Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
2000-05-26
2002-07-02
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C435S173100, C435S173300, C435S236000, C424S093210, C424S093720
Reexamination Certificate
active
06413714
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention concerns a process for rendering a biological composition substantially free of enveloped and non-enveloped viruses contained therein without substantial disruption or inactivation of cells contained therein and without significant loss of labile proteins or other valuable biological components also contained therein.
2. Description of Related Art
The problems associated with the application of virucidal procedures to biological compositions and the efforts to date to overcome these problems, including the application of light and chemical agents is reviewed briefly in U.S. Pat. Nos. 5,232,844 and 5,120,649, the disclosures of which are incorporated herein by reference. See column 1, line 26, through column 4, line 43, of U.S. Pat. No. 5,232,844 and column 1, line 27, through column 4, line 41, of U.S. Pat. No. 5,120,649.
Various photodynamic sterilization techniques have been evaluated for inactivating viruses in cellular components of blood. Although many of these appear promising for the treatment of red cell concentrates (Matthews et al., “Photodynamic therapy of viral contaminants with potential for blood banking applications”, in
Transfusion
, 28:81-83 (1988); O'Brien et al., “Evaluation of merocyanine 540-sensitized photoirradiation as a means to inactivate enveloped viruses in blood products”, in
J. Lab. Clin. Med
., 116:439-47 (1990); and Horowitz et al., “Inactivation of viruses in blood with aluminum phthalocyanine derivatives”, in
Transfusion
, 31:102-8 (1991)), photodynamic viral inactivation methods involving solely oxygen dependent reactions have so far proved inappropriate for the treatment of platelet concentrates (Proudouz et al., “Inhibition by albumin of merocyanine 540-mediated photosensitization of platelets and viruses”, in
Transfusion
, 31:415-22 (1991), Dodd et al., “Inactivation of viruses in platelet suspensions that retain their in vitro characteristics: comparison of psoralen-ultraviolet A and merocyanine 540-visible light methods”, in
Transfusion
, 31:483-90 (1991); and Horowitz et al., “Inactivation of viruses in red cell and platelet concentrates with aluminum phthalocyanine (AIPc) sulfonates”, in
Blood Cells
, 18:141-50 (1992)).
The use of psoralens together with UVA has demonstrated promise as a means of photoinactivating viral contaminants in platelet concentrates, although in most studies (Lin et al., “Use of 8-methoxypsoralen and long-wavelength ultraviolet radiation for decontamination of platelet concentrates”, in
Blood
, 74:517-525 (1989): and Dodd et al., supra, aminomethyl-trimethylpsoralen (AMT)), the combination of high levels of virus inactivation and the maintenance of platelet function were possible only when air was exchanged with nitrogen prior to UVA irradiation, a cumbersome procedure with inherent variability.
However, it was recently demonstrated (Margolis-Nunno et al., “Virus Sterilization in Platelet Concentrates with Psoralen and UVA in the Presence of Quenchers”
Transfusion
, 22:541-547 (1992)), that for the inactivation of ≧6.0 log
10
cell-free vesicular stomatitis virus (VSV) by AMT and UVA, the need for oxygen depletion as a means of protecting platelets could be obviated by inclusion of mannitol, a scavenger (quencher) of free radicals. (The addition of quenchers of type I (free radical mediated) or of type II (singlet oxygen mediated) photodynamic reactions is frequently used in other contexts to distinguish which active oxygen species produces a particular photodynamic effect.) Under the conditions used in that study, i.e., 25 &mgr;g/ml AMT and 30 minutes of UVA with 2 mM mannitol, the inactivation of cell-free VSV in air was in part oxygen dependent since equivalent virus kill (≧6.0 log
10
) with oxygen depleted required 3 to 4 times more UVA irradiation time (90 minutes to 2 hours).
However, while these methods achieved a high level of kill of cell-free lipid enveloped viruses, non-enveloped viruses and latent actively replicating and cell-associated viruses were not killed to a high extent under the conditions reported therein. Therefore, there was the need to effect the kill of these latter virus forms without causing significant damage to the desired, valuable components in the biological mixture. Conditions which result in the kill of ≧10
6
infectious doses of latent or non-enveloped virus have been shown to modify red blood cells and platelets and result in compromised recovery of labile proteins such as factor VIII.
In our copending application Ser. No. 08/069,235, the entire contents of which are hereby incorporated by reference, we demonstrated that superior viral inactivation could be achieved at the same time that superior protection of cells and labile proteins was also achieved by subjecting the biological composition, e.g., platelet concentrates to a virucidally effective amount of irradiation in the presence of (a) a mixture of a compound that quenches type I photodynamic reactions and a compound that quenches type II photodynamic reactions or (b) a bifunctional compound that is capable of quenching both type I and type II reactions for a period of time sufficient to inactivate any virus contained therein.
In spite of these advances, there continues to be a need for novel methods that achieve an even higher level of kill of both enveloped and non-enveloped viruses without significant loss of labile proteins or other valuable biological components.
SUMMARY OF THE INVENTION
The overall objective of the present invention was to achieve a high level of inactivation of both enveloped and non-enveloped viruses in biological compositions without incurring substantial disruption or inactivation of cells meant to be contained therein and without significant loss of labile proteins or other valuable biological components also contained therein. This objective was satisfied with the present invention, which relates generally to a process for inactivating extracellular and intracellular virus in a biological composition without incurring substantial disruption or inactivation thereof, said process comprising subjecting said composition to a virucidally effective amount of UVA1 irradiation alone or in the presence of an irradiation sensitizer compound for a period of time sufficient to inactivate any virus contained in said composition while retaining functionality of said composition. The inventive process can, thus, be used to inactivate viruses in whole blood, red blood cell concentrates and platelet concentrates, without adversely affecting red blood cell or platelet structure or function. Similarly, the inventive process can be used to inactivate viruses in biological compositions without incurring substantial inactivation of desired, soluble biological substances (e.g., coagulation factor concentrates, hemoglobin solutions) contained therein.
We have found that relatively more damage to cells and labile proteins is caused by shorter UVA wavelengths (<350 nm) and, therefore, cells and labile proteins can be better protected by using lamps that do not emit these shorter wave lengths or filters that eliminate these shorter wave lengths, yet viral inactivation levels are not compromised.
In accordance with another aspect of the invention, the inventive process is advantageously carried out in the presence of quencher compound. The quencher compound will be capable of quenching type I or type II photodynamic reactions or both, but preference is given to the use of (a) mixtures of a compound that quenches type I photodynamic reactions with a compound that quenches type II photodynamic reactions or (b) bifunctional compounds capable of quenching both type I and type II reactions.
In accordance with still another aspect of the invention, the inventive process is advantageously combined with a different virucidal method to enhance virus inactivation.
UV treatment alone of either plasma or AHF concentrates results in a relatively high loss of coagulation factor activity under conditions which kill ≧10
5
ID
50
Ben-Hur Ehud
Horowitz Bernard
Margolis-Nunno Henrietta
Amster Rothstein & Ebenstein
New York Blood Center Inc.
Weber Jon P.
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