WO1995000631A1

WO1995000631A1 - System for viral inactivation of blood

Info

Publication number: WO1995000631A1
Application number: PCT/US1994/007018
Authority: WO
Inventors: Henrietta Margolis-Nunno; Bernard Horowitz
Original assignee: New York Blood Center, Inc.
Priority date: 1993-06-23
Filing date: 1994-06-22
Publication date: 1995-01-05
Also published as: EP0707633A1; FI956078A0; JPH09500015A; AU698154B2; ZA944488B; FI956078A; KR960703167A; NO955291D0; CA2165065A1; NO955291L; EP0707633A4; AU7211394A

Abstract

A continuous-flow or semi-continuous flow system that accomplishes both virus inactivation of blood and virucidal reagent removal from treated blood. The system permits the pooling of blood prior to treatment with dyes together with visible light or compounds known to principally react with nucleic acids, the in-line use of filters or hydrophobic chromatography to remove unreacted virucidal compound together with its breakdown products, and the in-line distribution of the treated blood into single or multiple storage containers. It has been observed that any residual virucidal compound which cannot be separated from the blood by hydrophobic groups in filters or chromatography columns has lost the capacity to interact with nucleic acid and is no longer virucidal or mutagenic.

Description

SYSTEM FOR VIRAL INACTIVATION OF BLOOD

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a continuous-flow or semi-continuous flow system that accomplishes both virus inactivation of blood or a blood product and virucidal reagent removal from treated blood or a blood product.

2. Description of the Related Art

Blood can contain each of several different viruses including but not limited to hepatitis B virus (HBV), non-A, non-B hepatitis virus (NANBHV), cytomegalovirus, and immunodeficiency viruses. It is highly desirable to inactivate these viruses in the course of preparing blood products and prior to the therapeutic application of blood and blood fractions. The potential for transmission of these viruses presents a danger not only to potential recipients of blood and blood fractions, but can also pose a danger to persons involved in the preparation and administration of blood and blood products. Both physical (e.g., heat, irradiation) and chemical (e.g., aldehydes, organic solvents, detergents, etc.) methods have been used to inactivate viruses in mammalian blood and blood fractions.

Among the procedures for inactivating viruses are the use of lipid solvents with the addition of surface active agents (A.M. Prince, B. Horowitz, B. Brotman et al., "Inactivation of Hepatitis B and Hutchinson Strain non-A, non-B

Hepatitis Viruses by Exposure to Tween 80 and Ether", Vox Sang, (1984), 46, 36-43;

A.M. Prince, B. Horowitz, and B. Brotman, Sterilization of Hepatitis and HTLV- III Viruses by Exposure to Tri(n-Butyl)Phosphate and Sodium Cholate", The Lancet, 706-710, March 29, 1986, and U.S. Patent Nos. 4,481,189 and 4,540,573, the entire contents of which are incorporated herein by reference), and lipid solvents without the addition of surface active agents (S.M. Feinstone, K.B. Mihalik, T. Kamimura et al., "Inactivation of Hepatitis B Virus and non-A, non-B Hepatitis by Chloroform", Infectious Immunology, (1983), 41, 816-821; D.W. Bradley, J.E. Maynard, H. Popper et al., "Posttransfusion non-A, non-B Hepatitis: Physiochemical Properties of Two Distinct Agents", Journal of Infectious Diseases, (1983), 148, 254-265).

Solvent/ detergent treatment under appropriate conditions of temperature and contact time effectively disassembles viruses that have envelope proteins associated with lipid, while having negligible effect on the molecular conformations and biological activities of sensitive blood plasma proteins. Merocyanine, beta-propiolactone and cis-platin are among other agents that are applied to blood to inactivate viruses, though by mechanisms other than envelope disruption.

Current research into blood and blood component sterilization has emphasized the use of compounds, e.g., psoralens, phthalocyanines, etc., activatable by light or other forms of irradiation, e.g., X-rays. Despite the demonstration of kill of substantial quantities of virus(es) spiked into solution or cellular suspension being treated with maintenance of the structure /function of the desired blood component, substantial problems remain. First, because blood bags as currently manufactured have dead-ended ports which the virucidal compound may not enter completely, blood contained in the port may not be exposed to adequate virucidal conditions. Similarly, blood at the seam or outer edge of the blood bag may not fully equilibrate with virucidal compound. In either case, even though virus kill might be complete in the bulk of the blood or blood component, a single droplet of blood, if not adequately treated, contains sufficient virus to infect the recipient. Second, the activatable virucidal compounds and their breakdown products should be mostly or completely removed prior to infusion to reduce concerns about adverse toxicity and mutagenicity of these compounds. Third, unit-to-unit variation in treatment conditions should be minimized to ensure a uniformly high virucidal action, on the one hand, and unharmed blood components, on the other. Fourth, the virus sterilization system needs to be cost effective, both in terms of the cost of disposables and in labor costs.

Concerns about adverse toxicity and mutagenicity of these "photoactive" compounds appear to be mounting. Recently, at least one group has investigated the mutagenicity of the psoralen derivative aminomethyl-trimethyl psoralen (AMT) following treatment of platelet suspensions with the psoralen and UVA.

See S.J. Wagner, R. White, L. Wolf, J. Chapman, D. Robinette, T.E. Lawlor and

R.Y. Dodd, Photochemistry and Photobiology, 57(5), pp. 819-824 (1993). Significant mutagenesis was still observed for platelet suspensions irradiated with virucidal levels of UVA which maintain platelet in vitro function.

According to these investigators, the results suggested that residual available AMT is mutagenic in the Ames test and that the observed frameshift mutations may be caused by binding of AMT or its metabolites to nucleic acids in the absence of UVA light. The investigators advise caution before antiviral treatments with AMT are implemented. The investigators suggest that before antiviral treatments with AMT can be implemented, extensive toxicity and mutagenicity evaluation will be required and perhaps efforts will need to be made to achieve considerable reductions in residual psoralen levels in the treated platelet concentrates.

R. Bonomo, U.S. Patent No. 5,094,960, the entire contents of which are incorporated herein by reference, teaches a batch process for the removal of lipid soluble process chemicals, such as solvents, detergents, and phorbol esters, from labile biological mixtures by hydrophobic interaction chromatography, preferably using a resin comprising octadecyl (C18) chains coupled to a silica matrix. The process is advantageous because the biological activity of desirable components in such biological mixtures is substantially retained when the biological mixture is passed through the column, little or no desirable biological material is adsorbed to the column, and yet residuals of the lipid soluble process chemicals can be reduced to trace levels during the procedure. It is unclear whether the process is suitable for the removal of other types of virucidal compounds, such as dyes or photodynamic compounds, which, as mentioned above, are potentially toxic and/or mutagenic.

Judy et al., U.S. Patent No. 4,878,891, the entire contents of which are also incorporated herein by reference, teach an irradiation cell assembly, which operates on a flow principle and is useful for the inactivation of virus in blood and blood products. In practice, the blood or blood product to be treated is mixed with a photoactive porphyrin or hematoporphyrin compound. Then the mixture is fed to the irradiation cell assembly where the mixture is bathed in irradiation, thereby activating the photoactive porphyrin or hematoporphyrin compound and this, in turn, results in inactivation of any virus in the mixture. The amount of photoactive porphyrin or hematoporphyrin compound in the mixture is expressly limited to non-toxic amounts and the reference does not teach or suggest that it is advantageous to remove the photoactive porphyrin or hematoporphyrin remaining in the mixture after the irradiation treatment.

SUMMARY OF THE INVENTION

It was a principal object of the present invention to provide for a treatment system for blood and blood products that ensured complete sterilization of the blood and blood products so that the blood and blood products were not only safe for transfusion to patients, but also safe to prepare and handle.

It was another object of the present invention to provide for a treatment system for blood and blood products that ensured the most complete removal possible of virucidal and other added chemical reagents from treated blood and blood products.

It was another object of the present invention to provide for a means to remove mutagenic residue of the compounds employed to inactivate viruses.

It was also an object of the present invention to provide for a treatment system for blood and blood products that ensured a uniformly high virucidal action with little or no unit-to-unit variation, on the one hand, and maximum protection of labile biological components, on the other hand.

It was a further object of the present invention to provide for a treatment system for blood and blood products that was cost effective, both in terms of the cost of disposables and in labor costs.

These and other objects are met by the present invention, which relates broadly to a continuous or semi-continuous flow process for processing blood or a blood product to inactivate any virus contained therein, said process comprising:

(a) optionally flowing blood or a blood product over a predetermined flow path to a container suitable for pooling units of blood or blood products;

(b) mixing a virucidal reagent with said blood or blood product to yield a mixture of said blood or blood product and said virucidal reagent;

(c) optionally flowing said mixture over a predetermined flow path to a device capable of emitting irradiation and optionally subjecting said mixture to irradiation emitted from said device;

(d) flowing the mixture over a predetermined flow path through a substance containing hydrophobic ligands to remove substantially all residual virucidal reagent from the mixture to yield viral inactivated blood or blood product; and

(e) flowing the viral inactivated blood or blood product over a predetermined flow path into one or more storage containers.

The inventive virucidal treatment method is rapid, uniformly treats each droplet of blood or blood product, and is convenient. Whole blood as well as blood components (red blood cell concentrates, platelet concentrates, fresh frozen plasma, cryoprecipitates, etc.) can be virucidally treated, either as single units or preferably with the pooling of 2-25 units prior to treatment.

The invention furthermore relates broadly to a continuous or semi-

continuous flow system for processing blood or a blood product to inactivate any virus contained therein, said system comprising optically transparent tubing arranged in a predetermined flow path, said flow path being interrupted by:

(a) optionally a pooling container;

(b) optionally irradiation assembly means capable of irradiating a portion of said flow path;

(c) separation means for separating a virucidal reagent from said blood or blood product;

and said flow path terminating in:

(d) one or more storage containers.

The inventive flow system permits the pooling of blood or a blood product prior to treatment with dyes together with visible light or compounds known to react with nucleic acids, the in-line use of hydrophobic filters or hydrophobic resins to remove unreacted virucidal compound together with its breakdown products, and the in-line distribution of the treated blood or blood product into single or multiple storage containers.

Surprisingly, it has been found that in the case where the virucidal compound is a dye or photoactive compound or any other compound that interacts with nucleic acid, for example, merocyanine, beta-propiolactone, cis- platin, a phthalocyanine or a psoralen, e.g., aminomethyl-trimethyl psoralen (AMT), and after treatment such compound is removed by hydrophobic interaction chromatography or by a filter containing hydrophobic ligands, any residual virucidal compound which cannot be separated from the blood or blood product has lost the capacity to interact with nucleic acid and is no longer virucidal or mutagenic. This means that blood or blood products treated with such compounds in accordance with the teachings of the present invention will be particularly well tolerated by humans or other biological systems in which the treated blood or blood product is to be used, e.g., in tissue cultures. This also means that it is now possible to utilize such compounds in the context of a continuous or semi-continuous process for the processing of blood and blood products with reasonable certainty that the treated blood and blood products will be substantially free of virus and also well-tolerated by human patients. These advantages will also be realized with the use of such compounds in the context of a batch process for the processing of blood and blood products.

Accordingly, the invention also relates to a process for processing blood or a blood product to inactivate any virus contained therein, said process comprising:

(a) optionally pooling units of said blood or blood product;

(b) mixing with said blood or blood product a virucidal reagent capable of forming adducts with viral nucleic acid to yield a mixture of said blood or blood product and said virucidal reagent;

(c) optionally subjecting said mixture to irradiation;

(d) removing substantially all residual virucidal reagent from the mixture by passing the mixture through a substance containing hydrophobic ligands to thereby yield processed blood or blood product, which is substantially free of virus and which contains residual virucidal reagent, if any, in a form that is not virucidal or mutagenic.

Surprisingly, it has been discovered that virucidal reagents that pass through the hydrophobic ligand (i.e., that do not bind to it) are apparently altered (by a structural change which may or may not involve its covalent binding to protein or lipids) in such a way that the virucidal reagent has lost its ability to interact with nucleic acid and, thereafter, tests negative in an Ames test for mutagenicity and also is incapable, for example, of inactivating virus upon further irradiation with UVA. In addition, it has been discovered, quite surprisingly, that passage of blood or blood products through hydrophobic ligands proceeds without sacrifice of platelet or labile protein integrity even after the blood or blood product is subjected to treatment with virucidal compound and irradiation.

Thus, for example, without irradiation, >97% of AMT added to a platelet concentrate (PC) was removed with hydrophobic resin (C18) chromatography in the batch or column mode. Following C18 chromatography, platelet counts and aggregation responses to collagen in both untreated and AMT/UVA treated PCs were similar to those in samples without C18. With increasing time of UVA irradiation the amount of AMT that was associated with plasma or platelets and not bound by C18 increased to a value of >60% in 90 minutes. [When oxygen was depleted or when rutin was present, much less AMT (about 30%) passed through C18 when the same irradiation treatment was used.] Most of the AMT in the C18 pass-through appeared to be covalently bound to protein, and the portion which was not, was altered in structure as evidenced by the fact that the absorption spectrum of AMT in PBS which was irradiated with UVA and then passed through C18 was different from the absorption spectrum of AMT in PBS prior to irradiation. The AMT which passed through C18 after AMT/UVA treatment of plasma or platelets was not virucidal upon further irradiation with UVA.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be more fully described with reference to the figure, wherein:

Figure 1 is a schematic of the inventive component system.

DETAILED DESCRIPTION OF THE INVENTION

Blood is made up of solids (cells, i.e., erythrocytes, leukocytes, and platelets) and liquid (plasma). The cells contain potentially valuable substances

such as hemoglobin, and they can be induced to make other potentially valuable substances such as interferons, growth factors, and other biological response modifiers. The plasma is composed mainly of water, salts, lipids and proteins. The proteins are divided into groups called fibrinogen, serum globulins and serum albumin. Typical antibodies (immune globulins) found in human blood plasma include those directed against infectious hepatitis, influenza H, etc.

Cells found in blood include red cells, various types of leukocytes or white cells, and platelets. Fractionation of cell types typically utilizes centrifugation, but may involve other forms of differential sedimentation through addition of rouleaux enhancing agents such as hydroxyethyl starch, separations based on immunological specificity, etc. Leukocytes may be removed from other cells through the use of leukocyte removing filters.

Proteins found in human plasma include prealbumin, retinol-binding protein, albumin, alpha-globulins, beta-globulins, gamma-globulins (immune serum globulins), the coagulation proteins (antithrombin III, prothrombin, plasminogen, antihemophilic factor (factor VIII), fibrin-stabilizing factor (factor XIII), fibrinogen), immunoglobulins (immunoglobulins G, A, M, E and D), and the complement components. There are currently more than 100 plasma proteins that have been described. A comprehensive listing can be found in Th e Plasma Proteins, ed. Putnam, F.W., Academic Press, New York (1975).

Proteins found in blood cell fraction include hemoglobin, fibronectin, fibrinogen, enzymes of carbohydrate and protein metabolism, platelet derived growth factor, etc. In addition, the synthesis of other proteins can be induced, such as interferons and growth factors. A comprehensive list of inducible leukocyte proteins can be found in S. Cohen, E. Pick, J.J. Oppenheim, Biology of the Lymphokines, Academic Press, New York (1979).

For a discussion of the fractionation of blood into its various components, see Kirk-Othmer's Encyclopedia of Chemical Technology, Third Edition, Interscience Publishers, Volume 4, pp. 25-62. See also, the discussion in Bonomo, U.S. Patent No. 5,094,960, column 6, lines 24-64.

The present invention is particularly directed, inter alia, to producing a protein-containing composition such as blood plasma, cryoprecipitates, blood plasma fractions, etc., which is substantially free of infectious virus, yet which retains a substantial amount of enzymatically or biologically active (undenatured) protein and from which process chemicals have been removed so that the resultant composition contains no more than physiologically acceptable levels of such process chemicals.

Non-limiting examples of "blood or blood products" for use according to the present invention include whole blood, blood plasma, blood plasma fractions, precipitates from blood fractionation. Contemplated is the treatment of concentrates of whole blood cells, red cells, white cells (leukocytes), platelets, platelet rich plasma, platelet poor plasma, cryoprecipitate, serum, and concentrates of granulocytes, monocytes, or lymphocytes or other cells capable of producing interferon, tumor necrosis factor (TNF), or other immune modulators or lymphokines, or the media separated from such concentrates or suspensions.

According to the present invention, there is contemplated the preparation of a protein-containing composition, particularly whole blood plasma or whole blood serum having an extent of inactivation of virus greater than 6 logs of virus, such as AIDS virus (HIV I), hepatitis B virus and non-A, non-B hepatitis virus, having a retention of functional activity for particularly biologically active proteins of at least 45%, preferably at least 75%, more preferably at least 85%, even more preferably at least 95%, and most preferably 98% to 100%, and having no more than physiologically acceptable levels of process chemicals.

Coagulation factor activity is retained at more than 60% of its original level and preferably more than 75 to 85%, and most preferably at more than 90 to 98% of its original level.

The inventive flow system is designed as a system of component treatment stations or zones linked by conventional tubing (2 in Fig. 1) and the blood or blood product is made to flow through such tubing by the action of gravity or by a pump.

In either case, the blood or blood product will normally first be fed from a donor or satellite bag (1 in Fig. 1) to a pooling container (3 in Fig. 1), in which it is possible, if desired, to pool the blood or blood product, e.g., 2-25 units thereof, prior to treatment. Pooling of blood units is preferable since pooling of blood increases the overall cost efficiency of the process.

The virucidal reagent or other process chemicals can either be added upstream of the pooling container, to the pooling container, or downstream of the pooling container. The only requirement is that the virucidal reagent should be added to the blood or blood product before the blood or blood product is fed to an irradiation zone.

The virucidal reagent or other process chemicals will, of course, be chosen from among those known to be useful in the context of viral inactivation processes, particularly sterilization processes for blood or blood products. Non- limiting examples of virucidal reagents or other process chemicals include various dyes, e.g., benzporphyrin derivatives, merocyanine 540, methylene blue, and phthalocyanines, compounds known to principally react with nucleic acids, an example of which is the class of psoralen derivatives, di- or trialkylphosphates, detergents, surfactants, antioxidants, e.g., glutathione, and

quenchers (type I or type II or compounds, such as the flavonoids, which are known to quench both type I and type II reactions).

It has been found advantageous to take steps to ensure that the added virucidal reagent or other process chemicals become evenly distributed in the blood or blood product. This can be accomplished through the use of, for example, rockers or, preferably, a static mixer unit. Alternatively, the system can be maintained under turbulent flow conditions so long as the level of turbulence of the blood or blood product being treated is maintained below a level where shearing of the body fluid or cells would occur.

Once the blood or blood product is pooled and mixed with the virucidal reagent or other process chemicals, the mixture is then fed to an irradiation zone (4 in Fig. 1) where the mixture is then subjected to irradiation by a suitable irradiation source (5 in Fig. 1). The type and conditions of irradiation are well known to those of ordinary skill in the art. See, for example, U.S. Patent Application No. 08/069,235, filed May 28, 1993, the entire contents of which are incorporated herein by reference. The exposure time is controlled by the flow rate and the volume of tubing in the irradiation path. The exposure time should be set for that period of time conventionally deemed necessary to effect maximum virus inactivation. It has been found advantageous to use tubing optically transparent to the particular irradiation source utilized and to flow such tubing in a serpentine flow path through the irradiation zone. Alternatively, virucidal compounds can be added to the bag containing the blood component to be treated and the bag exposed to irradiation.

Following the irradiation treatment, the blood or blood product is fed to a station (6 in Fig. 1) whereby removal of the virucidal reagent and other process chemicals from the blood or blood product is effected. Preferably, the means for removing the virucidal reagent and other process chemicals comprises a filter or cartridge or chromatography column which contains hydrophobic groups capable of binding the virucidal reagent or process chemicals without adversely affecting labile components of the blood or blood product. More preferably, the filter or cartridge or chromatography column contains a compound containing C-6 to C- 24 chains. Particularly preferred is a compound containing octadecyl (C18) chains. In general, the compounds are those described in U.S. Patent No. 5,094,960 and the removal of the virucidal reagent and process chemicals is substantially as described therein. In a particularly advantageous configuration of the component system, a cartridge is utilized, which is in-line with or integrated into a leukocyte removal filter.

Once the virucidal reagents and other process chemicals are removed from the blood or blood product, the blood or blood product is then fed into single or multiple storage containers (7 in Fig. 1), e.g., blood bags, and then stored until use. Where the blood or blood product is fed into multiple bags, advantageous use can be made of a multi-position valve.

The present invention will now be described with reference to the

following non-limiting examples:

Example 1: Batch treatment of red blood cell concentrates (RBCC) with aluminum phthalocyanine tetrasulfonate (AIPCS₄) and removal of AIPCS₄ by hydrophobic filtration

AIPCS₄ and reduced glutathione (GSH) at final concentrations of 10 μM and 4 mM, respectively, are added aseptically to each of four units of ABO, Rh blood group matched RBCC. The units are exposed to 176 J/cm2 visible light either individually or on pooling into a pooling container. After irradiation, the units are aseptically connected to a nylon filter modified by addition of C18 hydrophobic ligands. The treated RBCC pass through the filter, which removes unreacted AIPCS₄ and its by-products, and is collected into a single blood bag of sufficient size (0.8-1 liter) to hold all four units. The blood bag is sealed and stored at 4°C until provision for transfusion.

Example 2: Continuous-flow treatment of RBCC with AlPcS

Eight units of ABO, Rh-matched RBCC are aseptically attached to a sterile manifold, which at the outlet contains a static mixing device into which both RBCC and AlPcS₄ flow. RBCC at a flow rate of 20-100 mL/min and 100 μM AIPCS₄ at 1/10 of this flow rate enter the static mixer, which is connected to a serpentine flow path through disposable tubing optically transparent to >630 nm visible light. Exposure to light is for that period of time necessary to kill hepatitis C virus, typically 1/2 to 5 minutes, depending on the intensity of the light. The exposure time is controlled by the flow rate and the volume of tubing in the light path. Following light exposure, the RBCC are passed through a hydrophobic filter, connected in series, and designed to remove the virucidal reagent and its break-down products. Finally, the RBCC are distributed into 1-4 blood bags by way of a multi-position valve, and the bags sealed.

Example 3: Continuous-flow treatment of platelet concentrates (PCs)

A similar system to that described in Examples 1 and 2 can be employed when treating a PC pool, substituting 50 μg/mL aminomethyl-trimethyl psoralen (AMT) or other psoralen for AlPcS₄, 0.5 mM rutin for GSH, and 60 J/ cm 2 UVA for visible light.

Example 4: Continuous-flow treatment of fresh frozen plasma

Fresh frozen plasma (FFP) is thawed and 1 μM methylene blue and 0.5 mM glutathione added by means of a static mixer. The FFP is then subjected to irradiation with fluorescent light at 150,000 lux for 2 hours. Antihemophilic factor (AHF) and fibrinogen recovery exceed 85%. Separate experiments indicate the inactivation of > 10⁵ infectious units of Sindbis virus, added as a model for hepatitis C virus.

Example 5: Continuous-flow treatment of cryoprecipitates

Cryoprecipitates (8) are thawed and aseptically pooled and 1% tri(n- butyl)phosphate and 1% Triton X-100 are added to the pooling bag. After 4 hours mixing at 30°C on a rocker, the cryoprecipitates are passed through a sterile hydrophobic filter containing C18 functional groups and collected into a final bag and frozen. AHF recovery is 75%.

Example 6: C18 Chromatography of AMT and UVA treated platelet concentrates: Effects on platelet integrity and AMT removal

Platelet counts and the aggregation response to collagen were not compromised by C18 chromatography of treated PCs in either the batch or column mode as shown in Table 1 below.

Table 1: Effect of C18 Chromatography on Platelets after Treatment with 50 μl/ml AMT and 0.5 mM Rutin

Mode UVA -C18 C18 (Minutes) % Control Platelet % Control Platelet Aggregation Count/ μl Aggregation Count/ μl Extent/Rate x 10-3 Extent/Rate x 10-3

Column 0 98/98 1152 100/100 879 90 95/95 1179 100/98 807

Batch 0 n.d. 652 n.d. 624 90 95/98 662 96/92 630

Prior to irradiation, virtually all of the AMT added to a PC was removed by C18, but with increasing time of UVA irradiation the AMT associated with plasma or platelets and not bound by C18 increased. With oxygen depleted or rutin present during irradiation, less AMT passed through C18 (e.g., with 90 minutes of UVA in air without quenchers, more than 60%; with oxygen depleted or rutin present, 40%) (Table 2). Most AMT in the C18 pass-through was covalently bound to macromolecules [trichloroacetic acid (TCA) precipitable] (Table 3), and the portion which was not, was altered in structure (absorption spectrum). Table 2: Removal of AMT by C18: Effect of Different Treatment Conditions

% total cpm (unbound)

(3H AMT) post-C18

AMT UVA Air N₂ Air

(μg/ml) (Minutes) No quencher No quencher 0.35 mM rutin

50 0 2% 3% ^" 2%

50 30 43% 19% 20%

50 90 66% 41% 42%

Table 3: TCA Precipitable [3H] AMT after PC Treatment: Effects of Quenchers and C18 Passage

No Quencher plus 0.35 mM rutin % total AMT in: % total AMT in:

AMT UVA C18 TCA^* C18/ C18 TCA^* C18/

(μg/mi) (Minutes) Pass TCA" Pass TCA"

50 0 <1% 15% <1% <1% 17% <1% 50 30 42% 27% 16% 16% 22% 9% 50 90 68% 36% 29% 31% 29% 16%

[3H] AMT cpm following TCA precipitation before C18 passage C18 passage following TCA precipitation on unbound fraction These results suggest that although a portion of the added AMT (30-60%) remains in PCs or plasma after AMT/UVA treatment and C18 passage, that AMT has been altered (by structural changes which may or may not involve covalent binding to proteins or lipids) so that it is no longer capable of interacting with nucleic acid, and would therefore not be virucidal or mutagenic.

Example 7: C18 passage eliminates the virucidal activity of residual AMT

PC samples containing 50 μg/mL AMT and 0.35 mM rutin were irradiated with UVA (7-10 mW/cm2) as indicated (UVAi). Samples were passed (+C18) or not passed (-C18) through C18 and vesicular stomatitis virus (VSV) was added (1:10) prior to the second irradiation (UVA₂).

The results of this experiment are shown in Table 4 below and demonstrate that although the total AMT remaining after UVA irradiation is virucidal, that portion which is passed through C18 (23-52%) is no longer capable of virus kill.

Table 4: VSV Kill following C18 Removal after AMT/UVA Treatment of Plasma

+C18 after UVA #1 -C18 After UVA #1

AMT Rutin UVA #1 UVA #2 % AMT logio % AMT log_o

(μg/ml) (mM) (Minutes) (Minutes) Unbound VSV Kill VSV Kill

50 0.35 0 90 2% <0.5 100% >5.3

50 0.35 30 90 23% <0.5 100% >5.3

50 0.35 90 90 40% <0.5 100% 25

50 None 0 30 2% <0.5 100% >5.3

50 None 30 30 52% <0.5 100% 1.8

VSV a Idded here

Example 8: Passage through C18 eliminates the capability of residual AMT to form adducts with nucleic acids

PC samples containing 4 μl/ml [3H]AMT (specific activity 0.4 Ci/mM; HRI Associates, Concord, California) in addition to 50 μg/ml "cold" AMT and 0.35 mM rutin, were irradiated with UVA (7-10 mW/cm2) as indicated (UVAi) and either passed (+C18) or not passed (-C18) through C18. Cells were then added to the treated PCs (2.5 x 10⁶ A549 cells /ml) and samples were again irradiated as indicated (UVA₂). Following UVA₂, cells were collected by centrifugation and DNA was phenol /chloroform extracted and ethanol precipitated. DNA concentration was determined spectrophotometrically and the number of AMT/DNA adducts per thousand base pairs calculated by a comparison of molar ratios.

Results of this experiment are shown in Table 5 below and demonstrate that the adduct forming ability of residual AMT is virtually eliminated by passage through C18.

Table 5: AMT/DNA Adduct Formation by Residual AMT- Effect of C18

AMT Rutin UVA #1 UVA #2 +C18 after UVA #1 -C18 after UVA #1

(μg/ml) (mM) (Minutes) (Minutes) #adducts/103bp #adducts/10³bp

50 0.35 30 0 0.05* 0.1

50 0.35 30 30 0.1* 2.5

50 0.35 30 90 0.4* 5.0

50 0.35 0 90 n.d. 8.4

50 0.35 90 0 0.02** 0.01

50 0.35 90 30 0.3* 2.1

50 0.35 90 90 0.4* 2.8

50 0.35 0 90 n.d. 9.1

Cells added here

* After C18, 18% [3H] AMT remains ** 48% remains after C18 Examples 7 and 8 illustrate that C18 chromatography of AMT/UVA treated blood or blood products removes all of the residual AMT that is capable of nucleic acid interaction and therefore strongly suggests that AMT remaining after C18 would no longer be mutagenic.

Example 9: Mutagenicity of residual AMT with and without C18 chromatography

PC or plasma samples, treated with 50 μg/ml AMT and 0.35 mM rutin with and without 90 minutes UVA and with and without passage through C18 were evaluated for their ability to induce frame-shift or base substitution mutations in bacterial "Ames tester" strains. (Ames test was performed by Microbiological Associates Inc., Rockville, Maryland.)

Irradiated samples caused frame-shift mutations in the absence of C18. However following C18 chromatography the samples were no longer mutagenic. (Base substitutions were not induced by any of the samples and frame-shift mutations were highest in unirradiated AMT containing samples.)

It will be appreciated that the instant specification and claims are set forth by way of illustration and not limitation, and that various modifications and changes may be made without departing from the spirit and scope of the present invention.

Claims

What is claimed is:

1. A continuous or semi-continuous flow system for processing blood or a blood product to inactivate any virus contained therein, said system comprising optically transparent tubing arranged in a predetermined flow path, said flow path being interrupted by:

(a) optionally a pooling container;

(b) optionally irradiation assembly means capable of irradiating a predetermined portion of said flow path; and

and said flow path terminating in:

(d) one or more storage containers.

2. The flow system according to claim 1, configured so that flow along the flow path is effected by gravity.

3. The flow system according to claim 1, further comprising a pump.

4. The flow system according to claim 1, further comprising means for ensuring even distribution of a virucidal reagent in said blood or blood product.

5. The flow system according to claim 4, wherein said means for ensuring even distribution of the virucidal reagent in said blood or blood product comprises a static mixer.

6. The flow system according to claim 1, wherein the separation means for separating a virucidal reagent from said blood or blood product comprises a filter or cartridge which contains hydrophobic groups capable of binding the virucidal reagent without affecting the blood or blood product.

7. The flow system according to claim 6, wherein the hydrophobic groups comprise C18 alkyl chains.

8. The flow system according to claim 6, wherein the separation means for separating a virucidal reagent from said blood or blood product further comprises a leukocyte removal filter.

9. A continuous or semi-continuous flow process for processing blood or a blood product to inactivate any virus contained therein, said process

comprising: (a) optionally flowing blood or a blood product over a predetermined flow path to a container suitable for pooling units of blood or a blood product;

10. The process according to claim 9, wherein blood or a blood product is pooled in said container suitable for pooling units of blood or a blood product.

11. The process according to claim 9, wherein the flow of blood or blood product over the flow path is effected by gravity.

12. The process according to claim 9, wherein the flow of blood or blood product over the flow path is effected by a pump.

13. The process according to claim 9, further comprising evenly distributing the virucidal reagent in said blood or blood product.

14. The process according to claim 13, wherein the virucidal reagent is evenly distributed in said blood or blood product by mixing with a static mixer.

15. The process according to claim 9, wherein the removal of said virucidal reagent comprises flowing said mixture through an in-line filter or cartridge which contains hydrophobic groups capable of binding the virucidal reagent without affecting the blood or blood product.

16. The process according to claim 15, wherein the hydrophobic groups comprise C18 alkyl chains.

17. The process according to claim 16, wherein the virucidal reagent comprises a psoralen.

18. The process according to claim 17, wherein the psoralen is aminomethyl- trimethyl psoralen.

19. The process according to claim 16, wherein the removal of said virucidal reagent further comprises flowing said mixture through a leukocyte removal filter.

20. A process for processing blood or a blood product to inactivate any virus contained therein, said process comprising:

(a) optionally pooling units of said blood or blood product;

(c) optionally subjecting said mixture to irradiation;

21. The process according to claim 20, wherein the hydrophobic ligands comprise C18 alkyl chains.

22. The process according to claim 20, wherein the virucidal reagent comprises a psoralen.

23. The process according to claim 22, wherein the psoralen is aminomethyl-

trimethyl psoralen.