JP2003527230A - Flow devices for the reduction of compounds from biological compositions and methods of use thereof - Google Patents

Abstract

  (57) [Summary] Provided are methods and devices for reducing the concentration of low molecular weight compounds in a biological composition, but substantially maintaining the desired biological activity of the biological composition. This device comprises very porous sorbent particles, and the sorbent particles are immobilized on an inert matrix.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application is co-pending application number 09/003, filed January 6, 1998.
113, a continuation-in-part application, which is hereby incorporated by reference.

TECHNICAL FIELD The present invention relates to methods and devices for the reduction of compounds from biological compositions. These compounds have a molecular weight in the range of about 100 g / mol to about 30,000 g / ml.

Background Art There is a major body of research relating to the removal of substances from blood products. Most of this study is concerned with leukopenia. For example, M. N. Boomgaard et al., Tran
sfusion 34: 311 (1994); Bertolini et al., Vo
x Sang 62:82 (1992); M. Youstra-Di
See jkhuis et al., Vox Sang 67:22 (1994). Platelet filtration is the most common method used for leukopenia of platelet concentrates.
For example, M. Bock et al., Transfusion 31: 333 (1991).
(Sepacell PL-5A, Asahi, Tokyo, Japan); J
． D. Sweeney et al., Transfusion 35: 131 (1995).
(Leukotrap PL, Miles Inc., Covina, CA);
And M.M. van Marwijk et al., Transfusion 30:34.
(1990) (Cellselect, NPBI, Emmer-Compasc
uum, The Netherlands; Immugard Ig-500,
See Terumo, Tokyo, Japan). However, these current filtration mechanisms are acceptable for the removal of relatively low molecular weight compounds, including, for example, psoralens, psoralen photochemical reaction products, and other compounds commonly used in the treatment of biological fluids. is not.

The process of adsorption has been used to isolate selective blood components on phospholipid polymers. For example, several copolymers with different charges have been evaluated for interactions with blood components, including platelet adhesion and protein adsorption. K. Ishihara et al. Biomed. Mat. Res. 28:13
47 (1994). However, such polymers are not designed for adsorption of low molecular weight compounds.

Various dialysis means are capable of removing low molecular weight compounds from plasma and whole blood. For example, dialysis can successfully remove low molecular weight toxins and pharmaceutical compounds. Thus, dialysis can be used to remove psoralens and psoralen photochemical reaction products from blood products, for example. Unfortunately, current dialysis procedures involve very complex and expensive devices. Thus, the use of dialysis equipment is not practical for decontaminating large volumes of blood products.

Polystyrene divinylbenzene for adsorption of methylene blue, silica gel,
And the use of acrylic ester polymers has already been described. For example, PC
T Pub. No. WO 91/03933 discloses free adsorbent resins (eg Am
Berlites (Rohm and Haas (Frankfrut, Ger
many) and Bio Beads (Bio-Rad Laboratori)
es (Munich, Germany)). But,
Without very careful removal of the adsorbent resin after exposure to blood products, these methods pose a risk of infusion of resin particles.

Furthermore, leukocytes and viral inactivators (eg psoralen, hypericin,
Also disclosed are devices and processes for the removal of methylene blue, toluidine blue, and dyes such as crystal violet). In particular, PCT Publication No. WO 95/18665 describes a filter comprising a knitted woven web comprising a mechanically stable polymeric substrate. The web itself comprises interlocking woven fibers forming a matrix with voids and fibrillated particles disposed within the voids. However, this device causes a significant reduction in Factor XI activity.

[0008] Thus, a simpler, safer, and more effective method for reducing the concentration of low molecular weight compounds in a biological composition, while substantially maintaining the biological activity of the treated composition. Economic means are needed.

DISCLOSURE OF THE INVENTION Devices for reducing the concentration of compounds from biological compositions are provided. The compound has a molecular weight in the range of about 100 g / mol to about 30,000 g / mol. The device is a flow device. An example of the flow device is shown in FIG.
Flow devices are known in the literature and are described, for example, in PCT Publication WO
96/40857, which is incorporated herein by reference. The flow device is constructed by perfusing a blood product (single) through the flow device,
Allows the reduction of the concentration of low molecular weight compounds from substances such as blood products.

Typical compounds include pathogen inactivating compounds, dyes, thiols, plasticizers,
And activated complement. Devices are provided that include a three-dimensional network of adsorbent particles immobilized by an inert matrix. This immobilization reduces the risk of leakage of free adsorbent particles into the blood product. Furthermore, immobilization of adsorbent particles with an inert matrix simplifies manufacturing by reducing the problems associated with handling free adsorbent particles.

The present invention provides a method for reducing the concentration of a biological response modifier in a biological composition, the method substantially reducing the desired biological activity of the biological composition. maintain. The method involves treating the biological composition with a device.

In one embodiment, the device comprises an inert matrix containing high adsorbent particles, wherein the adsorbent particles range in diameter from about 1 μm to about 200 μm, and the device comprises a flow process. For use in.

In another embodiment, the biological response modifier is activated complement.

[0014]   In another embodiment, the biological composition is plasma.

[0015]   In another embodiment, the adsorbent material has a length less than 3 times the width.

In another embodiment, the adsorbent particles comprise a supercrosslinked polystyrene network.

In another embodiment, the adsorbent particles are activated carbon particles, wherein the activated carbon particles have a surface area greater than about 1200 m ² / g.

In another embodiment, the activated carbon particles are formed by steam activation of coconut shell.

In another embodiment, the inert matrix of the device is a fiber network composed of cellulose.

In another embodiment, the inert matrix is a particle network formed by sintering particles of ultra high molecular weight polyethylene together with particles of an ultra crosslinked polystyrene network.

[0021] In another embodiment, the method further reduces the concentration of the psoralen derivative, acridine derivative, dye, or quencher in the biological composition.

Best Mode for Carrying Out the Invention A device is provided for reducing the concentration of a compound from a biological composition. The compound has a molecular weight in the range of about 100 g / mol to about 30,000 g / mol. The device is a flow device. An example of the flow device is shown in FIG.
Flow devices are known in the literature and are described, for example, in PCT Publication WO
96/40857, which is incorporated herein by reference. The flow device is constructed by perfusing a blood product (single) through the flow device,
Allows the reduction of the concentration of low molecular weight compounds from substances such as blood products.

Representative compounds include pathogen inactivating compounds, dyes, thiols, plasticizers,
And activated complement. Devices are provided that include a three-dimensional network of adsorbent particles immobilized by an inert matrix. This immobilization reduces the risk of leakage of free adsorbent particles into the blood product. Furthermore, immobilization of adsorbent particles with an inert matrix simplifies manufacturing by reducing the problems associated with handling free adsorbent particles.

Definitions The term “acridine derivative” means acridine (dibenzo “b, e” pyridine;
10-azaanthracene) refers to a chemical compound containing a tricyclic structure. The compound has (and can bind to) noncovalently bound nucleic acid by intercalation. The term "aminoacridine" refers to an acridine compound having one or more nitrogen-containing functional groups. Examples of aminoacridines include 9-aminoacridine and acridine orange (shown in Figure 11).

The term “adsorbent particles” broadly refers to any natural or synthetic particle that is capable of interacting with a molecule in a liquid, thus causing the molecule to be removed from the liquid. Examples of naturally occurring adsorbents include activated carbon, silica, diatomaceous earth, and cellulose,
It is not limited to these. Examples of synthetic adsorbents include, but are not limited to, polystyrene, polyacrylic acid, and carbonaceous adsorbents. Adsorbent particles are often porous, often have large surface areas, and have various functional groups (eg, ionic, hydrophobic, acidic, basic) that can affect the way the adsorbent interacts with the molecule. ).

The terms “aromatic”, “aromatic compound” and the like broadly refer to compounds having a ring of atoms having delocalized electrons. The monocyclic compound benzene (C ₆ H ₆ ) is a common aromatic compound. However, electron delocalization can occur on more than one adjacent ring, such as naphthalene (2 rings) and anthracene (3 rings). Various classes of aromatic compounds include aromatic halides (aryl halides), directional heterocyclic compounds, aromatic hydrocarbons (allenes), and aromatic nitro compounds (aryl nitro compounds). Not limited to.

The term “biocompatible coating” means that it is harmful when contacted with blood products,
A hydrophilic polymer that does not cause a toxic or immunological response and that makes the surface more biocompatible by reducing cell adhesion, reducing protein adsorption, or modifying cell function. Broadly refers to surface coating. Suitable coatings are biocompatible, even if they have a minimal effect on the biological material to which they are exposed. "Minimal" effect means that there is no significant biological difference compared to the control. In a preferred embodiment, the biocompatible coating modifies the surface blood compatibility of the polymeric structure. For example, poly (2-hydroxyethylmethacrylate) (pHEMA) is frequently used for coating materials used in medical devices (eg blood filters).

The term “biocompatible housing” broadly refers to a container, bag, container, receptacle, etc. that is suitable for containing a biological material such as plasma. A suitable container, if it has a minimal effect on the biological material it contains,
They are biocompatible. A "minimal" effect means, for example, that there is no significant biological difference in blood product function on plasma, as compared to the controls herein above. Thus, the biological composition can be stored in the biocompatible housing prior to infusion into the recipient.

The term “biological fluid” includes human or non-human whole blood, plasma, platelets, red blood cells,
Included are white blood cells, serum, lymph, saliva, milk, urine, or products derived from any of the above, alone or as a mixture, with or without chemically added solutions. Preferably the liquid is blood or blood products with or without chemically added solutions, most preferably plasma.

[0030]   The term "blood bag" refers to a blood product container.

The term “blood product” refers to elements of fluid and / or associated cells that pass through the circulatory system of the body (eg, red blood cells, white blood cells, platelets, etc.); blood products include blood cells, platelets, etc. Included, but not limited to, mixtures, serum, and plasma. The term "platelet mixture" refers to one type of blood product in which the cellular elements are primarily or exclusively platelets. Platelet concentrate (PC) is
Platelets are one type of platelet mixture that is combined with a subnormal portion of plasma. In blood products, synthetic media can be created such that the volume is normally occupied by plasma; for example, platelet concentrates can fix platelets suspended in 35% plasma / 65% synthetic medium. Frequently, the synthetic medium contains phosphate.

The term “blood separation means” broadly refers to devices, machines, etc. capable of separating blood into blood products (eg platelets and plasma). The apheresis system is one type of blood separation means. Apheresis systems generally include a blood separation device, a complex network of tubes and filters, collection bags, anticoagulants, and computerized means for controlling all components.

The term “crosslinked” broadly refers to linear molecules linked together to form a two or three dimensional network. For example, divinylbenzene (DVB) acts as a crosslinker in the formation of styrene-divinylbenzene copolymer. The term also includes "hypercrosslinking", where a hypercrosslinked network is created by cross-linking linear polystyrene chains with a bifunctional agent, either in solution or in the expanded state. Various bifunctional agents can be used for crosslinking (eg, Davan.
kov and Tsyurupa, Reactive Polymers 13:
24-42 (1990); Tsyurupa et al., Reactive Polym.
ers 25: 69-78 (1995)).

The term “cyclic compound” refers to a compound having a ring of one (ie, monocyclic compound) or more than one (ie, polycyclic compound) atoms. The term is not limited to compounds with rings containing the specified number of atoms. Most cyclic compounds contain a ring of 5 or 6 atoms, but rings containing other numbers of atoms (eg, 3 or 4 atoms) are also included in the invention. The identity of the atoms in the ring is not limited, but the atoms are usually primarily carbon atoms. Generally speaking, the rings of a polycyclic compound are adjacent to each other; however, the term "polycyclic" compound includes compounds that contain multiple rings that are not adjacent to each other.

The term “dye” broadly refers to compounds that impart color. Dyes generally include chromophore and auxochrome groups attached to one or more cyclic compounds. The color is due to the chromophore, but the staining affinity is due to the auxochrome. Dyes are grouped into many categories; azine dyes (eg, neutral red, safranine, and azocarmine B); azo dyes; azocarmine dyes; dephenimethane dyes; fluorescein dyes; ketoimine dyes; rosaniline dyes; triphenylmethane dyes. ;
Phthalocyanine; and hypericin. It is contemplated that the methods and devices of the present invention can be practiced with any dye that is a cyclic compound.

The term “fiberized resin” generally refers to the immobilization of adsorbent material, including, for example, resin entrapped or attached to the fiber network. In another embodiment, the fiber network is composed of polymer fibers. In another embodiment, the fibers are
It consists of a polymer core with a high melting point (eg polyethylene terephthalate [PET]) surrounded by a polymer sheath with a relatively low melting point (eg nylon or modified PET). Fiberized resins can be produced by heating the fiber network under conditions that do not deleteriously affect the adsorption capacity of the resin to a significant extent. If the resin comprises beads, heating is done so that the adsorbent beads adhere to the outer polymer sheath to produce "fiberized beads." Fiberizing for use in the removal of cyclic compounds (eg, psoralens and especially aminopsoralens) and other products by producing a fiberized resin containing a known amount of adsorbent beads per given area. A sample of the formed resin can be obtained by cutting a given area of fiberized resin, rather than weighing the adsorbent beads.

The term “filter” refers broadly to devices, materials, etc. that allow certain components of a mixture to pass while retaining other components. For example, the filter may comprise a mesh having pores sized to allow the passage of blood products, such as plasma, while retaining other components such as resin particles. The term "filter" is not limited to the means by which particular components are retained.

The term “flow adapter” refers to a device capable of controlling the flow of certain substances, such as blood products. The flow adapter may further function to prevent the passage of some of the adsorbent resin material.

The term “heterocyclic compound” broadly refers to cyclic compounds in which one or more rings contains more than one type of atom. Generally, carbon represents the main atom, but as other atoms,
Examples include nitrogen, sulfur, and oxygen. Examples of heterocyclic compounds include furan, pyrrole, thiophene, and pyridine.

The phrase “high temperature activation process” typically refers to a high temperature process that results in a change in surface area, porosity, and surface chemistry of a treated material by thermal decomposition and / or oxidation of a starting material.

The term “immobilized adsorbent device (IAD)” refers to immobilized adsorbent material entrapped or attached to an inert matrix. When the inert matrix is a fiber network, the term IAD can be used interchangeably with the term fiberized resin.

The term “inert matrix” refers to any synthetic or naturally occurring entity that can be used to immobilize adsorbent particles without substantially affecting the desired biological activity of the blood product. A fiber or polymer material. The matrix may contribute to reducing the concentration of small organic compounds, but typically does not substantially contribute to the adsorption or removal process. In addition, the inert matrix can interact with cellular or protein components that result in cell depletion (eg, leukopenia) or protein or other molecule elimination. The matrix can undergo surface treatments or coatings to enhance functionality. For example, the matrix may be subjected to a hydrophobic coating or glow discharge treatment to increase biocompatibility, increase wettability, and / or facilitate priming.

The term “in-line column” refers to a container, usually cylindrical in shape, having an inlet and an outlet and containing substances arranged to reduce the concentration of small organic compounds from blood products.

The term “isolate” refers to separating a substance from a mixture that contains more than one component. For example, platelets can be separated from whole blood. The product isolated is
It does not necessarily mean complete separation of the product from the other components.

The term “macropore” generally means that the diameter of the pore is greater than about 500Å. The term micropore refers to a pore with a diameter less than about 20Å. The term intermediate hole is about 20
A hole with a diameter greater than Å and less than about 500 Å.

The term “macroporous” is used to describe a porous structure having a substantial number of pores with a diameter greater than about 500Å.

The term “macroreticular” is a relative term meaning that the structure has high physical porosity (ie, a large number of pores are present), and the porous adsorbent structure is macropores and micropores. Have both.

The terms “mesh enclosure”, “mesh pouch” and the like refer to enclosures, pouches, bags, etc. manufactured to include a plurality of openings. For example, the present invention contemplates a pouch that comprises immobilized adsorbent particles having a blood product in contact with the immobilized adsorbent particles, but having pores sized to retain the immobilized adsorbent particles within the pouch.

The term “photochemical reaction product” refers to the product resulting from a photochemical reaction of a psoralen or other dye (eg, methylene blue, phthalocyanine) upon exposure to ultraviolet radiation.

The term “polyaromatic compound” refers to a backbone such as polyethylene terphalate,
Alternatively, it refers to a polymeric compound containing aromatic groups, either as pendant groups such as polystyrene, or both.

The term “polystyrene network” broadly refers to polymers containing styrene (C ₆ H ₅ CH═CH ₂ ) monomers; polymers are linear and composed of single-bonded alkane chains with phenyl substituents. Can be, or in general, m- or p
-Can be cross-linked with phenylene residues or other bifunctional or hyper-crosslinked structures to form a two-dimensional polymer backbone.

The term “psoralen removal means” refers to substances or devices capable of removing, for example, greater than about 80% psoralen from blood products; preferably greater than about 90%; most preferably greater than about 99%. The psoralen removal means may also remove other components of the blood product, such as psoralen photochemical reaction products.

The phrase “reduces concentration” refers to the removal of some portion of low molecular weight compounds from a biological composition. At the same time, the reduction in concentration is preferably greater than about 70%, more preferably on the order of about 90%, and most preferably on the order of about 99%.

The phrase “removing substantially all of the free compound (eg, psoralen, psoralen derivative, isopsoralen, acridine, acridine derivative, or dye) in solution” preferably means that in solution Removal of more than about 80% of free compounds,
More preferably more than about 85% removal, even more preferably more than about 90% removal, and most preferably about 99% or more removal.

The term “resin” refers to a solid support that can interact and adsorb with various small organic compounds, including psoralens, in solution or liquid (eg, blood products), thereby reducing the concentration of elements in solution. Refers to the body (for example, particles or beads). The removal process is not limited to any particular mechanism. For example, psoralens can be removed by hydrophobic or ionic interactions (ie, affinity interactions). The term "adsorbent resin" broadly refers to both natural organic and synthetic materials and mixtures thereof.

The term “sintered medium” applies heat and pressure to a powder or mixture of powders,
Thereby, a structure formed by partially fusing powders or powder mixtures such that a path for a flowing liquid exists through the structure. Porous structures can be prepared by mixing powders of relatively low melting point polymers and heating them so that the plastic particles partially coalesce, but the route to the liquid permeates the porous mass. It is even possible to: Sintered adsorbent media can be similarly prepared by incorporating carbon or other high or non-molten adsorbent particles with a low melting point powder and heating. Methods for preparing porous plastic materials are described in US Pat. Nos. 3,975,481 and 4,110,391.
Nos. 4,460,530, 4,880,843, and 4,9.
25,880 and incorporated herein by reference. This process causes the coalescence of powder particles resulting in the formation of a porous solid structure. Sintered media can be formed into various shapes by placing the polymer powder in a tool that is formed during the sintering process. The adsorbent particles can be introduced into the sintered media by mixing the adsorbent particles with the powdered thermoplastic polymer prior to undergoing the sintering process.

The term “stabilizer” refers to a compound or composition that can optimize the adsorption capacity of a particular adsorbent. Generally speaking, the available stabilizers are water and ethanol (
It should be soluble in other wetting agents), non-volatile in water and ethanol, and safe in infusion in small amounts. Examples of stabilizers include, but are not limited to, glycerol and low molecular weight PEG. A "wetting agent" is distinguishable from a "stabilizer" and thus the former is believed to reopen the adsorbent pores of a resin that is not supercrosslinked (eg, non-giant net resin). Wetting agent
Generally, it does not prevent pore collapse under dry conditions, but stabilizers do. For a general discussion of wetting and wetting agents, see US Pat. No. 5,501, Pall et al.
No. 795, which is incorporated herein by reference.

The phrase “substantially maintaining a desired biological activity of a biological composition” refers to a property of the biological composition that is believed to indicate the potential performance of the composition in a therapeutic environment. To substantially maintain. For example, in the case of plasma, the in vivo activity is the level of coagulation factors such as Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, or Changes in PT and PTT time are not destroyed or are significantly reduced when substantially maintained in plasma when treated by the methods described herein. For example, a change in the level of coagulation factors such as Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI in treated plasma is about 20. %, Preferably less than about 10%. The change in PT and PTT time for treated plasma can be, for example, less than about 3 seconds and greater than about 1 second; preferably 1.5 seconds. Phrases that are substantially maintained for each of the properties associated with the described biological compositions are also described, for example, in Klein H. et al. G. Hen, Standards for
Blood Banks and Transfusion Service
s, 17th edition, Bethesda, MD; American Associate.
It is further contemplated that values acceptable to those of skill in the art may be included, as described in the literature, including on of Blood Banks, 1996, which is incorporated herein by reference.

As used with respect to the devices of the present invention, the term “equivalent thereto” refers to devices that function equivalently with respect to maintaining the biological activity of the biological composition. For example,
An "equivalent" device or matrix containing adsorbent particles is one that also maintains appropriate coagulation factor levels.

The term “low molecular weight compound” refers to about 100 g / mol to about 30,000 g / mo.
Refers to organic or biological molecules having a molecular weight in the range of 1. Low molecular weight compounds include, but are not limited to, the following compounds: small organic compounds such as psoralens, acridines, or dyes; quenchers such as glutathione; plastic extractables such as plasticizers. About 100 g, like activated complement
Biological modifiers having a molecular weight between 1 / mol and about 30,000 g / mol; and polyamine derivatives.

The term “biological composition suitable for infusion” refers to its essential biological properties (
For example, it maintains the coagulation function in crystals) but has very low levels of any undesired compounds (eg, inactive compounds, response modifiers) so that the infusion provides the desired function without adverse side effects. Refers to a biological composition.

As used in phrases such as “against control”, the term “control” refers to experiments performed to study the relative effects of various conditions. For example,
When the biological composition is treated with the device, "untreated control" refers to the biological composition without treatment with the device. This also refers to a comparison between two different types of devices.

The term “4 ′-(4-amino-2-oxa) butyl-4,5 ′ ′, 8-trimethylpsoralen” is alternatively referred to as “S-59”.

The term “N- (9-acridinyl) -β-alanine” instead refers to “5-[(β
-Carboxyethyl) amino] acridine. Furthermore, instead of "S-300
".

The term “XUS-43493” is instead referred to as “Optipore 493”.

The term “non-fibrous adsorbent material” refers to an adsorbent material consisting essentially of particles having a long or longest dimension, a width less than five times, or the narrowest dimension.

Adsorbent Particles An adsorbent that reduces the concentration of a compound in a biological composition but is useful in a device for substantially maintaining the desired biological activity of the biological composition. Particles are provided. Typically, the compound that decreases in the biological composition is about 100 g /
It has a molecular weight in the range of mol to about 30,000 g / mol.

The adsorbent particles can be of any regular or irregular shape that provides for incorporation into the inert matrix, but are preferably approximately spherical. The particles have a diameter greater than about 1 μm; preferably the particles have a diameter greater than about 10 μm, more preferably sintered as a matrix when using a sintered medium as a matrix for the adsorbent. When using a medium, the particles are about 50 μm and about 150 μm
Is the diameter between. Preferably, the particles have a diameter of between about 1 μm and about 200 μm, in this case using a twisted fibrous medium as the matrix, more preferably between about 1 μm and about 50 μm, where As a matrix for the adsorbent, a wet, knitted fiber medium is used.

In one preferred embodiment, the adsorbent particles are activated carbon derived from either natural or synthetic sources. Non-limiting examples of activated carbon include: Picatiff Medicinal, which is PICA USA.
Inc. Norit RO available from (Columbus, OH)
X 0.8, which is available from Norit Americas, Inc. (Atlanta
, GA), Ambersorb 572, which is Rohm
& Haas (Philadelphia, PA), and G-277, which is available from PICA (Columbus, OH).

Preferred activated carbons are those that have been specifically cleaned and / or meet US Pharmacopoeia standards. In addition, activated carbons with a surface area greater than 950 m ² / g are preferred, as activated carbons with a larger surface area are available for low molecular weight compounds and generally show better adsorption, and activated carbons with a surface area greater than 1200 m ² / g Is more preferable. Activated carbons formed by steam activation tend to have a more hydrophobic surface, so for more hydrophobic low molecular weight compounds, these steam activated carbons often have better binding capacity. , And these carbons are preferred. Fewer macroporosity confers selectivity for lower molecular weight compounds over large proteins that mediate biological activity, so activated carbon with less macroporosity, such as activated carbon prepared from coconut shells, is also present. preferable.

In one preferred embodiment, the particles are made of Norit Americas, In.
c. Norit A Supr, available from (Atlanta, GA)
a. Norit A Supra is a USP grade activated carbon formed by steam activation of coconut shells. This activated carbon has a very high total surface area (2000 m ² / g) and is by nature very porous.

In another preferred embodiment, the particles can be a hydrophobic resin. Non-limiting examples of hydrophobic resins include the following polyaromatic adsorbents: Amberlit.
e (registered trademark) adsorbent (for example, Amberlite (registered trademark) XAD-2,
XAD-4, and XAD-16), Rohm and Haas (Phila)
Delphia, PA); Amberchrom® adsorbent, Toso Haas (TosoHaas, Montgomeryville)
, PA); Diaion® // Sepabeads® Adsorbent (eg, Diaion® HP20), M
Tsuchishi Chemical America, Inc. (Whit
e Plains, NY); Hypersol-Macronet
(Registered trademark) Sorbent Resin (for example, Hypersol-Mac
ronet (registered trademark) Sorbent Resin MN-200, MN-1
50, and MN-400), Purolite (Bala Cynwyd, P.
A); and Dowex® Adsorbent (
For example, Dowex (R) XUS-40323, XUS-43493, and XUS-40285), Dow Chemical Company (Mi.
land, MI).

Preferred particles are Dowex® XUS-43493 (Optipo).
commercially available as re (R) L493 or V493) and Purol
Hydrophobic resins that are polyaromatic adsorbents containing supercrosslinked polystyrene networks, such as ite MN-200.

Dowex® XUS-43493 and Purolite MN-
Supercrosslinked polystyrene networks such as 200 are nonionic macroporous and macroreticular resins. Non-ionic macroreticular and macroporous Dowex® XUS-43493 is for psoralen, including, for example, 4 ′-(4-amino-2-oxa) butyl-4,5 ′, 8-trimethylpsoralen. Have a high affinity and have excellent wetting properties. The phrase "excellent wetting properties" means that a dry (ie, essentially anhydrous) adsorbent is contacted with a blood product to effectively reduce the concentration of small organic compounds from the blood product for the adsorbent. This means that it is not necessary to moisten with a wetting agent (eg ethanol) before.

Dowex® XUS-43493 and Purolite MN-
Super-crosslinked polystyrene networks such as 200 are preferably about 10μ.
It is in the form of spherical particles having a diameter in the range of m to about 200 μm. For example, Dow
Adsorbent particles, including ex® XUS-43493, preferably have extremely high internal surface areas and relatively small pores (eg, 46Å). The internal surface area of the particles can be from about 300 to about 1100 m ² / g; preferably 1100 m ² / g. Pore size of particles is greater than 25Å and less than 800Å; preferably about 25Å
~ About 150Å; most preferably about 25Å to about 50Å. Although the present invention is not intended to limit the mechanism by which the reduction of small organic compounds occurs, hydrophobic interactions are believed to be the major mechanism of adsorption. Its porous nature selectively imparts small molecules in the adsorption process by allowing them to access a larger proportion of surface area relative to large molecules (ie proteins) and cells. Pu
Rolite® has many similar characteristics to Dowex® XUS-43493, such as high affinity for psoralen and excellent wetting properties, and is also a preferred adsorbent particle.

Polystyrene particles can be classified as i) conventional networks and ii) hypercrosslinked workpieces based on the mechanism of synthesis and physical and functional characteristics. Preferred adsorbents have large surface areas, non-collapsed pores, and do not require wetting. In addition, preferred adsorbents have low levels of extractable residual monomers, crosslinkers, and other organic extractables.

In the conventional network, divinylbenzene (DVB) is mainly used as a crosslinking agent (
That is, it is a styrene-divinylbenzene copolymer that acts as a reagent that binds with a linear polystyrene chain. These polymer networks include
Included are "gel-type" polymers. Gel-type polymers are homogeneous non-porous styrene-DVB copolymers obtained by copolymerization of monomers. Macroporous adsorbents represent the second class of conventional networks. These are obtained by copolymerization of the monomers in the presence of diluents which precipitate the growing polystyrene chains.
The polystyrene network formed by this procedure has a relatively large internal surface area (up to hundreds of square meters per gram of polymer); Amberl
ite® XAD-4 is prepared by such a procedure.

Contrary to the conventional networks described above, the preferred adsorbents of the present invention (eg Do
wex® XUS-43493) is a hypercrosslinked network.
These networks are prepared by crosslinking linear polystyrene chains either in solution or in the expanded state with bifunctional agents; preferred bifunctional agents have conformationally limited crosslinking. When formed and the adsorbent is essentially anhydrous (ie, "dry"), it is believed to inhibit pore collapse.

Hyperbranched networks are believed to have three main features that distinguish them from conventional networks. First, there are low density polymer chains due to cross-linking, which holds polystyrene chains apart. As a result, adsorbents generally have relatively large pore surface areas and pore diameters. Second, the network can expand; that is, the capacity of the polymer phase increases when it comes into contact with organic molecules. Finally, hypercrosslinked polymers are "distorted" when in the dry state, and the rigidity of the network in the dry state suppresses the chain-to-chain attraction. However, when the adsorbent becomes wet, the strain relaxes, increasing the ability of the network to expand in the liquid culture. Davank
ov and Tsyurupa, Reactive Polymers 13: 2.
7-42 (1990); Tsyurupa et al., Reactive Polymer.
rs 25: 69-78 (1995), which are incorporated herein by reference.

Several crosslinkers have been successfully used to create crosslinks between polystyrene chains, including p-xylene dichloride (XDC), monochlorodimethyl ether (MCDE), 1,4-bis-. Chloromethyldiphenyl (CMDP),
4,4'-bis- (chloromethyl) biphenyl (CMB), dimethylformal (DMF), p, p'-bis-chloromethyl-1,4-diphenylbutane (DP
B), and tris- (chloromethyl) -mesitylene (CMM).
Crosslinks are formed between polystyrene chains by reacting one of these crosslinkers with the styrene phenyl ring by the Friedel-Crafts reaction. Therefore, the resulting cross-links link the styrene phenolic rings present on two different polystyrene chains. See, eg, US Pat. No. 3,729,457, which is incorporated herein by reference.

Crosslinking is particularly important as it generally eliminates the need for “wetting” agents. That is, cross-linking occurs when the adsorbent is in an essentially anhydrous (ie, “dry”) state,
It prevents the pores from collapsing, and thus does not need to be "restarted" with the wetting agent before the adsorbent contacts the blood product. In order to prevent the pores from collapsing, conformationally restricted crosslinks should be formed. Some bifunctional agents, such as DPB, do not generally give rise to restricted conformations;
DPB contains four consecutive methylene units that are susceptible to conformational rearrangements. Therefore, DPB is not a preferred bifunctional agent for use in the present invention.

[0082]   Table A summarizes some of the structurally relevant features of the adsorbent particles described above.

[0083]

[Table 1] Adsorbent Particle Processing Adsorbent particles can be further processed to remove fine particles, salts, potential extractables, and endotoxins. Removal of these extractables is typically done by treatment with either organic solvents, steam, or supercritical liquids. Preferably the particles are sterilized.

Several companies currently sell “cleaned” (ie, processed) modified versions of commercially available adsorbent particles. In addition to processing adsorbent particles (eg, resin), these companies have tested adsorbents and the final adsorbent is certified sterile (USP XXI) and pathogen-free (LAL). ), And no detectable extractables (DVB and total organics).

Thermal processing (eg, steam) is an effective method for processing adsorbent particles. F. Rodriguez, Principles Of Po
lymer Systems, (Hemisphere Publishing
Corp. ), Pp. 449-53 (3rd edition, 1989). Supelco,
Inc. (Bellefonte, PA) is Dowex® XUS-
A non-solvent, heat-proprietary process is used to clean the 43493 and Amberlite adsorbents. The main advantage of using steam is that it does not add any potential extractables to the adsorbent. However, one major disadvantage is that this process can strip water from the pores of the resin beads; the effective performance of some adsorbents is to contact illuminated blood products. The beads need to be rewet before. Use of Wetting Agents and Stabilizers with Adsorbent Resins Ambe that loses some of its adsorption capacity under certain conditions (eg, drying).
Methods such as rlite (R) can be used to control the drying and loss of adsorption capacity of the particles.

In one method, the particulate material or device can be manufactured in a wet state that is sealed and non-drying. This method is associated with several important drawbacks. The shelf life of the product can be reduced as the level of extractables from the material can increase over time. Sterilization is typically done by gamma irradiation of wet polymers,
It can be limited to steam processes. Manufacturing devices that require the components to be maintained in a wet state is generally more difficult than manufacturing dry devices; for example, bioburden and endotoxin are associated with delay times between device assembly and heat sterilization. Can be significant if is long.

A second method for controlling the loss of adsorption capacity involves the use of adsorbents which do not have a detrimental effect on drying. As already mentioned, a giant reticulated adsorbent with a highly cross-linked porous structure (eg Dowex® XUS-43493).
And Purolite® MN-200) suppress the collapse of pores due to cross-linking and generally do not require a wetting agent. Unlike Amberlite® XAD-16, these macroreticular adsorbents retain very high levels of initial activity when dried.

In the third method, the loss of adsorptive capacity on drying was determined by Am Am in the presence of a non-volatile wetting agent.
berlite® XAD-16 and related adsorbents (eg Ambe).
It can be suppressed by hydrating rlite® XAD-4). For example, when using Amberlite® XAD-16 as the adsorbent, the adsorbent beads may be partially dried prior to use during handling, sterilization, and storage. When the water content of these adsorbents drops below a critical level, a rapid loss of adsorption capacity occurs (probably due to "collapse" of the pores); therefore, for optimal effect, the pores should be wet before use. Must be restarted with medication.

Stabilizers are effective in maintaining adsorption capacities near their maximum when certain adsorbent resins are subject to drying conditions. It is believed that the use of stabilizers acts to prevent collapse of the adsorbent pores.

Acceptable stabilizers should be soluble in water and ethanol, non-volatile in ethanol and water, and safe for infusion in small amounts. Glycerol and low molecular weight polyethylene glycols (eg PEG-200 and PEG-400) are examples of stabilizers with these characteristics. Glycerol has a positive hemocompatibility history. It is frequently added to blood as a cryopreservative in the cryopreservation of red blood cell preparations. For example, Chaplin et al., Transf.
usion 26: 341-45 (1986); Valeri et al., Am. J. V
et. Res. 42 (9) 1590-94 (1981). Solutions containing up to 1% glycerol are routinely infused, and glycerol solutions are commercially available (eg Glyerolite 57 Solution, Fe.
nwal Laboratories, Deerfield, IL). Ambe
Adsorbent beads such as rlite® XAD-16 can be stabilized in ethanol and glycerol.

Low molecular weight polyethylene glycols commonly used as pharmaceutical bases also include
It can be used as a stabilizer. PEG has the general chemical formula H (OCH ₂ CH ₂ ) _n OH
Liquid and solid polymers of n, where n is greater than or equal to 4.
PEG formulations are usually followed by a number corresponding to their average molecular weight;
EG-200 has a molecular weight of 200 and a molecular weight range of 190-210.
PEG is commercially available in many formulations (eg, Carbowax, Pol.
y-G, and Solbase).

(Inert Matrix for Immobilizing Particles) The adsorbent particles are immobilized by the inert matrix. The inert matrix is
It can be made from fibrous or particulate, synthetic or natural polymers. The inert matrix can be a sintered polymer. Like the other components of the device, the inert matrix is preferably biocompatible and does not substantially detrimentally affect the biological activity of the substance upon contact.

In an embodiment using synthetic fibers, the synthetic fibers are polyester fibers (A
ir Quality Filtration (AQF), a division
n of Hoechst Celanese (Charlotte, NC).
)). Other preferred examples of synthetic fibers are polyethylene or polyamide fibers. Other representative synthetic fibers include polyolefin, polyvinyl alcohol, and polysulfone fibers.

In a preferred embodiment, the synthetic polymeric fiber comprises a first polymeric core having a high melting point surrounded by a sheath having a lower melting point. The polymer core can be polyester (polyethylene terephthalate). The sheath can be nylon or modified polyester. The fibers are Unitika (Osaka, Ja
pan) and Hoechst Trevira GmbH & Co. (Au
gsberg, Germany).

For the fibrous matrix, the most preferred embodiment uses cellulosic fibers. These cellulosic fibers are derived from a variety of sources such as jute, kozu, kraft, and Manila hemp. Networks of synthetic or natural polymer fibers are described in US Pat. Nos. 4,559,145 and 5,639,376 (
These are used to make filters as described in (herein incorporated by reference).

Sintered matrices are also a preferred embodiment. Suitable synthetic polymers for the construction of such sintered particles are high density polyethylene, ultra high molecular weight polyethylene, polypropylene, polyvinyl fluoride, polytetrafluoroethylene, nylon 6. More preferably, the sintered particles are polyolefins such as polyethylene.

The polymer fiber as described above may be an adsorbent resin without adsorbent particles attached. Such fibers can be formed into a fiber network or fixed onto a fiber network of less adsorbent fibers. Such fibers are contemplated by the invention: such fibers are preferably large, porous, adsorbent surface areas, or other adsorbents, to facilitate reduction of the concentration of low molecular weight compounds. Including sexual means.

Immobilization of Particles In one embodiment, adsorbent particles are immobilized in an inert matrix to create an adsorbent medium for reducing the concentration of small organic compounds in the material. The inert matrix can be a three-dimensional network that includes a synthetic or natural polymer fiber network with adsorbent particles immobilized therein.

Preferably, the adsorbent medium comprises porous adsorbent particles having a highly porous structure and a very large internal surface area, fixed by an inert matrix, as described above. Preferably, when the biological material comes into contact with the adsorbent medium, the adsorbent medium does not substantially adversely affect the biological activity or properties of the other material.

Techniques for immobilizing adsorbent beads on a fiber network to build an air filter are described in US Pat. No. 5,486,410 and US Pat. No. 5,605.
No. 746, which is incorporated herein by reference. As shown in FIG. 1, a polymeric network 600 of fibers has a high melting point polymer core 602 (eg, polyethylene terephthalate (PET)) surrounded by a relatively low melting point polymer sheath 604 (eg, nylon). Consists of. See US Pat. No. 5,190,657 to Heagle et al., Which is incorporated herein by reference. The fiberized resin is prepared by first uniformly dispersing the adsorbent beads in the fiber network. The network is then rapidly heated (eg, 180 ° C. × 1 min) and the fibers 60, shown in FIG.
The zero polymer sheath melts and binds to the adsorbent beads 606 and other fibers forming a crosslinked fiber network. As shown in FIGS. 3 and 4 (not proportional), generally speaking, the fiber network comprises three layers; fiber 600.
And a less dense inner layer 609 containing two outer layers 607 and adsorbent beads 606 and less fibers 600 that are densely packed with. In a preferred embodiment, the ends of the fiberized resin may be sealed with polyurethane or other polymer. Alternatively, as shown in FIGS. 3 and 4, heat seals 608 can be made in fiberized resin obtained at predetermined intervals; for example, heat seals can be made in fiberized resin in a square pattern. Can be done. The fiberized resin is then a resin containing the desired mass (eg, preferably less than 5.0 g and more preferably less than 3.0 g) of adsorbent beads and of a size suitable for placement in a blood product container. Can be cut by heat sealing to form a sample of The heat seal is used to prevent the cut fibrous resin from fraying and serves to immobilize the adsorbent beads. However, the use of such heat seals is not required to practice the present invention. In the alternative embodiment shown in FIG. 4, the adsorbent beads 606 are not secured to the fibers themselves, but rather between the more dense outer layers 607 of the fibers and the heat seal 60.
Fixed at 8; this embodiment can also yield a sample of fiberized media containing a predetermined amount of adsorbent after being cut by heat sealing.

The present invention also includes an adhesive (eg, binder) to secure the adsorbent resin to the fibers.
Intended for use. Moreover, although the adsorbent beads are preferably chemically attached to the fiber network, the beads can also be physically entrapped within the fiber network; this can, for example, be sufficient to keep the beads in place. Can be achieved by surrounding the beads with

Other ways in which the adsorbent particles may be fixed to the fiber network are also contemplated. The particles may be dried-laminated as described in US Pat. Nos. 5,605,746 and 5,486,410 (AQF patents), incorporated herein by reference.
fixed) process. Particles are described in US Pat. No. 4,559,
It may be fixed using a wet-lamination process, as described in Nos. 145 and 4,309,247, incorporated herein by reference. The particles can be prepared as described in US Pat. No. 5,616,254, incorporated herein by reference,
It can be fixed using a melt-blown process. When the wet-lamination process is used to build a matrix from natural polymer fibers, the inert matrix preferably comprises a binder for binding the adsorbent particles to the fibers. Non-limiting examples of binders include melamine, polyamines, and polyamides. The matrix typically comprises 1% or less of such binder.

When the inert matrix is constructed from particles of synthetic polymer that are sintered with the adsorbent particles, it is important that the adsorbent particles have a higher melting point than the matrix.

In a preferred embodiment, the adsorbent particles are immobilized in the fiber matrix formed by thermal bonding of the biocomponent fiber network. An alternative embodiment is to immobilize adsorbent particles in non-biocomponent fibers, and a wet strong resin system, adhesive,
Or using additional fusible fibers to form a bond between the fibers and the adsorbent particles. Non-limiting examples of useful fibers include polyesters, nylons, and polyolefins. (Suppliers of fibers to the non-woven industry are
A Guide to Fibers for Nonwovens, Non
wovens Industry, June 1998, 66-87). Examples of wet strong resin systems include melamine / formaldehyde, epichlorohydrin based resins, polyamines, and polyamides. The use of heat fusible fibers to fix particles in a fiber matrix has been disclosed.
See, eg, US Pat. No. 4,160,059.

Preferably, the resulting adsorbent medium comprises a known amount of adsorbent per area. The adsorbent per area is about 300 g / m ² to about 1100 g / m ² , preferably about 50 g / m ² .
It is from 0 g / m ² to about 700 g / m ² . Thus, the appropriate amount of adsorbent intended for a particular purpose can be easily measured by cutting out a given area of fiberized resin (ie, there is no basis weight of fiberized resin).

The adsorbent medium is preferably biocompatible (ie, toxic, harmful,
Or produces no immunological response); has minimal impact on the properties of substances such as biological compositions (eg plasma); and does not associate with toxic extractables. The fixed adsorbent particles of the adsorbent medium preferably have a high mechanical stability (ie no fine particle formation). The adsorbent medium comprises about 20-70 wt% adsorbent, preferably 30-50 wt% adsorbent. Preferably, the adsorbent medium comprises about 30% by weight of the adsorbent particles when a fibrous matrix is used. When the sintered particulate matrix is used with milled polymeric adsorbent particles, the adsorbent medium preferably comprises about 50% by weight of the adsorbent particles.

Coating of Adsorbent Particles The surface hemocompatibility of the particles, matrix or adsorbent medium can be improved by coating its surface with a hydrophilic polymer. A typical hydrophilic polymer is poly (2-hydroxyethyl methacrylate) (pHEMA) (see, for example, Scientific Polymer Products, In).
c. (Available from Ontario, NY) and cellulose-based polymers such as ethyl cellulose, which is available from Dow Chemical (Mid.
land, MI)). For example, Andrade et al.
Trans. Amer. Soc. Artif. Int. Organs XVII
: 222-28 (1971). Other examples of coatings include polyethylene glycol and polyethylene oxide, which are also Sci
Entity Polymer Products, Inc. Available from. Polymer coatings can increase blood compatibility and reduce the risk of small particle formation due to mechanical degradation.

The adsorbent surface can also be modified with immobilized heparin. Further, the strong anion exchange polystyrene divinylbenzene adsorbent can be modified by heparin adsorption. Heparin is a polyanion and adsorbs very strongly on the surface of adsorbents with strong anion exchange characteristics. Various quaternary amine modified polystyrene divinylbenzene adsorbents are commercially available.

The coating can be applied in a number of different ways, as described in US Pat.
455, 040 (incorporated herein by reference), radio glow discharge polymerization, and Wurster coating process (Inter.
national Processing Corp. (Winchester
, KY)).

In one embodiment, the Wurster coating process suspends the adsorbent particles in the chamber such that a hydrophilic polymer such as pHEMA can be evenly sprayed onto all surfaces of the adsorbent particles. (Generally by air pressure) can be applied. As illustrated in Example 2, Dowex® XUS-43493 evenly sprayed with pHEMA demonstrated an increase in platelet yield as well as a dramatic effect of platelet shape change with increased amount of coating. . It has been found that the Wurster coating process selectively coats the outer surface of the adsorbent surface, leaving the inner porous surface largely unaffected.

In a preferred embodiment, the coating can be applied by dipping the adsorbent medium immobilized in a hydrophilic polymer (see Example 2). This process is simpler and less expensive than spraying adsorbent particles with a hydrophilic polymer.

The process is not limited to applying a coating of adsorbent medium at any particular time. For example, in one embodiment, the pHEMA coating comprises
Applied after production of the adsorbent medium but before heat sealing the adsorbent medium. In another embodiment, the adsorbent medium is first heat sealed and then the pHEMA coating is applied. In addition to coating adsorbent media, rinse processes associated with pHEMA application are used to remove loose particles and fibers.

As the amount of coating increases, it becomes more difficult for some compounds to cross the coating to reach the particle surface, resulting in reduced adsorption kinetics. Therefore, as the amount of coating increases, the increased amount of adsorbent must generally be used to achieve the same removal kinetics as the coated adsorbent. In one embodiment, the optimal level of pHEMA coating is the minimum coating at which a protective effect on plasma function is observed.

The coating can be sensitive to sterilization. For example, gamma sterilization can result in crosslinking and / or cleavage of the coating. Therefore, the type of sterilization (E-beam vs. gamma irradiation) and dose can affect the properties of the coated adsorbent. Generally, E-beam sterilization is preferred.

Devices Devices are provided for the reduction of the concentration of compounds from biological compositions. The compound has a molecular weight in the range of about 100 g / mol to about 30,000 g / mol. The device is a flow device. An example of the flow device is shown in FIG. Flow devices are known in the literature and, for example, PCT publication WO
96/40857, which is incorporated herein by reference.

The flow device allows the concentration of low molecular weight compounds from substances such as blood products to be reduced by perfusing the blood product through the flow device.

The adsorbent medium of the flow device is preferably about 3-30 mm thick in order to promote uniform flow of biological liquid without substantial pressure drop. Preferably, the media is about 3-15 mm thick. More preferably, the media is about 5-8 mm thick.

In one embodiment, the device is a disk placement flow device. A disk placement flow device is shown with reference to FIG. 14, which is an exploded view of the device implementation. The biological composition to be treated in the device flows through the tube collector to the housing inlet (1). The biological composition is then
Through the IAD (Immobilized Adsorption Device) housing inlet (2) and the IAD
It flows into the medium (3), which reduces the concentration of low molecular weight compounds in the biological composition. The biological composition may then flow through the prefilter (4),
This is optional. It then flows through the membrane (5) which removes particulate matter from the composition. Finally, the treated biological composition exits the device through the IAD housing outlet (6).

In another embodiment, the device is a drip chamber arrangement (Porex IA).
D). A drip chamber placement flow device is shown with reference to FIG. 15, which is an exploded view. The biological composition to be treated with the device is IAD
(Fixed adsorption device) flows through the housing inlet (60). Then
The biological composition flows to the IAD housing (drip chamber) (50) and further to the IAD medium (40), which reduces the concentration of low molecular weight compounds in the biological composition. The biological composition is then prefiltered (30).
Can flow through, which is optional. It then flows through a membrane (20) that removes particulate matter from the composition. Finally, the treated biological composition exits the device through the IAD housing outlet (10).

Preferred Embodiments for Biological Compositions In some embodiments, the present invention provides devices for reducing the concentration of a compound in a biological composition comprising cells. The device contains an adsorbent medium and is in a flow configuration. The compound is about 100 g / mol to about 30,000.
It has a molecular weight in the range of 0 g / mol. The biological activity of the biological composition is substantially maintained after contact with such a device.

Anaphylatoxin C3a and te (te
biological response modifiers (eg activated complement), such as the membrane attack complex SC5b-9, process (eg leukocyte filtration, pheresis, recovery of shed blood, etc.) and whole blood and its components. It has been shown to be produced by storage of the ingredients. These biological response modifiers are associated with adverse events in surgery and infusion.

In some embodiments, the devices of the invention reduce the concentration of activated complement in the biological composition. The concentration of activated complement in the composition is
It is reduced when treated with the device, as opposed to the composition which is not treated with the device. In one embodiment, the exposure to the device is more C3 than the control.
a complement fragment and SC5b-9 terminal component (terminal comp
onent) of at least about 30%. In another embodiment, exposure to the device exposes the C3a complement fragment and SC5b-9 more than the control.
It results in a reduction of at least about 50% of the end component. In another embodiment, exposure to the device results in a reduction of C3a complement fragment of at least about 90% over the control.

In one embodiment, the invention provides a device for reducing the concentration of a compound in a biological composition containing plasma. Treatment of the biological composition containing plasma with the device substantially maintains the biological activity of the plasma. The adsorbent medium comprises adsorbent particles immobilized by an inert matrix. Preferred particles are highly porous and have a surface area of greater than or equal to about 750 m ² / g.

Particularly preferred particles are those of Norit America, Inc. (Atlant
a, GA) Norit A Supra. Norit
A Supra is a US formed by steam activation of coconut shells
It is P grade activated carbon. This activated carbon has a very high total surface area (2000 m ²
/ G) and is very microporous in nature.

Further, the particles may be selected from any of the following particles, wherein the particles are preferably about 1 by either milling direct synthesis, or some other means.
having a size range from μm to about 200 μm, and Picactif Medi
activated carbon, such as cinal (Pica USA, Columbus, OH), A
mbersorb 572 (Rohm and Haas, Philadelp
hia, PA), synthetic carbonaceous adsorbents, Amberlite adsorbents (eg, Amberlite® XAD-2, XAD-4, and XAD-1).
6), available from Rohm and Haas (Philadelphia, PA); Amberchrom® adsorbent, Toso Haas (To
SoHaas, Montgomeryville, PA); Dia
ion (registered trademark) // Sepabeads (registered trademark) Adsorbents
(For example, Diaion (registered trademark) HP20), Mitsubishi Ch
electrical America, Inc. (White Plains, NY)
Available from Hypersol-Macronet® Sorben
t Resins (for example, Hypersol-Macronet (registered trademark)
Sorbent Resins MN-150 and MN-400), Puro
lite (Bala Cynwyd, PA), as well as Dowex
(Registered trademark) Advisors (for example, Dowex (registered trademark) XUS-
40323, XUS-43493, and XUS-40285), Dow Ch.
Hydrophobic resins such as those available from the Electronic Company (Midland, MI).

The inert matrix may be composed of synthetic or natural polymeric fibers or particles. In a preferred embodiment, the matrix is sintered particles of fibrous cellulose or ultra high molecular weight polyethylene.

Representative compounds that are reduced or controlled by the devices, materials, and methods of the present invention include psoralens, psoralen derivatives, isopsoralens, psoralen photochemical reaction products, methylene blue, phenothiazines, acridines, plastic extractables,
Biological response modifiers, quenchers, and polyamine derivatives.

When the device is used in a composition containing plasma, the device maintains a suitable level of clotting activity. Coagulation activity is measured by prothrombin time (PT), activated partial thromboplastin time (aPTT), as well as coagulation factor factors I and I.
Includes functional measurements of Factor I, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, and Factor XII. Suitable functional measurements of clotting factor activity are levels greater than about 80% prior to passage through the device, or in the case of clotting time, remain within the normal range established for each laboratory doing this type of test. Is. Preferred measures of clotting activity include PT and aPTT. These are the overall ability of plasma to coagulate, as well as Factor I, Factor II, Factor V, Factor VII, Factor X, Factor XI, and Factor XII, which are It is usually not replaced). It is preferred to retain more than about 90% of the coagulant activity of these factors relative to their level prior to passage through the device, and have PT and aPTT changes of less than about 1.5 seconds.

In one embodiment, the device may include an adsorbent medium and a housing.
The housing should promote an even flow of plasma to promote good media utilization, and when infused, pushes air ahead of it by the plasma, thereby contacting the plasma with the adsorbent medium. Eliminate area-reducing bubbles, thereby reducing media utilization. The housing may be flat, or may have a substantial depth to accommodate a non-flat adsorbent medium or particle retention medium (eg, a cylinder-shaped adsorbent medium). In the preferred embodiment, the housing is flat. The housing may have inlets and outlets in various directions, for example inlet top / outlet top or inlet bottom / outlet bottom. In a preferred embodiment, the outlet is at the bottom to promote good drainage and the inlet is at the top to facilitate media utilization.

In another embodiment, a device that includes an adsorbent medium and a housing may also include a particle retention medium. In a preferred embodiment, the device includes a particle retention medium downstream of the adsorbent medium to retain particles spilling from the adsorbent medium while maintaining high liquid flow and high recovery of proteins. The particle retention medium can be a membrane, a dry or wet matrix of fibers, a sintered polymer matrix, a woven matrix, a nonwoven material (polyester nonwoven), or a combination thereof.

The device housing holds the particle holding medium in a substantially parallel direction downstream of the adsorbent medium. (U.S. Pat. No. 5,660,731 (incorporated herein by reference) discloses an example of a filter housing). The housing can be constructed of any suitable, rigid, impermeable material that does not substantially adversely affect the biological activity of the liquid. Preferably the housing is constructed from synthetic polymers. Non-limiting examples of such polymers include polyacrylic acid, polyethylene, polypropylene, polystyrene, and polycarbonate plastics.

The adsorbent medium of the device containing particles immobilized in an inert matrix is 3 mm and 30 mm to facilitate even flow of biological liquid without substantial pressure drop.
Should be between the thickness of. Preferably the medium should be between 3 mm and 15 mm thick. More preferably, the media should be between 5 and 8 mm thick.

Gravity flow is preferred for the device. More preferably, the device is 1
Gravity flow device constructed to allow a flow rate of 0.1-10 mL / cm ² / min with 2-72 inch water pressure differential. More preferably, the device is 2
Allow a flow rate of 0.2-5 mL / cm ² / min with a pressure differential of 4-48 inches of water.

Applications The present invention contemplates reducing the concentration of low molecular weight compounds from biological compositions. The compound has a molecular weight in the range of about 100 g / mol to about 30,000 g / mol. Such compounds include, for example, photoactivated products, aminoacridines, organic dyes, and pathogen inactivating agents such as phenothiazines. Representative pathogen inactivating agents include furocoumarins such as psoralens and acridines. For example, after treatment of a blood product with a pathogen inactivating compound as described in US Pat. Nos. 5,459,030 and 5,559,250, which are incorporated herein by reference, The concentration of pathogen inactivating compound in the blood product can be reduced by contacting the treated blood product with the device of the invention.

In one embodiment, the present invention contemplates a method of inactivating a pathogen in solution, which method comprises: a) in any order, i) a cyclic compound, ii) contamination with the pathogen. Suspected solution, and iii) providing a fiberized resin; b) treating the solution with the cyclic compound to produce a treated solution product, the solution producing A step in which the pathogen is inactivated in the product; and c) contacting the treated solution product with the fibrous resin, and further reducing the concentration of small organic compounds in the blood product. But includes a device for substantially maintaining the desired biological activity of the blood product, the device comprising highly porous adsorbent particles, wherein the adsorbent particles comprise an inert matrix. Fixed by Is determined.

In addition to pathogen inactivating compounds, their reactive degradation products can be reduced from substances such as blood products prior to infusion.

The materials and devices disclosed herein can be used in apheresis methods. Whole blood can be separated into two or more specific components, such as red blood cells, plasma, and platelets. The term "apheresis" broadly refers to a procedure in which blood is removed from a donor and separated into various components, the component of interest is collected and retained, and other components are returned to the donor. The donor receives replacement fluid during the reinfusion process to help compensate for volume and blood pressure loss caused by component removal. Apheresis system is PCT publication WO96 / 4085
7, which is incorporated herein by reference.

Low Molecular Weight Compounds The devices of the present invention reduce the concentration of low molecular weight compounds in biological compositions. The term "low molecular weight compound" refers to about 100 g / mol to about 30,000 g
Refers to organic or biological molecules having a molecular weight in the range of / mol. Low molecular weight compounds include, but are not limited to, the following compounds: psoralens,
Acridine, or small organic compounds such as dyes; quenchers such as glutathione; plastic extractors such as plasticizers; activated complement such as
Biological modifiers having a molecular weight of between about 100 g / mol and about 30,000 g / mol; and polyamine derivatives.

Small Organic Compounds Various sets of small organic compounds can be adsorbed by the devices of the invention. The molecule can be cyclic or acyclic. In one embodiment, the compound is preferably a psoralen, acridine, or a cyclic compound such as a dye. In another embodiment, the compound is a thiol.

Non-limiting examples of cyclic compounds include actinomycin, anthracyclinone, mitomycin, anthramycin, and organic dyes, and benzodipyrone, fluorene, fluorenone, furocumarine, porphyrin, protoporphyrin, purpurin, Phthalocyanine, Hypericin, Monostral Fast Blue, Norphillin A, phenanthridine, phenazathionium (ph
enazothionium) salts, phenazine, phenothiazine, phenylazide, quinoline, and photoreactive compounds such as thiaxanthenone.
Preferably the compound is furocoumarin or an organic dye. More preferably,
The compound is furocmarin.

Non-limiting examples of furocoumarins include psoralens and psoralen derivatives. Specifically, 4'-aminomethyl-4,5 ', 8-trimethylpsoralen, 8-methoxypsoralen, halogenated psoralens, isopsoralens, and psoralens (linked to quaternary amines, sugars, or other nucleic acid binding groups) Is intended. Also contemplated are the following psoralens: 5'-bromomethyl-4,4 ', 8-trimethylpsoralen, 4'-bromomethyl-4,5', 8-trimethylpsoralen, 4 '-(4
-Amino-2-aza) butyl-4,5 ', 8-trimethylpsoralen, 4'-(4
-Amino-2-oxa) butyl-4,5 ', 8-trimethylpsoralen, 4'-(
2-Aminoethyl) -4,5 ', 8-trimethylpsoralen, 4'-(5-amino-2-oxa) pentyl-4,5 ', 8-trimethylpsoralen, 4'-(5-amino-2-) Aza) pentyl-4,5 ', 8-trimethylpsoralen, 4'-(6-
Amino-2-aza) hexyl-4,5 ', 8-trimethylpsoralen, 4'-(7
-Amino-2,5-oxa) heptyl-4,5 ', 8-trimethylpsoralen, 4
'-(12-amino-8-aza-2,5-dioxa) dodecyl-4,5', 8-
Trimethylpsoralen, 4 '-(13-amino-2-aza-6,11-dioxa)
Tridecyl-4,5 ', 8-trimethylpsoralen, 4'-(7-amino-2-aza) heptyl-4,5 ', 8-trimethylpsoralen, 4'-(7-amino-2-
Aza-5-oxa) heptyl-4,5 ', 8-trimethylpsoralen, 4'-(9
-Amino-2,6-diaza) nonyl-4,5 ', 8-trimethylpsoralen, 4'
-(8-Amino-5-aza-2-oxa) octyl-4,5 ', 8-trimethylpsoralen, 4'-(9-amino-5-aza-2-oxa) nonyl-4,5 ', 8
-Trimethylpsoralen, 4 '-(14-amino-2,6,11-triaza) tetradecyl-4,5', 8-trimethylpsoralen, 5 '-(4-amino-2-aza) butyl-4,4' , 8-Trimethylpsoralen, 5 '-(6-amino-2-aza) hexyl-4,4', 8-trimethylpsoralen, 5 '-(4-amino-2-oxa) butyl-4,4', 8 -Trimethylpsoralen. Preferably, the psoralen is
It is 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen.

(Acridine) Non-limiting examples of acridine include acridine orange, acriflavine, quinacrine, N1, N1-bis (2-hydroxyethyl) -N4- (6-chloro-2-methoxy-9-acridinyl)- 1,4-pentanediamine, 9- (3
-Hydroxypropyl) aminoacridine, N- (9-acridinyl) glycine, S- (9-acridinyl) -glutathione. In a preferred embodiment, the acridine is N- (9-acridinyl) -β-alanine, alternatively designated as 5-[(β-carboxyethyl) amino] acridine.

Dyes Non-limiting examples of dyes include phenothiazines such as methylene blue, neutral red, toluidine blue, crystal violet, and Azure A, phenothiazones such as methylene violet Bernthsen,
Examples include phthalocyanines such as aluminum 1,8,15,22-tetraphenoxy-29H, 31H-phthalocyanine chloride and silica analogs, and hypericin. Preferably the dye is methylene blue or toluidine blue. More preferably, the dye is methylene blue.

The term “thiazine dye” includes dyes containing ionic atoms in one or more rings. The most common thiazine dye is methylene blue (3,7-bis (dimethylamino) -phenothiazine-5-ium chloride). Other thiazine dyes include, but are not limited to, Azure A, Azure C, and thionine, as described, for example, in Schinazi US Pat. No. 5,571,666.

The term “xanthene dye” refers to a dye that is a derivative of the compound xanthene.
Xanthene dyes can fall into one of three main categories: i) fluorenes or aminoxanthenes, ii) rhodols or aminohydroxyxanthenes, and iii) fluorones or hydroxyxantheses. Examples of xanthene dyes contemplated for use in the present invention include rose bengal and eosin Y;
These dyes are available from many sources (eg, Sigma Chemical
Co. , St. Louis, MI), and Schinazi, US Pat.
571,666, incorporated herein by reference, and can be obtained commercially.

Quencher The concentration of various compounds can be reduced. Other representative compounds include quenching compounds. Methods of eliminating unwanted side reactions in pathogen inactivating compounds that are electrophilic groups or that include functional groups capable of forming electrophilic groups are described in co-pending US application “Methods” filed January 6, 1998. for Quen
ching Pathogen Inactivators in Biolo
“Gical Systems”, Incident Serial Number 282173000600, the disclosure of which is incorporated herein by reference. In this method, a substance such as a blood product is treated with a pathogen inactivating compound and a quencher, where the quencher comprises a nucleophilic functional group capable of covalently reacting with an electrophilic group. 1
In one embodiment, the pathogen inactivating compound comprises a nucleic acid binding ligand and a functional group such as a mustard group, which can react in situ to form an electrophilic group. Examples of quenchers include, but are not limited to, compounds containing a nucleophilic group. Representative nucleophilic groups include thiol, thioacid, dithioacid, thiocarbamate, dithiocarbamate, amine, phosphoric acid, and thiophosphoric acid groups. The quencher may be or include a nitrogen heterocycle such as pyridine. The quencher may be a phosphate containing compound such as glucose-6-phosphate. The quencher may also be a thiol-containing compound, such as glutathione, cysteine, N-acetyl cysteine, mercaptoethanol, dimercaprol, mercaptan, mercaptoethanesulfonic acid and its salts such as MESNA, homocysteine, aminoethanethiol, dimethyl. Included, but not limited to, aminoethanethiol, dithiothreitol, and other thiol-containing compounds. Representative aromatic thiol compounds include 2-mercaptobenzimidazole sulfonic acid, 2-mercaptonicotinic acid, naphthalenethiol, quinolinethiol, 4-nitrothiophenol, and thiophenol. Other quenchers include nitrobenzyl pyridine and inorganic materials such as selenide salts or organic selenides, thiosulfates, sulfites, sulfides, thiophosphates, pyrophosphates, hydrosulfides, and dithionitrites. Examples include nucleophiles. The quencher may be a peptide compound containing a nucleophilic group. For example,
The quencher can be a cysteine containing compound, for example a dipeptide such as GlyCys, or a tripeptide such as glutathione.

Compounds that can be removed by the devices of the present invention include methylthioglycolate,
It may include thiols such as thiolactic acid, thiophenol, 2-mercaptopyridine, 3-mercapto-2-butanol, 2-mercaptobenzothiazol, thiosalicylic acid, and thioctic acid.

Plastic Extractables The concentration of a group of low molecular weight compounds that are extractable from the plastic storage containers and tubes used to handle biological compositions are also biological using the devices of the invention. Can be reduced in the biological composition. Examples of extractables include, but are not limited to, plasticizers, residual monomers, low molecular weight oligomers, antioxidants, and lubricants. For example, R. Carmen, Transfusi
on Medicine Reviews 7 (1): 1-10 (1993). Sterilization of plastic components by steam, gamma irradiation, or electron beam can result in oxidation reactions and / or polymer cleavage, resulting in the formation of additional extractable species.

Plasticizers are commonly used to enhance the properties of plastics such as processability and gas permeability. The most common plasticizer found in blood storage containers is PV
Di (2-ethylhexyl) phthalate (DEHP) used in C formulation. DEHP has been identified as a potential carcinogen. Alternative plasticizers have been developed and include, but are not limited to, the following compounds: tri (2-
Ethylhexyl) trimellitate (TEHTM), acetyl-tri-n-hexyl citrate (ATHC), butyryl-tri-n-hexyl-citrate (BT
HC), and di-n-decyl phthalate.

The devices of the present invention can be used to reduce or control the concentration of plastic extractables in biological compositions in a variety of environments. Such environments include, but are not limited to: blood processing; blood storage;
And hemodialysis and extracorporeal membrane oxygenation.
In vitro applications, such as mbrane oxygenation).

Biological Response Modifiers (BRMs) Concentrations of a group of low molecular weight compounds broadly referred to as Biological Response Modifiers (BRMs) are also biological compositions using the devices of the invention. Can be reduced in. BRM is defined as "a broad spectrum of molecules that alter the immune response." Illustrated Dictionary of Immunolog
y, J. M. Cruise and R.C. E. Lewis. A general group of BRMs includes, but is not limited to, the following types of compounds: small molecules such as histamine and serotonin; lipids such as thromboxane, prostaglandins, leukotrienes, and arachidonic acid; Small peptides such as bradykinin; larger polypeptides containing additional groups containing activated complement fragments (C3a, C5a); cytokines such as IL-1, IL-6, and IL-8; and RANTES and Chemokines like MIP.

Accumulation of BRM in blood products during storage can adversely affect the desired biological activity of the biological composition. Complement activation has been demonstrated, for example, to occur during platelet storage under standard blood bank conditions. Complement activation is associated with a loss of platelet function and viability called "platelet storage disorders". For example, V
． D. Metic and O.M. Popovic, Transfusion 33
(2): 150-154 (1993). Accumulation of BRM in stored blood products can also have a deleterious effect, for example, on patients receiving blood products: Accumulation of BRM in platelet concentrates during storage is non-homogeneous hemolytic febrile infusion in patients receiving platelets. It is related to the reaction. For example, N. M. Heddle,
Current Opinions in Hematology 2 (6):
See 478-483 (1995).

Polyamine Derivatives The concentration of a group of low molecular weight compounds known as polyamine derivatives is also, for example,
It can be reduced in biological compositions using the device of the invention. The polyamine derivative is a compound containing a plurality of nitrogen atoms in the carbon skeleton.

Polyethylene Glycol Other representative compounds include activated polyethylene glycol (aPEG), which is used to provide immuno-masking properties or pacification to protein binding, respectively, to cells. Or it can be used for modification of the surface of a substance. The device can be obtained by reacting excess active polyethylene glycol, or active PEG with water or a small nucleophile (eg, phosphate, phosphate ester or thiol such as glutathione).
Can be used for the reduction of either of the inactive derivatives of Other compounds that can be removed include impurities in the active PEG preparation, which can affect the functioning of blood products or make them unsuitable for infusion (eg, toxic compounds). Finally, small molecules (leaving groups) such as N-hydroxysuccinimide released during the reaction of aPEG with cell surface nucleophiles can also be reduced.

Examples of compounds that can be removed by the device of the present invention include cyanuryl chloride, succinimidyl ester, oxycarbonyl imidazole derivatives, nitrophenyl carbonate derivatives, glycidyl ether derivatives, and activating moieties that can include aldehydes. Included are attached linear or branched polyethylene glycols.

EXAMPLES The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting its scope.

In the following experimental disclosure, the following abbreviations are used: eq (equivalent); M (1
Mol per liter); μM (micromol per liter); N (standard); mol (mol); mmol (mmol); μmol (micromol); nm
ol (nanomol); g (gram); mg (milligram); μg (microgram); Kg (kilogram); L (liter); mL (milliliter); μL (microliter); cm (centimeter); mm ( Mm); μm (micrometer) nm (nanometer); min (min); s and sec. (Seconds)
J (joules or watt seconds); ° C (degrees Celsius); TLC (thin layer chromatography); HPLC (high performance liquid chromatography); pHEMA and p (H
EMA) (poly [2-hydroxyethylmethacrylate]); PC (platelet concentrate); PT (prothrombin time); aPTT (activated partial thromboplastin time); TT (thrombin time); HSR (hypotonic shock response); FDA (Food and Drug Administration); GMP (Good Manufacturing Practice); DMF (Drug Master)
rfiles); SPE (solid phase extraction); Aldrich (Milwaukee,
WI); Asahi (Asahi Medical Co. Ltd., Tokyo)
Baker, JT Baker, Inc., Philli.
psburg, NJ); Barnstead (Barnstead / Therm
olyne Corp. , Dubuque, IA); Becton Dicki
nson (Becton Dickinson Microbiology S
systems; Cockeysville, MD); Bio-Rad (Bio-
Rad Laboratories, Hercules, CA); Cerus (
Cerus Corporation; Concord, CA); Chrono
-Log (Chrono-Log Corp .; Havertown, PA);
Ciba-Corning (Ciba-Corning Diagnostic)
s Corp. Oberlin, OH); Consolidated Pla
stics (Consolidated Plastics Co., Twin)
sburg, OH); Dow (Dow Chemical Co .; Midlan
d, MI); Eppendorf (Eppendorf North Amer
ica Inc. , Madison, WI); Gelman (Gelman S
Sciences, Ann Arbor, MI); Grace Davison (
W. R. Grace & Co. , Baltimore, MD); Helmer
(Helmer Labs, Noblesville, IN); Hoechst
Celanese (Hoechst Celanese Corp., Cha
rlotte, NC); International Processing
Corp. (Winchester, KY); Millipore (Milfo
rd, MA); NIS (Nicolet, a Thermo Spectra)
Co. , San Diego, CA); Poretics (Livermore)
, CA); Purolite (Bala Cynwyd, PA); Quidel
(San Diego, CA); Rohm and Haas (Chaony,
France); Saati (Stamford, CT); Scientific
c Polymer Products (Ontario, NY); Sigma
(Sigma Chemical Company, St. Louis, MO)
Spectrum (Spectrum Chemical Mfg. Corp.
． , Gardenia, CA); Sterigenics (Corona, CA)
); Tetko, Inc. (Depew, NY); TosoHaas (Toso
Haas, Montgomeryville, PA; Wallac (Wall
ac Inc. , Gaithersburg, MD); West Vaco (L
uke, W.A. Va); YMC (YMC Inc., Wilmington, NC)
); DVB (divinylbenzene); LAL (Limulus amoeba-like cell degradation product)
USP (USP); EAA (ethyl acetoacetate); EtOH (ethanol); HOAc (acetic acid); W (watts); mW (milliwatts); NMR (nuclear magnetic resonance; in a Varian Gemini 200 MHz Fourier transform spectrometer) Spectrum obtained at room temperature); ft ³ / min (cubic feet per minute); m. p. (Melting point); g / min and gpm (gallon per minute); UV (ultraviolet light); THF (tetrahydrofuran); DMEM (Dulbecco's modified Eagle medium); FBS (fetal bovine serum); LB (Luria Bro)
th); EDTA (ethylenediaminetetraacid); PMA (phorbol myristate acetate); PBS (phosphate buffered saline); AAMI (Associa)
tion for the Advancement of Medical
Instruments); ISO (International Organization for Standardization); EU (Endotoxin Unit); LVI (Large Volume Injectable Substance); GC (Gas Chromatography); M
(Mega-); kGy (1000 gray = 0.1 MRad); MΩ (M ohm);
PAS III (platelet addition solution III); dH ₂ O (distilled water); IAD (immobilized adsorption device); SCD (sterile binding device).

One of the following examples is referred to as HEPES buffer. This buffer contains 8.0 g of 13
7 mM NaCl, 0.2 g 2.7 mM KCl, 0.203 g 1 mM M
gCl ₂ (6H ₂ O), 1.0 g 5.6 mM glucose, 1.0 g 1 mg / m
bovine serum albumin (BSA) (available from Sigma, St. Louis, MO), and 4.8 g of 20 mM HEPES (Sigma, St. Lou.
is, available from MO).

[0159]   (Example 1)   Preparation of fiberized resin   (Preparation of fiberized resin and adsorbent beads)   Contains Amberlite® XAD-16 in a clean and hydrated state
Fixed adsorbent media (Rohm and Haas) were obtained from AQF. H
The fibers of the fiber network of oechst Celanese are polyethylene
It consists of a rephtalate core and a nylon sheath, which has a lower melting point than the core.
Had a point. The fiberized resin is first adsorbent beads in the fiber network.
Was prepared by evenly distributing Then add the fiber network quickly.
Heat to melt the polymer sheath of the fiber and bond it to the adsorbent beads and other fibers
, Formed a crosslinked fiber network. The fibrous resin formed is 130 g / m ² Amberlite® XAD-16 was included in the loading of
That is, each square meter of fiber contained 130 g of adsorbent beads).

The fiberized resin was cut into squares (14 cm x 14 cm) and the resulting pieces contained about 2.5 g of dry Amberlite® XAD-16. The Amberlite® XAD-16 beads were then pre-wet by soaking the fiberized resin in 30% ethanol for about 10 minutes. The remaining ethanol was then removed by rinsing twice with saline for 10 minutes. Alternative methods of wetting Amberlite® XAD-16 and other adsorbents are also effective and are encompassed by the present invention. Fiberized resins containing other types of beads (eg Dowex® XUS-4349).
It should be noted that crosslinked or hypercrosslinked resins such as 3) do not require a wetting step for effective psoralen removal.

Amberlite® XAD-16 HP (high purity) beads were also obtained directly from Rohm and Haas in the clean and hydrated state. Amber free (ie unfixed) prior to incorporation into the mesh pouch
For lite® XAD-16 HP beads, no pre-wetting was required; however, the amount of adsorbent was corrected to account for the water content of the beads (2
． 5g dry = 6.8g with 62.8% moisture). Dowex (registered trademark) XU
S-43493 beads were obtained from Dow, and dry beads did not require wetting, nor did the amount of beads correct for water. Polyester mesh pouch (7 cm x 7 cm square; 30 μm opening), then free Ambe
rlite (registered trademark) XAD-16 HP or Dowex (registered trademark) XU
2.5 g of any of the S-43493 beads (dry weight) was loaded.

The fiberized resin and adsorbent containing pouch were sterilized by autoclaving in a "wet" cycle at 121 ° C for 45 minutes. The fibrous resin and adsorbent-containing pouch were then separated and sterilized in 1 liter PL 2410.
It was inserted into a plastic container (Baxter). After insertion, PL 2410 plastic containers were heat sealed in a laminar flow hood using sterile scissors, hemostats, and impulse sealers.

Example 2 Preparation of pHEMA Coated Adsorbent Beads and Fibrous Resin Dowex® XUS-43493 (known under the trade name Optipore® L493) containing about 50% by weight water. From Dow,
Polymerized HEMA with viscosity and average molecular weight of 300 kD
Obtained from Polymer Products. Prior to coating
The adsorbent beads were dried to a water content of <5%. Polymer 95% denatured ethanol / 5
% E by dissolving in 50% water to achieve a pHEMA concentration of 50 mg / ml
A stock solution of MA was prepared.

The coating process is based on International Process
g Corp. In a 9-inch Wurster fluid bed coater at about 4 kg (dry) adsorbent charge. The coating process involved a pHEMA flow rate of 60-70 g / min, an inlet temperature of 50 ° C., and an air flow rate of about 200 ft ³ / min. A sample of coating adsorbent (50 g) was removed during the coating process, resulting in coating levels in the range of 3-18% (w / w) pHEMA. In the experiments below, pH of 3.7%, 7.3%, and 10.9%
Adsorbent beads coated with EMA (w / w) were used.

A 30 square μm polyester mesh pouch (7 cm
Non-immobilized dry (uncoated) Dow
An apparatus was prepared with ex® XUS-43493 (2.5 g) and pHEMA coated Dowex® XUS-43493 (3.0 g or 5.0 g). Pouch filled with adsorbent, separate sterile 1 liter PL2
It was inserted into a 410 plastic container (Baxter) and heat-sealed with an impulse sealer. Then, the pouch filled with the adsorbent and containing the PL-2410 plastic container was gamma irradiated (Ster) up to E-beam (NIS) or 2.5 MRad.
i Genetics). As mentioned above, E-beam sterilization is generally preferred.

Hoechst Celanese uses Amb according to the method described in Example 1.
A fibrous resin containing erlite® XAD-16 was prepared. This fibrous resin was cut into a square (14 cm × 14 cm). The obtained section is about 2.5
g dry Amberlite® XAD-16. The fibrous resin Amberlite® XAD-16 is simultaneously coated by wetting by immersion in a solution containing 50 mg / mL pHEMA in 95% ethanol / 5% distilled water. Residual ethanol was removed by rinsing twice with saline for 10 minutes. This procedure yielded a coating of about 6% (w / w) pHEMA. The fibrous resin was then sterilized by autoclaving at 121 ° C. for 45 minutes on a “wet” cycle. The fibrous resin was then sterilized individually
It was inserted into a liter PL2410 plastic container (Baxter) and heat fused into a laminar flow hood using sterile scissors, hemostats and impulse sealer.

Example 3 (Effect of Glycerol and Polyethylene Glycol on Adsorption Capacity) This example shows the effect as a stabilizer on the adsorption capacity of glycerol and polyethylene glycol and the kinetics of removal of aminopsoralen from plasma. Research science. The experiments in this example used free (ie, non-fibrillar) Amberlite® XAD-16 and Dowex® XUS-43493 adsorbent beads.

(Method) Amberlite (registered trademark) XAD-16HP (Rohm & Haas (P
hiladelphia, PA)) and Dowex® XUS-43.
493 (Supelco, Bellefonte, PA) was dried in an oven at 80 ° C. to a moisture content <5%. A known mass of adsorbent was added to 0-50% glycerol, 50% PEG-200 or 50% PEG-400 (glycerol, PE.
G-200, and PEG-400 were immersed in an ethanol solution containing Sigma). Following an incubation time of 15 minutes at room temperature, excess solvent was removed and the sample dried in an 80 ° C. oven overnight. Drying of the adsorbent at temperatures> 120 ° C was avoided. Previously, changes in the properties of the adsorbent (eg, melting of the pores) were observed at higher temperatures. After drying, a sample of adsorbent was weighed to determine the mass of stabilizer relative to the mass of adsorbent.

Several independent experiments were performed. Control samples, "non-wet" and "optimally wet" adsorbents were included in the experiment as described below. The non-wet adsorbent sample is a dry adsorbent without any pretreatment, while the optimum wet adsorbent sample is 30% ethanol / 70% adsorbent.
It was prepared by wet with% H ₂ O. The optimum wetting adsorbent was rinsed with dH ₂ O to remove residual ethanol. The adsorbent was prepared just before the adsorption experiment to ensure that no drying occurred.

Each of the adsorption experiments was performed using ³ H-4 ′-(4-amino-2-oxa) butyl-4.
100% with 150 μM 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen spiked with 5,5 ', 8-trimethylpsoralen
It was performed using human plasma. Plasma (6.0 mL) was added to vials containing adsorbent treated with different stabilizers. Adsorbent mass is glycerol or PEG
The volume was adjusted to add 0.2 g of adsorbent. The vial was placed on a rotator and stirred at room temperature. Removing the plasma samples at different time points, remaining ³ H-4 '- (4- amino-2-oxa) butyl-4,5', were determined 8-trimethyl psoralen. Samples (200 μL) were diluted with 5.0 mL of Optiphase HiSafe liquid scintillation cocktail (Wallac) and counted on a Wallac 1409 liquid scintillation counter (Wallac).

Adsorption Capacity of Amberlite® XAD-16 and Dowex® XUS-43493 Treated with Glycerol FIG. 7 shows Amberlite® XAD-1 in 100% plasma.
6 and related 4 '-(4- to Dowex® XUS-43493
Compare the effect of pretreatment with ethanol solutions containing different levels of glycerol on the adsorption capacity of amino-2-oxa) butyl-4,5 ', 8-trimethylpsoralen. The adsorbent sample was immersed in the ethanol / glycerol solution for 15 minutes and then dried at 80 ° C. for 48 hours. After contacting for 4 hours, one measurement of adsorption capacity was performed. Referring to FIG. 7, the glycerol volume shown on the x-axis is the weight / mass% of glycerol in ethanol. The adsorption capacity shown on the y-axis is the percentage of the adsorption capacity of the optimum wet adsorption sample. The adsorption capacity of XUS-43493 is represented by a square,
The adsorption capacity of XAD-16 is represented by a circle.

As the data in FIG. 7 show, the capacity of XAD-16 increased from 30% in the dry sample to over 90% in the sample wet with 20% glycerol. These results indicate that very low levels of glycerol are required to maintain high adsorption capacity after drying. 50% ethanol / 50% prior to drying
The control sample moistened with dH ₂ O (without glycerol) showed similar adsorption capacity to the dry untreated sample. On the other hand, the XUS-43493 sample did not show any effect of glycerol on the adsorption capacity, and the adsorption capacity reached 100% at all levels of glycerol. Although not important in the practice of the present invention, this observation supports the hypothesis that glycerol acts during drying to prevent the collapse of adsorbent pores. Due to the highly crosslinked structure of XUS-43493, it does not undergo sorbent pore collapse during drying.

Samples treated with glycerol appear to be very stable to drying. No change was observed in the adsorption capacity of the samples stored in the laminar flow hood for 7 days (data not shown).

(Amberlite® X treated with glycerol or PEG
Adsorption Capacity of AD-16 and Dowex® XUS-43493 Further experiments were performed with the low molecular weight polyethylene glycols PEG-200 and PEG-400, which are non-volatile and low toxicity drugs that dissolve in ethanol and water. did. Samples of adsorbent were treated with 50% solutions of PEG-200 and PEG-400 or glycerol in ethanol for 15 minutes. FIG. 8: Amberlite® XAD-16 (bottom) and Dow in 100% plasma.
For ex (R) XUS-43493 (top), using a dry adsorbent, 4 '
The effect of stabilizers on the adsorption capacity of-(4-amino-2-oxa) butyl-4,5 ', 8-trimethylpsoralen is compared. Samples that have not been wetted are
o Tx ". The adsorption capacity was reported as a percentage of the capacity of the optimum wetting adsorbent.

As demonstrated by the data in FIG. 8 and predictable based on its “macronet” structure, the capacity of Dowex® XUS-43493 is dry (“No Tx
Sample)). On the other hand, Amberlite (registered trademark) XA
D-16 had a maximum capacity of about 35% when dried. Glycerol,
Treating XAD-16 with PEG-200 and PEG-400
The capacity of the dry adsorbent was all improved. Each adsorption capacity is 90
%, Glycerol>PEG-200> PEG-400. Although it is not necessary to understand the exact mechanism of action for practicing the present invention, the difference in potency between the glycerol solution and the two PEG solutions may be due to the decrease in stabilizer penetration with increasing molecular weight. . That is, in the 15-minute addition procedure, glycerol (
MW = 92.1) is PEG-200 (MW = 190-210) or PEG-
It can penetrate the adsorbent pores more completely than any of the 400 (MW = 380-420) and diffuse more slowly due to its large size.

(Amberlite® X treated with glycerol or PEG
AD-16 Adsorption Kinetics Experiments were also performed to determine if plugging the pores of the adsorbent with glycerol or PEG caused reduced adsorption kinetics. FIG. 9 shows 4 ′-(4-amino-2-oxa) butyl-4,5 over 3 hours from 100% plasma using Amberlite® XAD-16 moistened with several different solutions. The adsorption of ', 8-trimethylpsoralen is compared. Specifically, FIG.
Data for i) wetted with 50% solution of glycerol before drying (open squares connected by solid lines), ii) wetted with 50% solution of PEG-400 before drying (shaded circles connected by broken lines) Iii) pre-wet, ie 50% ethanol / immediately before the start of the experiment
Represents XAD-16, moistened with 50% dH ₂ O (dotted triangles connected by dashed lines), and iv, without any treatment (shaded squares connected by solid lines, “No Tx”). The data in Figure 9 is for 50% glycerol / 50% ethanol, or 50% P
The Amberlite® XAD-16 sample wetted with EG-400 / 50% ethanol solution has adsorption kinetics very close to the optimally wetted ethanol sample (ie, the ethanol pre-wet sample). It has shown having.
The untreated dried sample of XAD-16 (No Tx) achieved only 30% removal in 3 hours.

The data presented in this example show that 50% ethanol and 50% glycerol, P
Amber as a stabilizer in solution form containing EG-200 or PEG-400
It has been shown that treating lite (R) XAD-16 can prevent the loss of adsorption capacity associated with drying. The results obtained with these stabilizers suggest that low molecular weight wetting agents represent a viable method for improved adsorption function.

Example 4 Removal of Methylene Blue from FFP This example relates to the ability of a variety of different polymeric adsorbent materials to remove methylene blue from fresh frozen plasma.

The experiments in this example evaluated "free" adsorbent resin (ie, not incorporated into a device containing non-immobilized adsorbent), and fibrous resin. The free adsorbent resin tested was Amberlite® XAD-16HP (Rohman).
d Haas), MN-200 (Purolite), and Dowex® XUS-43493 (Dow Chemical Co.). XA
D-16HP was obtained in the hydrated state, so no pretreatment was required (ie no need for wetting) and MN-200 was also supplied in the fully hydrated state. XUS-43493 was dry.

A fibrous resin containing XAD-16 was prepared as outlined in FIG. Briefly, 2 cm x 7 cm of a fibrous resin containing 130 g / m ² XAD-16 (ie 14 c
m ² ) pieces were first moistened with 70% ethanol and then rinsed thoroughly with distilled water.

U. S. P. A stock solution of methylene blue (10 mM) was prepared by dissolving methylene blue (Spectrum) in distilled water. This stock solution of methylene blue was added to a sample of 100% plasma to give a final concentration of 10 μM. For adsorption experiments, a sample of "free" adsorbent resin (ie, XAD-16HP, MN-200, and XUS-43493) was weighed into a 50 mL polyethylene tube. The water content of each adsorbent was determined by measuring the mass loss during drying. The mass of each adsorbent was corrected for water content such that an amount equal to 0.25 g of dry adsorbent was used each.

A 30 mL sample of 100% plasma containing 10 μM methylene blue was added to each vial. The vial was placed on a rotator at room temperature. Samples (200 μL) were removed from each vial at 15 minute intervals and assayed for residual methylene blue by HPLC. Samples of each plasma were diluted 5-fold with sample diluent (final concentration = 35% methanol, 25 mM KH ₂ PO ₄ , pH = 3.5). Proteins and other macromolecules were precipitated by incubating the samples for 30 minutes at 4 ° C. The sample was centrifuged and the supernatant was filtered (0.2 μm) to obtain 65% solvent A (25 mM KH ₂ P).
_{O 4, pH = 3.5),} 35% B ( by performing a linear gradient 20 min from methanol) to 80% B, C-18 reverse phase column (YMC ODS-AM,
4.6 mm × 250 mm). The limit of detection in the HPLC assay was about 0.5 μM methylene blue.

FIG. 10 compares the kinetics of adsorption of methylene blue from 100% plasma over 2 hours. Referring to FIG. 10, XAD-16HP data is represented by a white rhombus connected by a broken line, and MN-200 data is represented by a solid triangle connected by a solid line, and XUS-
The data of 43493 is represented by a white circle connected by a broken line, and is shown by a shaded square connecting a fibrous resin containing XAD-16 by a solid line. As the data show, XAD-16HP and MN-200 provided the fastest adsorption kinetics, followed by XUS-43493. The slightly slower kinetics of XUS-43493 may be the result of slower wetting due to its being used in the dry state. Finally, the fibrous resin containing XAD-16 had the slowest adsorption kinetics. This is due to the fibrous resin 14
Between the fibrous resin and the plasma during incubation due to the fact that the cm ² strips did not completely submerge in the plasma, thereby reducing the effective contact area between the adsorbent and the plasma. Could be caused by inadequate contact of the.

This data demonstrates that resins and fibrous resins envisioned for use in the present invention can be used to remove non-psoralen pathogenic inactive compounds such as phenothiazine dyes from blood products.

Example 5 This example compares the use of different types of powdered carbon as the active ingredient of the medium, to reduce the concentration of small organic compounds and to maintain the biological function of the fluid. ,
It shows how careful the selection of the active ingredient is. The five activated carbons shown in Example 5 reduce the concentration of psoralen (4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen) in plasma to about the same amount. Here, “A SUPRA” is used as the adsorbent particles, but the retention of the coagulation factor is substantially better than that of other adsorbents.

4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen was added to plasma at a concentration of 150 μM and the plasma was added in batches of 325 mL in batches of 3.
Pathogens were inactivated by irradiation with 0 J / cm ² UVA. 4 '-(4
The residual concentration of -amino-2-oxa) butyl-4,5 ', 8-trimethylpsoralen was about 90 μM in the resulting plasma pool. 325 mL of irradiated plasma was pumped at 20 mL / min through five different types of 90 mm Cuno ZetaPlus carbon pads. Plasma clotting factor level, clotting time and 4'-
The concentration of (4-amino-2-oxa) butyl-4,5 ', 8-trimethylpsoralen was assayed before and after flushing through a carbon pad.

[0187]

[Table 2] The flow rate of water for the R10S medium is 1.5 p. s. 2 gallons of water at i pressure difference
Minutes per square foot.

This example shows that the choice of activated carbon used can strongly influence the effect of IAD on biological activity.

Note: A Supra grade was listed in the same specification as the R1xS series, but with the only carbon changed. An increase in PTT indicates removal of coagulation factors. I, VII
I and IX indicate specific coagulation factors. All values are after photochemical treatment. 4 '-(4-Amino-2-oxa) butyl-4,5', 8-trimethylpsoralen was assayed by HPLC and clotting factors and clotting times were determined by an automated coagulation analyzer.
Assayed by.

Example 6 Effect of Particle Size on Removal of Low Molecular Weight Compounds and Biological Activity. In this example, I
It illustrates that the particle size of adsorbents used in AD can have a significant impact on the extent of removal of low molecular weight compounds and the measurement of biological activity.

Plasma subjected to photochemical treatment was prepared in the same manner as in Example 5. Dowex Opt
ipore L493 was ground using an Estro Model 480 grinder, sieved to about 100 μm and 50 μm, and 50 μm and 100 μm.
Two classes of particles were generated, a class between and and less than 50 μm. Filters such as ZetaPlus were prepared to contain either of these two classes of particles based on Cuno's R1xS specification.
Photochemically treated plasma (325 mL) was pumped through each pad at 20 mL / min. 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen and coagulation factors were analyzed as in Example 5.

[0192]

[Table 3] With this particular adsorbent, the smaller particle size resulted in substantially more effective removal, but had a greater effect or biological activity as measured by coagulation activity. This represents a trade-off often seen in choosing the particle size of the adsorbent particles.

Example 7 Effect of adsorbent loading on removal of low molecular weight compounds and biological activity.
This example compares the effect of changing the mass ratio of the active ingredients on decreasing the concentration of small organic compounds with a trade-off of the biological function of the fluid.

Plasma subjected to photochemical treatment was prepared in the same manner as in Example 5. The A Supra pad was prepared with a standard R1xS loading of approximately 60% carbon and a lower level loading of 30%. About 230 mL of plasma, 90 mm each
Pumped through the disc. 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen and coagulation factors were analyzed as in Example 5.

[0195]

[Table 4] This has the unexpected result that by reducing the loading of adsorbent particles, it is sometimes possible to reduce the effect of IAD on biological activity while substantially reducing the concentration of low molecular weight compounds. It is illustrated. Although the overall capacity of the IAD is reduced, this is not a problem as long as the IAD operates at much less than the theoretical capacity of the low molecular weight compound.

Example 8 Hemocompatible coating
Effect of using. This example illustrates that in some cases, treating the medium with a hydrophilic polymer can be effective for biological function.

Plasma subjected to photochemical treatment was prepared in the same manner as in Example 5. One 90 mm R14S pad was washed overnight with 50 mg / mL polyhydroxyethymethacrylate (pHEMA) in 95% ethanol and dried in an oven at 70 ° C. until there was no further weight change. Similarly, the second pad was washed with 95% ethanol without pHEMA and dried. About 325 mL of plasma was pumped through the disc at 5 mL / min. 4 '-(4-amino-2-
Oxa) butyl-4,5 ', 8-trimethylpsoralen and coagulation factor were used in Example 5
The same was analyzed.

[0198]

[Table 5] The coating showed efficacy on the biological activity of the biological composition, in this case the plasma clotting factor, but the low molecular weight compound used in this example, 4 '-(4-amino-2. The coating adversely affected the ability of the IAD to reduce the concentration of -oxa) butyl-4,5 ', 8-trimethylpsoralen. This represents a trade-off often seen when choosing a coating or surface treatment.

Example 9 Removal of low molecular weight compounds and effect of flow rate on biological activity. This example illustrates that flow rates can affect the reduction of the concentration of small organic compounds.

Photochemically treated plasma was prepared in the same manner as in Example 5. The A Supra pad was prepared with a standard R1xS loading of approximately 60% carbon and 325 mL of treated plasma was pumped through each of the 47 mm diameter disks at three different flow rates. 4 '-(4-amino-2-oxa) butyl-4,5', 8-
Trimethylpsoralen and coagulation factors were analyzed as in Example 5.

[0201]

[Table 6] The increase in flow rate showed a slight decrease in the extent of removal of low molecular weight compounds,
More substantial efficacy was conferred on biological activity as seen in the higher yields of Factor VIII and Factor IX coagulation activity. This indicates that adjusting the flux of biological compositions through IAD can confer selectivity of reduction of low molecular weight compounds on effects on biological activity.

Example 10 Effect of fluid volume on removal of low molecular weight compounds and biological activity. This example illustrates that fluid volume can also have a significant effect on conferring selectivity of low molecular weight compounds on mediators of biological activity.

Plasma subjected to photochemical treatment was prepared in the same manner as in Example 5. Filters such as ZetaPlus are ground to Dowex Optipore with particles smaller than 50 μm instead of powdered activated carbon according to the Cuno R1xS specification.
It was prepared using L493. Plasma was pumped through the filter at 20 mL / min and 180 mL and 325 mL samples were taken. 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen and coagulation factors were analyzed as in Example 5.

[0204]

[Table 7] Thus, by increasing the volume of treated fluid, additional selectivity can be imparted to the removal of lower molecular weight compounds than to the reduction of biological activity. Of course, this has limitations, and the selectivity decreases again as the IAD capacity approaches that of low molecular weight compounds.

Example 11 Use of Sintering Medium. Discs 90 mm diameter x 1/4 "thick were manufactured by Porex. Discs containing various weight ratios of finely ground Dowex Optipore L493 with particle size between 20 and 100 µm;
Small particles of ultra high molecular weight polyethylene (grade UF220) were sintered together.
Photochemically treated plasma was prepared in the same manner as in Example 5, and 200 mL of plasma was added to 1
Pumped through each disc at 6-18 mL / min. 4 '-(4-amino-
2-Oxa) butyl-4,5 ', 8-trimethylpsoralen and coagulation factors were analyzed as in Example 5.

[0206]

[Table 8] Although the use of a fibrous matrix containing activated carbon was seen in Example 7, changing the ratio of adsorbent in the IAD also affected both the removal of low molecular weight compounds and biological activity when using a sintered matrix. Exert. In this case, the 25% formulation would be 4 '-(4-amino-2-oxa) butyl-4,5, like the 35% and 50% formulations.
It does not give sufficient removal of ', 8-trimethylpsoralen. The 50% formulation loses more Factor VIII activity than the other formulations. 3 of these 3 media
The 5% formulation provides the highest selectivity of low molecular weight compound removal for reduced biological activity.

Example 12 In this example, the effect of increasing the mass of the adsorbent by increasing the diameter of the filter while keeping the thickness constant will be described.

Plasma subjected to photochemical treatment was prepared in the same manner as in Example 5. About 3 of plasma after treatment
Twenty-five mL was pumped at 5 mL / min through 90 mm diameter and 47 mm diameter R03S grade discs. 4 '-(4-amino-2-oxa) butyl-4,
5 ', 8-Trimethylpsoralen and coagulation factors were analyzed as in Example 5.

[0209]

[Table 9] Increasing the adsorbent diameter while maintaining the same thickness and the same flow rate has the same effect as reducing the flux and reducing the processing volume per unit cross-sectional area of the filter. Both of these effects tend to increase adsorption of low molecular weight compounds, but increase adsorption of biologically active mediators even more.
In this example, it can be seen that the adsorbent with the larger diameter adsorbs slightly more S-59 but substantially loses more Factor I activity.

Example 13 Flow studies with several forms of activated carbon media. P using a flow device
5-[(β-carboxyethyl) -amino] acridine and GS from RBC
Experiments were conducted to investigate the removal of H 2. PRBC (Erythrosol (eryt
hrosol), glucose, 63% HCT) at 300 μM degradable 5-[(β
-Carboxyethyl) amino] acridine derivative and 3 mM GSH were added and kept at room temperature without stirring for 20 hours before flushing through the device. The flow compound adsorption device media consists of various forms of activated carbon, such as a carbon / cellulose composite disc or carbon fiber felt. The media was sealed in a 90 mm diameter polycarbonate housing (Cuno, Meridien, CT). The added PRBC was pumped through the medium at a flow rate of 5 mL / min (Gilson M
inipuls, Middleton, Wis., PL146 plastic container (
Harvested in Baxter Healthcare). The results of this study are shown in Table 1.

[0211]

[Table 10] Example 14 Flow Compound Adsorption Device (CUNO Medium) vs. Batch Compound Adsorption Device (A
QF medium). In a flow compound adsorption device vs. a batch compound adsorption device,
Experiments were performed comparing the removal of 5-[(β-carboxyethyl) amino] acridine and GSH. Flow device is cellulose / Norit A Supr
a (Norit Americas, Inc. (Atlanta, GA)) carbon disk confined in a 90 mm housing. Two separate batch devices were used. That is, one is fibrous Pica G277 activated carbon (A
QF 500 g / m ² ) and the other is fibrous Purolite MN-2
00 (AQF 312 g / m ² ). Degradability of 300 μM 5
-[(Β-Carboxyethyl) -amino] acridine derivative and 3 mM GS
After the addition of H, PRBCs were pumped through the cellulose / carbon medium at a flow rate of 2 mL / min. After pumping, 5-[(β-carboxyethyl) -amino]
Acridine and GSH levels were reduced by 75% and 88%, respectively, and PRB
24 μM and 0.71 mM in C supernatant. PRB on the flow device
C was then transferred to a 6g MN-200 batch device. This makes 2
5-[(β-carboxyethyl) -amino] acridine and GS during 4 hours
H levels were further reduced by 5% and 1% to 20 μm and 0.67 mM. When PRBC is applied only to the 7g Pica G277 batch device, 5-
The level of [(β-carboxyethyl) -amino] acridine and GSH is 92.
% And 54%, resulting in concentrations of 8 μM and 3 mM. Therefore, a single pass through the flow device was more effective for GSH removal than a carbon batch device for 24 hours. However, the batch device alone was more effective at removing 5-[(β-carboxyethyl) -amino] acridine than the combination flow device and MN-200 device. Flowing through the device did not appear to adversely affect PRBC ATP concentration. The K + level in PRBC was lower after being subjected to the flow device compared to the carbon batch device for 24 hours.

Example 15 In this example, a Cuno consisting of the 30% Norit A Supra carbon (Norit Americas, Atlanta, GA) described in the above examples.
Figure 6 illustrates the typical performance of plasma-immobilized adsorption devices in flow mode with adsorbent media manufactured by (Meriden, CT).

R10S impregnated with 90 mm Cuno 30% Norit A Supra
IAD was assembled by connecting P medium and polysulfone membrane having 5 μm pores coated with 90 mm diameter Memtec hydroxypropylcellulose.

To complete as a disposable product, 1 LPL2410 bag is 1/8 "O
After docking with the D tube, the tube was connected to the IAD outlet so that the distance from the center line of the IAD to the bag was 40 cm. A tube of the same size was connected to the inlet of the SRD so that the centerline to bag distance was 30 cm when the irradiation bag was docked.

To 500 mL of plasma, 4 '-(4-amino-2-oxa) butyl-4,5',
8-Trimethylpsoralen was added to a final concentration of about 150 μM and plasma was added to
． Pathogens were inactivated by irradiation with ³ J / cm ² UVA. The concentration of 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen after irradiation was about 82 μM.

Table 2 provides the IAD with measurements of coagulation factor activity and 4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen levels after treatment with IAD. It is shown in comparison with the value before it was passed. The experiment was repeated 3 times. Mean values and standard deviations are also shown. The processing time was 14 minutes on average. Factor I, II, V, VII, Factor X or aggregate coagulation activity, prothrombin time (
It is clear that the measured values of PT) and activated partial thromboplastin time (aPTT) are virtually unchanged, and that Factors XI and XII change very little. Although Factor VIII and Factor IX show slightly large changes,
They are acceptable, especially when considering the formulation of recombinant proteins for these deficiencies. 4 '-(4-Amino-2-oxa) butyl-4,5', 8-trimethylpsoralen, while allowing only 0.9% of it to pass through, substantially all coagulation activity is retained. The very high selectivity of should be noted.

[0217]

[Table 11] Example 16 Reduction of activated complement by adsorption medium. This work illustrates the removal of biological response modifiers by cellulosic media impregnated with activated carbon and porous plastic media impregnated with milled polystyrene / divinylbenzene adsorbent.

Zymosan (Sigma Chemi), an Effective Activator of the Complement Cascade,
cal Company; St. Louis, MO) was added to plasma at a concentration of 10 mg / mL. Plasma was incubated for 1 hour at 37 ° C with gentle shaking. The plasma was then centrifuged and the supernatant was removed to remove solid zymosan. About 20 mL of this supernatant was added to two 600 mL plasma units, and samples were taken from each unit and subjected to C3a and SC5b-9 analysis. One of these units was fitted with a 90 mm disk of carbon impregnated cellulosic medium (Cat. # 2640ASP, Cellulo, Inc .; Fresno, CA.
) At 40 mL / min. The other unit was a sintered medium prepared as in Example 11 (Porex Technologies; Fairbu).
pumping at the same rate through rn, GA). A sample was taken from the filtrate of each unit and subjected to C3a and SC5b-9 analysis.

[0219]   Complement assays were performed using the Quidel assay kit.

[0220]

[Table 12] As shown in the table above, biological response modifiers may also be removed from the biological composition.

Example 17 Comparison of carbon fiber and carbon-impregnated cellulose. In this example, US Pat.
The use of the carbon fiber media disclosed in No. 731 is compared to the media disclosed herein.

Plasma subjected to photochemical treatment was prepared in the same manner as in Example 5. About 1 of plasma after treatment
75 mL was pumped through each medium at approximately 11 mL / min. S-59 and coagulation factors were analyzed as in Example 5.

[0223]

[Table 13] Supplier of medium: Kynol (American Kynol, Inc .; Ple
asantville, NY); Kuractive (Kuraray Che)
medical Co. Bizen City, Japan); Actitex (Pica USA; C)
olumbus, OH); Cuno A Supra (Cuno, Inc .; M
eridien, CT).

As shown in the table above, removal of low molecular weight compounds from biological compositions using carbon fiber media as disclosed in US Pat. No. 5,660,731, is described herein. Often, it does not sufficiently reduce the concentration of low molecular weight compounds in the biological composition as compared to the adsorbent media disclosed in the publication.

Example 18 Effect of pore size distribution of adsorbent on biological activity. This example illustrates that the pore size distribution of the adsorbent contained in the IAD can have a significant effect on the ability of the IAD to maintain its biological activity.

Porex Tec is a 90 mm diameter x 1/4 "thick porous plastic disk.
manufactured by hnologies (Fairburn, GA). The discs were made of Porolite (Bala Cynwyd) milled by Porex such that about 50% by weight of about 25 μm diameter particles of ultra high molecular weight polyethylene and more than 90 (wt)% of the particles had a diameter between 60 μm and 160 μm. , P
50% of one of the non-functionalized Hypersol-Macronet adsorbents of A).
% Of the sintered mixture. As described above, the S-59 solution was added to each of the three about 600 mL plasma units so that the concentration of S-59 was about 150 μM, and each unit was irradiated with UVA of 6.3 J / cm ² , Plasma was subjected to photochemical treatment. The treated units were pooled and about 600 mL of the pool was pumped through each disc at about 40 mL / min and analyzed for S-59 and coagulation factors as in Example 5. Equilibration step mercury penetration hole measurement (equilibra)
ted-step mercury intrusion porosimet
Surface area was analyzed by ry) (Micromeritics, Norcross, GA).

[0227]

[Table 14] IA as measured by aPTT, Factor IX activity, and Factor XI activity
The choice between these three specific adsorbents used in D had a strong effect on biological activity, but was strong against the extent of reduction of the low molecular weight compound S-59 used in this example. It had no effect.

The three adsorbents MN200, MN250 and MN270 used in the IAD in this example differ primarily in the pore size at which they each begin to have a quantifiable surface area and are not substantially different in surface chemistry. A consistent conclusion with the results is that the accessible surface area for low molecular weight compounds is similar among the three adsorbents, resulting in a similar reduction in S-59 for each IAD. Also, the reason for the better biological activity of IADs containing adsorbents with smaller surface areas associated with larger pores is due to the fact that larger pores tend to adsorb to the surface of the adsorbent. Specific mediator of biological activity (ie, I in this example)
X and Factor XI) are capable of adsorbing or deactivating to the surface of the larger pores, while the smaller pores exclude these mediators from the surface.

Example 19 This example demonstrates the use of I to remove low molecular weight compounds such as viral inactivators (psoralens) or biological response modifiers (activated complement-C3a) from whole blood.
Illustrates the usefulness of using AD. Pretreatment of whole blood with a cellulose acetate membrane resulted in activation of complement as seen in hemodialysis using a cellulose acetate membrane. Blood perfusion to remove activated complement and added psoralen was simulated by returning the effluent to a mixed pool of whole blood from the blood perfusion device (see Figure 13).

Two units of ABO-matched whole blood were added to Sacramento Blood.
d Center (Sacramento, CA). These units were kept at room temperature after donation. These two units, Millipore R
A type membrane (47 mm, Millipore, Marlborough, MA)
Two PL2410 plastic storage containers containing four (Baxter Heal
Thcare, Deerfield, IL). Cellulose acetate membrane (Mi
Whole blood was incubated for 24 hours at room temperature with llipore RA) to induce complement activation. Immediately before the start of blood perfusion, psoralen (150 μM S-5
9 (4 '-(4-amino-2-oxa) butyl-4,5', 8-trimethylpsoralen)) was added to each whole blood unit.

The IAD medium (300 g / m ² , MN-200, AQF) was cut into a circular disk shape having a diameter of 47 mm. The disks were housed in a 47 mm diameter polycarbonate housing with a stainless steel support screen (Cuno Inc., Meridi).
en, CT). Tube (3mm ID PharMed tube
ing) was attached to the housing. Peristaltic Pump (Masterfl)
ex) and calibrated the system for delivery at a flow rate of 75 mL / min. The tube inlet and outlet were attached to a 600 mL beaker (Nalgene) placed on a stir plate (see Figure 13). 500 mL throughout the study
The whole blood contained in the beaker was gently agitated with a Teflon-coated stir bar to simulate the mixing that would occur in a subject's body.

A solution containing 174 USP units of heparin per mL of saline (sodium salt, grade 1-A, 174 USP units / mg, Sigma Chemical).
Co. ) Rinsed the entire assembly. Saline was removed from the IAD assembly prior to introducing whole blood. A unit of whole blood (500 mL) was added to the beaker. Agitation was slowly increased until blood mixing was apparent. The whole blood flow was started. Whole blood was allowed to flow from the bottom of the IAD as shown in FIG. Samples of whole blood were taken from the beaker at 15 minute intervals. Immediate cell counts were performed using a Baker cell counter (product). Micro centrifuge (10,000 rpm, 5
Min), the sample was centrifuged, the supernatant removed and immediately frozen for later analysis.

Samples of the supernatant were thawed and analyzed for residual levels of psoralen using reverse phase HPLC. The level of activated complement fragment C3a was also determined using ELISA (Quidel, San Diego, CA). Finally, the level of allogeneic hemolysis was determined as described above.

Cell counts, hemolysis measurements, psoralen measurements, and C3a measurements are summarized in Table 3. In general, removal of both psoralen and C3a was shown. Moreover, white blood cell (WBC) and red blood cell (RBC) counts remained substantially constant throughout the study. Platelet counts showed a downward trend during this study with a slight increase in allogeneic hemolysis. Further studies with fresh whole blood will be needed to determine if the loss of platelets was due to the initial room temperature incubation performed on whole blood.

The advantages of using immobilized adsorbents in hemoperfusion devices include: That is, 1) the ability to independently control the particle size and pressure drop of the adsorbent-especially important at high flow rates or small particle adsorbents; The ability to control particle attrition by minimizing; 3) the ability to immobilize adsorbent particles to minimize small particle contamination and spills from the device; and 4) maintain a uniform and stable adsorption bed. ability.

[0236]

[Table 15]

[Brief description of drawings]

FIG. 1 schematically depicts a perspective view of one embodiment of a fiber, showing an inner core and an outer sheath, which form a fiber network of fiberized resin.

[Fig. 2]   FIG. 2 schematically illustrates a portion of one embodiment of the fiberized resin of the present invention.

FIG. 3 schematically illustrates a cross-sectional view of one embodiment of a fiberized resin, where:
The adsorbent beads are fixed to the fibers that make up the fiberized resin.

FIG. 4 schematically illustrates a cross-sectional view of one embodiment of a fiberized resin, where:
The adsorbent beads are fixed within a heat seal surrounding the fibers of fiberized resin and the sample of fiberized resin.

FIG. 5 shows Dowex® XUS-43493 and Amberlit.
e (R) XAD-16 HP free adsorbent beads and Amberlit
Figure 6 is a graph showing a comparison of adsorption kinetics for the removal of aminopsoralen from platelets using a fiberized resin containing e (R) XAD-16.

FIG. 6 is p (HEMA) coated Dowex® XUS-4349.
Figure 3 is a graph showing a comparison of adsorption kinetics for the removal of aminopsoralens from platelets using 3 beads and uncoated beads.

FIG. 7 shows Amberlite® XAD-16 and Dowex® XUS-43493 in the adsorption capacity of the relevant aminopsoralens.
3 is a graph showing a comparison of effects of glycerol content of pretreatment solutions.

FIG. 8 shows Amberlite® XAD-16 (bottom) and Dowe.
4 '-(4-amino-2-oxa) butyl-4,5', 8- on dried adsorbent in 100% plasma against x (R) XUS-43493 (top).
Figure 6 is a graph showing a comparison of the effect of wetting solution on trimethylpsoralen adsorption capacity; samples not wet with ethanol solution are labeled as "No Tx". Adsorption capacity is reported as a percentage of the capacity of the optimally moistened adsorbent.

FIG. 9 shows Amberlite® XA in several different solutions.
FIG. 8 is a graph showing a comparison of aminopsoralen adsorption from plasma over a period of 3 hours using D-16 Wet.

FIG. 10 is a graph showing a comparison of the adsorption kinetics of methylene blue from plasma over a 2 hour period.

FIG. 11 shows the chemical structures of acridine, acridine orange, 9-aminoacridine, and 5-[(β-carboxyethyl) amino] acridine.

FIG. 12 is a flow configuration diagram for a fixed adsorption device (IAD).

[Fig. 13]   FIG. 13 is an example of an experimental setup for a whole blood perfusion study.

FIG. 14 shows an exploded view of a disk shaped assembly made in accordance with the present invention.

FIG. 15 is an exploded view of a drip chamber assembly made in accordance with the present invention.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SZ, UG, ZW), EA (AM , AZ, BY, KG, KZ, MD, RU, TJ, TM) , AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, D K, EE, ES, FI, GB, GE, GH, GM, HR , HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, L V, MD, MG, MK, MN, MW, MX, NO, NZ , PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, U S, UZ, VN, YU, ZW F-term (reference) 4C058 AA22 BB07 JJ04                 4C077 AA12 BB03 CC06 KK30 MM07                       NN18                 4D017 AA11 BA04 CA03 CB03 DB01                 4G066 AA05B AC02C AC14B BA09                       BA16 BA20 BA26 CA20 DA12                       FA18

Claims (10)

[Claims] 1. A method of reducing the concentration of a biological response modifier in a biological composition, the method substantially maintaining a desired biological activity of the biological composition. , Treating the biological composition with a device, the device comprising an inert matrix enriched in adsorbent particles, the adsorbent particles ranging in diameter from about 1 μm to about 200 μm. , The device is used in a flow process. 2. The method of claim 1, wherein the biological response modifier is activated complement. 3. The method of claim 1, wherein the biological composition is plasma. 4. The method of claim 1, wherein the adsorbent material has a length less than 3 times the width. 5. The method of claim 1, wherein the adsorbent particles include a supercrosslinked polystyrene network. 6. The adsorbent particles are activated carbon particles, and the activated carbon particles are about 1 particle.
The method of claim 1 having a surface area of greater than 200 m ² / g. 7. The method of claim 6, wherein the activated carbon particles are formed by steam activation of coconut shell. 8. The method of claim 1, wherein the inert matrix of the device is a fiber network composed of cellulose. 9. The method of claim 1, wherein the inert matrix is a particle network formed by sintering particles of ultra high molecular weight polyethylene with particles of an ultra crosslinked polystyrene network. 10. The method of claim 1, further reducing the concentration of psoralen derivative, acridine derivative, dye or quencher in the biological composition.