biological activity of liquid basically.This equipment embodiment comprises and containing by fixed particulate adsorbing medium of inert base and granule retaining device optionally.
In one embodiment, the granule that is used for the equipment of erythrocyte compositions is an active carbon.Preferred this active carbon derives from synthetic source.The non-limitative example of active carbon comprises can be from PicaU.S.A. (Columbus, Ohio) the Picactif Medicinal of Huo Deing; Can be from NoritAmericas Inc. (Atlanta, GA) the Norit ROX 0.8 of Huo Deing; With can be from PicaU.S.A. (Columbus, OH) G-277 of Huo Deing.
In one embodiment, this adsorbent is preferably the active carbon that derives from synthetic source, as Ambersorb 572.These Ambersorb are Rohm ﹠amp; Haas (Philadelphia, PA) synthetic activation carbon containing (that is, the being rich in carbon) adsorbent of Sheng Chaning.Ambersorb is generally than the more competent big sphere of typical activity charcoal (300-900 μ m) granule.These Ambersorb are by handle synthetic production of expanded polystyrene pearl of height sulfonic acid esterification with the special-purpose high temperature activation method.These adsorbents do not need pre-the expansion to reach the optimal adsorption activity.
In another embodiment, the granule that is used for containing the equipment (" erythrocyte equipment ") of erythrocyte compositions can be a hydrophobic resin.The non-limitative example of hydrophobic resin comprises following polyaromatic adsorbent: Amberlite_ adsorbent (for example, Amberlite_XAD-2, XAD-4 and XAD-16), and can be from Rohm ﹠amp; (Philadelphia PA) obtains Haas; Can be from Toso Haas (TosoHass, Montgomeryville, PA) the Amberchrom_ adsorbent of Huo Deing; And Diaion_ //Sepabeads_ adsorbent (for example Diaion_HP20), can be from MitsubishiChemical America, (White Plains NY) obtains Inc..In a particularly preferred embodiment, these granules are can be from Purolite (Bala Cynwyd, PA) the Hypersol-Macronet_ absorbent resin of Huo Deing (Hypersol-Macronet_ absorbent resin MN-200 for example, MN-150 and MN-400) maybe can be from Dow Chemical Company (Midland, MI) the Dowex_ adsorbent of Huo Deing (for example XUS-43493 and XUS-40285).
Erythrocytic preferred inert base comprises synthetic or natural polymerization fibres.In a preferred embodiment, this inert base comprises synthetic polymeric fibers, and it comprises dystectic first polymer core that has that is had than the sheath encirclement of hanging down the fusion temperature.This polymer core can be polyethylene terephthalate or polyester core.This sheath can be nylon sheath or modified poly ester sheath.Fiber can from Unitika (Osaka, Japan) and Hoechst Trevira (Augsberg, Germany) commercially available.
In some embodiments, the adsorbing medium of erythrocyte equipment is in the sealing member.In one embodiment, this equipment comprises adsorbing medium and container.In another embodiment, this equipment that comprises adsorbing medium and container can comprise that also granule is detained medium.In one embodiment, this container comprises that volume is the blood bag of the about 1L of about 600ml-.In another embodiment, this container comprises that volume is the blood bag of the about 1L of about 800ml-.This granule keeps medium and can comprise the polyester system of knitting, polyester nonwoven or close film forming sealing member.
Preferred this equipment is in about 4 ℃ or about 22 ℃ (room temperatures) and under agitation contact 1-35 days with the erythrocyte compositions.In one embodiment, this erythrocyte compositions contacts with this equipment down at about 22 ℃ and is no more than about 36 hours.In another embodiment, this erythrocyte compositions contacts with this equipment down at about 22 ℃ and is no more than about 24 hours.In another embodiment, this erythrocyte compositions contacts with this equipment down at about 22 ℃ and is no more than about 12 hours.In another embodiment, this erythrocyte compositions contacts with this equipment down at about 22 ℃ and is no more than 6 hours.
In some embodiments, the erythrocyte compositions with change temperature after this equipment contacts.In one embodiment, this equipment contacted about 0.5-about 24 hours with this erythrocyte compositions down at about 22 ℃, was housed in then under about 4 ℃ up to about 5 weeks.In another embodiment, this equipment contacted about 0.5-about 12 hours with this erythrocyte compositions down at about 22 ℃, was housed in then under about 4 ℃ up to about 5 weeks.In another embodiment, this equipment contacted about 0.5-about 6 hours with this erythrocyte compositions down at about 22 ℃, was housed in then under about 4 ℃ up to about 5 weeks.
Use contains handles this erythrocyte compositions under the situation that is not had basically to lose in the erythrocyte function by the adsorbing medium of the fixed absorbent particles of inert base.Phrase " does not have loss " to be meant should suit to transfuse blood or it wants the product of purpose, and can be meant that in some cases haemolysis is lower than about 1% basically; It is about 0.8% that preferred haemolysis is lower than, and erythrocyte reclaims greater than about 80%; Preferred erythrocyte reclaims greater than 90%, and the variation of ATP concentration and no equipment contrast erythrocytic difference and is lower than approximately 10%, and the variation of extracellular potassium concentration and no equipment contrast erythrocytic difference less than about 15%.In 35 days, and compare without fixed granule, low at least by 10% for the hemolytic variation of IAD, preferably, low 20% more preferably 50%.Most preferably, and compare, for the hemolytic variation of IAD at least low 90% without fixed granule.
Can use the standard medicine box to estimate the erythrocyte function.Particularly, (MO) absorbance of the supernatant under 540nm measured haemolysis for (Sigma Chemical Company), St.Louis at Drabkin reagent can to pass through to measure erythrocyte.(Ciba-Corning Diagnostics, Medfield MA) can estimate potassium and spill to use the Na+/K+ analyser.(Sigma Diagnostics, St.Louis MO) can carry out the quantitative enzymatic determination of ATP in total erythrocyte sample and with the absorbance under the water background blank determination 340nm to use standard medicine box.
At pending biological composition is the place that contains erythrocytic compositions, and this equipment can reduce the concentration of low molecular weight compound in the erythrocyte sample.Preferred this equipment can reduce the concentration of acridine derivatives and mercaptan in the erythrocyte sample.More preferably this equipment can reduce 5-[(β-ethoxycarbonyl ethyl in the erythrocyte sample) amino]-concentration of acridine and glutathion.Can use the 5-[(β-carboxyethyl in HPLC test determination and the erythrocyte that this equipment contacts) amino] concentration of acridine and glutathion.Test mobile phase is the 10mM H in the HPLC water 3PO 4With the 10mMH in the acetonitrile 3PO 4Zorbax SB-CN and YMC ODSAM-303 post can be from MacMod Analytical, and (Chadds Ford, PA) and YMC, (Wilmingtion N.C.) obtains Inc. Inc..
The place that equipment contacts with celliferous biological composition under the stirring is being arranged, can should stir continuously or intermittently.Provide this stirrings by any suitable device that keeps the cell function degree, comprise the mechanical agitator of following type: reciprocal, track, three-dimensional rotation device and rotary apparatus type agitator.In one embodiment, provide this stirring by planetary agitator and be successive.In another embodiment, provide this stirring by planetary agitator and be intermittently.In another embodiment, provide this stirring by reciprocal agitator.
Use
The present invention attempts reducing the concentration of low molecular weight compound in the celliferous biological composition.These chemical compounds comprise, for example, and as psoralen inactivation reagent, aminacrine, organic dyestuff and the phenothiazine of photoactivation product.The psoralen inactivation reagent of illustration comprises the furan coumarin, for example psoralen and acridine.Then as described in US5459030 and 5559250 (being incorporated herein by reference), use psoralen deactivation compounds processing blood goods, can contact the concentration that reduces psoralen deactivation compounds in the blood products with equipment of the present invention by the blood products that will handle.
In one embodiment, the present invention attempts the method for psoralen in a kind of inactivation solution, and wherein this method comprises: ⅰ a) is provided successively) cycle compound, the ⅱ) suspension that pollutes with described psoralen, and ⅲ) Fibrized resin; B) handle described solution with described cycle compound, so that produce the solution product of the processing of wherein said psoralen inactivation; With c) solution product of described processing is contacted with described Fibrized resin, and comprise that further the concentration that is used to reduce the medium and small organic compound of blood products has kept the required bioactive equipment of blood products simultaneously basically, this equipment comprises very porous absorbent particles, and wherein these absorbent particles are fixed by inert base.
Except the psoralen deactivation compounds, for example before blood transfusion, can from the material of for example blood products, reduce its reaction catabolite.
Material and facility disclosed herein can be used for separation property blood transfusion method.Whole blood can be divided into two or more specific components (for example, erythrocyte, blood plasma and platelet).Term " separation property blood transfusion " broadly is meant and blood is taken out from donor and is divided into different component, collects and keeps interested component and other component is returned to the process of donor.This donor acceptance replacement liquid remedies because component is removed the volume and the pressure loss that causes with help in blood transfusion process again.The separation property blood transfusion system has description in the open WO96/40857 of PCT, this paper is incorporated herein by reference.
Low molecular weight compound
Equipment of the present invention reduces the concentration of low molecular weight compound in the celliferous compositions.Term " low molecular weight compound " is meant the organic or biomolecule of molecular weight at the about 30000g/mol of about 100g/mol-.Low molecular weight compound includes, but not limited to following chemical compound: little organic compound such as psoralen, acridine or dyestuff; Quencher as glutathion; Plasticity extractable as plasticizer; As the biological instrumentality of complement activation, it has the molecular weight of the about 30000g/mol of about 100g/mol-; And polyamine derivatives.
Little organic compound
Little organic compound on the same group can be not adsorbed by equipment of the present invention.These molecules can be ring-type or acyclic family.In one embodiment, these chemical compounds are preferably for example cyclic compound of psoralen, acridine or dyestuff.In another embodiment, these chemical compounds are mercaptan.
The non-limitative example of cyclic compound comprises D actinomycin D, anthracene nucleus ketone, mitomycin, antramycin and organic dyestuff and photoreaction chemical compound such as benzodipyrane ketone, fluorenes, Fluorenone, furan coumarin, porphyrin, protoporphyrin, alizarinopurpurin, phthalocyanine dye, hypericin, Monostral FastBlue, Norphillin A, phenanthridines, phenazathionium salt, azophenlyene, phenothiazine, triazobenzene, quinoline and thiaxanthone.Preferred these chemical compounds are the furan coumarin.
The non-limitative example of furan coumarin comprises psoralen and psoralen derivant.Pay special attention to be 4 '-aminomethyl-4,5 ', the psoralen that 8-trimethylpsoralen, 8-methoxypsoralen, halo psoralen, isopsoralen link to each other with quaternary ammonium, sugar or other nucleic acid conjugated group.Be also noted that following psoralen: 5 '-bromomethyl-4,4 ', the 8-trimethylpsoralen, 4 '-bromomethyl-4,5 ', the 8-trimethylpsoralen, 4 '-(4-amino-2-azepine) butyl-4,5 ', the 8-trimethylpsoralen, 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen, 4 '-(2-aminoethyl)-4,5 ', the 8-trimethylpsoralen, 4 '-(5-amino-2-oxa-) amyl group-4,5 ', the 8-trimethylpsoralen, 4 '-(5-amino-2-azepine) amyl group-4,5 ', the 8-trimethylpsoralen, 4 '-(6-amino-2-azepine) hexyl-4,5 ', the 8-trimethylpsoralen, 4 '-(7-amino-2, the 5-oxa-) heptyl-4,5 ', the 8-trimethylpsoralen, 4 '-(12-amino-8-azepine-2,5-two oxa-s) dodecyl-4,5 ', the 8-trimethylpsoralen, 4 '-(13-amino-2-azepine-6,11-two oxa-s) tridecyl-4,5 ', the 8-trimethylpsoralen, 4 '-(7-amino-2-azepine) heptyl-4,5 ', the 8-trimethylpsoralen, 4 '-(7-amino-2-azepine-5-oxa-) heptyl-4,5 ', the 8-trimethylpsoralen, 4 '-(9-amino-2, the 6-diaza) nonyl-4,5 ', the 8-trimethylpsoralen, 4 '-(8-amino-5-azepine-2-oxa-) octyl group-4,5 ', the 8-trimethylpsoralen, 4 '-(9-amino-5-azepine-2-oxa-) nonyl-4,5 ', the 8-trimethylpsoralen, 4 '-(14-amino-2,6,11-three azepines) myristyl-4,5 ', the 8-trimethylpsoralen, 5 '-(4-amino-2-azepine) butyl-4,4 ', the 8-trimethylpsoralen, 5 '-(6-amino-2-azepine) hexyl-4,4 ', 8-trimethylpsoralen and 5 '-(4-amino-2-oxa-) butyl-4,4 ', the 8-trimethylpsoralen.Preferred this psoralen is 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen.
Acridine
The non-limitative example of acridine comprises acridine orange, acriflavinium chloride, quinacrine, N1, N1-two (2-ethoxy)-N4-(6-chloro-2-methoxyl group-9-acridinyl)-1,4-pentanediamine, 9-(3-hydroxypropyl) aminacrine, N-(9-acridinyl) glycine, S-(9-acridinyl)-glutathion.This acridine is N-(9-acridinyl)-Beta-alanine in a preferred implementation, perhaps is called 5-[(β-carboxyethyl) amino] acridine.
Dyestuff
The non-limitative example of dyestuff comprises for example phenothiazine of methylene blue, dimethyl diaminophenazine chloride, toluidine blue, crystal violet and azure A; Phenthiazone as methylene-violet Bernthsen; As 1,8,15,22-four phenoxy groups-29H, the phthalocyanine dye of 31H-phthalocyanine aluminum chloride and Silicon stone analog; And hypericin.Preferred this dyestuff is methylene blue or toluidine blue.More preferably this dyestuff is a methylene blue.
Term " thiazine dye " is included in the dyestuff that contains sulphur atom in one or more rings.Modal thiazine dye is methylene blue [chlorination 3,7-two (dimethylamino)-phenothiazine-5-].Other thiazine dye includes, but not limited to azure A, aC and Lauth's violet, as described in the US5571666 of Schinazi.
Term " xanthene dye " is meant the dyestuff into chemical compound xanthene derivant.These xanthene dye branches can be gone into one of three main classification: ⅰ) fluorenes or amino xanthene, ⅱ) paramethylaminophenol or hydroxy amino xanthene and ⅲ) Fluorenone hydroxyl xanthene.The example that trial is used for xanthene dye of the present invention comprises rose-red and eosin W or W S; As described in the US5571666 of Schinazi, these dyestuffs can be from many sources (for example, Sigma Chemical Co_St.Louis, MI) commercially available, this paper is incorporated herein by reference.
Quencher
Can reduce the concentration of all cpds.Other illustration chemical compound comprises quenching compound.Method for quenching does not wish to comprise the side reaction of the psoralen deactivation compounds that contains the functional group that maybe can form, electrophilic group, total U.S. Patent application " Methods for Quenching Pathogen Inactivatorsin Biological Systems " as the summary of on January 6th, 1998 application numbers 28217300600 is incorporated herein its disclosed content.In the method, handle as the material of blood products with psoralen deactivation compounds and quencher, wherein this quencher comprises the nucleophilic functional group of can be covalently reacting with electrophilic group.In one embodiment, this psoralen deactivation compounds comprises linking ligand and for example nucleic acid of the functional group of Semen Sinapis group, and it can form electrophilic group by situ reaction.The example of quencher includes, but not limited to contain the chemical compound of nucleophilic group.The nucleophilic group of illustration comprises mercaptan, thionic acid, two sulfur carbonic acid, thiocarbamate, dithiocarbamate, amine, phosphate ester and D2EHDTPA ester group.This quencher can be or contain, for example the azacyclo-of pyridine.This quencher can be to contain for example phosphate ester of the chemical compound of G-6-P ester.This quencher also can be the mercaptan that contains following chemical compound (but being not limited to): glutathion, cysteine, N-acetylcystein, mercaptoethanol, dimercaptopropanol, BAL, mercaptan, sulfhydryl ethylsulfonic acid and its salt, for example, MESNA, homocysteine, aminoethyl mercaptan, dimethylaminoethyl mercaptan, dithiothreitol, DTT and other contain the chemical compound of mercaptan.The aromatic mercaptans chemical compound of illustration comprises 2-mercaptobenzimidazole sulfonic acid, 2-sulfydryl nicotinic acid, naphthalene alkene mercaptan, quinoline mercaptan, 4-nitro-phenylmercaptan. and phenylmercaptan..Other quencher comprises nitrobenzylpyridine and inorganic nucleopilic reagent such as selenides salt or organic selenides, thiosulfate, sulphite, sulfide, thiophosphate, pyrophosphate, sulfhydrate and dithio nitrite.This quencher can be the peptide compounds that contains nucleophilic group.For example, this quencher can be the chemical compound that contains cysteine, for example, and as the dipeptides of GlyCys or as the tripeptides of glutathion.
Can comprise mercaptan such as TGA methyl ester, thiolactic acid, phenylmercaptan., 2-mercaptopyridine, 3-sulfydryl-2-butanols, 2-mercaptobenzothiazole, thiosalicylic acid and thioctic acid by the chemical compound that equipment of the present invention is removed.
The plasticity extractable
Concentration from one group of low molecular weight compound of the extractable of plastics storage container that is used for adorning biological composition and pipe also can use equipment of the present invention to reduce from biological composition.The example of extractable includes, but not limited to plasticizer, residual monomer, low-molecular-weight oligomer, antioxidant and lubricant.Referring to for example R.Carmen, TransfusionMedicine Reviews 7 (1): 1-10 (1993).The sterilization of plastic component by steam, gamma-rays or electron beam can produce oxidation reaction and/or polymer splits, and causes forming the extractable of other type.
Plasticizer often is used to improve performance such as the machinability and the breathability of plastics.The most common plasticizer of finding in the blood storage container is phthalic acid two (the 2-ethyl the is own) ester that is used for the PVC goods.DEHP has been confirmed as potential carcinogen.Develop other plasticizer, included, but not limited to following chemical compound: 1,2,4-benzenetricarboxylic acid three (the 2-ethyl is own) ester (TEHTM), the just own ester of acetyl tributyl citrate three (ATHC), butyryl citric acid tri-n-hexyl ester (BTHC) and phthalate ester decanoate.
Equipment of the present invention can be used for reducing or control the concentration of the plasticity extractable in the biological composition of various environment.These environment include, but not limited to following: blood treatment; The blood storage; And for example external application of hemodialysis and external film oxidation.
Biological respinse modifier (BRMs)
One group of concentration that broadly is referred to as the low molecular weight compound of biological respinse modifier (BRMs) also can be used equipment of the present invention to reduce from biological composition and control.BRMs is meant " changing immunoreactive spectroscopic molecular ".Illustrated?Dictionary?of?Immunology，J.M.Cruse?and?R.E.Lewis。General BRMs group includes, but are not limited to: the micromolecule of histamine and serotonin for example; For example thromboxan, prostaglandin, leukotriene and arachidonic lipid; The little peptide of Kallidin I for example; The bigger polypeptide that contains other group, comprise the complement activation fragment (C3a, C5a); The cytokine of IL-1, IL-6 and IL-8 for example; And the chemotactic factor of RANTES and MIP for example.
May there be side effect gathering of BRMs to the required biological activity of biological composition in the blood products in storage.For example confirmed under standard blood bank condition, in the hematoblastic process of storage complement activation to take place.Complement activation be accompanied by and be referred to as " platelet storage infringement " platelet function and vigor forfeiture.Referring to for example V.D.Mietic and O.Popovic, Transfusion 32 (2): 150-154 (1993).May there be side effect gathering also of BRMs to the patient who for example accepts this blood products in the blood products of being preserved: being collected at of BRMs accepts to be attended by among the hematoblastic patient non-hemolytic heating transfusion reaction in platelet concentrate in storage.Referring to for example N.M.Heddle, Current Opinions inHematology 2 (6): 478-483 (1995).
Polyamine derivatives
One group of concentration that is known as the low molecular weight compound of polyamine derivatives for example also can use equipment of the present invention to reduce from biological composition.Polyamine derivatives is for containing the chemical compound of a plurality of nitrogen-atoms in carbon backbone chain.
Polyethylene Glycol
Other illustration chemical compound comprises activated polyethylene glycol (aPEG), and the surface that can use it for change cell or material is so that provide immune masking performance respectively or the stabilize proteins combination.This equipment can be used to reduce the unreacted derivant of excessive activated polyethylene glycol or PEG, thereby make activated PEG and water or for example phosphate, phosphate ester or as the little nucleopilic reagent reaction of the mercaptan of glutathion.Other chemical compound that can be removed comprises the impurity in the activated PEG goods, and it can influence the function of blood products or make them be not suitable for blood transfusion (for example toxic compounds).At last, also the micromolecule of the N-hydroxy-succinamide that discharges in for example aPEG and the cell surface nucleopilic reagent course of reaction can be reduced.
The example of the chemical compound that can remove by equipment of the present invention comprises linearity or the branched chair polymacrogol that sticks on the bioactive molecule, and these bioactive molecules comprise cyanuric chloride, succinimido ester, oxidation phosphinylidyne imdazole derivatives, carbonic acid Nitrobenzol ester derivant, glycidyl ether derivatives and aldehyde.
Be interpreted as the present invention be not confined to shown in and in described these accurate detail operations or precision compound, compositions, method or the step, various changes and equivalent should be conspicuous to those skilled in the art.As shown in the above, should point out clearly that these method and apparatus can and be used to process blood transfusion with the separation property blood transfusion system at present and mix with the miscellaneous equipment and the step of blood products.
Embodiment
The following examples are used to illustrate some of the preferred embodiment of the invention and feature, but do not constitute the restriction to its scope.
In following description of test, use following abbreviation: eq (equivalent); M (M); μ M (volume micromolar); N (equivalent); Mol (mole); Mmol (mM); μ mol (micromole); Nmol (nanomole); G (gram); Mg (milligram); μ g (microgram); Kg (kilogram); L (liter); ML (milliliter); μ L (microlitre); Cm (centimetre); Mm (millimeter); μ m (micron); Nm (nanometer); Min. (minute); S and sec. (second); J (joule, or watt. second); ℃ (degree centigrade); TLC (thin layer chromatography); HPLC (high pressure liquid chromatography); PHEMA and p (HEMA) (poly-[methacrylic acid 2-hydroxyl ethyl ester]); PC (s) (platelet concentrate); PT (prothrombin time); APTT (activated partial prothrombin time); TT (thrombin time); HSR (hypotonic shock response); FDA (U.S. food and drug administration); GMP (good production practices); DMF (EDMF); SPE (solid phase extractions); Aldrich (Milwaukee, WI); Asahi (Asahi Medical Co_Ltd_Tokyo, Japan); Baker (J.T.Baker, Inc_Phillipsburg, NJ); Barnstead (Barnstead/Thermolyne Corp_Dubuque, IA); BectonDickinson (Becton Dickinson Microbiology Systems; Cockeysville, MD); Bio-Rad (Bio-Rad Laboratories, Hercules, CA); Cerus (CerusCorporation; Concord, CA); Chrono-log (Chrono-Log Corp.; Havertown, PA); Ciba-Corning (Ciba-Corning Diagnostics Corp.; Oberlin, OH); Consolidated Plastics (Consolidated Plastics Co_Twinsburg, OH); Dow (Dow Chemical Co.; Midland, MI); Eppendorf (Eppendorf North America Inc_Madison, WI); Gelman (GelmanSciences, Ann Arbor, MI); Grace Davison (W.R.Grace ﹠amp; Co_Baltimore, MD); Helmer (Helmer Labs, Noblesville, IN); HoechstCelanese (Hoechst Celanese Corp_Charlotte, NC); InternationalProcesslng Corp. (Winchester, KY); Millipore (Milford, MA); NIS (Nicolet, a Thermo Spectra Co_San Diego, CA); Poretics (Livermore, CA); Purolite (Bala Cynwyd, PA); Rohm and Haas (Chauny, France); Quidel (San Diego, CA); Saati (Stamford, CT); Scientific Polymer Products (Ontario, NY); Sigma (Sigma ChemicalCompany, St.Louis, MO); Spectrum (Spectrum Chemical Mfg.Corp_Gardenia, CA); Sterigenics (Corona, CA); Tetko, and Inc. (Depew, NY); TosoHaas (TosoHass, Montgomeryville, PA); Wallac (Wallac Inc_Gaithersburg, MD); West Vaco (Luke, W.Va); YMC (YMC Inc_Wilmington, NC); DVB (divinylbenzene); LAL (Limulus AmoebocyteLystate); USP (American Pharmacopeia); EAA (ethyl acetoacetate); EtOH (ethanol); HOAc (acetic acid); W (watt); MW (milliwatt); NMR (nuclear magnetic resonance, NMR; The spectrum that on Varian Gemini200 MHz Fourier Transform Spectrometer, obtains at room temperature); Ft 3/ min (per minute cubic inch); M.p. (fusing point); G/min and gpm (gallons per minute); UV (ultraviolet); THF (oxolane); DMEM (the Eagles culture medium of Dulbecco ' s improvement); FBS (hyclone); LB (Luria Broth); EDTA (ethylenediaminetetraacetic acid); Phorbol MyristateAcetate (PMA); Phosphate buffered saline (PBS); AAMI (the pharmacy instrument improves association); ISO (International Standards Organization); EU (endotoxin unit); LVI (injected material in a large number); GC (gas chromatogram); M (million); KGy (1000 Grays=0.1 Megarad); M Ω (megohm); PAS III (platelet make-up solution III); DH 2O (distilled water); IAD (fixedly adsorption plant); SCD (aseptic connection device).
One of following examples are mentioned the HEPES buffer.This buffer contains the 1mM MgCl of 2.7mM KCl, 0.203g of 137mMNaCl, the 0.2g of 8.0g 2(6H 2O), the 20mM HEPES (can be from Sigma, St.Louis, MO acquisition) of the 1mg/ml bovine serum albumin of the 5.6mM glucose of 1.0g, 1.0g (can be from Sigma, St.Louis, MO acquisition) and 4.8g.
Embodiment 1
Fibrized resin with Amberlite_XAD-16
This embodiment utilizes Fibrized resin and contains kinetics and platelet function and the form of relatively removing the psoralen that deaminizes without the equipment of fixed adsorbent pearl from platelet.More particularly, the Fibrized resin that will contain immobilization Amberlite_XAD-16 is compared with the equipment that contains freedom (promptly without fixed) Amberlite_XAD-16 HP and Dowex_XUS-43493.
Preparation Fibrized resin and adsorbent pearl
Obtain the immobilization adsorbent medium that contains Amberlite_XAD-16 (Rohm and Haas) of clean and hydration status by AQF.The fiber of these Hoechst Celanese networks of fibers is made up of polyethylene terephthalate core and nylon sheath, and the melting temperature of this sheath is lower than this core.Being prepared as follows of this Fibrized resin: at first these adsorbent pearls are evenly distributed in the network of fibers.Next, this network of fibers Fast Heating made the polymer sheath fusion of fiber and adhere to the adsorbent pearl and other fiber on, thereby form crosslinked network of fibers.It is the 130g/m2 Amberlite_XAD-16 of (that is, every square metre of fiber contains 130g adsorbent pearl) that formed Fibrized resin contains load.
This Fibrized resin is cut into square, and (14cm * 14cm), the gained fragment contains the dried Amberlite_XAD-16 of about 2.5g.Then by this Fibrized resin being immersed in 30% ethanol about 10 minutes with the pre-moistening of this Amberlite_XAD-16 pearl.Other method of moistening Amberlite_XAD-16 and other adsorbent also are effectively and by the present invention to comprise.It should be noted that the Fibrized resin (for example, through bridge joint or super crosslinked resin such as Dowex_XUS-43493) that contains other type pearl does not need to be used for effectively removing the moistening step of psoralen.
Also can directly obtain Amberlite_XAD-16 HP (high-purity) pearl of clean and hydration status from Rohm and Haas.Before adding mesh bag, loose (promptly without fixed) Amberlite_XAD-16 HP pearl is not needed pre-profit temperature; But, the moisture (2.5g do agent=6.8g have 62.8% moisture) of amount to calculate pearl of proofreading and correct adsorbent.Obtain the Dowex_XUS-43493 pearl from Dow, this drying pearl does not need moistening and does not need water to proofread and correct the quality of this pearl.Then with polyester mesh bag (7cm * 7cm square; 30 μ m openings) fill with loose Amberlite_XAD-16 HP or the Dowex_XUS-43493 pearl of 2.5g (dry weight).
The bag of fibre-bearing resin and adsorbent " wets " to circulate under 121 ℃ by autoclaving and sterilized in 45 minutes.Afterwards, the bag with fibre-bearing resin and adsorbent inserts independent sterilization, 1-rises in PL 2410 plastic containers (Baxter).After the insertion, in the laminar flow fume hood, use sterile scissors, hemostat and impulse sealing device that PL 2410 plastic containers are sealed.
Fibrized resin contacts with the platelet concentrate that contains psoralen (PC) with the adsorbent pearl
2-3 unitary single donor Platelets Pheresis/Apheresis platelets is blended in 35% autologous plasma/65% platelet make-up solution (being synthetic medium) makes the platelet concentrate storehouse.The amino psoralen 4 of interpolation in this solution '-(4-amino-2-oxa-) butyl-4,5 ', 8-trimethylpsoralen (S-59), its amount realization 150 μ M4 '-(4-amino-2-oxa-) butyl-4,5 ', the ultimate density of 8-trimethylpsoralen.Gained PC solution is divided into the 300mL unit, then these unit is placed in PL 2410 plastic containers (Baxter) and uses 3J/cm 2UVA irradiation.After the irradiation, treated PCs is transferred in immobilization PL 2410 plastic containers that contain the Fibrized resin of being with Amberlite_XAD-16, loose Amberlite_XAD-16 HP or loose Dowex_XUS-43493, perhaps transfer in sky PL 2410 plastic containers and compare.Then these PL 2410 plastic containers (Baxter) are placed in the Helmer platelet calorstat under 22 ℃ and with about 70 rev/mins of stirrings.
In 8 hours storage at first, shifted out each PC sample every 1 hour, analyze by HPLC remaining 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen.With the diluents that contains trimethylpsoralen (TMP) (ultimate density=35% methanol, 25mMKH 2PO 4, pH=3.5) as internal standard with 5 times of every kind of diluted samples.By under 4 ℃ with these sample culturing 30 minutes, protein and other macromole precipitation.Then with these sample centrifugalize and with supernatant filtration (0.2 micron) and at C-18 reversed-phase column (YMC ODS-AM 4.6mm * 250mm) go up by in 20 minutes, flowing out from 65% solvent orange 2 A (25mM KH 2PO 4, pH=3.5), 35%B (methanol) analyzes to the linear gradient of 80%B.
By at Baker System 9118 CP (Baker Instrument Co.; Allentown, PA) monitoring platelet Counting every day is with the platelet yield of the 5-days duration of storage of Fibrized resin or a kind of loose pearl.Use Ciba-Corning 238 pH/Blood Gas Analyzer to estimate flood gas and pH.The equipment that use is used for test evaluation that form, change of shape, hypotonic shock response, gathering and GMP-140 (p-selects albumen) express and Fibrized resin or contains free adsorbent contacts the external platelet function after 5 days.Change of shape, gathering and hypotonic shock response are to use Lumi-Aggregometer (Chrono-Log) to estimate, and GMP-140 is to use Becton-Dickinson FACScan Fluorescence Analyzer (BectonDickinson) to measure by flow cytometry simultaneously.
Removing and platelet yield and function of psoralen
Fig. 5 compared with XUS-43493, XAD-16 HP and the Fibrized resin that contains XAD-16 from the platelet of 35% blood plasma/65% synthetic medium (PAS III), remove 4 '-(4-amino-2-oxa-) butyl-4,5 ', the adsorption dynamics adsorption kinetics of 8-trimethylpsoralen.Particularly, data represented the containing shown in the circle of solid line connection without fixed adsorbent XUS-43493 (2.5g pearl;＜5% moisture) equipment; Data represented shown in the triangle that dotted line connects contains (the 6.8g pearl without fixed XAD-16HP; 62.8% moisture) equipment; And (the Hoechst fiber that has the wet pearl of XAD-16 in 30% ethanol of the data represented Fibrized resin shown in the square that dotted line connects; 14cm * 14cm).Shown in data among Fig. 5, contain without 4 of the equipment of the equipment of fixed adsorbent and fibre-bearing resin '-(4-amino-2-oxa-) butyl-4,5 ', the adsorption dynamics adsorption kinetics of 8-trimethylpsoralen all is comparable.Therefore, fibrosis is handled does not have significantly big influence to removing kinetics.
In addition, the platelet yield and the function of the Fibrized resin of comparing have been studied with loose pearl.Specifically, ⅰ has been used in the test of this research) 6.8g XAD-16 HP (62.8% moisture); ⅱ) fibrosis XAD-16 (130g resin/the cm of the 14cm in 30% ethanol * 14cm temperature 2); And ⅲ) 2.5g XUS-43493 (＜5% moisture).XAD-16 HP and Fibrized resin sample preparation double platelet unit, but XUS-43493 only makes single part of platelet unit.The results are shown in Table 1.
As shown in table 1, compare with contrast in 5 days, contain 5 days pH and pO without the sample of fixed pearl (XAD-16 HP and XUS-43493) 2Value rises a little.Test with Fibrized resin has more comparable pH of relative comparison and pO 2Platelet count show with fibrotic mediators and contain contrast without the equipment of fixed adsorbent contact 5 days afterwards platelet lose 9-22%.Listed as table 1, Fibrized resin has better yield (5 days loss 9%), and with contain that all in-vitro evaluations present better when comparing without the equipment of fixed XAD-16 or XUS-43493 absorbent particles.
Table 1
Figure 9881384000421
Although in order to realize that the present invention does not need to understand observed pH of equipment and the pO with containing without fixed XUS-43493 and XAD-16 HP 2Higher mechanism, but high value it is believed that it is to reduce institute a little extremely by containing without platelet economy under the fixed adsorbent existence.By comparison, relative comparison, fibrotic mediators as one man has more comparable 5 days pH and pO than XUS-43493 or XAD-16 pearl 2Value.
With respect to XUS-43493 or XAD-16 HP adsorbent pearl, fibrotic mediators also has 5 days preferably platelet yields.The loss of observing the observation of XUS-43493 medium under several situations is 22-28%.But, it should be noted that with containing present preferred implementation and shift these platelet from this equipment after relating to 8 hours and exposing without the equipment of fixed adsorbent with XUS-43493; This process causes＜5% platelet loss.
Data declaration shown in the table 1 equipment that contains relatively without fixed adsorbent of fibrotic mediators have higher platelet yield.It is shocking that this fibrotic mediators also obtains to pass through pH/pO 2, the better 5 days platelet function shown in change of shape, gathering, form and the GMP-140.Although in order to realize that the present invention does not need to understand the principle of strengthening the property of this fibrotic mediators, can propose several hypothesis.At first, stick to these fibers on the adsorbent bead surface and can stop interaction between platelet and the bead surface.Secondly, fixing can the preventing of these pearls interacted and eliminate the deleterious mechanical influence of platelet.The 3rd, with these pearls fixing can enhance fluid in the shearing of bead surface, therefore reduced the interaction between platelet and the bead surface; By comparison, make the mobile reduction on surface of the relative pearl of fluid without fixed pearl and fluid free-flow.
The platelet loss
When the above-mentioned data of check, find out that there are some transmutabilities in once research and the loss of the platelet between the research next time.But the platelet loss of representing with the percentage rate of 5 days contrast countings in the higher research of initial platelet count is less.Study is in order to confirm whether the quantity that platelet is lost is constant to the available material area of given thrombocyte adhesiveness.About this research, two platelet unit are merged and should merge thing be divided into two samples.Sample is with one times of 35% autologous plasma/65% synthetic medium (PAS) dilution, so as this platelet count to be another unitary half.With these platelet mixture with 4 '-(4-amino-2-oxa-) butyl-4,5 ', the standard platelet holding conditions that 8-trimethylpsoralen+UVA handles and discusses in front contacts 5 days with the equipment that contains without fixed adsorbent (2.5g XUS-43493) down.
Although press the widely different of loss that percentage rate calculates, the total number of blood platelet that loses in two unit in fact is identical.Therefore, these presentation of results the loss of total over time platelet be constant basically; That is, although the platelet percentage of damage changes with initial platelet count, the sum of platelet loss almost is constant when reaching balance.Based on the result who proposes among this embodiment, Fibrized resin is free from side effects to external platelet function.
Embodiment 2
Fibrized resin with active carbon
This embodiment be to the Fibrized resin that contains Amberlite_XAD-16 and contain through the Fibrized resin of fixed active carbon relatively from platelet, remove 4 '-(4-amino-2-oxa-) butyl-4,5 ', the kinetics of 8-trimethylpsoralen and platelet function and form.
The preparation Fibrized resin
Hoechst Celanese preparation contains the Fibrized resin of Amberlite_XAD-16 HP (Rohm and Haas).This Fibrized resin that contains Amberlite_XAD-16 prepares as described in the previous embodiment, comprises 30% ethanol moistening step.Hoechst Celanese has also prepared with load 375g/m 2(AQF-375-B) and 500g/m 2(AQF-500-B) contain the Fibrized resin that passes through fixed active carbon (WestVaco).# in a preferred embodiment, this absorbent particles is the synthesizing activity charcoal, for example comprises Ambersorb and A-Supra.Because the synthesizing activity charcoal can be removed the particulate matter from coming off through fixed adsorbing medium in a large number, so they are preferred.This Fibrized resin is with the Fibrized resin similar methods preparation that is used to contain Amberlite_XAD-16.Every kind of Fibrized resin fibrous identical.
This Fibrized resin is cut into square, and (14cm * 14cm), the gained fragment contains the dried Amberlite_XAD-16 of about 2.5g.Next, with this Fibrized resin by autoclaving under 121 ℃, " wet " circulation 45 minutes the sterilization.Afterwards, the independent sterilization of this Fibrized resin insertion, 1-are risen in PL 2410 plastic containers (Baxter), and in the laminar flow fume hood, use sterile scissors, hemostat and impulse sealing device this container heat-sealing.
Fibrized resin contacts with the PC that contains psoralen
2-3 unitary single donor Platelets Pheresis/Apheresis platelets is blended in 35% autologous plasma/65% synthetic medium (PAS III) makes the platelet concentrate storehouse.Interpolation 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen, its amount realization 150 μ M 4 '-(4-amino-2-oxa-) butyl-4,5 ', the ultimate density of 8-trimethylpsoralen.Gained PC solution is divided into the 300mL unit, and is placed on these unit in PL 2410 plastic containers (Baxter) and uses 3J/cm 2UVA irradiation.After the irradiation, treated PCs is transferred in PL 2410 plastic containers of the Fibrized resin that contains band XAD-16, AQF-375-B, AQF-500-B, perhaps transfer in sky PL 2410 plastic containers and compare.Then these PL 2410 plastic containers are placed in the Helmer platelet calorstat under 22 ℃ and with about 70 rev/mins of stirrings.
According to the carrying out among the embodiment of front, in 8 hours storage at first, shifted out each PC sample every 1 hour, analyze by HPLC remaining 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen.With the diluents that contains trimethylpsoralen (TMP) (ultimate density=35% methanol, 25mM KH 2PO 4, pH=3.5) as internal standard with 5 times of every kind of PC diluted samples.By under 4 ℃ with these sample culturing 30 minutes, protein and other macromole precipitation.Then with these sample centrifugalize and with supernatant filtration (0.2 micron) and at C-18 reversed-phase column (YMC ODS-AM 4.6mm * 250mm) go up by in 20 minutes, flowing out from 65% solvent orange 2 A (25mM KH 2PO 4, pH=3.5), 35%B (methanol) analyzes to the linear gradient of 80%B.
By monitoring platelet Counting every day on Baker System 9118 CP with Fibrized resin or contain platelet yield without 5-days duration of storage of the equipment of fixed adsorbent.Use Ciba-Corning 238 pH/Blood Gas Analyzer to estimate flood gas and pH.Use is used for test evaluation that form, change of shape, hypotonic shock response, gathering and GMP-140 (p-selects albumen) express and Fibrized resin or contains equipment without fixed adsorbent contacting external platelet function after 5 days.Change of shape, gathering and hypotonic shock response are to use Chrono-Log Lumi-Aggregometer to estimate, and GMP-140 is to use Becton-Dickinson FACScan Fluorescence Analyzer to measure by flow cytometry simultaneously.
Removing and platelet yield and function of psoralen
Fig. 6 compared with the Fibrized resin that contains XAD-16 and Fibrized resin with two kinds of different activities charcoal load from the platelet of 35% blood plasma/65% synthetic medium (PAS III), remove 4 '-(4-amino-2-oxa-) butyl-4,5 ', the adsorption dynamics adsorption kinetics of 8-trimethylpsoralen.Particularly, 4 of the data represented use fibrosis XAD-16 pearl shown in the circle '-(4-amino-2-oxa-) butyl-4,5 ', the removing of 8-trimethylpsoralen; Data represented use fibrosis AQF-500-B shown in the square removes; And the data represented use fibrosis AQF-375-B shown in the triangle removes.Shown in data among Fig. 6,4 of different Fibrized resins '-(4-amino-2-oxa-) butyl-4,5 ', the adsorption dynamics adsorption kinetics of 8-trimethylpsoralen all is comparable, and this Fibrized resin all demonstrate extraordinary remove kinetics (after 4 hours≤remnants 4 '-(4-amino-2-oxa-) butyl-4 of 0.5 μ M, 5 ', the 8-trimethylpsoralen).
Also having estimated the platelet yield and the external platelet count certificate of every kind of Fibrized resin lists in the table 2.
Table 2
Figure 9881384000461
With reference to table 2, charcoal base Fibrized resin provides the good platelet yield that loses less than 10%; As the research of front embodiment, the Fibrized resin that contains XAD-16 has high slightly platelet loss (about 17%).As for pO 2, 5 days values of charcoal Fibrized resin can compare with contrast.Although in order to realize the present invention does not need to understand why the charcoal Fibrized resin has high slightly pH value, it may be from activation process by remaining extractable (for example phosphate) people for causing; The PC storage that has a charcoal base Fibrized resin is observed pH (pH=7.3-7.4) fast rise after 8 hours and has been supported this viewpoint.The USP charcoal that use is attended by considerably less extractable can be eliminated observed pH and raise at first.
The carbon based fibers resin all provides good result in change of shape and gathering test.Although the AQF-500-B Fibrized resin change of shape extremely be better than contrast, the platelet that accompanies with AQF-500-B presents poor slightly gathering test.
Embodiment 3
The pHEMA bag is by the influence to the compatibility of adsorbent blood
This embodiment be to relatively from platelet, remove 4 with the Dowex_XUS-43493 of pHEMA bag quilt and the Fibrized resin that contains Amberlite_XAD-16 '-(4-amino-2-oxa-) butyl-4,5 ', the kinetics of 8-trimethylpsoralen and platelet function and form.
The adsorbent pearl and the Fibrized resin of preparation pHEMA bag quilt
The Dowex_XUS-43493 (the commercial Optipore_L493 of being referred to as) that contains about 50wt% water obtains from Dow, and the polymerization HEMA with viscosity-average molecular weight of 300kD obtains from Scientific Polymer Products.Before the bag quilt, these adsorbent pearls are dried to moisture＜5%.By with this polymer dissolution in 95% denatured alcohol/5% water so that pHEMA concentration reaches 50mg/ml, thereby the preparation pHEMA stock solution.
Is that the 9-inch Wurster fluid bed bag of about 4kg (doing) adsorbent carries out this bag by process in by device by International Processing Corp. having load.This bag is related to the pHEMA flow velocity of 60-70g/min, 50 ℃ inlet temperature and about 200ft by process 3The air velocity of/min.The bag that shifts out 50g in wrapping by process is adsorbed the agent sample, so that the bag of acquisition 3-18% (w/w) pHEMA is by level; Will with 3.7%, 7.3% and the adsorbent pearl of 10.9%pHEMA (w/w) bag quilt be used for following research.
Contain without fixed do (through bag quilt) Dowex_XUS-43493 (2.5g) and be by the adsorbent of required quality being put into square 30 μ m polyester mesh bag (7cm * 7cm) make with the equipment of the Dowex_XUS-43493 (3.0g or 5.0g) of pHEMA bag quilt.The 1-that the bag of filling adsorbent is inserted sterilization separately rises in PL 2410 plastic containers (Baxter), and seals with the impulse sealing device.Afterwards, the PL-2410 plastic containers that contain the bag of filling adsorbent are sterilized to 2.5MRad by electron beam or gamma-rays (SteriGenics); As mentioned above, preferred electron bundle sterilization usually.
According to the method described in the embodiment 1, Hoechst Celanese preparation contains the Fibrized resin of Amberlite_XAD-16.This Fibrized resin is cut into square, and (14cm * 14cm), the gained fragment contains the dried Amberlite_XAD-16 of about 2.5g.The Amberlite_XAD-16 of this Fibrized resin is wrapped quilt by moistening gentleness in the solution that is immersed in 95% ethanol/5% distilled water that contains 50mg/mL pHEMA simultaneously.Flushing is removed residual ethanol twice in normal saline in 10 minutes.This process obtains the bag quilt of about 6% (w/w) pHEMA.Then this Fibrized resin " is wet " to circulate under 121 ℃ by autoclaving and sterilized in 45 minutes.Afterwards, the independent sterilization of this Fibrized resin insertion, 1-are risen in PL 2410 plastic containers (Baxter), and in the laminar flow fume hood, use sterile scissors, hemostat and the heat-sealing of impulse sealing device.
The adsorbent pearl of pHEMA bag quilt contacts with the PC that contains psoralen with Fibrized resin
Single donor Platelets Pheresis/Apheresis platelets unit is blended in 35% autologous plasma/65% synthetic medium (PAS III) makes the platelet concentrate storehouse.Interpolation 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen, its amount realization 150 μ M 4 '-(4-amino-2-oxa-) butyl-4,5 ', the ultimate density of 8-trimethylpsoralen.Then gained PC solution is divided into the 300mL unit, and is placed on these unit in PL 2410 plastic containers (Baxter) and uses 3J/cm 2UVA irradiation.After the irradiation, treated PCs is transferred in the PL2410 plastic containers that contain the equipment shown in the following gained fragment.Preparation does not have the control sample of adsorption plant yet.Then these PL2410 plastic containers are placed in the Helmer platelet calorstat under 22 ℃ and with about 70 rev/mins of stirrings.
In 8 hours storage at first, shifted out each PC sample every 1 hour, analyze by HPLC remaining 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen.With the diluents that contains trimethylpsoralen (TMP) (ultimate density=35% methanol, 25mMKH 2PO 4, pH=3.5) as internal standard with 5 times of every kind of PC diluted samples.By under 4 ℃ with these sample culturing 30 minutes, protein and other macromole precipitation.Then with these sample centrifugalize and with supernatant filtration (0.2 micron) and at C-18 reversed-phase column (YMC ODS-AM4.6mm * 250mm) go up by in 20 minutes, flowing out from 65% solvent orange 2 A (25mMKH 2PO 4, pH=3.5), 35%B (methanol) analyzes to the linear gradient of 80%B.
Measure with Fibrized resin or contain by platelet Counting on Baker System 9118 CP without the platelet yield after the 5-days duration of storage of the equipment of fixed adsorbent.Use Ciba-Corning 238 pH/Blood Gas Analyzer to estimate flood gas and pH.Use is used for test evaluation that form, change of shape, hypotonic shock response, gathering and GMP-140 (p-selects albumen) express and Fibrized resin or the contrast equipment that contains without fixed adsorbent contacts 5 days external platelet functions afterwards.Change of shape, gathering and hypotonic shock response are to use Chrono-Log Lumi-Aggregometer to estimate, and GMP-140 is to use Becton-Dickinson FACScan Fluorescence Analyzer to measure by flow cytometry simultaneously.
The pHEMA bag is by the influence of removing to psoralen
Fig. 7 compared use through pHEMA bag quilt and without the Dowex_XUS-43493 pearl of bag quilt from the platelet of 35% blood plasma/65% synthetic medium (PAS III), remove 4 '-(4-amino-2-oxa-) butyl-4,5 ', the adsorption dynamics adsorption kinetics of 8-trimethylpsoralen.Particularly, with 3.0g with the Dowex_XUS-43493 of 3.7% (w/w) pHEMA bag quilt remove 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen is represented with circle, with representing of 7.3% (w/w) pHEMA bag quilt with triangle, and representing with rhombus with 10.9% (w/w) pHEMA bag quilt; Square representative with 2.5g (doing) without the Dowex_XUS-43493 that wraps quilt remove 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen.Shown in data among Fig. 7,4 '-(4-amino-2-oxa-) butyl-4,5 ', the kinetics of 8-trimethylpsoralen absorption is reduced by the increase of level with the pHEMA bag.Although in order to realize that the present invention does not need to understand this mechanism, this reduction it is believed that be because 4 '-(4-amino-2-oxa-) butyl-4,5 ', the resistance that the 8-trimethylpsoralen is diffused in the absorbent particles increases.
Same Dowex_XUS-43493 mensuration 4 with 3.7%, 7.3% and 10.9% (w/w) pHEMA bag quilt '-(4-amino-2-oxa-) butyl-4,5 ', the adsorption dynamics adsorption kinetics that the 8-trimethylpsoralen is removed.(not shown) explanation as a result be coated with 10.9%pHEMA pearl remove kinetics be can with compare without (contrast) pearl of bag quilt.
The pHEMA bag is by the influence to the platelet yield
In two researchs, in every kind, use not commensurability adsorbent (3.0g and 5.0g) but the pHEMA bag of par is measured the pHEMA bag by the influence to the platelet yield by (3.7%, 7.3% and 10.9%[w/w]).Relative 5 days the platelet count calculating platelet yield of the treated PC that contact with the equipment that contains without fixed adsorbent.Shown in result in the table 3, when the XUS-43493 of test 3.0g, along with pHEMA bag by the dose response that exists on 5 days the platelet slightly that is increased in of level; Therefore these results advise that low-level pHEMA bag may be the most effective, this be because low-level pHEMA bag by to 4 '-(4-amino-2-oxa-) butyl-4,5 ', the littler while of effect of kinetics of removing of 8-trimethylpsoralen, it still suppressed platelet adhesion to adsorbent surface.Opposite with the influence slightly that XUS-43493 with 3.0g sees, observing dose response-pHEMA bag when test 5.0g has been increased by 5 days platelet yields by the increase of level.But it is low that yield is still observed when using 3.0g adsorbent pearl.
Table 3
Polymer coating Bag is by level (%) Quantity of sorbent (g) The platelet yield, 5 days (%) *
Do not have 0 2.5 73.1
pHEMA 3.7 3.0 91.4
pHEMA 7.3 3.0 87.7
pHEMA 10.9 3.0 89.0
pHEMA 3.7 5.0 71.8
pHEMA 7.3 5.0 80.2
pHEMA 10.9 5.0 86.6
Result shown in the table 3 advises using the adsorbent of low amount (for example, 2.5-3.0g) to wrap by (the platelet yield for example ,≤3.0%w/w) will offer the best with low-level pHEMA.As mentioned above, the optimum level of pHEMA bag quilt is the parcel quilt of observing the protection effect of platelet yield and external platelet function.
PHEMA bag quilt and sterilization are to the influence of platelet function
The front has illustrated because the pHEMA bag can or be split by cross-linking radiation, so sterilizing methods can have a significant impact adsorbant function.In order to estimate this influence, use through the pHEMA bag and test through 2.5 MRad electron beams or 2.5MRad γShe Xianmiejun by (3.7%w/w) with without the equipment without fixed XUS-43493 of bag quilt.To contain 2.5g (doing) without fixed bag by or pack square 30 μ m polyester mesh bags into (among the 7cm * 7cm) without each equipment of the XUS-43493 of bag quilt; The contrast that contains treated PC is housed in separately in PL 2410 plastic containers.The results are shown in Table 4.
Table 4
With reference to table 4,5 days pH value shows it is very stable compared with the control.With the pO that contains without the measuring apparatus of the fixed adsorbent that does not wrap quilt 2Value raises a little, and this hint metabolism reduces slightly; Be shown with pHEMA bag and reduce this influence, contain the more approaching contrast than the equipment of γShe Xianmiejun without the result of the equipment of the electron beam sterilization of fixed adsorbent.
Sample through pHEMA bag quilt all has extraordinary platelet yield, and the sample of electron beam sterilization presents better slightly.Change of shape presents the pattern similar to yield with assembling, and containing the equipment that is adsorbed agent without fixed bag provides minimum and provide and contrast similar high value through the sample of pHEMATF/ electron beam sterilization.Shine in identical aspect hypotonic shock response (HSR) test or comparison with the sample that contains without the device processes of fixed adsorbent with contrast.With containing without the sample comparison of the device processes of fixed adsorbent according to having lower form score, but the GMP-140 test demonstrates lower level activation.
Carry out another blood Study on Compatibility relatively to contain without fixed equipment (2.5g pearl without the XUS-43493 that wraps quilt; 30 μ m polyester mesh bags; 7cm * 7cm), contain (the 14cm * 14cm) and contain Fibrized resin of the Fibrized resin without the bag quilt of Amberlite_XAD-16 through the Amberlite_XAD-16 of pHEMA bag quilt.Result shown in the table 5 has shown the good result of pHEMA on Fibrized resin.
Table 5
Figure 9881384000531
By in the table 5 about the data declaration of external platelet function and platelet yield, with the pHEMA bag by the pO that brings to Fibrized resin 2It is viewed to be worth more approaching contrast.Fibrized resin through pHEMA bag quilt also presents higher platelet yield than the Fibrized resin without the bag quilt.The Fibrized resin that the result demonstrates through pHEMA bag quilt puts up a good show than the XUS-43493 pearl without the bag quilt in all external platelet function tests.And, put up a good show than XUS-43493 pearl in most of external platelet function tests without the Fibrized resin that wraps quilt without the bag quilt.
Embodiment 4
Glycerol and Polyethylene Glycol are to the influence of adsorbent power
This embodiment test glycerol and Polyethylene Glycol as stabilizing agent to from blood plasma, removing the adsorbent power and the effect of kinetics of the psoralen that deaminizes.In the test of this embodiment, use free (promptly without fibrosis) Amberlite_XAD-16 and Dowex_XUS-43493 adsorbent pearl.
Method
((Supelco, Bellefonte PA) are dried to moisture＜5% for Rohm and Haas (Philadelphia, PA)) and Dowex_XUS-43493 with Amberlite_XAD-16 HP in 80 ℃ baking oven.The adsorbent of known quantity is immersed in the alcoholic solution that contains 0-50% glycerol, 50%PEG-200 or 50%PEG-400 (glycerol, PEG-200 and PEG-400 are from Sigma).Then at room temperature cultivated 15 minutes, remove excessive solvent and these samples are descended dry a whole nights at 80 ℃; Avoid dry this adsorbent under＞120 ℃ temperature, this is to change because preferentially observe performance of the adsorbent (for example, hole fusion) under higher temperature.After the drying, the sample of sorbent of weighing is to determine the amount of stabilizing agent in the unit mass adsorbent.
Carry out several independent researchs.In research as described below, comprise the control sample of " without moistening " adsorbent and the adsorbent of " through best moistening ".Sample of sorbent without moistening is not stand any pretreated dry adsorbent, and is by with 30% ethanol/70%dH through the sample of sorbent of best moistening 2O moistening adsorbent is made.Will be through the adsorbent dH of the best profit temperature 2The O flushing is to remove residual ethanol.In order to ensure drying not taking place, this adsorbent of preparation before absorption research.
Use contain 150 μ M admixtures have 3H-4 '-(4-amino-2-oxa-) butyl-4,5 ', 4 of 8-trimethylpsoralen '-(4-amino-2-oxa-) butyl-4,5 ', 100% human plasma of 8-trimethylpsoralen carries out every kind of absorption research.Blood plasma (6.0mL) is added in the bottle that contains the adsorbent that useful different stabilizers handled.The amount of proofreading and correct adsorbent is so that glycerol or PEG content provide 0.2g adsorbent.Be placed on these bottles in the rotary apparatus and stirring at room temperature.Different time shift out plasma sample and measure remaining 3H-4 '-(4-amino-2-oxa-) butyl-4,5 ', the level of 8-trimethylpsoralen.Sample (200mL) is diluted among the OptiphaseHiSafe Liquid Scintillation Cocktail (Wallac) of 5.0mL and counting in Wallac 1409Liquid Scintillation Counter (Wallac).
Amberlite_XAD-16 that crosses with glycerin treatment and the absorbability of Dowex_XUS-43493
Fig. 8 compared with the alcoholic solution that contains varying level glycerol to the pretreatment of Amberlie_XAD-16 and Dowex_XUS-43493 to relevant 4 in 100% blood plasma '-(4-amino-2-oxa-) butyl-4,5 ', the influence of 8-trimethylpsoralen absorbability.80 ℃ down before dry 48 hours with sample of sorbent moistening 15 minutes in ethanol/glycerite.After 4 hours contact, carry out the single mensuration of absorbability.With reference to Fig. 8, the glycerol content shown on the X-axle is the weight/volume percentage rate of glycerol in ethanol.Absorbability shown on the Y-axle is relatively through the percentage rate of the absorbability of the sample of sorbent of best moistening.The absorbability of XUS-43493 represents that with square XAD-16 represents with circle.
Shown in data among Fig. 8, the ability of XAD-16 from drying sample about 30% be increased in the sample of moistening in 205 glycerites more than 90%.These presentation of results dry after in order to keep the very low-level glycerol of high absorption capacity needs.Before drying at 50% ethanol/50%dH 2The control sample of moistening demonstrates and the identical absorbability of the exsiccant undressed sample of process among the O (no glycerol).On the contrary, these XUS-43493 samples do not demonstrate any influence of glycerol to absorbability; Absorbability is near 100% under the glycerol of all levels.Although test of the present invention is not had decisive role, this discovery supports that glycerol plays the hypothesis that prevents in the dry run that the adsorbent hole from subsiding; Because XUS-43493 has highly cross-linked structure, it is subsided by or not the hole when carrying out drying.
Sample with glycerin treatment demonstrates drying highly stable.Sample is preserved the variation (data not shown) that did not observe absorbability in 7 days in the laminar flow fume hood.
In a preferred embodiment of the present invention, the dry adsorbent of 2.5g is used for removing psoralen and psoralen photosynthate from each unitary platelet.In 30% glycerol/70% ethanol, soak this adsorbent, then dry, make adsorbent contain about 50% glycerol.Therefore the sample of a 5.0g should contain dried adsorbent of 2.5g and 2.5g glycerol.Like this, the typical unitary platelet of 300mL should contain 0.8% glycerol, and one is considered to accept the level that is used to transfuse blood.
Amberlite_XAD-16 that handled with glycerol or PEG and the absorbability of Dowex_XUS-43493
Carry out other research with low molecular poly PEG-200 and PEG-400, non-volatile and hypotoxicity reagent that can be dissolved in the second alcohol and water.In 50% PEG-400, PEG-200 in ethanol or the glycerite sample of sorbent was handled 15 minutes.Fig. 9 compared stabilizing agent to dry adsorbent A mberlite_XAD-16 (following) and Dowex_XUS-43493 (top) in 100% blood plasma 4 '-(4-amino-2-oxa-) butyl-4,5 ', the influence of 8-trimethylpsoralen absorbability; Sample without moistening is designated as " No Tx ".Absorbability is to report with the relative percentage rate through the ability of the adsorbent of best moistening.
As shown in the data and with its " macroreticular " structure being basic forecast among Fig. 9, the ability of Dowex_XUS-43493 is not subjected to drying (" No Tx " sample) influence.On the contrary, dry back Amberlite_XAD-16 is about 35% of maximum capacity.Handle the ability that XAD-16 has improved dry adsorbent with glycerol, PEG-200 and PEG-400; Each adsorbent power is all greater than 90%, wherein glycerol＞PEG-200＞PEG-400.Although in order to realize that the present invention does not need to understand its accurate mechanism of action, the difference of the ability between glycerol and the two kinds of PEG solution may be that the infiltration of stabilizing agent reduces with the increase of molecular weight and causes.That is to say that in 15 minutes application process, glycerol (MW=92.1) may can be penetrated in the adsorbent hole more completely than therefore spreading slower PEG-200 (MW=190-210) or PEG-400 (MW=380-420) owing to its size is big.
The adsorption dynamics adsorption kinetics of the Amberlite_XAD-16 that handled with glycerol or PEG
Study equally to determine whether the hole of filling adsorbent with glycerol or PEG causes opposite kinetics to reduce.Figure 10 compared the Amberlite_XAD-16 that uses in several different solutions from 100% blood plasma through 3 hours 4 '-(4-amino-2-oxa-) butyl-4,5 ', the absorption of 8-trimethylpsoralen.Specifically, data represented XAD-16 ⅰ among Figure 10) dry preceding with 50% glycerite moistening (open squares that solid line connects), ⅱ) cross (the black circle that dotted line connects) with 50% PEG-400 solution-wet before the drying, ⅲ) pre-moistening, promptly before beginning one's study with 50% ethanol/50%dH 2O moistening (dotted line connect black triangle) is with ⅳ) do not pass through (the black square that solid line is connected of any processing; " No Tx ").Data acknowledgement among Figure 10 in 50% glycerol/50% ethanol or 50%PEG-400/50% alcoholic solution the adsorption dynamics adsorption kinetics of the Amberlite_XAD-16 sample of moistening very approaching with adsorption dynamics adsorption kinetics in ethanol through the sample (promptly using the sample of alcohol pre-treatment) of the best profit temperature.Drying but undressed XAD-16 sample (No Tx) are by only realizing about 30% remove in 3 hours.
Data declaration shown in the present embodiment handle Amberlite_XAD-16 with the stabilizing agent of the solution form that contains 50% ethanol and 50% glycerol, PEG-200 or PEG-400 and can prevent forfeiture with the dry absorbability that accompanies.The result who obtains with these stabilizing agents hints that the representative of low-molecular-weight wetting agent is used to strengthen the spendable method of adsorbant function.
Embodiment 5
From FFP, remove methylene blue
Present embodiment relates to various different polymeric adsorbant materials and freezes the ability of removing methylene blue the blood plasma from aquatic foods.
The test evaluation of present embodiment " freedom " absorbent resin (that is, not adding the equipment that contains without fixed adsorbent) and Fibrized resin.The free absorbent resin of these that tested is Amberlite_XAD-16 HP (Rohm and Haas), MN-200 (Purolite) and Dowex_XUS-43493 (Dow Chemical Co.).This XAD-16 HP comes in hydration status so that do not need pretreatment (that is, nonwetting), and this MN-200 also provides with the state of complete hydration; This XUS-43493 is what do.
The Fibrized resin that contains XAD-16 is normally with preparation described in the embodiment 1.Briefly, will contain 130g/m 22cm * 7cm of XAD-16 (is 14cm 2) the at first moistening in 70% ethanol of Fibrized resin bar, use the distilled water cleaning down then.
Methylene blue (10mM) stock solution is made by U.S.P. methylene blue (Spectrum) is dissolved in the distilled water.Add this methylene blue stock solution to obtain 10 μ M in 100% plasma sample ultimate density." freedom " absorbent resin sample (that is, XAD-16 HP, MN-200 and XUS-43493) is weighed into is used for absorption research in the 50mL polypropylene tube.Lose the moisture of determining every kind of adsorbent by dry quality measurement.Proofread and correct the quality of every kind of adsorbent so that every kind is equivalent to use the dried adsorbent of 0.25g with moisture.
In each bottle, add 100% plasma sample that 30mL contains 10 μ M methylene blue.At room temperature these bottles are placed on the rotary apparatus.From each bottle, to remove sample (200mL) at interval in 15 minutes and to identify the residue methylene blue by HPLC.With diluents (ultimate density=35% methanol, 25mM KH 2PO 4, pH=3.5) with 5 times of every kind of plasma sample dilutions.By under 4 ℃, protein and other macromole being precipitated these sample culturing.With the sample centrifugalize and with supernatant filtration (0.2 μ m) and at C-18 reversed-phase column (YMC ODS-AM, 4.6mm * 250mm) go up by in 20 minutes, flowing out from 65% solvent orange 2 A (25mM KH 2PO 4, pH=3.5), 35%B (methanol) to the linear gradient of 80%B by analysis.The detection limit of HPLC test is about 0.5 μ M methylene blue.
Figure 11 has compared the kinetics of adsorbing methylene blue in 2 hours from 100% blood plasma.With reference to Figure 11, XAD-16 HP data are by shown in the open diamonds of dotted line connection, shown in the black triangle that the MN-200 data are connected by solid line, shown in the empty circles that the XUS-43493 data are connected by dotted line, and shown in the black square that connects by solid line of the Fibrized resin that contains XAD-16.Shown in these data, this XAD-16 HP and MN-200 provide the fastest adsorption dynamics adsorption kinetics, then are XUS-43493.Use during as XUS-43493 with its drying regime, its kinetics may be a bit slightly slowly since the slow institute of its moistening extremely.At last, the Fibrized resin that contains XAD-16 has the slowest adsorption dynamics adsorption kinetics.This may be in batch incubation, because 14cm 2The part of Fibrized resin bar in whole absorption research, be not fully immersed in the blood plasma, therefore reduced the effective contact area between adsorbent and the blood plasma, cause between Fibrized resin and the blood plasma contact relatively poor.
These data declarations can use in the present invention and to attempt the resin that uses and Fibrized resin and from blood products, remove psoralen deactivation compounds as the non-psoralen of phenothiazine dyestuff.
Embodiment 6
From erythrocyte, remove the acridine chemical compound through packing
Present embodiment relates to various different resins materials are removed the acridine chemical compound from the erythrocyte (PRBC) through packing ability.More particularly, the test evaluation of present embodiment is removed this acridine chemical compound, 5[(β-carboxyethyl from PRBC) amino] acridine.
The chemical constitution of several acridines is listed among Figure 12.As shown in figure 12,9-aminoacridine and 5-[(β-carboxyethyl) amino] acridine is aminacrine.
Choice of Resin
With the resin of several types research chemical compound 5-[(β-carboxyethyl) amino] equilibrium adsorption of acridine.The polymeric adsorbant resin of being estimated is Amberlite_XAD-2, XAD-4, XAD-7 and XAD-16 HP (Rohm and Haas); Purolite_MN-150, MN-170, MN-200, MN-300, MN-400, MN-500 and MN-600; With Dowex_XUS-43493 and XUS-40285 (Dow Chemical Co.).In addition, test several Amberlite_ anion exchange resin (IRA-958, IRA-900, IRA-35, IRA-410 and IRA-120; Rohm andhaas) and Amberlite_ weak cation exchange resin (DP-1; Rohm and haas).And, estimate several charcoals, comprise Hemosorba_AC (Asahi), PICA G277 and Norit ASupra (all can be commercially available) from American Norit.At last, also test Porapak_RDx (Waters), a kind of styrene ethylene base ketopyrrolidine copolymer with nitro-aromatic compound affinity.
At first, estimate 5-[(β-carboxyethyl with every kind of resin sample) amino] ability of acridine and adenine (adenine) carries out equilibrium adsorption research.Weigh about 0.1g resin and changing in the 6mL polypropylene tube.Will be in distilled water contain 100 μ M5-[(β-carboxyethyls) amino] the 5.0mL aliquot of 25% blood plasma/75%Adsol_ (Baxter) of acridine adds in each pipe.Typically for example erythrocytic cell product is housed in the medium that contains low percentile blood plasma (10-35%) and residue synthetic medium; The example of the synthetic medium that one kind of Adsol_ is made up of the adenine in saline solution, glucose and mannitol.Certainly, the present invention attempts using acridine concentrate rather than the 100 μ M in the mixture of blood plasma and other synthetic medium.Next, these pipes are placed on the cylinder agitator and at room temperature cultivated 3 hours.After the cultivation, shift out the aliquot of every kind of sample and analyze remaining 5-[(β-carboxyethyl by HPLC) amino] acridine and adenine.
With regard to the HPLC process, with diluents (50% methanol, 25mM KH 2PO 4, pH=3.5) with 2 times of every kind of diluted samples, by under 4 ℃, protein and other macromole being precipitated these sample culturing.Then with these sample centrifugalize and with supernatant filtration (0.2 μ m) and at C-18 reversed-phase column (YMC ODS-AM, 4.6mm * 250mm) go up by in 20 minutes, flowing out from 75% solvent orange 2 A (25mM KH 2PO 4, pH=3.5), 25%B (methanol) analyzes to the linear gradient of 80%B.With regard to 5-[(β-carboxyethyl) amino] with regard to acridine removes, at C fEstimated capacity under=1 μ M (μ mole/g) is by having C 0The single determining adsorption of=100 μ M is determined; This results are shown in table 6 the 2nd row (ND=can not measure).With regard to adenine, at C fEstimated capacity under the=1mM (mmole/g) is by having C 0The single determining adsorption of=1.5mM is determined; This results are shown in table 6 the 3rd row (ND=can not measure).
Table 6
Figure 9881384000601
When attempt using any resin that can adsorb the acridine chemical compound, adenine absorption 5-[(β-carboxyethyl is crossed on preferred Choice of Resin ground) amino] acridine and present and hang down haemolysis.Figure 13 is the adenine ability (Y-axle) and the 5-[(β-carboxyethyl of different resins) amino] curve of acridine ability (X-axle) data.Shown in the data among table 6 and Figure 13, Dowex_XUS-43493 and Purolite_MN-200 resin have the highest 5-[(β-carboxyethyl) amino] the acridine ability; And, consider high 5-[(β-carboxyethyl when simultaneously) amino] when acridine ability and low adenine ability, Amberlite_XAD-16 HP is more satisfactory.
The result of the in vitro tests described in the present embodiment hints that Dowex_XUS-43493 and described resin Purolite_MN-200 are for removing the preferred resin of acridine chemical compound from PRBC.
Embodiment 7
From the erythrocyte of packing, remove the acridine chemical compound with styrene-divinylbenzene adsorbent
Present embodiment relates to various different styrene-divinylbenzene, and (styrene-DVB) adsorbent is removed the ability of aminacrine chemical compound from blood products.More particularly, the test evaluation of present embodiment is removed acridine orange and 9-aminoacridine (as described in Figure 12) from blood plasma/Adsol_ solution.
A. test procedure
With regard to the test of present embodiment, preparation 5-[(β-carboxyethyl in distilled water) amino] stock solution (10mM) of acridine (Cerus) and acridine orange (Aldrich), and in ethanol, prepare the stock solution (10mM) of 9-aminoacridine (Aldrich).With this 5-[(β-carboxyethyl) amino] acridine adds in the solution that contains 25% blood plasma/75% normal saline to reach ultimate density 100 μ M, this acridine orange and 9-aminoacridine chemical compound added to contain in 25% blood plasma/75%Adsol_ (Baxter) listerine simultaneously to reach final acridine concentration 100 μ M.
Sorbent used is Amberlite_XAD-16 HP (Rohm and Haas); Purolite_MN-200 and Dowex_XUS-43493 (Supelco).Lose the moisture of measuring every kind of adsorbent by measuring dry back quality; Proofread and correct moisture so that use the equivalent of every kind of dried adsorbent of 0.25g.Adsorbent is accurately taken by weighing in the 50mL Falcon pipe.In each pipe that contains adsorbent, add 25% blood plasma/75%Adsol_ solution that 30mL contains 100 μ M acridines.At room temperature these pipes are placed on the rotary apparatus then, and storage is used with post analysis.
By remaining acridine level in the HPLC analytic sample.With diluents (50% methanol, 25mMKH 2PO 4, pH=3.5) with 2 times of every kind of diluted samples, by under 4 ℃, protein and other macromole being precipitated these sample culturing.Then with the sample centrifugalize and with supernatant filtration (0.2 μ m) and at C-18 reversed-phase column (YMC ODS-AM, 4.6mm * 250mm) go up by in 20 minutes, flowing out from 75% solvent orange 2 A (25mMKH 2PO 4, pH=3.5), 25%B (methanol) analyzes to the linear gradient of 80%B.Use a diode arrays detector detecting 9-aminoacridine under the 400nm, detecting acridine orange under the 490nm and under 410nm, detecting 5-[(β-carboxyethyl by visible absorbance) amino] acridine.
B. adsorption dynamics adsorption kinetics
From 25% blood plasma/75% normal saline through 3 hours to 5-[(β-carboxyethyl) amino] the absorption adsorption dynamics adsorption kinetics of Dowex_XUS-43493, Purolite_MN-200 and Amberlite_XAD-16 HP relatively of acridine.Figure 14 A and Figure 14 B have shown remaining 5-[(β-carboxyethyl) amino] acridine and the function of time, Figure 14 B shows the data of logarithmic scale.In Figure 14 A and Figure 14 B, XAD-16 HP data are by shown in the black circle of dotted line connection, and the MN-200 data are by shown in the black square of dotted line connection, and the XUS-43493 data are by shown in the black triangle of solid line connection.By these data declarations, XUS-43493 and MN-200 have the fastest adsorption dynamics adsorption kinetics and almost of equal value.XAD-16 HP shows to have lower 5-[(β-carboxyethyl) amino] the acridine ability.
Figure 15 has compared the adsorption dynamics adsorption kinetics of removing 9-aminoacridine and acridine orange with Dowex_XUS-43493 from 25% blood plasma/75%Adsol_.With reference to Figure 15, the black square that dotted line connects is represented 9-aminoacridine, and the black circle that solid line connects is represented acridine orange.By these data declarations, surpass 3 hours and detect less than the 9-aminoacridine level.By comparison, Dowex_XUS-43493 is lower to the ability of acridine orange, and this may be because two seasons amino is arranged on acridine orange.
Embodiment 8
The PRBC that preparation usefulness-[(β-carboxyethyl) amino] acridine derivatives and glutathion were handled.Whole blood with fresh ABO-coupling prepares the PRBC storehouse.Use Beckman Sorvall RC3B centrifugal separator under 3800rpm with these whole blood unit centrifugalize 5 minutes.Use an expresser with blood plasma squeezing in another clean bag.During an above unit, these PRBC unit are incorporated in the Clintec Viaflex bag of 3.0L size if desired.Add the Erythrosol of 94mL to each unitary PRBC.Measured hematocrit (HCT) percentage rate in 5 minutes by blood products being filled in the capillary tube and with its rotation.Measure hematocrit and be not less than 55% to guarantee it.In the PRBC of every kind of 100mL, add 12% glucose of 3.3mL.Measure final hematocrit percentage rate.PRBC (300mL) is recharged among the plastic containers PL146 (Baxter Healthcare).In the blood bag, add the 30mM 5-[(β-carboxyethyl of 150mM glutathion of 6.0mL to reach the 3.0mM ultimate density and to add 3.0mL) amino] acridine derivatives to be to reach 300 μ M ultimate densities.Use wrist effect shaking machine (manufacturer) that this PRBC mixture was stirred 1 minute.Will be through 5-[(β-carboxyethyl) amino] PRBC that handled of acridine derivatives and glutathion at room temperature incubated overnight so that this 5-[(β-carboxyethyl) amino] acridine derivatives resolves into 5-[(β-carboxyethyl) amino] acridine.
Embodiment 9
5-[(β-carboxyethyl in the PRBC and the PRBC supernatant) amino] the HPLC test of acridine.With this sample (100 μ L) with 100 μ L normal saline dilutions and with gained mixture vortex.In this solution, add 300 μ L at CH 320mM H among the CN 3PO 4, with 15 seconds of this mixture vortex.Under 13200rpm with this sample centrifugalize 5 minutes.This supernatant (200 μ L) dilution is advanced among the cold 0.1M HCL of 800 μ L and through vortex.By using 0.2 μ m GelmanAcrodisc filter that this sample is filled in the automatic sampler.The HPLC condition is as follows: (manufacturer of post and conventional post and part number) post=Zorbax SB-CN, 4.6mm * 150mm, 3.5 μ m granules; Guard column=(4.6mm * 12.5mm, 5 μ m granules (Mac Mod Analytical, Inc. (Chadds Ford, PA)); Mobile phase A is the 10mM H in HPLC water 3PO 4Mobile phase C is the 10mM H in acetonitrile 3PO 4Temperature is 20 ℃; Sample volume is 100 μ L; Gradient condition is as follows:
Time A(%) C(%) Flow velocity (mL/min)
0.00 90.0 10.0 1.0
5.28 77.0 23.0 1.0
10.00 40.0 60.0 1.0
11.00 90.0 10.0 1.0
16.00 Stop Stop Stop
DAD is provided with as follows:
Label Detect wavelength Detection bandwidth Reference wavelength Reference bandwith
A 410 5 580 20
B 260 5 580 20
Embodiment 10
The HPLC test of the glutathion in the PRBC and the PRBC supernatant.This sample is according to HPLC test preparation as mentioned above.The HPLC condition is as follows: analytical column=YMC ODS-AM-303,250mm * 4.6mm, 5 μ m granules; Guard column=Brownlee, 15 * 3.2mm, 7 μ m granules; Mobile phase A is the 10mMH in HPLC water 3PO 4Mobile phase C is the 10mMH in acetonitrile 3PO 4Temperature is 15 ℃; Sample volume is 75 μ L; Gradient condition is as follows:
Time A(%) C(%) Flow velocity (mL/min)
0.00 95.0 5.0 0.5
6.00 95.0 5.0 0.5
8.00 10.0 90.0 1.0
9.00 95.0 5.0 1.0
20.00 Stop Stop Stop
DAD is provided with as follows:
Signal Detect wavelength Detection bandwidth Reference wavelength Reference bandwith
A 205 10 600 100
Embodiment 11
The method of screening adsorbent.The testing liquid that use contains 25% blood plasma and 75%Erythrosol represents the supernatant among the PRBC.Erythrosol is prepared as the germ-resistant separately stock solution part (solution C and solution D) of separating of two warps.With isopyknic solution C and the mixed final solution that gets of solution D.
Figure 9881384000644
With blood plasma-Erythrosol solution admixture 5-[(β-carboxyethyl) amino] ultimate density of acridine to 300 μ M.In this mixture, add glutathion (reduction form, Sigma Chemical Co.) to obtain the ultimate density of 3mM.Sample of sorbent (0.2-0.8g) is accurately taken by weighing (± 0.001g) in the bottle that advances to determine the 7mL band spiral gag that empty bottle is heavy.The sample of sorbent of the pre-moistening of needs is suspended in 70% ethanol, this adsorbent is left standstill and remove the supernatant.By being resuspended in adsorbent in the distilled water, making this adsorbent leave standstill also this supernatant of decant to remove residual ethanol.When calculating adsorbent power, proofread and correct adsorbent mass according to moisture.In each pipe, add 5.0mL blood plasma/Erythrosol aliquot.These pipes are placed on the rotary apparatus and at room temperature overturn 24 hours.From rotary apparatus, remove these bottles and adsorbent is left standstill.From each pipe, remove supernatant sample.The centrifugalize sample is to remove remaining adsorbent.By the HPLC analytic sample to measure 5-[(β-carboxyethyl) amino] the acridine residual level.Use aforesaid anti-phase test to detect the residual level of glutathion.The adsorbent results of screening is shown in table 7 and 8.
Table 7
Adsorbent screen-is removed 5-[(β-carboxyethyl from 25% blood plasma/75%Erythrosol) amino] acridine (" LOD " is detectable limit)
Figure 9881384000671
Figure 9881384000701
Figure 9881384000721
Table 8 adsorbent screening-from 25% blood plasma/75%Erythrosol, remove glutathion
Figure 9881384000722
Figure 9881384000741
Figure 9881384000761
Embodiment 12
The absorbability of adsorbent.Carry out absorbability (the μ mole5-[(β-carboxyethyl) amino of adsorption isotherm thread test to measure various types of adsorbents] acridine/g adsorbent).Figure 17 has shown and has compared the adsorption isotherm that is obtained by several Ambersorb with the adsorption isotherm of Purolite MN-200.Containing 0.2-3mM 5-[(β-carboxyethyl) amino] adsorb research in 25% blood plasma/75%Erythrosol solution of acridine and 0.6-10mM GSH.At room temperature sample was stirred 24 hours.Using absorbability in the table 7 (22 μ mole/g) to calculate measures in requisition for about 4gPurolite MN-200 5-[(β-carboxyethyl in the PRBC unit of 300mL) amino] level of acridine reduces to terminal level 1 μ M from 300 μ M.The Ambersorb572 (130 μ mole/g) that need be lower than 1g is to realize comparable removing.Simple calculate to estimate in requisition for the Ambersorb 572 that is lower than 1g the 6mM of level from the 150mL supernatant (50%HCT) of GSH in the PRBC unit of 300mL reduced to terminal level 500 μ M.
Embodiment 13
Removing for a long time of catabolite.
This experimental examination PRBC removes fibrosis PICA G-277 active carbon equipment (AQF, 7.3g.500m2/g) after 5-[(β-carboxyethyl wherein) amino after exposing 24 hours] level of acridine and GSH.This research is carried out in the place that has the continuous device exposure at PRBC abreast.Figure 18 has shown under the situation that is not having equipment in the storage in 1-4 week 5-[(β-carboxyethyl in the supernatant sample) amino] concentration of acridine and GSH is much higher.
Under the situation that the equipment of removing (MN-200) arranged after storage 35 days in the PRBC that shakes at first 5-[(β-carboxyethyl) amino] concentration of acridine reduces to 5 μ M.5-[(β-carboxyethyl is taking place in this explanation really in static holding conditions under 4 ℃) amino] acridine removes.
Embodiment 14
Closed material (film, braiding, nonwoven) is to the influence of IAD
Use closed material to surround adsorbent medium through investigation and be used for the initial purpose that granule is kept here.This initial purpose is to keep granule here.But, can improve the blood compatibility of equipment by preventing contact membranes between RBCs and the film.Can easily use hydrophilic polymer (PEO, PEG, HPL) to regulate film to improve the blood compatibility but do not change function.Surround fibrosis Pica G277 activated carbon media (the AQF 500g/m of about 10g by heat-sealing film, Woven polyester or non-woven polyester material 2).With PRBC unit (300mL) with the degradable 5-[(β-carboxyethyl of 300 μ M) amino] acridine derivatives and 3mMGSH batching, at room temperature in the platelet electromagnetic shaker, kept 20 hours, import IAD then.Monitoring 5-[(β-carboxyethyl in 24 hours) amino] concentration of acridine.Figure 19 has shown that 300mL is exposed to by fibrosis Pica G277 active carbon (500g/m 2) 5-[(β-carboxyethyl in the IAD that forms and the unitary supernatant of PRBC of sealing with film, braiding or non-woven material) amino] level of acridine.With the degradable 5-[(β-carboxyethyl of PRBC (Erythrosol, glucose, 62%HCT) with 300 μ M) amino] acridine derivatives and 3mM GSH batching, at room temperature stirred in the platelet electromagnetic shaker before input IAD.The IAD that at room temperature will contain PRBC stirred 24 hours.
These research explanations Tetko closure demonstrates 5-[(β-carboxyethyl in 24 hours) amino] the fastest kinetics of removing of acridine.The ultimate density that all closed materials reach after 2 weeks is similar, 5-[(β-carboxyethyl after 1 day) amino] acridine concentration is reduced to about 2 μ M, but liter is got back to 10 μ M about 8 days.
Embodiment 15
The chemical compound adsorption plant is to the influence of erythrocyte function.5-[(β-the carboxyethyl of erythrocyte function indicator monitoring distinct device configuration) amino] acridine and GSH remove the whole process of test.The parameter of measuring comprises dissolving percentage rate, ATP and K +Concentration.Table 9 demonstrates the existence that ATP concentration generally is not subjected to chemical compound to remove equipment to be influenced: use the roughly the same of the reduction of ATP concentration of MN-200 or Pica G277 equipment and contrast (not having IAD).Find K among the PRBC +Level pass in time and increase.Temperature when removing does not influence at chemical compound removes the K in 20 days the PRBC unit of exposure in the equipment +Level.But, when open-assembly time extends to 35 days, K among the PRBC +The speed that increases changes with the adsorbent type, and MN-200 and PICA G-277 equipment arrive 40 and the terminal level of 45mmol/L respectively, and do not have the contrast of equipment to arrive 39mmol/L.Usually found after 24 hours between the 0.1-1% at dissolved erythrocytic percentage rate in the contrast PRBC unit that exposed through equipment and do not have equipment.Shown in table 10 and table 11 and Figure 20 and 21 diagram, the dissolving percentage rate has very big variation with sorbent used type.Table 10 has shown that PRBC is exposed to the fixedly solubility value after 35 days in the adsorption plant of two classes.Table 11 has shown that PRBC is exposed to the solubility value that obtains after 42 days in the loose absorbent particles that is enclosed in the Woven polyester net.In 1 minute, use wrist effect electromagnetic shaker to prepare all PRBC unit, afterwards this PRBC was at room temperature left standstill 4 hours.These equipment were at room temperature kept on the platelet electromagnetic shaker 24 hours, during studying, they are left standstill under 4 ℃ afterwards.Fixedly MN-200 demonstrates lower hemolysis levels than fixing PICA G-277, and the discrete particles adsorbent compares that Ambersorb is synthetic to be contained carbon adsorbent and demonstrate minimum hemolysis levels simultaneously.MN-200IAD demonstrates than identical but without the low haemolysis of fixed adsorbent.To compare other IAD without fixed adsorbent and observe similar observation.
The concentration of ATP (μ mol/dL) among the PRBC in the whole process of table 9.
Figure 9881384000791
Table 10. is exposed to PRBC (56%HCT) the dissolving percentage rate among the different I AD in the whole time
Table 11. is exposed to PRBC (55%HCT) the dissolving percentage rate that is enclosed in the different discrete particles adsorbents of knitting in the system net
Figure 9881384000793
Understand that for example absorbent particles is in the decisive performance that keeps the erythrocyte function aspects.Listed the comparative study of five different adsorbents to erythrocyte hemolytic influence.Ambersorb 572 only produces 0.16% haemolysis in PRBC (55%) after 24 hours expose, and Darco AC (Norit Americas, the haemolysis of Inc. (Atlanta, GA)) generation 0.13%.These adsorbents make aspect the erythrocyte haemolysis more much better than Purolite MN-200, Hemosorba CH-350 and Pica G277 adsorbent.
Table 12 shown contain glutathion and 5-[(β-carboxyethyl) amino] comparative study that contacts with a large amount of different adsorbents of the supernatant of erythrocyte sample of acridine.Active carbon is can reduce 5-[(β-carboxyethyl basically) amino] acridine concentration can reduce the adsorbent that type is only arranged of glutathione concentrations again.Natural activity charcoal (Norit and PICA) and synthesizing activity charcoal (Ambersorb) are all proving effective aspect the chemical compound reduction.
The performance of several different adsorbents of table 12.
Figure 9881384000811
The value that @PS-DVB=polystyrene-divinylbenzene, AC=active carbon ^ are classified "＞" as be under test LOD to the single mensuration * of residual level by 25% blood plasma, 75%Erythrosol in the evaluation that obtains of single-point absorption research
+ the evaluation that obtains by multipoint adsorption research among 25% blood plasma, the 75%Erythrosol
Embodiment 16
(Charlotte NC) produces the fibrotic mediators of being made up of the Dowex OptiporeL-493 that sticks on the nonwoven polyester fibre substrate (Hoechst-L493) by the AQF portion of Hoechst Celanese.Remove the evaluation that equipment carries out the performance of this adsorbent medium being used for hematoblastic batch.
The platelet preparation
In 35% autologous plasma of 300mL, 65%PAS III, contain 3.5-4.5 * 10 11Hematoblastic single donor separation property blood transfusion platelet unit obtains from Sacramento Blood Bank Center.With 4 '-(4-amino-2-oxa-) butyl-4,5 ', 8-trimethylpsoralen (BaxterHealthcare) is added in each platelet unit to reach the ultimate density of 150 μ M.Be incorporated in the single PC-2410 plastic containers and fully mixing with platelet unit (4-5).This platelet mixed liquor is evenly divided to go in 4-5 the 1L PL2410 plastic containers, and each container contains 300mL platelet mixture approximately.With these unit 3.0J/cm 2UVA handles and moves into suitably to remove in the equipment and studies.All tests all comprise through photochemical treatment (150 μ M psoralens, 3.0J/cm 2UV-A) but the contrast platelet unit that does not contact with the equipment of removing.
The equipment preparation
The standard that contains 2.5g Dowex XUS 43493 is removed equipment and is prepared by Baxter HealthcareCorporation (Lot FX1032 D96F20042R).The IAD of test prepares with the Hoechst-L493 medium (Hoechst Celanese Corp.) of supply as the roll raw material.The suitable quantity of sorbent (5.0g and 7.5g) with each IAD is measured and cut into to medium.With the medium side of being cut in the bag that constitutes by impulse welding 30 μ m polyester webs (Tetko).The mesh bag that contains these media through autoclaving (121 ℃, 20min) and put into aseptic PL 2410 plastic containers.Perhaps, mesh bag is put into PL 2410 containers, sterilize by gamma-rays (SteriGenics) to 2.5MRad with these seals of vessel and with this whole device.Before shifting, use syringe surplus air is manual in equipment and find time through the platelet of photochemical treatment.
Adsorption dynamics adsorption kinetics
With 4 '-(4-amino-2-oxa-) butyl-4,5 ', after the photochemical treatment of 8-trimethylpsoralen+UVA, these platelet mixture are sent in the PL2410 plastic containers that have the equipment of removing.Be placed on these equipment in the standard platelet incubator (Helmer) and under 22 ℃ with 70 rev/mins of stirrings.Take out the platelet blend sample every 1 hour and be used at first storages in 8 hours.These samples 4 ℃ down storage and analyze by HPLC subsequently remaining 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen.This test relates to the dissolving platelet and dissolves 4 '-(4-amino-2-oxa-) butyl-4,5 ', 8-trimethylpsoralen and do not have the original sample goods of photosynthetic product.The supernatant of sample product is to be used in the ethanol that gradient increases in the KH2PO4 buffer to analyze on the C-18 reversed-phase column.
External platelet function
The platelet mixture stirred with the equipment of removing contact 7 days.In a research, the platelet mixture contacts 24 hours and uses sterile tube welding machine (Terumo SCD 312) to transfer in the aseptic 1L PL2410 plastic containers with IAD.Be put back in the platelet incubator platelet and the remaining time of preserving 7 days duration of storage.
After preserving 5 or 7 days, measure platelet count and pH in each platelet unit.Use CIBA-Corning model 238 Blood Gas Analyzer to measure pH and pO 2/ pCO 2Use Baker 9118+Hematology analyzer measures the platelet count in each sample.。Carry out several tests to estimate external platelet function.Use the change of shape of Chrono-Log model 500 VSwhole blood aggregometer monitoring platelet sample.Change of shape is to change recently quantitative with the variation of adding optical transmission after the EDTA to add the maximum of optical transmission after the ADP.
Platelet is estimated by hypotonic shock response (HSR) test the reaction of low osmotic pressure.Adding the hypisotonic solution variation of optical transmission afterwards is to use Chrono-Log model 500 VSwhole blood aggregometer to measure.Data are to reach after its minima 2 minutes with absorbance to be reported by the hypotonic percentage rate that pushes back receipts.
The ability that platelet is gathered ADP/ collagen agonist reaction is that the variation of the optical transmission measured by Chrono-Logmodel 500 VS whole blood aggregometer illustrates.
Hematoblastic state is estimated by platelet is given a mark.The score of each sample is hoodwinked and 100 hematoblastic morphology scores are added up to sample.The highlyest may be divided into 400 (discoid=4, intermediate=3, spherical=2, tree-shaped=1, balloon-like=0).
Biologically active pdgf is that (Granular Membrane Protein, expression GMP-140) illustrates by measuring p-selection albumen.CD62 antibody as test specimen, is contrasted IgG1 as negative contrast with mouse, and the CD62 antibody that will have acetic acid Semen Myristicae fluorine ripple alcohol (PMA) is used for over against photograph.Go up analytic sample at FACScan (Becton Dickinson).The report active percentage rate be with over against the photograph ratio and be the poor of test value and negative control value.
Adsorption dynamics adsorption kinetics
Investigation adsorbent pearl adds the influence in the fibre substrate.Contain in 35% blood plasma in 300mL, the 65%PAS III 4.0 * 10 11Individual hematoblastic platelet unit 150 μ M 4 '-(4-amino-2-oxa-) butyl-4,5 ', 8-trimethylpsoralen+3.0J/cm 2UVA handles.IAD is by having 450g/m 2The Hoechst-L493 preparation of adsorbent load.These IAD are through γShe Xianmiejun.After psoralen+UVA processing, these platelet are transferred in the equipment of removing.Shifted out sample every 1 hour and analyze by HPLC remaining 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen.The standard that Figure 22 has compared the IAD that contains 5.0g and 7.5gHoechst-L493 medium and contained the loose pearl of 2.5g remove equipment IAD 4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen remove kinetics.IAD or contain 5.0gHoechst-L493 (triangle), 7.5g Hoechst-L493 medium (square) or contain the loose Dowex L493 of 2.5g adsorbent pearl (circle).It is 450g/m that the Hoechst-L493 medium contains load 2The adsorbent pearl.
When with the loose adsorbent pearl of same amount relatively the time 4 of the Hoechst medium '-(4-amino-2-oxa-) butyl-4,5 ', the adsorption dynamics adsorption kinetics of 8-trimethylpsoralen is slower.That the equipment of removing that contains the loose pearl of 2.5g made in the short time (1 hour) is remaining 4 '-(4-amino-2-oxa-) butyl-4,5 ', the level of 8-trimethylpsoralen reaches minimum.But when the long period, it is better that the IAD of the Hoechst-L493 medium of 7.5g and 5.0g shows.In this research, in being lower than 4 hours contact all three adsorption dynamics adsorption kinetics of removing equipment all reach relatively soon＜0.5 μ M remnants 4 '-(4-amino-2-oxa-) butyl-4,5 ', the target of 8-trimethylpsoralen.
Notice the Hoechst-L493 that includes the higher amount adsorbent in long-time (6-8 hour) obtain remnants 4 '-(4-amino-2-oxa-) butyl-4,5 of reduced levels ', the 8-trimethylpsoralen.The slower adsorption dynamics adsorption kinetics of this observation hint Hoechst-L493 IAD makes the material conveying be restricted (flow rate of liquid and diffusion) and does not make because the functional adsorption area loss of fiber absorption.Other research explanation has reduced levels adsorbent load (200-300g/m 2) the Hoechst-L493 medium make fluid more easily be penetrated in the medium, this makes adsorption dynamics adsorption kinetics faster.The front is 160g/m to containing load 2The adsorption dynamics adsorption kinetics that studies show that of Hoechst medium of Amberlite XAD-16 adsorbent be not subjected to adding the influence of fibre substrate with low-level load.
Platelet yield and external platelet function
The data that in this joint, present two independent studies.Platelet unit in each research is obtained by single library, so that can clearly estimate IAD medium style, quantity of sorbent and the influence of time of contact.
First research evaluation fibrosis (Hoechst medium) is to prolonging the influence of contact (5 and 7 days) hematoblastic yield in back and function.Four platelet unit altogether that will obtain from a storehouse are used for this research.The contrast of no IAD is handled through psoralen+UVA.Two platelet unit are contacted with 5.0g Hoechst-L493.Unit contacted 24 hours with IAD before the PL2410 storage container of having transferred to.Remaining another unit contacts with IAD in research process always.With these Hoechst-L493 IAD wet sterilization (120 ℃, 20 minutes).Remove equipment (the loose Dowex XUS-43493 of 2.5g) through γShe Xianmiejun from the standard that Baxter obtains.Notice typically contact 8-16 hour in test with the equipment that utilizes loose absorbent particles after platelet do not removed the equipment and removed from standard.With storage after 5 and 7 days platelet count and the result of external function collect in respectively among table 13A and the table 13B.
Table 13A. (5 days)
Hoechst-L493 fibrotic mediators and standard are removed the comparison of equipment
Preserve platelet yield and external function after 5 days
Sample Platelet count (* 10 11/300mL) Yield (%) pH pCO 2 pO 2
Contrast (+PCT-RD) 3.50±0.11 100 6.91 18 120
5.0g Hoechst/L-493 shifted in the time of 24 hours 3.41±0.06 97±4 6.95 21 95
5.0g Hoechst/L-493 does not shift 3.33±0.08 95±4 6.93 24 87
2.5g Dowex Optipore L-493 Baxter Lot FX 1032 D96F20042R do not shift 2.60±0.07 74±3 7.04 13 146
Sample Change of shape HSR Gather Form MP-140
Contrast (+PCT-TAD) 0.83±0.26 0.31±0.08 69±4 273 67.2
5.0g Hoechst/L-493 shifted in the time of 24 hours 0.90±0.02 0.43±0.04 83±0 268 61.5
5.0g Hoechst/L-493 does not shift 0.83±0.12 0.40±0.01 80±2 280 58.9
2.5g Dowex Optipore L-493 Baxter Lot FX 1032 D9620042R do not shift 0.24±0.04 0.38±0.02 47±3 240 71.3
The relatively storage that table 13B. (7 days) Hoechst-L493 fibrotic mediators and standard are removed equipment is platelet yield and external function after 7 days
Sample Platelet count (* 10 11/300mL) Yield (%) pH pCO 2 pO 2
Contrast (+PCT-IAD) 3.45±0.06 100 6.97 13 126
5.0g Hoechst/L-493 shifted in the time of 24 hours 3.28±0.22 95±7 6.90 18 105
5.0g Hoechst/L-493 does not shift 3.11±0.15 90±5 6.88 21 100
2.5g Dowex Optipore L-493 Baxter Lot FX 1032 D96F20042R do not shift 2.29±0.07 66±2 7.04 9 161
Sample Change of shape HSR Gather Form MP-140
Contrast (+PCT-IAD) 0.54±0.05 0.25±0.03 48±0 284 80.1
5.0g Hoechst/L-493 shifted in the time of 24 hours 0.76±0.02 0.64±0.16 86±11 267 72.7
5.0g Hoechst/L-493 does not shift 0.67±0.00 0.29±0.04 79±7 280 66.1
2.5g Dowex Optipore L-493 Baxter Lot FX 1032 D96F20042R do not shift 0.31±0.09 0.28±0.06 29±11 261 77.1
The platelet yield of Hoechst-L493 IAD (5.0g) is removed the yield of equipment (2.5g) significantly better than standard.Realized loss＜10% in the storage that contacts 7 days continuously with Hoechst-L493 IAD (5.0g).The platelet unit of handling with Hoechst-L493 IAD all change of shape, gather and GMP-140 aspect present preferable performance than the contrast of no IAD.Keep and Hoechst-L493 IAD) (5.0g) contact whole 5 days platelet than contact 24 hours afterwards the platelet of transfer demonstrate comparable external function.What is interesting is that the platelet that keeps contacting with Hoechst-L493IAD shows better in the GMP-140 test.Both intermediate performances is widely different after 7 days.This observation hint has reduced the speed of expressing by hematoblastic p-protein isolate in the storage with contacting of IAD.
Second research is to seek to determine whether on platelet yield or external function bigger reduction is arranged with the medium of a large amount more in the IAD of the Hoechst-L493 medium that contains 5.0g and 7.5g.In this research, Hoechst-L493 IAD passes through γShe Xianmiejun.Standard is removed equipment (2.5g Dowex XUS-43493) and is included in this research.Platelet keeps contacting with the equipment of removing in the whole process of this research.With storage after 5 and 7 days platelet count and the result of external function collect in respectively among table 14A and the table 14B.
Table 14A. (5 days)
The evaluation of the Hoechst-L493 fibrotic mediators of γShe Xianmiejun
Preserve platelet yield and external function (not shifting) after 5 days
Sample Platelet count (* 10 11/300mL) Yield (%) pH pCO 2 pO 2
Contrast (+PCT-IAD) 3.69±0.19 100 6.89 25 88
5.0g Hoechst/L-493 3.68±0.16 93±6 6.89 27 70
7.5g Hoechst/L-493 3.44±0.11 87±5 6.87 26 84
2.5g Dowex Optipore L-493 Baxter Lot FX 1032 D96F20042R 2.85±0.13 72±5 7.04 13 145
Sample Change of shape SR Gather Form MP-140
Contrast (+PCT-IAD) 0.56±0.12 0.26±0.04 73±1 289 65.1
5.0g Hoechst/L-493 0.45±0.04 0.40±0.00 76±2 278 54.5
7.5g Hoechst/L-493 0.48±0.06 0.25±0.01 73±2 290 55.4
2.5g Dowex Optipore L-493 Baxter Lot FX 1032 D96F20042R 0.04±0.06 0.30±0.01 38±3 232 62.8
Table 14B. (7 days)
The evaluation of the Hoechst-L493 fibrotic mediators of γShe Xianmiejun
Preserve platelet yield and external function (not shifting) after 7 days
Sample Platelet count (* 10 11/300mL) Yield (%) pH pCO 2 pO 2
Contrast (+PCT-IAD) 3.88±0.18 100 6.99 18 93
5.0g Hoechst/L-493 3.67±0.08 95±5 6.92 22 81
7.5g Hoechst/L-493 3.48±0.07 90±4 6.91 20 91
2.5g Dowex Optipore L-493 Baxter Lot FX 1032 D96F20042R 2.65±0.22 68±6 7.04 9 159
Sample Change of shape HSR Gather Form
Contrast 0.53±0.12 0.23±0.02 65±0 260
5.0g Hoechst/L-493 0.54±0.00 0.30±0.01 83±8 266
7.5g Hoechst/L-493 0.53±0.09 0.29±0.01 81±13 280
2.5g Dowex Optipore L-493 Baxter Lot FX 1032 D96F20042R 0.23±0.04 0.25±0.03 33±9 228
The result who in table 14A and 14B, compiles with study at first in observed result similar.The hematoblastic yield that contacts with Hoechst-L493 IAD than and standard to remove the hematoblastic yield that equipment contacts much higher.7.5g the platelet yield of Hoechst-L493 IAD is a little less than the platelet yield of 5.0g IAD.The platelet of handling with Hoechst-L493 IAD in all in vitro testses presents and can compare with the contrast platelet.Gathering at the 7th day in the test and to compare the platelet that contacts with Hoechst-L493 IAD with the contrast of no IAD and demonstrate the performance of having improved.Do not carry out the GMP-140 test at the 7th day.
Remove equipment (the loose XUS-43493 of 2.5g) and compare when contact 5 days standard of storage with platelet, (5.0,7.0g) Zhi Bei IAD makes that external function is good with the Hoechst-L493 medium.And, through 150 μ M 4 '-(4-amino-2-oxa-) butyl-4,5 ', 8-trimethylpsoralen+3.0J/cm 2The platelet that UVA handles demonstrates by change of shape when contacting 5 and 7 days with Hoechst-L493 IAD (5.0g), gathers the external function with the indicated improvement of GMP-140.Relatively another storage that studies have shown that band IAD of preserving in hematoblastic 5-7 days with or without IAD (5.0g Hoechst-L493) can improve the platelet performance, shown in external function test.
Embodiment 17
AQF fibrosis pearl and the comparison of free pearl in PRBC.This research relatively uses the AQF fibrosis pearl of Ambersorb 572 and free pearl to remove S-300 (N-(9-acridinyl)-Beta-alanine) and glutathion from PRBC.
By under 2100rpm with whole blood centrifugalize 5 minutes and squeeze out blood plasma, every then unit adds 84mL Erythrosol and makes PRBC.6 unit that mated through ABO are incorporated in the Clintec Viaflex bag of a 3.0L.About 300mL turned back among 146 bags of each initial PL and with the 150mM glutathion of 6.0mL quantitatively to the ultimate density of 3.0mM and with the 30mM S-300 derivant of 3.0mL quantitatively to the ultimate density of 300 μ M.With its manual mixing and it was at room temperature cultivated 4 hours.
These PRBC are transferred to the 4.8g Ambersorb pearl (400g/m that is contained in the AQF fibrotic mediators 2) or the 1L PL1813 bag of one of two adsorption plants of the loose Ambersorb pearl of 4.8g in.These fibrotic mediators or loose pearl are enclosed in a Tekto knits in the system mesh bag.Each adsorption plant is repeated secondary and will be in the unit among 1813 bags of the PL that does not have adsorption plant in contrast.They were at room temperature preserved 24 hours on the platelet electromagnetic shaker, transfer to then under the quiescent conditions 4 ℃ of storages down.With S-300 derivant and glutathion batching before, handling after but before transferring to adsorption plant and 2,4,6,8,20 and 24 hours and transfer to adsorption plant after sampled in these unit in 1,2 and 5 weeks.Prepare the HPLC that these samples are used for S-300 and glutathion and analyze, the results are shown in the table 15.Also these samples are carried out percentage of hemolysis, ATP concentration, K +Concentration and pH analyze (referring to table 16).
The result of this test hints that Fibrized resin is better than free pearl.Compare with free pearl, this Fibrized resin demonstrates that equivalence is removed S-300 and glutathion has the benefit of improving the erythrocyte function.Table 15 has shown that it is similar that the S-300 of the adsorption plant of two kinds of chemical compounds removes.After 24 hours the glutathion of fibrosis pearl remove kinetics demonstrate good slightly but two equipment all in acceptable level.Table 16 shown the sample handled with regard to erythrocytic function test speech Fibrized resin can with contrast the unit and compare.Two equipment by comparison, ATP and pH are substantially the same.Percentage of hemolysis and K after 24 hours +The free pearl of level is much higher, and this explanation erythrocyte function has been lost basically.
Table 15. uses Ambersorb 572 pearls and the free pearl in fibrotic mediators to remove S-300 and glutathion from PRBC.The residual concentration of chemical compound in the PRBC (S-300) or the supernatant (glutathion) after handling.
Figure 9881384000911
Table 16. is used in percentage of hemolysis, the K of the PRBC that Ambersorb 572 pearls in the fibrotic mediators and Ambersorb 572 free pearls handle +, ATP and pH relatively.
Figure 9881384000921
Embodiment 18
Alr mode is to the influence of removing of the S-300 that uses fibrosis Ambersorb 572 and glutathion and to the influence of erythrocyte function.This research is conceived to stirring means used in the IAD processing procedure to the influence of removing S-300 (N-(9-acridinyl)-Beta-alanine) and to hematocrit percentage rate, percentage of hemolysis, ATP concentration, K +The influence of concentration and pH.
Preparation PRBC is as a storehouse and as handling among the embodiment 17.After at room temperature handling 4 hours, 6 unit are transferred to (AQF produces, 400g/m by the Ambersorb 572 that is closed in the 4.8g of the Tekto system of knitting in the mesh bag 2) among the IAD that forms.These IAD are included in the 1L PL1813 bag.The contrast unit is transferred to and do not had in the PL1813 of the IAD bag.Then with the following processing of they processes:
Test article Stir type at room temperature at first 24 hours 4 ℃ of holding conditions
1 Platelet electromagnetic shaker (72 rev/mins) Static
2 Platelet electromagnetic shaker (72 rev/mins) Track electromagnetic shaker (intermittently)
3 Track electromagnetic shaker (72 rev/mins) Static
4 Platelet rotary apparatus (6 rev/mins) Static
5 Nutator (three-dimensional rotation, 24 rev/mins) Static
6 Static (not stirring) Static
7 (contrasts) Platelet electromagnetic shaker (no medium or closure) Static
With similarly sampled in the unit among the embodiment 17.The result shows that preferred a certain stirring means is used to remove S-300 and glutathion.Although the track electromagnetic shaker demonstrates than the lower level haemolysis of other method, all modes obtain identical removing.Table 17 has shown S-300 and glutathione level.Table 18 has shown erythrocytic function.
Table 17 is handled with different alr modes and is had S-300 and a glutathione level in the unit of AQF fibrosis Ambersorb 572.
Figure 9881384000941
PS=platelet electromagnetic shaker, OS=track electromagnetic shaker, PR=platelet rotary apparatus, N=Nutator.Percentage of hemolysis, K that table 18 uses fibrosis AQF Ambersorb 572 and uses different alr modes to obtain +, ATP and pH result.
Embodiment 19
Removing of the activatory complement of handling by fibrosis Ambersorb 572 of PRBC.This research is conceived to then remove formation complement fragment C3a and SC5b-9 in PRBC afterwards with the AQF fibrotic mediators with S-300 derivant and glutathion processing.
PRBC is as a storehouse and by each is handled among the embodiment 17 in preparation.After at room temperature handling 4 hours, these unit are transferred to contain among 1813 bags of the following 1L PL.
Test article (PRBC unit #) Explanation
1 No IAD
2 No IAD-cultivates through ice
3 4.8g Ambersorb IAD(400g/m 2) (no closure)
4 7 acetyl cellulose films (diameter 47mm)
The acetyl cellulose film is known to be to make complement activation and be used as over against photograph.All unit were all at room temperature handled 24 hours before the static storage down at 4 ℃ except that unit 2.Unit 2 is 4 ℃ of storages continuously down.
Before IAD handles, in the processing procedure after 4,8 and 24 hours and 5 days, from each test article, take out three 1.5mL samples, with the centrifugalize 15 minutes and each supernatant of 450 μ L mixed with the cold 200mM EDTA of 50 μ L and under 2000xg of each sample through vortex.With them quick freezing and storage under-70 ℃ on dry ice.
Enzyme immunity test (Quidel) is used to detect the formation of complement fragment C3a and SC5b-9.These segmental existence are the identical activatory symbols of complement.This test relate to by with bond this target fragment and use the colour developing quality testing of this HRP to survey of the bonded mouse antibody of horseradish peroxidase (HRP).Working sample absorbance and according to the fragment concentrations in the standard curve calculation sample.With similarly also these samples are estimated S-300, glutathion and haemolysis among the embodiment 17.
The result shows and compares that complement activity reduces in the sample of handling with Ambersorb 572 pearls in the AQF medium.Table 19 demonstrates with regard to contrast, and complement activity is than 4 ℃ of following continuously low in the samples of storage.The sample of handling with IAD demonstrates than low in the level of the C3a of 5 days contrast and SC5b-9, and C3a is near detectable limit after 24 hours.Table 20 demonstrates removing by carrying out with the AQF medium is desirable of S-300 and glutathion.
Table 19 is through the complement fragment C3a and the SC5b-9 level of different disposal.
Table 20 is through S-300 concentration (PRBC), glutathione concentrations (supernatant) and the percentage of hemolysis of different disposal.
Figure 9881384000972
Embodiment 20
Improve the blood compatibility of adsorbent by inert particle substrate.The present embodiment proof is fixed on absorbent particles the blood compatibility and the removing basically less than influence low molecular weight compound of having improved adsorbent in the inert particle substrate.In addition, the present embodiment support is fixed on absorbent particles in the inert base (fiber or granule) argument of the conventional method that is the compatibility of raising adsorbent blood.Under the results are shown in of the IAD that forms by the absorbent particles that is fixed in fibre substrate and the particle matrix.
The medium of being studied comprises the Purolite MN-200 absorbent particles (200-1200 μ m) that is fixed in ultra-high molecular weight polyethylene (UHMWPE) matrix of microparticles in the present embodiment.Representational medium is that (Fairburn GA) produces by Porex.Mix the dish that forms the fixed absorbent medium by the UHMWPE that about 50% (w/w) PuroliteMN-200 (200-1200 μ m) and 50% (w/w) is had same particle size.This mixture is put into cylindrical cavity be heated under pressure, its condition is enough to make the UHMWPE particle fusion and surrounds absorbent particles.The diameter of gained dish is 3.50 inches and thickly is about 0.250 inch.These coil heavily about 24g, corresponding in each dish 12g MN-200 being arranged approximately.Dielectric disc is put into plastics storage container (PL2410, Baxter HealthcareCorp.) and the gamma-rays by having 25-40kGy, and (CA) whole device is sterilized in radiation, thereby makes IAD for Sterigenics, Hayward.
This fibre substrate IAD comprises adsorbent/m with 300g 2Load be fixed on Purolite MN-200 in the nonwoven polyester substrate.Fixedly Purolite MN-200 that will about 2.5g (quantity of sorbent) puts into that (Tetko, DePew is in the bag that NY) constitutes by 30 μ m Woven polyester materials.This bagging apparatus is put into plastics storage container (PL2410, Baxter Healthcare Corp.) and the gamma-rays by having 25-40kGy, and (CA) whole device is sterilized in radiation for Sterigenics, Hayward.
Comprise 35% autologous plasma that is suspended in about 300mL, platelet (3-5 * 10 in the 65% platelet interpolation solution 11Cell) the platelet concentrate unit through the ABO coupling is that (Sacramento CA) obtains from SacramentoBlood Band.15mM ammonification psoralen from 3mL to each unit that give (4 '-(4-amino-2-oxa-) butyl-4,5 ', the 8-trimethylpsoralen) before these platelet unit are compiled and separately.By in UVA irradiation apparatus (BaxterHealthcare Corp.), using 3.0J/cm 2UVA irradiation each unit is stood photochemical treatment.Two platelet unit through photochemical treatment are transferred in the PL2410 plastics storage container with two test SRD.A treated unit contacts with IAD in contrast and not.In process of the test, all unit are placed on platelet electromagnetic shaker under the room temperature (22 ℃) (Helmer, Noblesville, IN) in.
Measure the adsorption dynamics adsorption kinetics of the psoralen of each IAD embodiment.In storages in 8 hours at first, shifted out each platelet unit sample (about 1mL) at interval in 2 hours.Analyze remaining psoralen level in these samples by high pressure lipuid chromatography (HPLC) then.Then at room temperature preserved 5 days, measure platelet count, pH and dissolved gas in each unit.Also estimate platelet function, comprising by carrying out in vitro tests: change of shape, gather, hypotonic shock response, GMP-140 (expression of p-protein isolate) and form score.
Figure 23 has shown the psoralen adsorption dynamics adsorption kinetics.Two IAD remove psoralen to HPLC and test the quantitative limit in 8 hours incubation.With respect to fiber IAD (2.5g), because therefore the adsorbent (about 12g) of the more a large amount of granule IAD has adsorption dynamics adsorption kinetics faster.
Platelet count, pH mensuration and external platelet function test result are collected in the table 21.Fiber and particle matrix IAD have the recovery rate of blood platelet more than 90%.Consider that granule IAD contains about 12g MN-200, this observation has special influence to this granule IAD.Notice that containing 2.5g typically makes platelet loss in 5 days 25-35% without the equipment of removing of fixed absorbent particles.Therefore containing 12g removes＞50% platelet as scheduled without fixed particulate equipment.Obviously, adsorbent is fixed on greatly reduce in the particle matrix platelet loss simultaneously psoralen to remove kinetics still very fast.
The result of external platelet function research is collected in the table 21 time half.Equally, two kinds of IAD all demonstrate satisfied result.Because single high measurement value demonstrates big standard deviation, so a little higher than contrast of hypotonic shock response.The performance that other test shows with all is basic identical, and it is fine that these IAD show in gathering test really.
5 days the platelet yield of table 21. fibre substrate and particle matrix IAD and the comparison of external platelet function
Sample Platelet count (* 10 11/300mL) The % yield pH pCO 2 pO 2
Contrast 2.89±0.06 100 6.95 22 116
AQF fibre substrate IAD (300g/m 2MN-200) 2.71±0.11 94±4 6.96 23 112
Porex particle matrix IAD (50% MN-200) 2.61±0.10 90±4 6.90 22 123
Sample Change of shape HSR Gather Form GMP-140
Contrast 1.03±0.33 0.71±0.28 44±3 308 67.0
AQF fibre substrate IAD (300g/m 2MN-200) 0.96±0.14 0.53±0.06 60±0 311 63.9
Porex particle matrix IAD (50% MN-200) 1.00±0.05 0.42±0.01 64±1 304 66.2
Can be penetrated in the medium geometry more fully by making liquid by this configuration, thereby further this particle matrix IAD be optimized with the change dish.Thin dish can use the adsorbent of less amount to obtain the suitable kinetics of removing.
Except that the geometry of optimization dish, wetting apparatus and therefore can further improve this adsorption dynamics adsorption kinetics by the moistening that increases medium.In the initial period of removing, the inherent hydrophobic performance of UHMWPE substrate makes this equipment moistening slow.Can be used in the strategy that improves moistening comprises and uses wetting agent (for example, glycerol, poly-oxireme, Polyethylene Glycol, hydrophilic polymer) or handle through gaseous plasma glow discharge.Different with wetting agent, handle the surface chemistry that can be directly used in change UHMWPE adhesive stroma through glow discharge.

Claims (10)

1. method that reduces the concentration of biological respinse modifier in the celliferous biological composition, wherein this method has kept the required biological activity of biological composition basically, comprise with this biological composition of a kind of device processes, wherein this equipment comprises the inert base that contains high absorbent particles, and wherein the diameter range of absorbent particles is at the about 1500 μ m of about 100 μ m-, and wherein this equipment is used for batch process.
2. according to the process of claim 1 wherein that this biological respinse modifier is a complement activation.
3. according to the process of claim 1 wherein that the absorbent particles of this equipment is for having good wettable polyaromatic absorbent particles.
4. according to the process of claim 1 wherein that the absorbent particles of this equipment is the active carbon that is obtained by synthetic source.
5. according to the process of claim 1 wherein that the inert base of this equipment is a synthetic polymeric fibers, and wherein this synthetic polymeric fibers comprises the polymer core with high melting temperature that sheath with low fusion temperature surrounds.
6. according to the process of claim 1 wherein that the inert base of this equipment is a particle network, and wherein this particle network comprises polyethylene particle.
7. according to the process of claim 1 wherein that this biological composition comprises platelet.
8. according to the process of claim 1 wherein that this biological composition comprises erythrocyte.
9. according to the process of claim 1 wherein that this method also reduces the concentration of psoralen derivant in the biological composition or acridine derivatives.
10. according to the process of claim 1 wherein that this method also reduces the concentration of dyestuff in the biological composition or quencher.
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