WO1997015331A1 - Methods of preserving microorganisms - Google Patents
Methods of preserving microorganisms Download PDFInfo
- Publication number
- WO1997015331A1 WO1997015331A1 PCT/GB1996/002615 GB9602615W WO9715331A1 WO 1997015331 A1 WO1997015331 A1 WO 1997015331A1 GB 9602615 W GB9602615 W GB 9602615W WO 9715331 A1 WO9715331 A1 WO 9715331A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- micro
- amphiphile
- organisms
- virus particles
- polyoxyethylene
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 46
- 244000005700 microbiome Species 0.000 title claims abstract description 33
- 241000700605 Viruses Species 0.000 claims abstract description 31
- OGQYPPBGSLZBEG-UHFFFAOYSA-N dimethyl(dioctadecyl)azanium Chemical compound CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC OGQYPPBGSLZBEG-UHFFFAOYSA-N 0.000 claims description 27
- 239000002245 particle Substances 0.000 claims description 27
- 239000004094 surface-active agent Substances 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- -1 polyoxyethylene Polymers 0.000 claims description 17
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 16
- 230000002209 hydrophobic effect Effects 0.000 claims description 12
- 241000991587 Enterovirus C Species 0.000 claims description 11
- 238000004108 freeze drying Methods 0.000 claims description 10
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 10
- 150000003904 phospholipids Chemical class 0.000 claims description 10
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 claims description 10
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 9
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 8
- 239000004359 castor oil Substances 0.000 claims description 7
- 235000019438 castor oil Nutrition 0.000 claims description 7
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 claims description 7
- 229920000136 polysorbate Polymers 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 claims description 6
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 claims description 5
- 229930186217 Glycolipid Natural products 0.000 claims description 5
- 108010077895 Sarcosine Proteins 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 5
- 229960003237 betaine Drugs 0.000 claims description 5
- 239000012071 phase Substances 0.000 claims description 5
- 229940043230 sarcosine Drugs 0.000 claims description 5
- 229910021653 sulphate ion Inorganic materials 0.000 claims description 5
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 4
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 4
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000005642 Oleic acid Substances 0.000 claims description 4
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 4
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 4
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid group Chemical group C(CCCCCCC\C=C/CCCCCCCC)(=O)O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 4
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 239000003833 bile salt Substances 0.000 claims description 3
- PQLXHQMOHUQAKB-UHFFFAOYSA-N miltefosine Chemical compound CCCCCCCCCCCCCCCCOP([O-])(=O)OCC[N+](C)(C)C PQLXHQMOHUQAKB-UHFFFAOYSA-N 0.000 claims description 3
- 229960003775 miltefosine Drugs 0.000 claims description 3
- 229950004354 phosphorylcholine Drugs 0.000 claims description 3
- PYJNAPOPMIJKJZ-UHFFFAOYSA-N phosphorylcholine chloride Chemical compound [Cl-].C[N+](C)(C)CCOP(O)(O)=O PYJNAPOPMIJKJZ-UHFFFAOYSA-N 0.000 claims description 3
- 229920000642 polymer Polymers 0.000 claims description 3
- LDVVTQMJQSCDMK-UHFFFAOYSA-N 1,3-dihydroxypropan-2-yl formate Chemical compound OCC(CO)OC=O LDVVTQMJQSCDMK-UHFFFAOYSA-N 0.000 claims description 2
- 230000003028 elevating effect Effects 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 claims description 2
- 150000004668 long chain fatty acids Chemical group 0.000 claims description 2
- 230000028993 immune response Effects 0.000 claims 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 229960005486 vaccine Drugs 0.000 abstract description 5
- 230000000717 retained effect Effects 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 11
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 239000003921 oil Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- 230000035899 viability Effects 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 description 3
- WBWWGRHZICKQGZ-HZAMXZRMSA-N taurocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 WBWWGRHZICKQGZ-HZAMXZRMSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 2
- 241000274177 Juniperus sabina Species 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000003501 vero cell Anatomy 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229940118683 ox bile extract Drugs 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 210000000605 viral structure Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
-
- 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
-
- 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
- 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
Definitions
- the present invention relates to methods of preserving micro-organisms such that they retain their infectivity.
- the invention relates to methods of preserving viral particles.
- 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.
- certain viruses display reduced viability/infectivity when subjected to these processes .
- 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.
- preferred methods of storage involve freezing at -70°C or refridgeration at 4°C.
- such storage conditions are not particularly suitable for use in tropical countries or indeed countries where the required facilities and equipment are scarce.
- 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.
- 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 .
- micro-organisms particularly virus particles, such as polio virus particles
- virus particles such as polio virus particles
- 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.
- such compositions have particular advantages for use in countries where the ususal storage methods are less appropriate, and provide an effective means by which such viruses can be transported and stored without the need for extreme freezing or prolonged refridgeration.
- the present invention provides a method of storing micro-organisms such that they maintain infectivity, which method includes the steps of :-
- micro-organisms are virus particles 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.
- 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 (e.g. fluorinated) analogue thereof, or a polyoxyethylene-containing lipid.
- the hydrophobic solvent is a mono-, di- or tri-glyceride, or oleic acid.
- the method comprises : (i) co-dispersing the micro-organisms with an amphiphile in a liquid medium;
- the liquid medium can be water, and it can be removed by, e.g. freeze drying, centrifugal vacuum drying or any other suitable method.
- 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 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 phosphatidyl choline head group eg phosphatidyl choline (PC) , lysophosphati
- a bile salt a glycolipid, a polyoxyethylene containing surfactant, a lipophilic sulphate, betaine, a sarcosine containing surfactant, Solulan C24 , polyoxyethylene 40 stearate, one of the Tween series of surfactants, one of the Span series of surfactants or a pegolated castor oil derivative, e.g. Cremaphor EL35.
- the micro ⁇ organisms eg virus particles
- This array is then in turn coated with the hydrophobic solvent .
- access to the micro-organisms by water is restricted, which in turn accounts for the improved storage properties when the micro-organism preparation is reconstituted from a freeze-dried state.
- the present invention provides a method of storing micro-organisms such that they retain infectivity, which method includes the following steps:
- the water is removed by freeze-drying.
- 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 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 phosphatidyl choline head group eg phosphatidyl choline (PC) , lysophosphati
- a bile salt a glycolipid, a polyoxyethylene containing surfactant, a lipophilic sulphate, betaine, a sarcosine containing surfactant, Solulan C24, polyoxyethylene 40 stearate, one of the Tween series of surfactants, one of the Span series of surfactants or a pegolated castor oil derivative, e.g. Cremaphor EL35.
- the amphiphile is Solulan C24, polyoxyethylene 40 stearate, one of the Tween series of surfactants, one of the Span series of surfactants or a pegolated castor oil derivative, e.g Cremaphor EL35.
- the amphiphile is Solulan C24 or polyoxyethylene 40 stearate.
- 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.
- 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 40 stearate, one of the Tween series of surfactants, one of the Span series of surfactants or a pegolated castor oil derivative, e.g. Cremaphor EL35.
- a phospholipid for instance lecithin
- a glycolipid e.g., a polyoxyethylene containing surfactant, a lipophilic sulphate, betaine, a sarcosine containing surfactant, Solulan C24 , polyoxyethylene 40 stearate, one of the Tween series of surfactants, one of the Span series of surfactants or a pegolated castor oil derivative,
- a micro-organism composition obtainable by any of the methods described herein, particularly a micro- organism composition comprising virus particles, eg polio virus particles; and
- composition of the invention for the storage of virus particles.
- Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis .
- a suspension of IO 9 polio virus particles (Sabin strains, Types 1, 2, 3) per ml of culture was diluted 1000-fold in distilled water. 1ml of the diluted suspension was mixed with 1ml of adispersion of sonicated soya phospholipid
- control vial (at a concentration of lOOmg/ml) in distilled water.
- a control vial was prepared which contained virus only, without the addition of phospholipid.
- a control vial of polio virus was prepared as above. To this control vial, containing virus alone, was added lml of culture medium.
- lO ⁇ l of oil/virus preparation was transferred to a fresh vial, and lml of a 2% solution of ox bile extract (containing predominantly sodium taurocholate) was added.
- Example 2 A virus suspension (Sabin strains, Types 1, 2, 3) containing 5xlO e particles/ml (spun to remove contaminating protein) was diluted 50-fold by addition of 200 ⁇ l of the suspension to 9.9ml of distilled water, yielding a concentration of IO 7 particles/ml. The suspension was divided into four equal aliquots of 2.5ml, 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.
- sonicated phospholipid dispersion (lOOmg/ml) was added to the aliquot of diluted virus particles with gentle mixing. 200 ⁇ l of this mixture was dispensed into 20 freeze-drying vials, and the remainder was transferred, In lOO ⁇ l aliquots, into other tubes as "pre ⁇ drying" controls. The controls were stored overnight at +4°C. The freeze-drying vials were placed in the centrifugal rotor of the freeze-dryer and lyophilised overnight .
- the suspensions prepared above were used to perform 10- 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. Nature of sample Highest dilution at which 50% cytopathic effects were observed. Nature of sample Highest dilution at which 50% cytopathic effects were observed. Nature of sample Highest dilution at which
- 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 50% cytopathic effects were observed.
Abstract
Description
Claims
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1019980702966A KR19990067029A (en) | 1995-10-25 | 1996-10-25 | How to preserve microorganisms |
NZ320446A NZ320446A (en) | 1995-10-25 | 1996-10-25 | Preserving microorganisms by mixing them with an amphiphile and a hydrophobic solvent |
EP96935088A EP0857069A1 (en) | 1995-10-25 | 1996-10-25 | Methods of preserving microorganisms |
BR9610932-7A BR9610932A (en) | 1995-10-25 | 1996-10-25 | Process for storing microorganisms, microorganism composition and use |
JP9516410A JP2000501282A (en) | 1995-10-25 | 1996-10-25 | How to store microorganisms |
AU73183/96A AU714485B2 (en) | 1995-10-25 | 1996-10-25 | Methods of preserving microorganisms |
US09/065,734 US6165773A (en) | 1995-10-25 | 1998-04-24 | Methods of preserving viruses |
NO981865A NO981865L (en) | 1995-10-25 | 1998-04-24 | Microorganism Conservation Procedures |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9521806.1 | 1995-10-25 | ||
GBGB9521806.1A GB9521806D0 (en) | 1995-10-25 | 1995-10-25 | Preservation methods |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/065,734 Continuation US6165773A (en) | 1995-10-25 | 1998-04-24 | Methods of preserving viruses |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997015331A1 true WO1997015331A1 (en) | 1997-05-01 |
Family
ID=10782855
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1996/002615 WO1997015331A1 (en) | 1995-10-25 | 1996-10-25 | Methods of preserving microorganisms |
Country Status (12)
Country | Link |
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EP (1) | EP0857069A1 (en) |
JP (1) | JP2000501282A (en) |
KR (1) | KR19990067029A (en) |
CN (1) | CN1202829A (en) |
AU (1) | AU714485B2 (en) |
BR (1) | BR9610932A (en) |
CA (1) | CA2235495A1 (en) |
GB (1) | GB9521806D0 (en) |
NO (1) | NO981865L (en) |
NZ (1) | NZ320446A (en) |
WO (1) | WO1997015331A1 (en) |
ZA (1) | ZA969015B (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7645608B2 (en) | 2004-08-17 | 2010-01-12 | Pml Microbiologicals, Inc. | Microorganism specimen storage, hydrating, transfer and applicator device |
US9045728B2 (en) | 2010-12-02 | 2015-06-02 | Oncolytics Biotech Inc. | Liquid viral formulations |
US9044498B2 (en) | 2010-12-02 | 2015-06-02 | Oncolytics Biotech Inc. | Lyophilized viral formulations |
US9101607B2 (en) | 2010-03-31 | 2015-08-11 | Stabilitech Ltd. | Method for preserving alum adjuvants and alum-adjuvanted vaccines |
US10029007B2 (en) | 2011-10-05 | 2018-07-24 | Stabilitech Biopharma Ltd | Stabilisation of polypeptides |
US10206960B2 (en) | 2010-03-31 | 2019-02-19 | Stabilitech Biopharma Ltd | Stabilisation of viral particles |
US10716859B2 (en) | 2010-03-31 | 2020-07-21 | Stabilitech Biopharma Ltd | Excipients for stabilising viral particles, polypeptides or biological material |
US10806783B2 (en) | 2014-04-11 | 2020-10-20 | Stabilitech Biopharma Ltd | Vaccine compositions |
US10980871B2 (en) | 2017-05-08 | 2021-04-20 | Iosbio Ltd | Vaccine compositions |
US11530379B2 (en) | 2020-07-20 | 2022-12-20 | Stratix Labs Corporation | Devices and methods for inoculating a target |
Citations (4)
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WO1995010601A1 (en) * | 1993-10-12 | 1995-04-20 | Chiron Viagene, Inc. | Methods for preserving recombinant viruses |
WO1995013795A1 (en) * | 1993-11-16 | 1995-05-26 | Cortecs Limited | Hydrophobic preparations |
WO1996017594A1 (en) * | 1994-12-09 | 1996-06-13 | Cortecs Limited | Sequestration agents |
WO1996017593A1 (en) * | 1994-12-09 | 1996-06-13 | Cortecs Limited | Solubilisation aids for hydrophilic macromolecules |
-
1995
- 1995-10-25 GB GBGB9521806.1A patent/GB9521806D0/en active Pending
-
1996
- 1996-10-25 CN CN96198528A patent/CN1202829A/en active Pending
- 1996-10-25 JP JP9516410A patent/JP2000501282A/en active Pending
- 1996-10-25 WO PCT/GB1996/002615 patent/WO1997015331A1/en not_active Application Discontinuation
- 1996-10-25 AU AU73183/96A patent/AU714485B2/en not_active Ceased
- 1996-10-25 CA CA002235495A patent/CA2235495A1/en not_active Abandoned
- 1996-10-25 KR KR1019980702966A patent/KR19990067029A/en not_active Application Discontinuation
- 1996-10-25 EP EP96935088A patent/EP0857069A1/en not_active Withdrawn
- 1996-10-25 NZ NZ320446A patent/NZ320446A/en unknown
- 1996-10-25 ZA ZA9609015A patent/ZA969015B/en unknown
- 1996-10-25 BR BR9610932-7A patent/BR9610932A/en not_active Application Discontinuation
-
1998
- 1998-04-24 NO NO981865A patent/NO981865L/en not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995010601A1 (en) * | 1993-10-12 | 1995-04-20 | Chiron Viagene, Inc. | Methods for preserving recombinant viruses |
WO1995013795A1 (en) * | 1993-11-16 | 1995-05-26 | Cortecs Limited | Hydrophobic preparations |
WO1996017594A1 (en) * | 1994-12-09 | 1996-06-13 | Cortecs Limited | Sequestration agents |
WO1996017593A1 (en) * | 1994-12-09 | 1996-06-13 | Cortecs Limited | Solubilisation aids for hydrophilic macromolecules |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7645608B2 (en) | 2004-08-17 | 2010-01-12 | Pml Microbiologicals, Inc. | Microorganism specimen storage, hydrating, transfer and applicator device |
US9101607B2 (en) | 2010-03-31 | 2015-08-11 | Stabilitech Ltd. | Method for preserving alum adjuvants and alum-adjuvanted vaccines |
US10206960B2 (en) | 2010-03-31 | 2019-02-19 | Stabilitech Biopharma Ltd | Stabilisation of viral particles |
US10716859B2 (en) | 2010-03-31 | 2020-07-21 | Stabilitech Biopharma Ltd | Excipients for stabilising viral particles, polypeptides or biological material |
US9045728B2 (en) | 2010-12-02 | 2015-06-02 | Oncolytics Biotech Inc. | Liquid viral formulations |
US9044498B2 (en) | 2010-12-02 | 2015-06-02 | Oncolytics Biotech Inc. | Lyophilized viral formulations |
US9610309B2 (en) | 2010-12-02 | 2017-04-04 | Oncolytics Biotech Inc. | Liquid viral formulations |
US9610352B2 (en) | 2010-12-02 | 2017-04-04 | Oncolytics Biotech Inc. | Lyophilized viral formulations |
US10029007B2 (en) | 2011-10-05 | 2018-07-24 | Stabilitech Biopharma Ltd | Stabilisation of polypeptides |
US10806783B2 (en) | 2014-04-11 | 2020-10-20 | Stabilitech Biopharma Ltd | Vaccine compositions |
US10980871B2 (en) | 2017-05-08 | 2021-04-20 | Iosbio Ltd | Vaccine compositions |
US11530379B2 (en) | 2020-07-20 | 2022-12-20 | Stratix Labs Corporation | Devices and methods for inoculating a target |
Also Published As
Publication number | Publication date |
---|---|
CN1202829A (en) | 1998-12-23 |
NO981865D0 (en) | 1998-04-24 |
ZA969015B (en) | 1998-04-28 |
AU714485B2 (en) | 2000-01-06 |
NZ320446A (en) | 1999-05-28 |
CA2235495A1 (en) | 1997-05-01 |
EP0857069A1 (en) | 1998-08-12 |
BR9610932A (en) | 1999-12-21 |
GB9521806D0 (en) | 1996-01-03 |
AU7318396A (en) | 1997-05-15 |
KR19990067029A (en) | 1999-08-16 |
NO981865L (en) | 1998-06-24 |
JP2000501282A (en) | 2000-02-08 |
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