The present invention relates to a novel method of decontamination and sterilisation of such as, biological material, and the sterilisation of devices supporting such organic material, for example, medical devices, such as, delivery devices and medical implants.

In addition, the invention relates to sterile organic materials and/or sterile medical devices produced by such methods.

Background of the Invention

Sterile medical devices and/or organic materials are generally obtained either by operating under aseptic conditions or by sterilisation, e.g. with a sterilising agent, such as, ethylene oxide.

However, operating under aseptic conditions is expensive. Furthermore, ethylene oxide is known to be toxic, for example, when inhaled, for example, recent studies of women exposed to ethylene oxide whilst working in commercial sterilization facilities have shown an increased incidence of breast cancer.

Therefore, there is an increasing desire to find alternative methods of sterilisation, e.g. of organic materials. There have been many investigations into sterilisation by irradiation, e.g. y irradiation, and this is considered to be increasingly desirable. Thus. for example, US Patent application No. 2006/0115376 describes irradiating the enzyme trypsin, with e.g. a dose of 45 kGy at a rate of 1.9 kGy/hr, optionally in the presence of a sodium ascorbate stabiliser. The method describes a trypsin recovery of 97%. However, all the measurements disclosed therein are of activity and none describe the retention of the structural integrity of the trypsin enzyme. It is clear to the person skilled in the art that with an organic material, such as an enzyme, it is possible to retain the activity of the enzyme even if the structural integrity is destroyed, since the active site of the enzyme usually comprises only a small part of the whole of the enzyme, whereas damage to the structure of the enzyme can occur at all parts of the enzyme.

International Patent application No. WO 03/020325 describes a method of irradiating plasma protein fractions, such as albumin, at a dose of 45 kGy and a rate of 1.9 kGy/hr at ambient temperature, with various additives present, such as brain homogenates, albumin, etc. The irradiation is carried out at varying levels of residual solvent content and in the presence or absence of volatile stabilizers. However, samples exhibited some breakdown of albumin (plasma protein fraction) upon irradiation to 45 kGy. The use of some volatile stabilisers, such as, acetone and ethanol is disclosed, but there is little quantitative data disclosed. Furthermore, whilst there is reference to "quantitative recovery", no limits are given.

International Patent application No. WO 02/103029 describes a method of irradiation of anti-insulin monoclonal immunoglobulin using 45 kGy of low dose gamma irradiation from a ⁶⁰ Co source, in the presence of a stabiliser. The disclosed method provides a recovery of activity of anti-insulin monoclonal immunoglobulin of 76-83% after irradiation. WO '029 and assays the immunoglobulins for activity assays and therefore has inherent experimental errors, probably not better than +/- 7%. Furthermore, there is no quantitative data disclosed about retention of structural activity of the immunoglobulin. However, none of the prior art methods are considered to be satisfactory since, inter alia, the methods do not provide a sterilised organic material of close to 100% recovery, i.e. the sterilisation is either not complete or the sterilisation process itself may give rise to residual impurities. In the latter case a less than 100% recovery gives rise to the possibility that a proportion of the organic material may degrade upon irradiation and the degraded organic material may be an undesirable impurity in the final organic material. It is known that a major problem that occurs during sterilisation of an organic material by irradiation is oxidative damage of the organic material itself whilst it is exposed to the radiation and this may lead to a less than 100% pure sterilised organic material. It will be understood by the person skilled in the art that with prior art decontamination or sterilisation methods when an organic material is sterilised activity of the organic material may be retained but structural integrity of the organic material is not. This is because free radical damage to the organic material may occur at sites in the organic material, e.g. a protein or enzyme, other than at the active site of the organic material.

The problem may be mitigated, although not removed, by irradiating the organic material under a nitrogen atmosphere which is reported as generally producing a organic material with a purity of about 70% w/w. Therefore, there remains a need for a suitable method of sterilising organic material which retains the structural integrity of the organic material and avoids degradation of the organic material and, if appropriate, retains the clinical activity of the organic material. As hereinbefore described, an organic material, such as an enzyme, may retain its activity even if the structural integrity is destroyed, since the active site of the enzyme usually comprises only a small part of the whole of the enzyme, whereas damage to the structure of the enzyme can occur at all parts of the enzyme. Therefore, there is especially a need for an irradiation method of decontamination or sterilisation that satisfies the aforementioned criteria.

Summary of the Invention

Generally, previous methods for reducing free radical damage to organic material, etc. under irradiation conditions have focussed on scavenging the oxidant present in the system. With most systems comprising a organic material in an aqueous medium, this usually means scavenging the hydroxy radical which is produced by the irradiation of an aqueous system. However, in such aqueous systems the presence of free radicals will also lead to the production of hydrated electrons, often in substantially equal yield, to the hydroxy radicals. Importantly, the hydrated electrons are not removed by conventional scavengers of oxidants, such as ascorbate. Furthermore, hydrated electrons are extremely powerful reducing agents and react rapidly with organic materials, such as, proteins and enzymes, generally causing damage, for example, by de-amination, etc. to the structural integrity of and consequently the biological activity of the organic material. The most likely reason why the methods of the prior art are unsatisfactory, i.e. do not achieve 100% protection of the integrity of the organic material is because they have ignored the effect of the hydrated electron species. This is especially the case with the prior art methods which are carried out in a nitrogen atmosphere since the presence of a nitrogen atmosphere will not have any deleterious effect on the presence of the hydrated electron species.

It is the discovery that the damage to the integrity of radiation decontaminated or sterilised organic materials is due to the presence of hydrated electrons that forms the basis of the present invention.

In addition, we have found that certain oxygen containing species, such as, air, oxygen itself and oxides of nitrogen, especially, nitrous oxide, can act as very good scavengers of hydrated electrons. Furthermore, hydrated electron scavengers, such as oxygen, air and nitrous oxide are able to convert the hydrated electrons into hydroxy radicals which can then be scavenged by conventionally known hydroxy radical scavengers, such as, ascorbate, etc. Therefore, by scavenging the hydrated electron, the present invention is capable of removing a major source of free radical damage. Nitrous oxide does also scavenge hydrogen atoms, a free radical produced by the irradiation of water whose yield relative to the electron and hydroxyl is much less (at about 10%) but it is also capable of damaging organic material, such as, proteins.

Thus, according to a first aspect of the invention we provide a novel method of decontamination or sterilisation of an organic material which comprises of a system comprising the organic material, wherein the system comprises a scavenger of hydrated electrons and a scavenger of hydroxy radicals.

It will be understood herein that a reference to the decontamination or sterilisation of organic material shall include the decontamination or sterilisation of devices supporting such a organic material, for example, medical devices, such as, delivery devices and medical implants.

Furthermore, we have also found that the oxidative damage may also be temperature dependent. Therefore, desirably the decontamination or sterilisation method of the present invention is conducted at low temperature.

Since, as hereinbefore described, the present invention relies upon the removal of hydrated electrons from the organic material system any conventionally known hydrated electron scavenger or combination of scavengers may be used in the method of the invention. Thus, for example, one or more amino acid species may act as an hydrated electron scavenger. Alternatively, oxygen is a particularly good scavenger of hydrated electrons and leads to the formation of, for example, the superoxide radical, a species which is largely unreactive with many organic materials, e.g. proteins. Furthermore, oxygen may also "fix" the damage that may be caused by hydroxyl radicals even in the presence of ascorbate. Some organic materials, such as, proteins can be sensitive to damage by hydroxyl radicals and hence use of a nitrogen atmosphere has often been preferred to that of oxygen or air. However, some organic materials, such as proteins and enzymes, e.g. R ase, are protected by oxveen relative to nitrogen. However, a soluble, e.g. water soluble, hydrated electron preferred and nitrous oxide is a particularly preferred hydrated electron scavenger since, inter alia, it can react with the hydrated electron species to form hydroxyl radicals and harmless nitrogen. The hydroxyl radicals can then be scavenged by, for example, ascorbate. In this way all the primary free radicals produced by water radiolysis are scavenged and not just hydroxyl radicals, as would be the case if only ascorbate was used as scavenger.

It will be understood by the person skilled in the art that often when a organic material is sterilised activity of the organic material may be retained but structural integrity of the organic material is not. This is because free radical damage to the organic material may occur at sites in the organic material, e.g. a protein or enzyme, other than at the active site of the organic material. A particular advantage of the present invention is that both structural integrity and activity of the organic material can be retained. A variety of hydroxy radical scavenger may be used. A particularly desirable hydroxy radical scavenger is an hydroxy radical scavenger ion, e.g. an antioxidant ion, such as, the ascorbate ion, e.g. in the form of an ascorbate salt, such as, sodium ascorbate. However, it will be understood that a variety of ascorbates, i.e. mineral ascorbates, may be used in the method of the invention. Examples, of such mineral ascorbates include, but shall not be limited to, mono-ascorbates of the Group I alkali metals, such as, sodium ascorbate or potassium ascorbate; di-ascorbates of the Group II alkaline earth metals, such as, calcium diascorbate.

The order of the steps in the method of the invention may vary. Thu« ^h " lwHm™ radical scavenger may be introduced to the system simultaneously, sequentially with the hydrated electron scavenger. Desirably the hydroxy radical scavenger is added simultaneously with the hydrated electron scavenger. In addition, the hydroxy radical scavenger and or hydrated electron scavenger may be introduced to a solution in which the organic material is already present.

The low temperature irradiation may be conducted at a temperature below ambient temperature (e.g. below room temperature), preferably the temperature is such that the organic material or medium is frozen prior to and, preferably, during the irradiation. Thus, the sterilisation may desirably be conducted with the organic material or medium at a temperature of about 0°C or below. Desirably, the irradiation is carried out a temperature below -50°C, for example from -50°C to -80°C, such as -78.5°C, e.g. dry ice (solid C0 ₂ ) temperature or at lower temperatures, such as liquid nitrogen temperature, i.e. -196°C. However, it will be understood by the person skilled in the art that combinations such as dry ice and one or more organic solvents, e.g. an alcohol, such as methanol, or a ketone, such as acetone, etc. may be used to cool the biological sample prior to and, preferably, during the irradiation in the method of the invention herein described. Desirably, the sample of the organic material is maintained at the low temperature as hereinbefore described, throughout the irradiation process.

Furthermore, it will be understood by the person skilled in the art that the organic material may be present or, the method of sterilisation may be conducted on a device, such as a container prior to the organic material being placed in the container. However, a preferred aspect of the invention comprises the sterilisatio" ^{nf » οηΙ,,,ίηη} containing a organic material such as a protein or an enzyme. Thus, the method of decontamination or sterilisation as hereinbefore described desirably provides the decontaminated or sterilised organic material such that no changes in either the structural integrity of the organic material or in the activity of the organic material can be detected within experimental error. Thus, the maximum loss of structural integrity and/or activity is no greater than 1%.

The sample of the organic material may be in a medium e.g. in the form of a composition, e.g. a mixture, solution, emulsion or suspension, etc. of the organic material with a suitable adjuvant, diluent or carrier. Alternatively, the organic material medium may be in the form of a coating of, for example, on a medical device as hereinbefore described.

The nature, the dose, etc. of the radiation source may vary and it will be understood by the person skilled in the art that the radiation should be in an amount sufficient to destroy any microbial contamination of the organic material and optionally any other materials present in the composition. Generally, the radiation will vary depending upon, inter alia, the radiation source, the nature of the organic material, etc. as will be understood by the person skilled in the art. Thus, the irradiation may desirably comprise gamma (γ) radiation in an amount of at least 25 Gy (2,500 rads) to which the organic material is exposed. Thus, for example, the amount of irradiation may be from 25 Gy (2,500 rads) to about 80 kGy (8,000,000 rads) or from 1 kGy (100,000 rads) to 60 kGy (600,000 rads) or from 10 kGy (1,000,000 rads) to 40 kGy (4,000,000 rads), e.g. 25 kGy (2,500,000 rads). Generally, the rate of irradiation will vary depending upon, inter alia, the radiation source, the nature of the organic material, typical contaminants, the medium in which the organic material is present, etc. as will be understood by the person skilled in the art. Thus, the rate of irradiation may desirably be at a rate of from about 1 kGy hr to about 5 kGy hr, e.g. about 1.9 kGy/hr. Lower dose rates of irradiation may also be suitable such that the radiation may be from a low dose rate source, e.g. dose rates that are typical of, for example, ⁶⁰ Co or ¹³⁷ Cs. Alternatively, the irradiation may be from an electron source, which may, for example, generate free radicals wherein the free radicals generated can react with each other. At higher dose rates, such as those obtained from electron accelerators, significant amounts of the primary free radicals products of water radiolysis may react with each other at the expense of reactions with the biological/ biochemical substrate.

In the method of the invention the medium of the organic material, e.g. the mixture, solution, emulsion or suspension, etc. of the organic material, may desirably be substantially saturated, e.g. a saturated solution.

More than one hydrated electron scavenger may be used. The hydrated electron scavenger may be in the form of a gas and such gasses may comprise, but shall not be limited to, oxygen, oxides of nitrogen, especially nitrous oxide, air or a mixture of nitrous oxide and air, e.g. Entonox®, or mixtures of other gases containing a proportion of an oxidising gas, or a mixture of nitrous oxide and oxygen. When the free radical scavenger gas is a mixture of nitrous oxide and air or nitrous oxide and oxygen, the ratio of nitrous oxide, oxygen or nitrous oxide: air may, be in the range of from 1.4 to 4: 1, preferably 1:1. When a soluble hydrated electron scavenger, as hereinbefore described, is used, the concentration of the hydrated electron scavenger will generally be lower than the concentration of the organic material in an, e.g. aqueous solution. Thus, the concentration of the hydrated electron scavenger, e.g. nitrous oxide, can be described as a percentage of the concentration of the organic material. The concentration of the hydrated electron scavenger may be 50% w/w of the concentration of the organic material, preferably 40% w/w, more preferably 30% w/w, more preferably 20% w/w and especially about 5 to 15% w/w, e.g. about 10% w/w.

The method of the invention may also include the use of one or more stabilisers. Although a variety of stabilisers may be used exemplary stabilisers include, but shall not be limited to, fat soluble antioxidants, such as one or more of the tocopherols; and free radical scavengers, such as an alcohol, e.g. ethanol.

In addition, the method of the invention may benefit from the removal of trace metals/ions and/or restricting the molecular weight of the organic material.

According to a yet further aspect of the present invention there is provided a decontaminated or sterilised organic material as hereinbefore described. There is especially provided an irradiation, e.g. γ irradiation, decontaminated or sterilised organic material which has retained about 100% of its structural integrity. According to this aspect of the invention there is especially provided a decontaminated or sterilised organic material prepared by a method as hereinbefore describe According to this aspect of the invention the organic material may comprise any of the organic materials hereinafter described, such as, a biological material, for example, a peptide, polypeptide, protein or enzyme. When the organic material according to this aspect of the invention comprises a polypeptide any conventionally known polypeptides may be used, however, a preferred group of polypeptides is selected from the group comprising eptifibatide, exenatide, atosiban or nesiritide.

It will be understood by a person skilled in the art it is within the scope of the present invention to include a decontaminated or sterilised medical device, such as an implant or syringe, e.g. coated or impregnated, etc with a organic material. Indeed, it is an especially advantageous aspect of the invention that it offers a novel method of decontaminating or sterilising a medical device and consequently a medical device sterilised according to the method of the invention as hereinbefore described. By the term organic material we mean a variety of species, including, but not limited to, biological material such as proteins, enzymes, peptides, or other macromolecules, such as RNA, DNA, etc. The invention particularly relates to the sterilisation of organic materials which are known to be degraded by conventional sterilisation techniques.

As used herein, the term "organic material" is intended to mean, inter alia, a biological material, e.g. any substance derived or obtained from a living organism. Illustrative examples of organic materials include, but are not limited to, the following: cells; tissues; blood or blood components; proteins, including recombinant and transgenic proteins, and proteinaceous materials; enzymes, incli enzymes, such as trypsin, chymotrypsin, alpha-glucosidase and iduronodate-2- sulfatase, immunoglobulins, including mono and polyimmunoglobulins; botanicals; food; and the like. Preferred examples of organic materials include, but are not limited to, the following: ligaments; tendons; nerves; bone, including demineralised bone matrix, grafts, joints, femurs, femoral heads, etc.; teeth; skin grafts; bone marrow, including bone marrow cell suspensions, whole or processed; heart valves; cartilage; corneas; arteries and veins; organs, including organs for transplantation, such as hearts, livers, lungs, kidneys, intestines, pancreas, limbs and digits; lipids; carbohydrates; collagen, including native, afibrillar, atelomeric, soluble and insoluble, recombinant and transgenic, both native sequence and modified; enzymes; chitin and its derivatives, including NO-carboxy chitosan (NOCC); stem cells, islet of Langerhans cells and other cells for transplantation, including genetically altered cells, red blood cells; white blood cells, including monocytes; and platelets. Other illustrative examples of organic materials include, but are not limited to, the following.

• Aqueous-based formulations or aqueous based pharmaceutical drug-device and biologic products containing natural and synthetic, or their modified forms (such as cross-linked products or proteoglycans) of polysaccharides and glycosaminoglycans; oligosaccharides and fractionated or truncated forms of glycosaminoglycans; saccharides including the saccharide components of Glycosaminoglycans. Examples of glycosaminoglycans include hyaluronan, chondroitin sulphates, heparin, heparan sulphate, dermatan ^■ keratin sulphate. • Aqueous-based controlled drug delivery and controlled drug release systems, including those based on hydrogels, examples of the latter including: hydrogels based on agarose, methylcellulose, hyaluronan and other natural or modified polysaccharide and glycosaminoglycans; hydrogels based on synthetic polymers, examples of which include polyvinyl alcohol, polyacrylates and their co-polymers.

Hydrogels, including those based on both natural and synthetic polymers and their combination in products which have application in drug release systems, eye products, cosmetic products, wound-healing products, tissue engineering and in biosensors.

Aqueous-based therapeutic polypeptides and their combination in pharmaceutical products and formulations such as drug-device products and immunoregulatory polypeptides such as cytokines and growth factors.

Aqueous products containing pharmaceutical products such as drugs, biologies and in combinations with devices to produce drug-device combination products. The formulation or production of such products to include their combination with the above categories of aqueous-based materials and products.

As used herein, the term "sterilise" or "sterilisation" is intended to me"

in the level of at least one active or potentially active biological pathogen found in the organic material being treated according to the present invention.

As used herein, the term "biological contaminant or pathogen" is intended to mean a contaminant or pathogen that, upon direct or indirect contact with a organic material, may have a deleterious effect on a organic material or upon a recipient thereof. Such biological contaminants or pathogens include the various viruses, bacteria (including inter-and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multicellular parasites, and/or prions or similar agents responsible, alone or in combination, for TSEs known to those of skill in the art to generally be found in or infect organic materials. Examples of biological contaminants or pathogens include, but are not limited to, the following: viruses, such as human immunodeficiency viruses and other retroviruses, herpes viruses, filoviruses, circoviruses, paramyxoviruses, cytomegaloviruses, hepatitis viruses (including hepatitis A, B and C and variants thereof), pox viruses, toga viruses, Epstein- Barr viruses and parvoviruses; bacteria (including mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), such as Escherichia, Bacillus, Campylobacter, Streptococcus and Staphylococcus ; parasites, such as Trypanosoma and malarial parasites, including Plasmodium species; yeasts; molds; and prions, or similar agents, responsible alone or in combination for TSE (transmissible spongiform encephalopathies), such as scrapie, kuru, BSE (bovine spongiform encephalopathy), CJD (Creutzfeldt-Jakob disease), Gerstmann-Straeussler-Scheinkler syndrome, and fatal familial insomnia. As used herein, the term "active biological contaminant or pathogen" is intended to mean a biological contaminant or pathogen that is capable of causing a deleterious effect, either alone or in combination with another factor, such as a second biological contaminant or pathogen or a native protein (wild-type or mutant) or antibody, in the organic material and/or a recipient thereof.

As used herein, the term "biological contaminant or pathogen" is also intended to include biological degradation products which may be produced from the normal irradiation of the organic material of the invention.

As used herein, the term "blood components" is intended to mean one or more of the components that may be separated from whole blood and include, but are not limited to, the following, cellular blood components, such as red blood cells, white blood cells, and platelets; blood proteins, such as blood clotting factors, enzymes, albumin, plasminogen, fibrinogen, and immunoglobulins; and liquid blood components, such as plasma, plasma protein fraction (PPF), cryoprecipitate, plasma fractions, and plasma- containing compositions.

As used herein, the term "cellular blood component" is intended to mean one or more of the components of whole blood that comprises cells, such as red blood cells, white blood cells, stem cells, and platelets.

As used herein, the term "blood protein" is intended to mean one or more of the proteins that are normally found in whole blood. Illustrative examples of blood proteins found in mammals, including humans, include, but are not following: coagulation proteins, both vitamin K-dependent, such as Factor VII and Factor IX, and non-vitamin K-dependent, such as Factor VIII and von Willebrands factor; albumin; lipoproteins, including high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL); complement proteins; globulins, such as immunoglobulins IgA, IgM, IgG and IgE; and the like. A preferred group of blood proteins includes Factor I (fibrinogen), Factor II (prothrombin), Factor III (tissue factor), Factor V (proaccelerin), Factor VI (accelerin), Factor VII (proconvertin, serum prothrombin conversion), Factor VIII (antihemophiliac factor A), Factor ΓΧ (antihemophiliac factor B), Factor X (Stuart-Prower factor), Factor XI (plasma thromboplastin antecedent), Factor XII (Hageman factor), Factor XIII (protransglutamidase), von Willebrands factor (vWF), Factor la, Factor Ha, Factor Ilia, Factor Va, Factor Via, Factor Vila, Factor Villa, Factor IXa, Factor Xa, Factor XIa, Factor Xlla, and Factor Xllla. Another group of blood proteins includes proteins found inside red blood cells, such as haemoglobin and various growth factors, and derivatives of these proteins.

As used herein, the term "liquid blood component" is intended to mean one or more of the fluid, non-cellular components of whole blood, such as plasma (the fluid, non- cellular portion of the whole blood of humans or animals as found prior to coagulation) and serum (the fluid, non-cellular portion of the whole blood of humans or animals as found after coagulation).

The term 'polypeptide', 'protein', 'peptide' refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does exclude post-expression modifications of the polypeptide although chemical or post- expression modifications of these polypeptides may be included or excluded as specific embodiments. Therefore, for example, modifications to polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Further, polypeptides with these modifications may be specified as individual species to be included or excluded from the present invention. In one embodiment, the molecule is a polypeptide or their related analogues or derivatives thereof. Preferably, the polypeptide is a cyclic peptide. According to another preferred embodiment, the polypeptide is a non-cyclic peptide. In still another preferred embodiment, the polypeptide is selected from the group comprising eptifibatide, exenatide, atosiban or nesiritide.

Thus, according to a further aspect of the invention we provide a method of decontamination or sterilisation of a medical device, e.g. a coated medical device, as hereinbefore described, that is a medical device, such as an implant or syringe coated or impregnated, etc with a organic material.

The term organic material is intended to include functioning biochemical entities and biologically active molecules. More particularly, the invention relates to inactivation of potential biological contaminants e.g., viruses, bacteria, yeasts, molds, mycoplasmas and parasites, of compositions including antibodies, peripheral blood cells e.g. red blood cells and platelets, plasma protein fractions, e.g. albumin and clotting factors, collected from whole blood, e.g. the blood of virally infected persons, body fluids, including, but not limited to, urine, spinal fluids, amni( synovial fluids, ex vivo media used in the preparation of anti-viral vaccines, and cell culture media, e.g., foetal bovine serum and bovine serum, or products derived from such compositions, and solutions of sugars, amino acids, peptides, and lipids for intravenous nutrition. The present invention is further directed to blood based proteins and biologically derived proteins, including, but not limited to monoclonal antibodies, botulinum toxin and plant derived proteins, haemoglobin, within and independent of red cells, antibodies and vaccines.

Desirably the radiation treatment as hereinbefore described inactivates contaminants while rendering the organic material mostly unchanged, in both structure and biological function.

Such methods allow for the preservation of a organic material without the need for refrigeration or other treatment that would result in significant additional expense. In addition, the present invention teaches the sterilization and stabilization of biological proteins, such as sterilization of antibodies and other chemical components of the blood, so that the biological proteins may be stored safely at room temperature and subsequently used with greatly reduced risk of bacterial or specific viral contamination.

Many functional organic materials may be made in accordance with the method of the present invention. Such sterilized products may be used in a method for prophylaxis or treatment of a condition or disease, such that the organic material may be stored at ambient temperature prior to administering an effective amount of the organic material to a patient. Similarly, sterilized and stabilized products irradiated organic materials, biochemical entities and biologically active molecules may be incorporated into diagnostic test methods and kits and for use as elements in industrial and chemical processes. Likewise, nutritional solutions containing sugars, amino acids, peptides and lipids may be sterilized and prepared for storage at room (ambient) temperature by this method. Proteins may generally have a molecular weight of 40,000 Daltons or less. Peptides include hormones, such as natriuretic peptides, such as B-type natriuretic peptides, such as nesiritide.

The method of the invention may particularly comprise the irradiating one or more functional organic materials and biologically active molecules so as to preserve its function and permit storing the resulting decontaminated or sterilised material. Functional organic materials suitable for decontamination or sterilisation with the method of the present invention include, but are not limited to, blood or a blood components, such as red blood cells, white blood cells, monocytes, platelets, clotting factors, immunoglobulins, mono and polyimmunoglobulins, animal tissue (including those of mammals and other animal phyla), such as cartilage, bone marrow (including bone marrow cell suspensions), whole or processed ligaments, tendons, nerves, bone (including demineralised bone matrix), grafts, joints, femurs, femoral heads, teeth, skin grafts, heart valves, corneas, arteries, veins, lipids, carbohydrates, collagen (including native, afibrillar, atelomeric, soluble, and insoluble, recombinant and transgenic, both native sequence and modified) and organs (including organs for transplantation, such as hearts, livers, lungs, kidneys, intestines, pancreas, limbs and digits). Furthermore, the method of decontamination or sterilisation of the present invention may be applied to non-cellular material, such as proteins, such as, recombinant and transgenic proteins, proteinaceous materials, amino a sugars, lipids, enzymes (including digestive enzymes such as trypsin, chymotrypsin, alpha-glucosidase and iduronodate-2- sulphatase), antigens, marrow, chitin and its derivatives. The decontamination or sterilisation method of the invention as hereinbefore described may also provide for decontamination of viral pathogens, for example, viral pathogens, such as, Porcine Parvovirus (PPV), Adenovirus, Infectious Bovine Rhinotracheitis (IBR) virus, and Bovine Viral Diarrhoea (BVD) Virus, and Simian Immunodeficiency Virus (SIV).

Referring to the examples that follow, the preferred embodiments are carried out using the methods described herein, or other methods, which are known in the art.

It is understood that the invention is not limited to the embodiments set forth herein for illustration, but embraces all such forms thereof as come within the scope of the above disclosure.

EXAMPLES

The protection of the enzyme RNAse against a 25 kGy sterilisation dose of ionising radiation.

Experimental approach Preparation of enzyme solutions

Aqueous solutions of up to 1 mg ml of RNAse A ( molecular weight - 13.7 kDa) and 0.1 M ascorbate in 0.01 M phosphate buffer at pH values in the range 7.0-7.5 were saturated at room temperature with one of the following gases : a) nitrous oxide

b) nitrous oxide/ oxygen mixture

c) air

(it) Delivery of a 25 kGy sterilisation dose

The sealed samples were then frozen using dry ice and irradiated at a sterilisation dose of 25 kGy using a ⁵⁰ Co source at 90 Gy/minute.

(Hi) Determination of structural integrity

Following irradiation, SDS PAGE , using a 12% gel and by staining with Coomassie Blue, was used to determine the structural integrity of the enzyme. Replicate samples on a single gel were used to make quantitative measurements of the amount of enzyme and also to monitor any changes in molecular weight using DNA markers. Gels were scanned and analysed as blots using Quantiscan software. (iv) Determination of enzyme activity

RNAse activities were measured as described by Sakakibrara et al., (J.Biochem,I12,325-330,1992). Briefly, the amount of acid soluble nucleotides produced by hydrolysis of yeast RNA was measured. The standard assay system was as follows: Following dialysis of the enzyme solutions to remove ascorbate, the reaction mixture consisted of yeast RNA (50 μΐ, 5 mg/ml), 150 μΐ 50 mM Tris-HCl (pH7.5) and 50 μΐ of enzyme solution. After incubation for 15 minutes at 37 C, the reaction was stopped by addition of 250 μΐ of ice-cold 12% perchloric acid containing 20 mM lanthanum nitrate. The solution was kept on ice for 15 minutes and then centrifuged at 12000 x g for 15 minutes. 0.2 ml of the supernatant solution was then taken and diluted with 0.8 ml of water. The absorbance of this solution at 260 nm was then measured. RNAse activity which gave an increase in absorbance of 1.0 per minute was defined as one unit.

Experimental data

(i) Structural integrity of RNAse solutions after irradiation at a 25 kGy sterilisation dose When air- equilibrated solutions of RNAse (1 mg/ml), without any ascorbate, were irradiated at a sterilisation dose of 25 kGy , SDS PAGE showed that there was complete degradation of the enzyme.

In contrast, solutions of RNAse (lmg/ml) containing 0.1 M ascorbate and saturated with either, air , nitrous oxide or a nitrous oxide/oxygen mixture and wl sealed and frozen at dry ice temperatures, showed no degradation of the enzyme , as determined by comparison of the intensities of the enzyme bands in the gels measured at 0 kGy and 25 kGy doses of ionising radiation. The following Table shows the means of the intensities (arbitrary units) of the bands together with the standard deviations for samples where seven replicates were used.

*mean of duplicate samples

The same experiments confirmed also that there was no radiation-induced dimer formation, known to be a major reaction product in solutions of RNAse irradiated at ambient temperatures in the absence of any radio-protective agents. The detection limits of these experiments indicate that any dimer formation would be less than 1% of the enzyme concentration.

(ii) Enzyme activity determination after irradiation at a 25 kGy sterilisation dose

For the same set of enzyme solutions in the Table above, enzyme activities were determined using the method described by Sakakibrara, 1992, and as set out above.. The Table below summarises the data from triplicate measurements. Conditions Units/ml @ 0 kGy Units/ml @ 25 kGy

Ascorbate; air 34.0 (s.d. =2.8) 37.3 (s.d.= 0.4)

Ascorbate; nitrous oxide 37.5 (s.d =0.2) 35.0 (s.d.=3.4)

Ascorbate; nitrous oxide/oxygen 34.0 (s.d. = 0.4) 33.8 (s.d.=1.0)

The data in the Table again confirm that, within the detection limits of the experiments, the enzyme solution retains full activity.

The aqueous solutions of the enzyme RNAse can be protected, under certain conditions, from the damaging effects of a sterilising dose (25 kGy) of ionising radiation such that no changes in either the structural integrity of the enzyme, as measured in SDS PAGE experiments, or in its activity can be detected within the experimental error. Thus, the maximum loss of structural integrity and activity is no greater than 6%.

There is a substantial reduction of structural integrity in the nitrogen/ ascorbate experiment relative to the nitrous oxide data- in nitrogen the electrons are free to react with the enzyme to cause damage.