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WO1998015297A1 - Procede de sterilisation de matieres biologiques - Google Patents

Procede de sterilisation de matieres biologiques Download PDF

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Publication number
WO1998015297A1
WO1998015297A1 PCT/IB1997/001242 IB9701242W WO9815297A1 WO 1998015297 A1 WO1998015297 A1 WO 1998015297A1 IB 9701242 W IB9701242 W IB 9701242W WO 9815297 A1 WO9815297 A1 WO 9815297A1
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WO
WIPO (PCT)
Prior art keywords
reactive oxygen
biological
liquid
biological liquid
cell
Prior art date
Application number
PCT/IB1997/001242
Other languages
English (en)
Inventor
Peter Lea
Walter Dermott
Original Assignee
Lifetech Corporation
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Filing date
Publication date
Application filed by Lifetech Corporation filed Critical Lifetech Corporation
Priority to AU43934/97A priority Critical patent/AU4393497A/en
Publication of WO1998015297A1 publication Critical patent/WO1998015297A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
    • A61L2/0094—Gaseous substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/20—Gaseous substances, e.g. vapours
    • A61L2/202—Ozone

Definitions

  • the present invention relates to the decontamination and sterilization of microorganisms, such as viruses, bacteria, and fungi, particularly pathogenic microorganisms, present in biological materials, i.e. , protein preparations, carbohydrate solutions, lipid suspensions, or cells, e.g. , for administration into human or animal subjects, or for use, in vitro, for example in tissue culture systems.
  • microorganisms such as viruses, bacteria, and fungi, particularly pathogenic microorganisms
  • present in biological materials i.e. , protein preparations, carbohydrate solutions, lipid suspensions, or cells, e.g. , for administration into human or animal subjects, or for use, in vitro, for example in tissue culture systems.
  • the invention particularly relates to increasing the safety of the blood supply, and blood products.
  • Oxygen is an allotropic element, with the most common form of the gas being the di-atomic form (O 2 ).
  • Ozone (O 3 ) and singlet oxygen ( l 0 2 ) are other forms of the gas that occur naturally and that can be created artificially.
  • Ozone is the triatomic form of oxygen and is relatively unstable.
  • One method for generating ozone is to expose O 2 gas to electromagnetic radiation having a wave length of about 180 to 187 nm.
  • Other methods of forming ozone are well-known.
  • Singlet oxygen is the monatomic form of oxygen and is highly unstable.
  • One method for generating singlet oxygen is to expose ozone to ultra-violet light at a wavelength of about 253.7 nm. It is also known to expose a mixture of oxygen gas containing ozone and singlet oxygen to visible light at a wavelength of between about 500 and 800 nm to further induce the formation of singlet oxygen.
  • Ozone singlet oxygen, and even triplet oxygen are also well-known in the medical community as agents for treating blood and human tissue in order to fight disease or other pathogens.
  • ozone has been found, e.g. , to be effective against various viruses and fungi, and to inactivate a wide variety of bacteria including pseudomonas aeruginosa, staphylococcus aureus and mycobacterium tuberculosis.
  • ozone or singlet oxygen One very important use of reactive oxygen, e.g. , ozone or singlet oxygen, is in the sterilization of blood. Through the use of ozone and singlet oxygen, it is possible to deactivate a variety of bacterial and viral contaminants of blood, including HIV-1 and Hepatitis. Oxygenation in this manner may be used to treat blood prior to its transfusion. It is also known to oxygenate whole blood, by absorbing molecular oxygen into it, for various medical and surgical procedures such as coronary bypass operations.
  • the technique wherein the blood is contacted as a film is exemplified in this patent by the use of rotating vessels such as rotating bottles, which act to form the blood into a relatively thin film over most of the bottle interior surface, with the ozone being passed through the bottle interior.
  • the concentration of ozone in the treating atmosphere is said to be in the range of about 1 to 100 ppm, and usually in the range of from about 1 to 20 ppm.
  • the ozone level was at approximately 2 ppm, and apparently was even lower since the ozone was mixed with filtered air before being contacted with the blood in the rotating bottle.
  • the patentee emphasizes the need for what are referred to as "mild" contact conditions, apparently referring to the very low concentrations of ozone which are used.
  • a difficulty encountered in the method and apparatus contemplated in the 4,632,980 patent is that the treated film of blood is upon continued rotation of the bottle remixed with the untreated blood, thereby continually nullifying in whole or in part any salutary effects which are otherwise achieved. It will be further appreciated that the film of blood formed can at best be contacted at one surface thereof, i. e. , the film resides on the bottle surface and is not amenable to contact with the ozone from the support side.
  • a chamber is provided for receiving and collecting the liquid (such as blood) which is to be contacted with the oxygenating gas, such as oxygen containing ozone.
  • the oxygenating gas is introduced into a spray orifice in such a manner that the venturi effect generated by flow of the gas causes a flow of blood to be induced into and contacted with the gas stream, and to issue from the nozzle orifice as a dispersed spray of liquid droplets.
  • a spray collection surface is located within the chamber in the spray path of the liquid droplets, which collects substantially the entirety of the droplets of oxygenated liquid.
  • This collection surface is spaced from the spray orifice a distance selected so that the droplets impinge on the collection surface at relatively low velocity and form a film on the surface, where further contact with the gas can also be effected.
  • the method of contact achieved in the said apparatus is very effective in that high energy mechanical impact of the droplets with the collection surface is avoided, whereby hemolysis effects are eliminated or greatly reduced. Much higher concentrations of ozone can be safely utilized in this apparatus than have heretofore been taught.
  • the present invention advantageously provides a method for decontaminating a biological liquid while minimizing degradation of biological materials, such as proteins and cells.
  • the method of the invention comprises contacting the biological liquid with sufficient reactive oxygen to deactivate microorganisms that may be present in the biological liquid by taking account of a reactive oxygen demand of the biological liquid.
  • Critically, the maximum period of time, or effective treatment window, that the reactive oxygen is in contact with the biological liquid averts degradation of any biological material that may be present.
  • the reactive oxygen demand of the biological liquid is a function of a reactive oxygen demand of a liquid carrier and a reactive oxygen demand of any biological material that may be present in the liquid carrier.
  • the period of time of exposure of a biological liquid to reactive oxygen will depend on the reactive oxygen demand of the biological liquid, the nature and amount of microorganisms present, and the rate of transfer of reactive oxygen from the gas into the biological liquid.
  • the present invention advantageously provides for enhancing the rate of transfer of reactive oxygen from the gas into the liquid so that effective sterilization can occur with minimal degradation of biological material.
  • the invention provides for decontamination of viral pathogens.
  • the viral pathogen log titer can be decreased at least by a factor of 4; preferably, the viral pathogen log titer is decreased at least by a factor of 6.
  • the present invention provides for treatment to destroy envelope viruses, surprisingly, non-envelope viruses, and, retroviruses.
  • the viral pathogen is selected from the group consisting of Porcine Parvovirus (PPV), Adeno virus, Infectious Bovine Rhinotracheitis (IBR) virus, and Bovine Viral Diarrhea (BVD) Virus, and Simian Immunodeficiency Virus (SIV).
  • the biological material is a protein, such as, but not limited to, a blood protein, for example immunoglobulin useful for passive immunization.
  • the method of the invention is especially useful with a blood protein selected from the group consisting of serum albumin, clotting factor, and fibrinogen.
  • the biological material may be a biological liquid containing proteins, such as but not limited to serum, plasma, cell culture fluid, liquid nutrient fluid, or liquid nutrient extract, e.g. , Bovine Pituitary Extract (BPE).
  • BPE Bovine Pituitary Extract
  • the protein may be isolated from a fermentation culture.
  • the biological material comprises cells.
  • the invention provides for decontamination of any type of blood cell, such as but not limited to red blood cells, white blood cells, lymphocytes, or platelets.
  • the cells are bone marrow cells, or stem cells. More particularly, the stem cells may be selected from the group consisting of peripheral stem cells, autologous marrow cells, allogeneic marrow cells, umbilical chord blood cells, and tissue culture cells. In one particularly useful embodiment of the invention, the stem cells are a CD34 + or CD38 + hematopoietic progenitor cells.
  • FIGURE 1 Perspective, simplified schematic view of an apparatus for use in the method of the present invention.
  • FIGURE 2 Phosphate buffered saline (PBS) ozone saturation curves at 22.5 °C (open circles) and 21.5°C (open squares).
  • PBS Phosphate buffered saline
  • FIGURE 3 Ozone decay in saturated PBS at room temperature (22.5°C). The half-life was 273 sec (4 min, 33 sec); after 20 min, 3.1 % of the ozone remained; the curve fits a third order polynomial equation with a coefficient (R) of 0.99992.
  • FIGURE 4 Effects of ozone concentration on ozone transfer into PBS and human IgG in PBS.
  • Ozone concentration in PBS (y-axis, ⁇ g/ml) was determined by UV absorption at 260 nm after cumulative contact of 9 ⁇ g/ml ozone in air (ozone concentration) with PBS (solid circles); or 9 ⁇ g/ml ozone (solid squares), or 34 ⁇ g/ml ozone (solid triangles), with 0.5 ⁇ g/ml IgG in PBS.
  • FIGURE 5 Transfer efficiency of ozone in PBS and human IgG. Ozone transfer efficiency over time was determined from the data in Figure 4; the symbols are the same as in Figure 4. Ozone transfer efficiency was calculated as 100 x (amount of ozone in the liquid/ total amount of ozone delivered in the gas).
  • FIGURE 6 Effects of ozone on 1 % (w/v) human IgG in PBS.
  • the IgG solution was contacted with 36 ⁇ g/ml ozone (gas concentration) for the period of time indicated by a multiple pass technique.
  • Ozone concentration in the solution was measured spectrophotometrically by absorption at 260 nm (A 260 ).
  • FIGURE 7 Effects of ozone on carbonyl formation in 1 % human IgG/PBS solution in the presence of 1:5000 diluted octanol. Carbonyl concentration was determined by colorimetric reaction with 2,4-dinitrophenylhydrazine using standard techniques. The solution was contacted with 36 ⁇ g/ml ozone (gas concentration).
  • FIGURE 8 Effects of ozone on viral reduction of human IgG solutions in PBS, 10% maltose, pH 4.25, and 0.2 M glycine, pH 4.25.
  • Three different viruses were analyzed by incubating the sample on an indicator cell line in serum free media, and scoring for cytopathic effect (CPE).
  • the viruses tested were Adenovirus (vertical stripe bars), a non-enveloped virus; Infectious Bovine Rhinotracheitis (IBR) virus (horizontal stripe bars), an envelope virus; and Bovine Viral Diarrhea (BVD) virus (diagonal stripe bars), also an envelope virus.
  • IBR Infectious Bovine Rhinotracheitis
  • VBD Bovine Viral Diarrhea
  • FIGURE 9 Virus inactivation, as measured by viral titer, versus ozone concentration ( ⁇ g/ml).
  • A IBR inactivation versus ozone concentration.
  • B BVD inactivation versus ozone concentration.
  • C Adenovirus inactivation versus ozone concentration.
  • FIGURE 10 Virus inactivation, as measured by viral titer, versus increasing dilution in ozonated PBS.
  • A Dilution of IBR with ozonated PBS.
  • B Dilution of BVD.
  • C Dilution of Adenovirus.
  • D Dilution of Porcine Parvovirus (PPV).
  • FIGURE 11 Viral inactivation in 0.5 mg/ml IgG relative to pH in 0.2 M glycine containing octanol (1:5000). The ozone concentration was 34 ⁇ g/ml at a flow rate of 500 ml/min and a negative pressure of 2" of water.
  • A Inactivation studies with Bovine Viral Diarrhea (BVD) virus.
  • B Inactivation studies with Infectious Bovine Rhinotracheitis (IBR) virus. Viral inactivation, as determined by viral titer, was evaluated over time of exposure. Control viral samples were prepared in PBS at pH 7.2 (solid circles).
  • Samples containing IgG were prepared in pH 7.2 (open circles) and pH 4.25 glycine-PBS (closed squares). Note that the suppression of viral inactivation due to IgG ozone demand (open circles) was completely eliminated, and viral killing enhanced, in the lower pH solution.
  • FIGURE 12 PBS saturation using pressurized ozone over time.
  • Ozone 34 ⁇ g/ml
  • Ozone was contacted with PBS using a oxygenating device as shown in Figure 1 after multiple passes at ambient atmospheric pressure (data not shown), or with a back-pressure of + 1 psi (open circles with a dashed line), +2 psi (open squares, solid line), or +3 psi (open triangles, dotted line).
  • + 1 psi open circles with a dashed line
  • +2 psi open squares, solid line
  • +3 psi open triangles, dotted line
  • the present invention provides a method for sterilizing a biological liquid comprising contacting the biological liquid, preferably containing a surfactant and/or a cosurfactant, with a concentration and pressure of reactive oxygen for a time effective to deactivate contaminants present within the biological liquid.
  • the amount of reactive oxygen required to deactivate a microorganism in a biological liquid depends on the reactive oxygen quenching activity of the liquid carrier (e.g. , PBS, saline, or water), the quenching activity of any biological materials to be decontaminated or sterilized by reactive oxygen, and the type and amount of microorganism present. These quenching activities are referred to herein as reactive oxygen demand.
  • the invention further relates to the discovery that minimizing the time to transfer reactive oxygen into a biological liquid containing a biological material and a microorganism reduces the structural degradation of the biological material, particularly proteins, polypeptides, amino acids, and cells.
  • biological material examples include protein preparations and cells.
  • the protein is serum immunoglobulin obtained from an immunized subject for use in passive immunotherapy.
  • the biological material comprises CD34 + or CD38 + hematopoietic stem cells.
  • the term "sterilizing” refers to the deactivation of microorganisms present in a biological liquid.
  • degradation refers to an irreversible change in the physical-chemical or functional properties of a biological material.
  • degradation include denaturation, fragmentation, or derivitization by reaction with oxygen.
  • a protein may become unfolded; it may be cleaved at a peptide or other bond into fragments; or amino acid side chains may be oxidized. Under such circumstances, the protein may lose its physiological activity.
  • an immunoglobulin may fail to bind antigen, or may not be recognized for opsonization via Fc receptors.
  • biological liquids encompasses a wide variety of fluid substrates which may be oxygenated, and in some instances, sterilized by use of the invention.
  • the biological liquid may, but need not necessarily, contain a biological material in solution or suspension.
  • biological liquid includes, for example, blood and blood fractions, including whole blood, serum, plasma, packed red blood cells, platelets, and leukocytes; and as well, bone marrow and semen.
  • fluids that may be administered to mammals, including humans, such as saline or glucose intravenous solutions and the like; and fluid nutrient media such as are employed to preserve organs for transplantation, or to grow bacteria, viruses, cells (particularly cells for transplantation, fermentation to produce recombinant proteins, or hybridomas for production of monoclonal antibodies), parasites, and the like.
  • a biological material such as but not limited to proteins, carbohydrates, or cells suspended in a buffered solution, e.g. , immunoglobulin in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the term also refers to such liquid carriers without any biological material; i.e.
  • biological liquid also refers to a liquid intended for in vivo administration, such as a fluorocarbon liquid for artificial blood or direct oxygen transfer in the lungs.
  • biological material is used herein to refer to proteins (including glycoproteins, proteoglycans, recombinant proteins, etc.), polypeptides, amino acids, carbohydrates, lipids (including lip id structures such as liposomes, micelles, lipoprotein particles, etc.), cells, and combinations of two or more of the preceding, which may be dissolved or resuspended in a biological liquid.
  • biological material refers to the product to be sterilized, and thus excludes the microorganism being inactivated by oxygenation treatment.
  • microorganism refers to the contaminant to be inactivated by oxygenation treatment.
  • examples of microorganisms include the prion infectious element (which, though it may not be an "organism” in the traditional sense, clearly has pathogenic potential and can be transmitted through some mechanism), viruses (including envelope, and surprisingly, non-envelope viruses, and retroviruses), bacteria, fungi, and parasitic protozoa (such as plasmodium, trypanosomes, leishmania).
  • the microorganism is a known pathogen, such as HIV or Hepatitis.
  • microorganisms may be opportunistic pathogens, such as certain bacteria or viruses that are normally inactivated by the immune system but are pathogenic in immune-compromised individuals. In some instances, the microorganism may not be pathogenic or harmful, but is nevertheless an undesirable contaminant.
  • mycoplasma An example of this last sort of microorganism is mycoplasma, which is a highly undesirable contaminant of cell cultures.
  • oxygen as used herein is meant to encompass all of these forms of the gas, and references to “oxygenation” similarly encompasses treatment by any of such forms of the gas and the transfer and absorption of the treating oxygen to the biological liquid, e.g. , for the sterilization of one or more components of the biological liquid.
  • oxygenation treatment refers to treatment of a biological liquid and/or microorganism with a reactive oxygen molecule.
  • the reactive oxygen molecule can be supplied as a component of other gasses, such as normal (triplet) oxygen, nitrogen, air, etc.
  • the reactive oxygen molecule supplied to the biological liquid is ozone (O 3 ).
  • the reactive oxygen molecule is singlet oxygen (O 2 ).
  • Other examples of reactive oxygen molecules are well known in the art, and include molecules that can form oxygen radicals as well as activated oxygen, including biological or chemical substrates that can be treated to form reactive oxygen.
  • ozone can react to form reactive oxygen intermediates, e.g.
  • peroxides which may then form singlet oxygen, react on their own, or form other reactive oxygen intermediates.
  • the present invention contemplates supplementing the biological liquid with reagents that activate oxygen to a reactive state.
  • reagents that activate oxygen to a reactive state.
  • amino acids particularly cysteine
  • Unsaturated carbon compounds such as lipids and detergents, may enhance peroxide formation.
  • every protein has an intrinsic, composition dependent, singlet oxygen potential.
  • the singlet oxygen potential for a given biological liquid can be increased by including free amino acids with high efficiency for ozone to singlet oxygen conversion.
  • reactive oxygen demand (somewhat akin to a p[O 3 ]); or anti-oxidant potential.
  • reactive oxygen demand will be a function of the intrinsic reactive oxygen demand of the biological material plus the amount of reactive oxygen required for effective microorganism inactivation.
  • the present inventors have discovered that effective sterilization requires that a sample or preparation is contacted with a sufficient concentration or quantity of reactive oxygen to satisfy that demand and supply enough reactive oxygen to destroy microorganisms.
  • the invention applies rapid gas transfer kinetics to provide a pathotoxic dose of reactive oxygen in the presence of reactive oxygen quenchers (the liquid and a biological materials).
  • the invention advantageously avoids saturating the biological liquid and biological material with reactive oxygen, which will occur as the exposure time increases, while at the same time killing microorganism.
  • the amount of reactive oxygen will vary dramatically with the selection of different biological materials for sterilization, in addition to consideration of the reactive oxygen demand of the liquid carrier, and the kind and amount of microorganism to be destroyed. As described below, various means are available to effectively provide a sufficient reactive oxygen concentration for sterilization without denaturing or destroying the biological material being sterilized.
  • the present invention concerns enhancements to the efficiency of reactive oxygen transfer to a biological liquid to satisfy the reactive oxygen demand in the shortest period of time, thus effecting the inactivation of microorganisms without significantly destroying the biological material.
  • This exposure time is the "effective treatment window. " Shorter times fail to deactivate microorganisms. Longer times lead to unacceptable degradation.
  • any biological material preparation whether purified proteins (such as immunoglobulins or albumins), isolated proteins (such as plasma or serum), carbohydrates, lipids, cells, or combinations thereof, can be sterilized by determining the reactive oxygen demand for the preparation, and determining the permissible reactive oxygen contact time range and concentration range to achieve sterilization without degradation of the biological materials.
  • the present invention provides for adjusting variables such as temperature differential between the gas and the biological liquid; pH of the biological liquid; inclusion of a surfactant and/or cosurfactant in the biological liquid; gas pressure; and gas humidity to increase transfer efficiency.
  • the invention is directed to a method for sterilizing a protein preparation comprising contacting the protein preparation with a concentration of reactive oxygen for a period of time, which concentration and time period depend on the concentration of protein and the reactive oxygen demand of the protein and liquid carrier that makes up the preparation, such that a total amount of reactive oxygen with which the protein preparation is contacted is sufficient to destroy a microorganism (prion or prion-like proteins, virus, bacteria, fungus, protozoan parasite) but does not degrade all or most of the protein, and leaves the protein physiologically active.
  • a microorganism prion or prion-like proteins, virus, bacteria, fungus, protozoan parasite
  • the principles of the present invention can be applied to any current technology for introducing ozone into a biological liquid.
  • an apparatus for effectively oxygenating a biological liquid provides an open mesh surface having opposed accessible faces.
  • Flow means are disposed to establish a flow of the biological liquid along the mesh surface, thereby forming the fluid into a membrane at the mesh openings of the surface.
  • Means are additionally provided for establishing an oxygenating gas atmosphere in contact with the opposed faces of the mesh surface, for oxygenating the biological liquid membrane from both sides of the mesh surface; and means are provided for collecting the flow of oxygenated biological liquid from the mesh.
  • the preferred apparatus provides for maximum efficiency of contact of the biological material with oxygen or reactive oxygen by exposing both surfaces of a liquid membrane to the sterilization gas. Furthermore, in a preferred embodiment, the apparatus places minimal mechanical stress on the material, since the liquid moves by controlled gravity flow down the mesh. Spraying and other harsher techniques are thus avoided. More importantly, the time of exposure of the material to reactive oxygen is minimized because the apparatus provides for highly efficient reactive oxygen transfer.
  • the mesh surface may be formed as a cylinder, which can be mounted with its axis vertically directed.
  • the means for flowing the biological liquid can in this instance comprise a distribution cap secured at the top of the cylinder with its periphery engaging the end of the cylinder.
  • a flow of biological liquid is provided onto the cap to enable a gravitational flow toward the edge of the cap and thereby onto the mesh surface.
  • the cylinder and flow means are mounted within a reaction and fluid collection chamber.
  • the chamber is provided with a gas inlet for oxygenating gas and a spaced gas outlet for discharging the gas.
  • Also connected to the chamber are fluid inlet means for the biological liquid to be treated, and an outlet enabling the treated biological liquid to be collected.
  • the flow means may further comprise a fluid delivery conduit and a flow control means between the conduit and distribution cap for regulating the amount of flow to the mesh surface.
  • the mesh surface preferably comprises a material which is inert to the biological liquid and to the oxygenating gas used in the apparatus, and may be in the form of a screen, a webbing or the like.
  • Figure 1 shows an embodiment of the invention.
  • an example of the apparatus 10 comprises an outer substantially hollow cylindrical chamber 12.
  • the bottom of chamber 12 is provided with a liquid exit port 13 for withdrawing biological liquids following the oxygenation.
  • Mounted coaxially within chamber 12 is a an open mesh surface 14 which is formed as a hollow open-ended cylinder 15.
  • the cylinder may be supported in the manner shown by being secured to and suspended from a flow distribution cap 21.
  • the latter is in turn mechanically supported by the gas input conduit 19.
  • Mesh 14 is an open webbing or screen relatively resistant to the oxygenating gas being used and to the biological liquid, such as gauze comprised of natural or synthetic materials; plastics such as polyethylenes, PVCs and the like; as well as highly inert metals and alloys.
  • the mesh generally has uniform circular, rectangular or other openings, with a mean opening dimension in the range of from about .02 to 10 mm.
  • the chamber 12 is generally enclosed at its upper end by a cover 16 through which passes (in sealed relation) a liquid inlet port 17 and a gas exit port 18.
  • Input port 17 in turn supports in sealed relation the coaxially mounted gas input conduit 19 for oxygenating gas which is to be introduced into the apparatus.
  • Conduit 19 is joined to a gas dispersing nozzle 20 which disperses the oxygenating gas to the upper interior of cylinder 15.
  • the oxygenating gas is provided to the conduit 19 from a conventional source as, for example, the apparatus described in commonly assigned Dunder, U.S. Patent No. 5,094,822. It passes into chamber 12 at the interior of cylinder 15, and flows as shown by arrows 22 in an axial direction toward the bottom of cylinder 15, thence passes upwardly between the cylinder 15 and the interior wall of chamber 12, and exits from port 18.
  • the flowable biological liquid to be treated which may representatively be considered as whole blood, is provided to inlet port 17 and flows to and over flow distribution cap 21, the periphery of which is smoothly adjoined by mechanical, adhesive or other means to the upper edge 24 of the mesh 14 comprising cylinder 15.
  • a liquid flow control or regulator 25 is positioned between inlet 17 and distributor cap 21.
  • Body 26 may be threadingly mounted on inlet 17 to enable this action or may be vertically displaced by simple electromechanical means such as solenoid actuation or the like, which opens into the interior of chamber 12.
  • this membrane can be of the order of 50 to 750 microns thickness - in contact with the gas on both surfaces. In consequence a very effective degree of gas mass transfer is achieved.
  • Reactive Oxygen Demand Anti-oxidant Potential
  • Reactive oxygen demand is, as described above, a concentration of reactive oxygen adsorbed or neutralized by a biological material, such as a protein, protein preparation, or a cell preparation.
  • the value for reactive oxygen demand varies with the makeup of each biological material. Different proteins or protein preparations, for example, have unique values for reactive oxygen demand.
  • reactive oxygen demand of a biological material can be empirically determined by titrating the amount of reactive oxygen required to reach saturation of a solution or suspension. In any system, this will depend on time of exposure and the concentration and pressure of reactive oxygen, as well as temperature, pH, and the presence of surfactant and/or cosurfactants. As discussed below, where the reactive oxygen demand of a biological liquid requires exposure times that exceed safe limits to avoid degradation of the biological material, various means to increase transfer efficiency can be employed.
  • the present invention provides a number of different means for increasing reactive transfer efficiency, including adjusting the pH of the biological liquid; providing a temperature differential between the gas and the liquid; increasing the pressure of the gas and the concentration of reactive oxygen in the gas, or both; and including a surfactant and/or cosurfactant in the biological liquid.
  • These strategies may be employed individually, i.e. , in isolation, or two or more can be employed simultaneously. It should be noted that merely adding a biological material to the biological liquid facilitates reactive oxygen transfer.
  • Temperature differential It has been found that maintaining a temperature differential of up to 5 °C between the gas phase (at the higher temperature) and the biological liquid increases reactive oxygen transfer efficiency.
  • the absolute temperature of the system is regulated between about 4°C and about 40°C; it may be up to 42°C.
  • the gas and liquid temperatures are within about 5°C of room temperature (22.5°C); in another embodiment, gas and liquid temperatures are within about 5°C of 37 °C.
  • the protein preparation includes a surfactant and/or cosurfactant.
  • a surfactant and/or cosurfactant greatly facilitates transfer of reactive oxygen into the substrate, i.e. , the protein preparation. It has been found that the transfer rate efficiency reaches a plateau after the first one to three passes in a multiple run system, then generally decreases. By including a surfactant and/or cosurfactant, the transfer rate efficiency remains steady at the plateau level; there is no apparent decrease.
  • the cosurfactant is octanol.
  • Octanol is closely related to octanoic acid, a blood resident fatty acid.
  • the octanol may be oxidized to octanoic acid by the reactive oxygen.
  • Other surfactants particularly C-6 to C-18 alcohols or acids, or lecithins, can also be used.
  • the surfactant is sodium dodecyl sulfate (SDS). Any surfactant that is inert to reactive oxygen or that does not adversely affect the biological material can be employed.
  • the surfactant chosen corresponds to, or may react with reactive oxygen to form, a naturally occurring compound.
  • a surfactant and/or cosurfactant prevents protein (or other molecular) clumping, thus maintaining protein accessibility and avoiding excessive reactive oxygen demand effect.
  • the surfactant also decreases surface tension of the biological liquid, permitting faster gas transfer into the liquid.
  • Surfactants are especially useful in film-type reactive oxygen contactor apparatuses, e.g. , as described above and shown in Figure 1.
  • the presence of a surfactant advantageously enhances liquid flow characteristics.
  • the various kinds of protein that can be treated according to the invention include proteins produced in fermentation of recombinant or non-recombinant microorganisms; animal proteins; and preferably, human proteins ⁇ more preferably, blood proteins and blood products.
  • suitable proteins include immunoglobulin (as shown in specific examples, infra), serum albumin, insulin, clotting factors (Factor VIII, Factor IX, Factor X), fibrinogen, prothrombin and thrombin, interferons, lymphokines, cytokines, and the like.
  • a protein preparation is treated with reactive oxygen in an appropriate apparatus for a time and at a concentration to saturate the preparation without denaturing the protein.
  • Effective reactive oxygen transfer occurs when the amount of reactive oxygen is sufficient to deactivate or destroy microorganisms, such as viruses, but does not destroy protein function.
  • effective reactive oxygen transfer for serum IgG destroys viral pathogens present in the preparation, while retaining IgG antigen-binding activity.
  • this is accomplished in a single run of less than three minutes exposure to ozone, and more preferably less than two minutes exposure.
  • the optimum range for reactive oxygen treatment is different for each type of protein or protein preparation. It can be readily determined by contacting the protein with reactive oxygen to the saturation point for various time periods, and testing for protein activity. Once the reactive oxygen demand of the protein or protein preparation is determined, the invention provides for effecting reactive oxygen transfer within the required time period to avoid protein denaturation, as described in detail, infra.
  • Protein structural integrity or activity can be analyzed by a number of criteria, including but not limited to: polyacrylamide gel electrophoresis, isoelectric focusing, amino acid content, functional activity (such as enzymatic activity for an enzyme, antigen binding or complement activation for an immunoglobulin), antigenicity (i.e. , recognition by an antibody), HPLC analysis, sequence analysis, differential scanning calorimetry, and other techniques for evaluating protein activity.
  • polyacrylamide gel electrophoresis isoelectric focusing
  • amino acid content amino acid content
  • functional activity such as enzymatic activity for an enzyme, antigen binding or complement activation for an immunoglobulin
  • antigenicity i.e. , recognition by an antibody
  • HPLC analysis sequence analysis
  • differential scanning calorimetry and other techniques for evaluating protein activity.
  • Specific decontamination conditions can be determined for proteins by considering the concentration of protein in the preparation, the inherent reactive oxygen demand of the protein, the actual reactive oxygen demand (inherent demand times concentration), and the reactive oxygen requirement for decontamination of microorganisms, e.g. , viruses. Examples of decontamination conditions and analytical criteria for confirming retention of protein function for various proteins preparations are provided in the specific examples, infra.
  • Various cell preparations are suitable for decontamination according to the claimed invention, including but not limited to whole blood, packed red blood cells (RBCs), white blood cells, lymphocytes, platelets, bone marrow, and stem cells.
  • RBCs packed red blood cells
  • the present invention provides for decontamination of stem cells, such as peripheral blood or whole blood stem cells, autologous stem cells, allogeneic stem cells, and chord blood stem cells.
  • the invention provides for decontamination of a cell preparation comprising contacting the cells with a concentration of reactive oxygen for a period of time, which concentration and time period depend on the density of cells and the reactive oxygen demand of the cells, such that the total amount of reactive oxygen with which the cell preparation is contacted is sufficient to destroy a viral pathogen or other contaminant but does not denature or destroy the cell.
  • the apparatus shown in Figure 1 is preferred. An apparatus of the invention for the decontamination of cells must avoid mechanical damage to the cells. The apparatus of Figure 1 is particularly suited for this.
  • various anti-oxidant enzymes such as glutathione in red blood cells, and catalase in serum, may be present that increase the anti-oxidant potential, and hence the reactive oxygen demand, of such preparations.
  • the present invention advantageously provides for neutralizing the anti-oxidant effects of these enzymes to achieve inactivation of microorganisms without destruction of the cells.
  • the role of reactive oxygen demand in reactive oxygen-mediated decontamination has important implications for sterilization of cells as well.
  • the present invention provides for determining the reactive oxygen pressure, exposure time, cell concentration, and other factors, accounting for any inherent cellular reactive oxygen demand, to decontaminate cells.
  • various means can be employed to ensure adequate reactive oxygen transfer efficiency to meet that demand without incurring cellular degradation.
  • CD34 + or CD38 + hematopoietic stem cells are particularly preferred for treatment according to the invention. Such cells may be obtained and stored prior to radiation or chemotherapy, which destroys these types of cells in a patient. The harvested CD34 + or CD38 + cells are then treated to decontaminate any viral pathogens or other microorganisms before transplantation into the immune-compromised patient.
  • Decontamination of cells involves treatment of complex structures with many variables. Accordingly, as discussed herein, in preferred aspects of the invention, various strategies may be employed to enhance reactive oxygen treatment of cells.
  • a biological liquid containing cells is supplemented with an effective concentration of amount of an antioxidant.
  • an antioxidant will, of course, affect the reactive oxygen demand of the biological liquid. Its presence can serve to protect cells to a greater degree than any microorganisms that might be present. As shown in a specific example, infra, the presence of an antioxidant protects platelets from degradation during ozone treatment.
  • antioxidants for use in the present invention include, but are not limited to, vitamin-C, vitamin-E (preferably a water soluble form of vitamin-E), glutathione, and D-mannitol.
  • the antioxidant is D- mannitol.
  • concentration of antioxidant to be used will depend on the concentration and nature of the cells in the biological liquid, but will generally be within accepted effective concentration ranges, which are well known for these antioxidants. Alternatively, an effective concentration can be determined by simple titration analysis.
  • Humidity High humidity of the gas is important for the decontamination or sterilization of a cell preparation (it may also be of value for a highly concentrated protein or lipid preparation), to prevent drying of the cells or denaturation of the proteins.
  • decontamination of a biological liquid containing cells in accomplished by contacting the biological liquid with a gas having 100% humidity.
  • Increased humidity has also been found to be necessary to compensate for increasing the ozone concentration, which has a drying effect on the biological material.
  • Increased humidity has little effect, however, on liquids containing up to 20% of most proteins.
  • total nutrient admixtures for total parenteral nutrition, e.g. , as described in Deitel et al. [CJS 32:240-243 (1989)] .
  • These mixtures contain lipid particles, liposomes, proteins (e.g. , heparin), amino acids, carbohydrates, vitamins, and minerals.
  • the reactive oxygen demand of such a total nutrient admixture can be determined.
  • the admixture can be treated to deactivate any microorganisms under conditions that protect the components. If necessary, pH can be easily adjusted after treatment with appropriate buffers or salts.
  • viruses Various types of viruses are contemplated for deactivation or destruction using the methods of the present invention, including envelope (lipid) and, surprisingly, non- envelope (non-lipid) viruses.
  • the invention advantageously provides for deactivation of retroviruses.
  • viruses, and the log viral titers of deactivation achieved with the present invention include the non-envelope viruses Procine Parvovirus (PPV) (5.00) and Adenovirus (8.00); the envelope viruses Infectious Bovine Rhinotracheitis (IBR) virus (8.10), and Bovine Viral Diarrhea (BVD) virus (7.00); and the retrovirus Simian Immunodeficiency Virus (SIV) (6.00).
  • PSV Procine Parvovirus
  • IBR Infectious Bovine Rhinotracheitis
  • BBD Bovine Viral Diarrhea
  • SIV Simian Immunodeficiency Virus
  • the present invention provides for deactivation of Human Immunodeficiency Virus (HIV), including HIV-1 and HIV-2; Human T- Lymphotrophic Virus (HTLV), including HTLV-1 and HTLV-2; Hepatitis Virus, including Hepatitis-B, Hepatitis-C, and all Hepatitis strains; Adenovirus; Herpes Virus, including all strains; Cytomegalovirus; Epstein-Barr Virus; etc.
  • HIV Human Immunodeficiency Virus
  • HTLV Human T- Lymphotrophic Virus
  • Hepatitis Virus including Hepatitis-B, Hepatitis-C, and all Hepatitis strains
  • Adenovirus Herpes Virus, including all strains
  • Cytomegalovirus Epstein-Barr Virus
  • Ozone was generated in an apparatus as described in U.S. Patents No. 5,094,822 and No. 5,443,800. This patented system is microprocessor controlled, and provides significant control over ozone concentration in the gas. All ozone gas concentrations reported in the following examples were calculated from the settings on the apparatus. Unless otherwise stated, materials were contacted with 34 ⁇ g/ml of ozone (gas concentration) in oxygen.
  • the biological liquid was contacted with ozone in an apparatus as shown in Figure 1.
  • the liquid flow rate in the device was 25 ml/minute.
  • Poly vinyl chloride (PVC) mesh having roughly square pores of an average size of 3 mm was used to form liquid membranes.
  • Total contact time with ozone was 1 minute. Longer times were achieved using multiple runs.
  • the apparatus was fitted with a back-pressure device to increase the gas pressure by up to 3 psi.
  • ozone demand For a basic determination of ozone demand, PBS or PBS containing a material (e.g. , a protein, such as IgG) is contacted with ozone. Extended ozone contact was achieved by passing the biological liquid through the device multiple times. Ozone concentration in the PBS was determined by absorbance at 260 nm (A 260 ). The ozone concentration saturation reflects the ozone demand.
  • a material e.g. , a protein, such as IgG
  • Figure 3 shows the ozone decay curve in PBS at room temperature. This information is useful to evaluate the ozone contact time for achieving an effective concentration in a biological liquid within the treatment window. Rapid ozone decay would mandate a longer exposure time to achieve effective ozone contact. Temperature greatly affects ozone decay rate, and must be considered when calculating contact times.
  • Immunoglobulins in the plasma are important humoral factors to maintain immunity.
  • the infusion of immune globulins into humans has led to immunologic improvement [Christensen et al , J. Pediatr. , 118:606-14 (1991)].
  • intravenous IgG has to be sterile.
  • IgG can be successfully sterilized with ozone under controlled conditions.
  • the actions of free radicals produced by oxidative stress and ozonolysis on IgG suspended in biological fluids have to be determined [Pry or and Uppu, J. Biol. Chem. , 268:3120-6 (1993)].
  • the ozone effects of protein degradation in human IgG were examined by polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and relative changes in amino acid composition of IgG. Carbonyl groups resulting from various oxidative conditions were also determined.
  • the nitrocellulose was washed, and then incubated with an anti-human-Ig conjugated with alkaline phosphatase.
  • the nitrocellulose was washed again and developed with alkaline phosphatase substrate solution (5-bromo-chloro-3-indoyl phosphate and nitroblue tetrazolium) to reveal bound antibodies.
  • Results Conditions for optimal ozone transfer efficiency were determined within an operating window of approximately two minutes.
  • Figure 6 shows that ozone saturation of a solution of 1 % IgG in PBS is achieved after about three minutes. Human IgG retained both structural and functional integrity, as demonstrated by SDS-PAGE, immunoblotting (with nitrocellulose-associated antigen), and amino acid analysis. There was no complement activation by IgG in this treatment window, indicating retention of Ig structural integrity.
  • Immunoglobulins confer humoral immunity in man. They have been widely used for infusions of IVIg (intravenous immunoglobulin) in therapy and prophylaxis as well as in patients with autoimmune diseases and immune deficiencies. To guarantee high purity, efficacy and safety of IVIg requires stringent viral and bacterial inactivation which is becoming a global concern.
  • a microprocessor controlled system as described in Example 2, was used to deliver the exact, required concentrations of ozone into biological liquids. Treatment with ozone inactivates infectious agents present in IgG under conditions that retain the intrinsic protein biological activities. Using an operating window of from one to three minutes, IgG remained functionally and structurally intact as demonstrated by SDS-PAGE, immunoblotting and amino acid analysis with no evidence of IgG aggregation.
  • BVD and 1 non-enveloped virus, Adenovirus were inactivated at concentrations of 10 7 , 10 6 , and 10 6 log reductions for IBR, BVD and Adenovirus respectively using the glycine, pH 4.25 buffer ( Figure 8).
  • the maltose buffer was less effective in viral inactivation probably as a result of the higher ozone demand by the disaccharide, and thereby reducing the sterilizing power of ozone ( Figure 8).
  • EXAMPLE 4 Inactivation of Enveloped and Non-enveloped Viruses by Oxidative Stress
  • Ozone an energized form of oxygen
  • Endothelial cells compose the lining of arteries and veins and are subject to a variety of insult from toxins circulating in the blood.
  • This study was designed to study the in vitro effects of ozonated PBS on human umbilical vein endothelial cells (HUVEC) grown in culture.
  • PBS was ozonated at 4°C, using two temperature conditions monitored continuously by digital thermometers.
  • Endothelial cell monolayer 1 was kept on ice with a starting temperature of 4°C warming up to 8°C.
  • Endothelial cell monolayer 2 was monitored at 10 °C to 19 °C. Control monolayers were exposed to 4°C PBS with no ozone.
  • EXAMPLE 7 Effects of Ozone On The Hematopoietic Activity of Human Pluripotent Progenitor Cells
  • Pluripotent progenitor cells are capable of differentiating into a variety of cell lineages. Stem cell viability and differentiation is therefore of great importance in treatment of whole blood with ozone.
  • ozone treatment on normal hematopoiesis (colony formation, proliferation and multi-lineage cell differentiation) was examined using standard progenitor colony methylcellulose protocols.
  • An enriched cell population was prepared using CD34 + magnetic cell sorting (MACS). Colonies were scored and the plating efficiency calculated as compared to peripheral blood mononuclear cells (PBMC's) controls.
  • PBMC's peripheral blood mononuclear cells
  • Colony progenitors were also analyzed for CD34 and CD38 surface markers as well as cell viability using two color flow cytometry. Preliminary results have demonstrated extremely low levels of cellular damage at concentrations of ozone that achieve virtually 100% viral and bacterial deactivation.

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Abstract

La présente invention concerne la décontamination et la stérilisation d'agents pathogènes tels que des virus, des bactéries et des champignons présents dans des matières biologiques, c'est à dire des préparations protéiques, des solutions glucidiques, des suspensions lipidiques ou des cellules, par exemple, destinées à une administration à des sujets humains ou animaux, ou destinées à une utilisation dans des systèmes de culture tissulaire. L'invention concerne notamment une méthode de décontamination d'un liquide biologique tout en réduisant au minimum la dégradation de matières biologiques, telles que des protéines et des cellules. La méthode de l'invention consiste à mettre le liquide biologique en contact avec suffisament d'oxygène réactif pour désactiver des microorganismes pouvant être présents dans le liquide biologique par prise en compte d'une demande d'oxygène réactif du liquide biologique. La durée maximale ou la fenêtre de traitement effectif critique de contact de l'oxygène réactif avec le liquide biologique évite la dégradation de la matière biologique pouvant être présente dans le liquide biologique. La demande d'oxygène réactif du liquide biologique est fonction d'une demande d'oxygène réactif d'un vecteur liquide et d'une demande d'oxygène réactif de n'importe quelle matière biologique pouvant être présente dans le vecteur liquide. Plus la demande d'oxygène réactif du liquide biologique est grande plus le transfert d'oxygène réactif requis pour désactiver des microorganismes dans la fenêtre de traitement effectif est rapide. Dans des modes de réalisation spécifiques, l'invention prévoit la stérilisation de l'immunoglobuline et de diverses cellules sans les dégrader.
PCT/IB1997/001242 1996-10-09 1997-10-07 Procede de sterilisation de matieres biologiques WO1998015297A1 (fr)

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US6517855B2 (en) 1999-06-01 2003-02-11 The Regents Of The University Of California Method of sterilizing
WO2003031552A1 (fr) * 2001-10-05 2003-04-17 Steris Inc. Decontamination de surfaces contaminees par un materiau infecte par les prions, au moyen de preparations a base d'agents oxydants
WO2003031551A1 (fr) * 2001-10-05 2003-04-17 Steris Inc. Decontamination de surfaces contaminees par une substance infectee par des prions au moyen d'agents oxydants gazeux
US6627426B2 (en) * 1997-02-14 2003-09-30 Invitrogen Corporation Methods for reducing adventitious agents and toxins and cell culture reagents produced thereby
US6719988B2 (en) 1997-02-21 2004-04-13 The Regents Of The University Of California Antiseptic compositions for inactivating prions
US6720355B2 (en) 1997-02-21 2004-04-13 The Regents Of The University Of California Sodium dodecyl sulfate compositions for inactivating prions
US7071152B2 (en) 2003-05-30 2006-07-04 Steris Inc. Cleaning and decontamination formula for surfaces contaminated with prion-infected material
US7129080B2 (en) 2001-10-05 2006-10-31 Steris Inc. Vitro model of priocidal activity
WO2008154628A2 (fr) * 2007-06-12 2008-12-18 Musculoskeletal Transplant Foundation Procédé pour la stérilisation d'un tissu mou acellulaire sous vide
US7572632B2 (en) 1997-02-14 2009-08-11 Life Technologies Corporation Dry powder cells and cell culture reagents and methods of production thereof
EP2172227A1 (fr) * 1998-06-30 2010-04-07 Life Technologies Corporation Procédés servant à reduire des agents adventicés ou des toxines dans un échantillon et réactifs de cultures cellulaires obtenus par ce procédé

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Cited By (20)

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US7572632B2 (en) 1997-02-14 2009-08-11 Life Technologies Corporation Dry powder cells and cell culture reagents and methods of production thereof
US6627426B2 (en) * 1997-02-14 2003-09-30 Invitrogen Corporation Methods for reducing adventitious agents and toxins and cell culture reagents produced thereby
US6719988B2 (en) 1997-02-21 2004-04-13 The Regents Of The University Of California Antiseptic compositions for inactivating prions
US6720355B2 (en) 1997-02-21 2004-04-13 The Regents Of The University Of California Sodium dodecyl sulfate compositions for inactivating prions
US7307103B2 (en) 1997-02-21 2007-12-11 The Regents Of The University Of California Sodium dodecyl sulfate compositions for inactivating prions
US7226609B2 (en) 1997-02-21 2007-06-05 The Regents Of The University Of California Sodium dodecyl sulfate compositions for inactivating prions
EP2172227A1 (fr) * 1998-06-30 2010-04-07 Life Technologies Corporation Procédés servant à reduire des agents adventicés ou des toxines dans un échantillon et réactifs de cultures cellulaires obtenus par ce procédé
US6419916B1 (en) 1999-06-01 2002-07-16 The Regents Of The University Of California Assay for compounds which affect conformationally altered proteins
US6517855B2 (en) 1999-06-01 2003-02-11 The Regents Of The University Of California Method of sterilizing
US7129080B2 (en) 2001-10-05 2006-10-31 Steris Inc. Vitro model of priocidal activity
US7001873B2 (en) 2001-10-05 2006-02-21 Steris Inc. Decontamination of surfaces contaminated with prion-infected material with oxidizing agent-based formulations
JP2005505359A (ja) * 2001-10-05 2005-02-24 ステリス インコーポレイテッド プリオンに感染した物質で汚染された表面のガス状の酸化剤を用いた除染
WO2003031551A1 (fr) * 2001-10-05 2003-04-17 Steris Inc. Decontamination de surfaces contaminees par une substance infectee par des prions au moyen d'agents oxydants gazeux
WO2003031552A1 (fr) * 2001-10-05 2003-04-17 Steris Inc. Decontamination de surfaces contaminees par un materiau infecte par les prions, au moyen de preparations a base d'agents oxydants
US7803315B2 (en) 2001-10-05 2010-09-28 American Sterilizer Company Decontamination of surfaces contaminated with prion-infected material with gaseous oxidizing agents
US7071152B2 (en) 2003-05-30 2006-07-04 Steris Inc. Cleaning and decontamination formula for surfaces contaminated with prion-infected material
US7217685B2 (en) 2003-05-30 2007-05-15 Steris Inc. Cleaning and decontamination formula for surfaces contaminated with prion-infected material
US7393818B2 (en) 2003-05-30 2008-07-01 Steris Inc. Cleaning and decontamination formula for surfaces contaminated with prion-infected material
WO2008154628A2 (fr) * 2007-06-12 2008-12-18 Musculoskeletal Transplant Foundation Procédé pour la stérilisation d'un tissu mou acellulaire sous vide
WO2008154628A3 (fr) * 2007-06-12 2009-02-12 Musculoskeletal Transplant Procédé pour la stérilisation d'un tissu mou acellulaire sous vide

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