WO2001087357A2

WO2001087357A2 - Gamma irradiation of protein-based pharmaceutical products

Info

Publication number: WO2001087357A2
Application number: PCT/US2001/015864
Authority: WO
Inventors: William Drohan; Shirley Miekka; Thomas Busby; Annemarie Ralston
Original assignee: The American National Red Cross
Priority date: 2000-05-17
Filing date: 2001-05-16
Publication date: 2001-11-22
Also published as: EP1286706A2; WO2001087357A3; AU2001263200A1

Abstract

Methods are disclosed for inactivation of microbial contaminants in proteinaceous materials while preserving the activity of the materials. The proteinaceous material is exposed to gamma radiation at a high dose rate (e.g., ⊃ 3 kiloGray/hour) while in a lyophilized state under conditions which preserve activity of the material. The material may be formulated to contain one or more stabilizers, and/or be lyophlized to a low moisture content (e.g., less than 1.5%) prior to exposure to the gamma radiation.

Description

GAMMA IRRADIATION OF PROTEIN-BASED PHARMACEUTICAL

PRODUCTS

TECHNICAL FIELD The invention relates to the field of inactivation of microbial contaminants using gamma irradiation, and more particularly to inactivation of microbial contaminants in lyophilized, protein-based pharmaceutical and biomedical device products.

BACKGROUND ART Biologically-derived products such as blood, blood products, and tissue extracts frequently find use in therapeutic products, diagnostic reagents, and in biological research.

However, products derived from living organisms can be contaminated with mycoplasmal, viral, bacterial, fungal, or other microbial contaminants. While the chances of infection by a pathogen contained in a biological product are generally low, a contaminated biological product can infect research or health workers by as a result of events such as aerosolization and accidental injections (e.g., "needle sticks").

One common class of biologically-derived materials used in therapeutic settings is blood and blood products. Whole and fractionated blood are used in a variety of clinical settings and, if contaminated, can pose a substantial risk of disease transmission, as a number of dangerous pathogens are known to be capable of being spread by transfer of blood and blood products (e.g., hepatitis and HIV).

Once a pathogen is identified, testing procedures can be created which identify the presence of the pathogen or a marker of the presence of the pathogen. If the pathogen is detected in a material, the material can be discarded, quarantined or further treated to inactivate the pathogen. Most commonly, tests used in screening biologically-derived materials use immunoassay technology. This technology relies on the presence of either antibodies to the pathogen or epitopes associated with the pathogen being present in the sample, although assays which detect genetic material from the pathogen (e.g., by detecting pathogen genetic material using polymerase chain reaction- or branched DNA-based assays) are coming into increasing use. However, such testing technology cannot detect unknown pathogens, and tests which rely on the presence of anti-pathogen antibodies are ineffective for a certain period after initial infection by the pathogen (which can be up to several months). Microbial contaminants can be grouped into two general classes: cellular (bacteria, mycoplasma, and protozoans) and acellular (prions, enveloped viruses, and nonenveloped viruses). Nonenveloped viruses, particularly small nonenveloped viruses (e.g., the Parvoviridae) are significantly more difficult to inactivate because they are resistant to detergent treatment (due to their lack of a lipid membrane) and their small size makes them relatively resistant to ionizing radiation. Because small nonenveloped viruses such as the Parvoviridae are difficult to inactivate, they are a commonly used model contaminant, particularly nonhuman species of Parvoviridae (e.g., porcine parvovirus or PPV).

Heat treatment has been used for inactivating microbial contaminants in albumin and other biologically-derived substances. Heat treatment is used in two basic forms: wet heat (a.k.a. pasteurization), in which a protein solution is heated in liquid form; and dry heat, in which a dry formulation (e.g., lyophilized material) is heated. Pasteurization has been successfully used on liquid formulations of albumin; the albumin solution is heated to at least 60° C for about ten hours. This has been found to be effective for inactivating microbial contaminants in albumin, although in many cases it may not destroy all known bacterial and viral forms. Dry heat processes typically require higher temperatures (e.g., about 80° C) and longer incubations (e.g., about 72 hours) than pasteurization. However, heat treatment techniques are not feasible for use on many proteins due to loss of protein activity upon prolonged exposure to heat. Solvent/detergent (S/D) treatment is commonly used for inactivation of enveloped viruses. An organic solvent (tri-N-butyl-phosphate, TNBP) and a nonionic detergent are added to the material, disrupting the lipid bilayer coat of enveloped viruses, rupturing the virus particles and rendering them non-infective. This method is simple and robust, but has no effect on nonenveloped viruses. Filtering is commonly used to remove bacterial contaminants from solutions.

However, due to the small size of viral particles and some mycoplasma, filters which are sufficient to remove viral and/or mycoplasma contaminants may also retain substantial amounts of proteins from the material.

U.S. Patent No. 4,620,908 discloses methods for inactivating microbial contaminants in proteinaceous materials using gamma irradiation. The patent teaches that proteinaceous material exposed to gamma irradiation must be at a reduced temperature during irradiation or "the material would be almost completely destroyed; that is the activity of the material would be rendered so low as to be virtually ineffective." However, the need for temperature reduction during irradiation renders the process substantially more complicated, as well as increasing the costs of the process.

Sterways Pioneer, Inc., has disclosed a process for terminal sterilization of blood products using gamma radiation (Reid, 1998, Biologicals 26 (2):125-130). The "Sterways Process" uses low dose rate gamma irradiation, where the dose rate is constant throughout the process. The requirement for low, constant rate irradiation is stated to minimize loss of biological activity in the product.

Miekka et al. (1998, Haemophilia 4:402-408) disclose pathogen inactivation in blood products using gamma irradiation. This reference discloses the use of low rates of irradiation (0.21-1.5 kGy/hr) for inactivation of bacterial and viral contaminants.

U.S. Patent No. 5,362,442 teaches methods for sterilizing biological materials having low solids contents. Material having a solids content of less than 20% is exposed to gamma irradiation at low rates of exposure (i.e., 0.5 to 3 kiloGray per hour). The patent teaches that delivery of gamma irradiation over an extended period of time (i.e., at a low dose rate) reduces damage to the biological materials.

There remains a need in the art for simple, commercially viable methods of inactivating pathogens in biological materials.

DISCLOSURE OF THE INVENTION The invention provides new methods of inactivation of microbial contaminants in proteinaceous materials, particularly proteinaceous materials derived from biological organisms. The materials are lyophilized, then exposed to a contaminant-inactivating dose of gamma irradiation under conditions which preserve biological activity of the materials. The lyophilized materials are exposed gamma radiation at high dose rates (i.e., greater than 3 kiloGray/hour). Conditions which preserve biological activity may be achieved by a number of routes, such as inclusion of one or more stabilizers in the lyophilized material and/or by lyophilizing the material to a low residual moisture content. Biological materials processed according to the methods of the invention retain biological activity and are suitable for their intended use following gamma irradiation. Accordingly, the invention provides methods for inactivation of microbial contaminants in a proteinaceous material, comprising exposing lyophilized proteinaceous material to an effective amount of gamma radiation at a dose rate of greater than 3 kiloGray per hour (kGy/hr) under conditions which preserve biological activity of the lyophilized proteinaceous material, thereby inactivating microbial contaminants in the lyophilized proteinaceous material.

The proteinaceous material may be any material containing protein(s), including protease inhibitors such as alpha- 1 proteinase inhibitor (API), or a blood clotting factor such as fibrinogen, Factor II, Factor VIII, Factor IX, Factor X, or Factor XIII.

In one aspect, the lyophilized proteinaceous material includes a first stabilizer, and may optionally contain additional (e.g., a second, third, etc. stabilizer). Useful stabilizers include antioxidants and free radical scavengers of Type I and/or Type II. Exemplary stabilizers include ascorbic acid, glutathione, mannitol, 6-hydroxy-2,5,7,8- tetramethylchroma-2-carboxylic acid, rutin, and salts thereof.

In another aspect the lyophilized proteinaceous material has a low residual moisture content, such as a residual moisture content of less than about 1.5%, or 1%. Low residual moisture content lyophilized proteinaceous materials may further comprise a stabilizer. Low residual moisture content may be achieved by, for example, elevated lyophilizer shelf temperatures during secondary drying (e.g., at least about 30° or 40° C), or by inclusion of a material which replaces bound water in the proteinaceous material.

The methods of invention may be utilized to inactivate any type of microbial contaminant, and preferably utilize a total gamma radiation dose sufficient to inactivate at least about 2, 3, or 4 log₁₀ of viral microbial contaminants. Exemplary total gamma radiation doses useful in the invention include about 18, 30, 40, 45, 50, and 60 kGy.

Contaminants inactivated by the present invention include cellular contaminants such as bacteria and protozoans and acellular contaminants such as enveloped viruses and nonenveloped viruses. Because the methods of the invention preserve the activity of the proteinaceous material (as measured after reconstitution), they are particularly advantageous for inactivation of difficult to inactivate contaminants such as small nonenveloped viruses (e.g., Parvoviridae).

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 represents clotting assay data for lyophilized fibrinogen irradiated in the presence of ascorbate. The data is plotted as clotting time (y axis) vs. radiation dose rate (x axis). The dashed line indicates the clotting time value for control (non-irradiated) samples. FIG. 2 summarizes blood loss data from a rat renal hemorrage model. "TFC" indicates data from non-irradiated fibrinogen (non-irradiated standard, in 0.15 M NaCl, 20 mM histidine, pH 7.4); "TFC/ASC" indicates data from non-irradiated fibrinogen in ascorbate (non-irradiated ascorbate control, in 0.15 M NaCl, 200 mM sodium ascorbate, 20 mM histidine, pH 7.4); "18kGy" indicates data from fibrinogen in ascorbate irradiated to a total dose of 18 kGy; "30kGy" indicates data from fibrinogen in ascorbate irradiated to a total dose of 30 kGy; and "45kGy" indicates data from fibrinogen in ascorbate irradiated to a total dose of 45 kGy. The horizontal bar in each column indicates the mean blood loss for each group. FIG. 3 summarizes survival data from a rat renal hemorrage model. Open triangles indicate data from untreated control; open circles indicate data from non-irradiated fibrinogen (non-irradiated standard, in 0.15 M NaCl, 20 mM histidine, pH 7.4); closed circles indicate data from non-irradiated fibrinogen in ascorbate (non-irradiated ascorbate control, in 0.15 M NaCl, 200 mM sodium ascorbate, 20 mM histidine, pH 7.4); open diamonds indicate data from fibrinogen in ascorbate irradiated to a total dose of 18 kGy; closed diamonds indicate data from fibrinogen in ascorbate irradiated to a total dose of 30 kGy; and open squares indicate data from fibrinogen in ascorbate irradiated to a total dose of 45 kGy.

BEST MODE FOR CARRYING OUT THE INVENTION

We have discovered new methods of pathogen inactivation of lyophilized proteinaceous materials. We have found, contrary to the expectations in the art, that microbial contaminants in proteinaceous materials can be inactivated using high dose rate gamma radiation without destroying the activity of the proteinaceous material. Lyophilized proteinaceous materials are exposed to gamma radiation at high dose rates (i.e., at least 3 kiloGray per hour) under conditions which preserve the functionality of the materials for their intended purpose.

Contaminant inactivation in accordance with the methods of the invention also allows the delivery of relatively high total doses of gamma radiation to lyophilized proteinaceous materials without destroying the functionality of the materials for their intended purpose. Accordingly, the methods of the invention are well suited to demanding contaminant inactivation requirements, such as the inactivation of small, nonenveloped viruses. We have found that inclusion of one or more stabilizers, particularly stabilizers which are free radical scavengers, protect the biological activity of proteinaceous materials during high dose rate gamma irradiation. We have also found that low residual moisture content in the lyophilized material protects biological activity during high dose rate irradiation.

Accordingly, the invention provides methods for inactivating microbial contaminants in a proteinaceous material by exposing lyophilized proteinaceous material to gamma radiation at a dose rate of greater than 3 kiloGray per hour (kGy/hr) under conditions which preserve the biological activity of the material. Conditions which preserve the biological activity of the proteinaceous material include the presence of one or more stabilizers. In certain embodiments, a single stabilizer is included with the lyophilized material. In other embodiments two or more stabilizers are included in the lyophilized material. The lyophilized material comprising stabilizer(s) is exposed to high dose rate gamma radiation, thereby inactivating microbial contaminants. Other conditions which preserve the biological activity of the proteinaceous material are low residual moisture content in the lyophilized material. The lyophilized material with low residual moisture content is exposed to high dose rate gamma radiation, thereby inactivating microbial contaminants.

The incorporation of stabilizers into the lyophilized material may be combined with low residual moisture content to preserve the biological activity of the proteinaceous material. The low residual moisture content lyophilized material including one or more stabilizers is exposed to high dose rate gamma radiation, thereby inactivating microbial contaminants.

Definitions

The term "proteinaceous material," as used herein, refers to an acellular preparation comprising protein and/or peptides as a component. Preferably, a proteinaceous material comprises protein and/or peptides as the majority (by mass) of the organic, non-solvent components in material. Proteinaceous material may be derived from a biological organism or it may be a synthetic or recombinant functional equivalent thereof. Examples of biological materials include blood products such as albumin (Cohn Fraction V), immunoglobulins and hyperimmune globulins, purified blood clotting factors such as fibrinogen, thrombin, factors II, VII, Vila, VIII, IX, X, XI, and XIII, protease inhibitors such as alpha- 1-proteinase inhibitor and antithrombin III, enzymes (e.g., butyryl cholinesterase, Protein C, activated Protein C), purified growth factors, peptide hormones, tissue culture sera, vaccines, cell or tissue extracts, and the like. The term proteinaceous materials encompasses synthetic or recombinant functional equivalents of proteinaceous materials prepared from natural sources, such as recombinant versions of thrombin, fibrinogen, clotting factors, growth factors or antibodies.

The term "microbial contaminant", as used herein, refers to an undesirable cellular or acellular material which may be found in a proteinaceous material of interest. Cellular microbial contaminants include bacteria, fungi, molds and protozoans. Acellular microbial contaminants include viruses and prions. Viruses may be categorized according to a number of criteria, including genome (DNA or RNA; single or double stranded), presence or absence of a lipid envelope ("enveloped" and "nonenveloped" viruses, respectively), size, and family (e.g., Parvoviridae, Hepadnaviridae, etc.). A microbial contaminants is considered "inactivated" when proliferation of the contaminant cannot be detected in a standard assay for the contaminant. The identity and manner of conducting the assay will, of course, vary depending on the contaminant. Generally, such assays incubate a sample from the proteinaceous material (normally after reconstitution with a sterile liquid) under physical and nutritional conditions which would normally permit reproduction (e.g., in the presence of the appropriate nutrients, temperature, dissolved gases, necessary host cells and the like required by the particular contaminant).

A "stabilizer" is a compound which increases the resistance of a proteinaceous material to damage by gamma irradiation. Stabilizer activity may be measured by testing activity of a proteinaceous material exposed to an effective amount of gamma radiation in the presence or absence of the stabilizer. Stabilizers useful in the methods of the invention promote increased activity of the proteinaceous material after gamma irradiation as compared to proteinaceous material irradiated in the absence of a stabilizer.

A lyophilized material is considered to have a "low residual moisture content" if it has less than about 1.5% residual water. Residual water content may be measured using any method known in the art. It should be noted that, as is known in the art, residual moisture is calculated in relation to the lyophilized material, rather than to the original quantity of water in the solution which was lyophilized.

The term "effective amount of gamma radiation" refers to a dose of gamma irradiation effective to inactivate microbial contaminants in a material. An effective amount of gamma radiation is sufficient to reduce microbial burden in a biological material by a factor of at least about 10^'2 (e.g., at least 2 Iog₁₀ of inactivation), more preferably by a factor of at least about 10^"3 or 10^"4. A preferred effective amount of gamma radiation is a dose sufficient to provide at least about 3.5 log₁₀ of inactivation. The total dose of gamma radiation that is an effective amount will depend on the microbial contaminant that is the target of the inactivation process. Cellular microbial contaminants are sensitive to inactivation with gamma irradiation. Accordingly, an effective amount of gamma radiation to inactivate cellular microbial contaminant(s) is typically about 10-25 kGy. Acellular microbial contaminants such as viruses are typically more resistant to inactivation by gamma radiation. An effective amount of gamma radiation is normally at least about 15 kGy for most viruses (e.g., enveloped viruses), preferably about 20-35 kGy. For small nonenveloped viruses, such as the Parvoviridae, an effective amount of gamma radiation is normally about 35 kGy, typically about 40 to 50 kGy or 40 to 45 kGy total dose of gamma radiation. An "effective amount of a stabilizer" refers to an amount of a stabilizer which reduces gamma radiation damage to proteinaceous materials, such that the proteinaceous materials remain suitable for their intended purpose following the gamma radiation treatment. Preferably, an effective amount of a stabilizer is sufficient to reduce gamma radiation damage to proteinaceous materials by at least about 10% (i.e., the material irradiated in the presence of stabilizer is at least 10% more active than material irradiated under the same conditions but in the absence of stabilizer). Reduction in gamma radiation damage to proteinaceous materials is measured using an appropriate assay (e.g., solubility, binding activity, enzyme activity, and the like). When the composition comprises more than one stabilizer, the term "effective amount of a stabilizer" refers to an effective amount of the combined stabilizers ( . e. , the combined amount of stabilizers is effective to reduce gamma radiation damage by at least 10%).

The term "preserve biological activity" or "preserving biological activity" refers to maintaining the biological activity of a material such that the material continues to be suitable for its intended use. The term "suitable for its intended use", as used herein, means that the material has sufficient activity to be effective for its intended use. For example, a fibrinogen material is suitable for its intended use if it can be readily resolubilized to form a high concentration solution (e.g., a solution > 100 mg/mL in less than about 30 minutes) and it readily forms a clot upon addition of active thrombin (e.g., within about a three-fold range of the clotting time of a normal, non-irradiated control sample) under the appropriate conditions.

The term "activity", as applied to a proteinaceous material herein, refers to activity of the protein in its intended use. The "activity" of a proteinaceous material, refers to the property of the material which is necessary for the intended use of the material. For example, for a blood clotting factor which must have enzymatic activity (such as proteolytic activity) to be useful for its intended use, the enzymatic activity is the "activity" of the material, while solubility is considered the "activity" of a proteinaceous material such as serum albumin. The term "comprising" and its cognates, as used herein, is intended in its inclusive sense (i.e., synonymous with "including" and its cognates).

As used herein, the singular form "a", "an", and "the" includes plural references unless indicated otherwise.

Proteinaceous material is typically formulated with one or more pharmaceutically acceptable excipients, such as salts (e.g., NaCl), pH buffers (e.g., di- and mono-basic sodium phosphate or sodium citrate), bulking agents (e.g., dextrose, fructose, dextrans and the like), and the like, and may be diluted or concentrated to achieve a target concentration. Preferably, and particularly where the proteinaceous material is intended for parenteral administration, the proteinaceous material is formulated to be isotonic and at a physiologically acceptable pH upon reconstitution. As will be understood by one of skill in the art, the osmotic strength and pH of a formulation will depend on the intended route of administration and properties of the material. For example, proteinaceous materials intended for subcutaneous or intramuscular injection are typically formulated to be isotonic and near neutral pH (e.g., pH of about 6.8 to about 7.5), to avoid irritation at the site of injection. As is understood in the art, a broader range of tonicities and pH's are acceptable for materials intended for intravenous administration.

An effective amount of a stabilizer may also be included in the formulation. Acceptable stabilizers include anti-oxidants and free radical scavengers, and may be "small molecules" (non-peptide compounds typically less than about 2000 Daltons in molecular mass), peptides, enzymes or derivatives thereof.

Stabilizers for use in the invention include vitamins such as ascorbic acid, α- tocopherol, TROLOX™ (6-hydroxy-2,5,7,8-tetramethylchroma-2-carboxylic acid) and salts thereof; enzymes including catalase and superoxide dismutase; flavonoids such as amentoflavone, apigenin (4',5,7-trihydroxyflavone), catechin, epicatechin, gardenin D, hispidulin, 7,8-dihydroxy flavone, kaempferol, ketanserin, magnolol, myricetin, paroanthocyanidin, silymarin, pycnogenol, rutin, silybin and salts thereof; flavonoid phosphates including phosphoric acid half-esters of hydroxyfiavanes, e.g., 6-hydroxy-4'- methoxyflavane, 6-hydroxy-3,4'-dimethoxyflavane, 6-hydroxy-4'-methoxy-3- methylflavane, catechol ((+)-3,3',4',5,7-pentahydroxyflavane) and leucocianidol (S^'^^'jS -hexahydroxyflavane) and glycosides thereof, e.g., 2,3,3',4,4',5,7- heptahydroxyflavane glucoside and salts thereof; hydroxyflavanones, e.g., liquiritigenin (4',7-dihydroxyflavanone), pinocembrin (dihydrochrysin, 5,7-dihydroxyflavanone), naringenin (4^,,5,7-trihydroxyflavanone), eriodictyol (3^l,4',5,7-tetrahydroxyflavanone), dihydroquercetin (taxifolin, 3,3',4',5,7-pentahydroxyflavanone), 6-hydroxy-4'- methoxyflavanone, sacuranetin (4',5-dihydroxy-7-methoxy-flavanone), isosacuranetin (5,7- dihydroxy-4'-methoxy-flavanone), hesperetin (3',5,7-trihydroxy-4^,-methoxyflavanone) and silibinin (2- [trans-2-(4-hydroxy-3 -methoxyphenyl)-3 -hydroxymethyl- 1 ,4-benzodioxan-6- yl]-3,5,7-trihydroxychroman-4-one) and glycosides thereof, e.g., pinocembrin 7-rutinoside, sarothanoside (pinocembrin 7-neohesperidoside), salipurposide (naringenin 5 glucoside), prunin (naringenin 7-glucoside), narirutin (naringenin 7-rutinoside), naringin (naringenin 7- neohesperiodoside), eriodictin (eriodictyol 7-rhamnoside), eriocitrin (eriodictyol 7- rutinoside), eriodictyol 7-neohesperidoside, didymin (isosacuranetin 7-rutinoside), poncirin (isosacuranetin 7-neohesperidoside), persicoside (hesperitin glucoside), hesperidin

(hesperetin 7-rutinoside), and neohesperidin (hesperetin 7-neohesperidoside) and salts thereof; hydroxyflavones, e.g., chrysin (5, 7-dihydroxy flavone), primetin (5,8- dihydroxyflavone), galangin (3,5,7-trihydroxyflavone), baicalein (5,6,7-trihydroxyflavone), datiscetin (2',3,5,7-tetrahydroxyflavone), lotoflavin (2^,,4',5,7-tetrahydroxyflavone), caempferol (3,4',5,7-tetrahydroxyflavone), fisetin (3,3',4',7-tetiahydroxyflavone), luteolin

(3\4^5,7-tetrahydroxyflavone), scutellarein (4',5,6,7tetrahydroxyfiavone), morin (2^l,4,4^,,5,7-pentahydroxyflavone), robinetin (3,3',4'5',7-pentahydroxyflavone), quercetin (3,3',4',5,7-pentahydroxyflavone), tectochrysin (5-hydroxy-7-methoxyflavone), genkwanin (4',5-dihydroxy-7-methoxyflavone), acacetin (5,7-dihydroxy-4^,-methoxyflavone), diosmetin (3',5,7-trihydroxy-4^,-methoxyflavone), chrysoeriol (4',5,7-trihydroxy-3'- methoxyflavone), rhamnetin (3,3',4',5-tetrahydroxy-7-methoxyflavone), isorhamnetin (S^'^ -tetrahydroxy-S'-methoxyflavone), chloroflavonin (3^,-chloro-2^l,5-dihydroxy-3,7,8- trimethoxyflavone) and eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) and glycosides thereof, e.g., chrysin 7-rutinoside, chrysin 7-neohesperidoside, apiin (apigenin 7- apiosylglucoside), rhoifolin (apigenin 7-neohesperidoside), isorhoifolin (apigenin 7- rutinoside), nicotiflorin (caempferol 3-rutinoside), lespedin (caempferol 3,7-dirhamnoside), robinin (caempferol 3-robinoside 7-rhamnoside), scolymoside (lonicerin, luteolin 7- rutinoside), veronicastroside (luteolin 7-neohesperidoside), quercitrin (quercetin 3- rhamnoside), isoquercitrin (quercetin 3-glucoside), hyperoside (quercetin 3-galactoside), rutoside (rutin, quercetin 3-rutinoside), 6-hydroxymethylrutoside, monoxerutin [7-(2- hydroxyethyl)-rutoside], ethoxazorutoside [4'-O-(2-morpholinoethyl)-rutoside], troxerutin [3',4',7-tris-(2-hydroxyethyl)-rutoside], acaciin (linarin, acacetin 7-rutinoside), fortunellin (acacetin 7-neohesperidoside), diosmin (diosmetin 7-rutinoside), neodiosmin (diosmetin 7- neohesperidoside) and narcissin (isorhamnetin 3-rutinoside) and salts thereof; hydroxyflavylium salts, e.g., cyanidin and glycosides thereof, e.g., keracyanin (cyanidin 3- rutinoside) and salts thereof; fatty acids such as linoleic acid, oleic acid, palmitic acid, and furan fatty acid and salts thereof; as well as other compounds including probucol (C₃₁H₄₈O S₂), butylated hydroxytoluene, mannitol, tryptophan, DL-methionine, glutamic acid, N-acetylcysteine, 1,3 dimethyluric acid, cysteine, cysteamine, reduced or oxidized glutathione, uric acid, allopurinol; deferoxamine and salts thereof. Preferred stabilizers include ascorbic acid, glutathione, mannitol, TROLOX™, rutin and salts thereof. Where a stabilizer is a salt, the salt is preferably a pharmaceutically acceptable salt. Stabilizers which are free radical scavengers are preferably Type I and/or Type II free radical scavengers. Type I free radical scavengers neutralize free radicals formed as a result of the absoφtion of ionizing radiation by water molecules (i.e., hydrogen, H^", and hydroxyl, OH^", radicals). Type II free radical scavengers neutralize radicals formed as a result of the absoφtion of ionizing radiation by molecular oxygen (i.e., superoxide, 0 ^", and singlet oxygen, ¹0₂ ^", radicals). Many free radical scavengers, such as ascorbic acid and rutin, are both Type I and Type II scavengers.

An effective amount of a stabilizer is an amount of stabilizer that is effective to reduce gamma radiation damage by at least 10%, preferably at least 20, 30, 40 or 50%. The amount of a stabilizer which is an effective amount will vary according to the identity of the particular stabilizer, as will be apparent to one of skill in the art. The effective amount of a stabilizer may be easily determined by routine testing of a range of concentrations of the stabilizer. For example, 50-200 mM sodium ascorbate is generally considered an effective amount of this stabilizer, when ascorbate is used as the sole stabilizer, although reduced concentrations of ascorbate (e.g. about 25 to 100 mM) are considered effective when ascorbate is one component in a multi-stabilizer formulation.

Combinations of various stabilizers may also be used. Combinations may be desirable in that the required concentration of one stabilizer may be reduced by addition of second stabilizer. For example, we have found that 200 mM sodium ascorbate is effective in reducing gamma radiation damage of proteinaceous materials. However, 100 mM sodium ascorbate is just as effective if combined with 20 mM TROLOX™ or 0.5 mM rutin.

Contaminant inactivation in accordance with the methods of the invention is carried ^' out on lyophilized proteinaceous material. Lyophilization is well known in the art, and is accomplished by freezing a solution of the proteinaceous material, then removing the solvent (e.g., water) in a two stage process. The first stage (primary drying) sublimates moisture from the frozen material and is accomplished by subjecting the frozen material to reduced atmospheric pressure (e.g., less than about 150 milliTorr, but more preferably less than about 100 milliTorr) at low temperatures (e.g., temperatures sufficient to maintain the material in a frozen state). The second stage (secondary drying) is a desoφtion process where moisture is removed from the product at elevated temperatures at reduced atmospheric pressure. Due to the low atmospheric pressure within the lyophilization apparatus, convective heat transfer is poor, and any heating or cooling of the material is accomplished by conduction from the "shelf holding the containers of the material. Most commonly, the "shelves" of a lyophilizer contain channels through which a fluid is passed; heating or cooling of the fluid regulates the temperature of the shelf. Temperatures in a lyophilization apparatus are normally regulated according to "shelf temperature", which is the temperature of the shelf, rack or other means for holding the containers of the product to be lyophilized.

Materials for lyophilization are typically dispensed into unit dose or multi dose containers for lyophilization, although lyophilization may also be carried out in bulk using trays, pans and the like. When the proteinaceous material is to be incoφorated into a bandage, wound dressing or other device, the material may be incoφorated into or applied onto the device prior to lyophilization, and lyophilized in situ. Preferably, the solution comprising the proteinaceous material is dispensed into unit dose or multi dose vials for lyophilization. Freezing may be accomplished using any appropriate method known in the art. Typically, the lyophilization containers are temporarily covered, then placed in a freezing apparatus such as a chest freezer or a freezing bath. Alternately, the proteinaceous solution, dispensed into the lyophilization containers, may be placed into the lyophilization apparatus and frozen as a preliminary step in the lyophilization process.

Lyophilization is typically carried out using a temperature "profile" which dictates the shelf temperature of the lyophilization apparatus at various time points in the lyophilization process. Preferably, the primary drying stage of the lyophilization protocol utilizes one or more stages at sub-freezing temperatures (i.e., below 0° C), followed by secondary drying at a temperature above freezing, typically 20-40° C, to dry the material to the desired residual moisture content. Generally, increased shelf temperatures during the final stage of lyophilization result in decreased residual moisture contents.

When the proteinaceous materials are lyophilized in unit dose or multi dose vials, the vials are preferably sealed ("stoppered" when a resilient vial stopper is used as a closure) while under reduced atmospheric pressure. The vials may be additionally or alternately flushed with a relatively non-reactive gas (e.g., nitrogen) prior to sealing. Lyophilization equipment which can carry out automated flushing and/or sealing operations following completion of the lyophilization cycle are commonly available.

Lyophilized materials typically have a residual moisture content of about 1.5-3%. In certain embodiments of the invention, the proteinaceous material is lyophilized to a low residual moisture content. A low residual moisture content is less than about 1.5% residual moisture, but may also be less than about 1.25%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, or 0.6% and greater than about 0.1%, 0.2%, 0.3%, or 0.4% residual moisture, wherein the upper and lower limits are independently selected. Low residual moisture may be accomplished by a variety of methods known in the art. Most commonly, low residual moisture content is achieved by adjusting the lyophilization process to result in a low residual moisture content in the lyophilized product, preferably by increasing the shelf temperature during the secondary drying phase of lyophilization. Secondary drying is preferably carried out at a shelf temperature greater than 30° C, more preferably about 35° to about 50° C, about 35° to about 45° C, or about

40° C. It is preferred that the lyophilized proteinaceous material be sealed in its package (e.g., unit or multi-dose vial, or in a secondary package if the material is bulk lyophilized) as soon as possible upon completion of the secondary drying phase of the lyophilization process, to prevent readsoφtion of water into the lyophilized proteinaceous material.

One preferred lyophilization protocol for producing lyophilized materials with low residual moisture content utilizes a 100 milliTorr vacuum with a primary drying stage of 24 ^• hours at -30° C, followed by secondary drying for 24 hours at -10° C and 18 hours at +40° C.

Reducing residual moisture becomes increasingly difficult below about 1% residual moisture because much of the residual water is "bound water", or water which is involved in hydrogen bonding with the proteinaceous material. Accordingly, low residual moisture content in the proteinaceous material may be achieved by formulating the proteinaceous material with a compound which replaces bound water, such as trehalose.

Residual moisture in the lyophilized proteinaceous material may be tested by any method known in the art. Residual moisture may be measured by, for example, weighing a sample of the material before and after exhaustive drying, or by using a method which measures water as a result of a chemical reaction (e.g. , a method utilizing the Karl Fisher reagent).

One preferred method of testing residual moisture utilizes the Karl Fisher (KF) reagent. This method involves the extraction of the moisture from the sample using an evaporator, carried to a titration chamber with a nitrogen gas stream, and titrated with KF reagent. During the titration of water several reactions occur which are summarized as: H₂0 + 1₂ + (RNH)SO₃CH₃ + 2RN ^• (RNH)SO₄CH₃ + 2 (R H) I. The amount of water in the sample is directly proportional to the amount of iodine consumed. Coulometric KF titration produces the iodine required for the KF reaction by the anodic oxidation of iodide, summarized as: 21^" + 2e^"T₂. The iodine, coulometrically generated at the anode, together with the solvent, form

KF reagent which reacts quantitatively with water. When all of the moisture has reacted, the voltage at the sensing electrode drops, the coulometer stops and the electrical charge integrated during the titration process is converted to micrograms of water. Percentage moisture is determined using the formula % Moisture = (mg water/mg sample) x 100. One particularly preferred method of determining residual moisture utilizes solutions and equipment from EM Science; AQUASTAR® Coulomat A anode solution (AX1697A; EM Science, an anode solution comprising chloroform, imidazole, sulfur . dioxide and iodine in methanol) and AQUASTAR® Coulomat C cathode solution (AX1697C; EM Science, a cathode solution comprising carbon tetrachloride, sulfur dioxide, and 2,2-iminodiethanol in methanol), an AQUASTAR® C3000 coulometric titrator, and a solid evaporator with nitrogen carrier stream. The sample is placed into the evaporator, which heats the sample in the presence of a nitrogen gas stream. The sample is heated to above the boiling point of water, typically about 120° to 150 ° C for most proteinaceous materials, and any evaporated water is carried to the titration chamber in the nitrogen gas stream. Water in the nitrogen gas stream is measured coulometrically by reaction with the Karl Fisher reagent (the anode solution), and integrated by the coulometric instrument to give total water evaporated from the sample. Lyophilized product having a low residual moisture content is exposed to an effective amount of gamma radiation. Typically, this is accomplished by exposing the lyophilized material to a ⁶⁰Co source. Gamma irradiation is carried out at a dose rate of at least 3 kGy/hr, but may also be at least 4, 5, 6, 7, or 8, kGy/hr. The maximum dose rate will vary depending on the gamma source, as will be apparent to one of skill in the art. When a ⁶⁰Co source is utilized, the maximum dose rate is up to about 17 kGy/hr. We have found no upper limit to the dose rate that may be used for inactivation of contaminants according to the invention.

Gamma irradiation may be carried out at any temperature, but preferably not higher than normal ambient temperature (e.g., not more than about 25° C, more preferably not more than about 20° C). The material may or may not be chilled, refrigerated or frozen during irradiation, according to the preference of those carrying out the method of the invention. We have found that the temperature of the material during irradiation does not affect contaminant inactivation or activity of proteinaceous material after irradiation when proteinaceous materials are treated in accordance with the invention. The proteinaceous material is exposed to the gamma radiation source for an appropriate time to deliver an effective amount of gamma radiation. The total dose that is an effective amount of gamma radiation depends on the type of contaminant that is the target of inactivation. Gamma radiation inactivates microbial contaminants by direct and indirect damage. Direct damage is caused when gamma radiation is directly absorbed by genetic material in a contaminant and typically takes the form of strand breakage, which can block subsequent genetic replication. The sensitivity of a microbial contaminant to direct damage from gamma radiation is related to the size of the genome of the contaminant, because a large genome is more likely to be "hit" by gamma rays than a small genome. Indirect damage is caused by free radicals, which are generated when gamma radiation energy is absorbed by compounds such as oxygen and water.

Where a terminal sterilization of the proteinaceous material is desired, an effective amount of gamma radiation is a total dose that inactivates cellular microbial contaminants, preferably at least about 10 kGy, more preferably ranging from a lower limit of about 10, 15, 18 or 20 kGy to an upper limit of about 20, 22, or 25 kGy, total dose, wherein the upper and lower limits are independently selected except that the lower limit is always less than the upper limit. When the microbial contaminant is an enveloped viral contaminant, an effective amount of gamma radiation is preferably at least 15 kGy, more preferably ranging from a lower limit of about 15, 18, 20, 22, or 25 kGy to an upper limit of about 25, 30 or 35 kGy, wherein the upper limit and lower limit are independently selected except that the lower limit is always less than the upper limit. When the microbial contaminant is an nonenveloped viral contaminant such as a parvovirus (e.g., B19 human parvovirus or PPV), an effective amount of gamma radiation is preferably at least 20 kGy, more preferably ranging from a lower limit of about 20, 25, 30, or 35 kGy to an upper limit of about 30, 35,

40, 45, 50, or 60 kGy, wherein the upper limit and lower limit are independently selected except that the lower limit is always less than the upper limit.

As will be apparent to one of skill in the art, the time required to deliver an effective dose of gamma radiation to the proteinaceous material will depend on the type of gamma radiation source and the rate at which the source is emitting gamma radiation. Gamma radiation sources which utilize radioactive elements as the gamma radiation source emit ever decreasing rates of gamma radiation over time, as a consequence of radioactive decay. Accordingly, the total time of exposure to the gamma radiation source is adjusted according to the emission rate of the source. Gamma radiation sources may be calibrated using any appropriate method known in the art, such as free air or thimble cavity ionization chambers or chemical dosimeters such as the Fricke dosimeter, which measures conversion of ferrous ion to ferric ion.

Inactivation of microbial contaminants may be assayed using any method known in the art. Commonly, samples of the material taken before and after gamma irradiation are assayed for presence of microbial contaminants. Such assays typically measure the ability of a microbial contaminant to reproduce, such as by placing samples in conditions under which a microbial contaminant would be expected to proliferate. The exact design of the assay will, of course, depend on the identity of the microbial contaminant of interest. Where the microbial contaminant is cellular, the samples are typically incubated under conditions appropriate for growth (e.g., permissive medium, temperature and the like) of the target contaminant. Acellular contaminants generally require the inclusion of a permissive host cell in which the contaminant can proliferate. Preferably, microbial contaminant inactivation is assessed using a validation approach, where samples are "spiked" with a known amount of a microbial contaminant (e.g., porcine parvovirus, a model for the human pathogenic contaminant B19 parvovirus), subjected to the inactivation methods of the invention, then assayed for the ability of a microbial contaminant to reproduce under the appropriate conditions. In the case of PPV, contaminant survival is preferably assayed by adding resolubilized samples to a culture of host cells (e.g., porcine Kidney-13 cells) and assaying for cytopathic effects. Viral titer in the sample may be calculated using any appropriate method in the art, such as the Spearman-Karber method.

Validation is a preferred method of assessing microbial inactivation, as testing for contaminant levels in every material subjected to the methods of the invention would be burdensome and inefficient.

Microbial inactivation in accordance with the invention does not render the proteinaceous material unsuitable for its intended use. Preferably, the proteinaceous material will maintain at least about 40% of its activity, more preferably at least about 50%, 60%, 70%, 80% or 90%. Activity is preferably measured compared to a proteinaceous material which has been lyophilized but not irradiated.

The exact measure of the "activity" of a proteinaceous material will depend on the identity of the proteinaceous material. As will be apparent to one of skill in the art, the manner of measuring the activity of a proteinaceous material will depend on the material, its intended use, and the function of the material in the intended use. Because proteinaceous materials generally must be in aqueous solution to be useful, solubility in aqueous solvents is one measure of activity. Other properties which can be used as generally applicable "activities" relate to chemical identity, such as size (determination of which measures breakdown and/or aggregate formation), charge (e.g., isoelectric point), and the like. Suitability as a substrate may be measured to find the activity for proproteins which are processed during or in preparation for their intended use, for example fibrinogen and plasminogen. Antigen binding is a preferred "activity" for antibodies and immunoglobulin preparations. For proteinaceous materials which require an enzymatic activity for their intended puφose, enzymatic activity is a preferred indicator of activity. The activity of clotting factors may be conveniently measured using an appropriate clotting assay.

EXAMPLES

Example 1: Validation of microbial contaminant inactivation

Proteinaceous material are 'spiked' with model contaminants representing enveloped (bovine viral diarrhea virus, BVDV) and nonenveloped (porcine parvovirus, PPV) viruses, irradiated, and tested for contaminants which survive irradiation. Cellular contaminants such as bacteria are more easily inactivated than viral contaminants, and so it is assumed that conditions sufficient to inactivate viral contaminants will also inactivate cellular contaminants

Lyophilized, partially purified fibrinogen is redissolved in water or water containing stabilizer(s). The dissolved fibrinogen is dispensed into aliquots, each of which is spiked with a viral stock suspension of PPV or BVDV. The spiked material is lyophilized and divided into control (non-irradiated) and experimental (irradiated) groups.

Experimental group samples are exposed to gamma radiation at high dose rates (> 3 kGy/hr) to varying total doses. The samples are redissolved, diluted, and tested for BVDV and PPV titer using TCID50 assays.

BVDV titer is assayed using bovine turbinate cells (ATCC CRL-1390). The cells are grown to 90-100% confluence in MEM plus 10% horse serum and 1% glutamine, then infected by addition of diluted samples (fibrinogen and controls). Infections are performed with a series of five-fold dilutions in quadruplicate, and the cells are incubated at 37° C for 3-7 days. At the end of the incubation, cell layers are analyzed for cytopathic effect and viral titer is calculated by the Karber method (1931, Arch. Exp. Pathol. Pharmakol. 162:480-483).

PPV titer is assayed using porcine Kidney- 13 cells (ATCC CRC-6489). The cells are grown to 60-70% confluence in MEM plus 10% fetal bovine serum and 1% glutamine, then infected by addition of diluted samples. Before the samples containing PPV can be assayed, the fibrinogen and the cytotoxic ascorbate are removed. The samples are digested with trypsin (a 1 :200 w/w ratio of trypsin to fibrinogen) at 37°C for 30 minutes. They are then centrifuged in a Beckman TL-100 ultracentrifuge (TLS-55 Beckman rotor) for 20 minutes at 50,000 RPM (4°C). The supernatant is removed and the viral pellet is re- suspended in the original volume of mediainfections are performed with a series of fivefold dilutions in quadruplicate, and the cells are incubated at 37° C for 3-7 days. At the end of the incubation, cell layers are analyzed for cytopathic effect and viral titer is calculated by the Karber method (1931, Arch. Exp. Pathol. Pharmakol. 162:480-483).

Titers of control and irradiated samples are compared to determine the amount of inactivation at each total dose level.

Example 2: Inactivation of PPV Spiked, partially purified human plasma fibrinogen was used to test PPV inactivation at a range of temperatures and dose rates. Partially purified human plasma fibrinogen (approximately 85% fibrinogen, 10% albumin and 5% assorted proteins including fibronectin and Factor XIII) in 200 mM sodium ascorbate was spiked with viral stocks of PPV, then lyophilized. The lyophilized samples were then irradiated at 0.8, 2.4, 8.8 or 16 kGy/hr at ambient (25° C) or reduced (4°, -20°, or -78° C) temperature.

After irradiation, PPV inactivation was measured using a TCID50 assay as described in Example 1. Inactivation of PPV (log₁₀) was not significantly affected by temperature or dose rate, except that the samples irradiated at 16 kGy/hr showed significantly less inactivation than other dose rates. However, the reduced inactivation at this high dose rate may be due to inaccurate calibration of the gamma source used for this very high dose rate. Results are summarized in Table 1.

TABLE 1

Dose (kGy) Temp (° C) Rate (kGy/h) PPV Inactivation (lo^n)

0 25 na 0

40 25 2.4 4.15

40 4 2.4 3.98

40 -20 2.4 3.98

40 25 0.8 4.15

40 25 2.4 4.15

40 25 8.8 4.59

40 -78 16 2.93 Example 3: Irradiation of fibrinogen with ascorbate at high dose rates

Partially purified, freeze-dried, human plasma fibrinogen was used in this study. Each vial contains approximately 400 mg of total protein which is approximately 85% fibrinogen, 10% albumin and 5% assorted proteins including fibronectin and Factor XIII. The fibrinogen material was dissolved in 3.3 mL of 37° C water.

3.3 mL of 200 mM sodium ascorbate was added to those vials being tested with stabilizer. The vials were partially stoppered and frozen at -80° C for at least two hours.

All samples were freeze dried in a lyophilizer equilibrated to -30° C shelf temperature. The atmosphere was reduced to 100 milliTorr and the shelf temperature was held at -30° C for 24 hours, followed by 24 hours at -10° C, and 18 hours at +40° C. The vials were fully stoppered, then the vacuum was released. Testing of samples lyophilized under these conditions indicates that the lyophilized materials should contain approximately 0.8% residual moisture.

Samples were stored at 4° C until irradiated. Irradiation was performed using a ⁶⁰Co source at ambient temperature (approximately 20° C). A calculated dose of 40 kGy was delivered at dose rates of 0.8, 2.4, 8.8 or 16 kGy/hr.

Samples were redissolved in 3.3 mL of water. Activity of the fibrinogen material was measured in a clotting assay. The clotting assay was conducted by diluting resolubilized fibrinogen 200:1, then mixing with 0.5 volumes of human thrombin (25 U/mL in 25 mM CaCl₂). Clot formation is measured optically. The assays were conducted using an Electra 900 coagulation assay instrument (MLA, Pleasantville, NY), which automatically mixes the sample and thrombin, then measures clotting time.

Samples irradiated at 0.8, 2.4, and 8.8 kGy/hr showed approximately equal increases in clotting time (to about 5.5. seconds, as compared to about 3.5 seconds for non- irradiated) indicating reduced activity. However, the samples were well within the acceptable range for fibrinogen material. The samples irradiated at 16 kGy/hr showed clotting times near those of the non-irradiated control. This may be due to an incorrect dose rating for the gamma source used for the 16 kGy/hr samples which resulted in a total dose of less than 40 kGy. Results are summarized in FIG. 1.

Example 4: Irradiation of API at high dose rates

Alpha- 1-proteinase inhibitor (API) was formulated in 20 mM NaP0 , pH 6.7, and 0.1 M NaCl (Buffer A), 300 mM glycine, pH 6.4, 0.15 M NaCl (Buffer C), or 10 mM histidine, pH 6.8, 0.1 M NaCl (Buffer E), with or without 50 mM ascorbate. The protein samples were lyophilized, then irradiated at 6.8 kGy/hr to a total dose of 45 kGy.

No differences were observed between irradiated samples and unirradiated controls in any of the buffer formulations, or in formulations with or without buffers, suggesting that lyophilized API can be irradiated at high dose rates of gamma radiation in the absence of stabilizers without becoming unsuitable for use as a protease inhibitor.

Example 5: Irradiation of Factor VIII at high dose rates

Freeze-dried Factor VIII (Antihemophilic Factor, Human, Method M, Solvent and Detergent Treated, obtained from American Red Cross) was dissolved in water, then re- lyophilized with a final shelf temperature of 40° C. Controls were not dissolved. Residual moisture content was measured in each sample.

Factor VIII samples were irradiated at 8.8 kGy/hr to a total dose of 40 kGy. Samples were dissolved in water, then tested in a clotting assay. Dissolved Factor VIII was diluted to approximately 1 unit per milliliter (U/mL) with BAT buffer (0.05 M imidazole, 0.10 M NaCl, 0.1% (w/v) bovine serum albumin and 0.01% (v/v) Tween-20, pH 7.4), then 1:10, 1:20, 1:40 and 1:80 dilutions were prepared. Factor VIII activity was measured by one-stage APTT assay using Factor VIII deficient plasma in a MLA ELECTRA 900 clotting assay machine.

Results were calculated as percent activity compared to an unirradiated control. Material dissolved and redried at increased final shelf temperature (40° C), which normally results in reduced residual moisture, retained substantially more activity than irradiated control. Results are summarized in Table 2.

TABLE 2

Example 6: Preservation of Factor XIII activity in high rate irradiation

Factor XIII is an important component of fibrinogen preparations used in thrombin/fibrin sealants. Factor XIII does not contribute to clot formation, but rather stabilizes the clot by crosslinking fibrin within the clot. This activity is very important for effective hemostasis. It should be noted that some Factor XIII activity can be lost during contaminant inactivation without decreasing the effectiveness of the fibrinogen preparation, because the Factor XIII levels in fibrinogen preparations are proportionately greater than in blood, do to co-purification of the Factor XIII and fibrinogen.

Partially purified fibrinogen in freeze-dried form is utilized. The fibrinogen material is dissolved in 37° C water, reformulated to add stabilizer(s), then re-lyophilized. The lyophilized material is irradiated at high dose rate, then resolubilized once again.

Factor XIII activity is measured by a modification of the method of Lorand et al. (1971, Analytical Biochemistry 44:221-231). A fluorescent compound dansyl cadaverine (5-dimethylaminonaphthalene-l-(N-(5-aminopentyl))sulfonamide; Molecular Probes; Eugene, Oregon) is attached by Factor XIII through its free ε-amino group to the γ- carboxamide groups of glutamine residues of N,N-dimethylated α-casein. This attachment causes a shift and an increase in the intensity of the dansyl fluorescence when excited at 360 nm. As the reaction progresses, the change in fluorescence intensity at 500 nm is monitored. The increase is linear during the first 30 minutes of the reaction and the slope of the increase is proportional to the amount of Factor XIII in the test solution. A series of dilutions of each test sample and controls is assayed and their slopes are plotted against the dilution to determine the percent of residual Factor XIII activity.

Factor XIII activity is compared between non-irradiated control, irradiated control (no stabilizer) and irradiated samples with stabilizer(s).

Example 7: Irradiation of fibrinogen in multi-stabilizer formulations

Fibrinogen was reconstituted and reformulated by addition of 100 mM or 200 mM sodium ascorbate in combination with a second stabilizer. The resulting formulations were re-lyophilized and separated into two groups. One group was irradiated at 6.7 kGy/hr to a total dose of 45 kGy, and the other group was not irradiated (control).

Time to complete solubilization was measured, and resolubilized samples were also tested in a clotting assay as described in Example 2. All stabilizer formulations substantially improved the solubility and clotting activity of the fibrinogen samples as compared to lyophilized fibrinogen irradiated in the absence of any stabilizer. Results are summarized in Table 3.

TABLE 3

Example 8: In vivo testing of irradiated fibrinogen

Fibrin sealant preparations are a promising technology for prevention of blood loss due to trauma or surgery. The natural clotting process involves the action of thrombin on the protein fibrinogen, which is found in the blood. When tissue is injured, a cascade of biochemical events take place of which the end result is the action the proteolytic enzyme thrombin on fibrinogen. When this occurs, the fibrinogen polymerizes into an insoluble, sticky mesh called fibrin. This meshwork adheres to the tissues and entraps blood cells and platelets, producing a blood clot to prevent further blood loss and initiate the wound healing process. This natural clotting process can be exploited to stop bleeding due to trauma or surgery by applying concentrated preparations of thrombin and fibrinogen

(thrombin/fibrinogen sealant) to the site of bleeding.

In vivo testing in an animal model of parenchymal organ hemorrhage was used to assess the activity of fibrinogen preparations in thrombin/fibrinogen sealants. The in vivo test for the hemostatic effectiveness of the irradiated fibrinogen was performed in a challenging high-pressure hemorrhage model. This model was designed so rats receiving the untreated control fibrinogen will not survive 100% of the time. With the control samples being challenged this way, more subtle differences between the untreated and treated fibrinogen can be distinguished. To achieve these demanding conditions, the blood pressure of the test rats are maintained at a relatively high value and the rats received a high dose of the anticoagulant heparin. Rats were randomly assorted into groups of 8-12 rats per group.

The animals were anesthetized with urethane. Each animal was given the low end of the urethane dose initially, and an additional dose up to the high end of the range was delivered only as needed. The level of anesthesia was monitored by standard muscle reflexes and respiratory rate. The surgical site was shaved and cleaned with alcohol. A small lubricated thermoprobe was placed rectally to monitor the animal's core body temperature, which is supported by a heating pad, and a heating lamp coupled to a thermoregulator. Surgery was begun once the limb, tail, panniculus, and corneal reflexes were negative, and the respiratory rate was less than 100 per minute. The ventral neck region was incised longitudinally and the right carotid artery bluntly dissected and exteriorized. A small pressure-transducer catheter was threaded 2-3 cm proximally into the carotid artery and tied into place. The incision is closed or covered. The transducer was attached to a monitor that measures blood pressure (systolic, diastolic, and mean arterial pressures), and heart rate. Any blood loss during the catheterization procedure was documented. The animal was then allowed to stabilize for 5 minutes.

The kidney was exposed by a midline abdominal or a left flank incision. The rat was then allowed to stabilize for 5 minutes, as its body temperature was brought up to 39° - 40° C by use of the heating pad, and a heat lamp if necessary. At least five minutes after the laparotomy was complete, but not before the animal's core temperature reached 38° C, the animal was given an intravenous injection of 2000 units/Kg Heparin Sodium Injection (5,000 IU/ml, USP) in the dorsal tail vein. The injection site was immediately bandaged with tape and gauze to prevent bleeding. A partial nephrectomy was then performed. The re-dissolved fibrinogen (at approximately 120 mg/mL) was placed in one half of a double-barreled syringe. The other half of the syringe was filled with thrombin (300 units/mL). The two solutions were applied to the surgically created wound through a mixing tip on the syringe.

Time to hemostasis was recorded. Blood loss was measured by swabbing the surgical area with pre-weighed gauze pads and re-weighing the pads. Blood pressure and heart rate were monitored throughout the experiment. Survival times were recorded, up to a maximum of sixty minutes after the last application of pressure or product to the injury site, although survival is considered a less informative endpoint than blood loss. Any animals surviving at 60 minutes post-nephrectomy were euthanized. Analysis of blood loss indicates that fibrinogen, fibrinogen in ascorbate (200 mM sodium ascorbate), and fibrinogen in ascorbate irradiated at 8 kGy/hr to a total dose of 18, 30 or 45 kGy were approximately equivalent (p >0.05 by one-way ANOVA and Mann- Whitney two-tailed tests of fibrinogen control vs. material irradiated to 45 kGy as well as fibrinogen in ascorbate vs. material irradiated to 45 kGy), indicating that fibrinogen irradiated at a high dose rate in the presence of a stabilizer is suitable for its intended use as a hemostat/sealant. This data is summarized in FIG. 2. The survival curves (FIG. 3) showed a statistically significant increase in survival with all treatments.

Claims

CLAIMS We claim:

1. A method for inactivation of microbial contaminants in a proteinaceous material, comprising: exposing a lyophilized proteinaceous material to an effective amount of gamma radiation at a dose rate of greater than 3 kiloGray per hour (kGy/hr) under conditions which preserve biological activity of said lyophilized proteinaceous material, thereby inactivating microbial contaminants in said lyophilized proteinaceous material.

2. The method of claim 1 , wherein said lyophilized proteinaceous material retains at least 40% biological activity upon reconstitution.

3. The method of claim 2, wherein said lyophilized proteinaceous material retains at least 50% biological activity upon reconstitution.

4. The method of claim 3, wherein said lyophilized proteinaceous material retains at least 60% biological activity upon reconstitution.

5. The method of claim 2, wherein said lyophilized proteinaceous material further comprises a first stabilizer.

6. The method of claim 5, wherein said first stabilizer is an antioxidant.

7. The method of claim 5, wherein said first stabilizer is a free radical scavenger.

8. The method of claim 7, wherein said free radical scavenger is a type I free radical scavenger.

9. The method of claim 7, wherein said free radical scavenger is a type II free radical scavenger.

10. The method of claim 5, wherein said first stabilizer is selected from the group consisting of ascorbic acid, glutathione, mannitol, 6-hydroxy-2,5,7,8-tetramethylchroma-2- carboxylic acid, rutin, and salts thereof.

11. The method of claim 10, wherein said lyophilized proteinaceous material further comprises a second stabilizer.

12. The method of claim 11, wherein said second stabilizer is selected from the group consisting of ascorbic acid, glutathione, mannitol, 6-hydroxy-2,5,7,8- tetramethylchroma-2-carboxylic acid, rutin, and salts thereof

13. The method of claim 5, wherein said first stabilizer is ascorbic acid or a salt thereof.

14. The method of claim 13, wherein said lyophilized proteinaceous material further comprises a second stabilizer

15. The method of claim 14, wherein said second stabilizer is selected from the group consisting of glutathione, mannitol, 6-hydroxy-2,5,7,8-tetramethylchroma-2- carboxylic acid, rutin, and salts thereof.

16. The method of claim 5, wherein said lyophilized proteinaceous material has a moisture content of less than about 1.5%.

17. The method of claim 16, wherein said lyophilized proteinaceous material has a moisture content of less than about 1%.

18. The method of claim 2, wherein said lyophilized proteinaceous material has a moisture content of less than about 1.5%.

19. The method of claim 18, wherein said lyophilized proteinaceous material has a moisture content of less than about 1%.

20. The method of claim 2, wherein said lyophilized proteinaceous material is a lyophilized blood product.

21. The method of claim 20, wherein said lyophilized blood product is a clotting factor.

22. The method of claim 21, wherein said clotting factor is selected from the group consisting of fibrinogen, Factor II, Factor VIII, Factor IX, Factor X, and Factor XIII.

23. The method of claim 22, wherein said clotting factor is fibrinogen.

24. The method of claim 22, wherein said clotting factor is Factor VIII.

25. The method of claim 22, wherein said clotting factor is Factor XIII.

26. The method of claim 22, wherein said clotting factor is Factor IX.

27. The method of claim 22, wherein said clotting factor is Factor II.

28. The method of claim 22, wherein said clotting factor is Factor X.

29. The method of claim 2, wherein said proteinaceous material is alpha- 1- proteinase inhibitor.

30. The method of claim 2, wherein said dose rate is greater than about 4 kGy/hr.

31. The method of claim 2, wherein said proteinaceous material has been lyophilized at a final shelf temperature of at least about 30° C.

32. The method of claim 31, wherein said final shelf temperature is at least about 40° C.

33. The method of claim 2, wherein said effective amount of gamma radiation is sufficient to inactivate at least about 2 logs of viral microbial contaminants.

34. The method of claim 33, wherein said effective amount of gamma radiation is sufficient to inactivate at least about 3 logs of viral microbial contaminants.

35. The method of claim 34, wherein said effective amount of gamma radiation is sufficient to inactivate at least about 4 logs of viral microbial contaminants.

36. The method of claim 33, wherein said viral microbial contaminants are nonenveloped viral microbial contaminants.

37. The method of claim 36, wherein said nonenveloped viral microbial contaminants are members of the Parvoviridae.

38. The method of claim 2, wherein said effective amount of gamma radiation is at least about 18 kGy.

39. The method of claim 38, wherein said effective amount of gamma radiation is at least about 30 kGy.

40. The method of claim 39, wherein said effective amount of gamma radiation is at least about 40 kGy.

41. The method of claim 40, wherein said effective amount of gamma radiation is at least about 45 kGy.

42. A method for inactivation of microbial contaminants in a proteinaceous material, comprising: lyophilizing said proteinaceous material; and exposing a lyophilized proteinaceous material to an effective amount of gamma radiation at a dose rate of greater than 3 kiloGray per hour (kGy/hr) under conditions which preserve biological activity of said lyophilized proteinaceous material, thereby inactivating microbial contaminants in said lyophilized proteinaceous material.

43. The method of claim 42, wherein said lyophilized proteinaceous material retains at least 40% biological activity upon reconstitution.

44. The method of claim 43, wherein said lyophilizing comprises lyophilizing said proteinaceous material at a final shelf temperature of at least about 30° C.

45. The method of claim 43, wherein said final shelf temperature is at least about

40° C.