AU714485B2 - Methods of preserving microorganisms - Google Patents

Methods of preserving microorganisms Download PDF

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AU714485B2
AU714485B2 AU73183/96A AU7318396A AU714485B2 AU 714485 B2 AU714485 B2 AU 714485B2 AU 73183/96 A AU73183/96 A AU 73183/96A AU 7318396 A AU7318396 A AU 7318396A AU 714485 B2 AU714485 B2 AU 714485B2
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micro
polyoxyethylene
amphiphile
organisms
virus particles
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AU7318396A (en
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Charles Anthony Hart
Roger Randal Charles New
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Cortecs Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/13—Poliovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Genetics & Genomics (AREA)
  • Communicable Diseases (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)

Description

WO 97/15331 PCT/GB96/02615 1 METHODS OF PRESERVING MICROORGANISMS The present invention relates to methods of preserving micro-organisms such that they retain their infectivity.
In particular the invention relates to methods of preserving viral particles.
Storage/viability problems occur in relation to microorganism storage. In particular problems occur in relation to viral storage where the virus particles are employed for uses such as:viral vectors for use in, e.g. gene therapy; storage of viruses for general research progress, e.g. in culture banks; viruses to be used for release into the environment for control of agricultural pests; and vaccines.
Vaccines comprising viral particles have been in use for a number of years. It is, however, essential that such vaccines can be stored, sometimes for long periods, without the viral component losing its infectivity.
Common storage methods include freezing or freeze-drying, the latter usually involving reconstitution using water at a later stage. Unfortunately, certain viruses display reduced viability/infectivity when subjected to these processes.
WO 97/15331 PCT/GB96/02615 2 One virus which is not suitably stored as described above is polio virus. This virus is readily degraded at room temperature in aqueous suspension, is stable for only two weeks at 0°C and is destroyed by lyophilisation. For this particular virus preferred methods of storage involve freezing at -70 0 C or refridgeration at 4 0 C. However, such storage conditions are not particularly suitable for use in tropical countries or indeed countries where the required facilities and equipment are scarce.
International Application No PCT/GB94/02495 discloses compositions comprising a hydrophilic species solubilised in a hydrophobic phase, as well as methods for their preparation. UK application no. 9424901.8 discloses compositions aS described in PCT/GB94/02495 which incorporate additional components which aid retention of the hydrophilic species in the hydrophobic phase. UK application no.9424902.6 discloses compositions as described in PCT/GB94/02495 which incorporate moieties which aid formation of the composition.
In addition, UK patent application no. 9422990.3 discloses immunogenic compositions which comprise an immunogen solubilised, suspended or otherwise dispersed in a hydrophobic phase. The immunogen can be a virus and the compositions are useful as vaccines.
It has now been found that micro-organisms, particularly virus particles, such as polio virus particles, may be converted to a form suitable for long term storage at ambient temperature, with retention of infectivity after reconstitution in aqueous medium. Thus, such compositions have particular advantages for use in countries where the ususal storage methods are less appropriate, and provide 4 a @5 5D 4 4 @54 an effective means by which such viruses can be transported and stored without the need for extreme freezing or prolonged refrigeration.
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.
Summary of the Invention In a first aspect, the present invention a method of storing micro- S organisms such that they retain infectivity, which method includes the steps of: mixing the micro-organisms with an amphiphile in a liquid medium; 15 (ii) removing the liquid medium to leave an array of amphiphile molecules with their hydrophilic head groups orientated towards the micro-organism; (iii) mixing the micro-organisms/amphiphile array with a hydrophobic solvent; and (iv) storing the product of step (iii).
In one preferred embodiment the micro-organisms are virus particles S particularly polio virus particles.
Suitable methods for carrying out the above method are those described in PCT/GB94/02495, UK 9424901.8, UK 9424902.6 and UK 9422990.3.
25 The hydrophobic solvent could for example be a long chain fatty acid, a medium chain alcohol, a branched long chain alcohol, a monoglyceride, a diglyceride, a medium chain triglyceride, a long chain triglyceride, a halogenated fluorinated) analogue thereof, or a polyoxyethylenecontaining lipid.
In particular embodiments the hydrophobic solvent is a mono-, di- or tri-glyceride, or oleic acid.
In one preferred embodiment the method comprises: 4 4 4 0* 0 4 00 @0 9 0 *9 0 *r 0 0 00 00 00 0 4 co-dispersing the micro-organisms with an amphiphile in a liquid medium; (ii) removing the liquid medium to leave an array of amphiphile molecules with their hydrophilic head groups orientated towards the micro-organism; and (iii) providing a non-aqueous solvent around the micro-organisms/amphiphile array.
The liquid medium can be water, and it can be removed by, e.g. freeze drying, centrifugal vacuum drying or any other suitable method.
Suitably, in the above methods the amphiphile will be a phospholipid, for instance one with a phosphatidyl choline head group, eg phosphatidyl choline (PC), lysophosphatidyl choline(lyso-PC), sphingomyelin or a derivative of one of these such as hexadecyl phosphocholine or an amphiphile polymer containing phosphoryl choline. A bile salt, a glycolipid, a polyoxyethylene containing surfactant, a lipophilic @00 0000 0' 2 0 0 2 00 0 sulphate, betaine, a sarcosine containing surfactant e.g. Solulan C24, polyoxyethylene 40 stearate, a lipid with a polyoxyethylene sorbitan head group e.g. one of the Tween series of surfactants, a lipid with a sorbitan head group e.g. one of the Span series of surfactants or a pegolated castor oil derivative, e.g. Cremaphor Without wishing to be bound by the following, it is believed that in the methods described above the micro-organisms, e.g. virus particles, first form an array with the amphiphile molecules. This array is then in turn coated with the hydrophobic solvent. In this way access to the micro-organisms by 10 water is restricted, which in turn accounts for the improved storage properties when the micro-organism preparation is reconstituted from a :freeze-dried state.
In a second aspect, the present invention provides a method of storing micro-organisms such that they retain infectivity, which method includes the following steps: mixing the micro-organisms with an amphiphile in an aqueous solvent; (ii) removing the water; and (iii) mixing the product from step (ii) with a hydrophobic solvent.
Preferably, the water is removed by freeze-drying.
20 The amphiphile can be a phospholipid, for instance one with a phosphatidyl choline head group, eg phosphatidyl choline (PC), lysophosphatidyl choline (lyso-PC), sphingomyelin or a derivative of one of these such as hexadecyl phosphocholine or an amphiphile polymer containing phosphoryl choline. A bile salt, a glycolipid, a polyoxyethylene containing surfactant, a lipophilic sulphate, betaine, a sarcosine containing surfactant, cholesterol linked to a single polyoxyethylene chain e.g. Solulan C24, polyoxyethlene 40 stearate, a lipid with a polyoxyethylene sorbitan head group e.g. one of the Tween series of surfactants, a lipid with a sorbitan head group e.g. one of the Span series of surfactants, or a pegolated castor oil derivative e.g. Cremaphor In a particularly preferred embodiment of this aspect the amphiphile is cholesterol linked to a single polyoxyethylene chain, polyoxyethlene stearate, a lipid with a polyoxyethylene sorbitan head group, a lipid with a sorbitan head group or a pegolated castor oil derivative e.g. Cremaphor In particularly preferred WO 97/15331 PCT/GB96/02615 6 embodiments the amphiphile is Solulan C24 or polyoxyethylene 40 stearate.
It is possible that upon removal of the water the amphile/micro-organism array will be in an "open" form.
Thus, upon reconstitution water may still have access to the micro-organisms and this will lead to loss of infectivity. Therefore, in another preferred embodiment of this aspect of the invention the method also includes the step of elevating the temperature of the mixture after removal of the water. This ensures that the structure adopted by the amphiphile/micro-organism array is more condensed, which in turn results in more restricted access for water upon reconstitution.
When the heating step is employed, the amphiphile will be one which remains solid after the water removal step, eg it can be chosen from a phospholipid, for instance lecithin, a glycolipid, a polyoxyethylene containing surfactant, a lipophilic sulphate, betaine, a sarcosine containing surfactant, Solulan C24, polyoxyethylene stearate, one of the Tween series of surfactants, one of the Span series of surfactants or a pegolated castor oil derivative, e.g. Cremaphor In other aspects the invention provides: i) a micro-organism composition obtainable by any of the methods described herein, particularly a microorganism composition comprising virus particles, eg polio virus particles; and ii) the use of a composition of the invention for the storage of virus particles.
WO 97/15331 PCT/GB96/02615 7 Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis.
The invention will now be described with reference to the following example, which should not be construed as in any way limiting the invention.
Example 1 A suspension of 109 polio virus particles (Sabin strains, Types 1, 2, 3) per ml of culture was diluted 1000-fold in distilled water. Iml of the diluted suspension was mixed with Iml of adispersion of sonicated soya phospholipid (at a concentration of 100mg/ml) in distilled water. A control vial was prepared which contained virus only, without the addition of phospholipid.
The contents of both vials were shell-frozen in liquid nitrogen and lyophilised overnight. The following day, Iml of oleic acid was added to the vial containing virus and phospholipid, and the contents of the vial wre then mixed on a roller mixer for several hours. A clear solution was obtained.
A control vial of polio virus was prepared as above. To this control vial, containing virus alone, was added iml of culture medium.
of oil/virus preparation was transferred to a fresh vial, and iml of a 2% solution of ox bile extract (containing predominantly sodium taurocholate) was added.
The mixture was shaken well to disperse the oil in water, with the intention of releasing particles into the aqueous phase. Ten-fold serial dilutions were made in culture medium, and 0.5ml of each dilution was added to WO 97/15331 PCT/GB96/02615 8 confluent monolayers of Viro cells, and incubated for four days, to test for the presence of intact virus. An identical procedure was followed for the contents of the control vial. Growth was assessed by visual observation of virus-induced cell lysis in each monolayer. Growth was recorded in the two series of dilutions as follows: Dilution of lyophilisate 102 103 104 105 106 Virus particles present 104 103 102 10 1 (per ml) Oil-based lyophilisate Oil-free lyophilisate These results indicate that the method of the present invention clearly improves the viability of stored viral preparations, when compared to lyophilisation alone.
Example 2 A virus suspension (Sabin strains, Types 1, 2, 3) containing 5x10 8 particles/ml (spun to remove contaminating protein) was diluted 50-fold by addition of 200il of the suspension to 9.9ml of distilled water, yielding a concentration of 107 particles/ml. The suspension was divided into four equal aliquots of and dispensed into 7ml screw-capped glass vials. One aliquot was employed in the experiment described herein, while two were used in the experiment described in example 3.
of sonicated phospholipid dispersion (100mg/ml) was added to the aliquot of diluted virus particles with gentle mixing. 200l of this mixture was dispensed into freeze-drying vials, and the remainder was WO 97/15331 PCT/GB96/02615 9 transferred, In 100l aliquots, into other tubes as "predrying" controls. The controls were stored overnight at +4 0 C. The freeze-drying vials were placed in the centrifugal rotor of the freeze-dryer and lyophilised overnight.
On the following day 100pl of culture medium was added to the contents of ten of the vials freeze-dried overnight, while 1001 of oleic acid was added to the other ten. The groups were labelled and respectively.
of samples from two labelled tubes were transferred to fresh iml vials, and Iml of 0.1M bicarbonate solution containing 25mg/ml sodium taurocholate was added and mixed well. Under these conditions the oil was dispersed well to give a clear solution.
4 x 201l aliquots of sample were transferred from the pre-drying control group stored overnight at +4 0 C to fresh iml vials. To two of these vials was added iml of medium, while to the other two was added 1ml of 0.1M bicarbonate solution containing 25mg/ml sodium taurocholate. The contents of each of the vials was mixed well.
The suspensions prepared above were used to perform fold dilutions in Vero cell monolayer cultures, in order to measure the viability of the polio virus present. the results were expressed as the highest dilution at which 50% cytopathic effects were observed.
WO 97/15331 PCT/GB96/02615 Nature of sample Highest dilution at which CPE observed Non-dried control in medium 10-4/10 Non-dried control in taurocholate 10-3/10 3 Oil-free lyophilate in medium 10-1/100 Oil-free lyophilate in taurocholate 10-/10- 1 Oil-based lyophilate in taurocholate 10-/10 6 Example 3 of distilled water was added to one aliquot of virus particles prepared as described in example 2, and this group was labelled 2.5ml of Solulan C24 (100mg/ml) was added to another aliquot and mixed gently.
This group was labelled 2001 of each preparation was dispensed into 10 freezedrying vials, and the remainder in 1009l aliquots into other tubes as "pre-drying" controls. The controls were stored overnight at +4 0 C. The freeze-drying vials were placed in the centrifugal rotor of the freeze-dryer and lyophilised overnight.
On the following day 100l of culture medium was added to each vial in group and mixed gently. The vials in group were sealed and heated to 60 0 C in a hot water bath for 5 seconds to melt the Solulan C24, which resulted in a claer solution. Upon cooling to room temperature this material solidified. 90l of medium was added to the vials of the group to make the total volume up to 100l. 10l of sample was then transferred from each of groups and to fresh iml vials and iml of medium was added to each and mixed well.
WO 97/15331 PCT/GB96/02615 11 To fresh iml vials was added 4 x 20cl of samples from each of the pre-drying groups and iml of medium was added to each. The contents of each vial were mixed well.
The suspensions prepared as described herein were used to perform 10-fold dilutions in Vero cell cultures,to measure the viability of the polio virus present. The results were expressed as the highest dilution at which cytopathic effects were observed.
Nature of Sample Highest Dilution at which 50% CPE observed Non-dried control water Non-dried control Solulan C24 Freeze-dried control water Freeze-dried control Solulan C24 10-4/10- 10-2/10-2 10-2/10-2 10-6/10-1

Claims (18)

1. A method of storing micro-organisms such that they retain infectivity, which method includes the steps of: mixing the micro-organisms with an amphiphile in a liquid medium; (ii) removing the liquid medium to leave an array of amphiphile molecules with their hydrophilic head groups orientated towards the micro-organism; (iii) mixing the micro-organisms/amphiphile array with a hydrophobic solvent; and (iv) storing the product of step (iii). *.10 2. A method as claimed in claim 1 wherein the micro-organisms are virus particles. S 3. A method as claimed in claim 2 wherein the virus particles are polio virus particles.
4. A method as claimed in any one of claims 1 to 3 wherein the hydrophobic solvent is a long chain fatty acid, a medium chain alcohol, a branched long chain alcohol, a monoglyceride, a diglyceride, a medium chain *0 triglyceride, a long chain triglyceride, a halogenated analogue thereof or a polyoxyethylene-containing lipid. A method as claimed in claim 4 wherein the hydrophobic solvent is a :20 mono-, di- or tri-glyceride. S 6. A method as claimed in claim 4 wherein the hydrophobic solvent is oleic acid.
7. A method as claimed in any one of claims 1 to 6 wherein the amphiphile is a phospholipid, a bile salt, a glycolipid, a polyoxyethylene- 25 containing surfactant, a lipophilic sulphate, betaine, a sarcosine containing S surfactant, cholesterol linked to a single polyoxyethylene chain, polyoxyethylene 40 stearate, a lipid with a polyoxyethylene sorbitan head group, a lipid with a sorbitan head group or a pegolated castor oil derivative.
8. A method as claimed in claim 7 wherein the amphiphile is a phospholipid which is phosphatidyl choline lyso-phosphatidyl choline (lyso-PC), sphingomyelin, hexadecyl phosphocholine or an amphiphile polymer containing phosphoryl choline.
9. A method of storing micro-organisms such that they retain infectivity, which method includes the following steps: mixing the micro-organisms with an amphiphile in an aqueous solvent; (ii) removing the water; and I i0 -1 (iii) mixing the product from step (ii) with a hydrophobic solvent. A method as claimed in claim 9 wherein the water is removed by freeze drying.
11. A method as claimed in claim 9 or claim 10 wherein the mixture of amphiphile and micro-organisms is converted to a condensed form by elevating the temperature of the mixture after removal of the water.
12. A method as claimed in any one of claims 9 to 11 wherein the micro- organisms are virus particles.
13. A method as claimed in claim 12 wherein the virus particles are polio 10 virus particles. S14. A method as claimed in any one of claims 9 to 13 wherein the I hydrophobic solvent is a long chain fatty acid, a medium chain alcohol, a branched long chain alcohol, a monoglyceride, a diglyceride, a medium chain triglyceride, a long chain triglyceride, a halogenated analogue thereof or a .15 polyoxyethylene-containing lipid.
15. A method as claimed in any one of claims 9 to 14 wherein the amphiphile is a phospholipid, a bile salt, a glycolipid, a polyoxyethylene- containing surfactant, a lipophilic sulphate, betaine, a sarcosine containing surfactant, cholesterol linked to a single polyoxyethylene chain, 20 polyoxyethylene 40 stearate, a lipid with a polyoxyethylene sorbitan head group, a lipid with a sorbitan head group or a pegolated castor oil derivative.
16. A method as claimed in claim 15 wherein the amphiphile is cholesterol linked to a single polyoxyethylene chain, polyoxyethylene stearate, a lipid with a polyoxyethylene sorbitan head group, a lipid with a sorbitan head group or a pegolated castor oil derivative.
17. A method as claimed in claim 16 wherein the amphiphile is polyoxyethylene 40 stearate.
18. A method as claimed in claim 16 wherein the amphiphile is cholesterol linked to a single polyoxyethylene chain.
19. A micro-organism composition obtainable by a method as defined in any one of claims 1 to 18. A micro-organism composition as claimed in claim 19 comprising virus particles.
21. A micro-organism composition as claimed in claim 20 comprising polio virus particles. E- AM ff 14
22. A composition as claimed in claim 20 or claim 21 when used for the storage of virus particles.
23. A composition as claimed in any one of claims 19 to 21 when used to induce an immune response in a subject.
24. A composition as claimed in any one of claims 19 to 21 when used to prepare an agent capable of inducing an immune response in a subject. A method substantially as herein described with reference to any one of the foregoing Examples.
26. A micro-organism composition substantially as herein described with 10 reference to any one of the foregoing Examples. DATED this 27th day of October 1999 CORTECS (UK) LIMITED S Patent Attorneys for the Applicant: R.. F.B. RICE CO. a. a 00• **a
AU73183/96A 1995-10-25 1996-10-25 Methods of preserving microorganisms Ceased AU714485B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9521806.1A GB9521806D0 (en) 1995-10-25 1995-10-25 Preservation methods
GB9521806 1995-10-25
PCT/GB1996/002615 WO1997015331A1 (en) 1995-10-25 1996-10-25 Methods of preserving microorganisms

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CN102892427A (en) 2010-03-31 2013-01-23 稳定性科技有限公司 Excipients for stabilising viral particles, polypeptides or biological material
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TW201233803A (en) 2010-12-02 2012-08-16 Oncolytics Biotech Inc Lyophilized viral formulations
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