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WO1999051279A1 - A method for treating contaminated products and articles - Google Patents

A method for treating contaminated products and articles Download PDF

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Publication number
WO1999051279A1
WO1999051279A1 PCT/AU1999/000249 AU9900249W WO9951279A1 WO 1999051279 A1 WO1999051279 A1 WO 1999051279A1 AU 9900249 W AU9900249 W AU 9900249W WO 9951279 A1 WO9951279 A1 WO 9951279A1
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WO
WIPO (PCT)
Prior art keywords
denaturing agent
article
proteins
dna
gelatine
Prior art date
Application number
PCT/AU1999/000249
Other languages
French (fr)
Inventor
Hans Klieber
Original Assignee
Nokuta Pty. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nokuta Pty. Ltd. filed Critical Nokuta Pty. Ltd.
Priority to NZ507037A priority Critical patent/NZ507037A/en
Priority to AU31305/99A priority patent/AU746247B2/en
Priority to JP2000542049A priority patent/JP2002510657A/en
Priority to EP99913002A priority patent/EP1066062A4/en
Publication of WO1999051279A1 publication Critical patent/WO1999051279A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A23J3/06—Gelatine
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09H—PREPARATION OF GLUE OR GELATINE
    • C09H3/00—Isolation of glue or gelatine from raw materials, e.g. by extracting, by heating
    • C09H3/02—Purification of solutions of gelatine

Definitions

  • the present invention relates to a method for treating products and articles contaminated or potentially contaminated with contagious pathogenic agents.
  • surfaces of articles, such as surgical apparatus, which come into contact with a contagious pathogenic agent may be the source of infection to other animals/humans if they are not suitably decontaminated and sterilised.
  • CJD Creutzfeldt-Jakob disease
  • BSE Bovine Spongiform Encephalopathy
  • the temperatures routinely used to sterilise surgical instruments in hospitals may help to spread CJD. Further, increasing the temperatures of the autoclave used to disinfect instruments may actually make it harder to destroy the CJD prions.
  • Pathogens are disease causing parasites which include DNA and/or proteins in their structural make-up. They cause disease by invading and multiplying in the living tissue of a host cell. Contagious pathogenic agents such as very small viruses, plasmids or prions have been known to cause brain diseases such as. "Mad Cow” disease (BSE), Creutzfeldt-Jacob disease (CJD) or Scrapie. Prions have been defined as proteins which have become sticky clumps and cause neurological havoc and are thought to be the cause of the brain diseases. Recent research postulates that prions may play a role in reproduction complimentary or additional to genes.
  • Native globular proteins and solutions of double helical DNA undergo significant changes in a number of physical properties when subjected to: heat; extremes of pH; exposure to strong solutions of amides such as urea or urea derivatives such as guanidine hydrochloride; organic solvents; radiation; enzymes; and detergents. This physical change is called denaturation. Denaturation of proteins and DNA yields unfolded, random conformations of their corresponding polypeptide and DNA chains and results in the loss of the proteins biological activity.
  • bonds which are affected by the denaturation process include hydrogen bonds; hydrophobic bonds; salt bridges or ionic bonds between groups which are positively and negatively charged; and intramolecular bonds such as are found in cross linkages due to the disulfide bond groups of cystine.
  • Urea and guanidine are examples of denaturation agents. Although the mechanism of action of these denaturing agents is not fully understood, it is believed that they disrupt non-covalent interactions.
  • polypeptide chains devoid of cross-links usually assume a random-coil confirmation in 8 M urea or 6 molar guanidine hydrochloride, as evidenced by physical properties such as viscosity and optical rotary spectra. These compounds break hydrogen bonds in the protein, presumably by forming hydrogen bonds of its own due to its peptide like character.
  • Disulfides bonds can be cleaved reversibly by reduction with a reagent such as beta-mercaptoethanol.
  • a reagent such as beta-mercaptoethanol.
  • the protein ribomiclease cannot be readily reduced by beta-mercaptoethanol unless the protein is partially unfolded by denaturing agents such as urea or guanidine hydrochloride.
  • the contagious pathogenic agents causing brain diseases such as "Mad Cow” disease, Creutzfeldt-Jacob disease or Scrapie are not destroyed by commonly used methods such as heat or radiation treatment.
  • the present inventor has surprisingly found that this can be achieved by treating the animal derived product or the surface of an article with denaturing agents such as urea derivatives.
  • the present invention relates to a method for the denaturation of proteins and/or DNA present in gelatine or other polypeptide products derived from animals, comprising treating the gelatine or other polypeptide product with a denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the gelatine or other polypeptide product.
  • the present invention relates to a method of treating gelatine or other polypeptide products derived from animals potentially contaminated with contagious pathogenic agents, comprising treating the gelatine or other polypeptide products with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the gelatine or other polypeptide products.
  • the present invention relates to use of a denaturing agent for the denaturation of proteins and/or DNA present in gelatine or other polypeptide products derived from animals, wherein the gelatine or other polypeptide product is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the gelatine or other polypeptide product.
  • the present invention is directed to the use of a denaturing agent for treating gelatine or other polypeptide products derived from animals potentially contaminated with contagious pathogenic agents. wherein the gelatine or other polypeptide products are treated with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the gelatine or other polypeptide products.
  • the present invention relates to a denaturing agent when used to denature proteins and/or DNA present in gelatine or other polypeptide products derived from animals, wherein the gelatine or other polypeptide product is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the gelatine or other polypeptide product.
  • the other polypeptide products derived from animals may be animal extracts or meat for animal or human consumption.
  • the present invention relates to a method for the denaturation of proteins and/or DNA present on the surface of an article, comprising treating the surface of the article with a denaturing agent in an amount effective to denature the proteins and/or DNA of the contagious pathogenic agent without substantially affecting the surface of the article.
  • the present invention relates to a method of treating a surface of an article potentially contaminated with contagious pathogenic agents, comprising treating the surface of the article with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the surface of the article.
  • the present invention relates to use of a denaturing agent for the denaturation of proteins and/or DNA present on the surface of an article, wherein the surface of the article is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the surface of the article.
  • the present invention is directed to the use of a denaturing agent for treating a surface of an article potentially contaminated with contagious pathogenic agents, wherein the surface of the article is treated with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the surface of the article.
  • the present invention relates to a denaturing agent when used to denature proteins and/or DNA present on the surface of an article, wherein the surface of the article is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the surface of the article.
  • the surface of the article to be treated may be soft or hard.
  • Soft surfaces includes surfaces which are malleable and have little or no resistance to pressure or weight and includes natural and synthetic fabrics.
  • Hard surfaces include surfaces which are firm or rigid and include steel and steel alloys such as stainless steel.
  • the surface of the article to be treated is the stainless steel surface of a surgical apparatus.
  • the proteins and/or DNA to be denatured are contained in or derived from contagious pathogenic agents.
  • the contagious pathogenic agents may be very small viruses, plasmids or prions.
  • the properties of the animal derived products or the surface of the article which are contaminated with the contagious pathogenic agent are not substantially affected when treated with the denaturing agent, such that the denaturing agent reacts with the proteins and/or DNA of the contagious pathogen rather than the animal product or the surface of the article.
  • Varying factors such as the concentration of the denaturing agent and the time of reaction between the denaturing agent and the contaminated animal product or surface of article may be used to control the reaction between the denaturing agent and the animal derived product and surface of the article.
  • the denaturing agent is the urea analogue compound according to formula (I):
  • X is selected from nitrogen or oxygen
  • the salt derivative of Formula (I) is preferably selected from the chloride, nitrate or aluminium sulphate salt.
  • the denaturing agent is non-toxic and selected from the group consisting of urea, guanidine, L(+)-Arginine, creatine and creatinine or salts thereof. More preferably, the urea derivative is guanidine or guanidine hydrochloride.
  • the animal derived product or surface of the article contaminated (or possibly contaminated) with the contagious pathogenic agent is treated with a 0.1% w/v up to a saturated solution, preferably a 5 to 10% w/v solution, of the denaturing agent.
  • the solution may be based on organic or non-organic solvents which dissolve the denaturing agent.
  • the solvent is selected from an alcohol, such as ethanol, water or an aqueous alcoholic mix.
  • the animal derived product contaminated with a contagious pathogenic agent may be treated in a dry state, as a gel or in a liquid, such as water, which is preferably heated to a temperature of over 40°C.
  • a dry animal derived product or surface of the article contaminated with a contagious pathogenic agent is washed with an alcoholic solution of the denaturing agent such that the denaturing agent does not dissolve the animal derived product or the surface of the article but allows reaction of the guanidine with the contagious pathogenic agent.
  • the contagious pathogen may be in the form of CJD prions or BSE prions.
  • the denaturing agent is guanidine or its hydrochloride salt and the solvent is ethanol, water or a water-ethanol mix.
  • the solvent may be removed by usual methods such as reduced pressure evaporation or drying at approximately 50°C to 80°C.
  • the remaining animal derived product and the residual denaturing agent may be treated in any one of the following manners:
  • the gelatine may be further purified by extraction into water, washing with ethanol and drying:
  • the denaturing agent provided it is non-toxic, can be left in the animal derived product (guanidine and its salts, for example, are generally non-toxic);
  • the amount of denaturing agent contaminating the animal derived product can be reduced by drying the gelatine and further purifying it by usual methods such as centrifugation, spray drying, adsorption etc:
  • a solution of the animal derived product or dry gelatine still containing a denaturing agent such as guanidine and or its salts can be treated in an autoclave type container with heat under pressure (equivalent to 160°C at normal pressure) to convert guanidine into ammonia (which evaporates or can be evaporated) and melamine.
  • the melamine which is almost insoluble in water can be separated (eg by filtration or centrifugation), from a solution of the animal derived product in a non-organic solvent.
  • the treated animal derived product can be further purified by usual processes, such as extraction into water, washing with ethanol and drying.
  • the surface of articles contaminated or potentially contaminated with BSE prions may be treated by immersing the article in a 0.1% w/v up to a saturated solution, preferably a 5 to 10% w/v solution, of the denaturing agent in a solvent such as an aqueous or alcoholic solution.
  • a solvent such as an aqueous or alcoholic solution.
  • the article is immersed in 5 to 10% w/v guanidine or guanidine hydrochloride in either ethanol or water for one hour at approximately 40-50°C or in an autoclave for 10-20 minutes at 130-140°C. Any other sterilising processes can be additionally applied in the conventional manner.
  • the denaturing process renders the pathogen harmless.
  • the denaturing agent and the solvent may be removed by separating the treated material from the solution.
  • the treated material may then be washed with pure solvent such as water or alcohol followed by drying.
  • the utilised denaturing agent is guanidine or its salts and a method such as autoclaving has been used which brings the conditions to an equivalent of 160°C or more the guanidine is converted to melamine and ammonia. Both can be easily separated from the treated material by washing or a similar process. The denatured and rendered harmless pathogen no longer needs to be separated from the treated material.
  • Example 1 Treatment of Gelatine Gelatine is water soluble at > 40°C and is a mix of proteins derived by partial hydrolysis of animal collagen. According to the invention, gelatine contaminated with a contagious pathogenic agent, such as BSE prions, may be treated according to any one of the following methods:
  • a 10 micron to 200 micron layer of gelatine contaminated with BSE prions may be treated with 10% w/v guanidine hydrochloride in water.
  • the solvent from the mixture may then be removed under reduced pressure evaporation to leave the decontaminated gelatine and residual guanidine hydrochloride.
  • the gelatine may be further purified by extraction into water, washing with ethanol and drying.
  • a 300 micron to 1 mm layer of gelatine contaminated with BSE prions may be treated with 5% w/v guanidine in ethanol.
  • the solvent from the mixture may be removed by reduced pressure evaporation.
  • the gelatine residue may be further purified by extraction into water, washing with ethanol and drying.
  • the gelatine may be further purified by centrifugation to reduce the contamination by guanidine.
  • a solution of 20% w/v gelatine in water at > 40°C may be treated with 10% w/v guanidine hydrochloride in water.
  • the solvent from the mixture may be removed by reduced pressure evaporation or drying at 50°C to 80°C.
  • guanidine hydrochloride may be eliminated from the gelatine by utilising the different solubilities of the two substances.
  • the resulting gelatine residue may be further purified by washing with ethanol and drying; or
  • a gel of 30% w/v gelatine in water at room temperature may be treated with 15% w/v guanidine hydrochloride in water.
  • the gelatine solution or the dry gelatine still containing guanidine hydrochloride may be treated in an autoclave type container with heat under pressure (equivalent to 160°C at normal pressure) to convert guanidine into ammonia and melamine.
  • the ammonia may be evaporated.
  • the melamine may be separated by filtration from a solution of gelatine in a non-organic solvent.
  • the resulting gelatine residue may be further purified by washing with ethanol and drying.
  • Example 2 Treatment of other animal derived article
  • the treatment of other material (besides gelatine) derived from animal material contaminated with a contagious pathogenic agent, such as, animal extracts or meat can be carried out according to any one of methods 1 to 4 outlined in Example 1 although the chosen method may depend on the different solubilities and adsorption characteristics of the material to be treated.
  • Example 3 Treatment of surface of surgical instruments:
  • surfaces of articles contaminated with a contagious pathogenic agent such as BSE or CJD prions, may be treated according to either one of the following methods: 10
  • the surgical instruments were removed and rinsed in water. Additionally, in (2), the instruments may be further sterilised in the conventional manner, such as. in an autoclave.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
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  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
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Abstract

A method for the denaturation of proteins and/or DNA present in products derived from animals or on the surface of an article, comprising treating the animal derived product or the surface of the article with a denaturing agent in an amount effective to denature the protein and/or DNA.

Description

A Method For Treating Contaminated Products and Articles.
Technical Field
The present invention relates to a method for treating products and articles contaminated or potentially contaminated with contagious pathogenic agents. Background Art
There is a high level of awareness throughout the world that human illness and fatality can result from a variety of sources including the consumption of contaminated animal derived products wherein the animal has become the host for a contagious pathogenic agent and from infections obtained from articles contaminated with contagious pathogenic agents. When an animal infected with a contagious pathogenic agent is slaughtered the pathogenic agent is present in all the cellular body parts which are usually utilised for human use. The end products of the animal used foi¬ human consumption, such as meat and gelatine, therefore, can be potentially contaminated with contagious pathogenic agents.
Similarly, surfaces of articles, such as surgical apparatus, which come into contact with a contagious pathogenic agent may be the source of infection to other animals/humans if they are not suitably decontaminated and sterilised. The deadly brain disease Creutzfeldt-Jakob disease (CJD). the human form of Bovine Spongiform Encephalopathy (BSE), is an infection which is known to be present in both the central nervous system and throughout the lymph tissue of victims. At present, there is no effective way to sterilise surgical apparatus contaminated with prions that cause the disease CJD. There is concern that the temperatures routinely used to sterilise surgical instruments in hospitals may help to spread CJD. Further, increasing the temperatures of the autoclave used to disinfect instruments may actually make it harder to destroy the CJD prions.
Pathogens are disease causing parasites which include DNA and/or proteins in their structural make-up. They cause disease by invading and multiplying in the living tissue of a host cell. Contagious pathogenic agents such as very small viruses, plasmids or prions have been known to cause brain diseases such as. "Mad Cow" disease (BSE), Creutzfeldt-Jacob disease (CJD) or Scrapie. Prions have been defined as proteins which have become sticky clumps and cause neurological havoc and are thought to be the cause of the brain diseases. Recent research postulates that prions may play a role in reproduction complimentary or additional to genes.
Native globular proteins and solutions of double helical DNA undergo significant changes in a number of physical properties when subjected to: heat; extremes of pH; exposure to strong solutions of amides such as urea or urea derivatives such as guanidine hydrochloride; organic solvents; radiation; enzymes; and detergents. This physical change is called denaturation. Denaturation of proteins and DNA yields unfolded, random conformations of their corresponding polypeptide and DNA chains and results in the loss of the proteins biological activity.
The bonds which are affected by the denaturation process include hydrogen bonds; hydrophobic bonds; salt bridges or ionic bonds between groups which are positively and negatively charged; and intramolecular bonds such as are found in cross linkages due to the disulfide bond groups of cystine.
Urea and guanidine are examples of denaturation agents. Although the mechanism of action of these denaturing agents is not fully understood, it is believed that they disrupt non-covalent interactions. In proteins, polypeptide chains devoid of cross-links usually assume a random-coil confirmation in 8 M urea or 6 molar guanidine hydrochloride, as evidenced by physical properties such as viscosity and optical rotary spectra. These compounds break hydrogen bonds in the protein, presumably by forming hydrogen bonds of its own due to its peptide like character.
Disulfides bonds can be cleaved reversibly by reduction with a reagent such as beta-mercaptoethanol. However, the protein ribomiclease cannot be readily reduced by beta-mercaptoethanol unless the protein is partially unfolded by denaturing agents such as urea or guanidine hydrochloride.
The contagious pathogenic agents causing brain diseases such as "Mad Cow" disease, Creutzfeldt-Jacob disease or Scrapie are not destroyed by commonly used methods such as heat or radiation treatment.
There is clearly a need for a method of treating animal derived products such as gelatine or other polypeptide containing products derived from animals which are contaminated or potentially contaminated with infectious pathogenic agents. As the gelatine or other polypeptide product will ultimately be consumed by an animal or human, they are required in their purest uncontaminated state. There is also clearly a need for a method of treating surfaces of articles which are contaminated or potentially contaminated with infectious pathogenic agents.
The present inventor has surprisingly found that this can be achieved by treating the animal derived product or the surface of an article with denaturing agents such as urea derivatives.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Disclosure of the Invention
In a first aspect, the present invention relates to a method for the denaturation of proteins and/or DNA present in gelatine or other polypeptide products derived from animals, comprising treating the gelatine or other polypeptide product with a denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the gelatine or other polypeptide product.
In a second aspect, the present invention relates to a method of treating gelatine or other polypeptide products derived from animals potentially contaminated with contagious pathogenic agents, comprising treating the gelatine or other polypeptide products with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the gelatine or other polypeptide products.
In a third aspect, the present invention relates to use of a denaturing agent for the denaturation of proteins and/or DNA present in gelatine or other polypeptide products derived from animals, wherein the gelatine or other polypeptide product is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the gelatine or other polypeptide product.
In a fourth aspect, the present invention is directed to the use of a denaturing agent for treating gelatine or other polypeptide products derived from animals potentially contaminated with contagious pathogenic agents. wherein the gelatine or other polypeptide products are treated with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the gelatine or other polypeptide products.
In a fifth aspect, the present invention relates to a denaturing agent when used to denature proteins and/or DNA present in gelatine or other polypeptide products derived from animals, wherein the gelatine or other polypeptide product is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the gelatine or other polypeptide product.
The other polypeptide products derived from animals may be animal extracts or meat for animal or human consumption.
In a sixth aspect, the present invention relates to a method for the denaturation of proteins and/or DNA present on the surface of an article, comprising treating the surface of the article with a denaturing agent in an amount effective to denature the proteins and/or DNA of the contagious pathogenic agent without substantially affecting the surface of the article.
In a seventh aspect, the present invention relates to a method of treating a surface of an article potentially contaminated with contagious pathogenic agents, comprising treating the surface of the article with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the surface of the article.
In an eighth aspect, the present invention relates to use of a denaturing agent for the denaturation of proteins and/or DNA present on the surface of an article, wherein the surface of the article is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the surface of the article.
In a ninth aspect, the present invention is directed to the use of a denaturing agent for treating a surface of an article potentially contaminated with contagious pathogenic agents, wherein the surface of the article is treated with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the surface of the article.
In a tenth aspect, the present invention relates to a denaturing agent when used to denature proteins and/or DNA present on the surface of an article, wherein the surface of the article is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the surface of the article.
The surface of the article to be treated may be soft or hard. Soft surfaces includes surfaces which are malleable and have little or no resistance to pressure or weight and includes natural and synthetic fabrics.
Hard surfaces include surfaces which are firm or rigid and include steel and steel alloys such as stainless steel. Preferably, the surface of the article to be treated is the stainless steel surface of a surgical apparatus.
Generally, the proteins and/or DNA to be denatured are contained in or derived from contagious pathogenic agents. The contagious pathogenic agents may be very small viruses, plasmids or prions.
Preferably, the properties of the animal derived products or the surface of the article which are contaminated with the contagious pathogenic agent are not substantially affected when treated with the denaturing agent, such that the denaturing agent reacts with the proteins and/or DNA of the contagious pathogen rather than the animal product or the surface of the article. Varying factors such as the concentration of the denaturing agent and the time of reaction between the denaturing agent and the contaminated animal product or surface of article may be used to control the reaction between the denaturing agent and the animal derived product and surface of the article.
Preferably, the denaturing agent is the urea analogue compound according to formula (I):
Ri
R, -N.
'C-X— (R5)n (I)
.(CH^
R, •N '
R4 wherein,
X is selected from nitrogen or oxygen; 6
R5 is selected from the group including hydrogen or an alkyl group optionally substituted with one or more amine groups and wherein when X is oxygen, n = 0; m=0 to 1 Rl5 R2, R3 and R4 may each represent hydrogen, lower alkyl, carboxyl or an alkyl carboxyl group optionally substituted with one or more amine groups and wherein either R1? or R2 may be taken together with either R3 or R4 as -CO-CH2- to form a five-membered ring; or its salt derivative thereof. The salt derivative of Formula (I) is preferably selected from the chloride, nitrate or aluminium sulphate salt.
Preferably, the denaturing agent is non-toxic and selected from the group consisting of urea, guanidine, L(+)-Arginine, creatine and creatinine or salts thereof. More preferably, the urea derivative is guanidine or guanidine hydrochloride.
Preferably, the animal derived product or surface of the article contaminated (or possibly contaminated) with the contagious pathogenic agent is treated with a 0.1% w/v up to a saturated solution, preferably a 5 to 10% w/v solution, of the denaturing agent. The solution may be based on organic or non-organic solvents which dissolve the denaturing agent.
Preferably, the solvent is selected from an alcohol, such as ethanol, water or an aqueous alcoholic mix. The animal derived product contaminated with a contagious pathogenic agent may be treated in a dry state, as a gel or in a liquid, such as water, which is preferably heated to a temperature of over 40°C.
In one embodiment, a dry animal derived product or surface of the article contaminated with a contagious pathogenic agent is washed with an alcoholic solution of the denaturing agent such that the denaturing agent does not dissolve the animal derived product or the surface of the article but allows reaction of the guanidine with the contagious pathogenic agent. The contagious pathogen may be in the form of CJD prions or BSE prions. Preferably, the denaturing agent is guanidine or its hydrochloride salt and the solvent is ethanol, water or a water-ethanol mix.
Following, treatment of the animal derived product in dry or solution form with a solution of the denaturing agent, the solvent may be removed by usual methods such as reduced pressure evaporation or drying at approximately 50°C to 80°C. The remaining animal derived product and the residual denaturing agent may be treated in any one of the following manners:
(a) the gelatine may be further purified by extraction into water, washing with ethanol and drying:
(b) the denaturing agent, provided it is non-toxic, can be left in the animal derived product (guanidine and its salts, for example, are generally non-toxic);
(c) the amount of denaturing agent contaminating the animal derived product can be reduced by drying the gelatine and further purifying it by usual methods such as centrifugation, spray drying, adsorption etc:
(d) most of or all of the denaturing agent may be eliminated from the animal derived product by using additional method steps, such as, the utilisation of different solubilities or adsorption methods;
(e) a solution of the animal derived product or dry gelatine still containing a denaturing agent such as guanidine and or its salts can be treated in an autoclave type container with heat under pressure (equivalent to 160°C at normal pressure) to convert guanidine into ammonia (which evaporates or can be evaporated) and melamine. The melamine which is almost insoluble in water can be separated (eg by filtration or centrifugation), from a solution of the animal derived product in a non-organic solvent. The treated animal derived product can be further purified by usual processes, such as extraction into water, washing with ethanol and drying.
The surface of articles contaminated or potentially contaminated with BSE prions, such as the surface of stainless steel surgical instruments, may be treated by immersing the article in a 0.1% w/v up to a saturated solution, preferably a 5 to 10% w/v solution, of the denaturing agent in a solvent such as an aqueous or alcoholic solution. Preferably, the article is immersed in 5 to 10% w/v guanidine or guanidine hydrochloride in either ethanol or water for one hour at approximately 40-50°C or in an autoclave for 10-20 minutes at 130-140°C. Any other sterilising processes can be additionally applied in the conventional manner. The denaturing process renders the pathogen harmless. As indicated above, the denaturing agent and the solvent may be removed by separating the treated material from the solution. The treated material may then be washed with pure solvent such as water or alcohol followed by drying. If the utilised denaturing agent is guanidine or its salts and a method such as autoclaving has been used which brings the conditions to an equivalent of 160°C or more the guanidine is converted to melamine and ammonia. Both can be easily separated from the treated material by washing or a similar process. The denatured and rendered harmless pathogen no longer needs to be separated from the treated material.
In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples.
Modes for Carrying Out the Invention
Example 1: Treatment of Gelatine Gelatine is water soluble at > 40°C and is a mix of proteins derived by partial hydrolysis of animal collagen. According to the invention, gelatine contaminated with a contagious pathogenic agent, such as BSE prions, may be treated according to any one of the following methods:
(1) A 10 micron to 200 micron layer of gelatine contaminated with BSE prions may be treated with 10% w/v guanidine hydrochloride in water.
The solvent from the mixture may then be removed under reduced pressure evaporation to leave the decontaminated gelatine and residual guanidine hydrochloride. The gelatine may be further purified by extraction into water, washing with ethanol and drying.
(2) A 300 micron to 1 mm layer of gelatine contaminated with BSE prions may be treated with 5% w/v guanidine in ethanol.
The solvent from the mixture may be removed by reduced pressure evaporation. The gelatine residue may be further purified by extraction into water, washing with ethanol and drying.
The gelatine may be further purified by centrifugation to reduce the contamination by guanidine. (3) A solution of 20% w/v gelatine in water at > 40°C may be treated with 10% w/v guanidine hydrochloride in water.
The solvent from the mixture may be removed by reduced pressure evaporation or drying at 50°C to 80°C.
Most of or all of the guanidine hydrochloride may be eliminated from the gelatine by utilising the different solubilities of the two substances. The resulting gelatine residue may be further purified by washing with ethanol and drying; or
(4) A gel of 30% w/v gelatine in water at room temperature may be treated with 15% w/v guanidine hydrochloride in water.
The gelatine solution or the dry gelatine still containing guanidine hydrochloride may be treated in an autoclave type container with heat under pressure (equivalent to 160°C at normal pressure) to convert guanidine into ammonia and melamine. The ammonia may be evaporated. The melamine may be separated by filtration from a solution of gelatine in a non-organic solvent. The resulting gelatine residue may be further purified by washing with ethanol and drying.
Example 2: Treatment of other animal derived article The treatment of other material (besides gelatine) derived from animal material contaminated with a contagious pathogenic agent, such as, animal extracts or meat, can be carried out according to any one of methods 1 to 4 outlined in Example 1 although the chosen method may depend on the different solubilities and adsorption characteristics of the material to be treated.
Example 3: Treatment of surface of surgical instruments:
According to the invention, surfaces of articles contaminated with a contagious pathogenic agent, such as BSE or CJD prions, may be treated according to either one of the following methods: 10
(1) Stainless steel surgical instruments contaminated or potentially contaminated with BSE or CJD prions were immersed in 10% w/v guanidine in water for 10 hours at 50°C.
(2) Stainless steel surgical instruments contaminated or potentially contaminated with BSE or CJD prions were immersed and autoclaved in 5% w/v guanidine hydrochloride in ethanol for 20 minutes at 135°C.
In both (1) and (2), the surgical instruments were removed and rinsed in water. Additionally, in (2), the instruments may be further sterilised in the conventional manner, such as. in an autoclave.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

11CLAIMS
1. A method for the denaturation of proteins and/or DNA present in gelatine or other polypeptide products derived from animals, comprising treating the gelatine or other polypeptide products with a denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the gelatine or other polypeptide product.
2. A method according to claim 1 wherein the proteins and/or DNA to be denatured are contained in or derived from contagious pathogenic agents.
3. A method according to claim 2 wherein the contagious pathogenic agents are viruses, plasmids or prions.
4. A method for the denaturation of proteins and/or DNA present on the surface of an article, comprising treating the surface of the article with a denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the surface of the article.
5. A method according to claim 4 wherein the proteins and/or DNA to be denatured are contained in or derived from contagious pathogenic agents.
6. A method according to claim 5 wherein the contagious pathogenic agents are viruses, plasmids or prions.
7. A method of treating gelatine or other polypeptide products derived from animals potentially contaminated with contagious pathogenic agents. comprising treating the gelatine or other polypeptide products with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the gelatine or other polypeptide products.
8. A method of treating a surface of an article potentially contaminated with contagious pathogenic agents, comprising treating the surface of the article with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the properties of the surface of the article.
9. A method according to any one of claims 2, 3 or 7 wherein the contagious pathogenic agent is BSE prions.
10. A method according to any one of claims 5. 6 or 8 wherein the contagious pathogenic agent is selected from BSE or CJD prions.
11. A method according to any one of claims 4, 5, 6, 8 or 10 wherein the surface of the article is soft. 12
12. A method according to any one of claims 4, 5, 6. 8 or 10 wherein the surface of the article is hard.
13. A method according to any one of claims 4, 5, 6, 8 or 10 wherein the surface of the article is the surface of a stainless steel surgical apparatus.
14. A method according to any one of claims 1, 2, 3. 7 or 9 wherein the polypeptide products derived from animals are animal extracts or meat for human or animal consumption
15. A method according to any one of claims 1 to 14 wherein the denaturing agent is the urea analogue compound according to formula (I) :
Ri
R, -N.
C=X-(R5)n (I)
. (CH,),
Ra ΓÇóN-
R,
wherein,
X is selected from nitrogen or oxygen;
R5 is selected from the group including hydrogen or an alkyl group optionally substituted with one or more amine groups and wherein when X is oxygen, n = 0; m=0 to 1;
R R2, R3 and R4 may each represent hydrogen, lower alkyl. carboxyl or an alkyl carboxyl group optionally substituted with one or more amine groups and wherein either R,, or R2 may be taken together with either R3 or R4 as -CO-CH2- to form a five-membered ring: or a salt thereof.
16. A method according to claim 15 wherein the salt of the urea analogue compound is a chloride, nitrate or aluminium sulphate salt.
17. A method according to claim 15 wherein the urea derivative is selected from the group consisting of urea, guanidine. guanidine hydrochloride. 13
L(+)-Arginine, creatine and creatinine.
18. A method according to claim 15 wherein the denaturing agent is guanidine or guanidine hydrochloride.
19. A method according to claim 15 wherein the denaturing agent is in a solution containing from 0.1% w/v up to a saturated solution, preferably a 5 to 10% w/v solution, of denaturing agent.
20. A method according to claim 19 wherein the denaturing agent is in a solvent selected from an alcoholic solution, water or an aqueous alcoholic mix.
21. Use of a denaturing agent for the denaturation of proteins and/or DNA present in gelatine or other polypeptide products derived from animals, wherein the gelatine or other polypeptide product is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA of the contagious pathogenic agent without substantially affecting the gelatine or other polypeptide product.
22. Use according to claim 21 wherein the proteins and/or DNA to be denatured are contained in or derived from contagious pathogenic agents.
23. Use according to claim 22 wherein the contagious pathogenic agents are viruses, plasmids or prions.
24. Use of a denaturing agent for the denaturation of proteins and/or DNA present on the surface of an article, wherein the surface of the article is treated with the denaturing agent in an amount effective to denature proteins and/or DNA without substantially affecting the surface of the article.
25. Use according to claim 24 wherein the proteins and/or DNA to be denatured are contained in or derived from contagious pathogenic agents.
26. Use according to claim 25 wherein the contagious pathogenic agents are viruses, plasmids or prions.
27. Use of a denaturing agent for treating gelatine or other polypeptide products derived from animals potentially contaminated with contagious pathogenic agents, wherein the gelatine or other polypeptide products is treated with a denaturing agent in an amount effective to denature proteins and/or DNA of the contagious pathogenic agent without substantially affecting the gelatine or other polypeptide products.
28. Use of a denaturing agent for treating a surface of an article potentially contaminated with contagious pathogenic agents, wherein the surface of the article is treated with a denaturing agent in an amount effective to denature 14
proteins and/or DNA of the contagious pathogenic agent without substantially affecting the surface of the article.
29. Use according to any one of claims 22, 23 or 27 wherein the contagious pathogenic agent is BSE prions.
30. Use according to any one of claims 25. 26 or 28 wherein the contagious pathogenic agent is selected from BSE or CJD prions.
31. Use according to any one of claims 24, 25, 26, 28 or 30 wherein the surface of the article is soft.
32. Use according to any one of claims 24. 25. 26, 28 or 30 wherein the surface of the article is hard.
33. Use according to any one of claims 24, 25. 26. 28 or 30 wherein the surface of the article is the surface of a stainless steel surgical apparatus.
34. Use according to any one of claims 21, 22, 23, 27 or 29 wherein the polypeptide products derived from animals are animal extracts or meat for human or animal consumption
35. Use according to any one of claims 21 to 34 wherein the denaturing agent is the urea analogue compound according to formula (I):
Ri
R, -N.
C=X-(R5)11 (I)
,(CH2)r
R, -N-
R,
wherein, X is selected from nitrogen or oxygen;
R5 is selected from the group including hydrogen or an alkyl group optionally substituted with one or more amine groups and wherein when X is oxygen, n = 0; m=0 to 1: 15
R╬▒, R2. R3 and R4 may each represent hydrogen, lower alkyl, carboxyl or an alkyl carboxyl group optionally substituted with one or more amine groups and wherein either R,, or R2 may be taken together with either R3 or R4 as -CO-CH2- to form a five-membered ring; or a salt thereof.
36. Use according to claim 35 wherein the salt of the urea analogue compound is a chloride, nitrate or aluminium sulphate salt.
37. Use according to claim 35 wherein the urea derivative is selected from the group consisting of urea, guanidine, guanidine hydrochloride, L(+)-Arginine, creatine and creatinine.
38. Use according to claim 35 wherein the denaturing agent is guanidine or guanidine hydrochloride.
39. Use according to claim 35 wherein the denaturing agent is in solution containing from 0.1% w/v up to a saturated solution, preferably a 5 to 10% w/v solution, of denaturing agent.
40. Use according to claim 39 wherein the denaturing agent is in a solvent selected from an alcoholic solution, water or an aqueous alcoholic mix.
41. A denaturing agent when used to denature proteins and/or DNA present in gelatine or other polypeptide products derived from animals, wherein the gelatine or other polypeptide product is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the gelatine or other polypeptide product.
42. A denaturing agent according to claim 41 wherein the proteins and/or DNA to be denatured are contained in or derived from contagious pathogenic agents.
43. A denaturing agent according to claim 42 wherein the contagious pathogenic agents are viruses, plasmids or prions.
44. A denaturing agent when used to denature proteins and/or DNA present on the surface of an article, wherein the surface of the article is treated with the denaturing agent in an amount effective to denature the proteins and/or DNA without substantially affecting the surface of the article.
45. A denaturing agent according to claim 44 wherein the proteins and/or DNA to be denatured are contained in or derived from contagious pathogenic agents.
46. A denaturing agent according to claim 45 wherein the contagious pathogenic agents are viruses, plasmids or prions. 16
47. A denaturing agent according to any one of claims 42 or 43 wherein the contagious pathogenic agent BSE prions.
48. A denaturing agent according to any one of claims 45 or 46 wherein the contagious pathogenic agent is selected from BSE or CJD prions.
49. A denaturing agent according to any one of claims 44, 45, 46 or 48 wherein the surface of the article is soft.
50. A denaturing agent according to any one of claims 44, 45, 46 or 48 wherein the surface of the article is hard.
51. A denaturing agent according to any one of claims 44, 45, 46 or 48 wherein the surface of the article is the surface of a stainless steel surgical apparatus.
52. A denaturing agent according to any one of claims 41, 42, 43 or 47 wherein the polypeptide products derived from animals are animal extracts or meat for human or animal consumption
53. A denaturing agent according to any one of claims 41 to 52 wherein the denaturing agent is the urea analogue compound according to formula (I):
R,
R, N^
" C = X-(R╬┤)n (I)
.(CH2)m
R3 N^
R I4
wherein,
X is selected from nitrogen or oxygen;
R5 is selected from the group including hydrogen or an alkyl group optionally substituted with one or more amine groups and wherein when X is oxygen, n = 0; m=0 to 1;
Rl5 R2, R3 and R4 may each represent hydrogen, lower alkyl, carboxyl or an alkyl carboxyl group optionally substituted with one or more amine 17
groups and wherein either R or R2 may be taken together with either R3 or R4 as -CO-CH2- to form a five-membered ring; or a salt thereof.
54. A denaturing agent according to claim 53 wherein the salt of the urea analogue compound is a chloride, nitrate or aluminium sulphate salt.
55. A denaturing agent according to claim 53 wherein the urea derivative is selected from the group consisting of urea, guanidine, guanidine hydrochloride,
L(+)-Arginine, creatine and creatinine.
56. A denaturing agent according to claim 53 wherein the denaturing agent is guanidine or guanidine hydrochloride.
57. A denaturing agent according to claim 53 wherein the denaturing agent is in solution containing from 0.1% w/v up to a saturated solution, preferably a 5 to 5% w/v solution, of denaturing agent. 5 , A denaturing agent according to claim 57 wherein the denaturing agent is in a solvent selected from an aqueous alcoholic solution, water or an non- organic solution.
PCT/AU1999/000249 1998-04-01 1999-04-01 A method for treating contaminated products and articles WO1999051279A1 (en)

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NZ507037A NZ507037A (en) 1998-04-01 1999-04-01 A method for treating products and articles contaminated with contagious pathogens by treatment with a urea analogue
AU31305/99A AU746247B2 (en) 1998-04-01 1999-04-01 A method for treating contaminated products and articles
JP2000542049A JP2002510657A (en) 1998-04-01 1999-04-01 Methods of treating contaminated products and articles
EP99913002A EP1066062A4 (en) 1998-04-01 1999-04-01 A method for treating contaminated products and articles

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AUPP2760A AUPP276098A0 (en) 1998-04-01 1998-04-01 A method for treating gelatine
AUPP2760 1998-04-01

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EP1066062A4 (en) 2002-10-16

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