CA2056619C - Method of inactivation of viral and bacterial blood contaminants - Google Patents

Abstract

A method is provided for inactivating viral and/or bacterial contamination in blood cellular matter, such as erythrocytes and platelets, or protein fractions. The cells or protein fractions are mixed with chemical sensitizers and irradiated with, for ex-ample, gamma or X-ray radiation.

Description

pL'T/ h'S91 /02504 «'O 91/16060 HiETHOD OF INACTIVATION OF VIRAL AND
BACTERIAL BLOOD CONTAMINANTS
FIELD OF THE Ii~IVENTION
This invention relates to the general field of biochemistry and medical sciences, and specif:.cally to inactivating viral/bacterial contamination of lyophilized or reconstituted blood cell compositions comprising erythrocytes, platelets, etc, or protein fractions.
BACKGROUND OF THE INVENTION
A major concern in the use of stored or donated homologous blood or plasma protein preparations derived from human blood is the possibility of viral and bacterial contamination.
Viral inactivation by stringent sterilization is not acceptable since this could also destroy the functional components of the blood, particularly the erythrocytes (red blood cells) and the labile plasma proteins. Viable RBC's can be characterized by one z0 ar more of the following: capability of synthesizing ATP; cell morphology; PSp values; oxyhemoglobin, methemoglobin and hemichrome values; MCV, MCH, and MCHC values; cell enzyme activity; and j~ vivo survival. Thus, if lyophilized then reconstituted ~~'O 91/16060 PCT/US91/02504 2L55E~1:
_Z_ and virally inactivated cells are damaged to t::e extent that the celis are not capable of metabc'«z~::~
er sy~,t~psizing ATP, or the cell circulation is co-pro:~ised, then their utility in transfusion ;.iedicine is compromised.
There is an immediate need to develop protocols for the deactivation of viruses that can be present ir, the human red blood supply. For example, only recently has a test been developed for Non A, Non B
hepatitis, but such screening methods, while reducing the incidence of viral transmission, do not make the blood supply completely safe or virus free..~.Current statistics indicate that the transfusion risk per unit of transfused blood is as high as 1:100 for Dlon A, Non B hepa:.itis, and ranges from 1:40,.000 to 1:1,000,000 for HIV, depending on geographic location. Clearly, it is desirable to develop a method which inactivates or removes virus indiscriminately from the blood.
Contamination problems also exist for blood plasma protein fractions,~such as plasma fractions containing immune globulins and clotting factors.
For example, new cases of non A, non B hepatitis have occurred in hemophilia patients receiving protein fractions containing Factor VIII which have been treated for viral inactivation according to approved methods. Therefore, there is a need for improved viral inactivation treatment of blood protein fractions.
The present invention thus provides a method for the inactivation of viral and bacterial contaminants present in blood and blood protein fractions.

WO 91/16060 ;~~5~~,19 PCT/L'S91/02504 c ~ -~~ ~ ~ ~ : ~w r ~ V': r "1 T T 0!J
:he present invention provides a method °or virai;~a~~erial inactivation ef dried or reconstituted cells (erythrocytes, platelets, he:~csc-es and other cellular or cell-like c:,mpone.~.ts or blood protein fractions, which allows for the cells or protein fractions to be useful in a transfusable state, while still maintaining relatively high cell viability, ATP synthesis and oxygen transport, in the case of cellular components, and therapeutic efficacy, in the case of protein' fractions.
The lyophilization and reconstitution media according to the present invention may be utilized to lyophilize and reconstitute proteins, particularly, blood plasma protein fractions. The protein fraction may be virally/bacterially deactivated by mixing with a chemical sensitizer, lyophilized (freeze-dried), then irradiated. If the lyophilization media of the invention is used, it is contemplated that the constituents of the media also serve to provide same degree of protection of the dry proteins during irradiation.
A preferred embodiment comprises reducing viral and bacterial contamination of dried or reconstituted cells with washing solutions containing a polymer or mixture of polymers having a molecular weight in the range of about 1K to 360 K, followed by one or more additional wastw cycles using a wash of a dextrose-saline solution at a pH in the range of about 7.0-7.4. The dextrose-saline solution will also contain a polymer having a molecular weight in the range of about 1K to 40K, and preferably about 2.5K.

PCf/L:Sg 1 /02504 'W'U 91/i6060 , ;~S~L~'~9 :he co-.position of reconstitut a ce'_ls wi;; a:.so preferabll contain a menosaccharide.
Preferably the cells will have beer, previous'~r :lcYi:'_'~ized using a lyophilization solut_or, bu:fe:e~
in the range of pH of 7.0 to 7.4 preferabil by a phosphate-buffered solution. A typical phosphate-buffered lyophilization solution will comprise mone-and di-basic potassium and sodium phosphate (usually in the range of 1-10 mM each) and 5-10 m.M adenine.
This solution maintains the pH at around 7.2.
A preferred phosphate-buffered solution to be used as the lyophilization buffer will comprise nicotinic acid, reduced glutathione, glutamine, inosine, adenine, monopotassium phosphate, magnesium chloride disodium phosphate all of which will serve as a basic salt buffer at a pH of about 7.2. 1n addition this lyophilization buffer will contain a final concentration of about 26% weight by volume of a monosaccharide, preferably 1.7 M glucose, and a final concentration of about 3.0% Weight by volume of polyvinylpyrrolidone (average molecular weight of 360K), and a final concentration of about 15% weight by volume of hydroxyethyl starch (average molecular weight of 500K).
The term lyophilization is broadly defined as freezing a substance and then reducing the concentration of the solvent, namely water, by sublimation and desorption, to levels which will no longer support biological or chemical reactions.
Usually, the drying step is accomplished in a high vacuum. However, with respect to the storage of cells and particularly erythrocytes, the extent of Wp 91/160b0 ~c~ss~~~ h P~/L'S91/02504 drying ; t:~~e a",ount cf residual moistsre; is o:
~. J
critica'~ i-portance in the ability o: ce'_ls w i the tar.d long-ter:~ storage a t roor, ter,:per a tur a .
Lsi.~.g t;:e procedure described herein, cells :nay be lyophilized to a residual water content of less than to weight %, preferably less than 3%, and sail' be reconstituted to transfusable, therapeutically usefu;
cells. Cells with about 3 weight % water content using this procedure may be stored for up to two weeks at room temperature, and at 4°C for longer. than eight months, without decomposition. This far exceeds the current A.A.B.B. standard for refrigerated storage of red blood cells of six weeks at 4°C or less than one day at room temperature without decomposition. These dried cells may be deactivated using a chemical sensitizes described herein.
According to the preferred embodiment of the present invention the washed packed red blood cells are mixed With a chemical sensitizes, then washed to remove excess sensitizes not bound to viral or bacterial nucleic acid, and the treated cel7a are then lyophilized. The dry cell and sensitizes mixture will then be irradiated, typically with gamma radiation, at an intensity of about 3K-50K rads, for a period of time sufficient to destroy viruses (in particular, the single-stranded or double-stranded RNA/DNA viruses), without any substantial adverse effect on the recovery and usefulness of the cells.
Other wavelengths of electromagnetic radiation such as X-rays, may be used.
In another preferred embodiment, the chemical sensitizers may be added to liquid protein w'0 91/16060 '~~'S~~~- ~ PCT/L;S91/0250.1 -b-preparations, then lyophilized and :.rradiated.
Particularly preferred are blood protein preparations, including but not limited to, plasma proteins, blood protein extracts, clotting fac:.ors (such as Factors VIII and IX), immune globulins and serum albu:ain.
Dry (lyophilized) cells or protein fractions may be directly mixed with the chemical sensitizes, then irradiated.
From the foregoing description, it will be realized that the invention can be used to selectively bind a metal atom or a metal atom containing chemica l sensitizes to blood-transmitted viruses, bacteria, or parasites. Also monoclonal or polyclonal antibodies 1~ directed against specific viral antigens (either coat proteins or envelope proteins) may be covalently coupled with either a metal atom or a metal atom-containing sensitizes compound, thereby increasing the effective cross-section of the contaminant to penetrating or other forms of radiation energy.
Since cell compositions also comprise a variety of proteins, the method of decontamination of cells described herein is also applicable to protein fractions, particularly blood plasma protein fractions, including, but not limited to, fractions containing clotting factors (such as Factor VIII and Factor IX), serum albumin and/or immune globulins.
The viral and bacterial inactivation may be accomplished by treating a protein fraction With a sensitizes as described herein. A protein fraction which has been lyophilized and reconstituted may be sensitized and irradiated to deactivate possible w'0 91/16060 ~css6l.~ PCT/US91/0:,504 ce~ta-:i.~,ation. It is contemplated that iiq~is a;,~
froze:, protein fractions may also be deconta:,t:.~.ated ac~:,r~~:::, to the present invention.
Deper.:i::~ upon the nature of the presumed radio:r t.._ 3 mec:~anis;,~ of the sensitizes reaction with the virus, other types of radiation may be used, such as X-ray, provided the intensity and power utilized is sufficient to inactivate the viral contamination without adverse effect on the cells. Mature human red blood cells and platelets lack nucleic acids;
therefore the nucleic acid binding sensitizers selectively target contaminating viruses and bacteria. Although described in connection wits.
viruses, it will be understood that the methods of the present invention are generally also useful to any biological contaminant found in,stored blood or blood products, including bacteria and blood-transmitted parasites.
DETAIL°D D~SCgIPT~ON ~F T'~rF -_TtwENT~ON
2o The cells are preferably prepared by immersing a plurality of erythrocytes, platelets and/or hemosomes, etc. in a physiologic buffered aqueous solution containing a carbohydrate, and one or more biologically compatible polymers, preferably having amphipathic properties. By the term amphipathic it is meant there are hydrophobic and hydrophilic portions on a single molecule. This immersion is followed by freezing the solution, and drying the frozen solution to yield novel freeze-dried erythrocytes containing less than 10~, and preferably about 3% or less by weight of moisture, which, when reconstituted, produce a significant percentage of viable, transfusably useful red blood cells, ~CSfiEil~
W091/16060 ~ . PCTIL'S91/0250.1 platelets or hemosomes. Preferred me_hods of reconstitution of the lyophilized cos,"position are descr_'cad below. Although described in connec t""
with red blood cells, it will be understccd t;~a= t:~e :,~e~:~.cs are generally also useful to lyophilize platelets, hemosomes, and blood protein fractions.
The carbohydrate utilized to prepare erythrocyte, platelet and/ or hemosome compositions according to the invention is biologically compatible with the erythrocytes, platelets or hemosomes, that is, nbn-disruptive to the cells or hemosome membrane, and one which permeates, or is capable of permeating.,., the membrane of the erythrocytes, platelets or hemosomes.
It is also advantageous to stabilize proteins, especially labile blood proteins, with~the carbohydrates during lyophilization and irradiation according to the invention. The carbohydrate may be selected from the group consisting of monosaccharides, since disaccharides do not appear to permeate the membrane to any significant extent.
Monosaccharide pentoses and hexoses are preferred as is a final concentration of from about 7.0 to 37.5 weight % in phosphate buffered saline (PBSy or a phosphate buffered solution, preferably about 26%.
Xyiose, glucose, ribose, mannose and fructose are employed to particular advantage.
It will be understood that the cells may be lyophilized using other protocols and irradiated as described below. Although viral inactivation will be attained, the advantage of retaining a significant percentage of viable useful red blood cells is lost if the described lyophilization procedure is not followed.

WO 9116060 ~~5~6~~ PCT~US9l/02504 _y_ The invention will be hereafter described :n connection with erythrocytes (RBC's) but it will be unders=ocd it is also applicable to plate'~ets, he:~oso-es or other blood cell types or bioiogoca~
cells, as well as protein fractions, par~icuiar~l plas,:.a protein fractions.
The erythrocytes will preferably be prepared from whole blood centrifugation, removal of the plasma supernatant and resuspending the cells in PBS or a phosphate buffered solution or a commercial dextrose-saline solution. This wash cycle may be repeated 2-3 times preferably using a commercial dextrose-saline solution, then the packed cells are diluted with the lyophilization buffer described above so that the final diluted concentration of carbohydrate and polymer are maintained in the necessary ranges.
Alternatively, commercially available packed blood cells may be used, which typically are prepared in cPDA (commercial solution containing citrate, 2o phosphate, dextrose and adenine).
Upon lyophilization to a moisture content of less than 10%, and preferably less than 3%, the lyophilized cells may be maintained under vacuum in vacuum-tight containers, or under nitrogen or other inert gas, at room temperatures for extended periods of time in absence of or without significant degradation of their desirable properties when reconstituted for use as transfusable cells. In using the preferred lyophilization method disclosed herein, a particular advantage of the present invention is that the lyophilized cells may be stored at room temperature for extended periods of time, WO 9i/16060 ' ~CSf s1.9 PCT/L;S91/02504 _sp_ thus obviating the need for low te~;,oerature refrigeration which is required for storing liqu:
CPDA preserved red blood cells prepared by methods c~
the prior art. The present invention also obviates :, the need for very low temperature (-80°C) frozen storage of red blood cells in glycerol.
By using the preferred reconstitution method disclosed herein it is a further advantage that the lyophilized red blood cells may be reconstituted at normal temperatures, ~.g. greater than about 4°C up to about 37°C, Which corresponds to normal human body temperature, and preferably at room temperature , (about 22°C). The reconstitution medium is preferably a solution comprising a polymer~or mixture of polymers having a molecular weight of from about 2.5K to 360 K, preferably 5K to about 360K, present in a concentration in the range of about 12 to 30%
weight by volume. This polymer may be the same polymer utilized to lyophilize the red blood cells as described above. Hence the polymers polyvinylpyrrolidone, hydroxyethyl starch, and dextran are particularly preferred and most preferred is polyvinylpyzrolidone (preferably molecular weight about 10K) present in a concentration of about 19%
weight by volume in the reconstitution solution. The reconstitution solution will be buffered again typically by phosphate-buffered solution comprising monopotassium phosphate and disodium phosphate as described hereinabove to maintain a pH within the 3o range of about 7.o to 7.4. The most particularly pref erred polymer is polyvinylpyrrolidone of an average molecular weight of about 10K. The most preferred reconstitution buffer will also contain ~:~.~~sszg WO 91/16060 ~ ~ PCT/US91/02502 -yl' adencs~.~,e tr iphosphate (ATP) in a f final conce~=ra=~c.'.
of abc::= S;~~t.
The p~?y;.~ers may be present in the various sol~.:_ions fr:;:~ a :final concentration of about 3,6K weight % ur to saturation, and have a molecular weight in the range o: from about 2.5K to about 360K. Preferably, the polymers have molecular weights in the range of from about 2.5K to about 500K, most preferably from about 2.5K to 50K, and are present in a concentration l0 of from about 3.6 weight % up to the limit of solubility of the polymer in the solution. Polymers selected from the group consisting of polyvinylpyrrolidone (PVP) and polyvinylpyrrolidone derivatives, and dextran and dextran derivatives provide significant advantages. Most preferred is the use of polyvinylpyrrolidone (an amphipathic polymer) of average molecular weight in the range of 2.5-360K in an amount in the range of 3-20% weight by volume in the solution prior to lyophilization.
Amino acid based polymers (~,.g., proteins), dextrans or hydroxyethyl starch may also be employed. In the lyophilization buffer hydroxyethyl starch (M-HES) with an average molecular weight of about 500K is employed in a 15% weight by volume final concentration. Other amphipathic polymers may be used, such as poloxamers in any of their various forms. The use of the carbohydrate-polymer solution in the lyophilization of red blood cells allows for the recovery of intact cells, a significant percentage of which contain biologically-active hemoglobin.
The most preferred reconstitution buffer will be a solution comprising monopotassium phosphate, disodium w'0 91/16060 PCT/L'S91/02504 ~L~~3fi~~,:j phosphate and ATP, all of which form a basic sa:.t buffer at a pH of about 7.2, which also contains about 19% weight by volume of poiyvinylpyrrolidone (average molecular weight about 10K).
:, The reconstitution solution may also optionally contain a monosaccharide, preferably present :.~. t:~.e concentration range of about 7.0 to 37.5% weight by volume. The preferred manosaccharides are xylose, glucose, ribose, mannose and fructose.
In the most preferred embodiment, the lyophilized erythrocytes can be reconstituted by mixing with an equal volume of the reconstitution buffer at a temperature of about 37°C and mixed. By "equal" it is meant that the volume is the same as the starting volume prior to lyophilization. After initial reconstitution, the solution is preferably diluted 1:1 with 1-4 additional volumes of the reconstitution buffer at a temperature of about 37°C with added mixing until fully hydrated.
Then, it is preferred that the rehydrated cells be washed according to the following procedure. It is realized, however, that once the cells are reconstituted With reconstitution buffer they are in a hydrated and useful form, but the combination of washings described hereinafter are preferred, specifically for clinical purposes.
After separating the cells from the reconstitution buffer by centrifugation, the resulting packed cells are preferably resuspended at room temperature in (approximately the volume used in the initial reconstitution) a wash buffer comprising nicotinic WO 91/16050 2es~~l~ PC'f/CS91/02504 acid, i.~.osine, adenine, glutamine, and ma:,nesm:~
chloride, all present at about 0.4-lOm.~! f~.:rther co:"prise~:g sodium chloride and potassium c:zloride each at about 30mM, buffered by lOmM disodium ., p,~.osp'.~.ate to pH 7 . 2 . This wash buf for fur ther co~prises a monosaccharide, preferably glucose a~ a concentration of about 20mM, and a polymer, preferably polyvinylpyrroltidone, of a molecular weight 40K and present at a concentration of about l0 16% weight by volume. Separation by centrifugation completes the first post-rehydration step, a washing step.
After the washing step the rehydrated cells may be suspended in a dextrose-saline transfusion buffer at 15 room temperature which preferably contains polyvinylpyrrolidone at a 10% weight by volume final concentration, with an average 2.5K molecular weight.
The cells can be used as is or be returned to autologous plasma. Additional wash steps in a 20 phosphate-buffered diluent buffer can further remove viruses, but this step is optional for preparation of rehydrated, transfusible cells.
The reconstitution and washings described above will in most instances achieve about 4 log reduction of 25 any viral and bactErial contamination, where 1 log reduction is achieved by drying and 3 log reduction is achieved by washing. Of course, different viruses may respond differently, potentially resulting in more than 4 iog reduction of contamination.
30 The reconstituted cells have characteristics which render them transfusable and useful for therapeutic WO 91 / 16060 ~ic.'' ~: rJ~b~,~ PCT/l.'S91 /02504 purposes in that their properties are si",ilar ..., _::a=
of fresh (~,.e_. not previously lyophilized; red biccd cells. Typically reconstituted red blood cells according to the present invention have a.~, :~ oxyhe:~,oglobin content greater than about 90% c° t::a~
in normal red blood cells. Hemoglobin recovery prior to any Washing step is typically in the range o: ~~
to 85%. The overall cellular hemoglobin recovery including the post-hydration washing steps is about 20 to 30%. The morphology of the reconstituted cells according to the present invention (by scanning electron microscope) typically shows no holes or gaps, and primarily discocytic with some stoma,tocytic morphology. The oxygen carrying capacity of.fresh red blood cells (as measured by Psp, the oxygen partial pressure at which 50% of the oxygen molecules are bound) was measured to be in the range of about 26 to 28 (average 26.7); with an average Hill coefficient (a measure of the cooperative binding of oxygen molecules to native hemoglabin) of 1.95. The typical P5o for erythrocytes lyophilized and reconstituted according to the present invention is about 27.5 (average) with an average Hill coefficient of 2.08. Assays of ATP in the reconstituted cells indicate ATP levels suggesting normal ATP to ADP
metabolism. Normal hemagglutination by readily available blood typing antisera of red blood sells made according to the present invention is also typically found.
This lyophilization and reconstitution procedure advantageously and significantly diminishes viral/
bacterial contamination in cell°like material (such as hemosames), and protein fractions. The contamination can be further reduced by the radiation ~ess6~~ p~/US91/02504 W'O 91/16060 .
-15- ' ' sensitizing and treatment, particularly while t:~.e cells cr protein fractions are in the dry state.
:he starting packed red blood cells or proteins (;~;;ic~ may initially be in a liquid or lyophilized state) are mixed with a sufficient amount (based or.
total wet weight of cells) of a chemical sensitizes.
Preferably, in a composition of packed red blood cells (about 10% hematocrit) about 0.1 to 1 mg of the chemical sensitizes will be used per ml of packed cells. Preferably, the mixture will be irradiated with gamma radiation in the range of 3K-50K rads, typically about 3K rads. Preferred exposure is from 1-10 minutes, if using gamma radiation. .
Alternatively, W light (320 nm) may be used, particularly for protein fractions. Preferred exposure is from 1-10 minutes, preferably 3 minutes, if using W radiation. By this irradiation in presence of a sensitizes, there will be about a 6 log reduction of viral and bacterial contamination, based on contamination present prior to washing and irradiation.
The present invention provides a selective method of generating free, radicals derived from chemical sensitizers only in the vicinity of viral RNA or DNA.
Indiscriminate radiolysis of blood containing virus in a hydrated state produces hydroxyl radical.
However, the hydroxyl radical will damage both the red blood cells and associated proteins as well as the viral target. Thus, viral inactivation would be aehieved at the sacrif ice of red cell viability.
Therefore, sensitizers which bind to DNA and/or RNA
and Which can be selected to generate radicals upon irradiation, are required. Since the radiolysis can VfO 91/16060 . PCT/1:S91/02504 ',~~rJ~ib~.=j '16-be performed in the dry state (preferabl= less ~~a:-.
10% residual moisture), generation cf hydroxy=
radicals from ;:ater is greatly reduced. ~.~, t~.s manner indiscriminate radical damage is °urther preve~ted. Exemplary compounds include:
I /, ~ ~: ~rl t N=1'~ '~~ He C~
'~r ~ ! t ~~ I ~ C t~ ~
N J Nw t . i~ . Hc~
H ~co t~~
~Ht1 The preparations of these compounds are known. See Martin, R.F. and Kelly, D.P., Aust. T~
2637-46 (1979); Firth, W., and Yielding, L.W., Wig. Chem., ~, 3002 (1982). Other radical-generating reagents which generate radicals upon irradiation are disclosed by Platz g~ ~.I,,~, oc.
~pl~ ~r,~ _ Oot. Ena. $~,, 57-60 ( 1988 ) and Kanakarajan gt s3.1.~ ~ JACS 11Q 6536-41 ( 1988 ) .
The radiation-sensitizing compound (which may also be modified to bear a metal atom substituent) may also be selected from the class consisting of DNA-binding drugs, including, but not limited to, netropsin, BD
peptide (a consensus peptide from HMG-1), S2 peptide, and the like. These and other DNA-binding drugs are pC?/LS91102504 ~:5~____~ ... . , .3. . . _. , ~~ ~25.C~' :3r. ' . 3.'".~
...~:C°_=~='".. ..._ ._.. , - ~- ~' ::= . -' a-._ ._. . '!. , _s-.a-. ~:. , .:e~=_=__~. ~.... a-._ ~a.~.-.
......~, .;o " ,.. ~~.- ?.~ ~s =~s. - .._ : ~....
_ .:~.e ra~~3=:.c~ sens:,=:zing com,po~:nd .:'a'_.... gay a_s~
tear a -,etai atom; can also comprise a class z° 2':
c~.~,3ir.g proteins and/or polypeptides and~cr peco_:e=.
~xa-pies o: this class of DNA-bindi:,g pr:,te~ns any =-pol;peptides and/or peptides are disclosed in E.A. and Travers, A.A. (1991; :rends _..
_ J ,....._ _.. - 1 I :vi .
Bicc'.~.e.~..ical Sciences ~, 92-97. Specific examples Diva-binding peptides include the SE peptide and 3~
peptide disclosed in the reference herein.
The DNA-binding specificity can be achieved by 15 covalently coupling the radiation sensitizing compaund and/or metal atom to either a DNA-binding drug or to a DNA-binding protein or polypeptide or peptide.
VI ,~ ~.: NN"
20 Netropsin W ~~ ~N~~
H
H
tJ~~~~t~ ~0 C H
"2+~~ ~~ 3 0' VII
H
~H

pC,T/LS91 /02504 other sensitizers include specia'_:.y designed molec~~les which for:,i triplex DNA, such as those disclosed by Youngquist and Dervan ~1~5 3- 2565 (1935); Van Dyke and Dervan, Scie.~.ce 225 i~22 (:.93~~%
;; 'pan Cyke and Dervan, Nuc. Acids Res. ~ 5555 !1900 %
Barton and Raphael, gNAS $~, 6460 (1985); Barton e-JAGS X06 2172 (1984); and Barton, PNAS 3~' 19c1 (1984). These molecules bind to DNA and RNA, site specifically, if desired, and carry reactive moieties which can generate free radicals in the proximity of the DNA or RNA.
R-I + e- - R ~ I-R-I'~ + Guanine ~ R-I + Guanine "
While not intending to be bound by a theory, it is believed that the ejected electron will be captured by that site with the most favorable electron affinity, which is most likely a second molecule of sensitizes elsewhere in the sample. Electron capture by R-I (or R-Hr) leads to dissociation of RX with the formation of a radical. The radical so generated will abstract a C-H hydrogen atom from a sugar moiety of a nearby nucleic acid which in turn will lead to DNA or RNA cleavage and viral inactivation.
The radical cation of the sensitizes (R-X'~) will eventually abstract an electron from that component of the sample with the most favorable oxidation potential. This is most likely guanine. The electron transfer reaction forms guanine radical cation. This substance will react with 02 upon reconstitution with aerated 1120. This process also leads to DNA cleavage and viral inactivation.
Unreacted material and reaction by-products will be ~C5s6~9 PCT~US91/02504 WO 9'1/16060 _1c_ re..~..oved during the washing steps involved in the reconstitution of the lyophilized ceps (Table 2).
This process will also further re::,ove any virus nct inactivated by the treatment described above.
S Co.~"pounds (1) and (2) bind tightly to DNA and RNA b~r either intercalation and/or by electrostatic interactions between positively charged ammonium ion groups and the negatively charged phosphate groups of the nucleic acid target. Red blood cells do not to contain nucleic acids and accordingly will not bind to such compounds by intercalation.
The best mode for using the invention is to add the sensitizes to potentially contaminated blood solutions, and to expose to gamma radiation or x-15 rays. Fluid solutions of blood are preferably exposed to 3000 rads, and dried lyophilized solid formulations are preferably exposed to 10,000 rads of radiation. It is known that the red cells will survive these doses of radiation in the absence of a 20 sensitizes. Lyophilized blood can withstand higher dosage levels of radiation than hydrated blood.
The gamma radiation or x-ray will be absorbed primarily by the heavy atom of the sensitizes, which will be bound to viral DNA or RNA. The radiation 25 will ionize the sensitizes as follows:
R-I + Y-Ray ~ R-I'~ + e' - (X-Ray) In some instances, particularly if the sensitizes and red blood cells are allowed to stand together for 3o more than several minutes, sensitizers may diffuse into the red blood cells prior to lyophilization.

W0 91/16060 . ~. . . ,,"
~'C5~76~..:j -20-Antioxidants such as glutathione (an exceilen:.
hydrogen atom donor) may be added to the prepara_:.c::
to aug;.~en~ the red cell defenses against free ra~i~a~
initiated damage. It will be understood Thai inccrrcration of the sensitizes into cells will also allow inactivation of intracellular viruses, especially viruses thought to reside inside white blood cells (most packed red blood cell units contain residual white cells), or intracellular blood parasites, such as malaria parasite which infects red blood cells.
The sensitizers are removed from the reconstituted blood serum or protein fraction by the washing protocol described above for lyophilized cells.
It is preferred that gamma or X-ray radiolysis take place in a dried lyophilized blood (or protein), virus, and sensitizes formulation rather than in a wet, fluid material for several reasons. Firstly, the dry material is less sensitive to radiation and can be exposed to larger doses of y-rays or other penetrating radiation without damage to red blood cells (Table 1y. This increases the extent of radiolysis of the sensitizes. Secondly, sensitizes radicals bound to DNA or RNA in the dry state can not dissociate from the virus due to the lack of diffusion in the solid material. This will force the sensitizes radical to react with viral RNA or DNA.
Thirdly, the solid state conditions will enhance hydrogen atom transfer reactions of the sensitizes radical with the viral nucleic acid, perhaps by quantum mechanical tunneling. Fourthly, the reconstitution and washing protocol used With lyophilized blood or protein fraction serves as a WO 91/16060 ~~SS~lg PCT/L'S91/02504 __21_ means tc remove unreacted material or reac~.ic.~, by-products, and further removes any virus not affect=_~ by the treatment (Table 2).
Ct;.e: Apes of radiation may be used inc~udir~g ionizing radiation in general, such as X-ray radiation. In one embodiment a metal ator may be a substituent on a chemical radiation sensitizes molecule which binds to nucleic acids, thereby targeting the embodiments such as bacteria, parasites and viruses. Metal atom substituents of chemical sensitizers for this purpose include Br, I, Zn, C1, Ca and F. The X-ray source is preferably a tunable source, so that the radiation may be confined to a narrow wavelength and energy band, if so desired.
The tunable feature allows for optimization of energy absorption by the metal atoms, thereby directing the absorbed penetrating radiation energy to the production of radicals by a chemical sensitizes bound to nucleic acid.
The present invention is applicable to contaminants which comprise single or double-stranded nucleic acid chains, including RNA and DNA, and viruses, bacteria or other parasites comprising RNA and/or DNA.
To illustrate the invention, red blood cells were lyophilized as described above, irradiated, and tested for erythrocyte characteristics measured. The results are shown in Table 1. The same procedure was then used, except that the bacteriophage T4 (in dextrose saline) was mixed with the cells and then washed successively With four different wash buffers.
The results are shown in Table 2.

w0 91 / 16060 y . , PC'f/L;S91 /0250a ~.:~: J~b~.
:able 1: Influence of irradiation on lyoghi~:zed reconstituted red blood cells. Doses as high as 20,000-50,000 tads do not affect cells in the d.Y
state according to the parameters assayed after reconstitution and listed below.
7~ CSLI''° o,~, T "~nh; 1 , zed Cep is to Gamma TYradia * Percentage of Contro' Dosage Level X0,~000 cads 50.000 tads Hb Recovery 100 Oxy Hb No Change from No Change from starting value starting value Cell Indices MCH 100 . 100 Metabolism (~mol/g Hb) Lactate 86 79 (~mol/g z0 Hb/Hr) * Control cells were non-irradiated, lyophilized reconstituted cells.

pCT/ i.'S9l /02504 W'O 91/16060 _23_ Table 2: Reduction in viral titre as a func~~cn ~.
washing of the red cells. The procedure used reconstit~,:ting the lyophilized cells invclves several washing steps which also reduce the viral titre. ,'_~:e :: extent o~ reduction with each wash decreases ~.:~=~- a prat=ical limit is attained. This represents a.~;
approxi:~ate 4 log reduction in viral titre.
wash'ra Protocol Ppduc~~on of Vira' Load '~ °lood Total Amouat Buffer wash Step of yirus Loa Reduction Experiment 1 (non-lyophilized cells) Reconstitution 7.3 Ox 10~ 0 Wash 4.80 X 104 3.2 Diluent 2.08 x 104 3,5 Transfusion 3.50 x 104 3.3 Experiment 2 (lyophilized cells) Lyophilization 3.68 x 108 0 Reconstitution 2.11 x 107 12"

Wash 2.38 x 104 4.2 Diluent 2.00 x 104 4.3 Transfusion 4.06 x 104 4.0 In Experiment 1, the effects of lyophilization on viral reduction are not included. In Experiment 2, these effects are included. The marker virus used in these cases was bacteriophage T4. The extent of reduction was determined using the plaque assay.
*'This shows an additional about 1 log reduction of contamination due to the drying step.

WO 91/16060 PCT/US91l02504 y~5~63..~
E.~CAMPLE 1 Packed human red blood cells purified fro,:, donated whole blood are washed free of the anticoagulant storage solution (commercially available CPDA, co;,taining citrate/phosphate/dextrose/adenine), and suspended in dextrose-saline at a 10% hematocrit.
Approximately 10 ml of washed packed red cells is placed in a quartz chamber and exposed to L;.v. light, preferably at 320 nm, for 2 minute time intervals, up to a 10 minute total exposure. At each 2 minute, interval the suspension is mixed and a small sample of red cells (10 microliters) is removed and diluted into 2 ml of water for spectrophotometric assay of hemoglobin. At each step the temperature of the irradiated red cell suspension is measured, to ensure that the suspension did not overheat. At no point did the suspension exceed 26 degrees C (normal body temperature is 37 degrees C). Untreated red cells contain a high proportion of functional oxyhemoglabin (oxyHb), usually in the range of 96% or higher.
Oxidation damage can form a semi-stable methemoglobin species (metHb), which can normally be reduced back to oxyhemoglobin by a cellular repair enzyme.
Hemichrome represents a more severely damaged form, and can be irreversible. Normal red cells can tolerate a moderate level of methemoglobin.
Hemichrome degradation can produce free heme, the iron-porphyrin component of native hemoglobin, which is damaging to cell membranes. Thus it is desirable to minimize hemichrome levels. Each hemoglobin species can be detected at a specific wavelength, using a standard spectrophotometer.
The following data show the sensitivity of the hemoglobin to damage by the increased U.V. exposure.

V4'O 91/16060 ~~~ss2~ PCT/L:S91/02504 An exposure of 3 minutes was nudged to be usable viral inactivation using a radiation sensitizes, wit:zout :r,~lic~ing excessive danage to red blood cells.
EXPOSURE % % %
~'M
(?V!;.nutes) O
p 96.6 3.4 0 2 90.2 7.5 2.3 4 84.5 13.4 2.1 6 76.7 22.5 0.9 g 72.6 27.4 0 10 66.4 33.6 0 EXAM LP E 2 , A suspension (0.1 ml) of bacteriophage lambda or bacteriophage phi-X174, of at least lOEV PFU/ml, is separately added to 4 ml of dextrose-saline containing 1 mg/ml of compounds I or II or III. Each suspension of bacteriophage with a radiation sensitizing compound is then exposed to U.V.
radiation of the preferred wavelength (320 nm) in a quartz chamber for the preferred time (3 minutes). A
control sample of each bacteriophage suspension, containing a sensitizes, is not exposed to U.V.
light. Serial dilutions are performed to quantitate the level of infectious titer, and aliquots of the various bacteriophage samples are then mixed with host bacteria and spread on nutrient agar. Following a normal growth period, the plates are assayed for plaques. Other bacteriophage suspensions are separately irradiated as above, but without added sensitizes, to demonstrate the effect of this dose of U.V. alone.

w0 ~it/16060 PCT/US91/0250a i~~ rJ~~~.~
Lost' 0 Red~~r°:o~ ~° ~ ~.:s '' ~°_ phi_u174 T a:."..~,da 1 (X=iy; >6. o >6 . c (X=I> 4.0 ><.o I6 1.7 >5.G
J
No compcund 2°3 2°3 From these data it can be seen that all three tested compounds significantly increase the sensitivity of double-stranded DNA virus (lambda) to L1.V. of the preferred exposure. Compound I is also effective against a single-stranded DNA virus, phi-X174.
Compound I is most preferred, showing a high~-(at least 6 log reduction) inactivation efficacy against both single-strand and double-strand DNA viruses.

Claims (17)

CLAIMS:

1. A process for reducing viral and/or bacterial and/or parasitical contaminations in a biological composition comprising the steps of contacting said composition with at least one chemical sensitizer selected from the group consisting of compounds which bind to DNA and/or RNA and are capable of selectively generating free radicals upon exposure to radiation, lyophilizing the composition, and exposing said composition to radiation of sufficient wavelength and intensity for a period of time sufficient to cause said sensitizer to reduce viral, bacterial and parasitical contamination in said composition.

2. A process according to claim 1 wherein said radiation comprises gamma radiation.

3. A process according to claim 1 wherein said chemical sensitizer comprises a metal atom.

4. A process according to claim 1 wherein said chemical sensitizer is sensitized by penetrating, ionizing radiation.

5. A process according to claim 1 wherein said chemical sensitizer is sensitized by gamma radiation or X-rays.

6. A process according to claim 1 wherein said one chemical sensitizer is selected from the group consisting of compounds of the formula:
X = -N3, -I

7. A substantially virally and bacterially and parasitically decontaminated lyophilized composition comprising red blood cells, platelets and/or proteins and containing inactive viral and/or bacterial contaminants which have been deactivated by binding of the viral and/or bacterial and/or parasitical DNA or RNA to at least one chemical sensitizer capable of selectively generating free radicals upon exposure to electromagnetic radiation, lyophilizing the composition, and by exposing said bound sensitizer to electromagnetic radiation of sufficient wavelength and intensity and for a period of time sufficient to cause said sensitizer to deactivate said RNA
and/or DNA.

8. The process according to any one of claims 1 to 6, wherein the step of lyophilizing comprises a step of contacting the composition with a lyophilizing solution buffered in the range of pH 7.0 to 7.4 prior to lyophilizing the composition.

9. The process according to claim 8, wherein the lyophilizing solution is a phosphate-buffered solution with a pH of about 7.2 and which comprises mono- and di-basic potassium and sodium phosphate in the range of 1-10 mM and adenine in the range of 5-10 mM.

10. The process according to claim 9, wherein the lyophilizing solution further comprises one or more components selected from the group consisting of: nicotinic acid, reduced glutathione, glutamine, inosine, magnesium chloride, a monosaccharide at a final concentration of about 26% weight by volume, polyvinylpyrrolidone at a final concentration of about 3.0% weight by volume, and hydroxyethyl starch at a final concentration of about 15% weight by volume.

11. The process according to any one of claims 1 to 6 or 8 to 10, wherein the step of lyophilizing the composition reduces the water content of the composition to less than 10%
by weight.

12. The process according to claim 11 wherein the water content of the composition is reduced to less than 3% by weight.

13. The substantially virally and bacterially and parasitically decontaminated lyophilized composition according to claim 7, wherein the composition has been contacted with a lyophilizing solution buffered in the range of pH 7.0 to 7.4 prior to lyophilizing.

14. The composition according to claim 13, wherein the lyophilizing solution is a phosphate-buffered solution with a pH of about 7.2 and which comprises mono- and di-basic potassium and sodium phosphate in the range of 1-10 mM and adenine in the range of 5-10 mM.

15. The composition according to claim 14, wherein the lyophilizing solution further comprises one or more components selected from the group consisting of: nicotinic acid, reduced glutathione, glutamine, inosine, magnesium chloride, a monosaccharide at a final concentration of about 26% weight by volume, polyvinylpyrrolidone at a final concentration of about 3.0% weight by volume, and hydroxyethyl starch at a final concentration of about 15% weight by volume.

16. The composition according to any one of claims 7 and 13 to 15, wherein the composition has a final water content of less than 10% by weight.

17. The composition according to claim 16, wherein the composition has a final water content of less than 3% by weight.