WO1997018844A1

WO1997018844A1 - Inactivation method and system in biological fluids

Info

Publication number: WO1997018844A1
Application number: PCT/US1996/019047
Authority: WO
Inventors: Raleigh A. Carmen; Thomas J. Bormann; Frank R. Pascale
Original assignee: Pall Corporation
Priority date: 1995-11-21
Filing date: 1996-11-15
Publication date: 1997-05-29
Also published as: AU1085697A

Abstract

Methods and systems for inactivating potential pathogens in biological fluids such as blood or blood components are disclosed, e.g., by adding photoactivating agents and separating them after photoactivation.

Description

INACTIVATION METHOD AND SYSTEM IN BIOLOGICAL FLUIDS This application is a continuation-in-part application of copending application Serial No. 08/561 ,621 , filed November 21 , 1995, which is incorporated by reference in its entirety.

Technical Field

This invention relates to inactivation protocols, especially involving the removal of inactivating agents, such as photoactive agents, from fluids, particularly biological fluids such as blood or blood components.

Background of the Invention The presence of potentially pathogenic material such as viruses and/or bacteria in biological fluid is of great concern during many protocols, particularly those involving the processing of blood and/or blood components, e.g. , to prepare transfusion products to be administered to a patient. For example, the introduction of disease causing material such as microorganisms, viruses and/or endotoxins into a patient, e.g., through administration of a contaminated fluid, may have serious, and possibly fatal, ramifications for the patient. Additionally, those who may come in contact with the contaminated fluid, e.g. , laboratory and/or medical technicians, may also face health risks resulting from exposure to the pathogens.

Some protocols for inactivating potentially pathogenic material include exposing the material to light (or other forms of radiation), in the presence of an inactivating material such as psoralen or methylene blue. It is believed that these inactivating materials are photoactive, so that in the presence of light they become activated and will react with the membrane structures and/or nucleic acids of the material in such a manner that the material, e.g. , viruses and/or microorganisms such as bacteria, may be killed, or prevented from replicating.

However, some inactivation protocols have suffered from several drawbacks. For example, the fluid to be treated may include components or constituents such as, for example, plasma proteins and/or leukocytes, that reduce the efficiency of inactivation, e.g. , by binding to the photoactive agent and/ or interfering with the materials' exposure to radiation. Since the material is bound, and/or fails to receive sufficient radiation, the inactivation efficiency may be reduced. As a result, it may be necessary to increase the level or concentration of inactivating material in the fluid, to attempt to overcome the interference. However, the increased level or concentration of inactivating material may have adverse effects on the desirable constituents of the fluid to be treated. Additionally, or alternatively, since the level of inactivating material is increased, the period for radiation exposure may have to be extended to allow for more complete activation. Moreover, as noted below, it may be desirable to remove the inactivating material from the fluid after treating the fluid, and it may be difficult to efficiently remove this additional amount of material.

It is desirable to remove the material from the fluid before administering the fluid to a patient, since the material and/or its byproducts is "foreign" to the recipient's system, and could cause an adverse result. Additionally, since some inactivating materials are thought to bind to and/or damage nucleic acids, i.e. , DNA and RNA, and damage to nucleic acids could lead to mutations, and possibly disease and/or birth defects, it would be desirable to minimize a patient's exposure to materials that could bind and/or damage nucleic acids. Accordingly, some fluid treatment or processing protocols include passing the inactivating material-including fluid through a filter or over a column to remove the material. However, these filters or columns may fail to efficiently remove the inactivating material and material byproducts, particularly when an increased amount of material is used.

In view of this, there is a need in the art for a method and system that provides efficient inactivation, and removal of the inactivation material(s) and/or inactivation material byproducts. There is also an unaddressed need in the art for a system and method that allows the use of a reduced amount or concentration of inactivating agent(s) such as photoactive agent(s), particularly when processing biological fluids such as blood and blood components.

Additionally, since biological fluids such as blood and blood components may also contain varying numbers of white blood cells (leukocytes), which may cause undesirable effects when administered to a patient, it would be highly desirable to provide a system and method for removing leukocytes, and inactivation materials(s) . The present invention provides for ameliorating at least some of the disadvantages of the prior art inactivation protocols, particularly for treating biological fluids such as blood and blood components. These improvements, and other advantages of the present invention, will be apparent from the description as set forth below.

Summary of the Invention

In accordance with the present invention, a biological fluid is depleted of a portion of fluid, e.g. , plasma, and an inactivating agent (such as a photoactive agent) and an additive solution are added to the biological fluid before inactivation is carried out, e.g. , by activating the inactivating agent. The depletion of plasma and the use of the additive solution allows inactivation of viruses and/or microorganisms in the biological fluid to be efficiently carried out while reducing or minimizing the amount or concentration of inactivating agent utilized. The invention also provides for the removal of the inactivating agent (and byproducts thereof) from the biological fluid combined with additive solution, preferably by passing the fluid through a inactivating agent binding filter assembly. The biological fluid, which has been treated to inactivate potential pathogens, and subsequently depleted of inactivating agent, is especially suitable for administration to a patient.

The present invention also provides sets for processing a biological fluid to inactivate potentially pathogenic material that may be present in the biological fluid. In one embodiment, the set includes a plurality of containers, including a container suitable for holding an inactivating agent (e.g. , a photoactive agent) and an additive solution, and an inactivating agent binding filter assembly comprising an inactivating agent binding medium disposed in a housing having an inlet and an outlet and defining a fluid flow path between the inlet and the outlet and through the binding medium. In another embodiment, the set includes separate containers for the inactivating agent and the additive solution. In a more preferred embodiment, the filter assembly also includes a bypass that allows a fluid, e.g. , including inactivating agent and/or additive solution, to avoid passing through the inactivating agent binding medium.

The present invention is compatible with a variety of fluid treatment protocols that include the use of inactivating agents such as photoactive agents for inactivating potential pathogens such as viruses and/or bacteria. Additionally, inactivation can be carried out in a closed sterile system. In some embodiments, inactivation can be carried out as part of an automated protocol.

In describing the present invention, the following terms are used as defined below. (A) Biological Fluid. In accordance with the invention, biological fluid includes any treated or untreated fluid associated with living organisms, particularly blood, including whole blood, warm or cold blood, and stored or fresh blood; treated blood, such as blood diluted with a physiological solution, including but not limited to saline, nutrient, and/or anticoagulant solutions; one or more blood components, such as platelets suspended in plasma, platelet concentrate (PC), platelet- rich plasma (PRP), plate let- free plasma, platelet-poor plasma (PPP), plasma, packed red cells (PRC), transition zone material, buffy coat; analogous blood products derived from blood or a blood component or derived from bone marrow; red cells suspended in physiological fluid; and platelets suspended in physiological fluid. The biological fluid may include leukocytes, or may be treated to remove leukocytes. As used herein, biological fluid refers to the components described above, and to similar blood products obtained by other means and with similar properties.

(B) Photoactive agent. A photoactive agent is a material that is capable of undergoing a chemical reaction when activated by radiation, e.g. , light. Preferably, the photoactive agent is activated in the presence of at least one nucleic acid, i.e. , DNA and/or RNA, and the chemical reaction leads to damage and/or binding with the nucleic acid. Typical photoactive agents include, but are not limited to at least one of porphyrins, and their derivatives; furocoumarins such as psoralens and their derivatives, such as 8-methoxypsoralen, 5-methoxypsoralen, 4'-aminomethyl-4,5'8-trimethylpsoralen (AMT); phthalocyanines, such as aluminum phthalocyanine; merocyanines such as MC540; and other photoactive dyes such as acridine dyes; xanthene dyes, e.g. , rose bengal and eosin Y, and thiazine dyes, such as phenothiazine dyes. In one embodiment, the photoactive agent may comprise a drug such as a member of the family of light- activated drugs derived from benzoporphyrin. These derivatives are sometimes referred to as BPDs. In an embodiment, the photoactive dye is a thiazine dye, such as, but not limited to, at least one of thionine, toluidine blue, neutral red, and methylene blue. The photoactive agent may be activated by visible light, sunlight, ultraviolet radiation, x-ray, light emitting diodes, and other forms of radiation.

The photoactive agent is capable of inactivating a wide variety of undesirable matter, particularly potential pathogens. For example, the photoactive agent is capable of inactivating viruses and/or microorganisms such as bacteria and protozoa. Photoactive agents may be utilized individually, sequentially, or in combination.

One preferred photoactive agent, methylene blue, is 3,7- Bis(dimethylamino)phenothiazin-5-ium chloride, C₁₆H₁₈C1N₃S. Other preferred photoactive agents, include, for example, at least one psoralen or psoralen derivative.

(C) Photoactive agent binding medium. A photoactive agent binding medium is a medium that removes or separates photoactive agent(s) (and byproducts thereof) from a photoactive agent- including fluid. During typical use, a fluid including photoactive agent, biological fluid, and additive solution is placed in contact with the photoactive agent binding medium, and the medium removes the agent (and byproducts thereof) from the fluid, e.g. , by binding the agent and byproducts. Preferably, the binding medium has little or no effect on the fluid and/or the desirable constituent(s) or component(s) of the fluid. For example, in one embodiment, the binding medium can remove activated photoactive agent and its byproducts from a suspension of platelets (typically also including additive solution), and the platelets remain suitable for use as a transfusion product. In another embodiment, the binding medium can remove activated photoactive agent and its byproducts from a suspension of red blood cells, and the red blood cells remain suitable for use as a transfusion product.

The binding medium is capable of removing the photoactive agent and/or the byproducts from fluid before and/or after activation. In a preferred embodiment, the binding medium removes or separates photoactive agent from biological fluid after the agent has been activated.

The photoactive agent binding medium may be in a variety of forms, e.g. , particles, fibers, sheets. The medium can be in the form of a screen, a depth filter, hollow fibers, a web, or a film. The photoactive agent binding medium can comprise a porous medium, e.g., in a filter housing. Alternatively, the photoactive binding material can comprise particles, e.g. , in a filter housing, bed, or column.

In an embodiment, the photoactive agent binding medium removes or separates other types of inactivating agents, and byproducts thereof, from an inactivating agent-including fluid. Other types of inactivating agents that can be removed from by the binding medium include, for example, those agents that do not require radiation for activation.

(D) Additive solution. An additive solution is a fluid, typically a synthetic fluid that is essentially free of plasma, that is compatible with a biological fluid and is usually also compatible with an inactivating agent such as a photoactive agent. A variety of suitable additive solutions are commercially available and/or are described in the literature.

Typically, the additive solution comprises at least one nutrient (e.g. , glucose) and/or buffer (e.g. , bicarbonate or phosphate). The nutrient can also be a buffer (e.g. , acetate). In some embodiments, the additive solution comprises at least one nutrient and a buffer in a physiological solution such as an isotonic carrier. Illustrative additive solutions include saline solutions, e.g. , comprising about 0.9% saline; and solutions containing glucose and bicarbonate or phosphate in an isotonic carrier. One example of a suitable isotonic carrier is saline.

Typically, additive solutions for use with biological fluids including red blood cells include adenine. Illustrative solutions for use with biological fluids including red blood cells include solutions such as SAGM, AS-3, AS-1 , and AS-5. Illustrative solutions for use with biological fluids including platelets include, for example, those disclosed in U.S. Patent Nos. 4,447,415 and 4,695,460; as well as those disclosed in Blood, vol. 67, pp. 672-675 (1986) and British Journal of Haematology, vol. 66, pp. 233-238 (1987).

In accordance with preferred embodiments of the invention, an additive solution is utilized to form a fluid mixture comprising plasma-depleted biological fluid, additive solution, and photoactive agent, and the photoactive agent is activated. Subsequently, essentially all of the photoactive agent (and/or byproducts thereof) is removed from the mixture, leaving additive solution combined with plasma-depleted biological fluid. In some embodiments, this additive solution/biological fluid mixture (which has been depleted of photoactive agent and/or photoactive agent byproducts) is stored for an extended period, such as several days or more. For example, an additive solution/platelet mixture may be stored for about 5 days, or more, and an additive solution/red blood cell mixture can be stored about 42 days, or more. (E) Fluid mixture. A fluid mixture comprises biological fluid (typically plasma-depleted biological fluid), at least one additive solution, and at least one inactivating agent such as a photoactive agent. In accordance with the invention, a mixture can be formed once the biological fluid, the additive solution, and the photoactive agent are in the same container or conduit. For example, the mixture is formed once the fluid, solution, and agent are combined in the same container. If desired, the contents of the container can be stirred or agitated.

Brief Description of the Drawings

Figure 1 is an embodiment of a system according to the present invention, including a plurality of containers, and an inactivating agent binding filter assembly including a bypass.

Figure 2 is another embodiment of a system according to the present invention including a plurality of containers, an inactivating agent binding filter assembly including a bypass, a leukocyte depletion filter assembly, and a red blood cell barrier filter assembly.

Figure 3 is an embodiment of an inactivating agent binding filter assembly according to the invention.

Specific Description of the Invention

Each of the components of the invention will now be described in more detail below.

The inactivating agent binding medium (hereinafter referred to as the photoactive agent binding medium) 1 is capable of binding at least one inactivating agent and/or byproduct thereof when the agent (typically combined or mixed with a biological fluid and an additive solution) is placed in contact with the binding medium. In a preferred embodiment, the binding medium provides for substantially complete removal of the photoactive agent and byproduct thereof from the biological fluid/additive solution, i.e. , the amount of photoactive agent and byproduct(s) that may be present is below the minimum detectable by conventional protocols routinely utilized in the art, including, for example, high pressure liquid chromatography (HPLC) and spectrophotometry. The desirable components of the biological fluid which have been depleted of the inactivating agent, may be efficiently recovered in an amount suitable for further use. The desirable components of the biological fluid can be at least one of the following: platelets, red blood cells, plasma, plasma proteins and coagulation factors. Exemplary plasma proteins and coagulation factors include fibrinogen (Factor I), prothrombin (Factor II), Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, plasminogen, antithrombin III, and Cl-inactivator. Embodiments of the invention include recovering at least one plasma protein depleted of the photoactive agent. Preferred plasma proteins and coagulation factors that can be recovered in accordance with the invention include prothrombin, fibrinogen, and Factor X. Preferably, the photoactive agent binding medium 1 comprises a porous medium, e.g. , a fibrous web or mat, or a porous film. A variety of media are suitable for use as a photoactive agent binding medium. In some embodiments, the binding medium includes carbon fibers, preferably activated carbon fibers. Suitable carbon fibers include, for example, phenolic fibers and novoloid fibers. In one embodiment, the binding medium comprises a self-supporting porous medium including activated carbon fibers, and the activated carbon fibers are themselves porous.

The binding medium may have a variety of configurations, including, for example, one or more of the following: a column, a web, sheet, a cylinder, and a depth filter. Of course, in some embodiments, for example, including at least one web, the configuration may also provide for depth filtration. The binding medium can have a planar configuration, or a pleated configuration. However, the medium may be formed into any geometric shape or form suitable for contacting a biological fluid. More preferably, the binding medium is in a shape or form suitable for passing a biological fluid containing at least one photoactive agent through the medium. Typically, the medium is disposed in a housing to provide a filter assembly. A variety of suitable housings are known in the art.

The medium may include two or more layers and/or media. Layers and/or media may be fibrous and/or membranous. Layers and/or media may provide prefiltration, support and/or better drainage. The binding medium may comprise layers and/or media each having a different pore structure.

In some embodiments, the medium 1 may be arranged within a container such as a flexible blood bag. Typically, however, as illustrated in Figure 3, the photoactive agent binding medium 1 is arranged within a housing to form a filter assembly or device 100. For example, in a preferred embodiment, as illustrated in Figure 3, filter assembly 100 comprises a housing having a first portion 102 including an inlet 106, and a second portion 101 including an outlet 105, and defining a fluid flow path between the inlet and the outlet, with the photoactive agent binding medium arranged across the flow path between the inlet 106 and the outlet 105. Accordingly, as a biological fluid or fluid mixture including at least one inactivating agent such as a psoralen or methylene blue (and/or a byproduct thereof) is passed through the assembly, photoactive agent contacts the binding medium, and is separated from the biological fluid or fluid mixture. In some embodiments, the filter assembly is capable of allowing bidirectional fluid flow therethrough. For example, fluid can flow from a first container through the assembly 100 (through inlet 106, medium 1, and then outlet 105) into a second container, and from the second container through the assembly into the first container (through outlet 105, medium 1 , and then inlet 106). Accordingly, the inlet can also be an outlet, and the outlet can also be an inlet.

However, in some embodiments, it may be desirable to allow unidirectional flow through medium 1. Accordingly, assembly 100 may also include a bypass 2, such as a conduit, that allows fluid to avoid passing through photoactive binding medium 1. For example, as illustrated in Figure 3, photoactive agent binding assembly 100 includes a bypass 2, with the bypass 2 being in fluid communication with inlet 106 and outlet 105 via connector 13 and conduit 120, and connector 12 and conduit 110, which are respectively below and above assembly 100. Of course, in some embodiments "inlet" 106 will function as an outlet, and "outlet" 105 will function as an inlet.

Flow control devices 7 such as clamps, valves, and the like, may be used to control the flow through the bypass 2 or through the medium 1, as desired. For example, a flow control device such as a clamp or valve can be in or on bypass 2, and additional flow control devices can be in or on conduits 110 and/or 120. Selectively opening and closing the flow control devices allows fluid to flow in the desired direction through the bypass 2 or the medium 1. Biological fluid, with or without inactivating agent (e.g. , photoactive agent) and/or additive fluid, may be collected, recovered and/or stored in at least one container, such as containers 5, 10, 15, 20 and 25 as illustrated in Figures 1 and 2. While a variety of containers, including flexible and non-flexible containers, are suitable, in some embodiments, the containers 5, 10, 15, 20 and 25 are commercially available blood collection and/or satellite bags. Preferably, at least one container is transparent to allow the contents of the container (e.g. , biological fluid and photoactive agent) to be more efficiently exposed to radiation. In some embodiments, the container utilized to allow the contents to be exposed to radiation comprises a plastic material such as poly (ethylene-vinyl acetate) (EVA).

Typically, the biological fluid, with or without inactivating agent and/or additive fluid, is passed from one container to another via conduits, e.g. , conduits 110, 120, 130, 140, 150, 160, 170, 180, 190, 210 and 220 and connectors, e.g. , connectors 12, 13, 14, and 16. A variety of conduits and connectors can be utilized. For example, commercially available flexible tubing and connectors, e.g. , as used in conventional blood processing systems, are suitable for use in the invention.

In the embodiments illustrated in Figures 1 and 2, conduits 110, 120, 130, 140, and 150, and connectors 12, 13, and 14, provide fluid communication with photoactive agent binding filter device 100, interposed between containers 5, 10 and 15.

In some embodiments, at least one conduit and/or container may have at least one inactivating agent contained therein. For example, at least one conduit may include at least one photoactive agent, e.g. , sealed between a first end and a second end of the conduit. Additionally, or alternatively, at least one container may contain at least one photoactive agent. If desired, the conduit and/or container may be steam sterilizable. In some embodiments, a steam sterilizable conduit and/or container may comprise a non- PVC plastic material, e.g. , at least one of polypropylene, styrene-ethylene-butylene- styrene (SEBS), poly(ethylene-vinyl acetate) (EVA), polyester, and polyurethane. A system 200 in accordance with the present invention includes a photoactive agent binding medium 1, at least one container, and at least one conduit. While the photoactive agent binding medium 1 may be located within a container such as a blood bag, in a more preferred embodiment, the medium 1 is positioned in a housing to provide a filter assembly 100, as illustrated in Figure 3, and as described above. A preferred system according to the invention includes a plurality of containers, conduits, connectors, and a filter assembly 100 in fluid communication with at least two containers. For example, the system illustrated in Figure 1 includes three containers, 5, 10, and 15, conduits 110, 120, 130, 140, 150, and 160; connectors 12, 13, and 14; and photoactive agent filter assembly 100 comprising photoactive agent binding medium 1 positioned in a housing. The illustrated system also includes a plurality of flow control devices 7 such as clamps or valves.

In some embodiments, the system includes additional devices such as at least one leukocyte filter device and/or at least one red cell barrier filter device. For example, the system illustrated in Figure 2 has some components as described with respect to Figure 1 (like components have like reference numbers), and also includes leukocyte filter device 21 ; and leukocyte filter device, red cell barrier filter device, or combined red cell barrier filter/leukocyte depletion device 11. Illustrative leukocyte filter devices and/or red cell barrier filter devices include those disclosed in, for example, U.S. Patent Nos. 4,880,548; 4,925,572; 5, 152,905; and 5,217,627; as well as International Publication Nos. WO 93/25295 and WO 93/04763. The housings disclosed in these patents and publications can also be suitable for use with a photoactive agent binding medium 1 to form an assembly 100.

The system illustrated in Figure 2 also includes additional containers 20 and 25, as well as conduits 170, 180, 190, 210, and 220, and connector 16.

The system 200 according to the invention may be open or closed. In a preferred embodiment, the system is closed. As used herein, the term "closed" refers to a system that allows the collection, processing, filtration, storage, and preservation of donor blood or blood components without the need to enter the system (and risk contamination of the system). A closed system can be as originally made, or result from the connection of the individual (or partially connected) components of such a system using what are known as "sterile docking" devices. Illustrative sterile docking devices are disclosed in U.S. Patent No. 4,507, 119.

The system, which may be part of system for automated processing as disclosed in, for example, International Publication WO 94/01193, is suitable for use with other devices including, for example, leukocyte filter and/or red cell barrier filter devices (as noted above), and/or gas processing devices. The system may include gas processing devices such as gas inlets and/or gas outlets as disclosed in U.S. Patent Nos. 5,126,054, 5,217,627 and 5,451 ,321 ; and International Publication WO 91/17809, as well as gas collection and displacement devices as disclosed in International Publication No. WO 93/25295.

In accordance with a method provided by the invention, a portion of fluid, typically plasma, is depleted from the biological fluid that is to be exposed to the inactivating agent(s) such as photoactive agent(s). Additive solution and photoactive agent are mixed with the plasma-depleted biological fluid to produce a fluid mixture. The method can include depleting the majority of the plasma from the biological fluid, and replacing most, if not all, of this volume of plasma with an equivalent volume of additive solution and inactivating agent. If desired, inactivating agent can be mixed with the biological fluid along with the additive solution to form a fluid mixture, or the inactivating agent and additive solution can be added separately to form the mixture. After the fluid mixture is formed, the inactivating agent is typically activated. For example, a photoactive agent can be exposed to radiation. In preferred embodiments of the method, the photoactive agent and/or byproducts thereof are subsequently removed from the fluid mixture, e.g., by contacting a photoactive agent binding medium with the photoactive agent and/or byproducts.

For example, the fluid mixture can be passed through a porous photoactive agent binding medium, to deplete the fluid mixture of photoactive agent and byproducts. The fluid that passes through the medium, which is depleted of photoactive agent and agent byproduct(s), is recovered for later use. The desirable components of the biological fluid which have been depleted of the photoactive agent and byproducts are efficiently recovered in an amount suitable for further use. The recovered photoactive agent depleted biological fluid is suitable for further processing or treatment, e.g. , fractionation and/or administration to a patient. The desirable components of the biological fluid can be at least one of the following: platelets, red blood cells, plasma, plasma proteins and coagulation factors. Exemplary plasma proteins and coagulation factors include fibrinogen (Factor I), prothrombin (Factor II), Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, plasminogen, antithrombin III, and Cl-inactivator. Embodiments of the invention include recovering at least one plasma protein depleted of the photoactive agent. Preferred plasma proteins and coagulation factors that can be recovered in accordance with the invention include prothrombin, fibrinogen, and Factor X. In some embodiments, for example, involving the processing of a platelet- containing fluid mixture, the mixture is placed in contact with the photoactive agent binding medium, and the photoactive agent is bound. The additive solution containing biological fluid, depleted of photoactive agent and its byproducts, is recovered, to provide platelets suitable for transfusion. Typically, a plurality of platelet-containing fluid mixtures are processed as described above, and the recovered platelets are pooled before transfusion.

Specific embodiments of the method are described in more detail below, using the systems illustrated in the Figures for general reference. For example, with respect to Figure 1, a unit of biological fluid can be held in container 15, and additive solution combined with inactivating agent can be held in container 5. Typically, flow control devices 7 are initially closed.

If desired, components of the biological fluid in container 15 can be concentrated according to their differences in specific gravities. For example, the container 15, holding a unit of biological fluid, can be centrifuged to separate components of the biological fluid by differential sedimentation. A portion of plasma can be depleted from the biological fluid in container 15, and passed through conduit 150 to container 10. If desired, conduit 150 can be subsequently clamped and/or heat sealed.

If the system includes a bypass, e.g. , bypass 2 (and the associated flow control device 7) as illustrated in Figure 1 , once the appropriate flow control devices 7 are opened and closed, the additive solution/inactivating agent can be passed from container 5 to container 15 through the bypass 2, and mixed with the plasma -depleted biological fluid to form the fluid mixture. For example, the flow control devices 7 associated with bypass 2 and conduit 130 can be opened, to allow the passage of additive solution/inactivating agent into container 15.

In those embodiments wherein the inactivating agent is a photoactive agent, the fluid mixture in the container 15, which is typically in a transparent container, is exposed to radiation, e.g., light, to activate the agent. The photoactive agent and its byproducts are then separated from the biological fluid/additive solution by placing the mixture in contact with the photoactive agent binding medium 1. In a typical embodiment, wherein the medium 1 is arranged within a housing to form a filter assembly 100 as illustrated in Figure 3, and used in a system such as that illustrated in Figure 1 , the photoactive agent and byproducts are separated by passing the mixture from container 15 along a fluid flow path from the inlet 106 through the photoactive agent binding medium 1 and the outlet 105 into container 5. For example, flow control device 7 associated with bypass 2 can be opened, to allow fluid to flow from container 15 and through medium 1 (in assembly 100) into container 5.

In the illustrative embodiments described above, the fluid mixture is formed in container 15. However, it should be clear that the fluid mixture could be formed in container 5, and the photoactive agent and byproducts can be separated by passing the mixture from container 5 through assembly 100 into container 15. Additionally, in other embodiments, e.g. , wherein the additive solution and the photoactive agent are held in separate containers (not shown), the mixture can be formed in any of the containers, and then passed through a photoactive agent binding medium 1 and into any container that is suitable for holding fluid depleted of photoactive agent(s) and byproducts thereof. A variety of additive solutions may be used in accordance with the invention, as long as the solutions are compatible with the biological fluid and the inactivating agent(s). As noted earlier, the additive solution can be mixed with the inactivating agent and then mixed with the biological fluid, or the additive solution can be mixed with the plasma depleted biological fluid before adding the inactivating agent. Accordingly, in a variation of the system according to Figure 1, additive solution, inactivating agent, and biological fluid, can be held in separate containers, to allow any combination of fluids before forming the fluid mixture.

In some embodiments, biological fluid can be combined with additive solution, and passed in one direction tlirough photoactive agent binding medium into a container of photoactive agent to form the fluid mixture. After the fluid mixture is formed, the mixture can be passed in the other direction through the photoactive agent binding medium into a container that is suitable for holding fluid depleted of photoactive agent(s) and byproducts thereof.

In accordance with the invention, a variety of biological fluids can be processed utilizing the system illustrated in Figure 1. In other embodiments, for example, using the system 200 illustrated in Figure 2 for reference, a biological fluid can be collected in container 25. In an illustrative embodiment, a biological fluid such as whole blood is collected in container 25, and centrifuged to form a supernatant layer of platelet-rich- plasma, and a sediment layer of concentrated red cells. The supernatant layer can be passed from container 25 into container 15. In preferred embodiments, a filter device 11 comprising a leukocyte filter device, red cell barrier filter device, or a combined red cell barrier filter/leukocyte depletion device, is inteφosed between container 25 and container 15. Accordingly, in some embodiments, the supernatant layer collected in container 15 is depleted of leukocytes and is essentially free of red blood cells. This supernatant layer in container 15 can be further processed as described earlier, e.g. , with reference to Figure 1. If desired, the concentrated red blood cells in container 25 can be further processed. For example, an additive or storage solution, e.g. , in container 20, can be passed into container 25, or the red blood cells can be passed into container 20, and the red blood cells can be stored for later use. In some embodiments, die red blood cells are depleted of leukocytes, e.g. , by passing the red blood cells through a leukocyte depletion filter device. Accordingly, as illustrated in Figure 2, a leukocyte depletion filter device 21 can be interposed between containers 25 and 20 to allow the red blood cells to be leukocyte depleted and passed into container 20.

A variety of protocols for treating fluid with at least one inactivating agent are known in the art, and the invention is not to be limited thereby. Illustrative protocols for contacting the fluid with photoactive agent include adding at least one photoactive agent to the biological fluid/additive solution, or by placing photoactive agent in a container and then passing the biological fluid/additive solution into the container. The photoactive agent utilized, and other parameters (e.g. , concentration of the agent, the amount of contact time before exposure to radiation) will depend upon the biological fluid to be treated and the characteristics of the photoactive agent used, as is known in the art.

Any biological fluid, especially any plasma-containing biological fluid, can be processed according to the invention. Moreover, components of the biological fluid, e.g. , red blood cells or platelets, may be separated from each other before or after contacting the fluid with photoactive agent. For example, a portion of plasma can be removed from whole blood and replaced with additive solution, and photoactive agent can be added to form a mixture. Subsequently, e.g. , after activation and depletion of the photoactive agent and byproducts, the red blood cells can be separated from the platelets.

Alternatively, whole blood can be processed to separate platelets from red blood cells, and a portion of plasma can be removed from the red blood cell and/or platelet suspensions. Additive solution and photoactive agent can be added to either or both suspensions to form mixtures, which can be subsequently processed as described above. The fluid mixture, i.e. , containing photoactive agent, biological fluid and additive solution, is exposed to radiation according to exposure protocols that are known in the art. Accordingly, the radiation source, the band of radiation utilized, and other parameters (e.g. , radiation intensity, length of exposure period) will depend upon the biological fluid to be treated and the characteristics of the photoactive agent used.

The fluid mixture is subsequently depleted of photoactive agent and byproducts as described previously, e.g. , by placing the photoactive agent and byproducts in contact with a photoactive agent binding medium. Typically, the fluid mixture is depleted of photoactive agent as it is passed through a photoactive agent binding device.

Illustratively, a mixture containing the photoactive agent methylene blue is passed through the binding device at a flow rate in the range of from about 5 ml/min to about

100 ml/min. In one embodiment, the flow rate is in the range from about 10 ml/min to about 80 ml/min. In some embodiments, the inactivating agent-depleted fluid may be washed, filtered, and/or stored, before further use. If desired, at least some of the additive solution can be removed from the inactivating agent-depleted fluid before further use. In some embodiments, leukocytes are removed from the fluid mixture or the biological fluid. For example, leukocytes may be removed before, after, or while passing the fluid mixture through the photoactive agent binding medium. Leukocytes can be removed from biological fluid before or after depleting plasma therefrom.

Typically, leukocyte depletion is accomplished by passing the biological fluid, or fluid mixture, through a leukocyte depletion medium, more preferably, a fibrous leukocyte depletion medium. Illustrative leukocyte depletion media, which are typically positioned in housings to form leukocyte depletion filter devices (e.g., devices 11 and 21 as described above), are disclosed in the U.S. Patents and International Publications referenced above. In an embodiment of the invention, the photoactive agent binding medium includes two or more layers, wherein at least one layer includes activated carbon fibers, and at least one layer includes a leukocyte depletion medium, so that the fluid passing through the medium may be depleted both of leukocytes and a photoactive agent such as methylene blue. In some embodiments, the photoactive agent binding medium includes a layer of activated carbon fibers inteφosed between layers of leukocyte depletion media. Such an configuration may provide for prefiltration (e.g. , removal of microaggregates before the fluid contacts the carbon) as well as removal of photoactive agent and leukocytes. This configuration may also prevent carbon fines from passing through the photoactive agent binding device.

Of course, as noted earlier, since the system may include at least one photoactive agent in a container or conduit, the blood, or at least one blood component, may be exposed to the photoactive agent at any desired point during processing. Since the conduits and containers are preferably transparent, the photoactive agent may be activated when desired.

During the processing of biological fluid in accordance with the invention, with or without inactivating agent, air or gas may be present and/or fluid may be trapped or retained in various elements of the processing system. It may be desirable to minimize the presence of air or gas and/or to maximize the recovery of the retained biological fluid. Accordingly, at least one of a gas inlet, gas outlet, and a gas collection and displacement loop as disclosed in, for example, U.S. Patent Nos. 5, 126,054, 5,217,627 and 5,451,321; and International Publication Nos. WO 91/17809, and WO 93/25295, may be used to separate gas from the biological fluid and/or to recover biological fluid trapped or retained in various elements of the system. As noted earlier, in some embodiments, the photoactive agent may be placed in contact with the binding medium by, for example, placing the medium (e.g. , without a housing) in a container including biological fluid and the photoactive agent. The fluid need not be passed along a defined fluid flow path through the binding medium to place the agent in contact with the binding medium and thereby bind the agent. Alternatively, or additionally, handling the container, e.g. , transporting, inverting and/or rocking the container, may cause additional fluid and agent to contact the binding medium, and additional agent may be bound. Accordingly, once the biological fluid and the binding medium are separated from one another, e.g. , by passing the fluid to a separate container, and/or by removing the fluid from the container holding die binding medium, the biological fluid is depleted of photoactive agent. In some embodiments, biological fluid including inactivating agent is placed in contact with the photoactive agent binding material more than once. For example, biological fluid including photoactive agent may be recirculated through the system to remove or deplete additional photoactive agent upon repeated contact with the binding medium. Illustratively, the biological fluid including photoactive agent may be repeatedly passed unidirectionally thorough the photoactive agent binding filter assembly. Alternatively, the fluid can be passed bidirectionally therethrough, e.g. , from inlet to outlet and outlet to inlet.

All of the references cited herein, including publications, patents, and patent applications, are hereby incoφorated in their entireties by reference. While the invention has been described in some detail by way of illustration and example, it should be understood that the invention is susceptible to various modifications and alternative forms, and is not restricted to the specific embodiments set forth. It should be understood that these specific embodiments are not intended to limit the invention but, on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

We claim:

1. A method for processing a biological fluid comprising: depleting plasma from a biological fluid to produce a plasma-depleted biological fluid; adding photoactive agent and additive solution to the plasma-depleted biological fluid to produce a fluid mixture, and; activating the photoactive agent.

2. The method of claim 1 further comprising separating the photoactive agent from the fluid mixture.

3. The method of claim 2 wherein separating the photoactive agent from the fluid mixture comprises placing the mixture in contact with a photoactive agent binding medium.

4. The method of claim 3 wherein placing the mixture in contact with the photoactive agent binding medium comprises passing the mixture through a photoactive agent binding filter assembly.

5. The method of claim 1 wherein adding additive solution to the plasma-depleted biological fluid comprises adding platelet additive solution to the fluid.

6. The method of claim 5 wherein the photoactive agent and the platelet additive solution are combined together before adding them to the plasma-depleted biological fluid.

7. The method of claim 1 wherein the photoactive agent comprises psoralen.

8. A method for processing a biological fluid comprising: separating a portion of plasma from a biological fluid including plasma and platelets to form a plasma-depleted platelet-containing biological fluid; adding a photoactive agent and a platelet additive solution to the platelet-containing fluid to produce a fluid mixmre including photoactive agent, additive solution, and platelets; activating the photoactive agent; and, separating the photoactive agent from the fluid mixmre to produce a photoactive agent-depleted fluid mixmre.

9. A method for processing a biological fluid comprising: treating a biological fluid to form a first sediment layer including red cells, and a first supernatant layer including platelets and plasma; separating the first sediment layer from the first supematant layer; treating the first supernatant layer to form a second sediment layer including platelets and a second supernatant layer including plasma; separating at least a portion of the second supematant layer including plasma from the second sediment layer including platelets; adding a photoactive agent and a platelet additive solution to the second sediment layer to produce a fluid mixmre including photoactive agent, additive solution, and platelets; activating the photoactive agent; and, separating the photoactive agent from the fluid mixmre to produce a photoactive agent-depleted fluid mixmre.

10. The method of claim 9 wherein separating the first sediment layer from the first supernatant layer comprises passing the first sediment layer through a porous medium.

11. The method of claim 9 or 10 further comprising adding a red blood cell additive solution to the separated first sediment layer to form a red blood cell additive mixmre.

12. The method of any one of claims 1-11 comprising processing the biological fluid in a closed system.

13. A method for processing a biological fluid comprising: depleting plasma from a biological fluid to produce a plasma-depleted biological fluid; adding inactivating agent and additive solution to the plasma-depleted biological fluid to produce a fluid mixmre, and; separating the inactivating agent from the fluid mixmre.

14. A system for processing a biological fluid comprising: a first container suitable for holding a platelet additive solution; a second container suitable for holding a platelet-containing solution, said container in fluid communication with the first container; and, inteφosed between the first container and the second container, an inactivating agent binding filter assembly comprising an inactivating agent binding medium disposed in a housing having an inlet and an outlet and defining a fluid flow path between the inlet and the outlet and through the medium.

15. The system of claim 14 wherein the filter assembly includes a bypass having a first end in fluid communication with the inlet of the housing and a second end in fluid communication with the outlet of the housing, said bypass comprising at least one conduit.

16. The system of claim 14 or 15 comprising a closed system.

17. The system of claim 16 further comprising a leukocyte depletion filter assembly.

18. The method of claim 1 wherein the biological fluid includes platelets, and the method includes separating photoactive agent, and byproducts thereof, from the fluid mixmre.

19. The method of claim 18 wherein separating photoactive agent and byproducts thereof comprises passing the fluid mixmre through a photoactive agent binding filter assembly.