JP2000501282A - How to store microorganisms - Google Patents
How to store microorganismsInfo
- Publication number
- JP2000501282A JP2000501282A JP9516410A JP51641097A JP2000501282A JP 2000501282 A JP2000501282 A JP 2000501282A JP 9516410 A JP9516410 A JP 9516410A JP 51641097 A JP51641097 A JP 51641097A JP 2000501282 A JP2000501282 A JP 2000501282A
- Authority
- JP
- Japan
- Prior art keywords
- amphiphile
- polyoxyethylene
- microorganisms
- surfactant
- virus
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 244000005700 microbiome Species 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 47
- 241000700605 Viruses Species 0.000 claims abstract description 32
- 239000002245 particle Substances 0.000 claims description 27
- 239000000203 mixture Substances 0.000 claims description 24
- 239000004094 surface-active agent Substances 0.000 claims description 22
- OGQYPPBGSLZBEG-UHFFFAOYSA-N dimethyl(dioctadecyl)azanium Chemical compound CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC OGQYPPBGSLZBEG-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 19
- -1 polyoxyethylene Polymers 0.000 claims description 19
- 230000002209 hydrophobic effect Effects 0.000 claims description 12
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 11
- 241000991587 Enterovirus C Species 0.000 claims description 9
- 230000000813 microbial effect Effects 0.000 claims description 9
- 150000003904 phospholipids Chemical class 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 8
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-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
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical group CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 229920000136 polysorbate Polymers 0.000 claims description 6
- 229930186217 Glycolipid Natural products 0.000 claims description 5
- 239000012071 phase Substances 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
- 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
- 108010077895 Sarcosine Proteins 0.000 claims description 4
- 229960003237 betaine Drugs 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
- 229940043230 sarcosine Drugs 0.000 claims description 4
- 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 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 239000003613 bile acid Substances 0.000 claims description 3
- 150000002632 lipids Chemical class 0.000 claims description 3
- 229960003775 miltefosine Drugs 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 150000004668 long chain fatty acids Chemical group 0.000 claims description 2
- 150000004667 medium chain fatty acids Chemical class 0.000 claims description 2
- PQLXHQMOHUQAKB-UHFFFAOYSA-N miltefosine Chemical compound CCCCCCCCCCCCCCCCOP([O-])(=O)OCC[N+](C)(C)C PQLXHQMOHUQAKB-UHFFFAOYSA-N 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 claims 3
- 230000028993 immune response Effects 0.000 claims 2
- 230000001939 inductive effect Effects 0.000 claims 2
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims 1
- 239000003833 bile salt Substances 0.000 claims 1
- 229910052698 phosphorus Inorganic materials 0.000 claims 1
- 239000011574 phosphorus Substances 0.000 claims 1
- 229950004354 phosphorylcholine Drugs 0.000 claims 1
- PYJNAPOPMIJKJZ-UHFFFAOYSA-N phosphorylcholine chloride Chemical compound [Cl-].C[N+](C)(C)CCOP(O)(O)=O PYJNAPOPMIJKJZ-UHFFFAOYSA-N 0.000 claims 1
- 229960005486 vaccine Drugs 0.000 abstract description 5
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 5
- 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 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 230000035899 viability Effects 0.000 description 4
- 238000001816 cooling Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 239000003921 oil Substances 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
- 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
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 125000003827 glycol group Chemical group 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 210000003501 vero cell Anatomy 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- 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
- SGTNSNPWRIOYBX-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-{[2-(3,4-dimethoxyphenyl)ethyl](methyl)amino}-2-(propan-2-yl)pentanenitrile Chemical compound C1=C(OC)C(OC)=CC=C1CCN(C)CCCC(C#N)(C(C)C)C1=CC=C(OC)C(OC)=C1 SGTNSNPWRIOYBX-UHFFFAOYSA-N 0.000 description 1
- DEXFNLNNUZKHNO-UHFFFAOYSA-N 6-[3-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-3-oxopropyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)C(CCC1=CC2=C(NC(O2)=O)C=C1)=O DEXFNLNNUZKHNO-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- VCUFZILGIRCDQQ-KRWDZBQOSA-N N-[[(5S)-2-oxo-3-(2-oxo-3H-1,3-benzoxazol-6-yl)-1,3-oxazolidin-5-yl]methyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C1O[C@H](CN1C1=CC2=C(NC(O2)=O)C=C1)CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F VCUFZILGIRCDQQ-KRWDZBQOSA-N 0.000 description 1
- 208000000474 Poliomyelitis Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 150000001298 alcohols Chemical class 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
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 210000000941 bile Anatomy 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
- 238000005119 centrifugation Methods 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 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
- 210000004185 liver Anatomy 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 229940057917 medium chain triglycerides Drugs 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- APVPOHHVBBYQAV-UHFFFAOYSA-N n-(4-aminophenyl)sulfonyloctadecanamide Chemical compound CCCCCCCCCCCCCCCCCC(=O)NS(=O)(=O)C1=CC=C(N)C=C1 APVPOHHVBBYQAV-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- LFGREXWGYUGZLY-UHFFFAOYSA-N phosphoryl Chemical group [P]=O LFGREXWGYUGZLY-UHFFFAOYSA-N 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Mycology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (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)
Abstract
(57)【要約】 感染能を維持するような、ウィルスなどの微生物の保存方法、および例えばワクチンを調製する際の上記方法の使用が提供される。 (57) [Summary] Provided are methods for preserving microorganisms, such as viruses, and the use of the methods described above, for example, in preparing vaccines, so as to maintain infectivity.
Description
【発明の詳細な説明】 微生物の保存方法 本発明は、微生物がその感染能を維持するような微生物の保存方法に関するも のである。特に、本発明は、ウィルス粒子の保存方法に関するものである。 貯蔵/生育可能性の問題は微生物の貯蔵に関連して生じる。特に、ウィルス粒 子が以下などの用途を目的として使用されるウィルスの貯蔵に関連して問題が生 じる: (a)例えば、遺伝子治療に使用されるウィルスベクター; (b)例えば、培養物バンク(culture bank)の、一般的な研究の発達を目的と するウィルスの貯蔵; (c)農業での害虫の制御を目的として環境中に放出するのに使用されるウィ ルス;および (d)ワクチン。 ウィルス粒子からなるワクチンは、長年の間使用されてきた。しかしながら、 このようなワクチンは、ウィルス成分がその感染能を失わずに、場合によっては 長期間、貯蔵できることが必須である。一般的な貯蔵方法としては、凍結または 凍結肝臓が挙げられ、後者は一般的に後の段階で水を使用した再構築を伴う。残 念なことに、ウィルスによっては、上記プロセスにかけると生育可能性/感染能 の減少を示すものがある。 上記したように適切に貯蔵されないウィルスとしては、ポリオウィルスがある 。このウィルスは、水性懸濁液中で室温で容易に分解し、0℃で2週間しか安定 せず、凍結乾燥によって破壊される。上記特定のウィルスでは、好ましい貯蔵方 法としては、−70℃で凍結するかまたは 4℃で冷却することが挙げられる。しかしながら、このような貯蔵条件は、熱帯 の国々や必要とされる設備や備品が不足するような国々で使用するのには特に適 当でない。 国際出願番号PCT/GB94/02495号には、疎水性相に可溶化される 親水性物質からなる組成物、およびその調製方法が開示される。また、英国出願 番号9424901.8号には、疎水性相における親水性物質の保持を補助する 成分をさらに含ませるPCT/GB94/02495号に記載されるのと同様の 組成物が開示される。さらに、英国出願番号9424902.6号には、組成物 の形成を補助する部分を導入するPCT/GB94/02495号に記載される のと同様の組成物が開示される。 加えて、英国出願番号9422990.3号には、疎水性相中に可溶化され、 懸濁されまたは分散される免疫原からなる免疫原性組成物が開示される。この免 疫原はウィルスであってもよく、また上記組成物はワクチンとして有用である。 微生物、特にポリオウィルス粒子等のウィルス粒子は、水性培地内で再構築さ れた後に感染能を維持しつつ、周囲温度での長期間の貯蔵に適した形態に変換さ れることが発見された。したがって、このような組成物は、一般的な貯蔵方法が あまり適当でない国で特に好ましく使用され、このようなウィルスを極度に凍結 あるいは長期間冷却することを必要とせずに輸送及び貯蔵できる効果的な手段を 提供する。 したがって、第一の概念によると、本発明は、以下の段階からなる、感染能を 維持するような微生物の貯蔵方法を提供するものである: (i) 微生物を両親媒性物質(amphiphile)と結合させ;および (ii)疎水性相中に微生物を可溶化、懸濁または分散する。 好ましい実施態様によると、上記微生物はウィルス粒子、特にポリオ ウィルス粒子である。 上記方法を行うための適当な方法としては、PCT/GB94/02495号 、UK 9424901.8号、UK 9424902.6号及びUK 942 2990.3号に記載される方法がある。 疎水性溶媒は、例えば、長鎖の脂肪酸、中鎖の脂肪酸、枝分れ鎖のアルコール 、モノグリセリド、ジグリセリド、中鎖のトリグリセリド、長鎖のトリグリセリ ド、これらのハロゲン化(例えば、フッ素化)類似体、またはポリオキシエチレ ン含有脂質である。 好ましい実施態様によると、疎水性溶媒は、モノ−、ジ−若しくはトリ−グリ セリド、またはオレイン酸である。 好ましい実施態様によると、上記方法は以下からなる: (i) 液状媒体中に微生物を両親媒性物質と一緒に分散し; (ii) 液状媒体を除去して、微生物に配向する親水性へッド基(head grou p)を有する両親媒性分子のアレイを残し;および (iii)微生物/両親媒性物質アレイ周辺に非水性溶媒を提供する。 上記液状媒体は水であってもよく、例えば、凍結乾燥、遠心真空乾燥または他 の適当な方法によって、除去できる。 好ましくは、上記方法において、両親媒性物質は、リン脂質であり、例えば、 ホスファチジルコリン(PC)、リゾホスファチジルコリン (lyso−PC )、スフィンゴミエリンまたはヘキサデシルホスホコリン等のこれらのうちのー の誘導体またはホスホリルコリンを含む両親媒性ポリマーなどの、ホスファチジ ルコリンヘッド基を有するものである。胆汁酸、糖脂質、ポリオキシエチレン含 有界面活性剤、親油性スルフェート、べタイン、サルコシン含有界面活性剤、ソ ルランC24(Solulan C24)、ポリオキシエチレン40ステアレート(polyoxye thylene40 stearate)、ツィーンシリーズの界面活性剤の一種、スパン(Span) シ リーズの界面活性剤の一種またはペゴレイトされた(pegolated:ポリエチレング リコール部分の付加によって修飾された)ヒマシ油誘導体、例えばクレマフォー イーエル35(Cremaphor EL35)。 以下に限定されることを意図するものではないが、上記方法において、微生物 、例えば、ウィルス粒子は、まず両親媒性分子とアレイを形成すると考えられる 。次に、このアレイは順番に疎水性溶媒で被覆される。このようにして、水によ る微生物への接近が制限されるが、これは微生物調製物が凍結乾燥状態から再構 築される際の貯蔵特性が向上されることを説明するものである。 第二の概念によると、本発明は、以下の段階からなる、感染能を維持するよう な微生物の貯蔵方法を提供するものである: (i) 水相において微生物を両親媒性物質(amphiphile)と結合させ;および (ii)水を除去する。 好ましくは、水は凍結乾燥によって除去される。 両親媒性物質は、リン脂質であってもよく、例えば、ホスファチジルコリン( PC)、リゾホスファチジルコリン(lyso−PC)、スフィンゴミエリンま たはへキサデシルホスホコリン等のこれらのうちの一の誘導体またはホスホリル コリンを含む両親媒性ポリマーなどの、ホスファチジルコリンヘッド基を有する ものである。胆汁酸、糖脂質、ポリオキシエチレン含有界面活性剤、親油性スル フェート、べタイン、サルコシン含有界面活性剤、ソルランC24(Solulan C24 )、ポリオキシエチレン40ステアレート(polyoxyethylene 40 stearate)、ツイ ーンシリーズの界面活性剤の一種、スパン(Span)シリーズの界面活性剤の一種ま たはペゴレイトされた(pegolated:ポリエチレングリコール部分の付加によって 修飾された)ヒマシ油誘導体、例えばクレマフォー イーエ ル35(Cremaphor EL35)。 上記概念の特に好ましい実施態様によると、両親媒性物質は、ソルランC24 (Solulan C24)、ポリオキシエチレン40ステアレート(polyoxyethylene 40 st earate)、ツィーンシリーズの界面活性剤の一種、スパン(Span)シリーズの界面 活性剤の一種またはペゴレイトされた(pegolated:ポリエチレングリコール部分 の付加によって修飾された)ヒマシ油誘導体、例えばクレマフォー イーエル3 5(Cremaphor EL35)である。特に好ましい実施態様によると、両親媒性物質は、 ソルランC24(Solulan C24)またはポリオキシエチレン40ステアレート(p olyoxyethylene 40 stearate)である。 水を除去すると、両親媒性物質/微生物のアレイは「開放(open)」形態を有す ることが可能である。したがって、再構築時には、水は依然として微生物に接近 しており、これにより感染能が失われるであろう。したがって、本発明の上記概 念の他の好ましい実施態様によると、上記方法はまた、水の除去後に混合物の温 度を上昇する段階を含む。これにより、確実に両親媒性物質/微生物のアレイに よってとられた構造がより凝縮し、さらにこれにより再構築時に水に対する接近 がより制限される。 加熱段階を使用する際には、両親媒性物質は水の除去後に固体を維持するもの であってもよく、例えば、レシチン、糖脂質、ポリオキシエチレン含有界面活性 剤、親油性スルフェート、べタイン、サルコシン含有界面活性剤、ソルランC2 4(Solulan C24)、ポリオキシエチレン40ステアレート(polyoxyethylene 40 s tearate)、ツイーンシリーズの界面活性剤の一種、スパン(Span)シリーズの界面 活性剤の一種またはペゴレイトされた(pegolated:ポリエチレングリコール部分 の付加によって修飾された)ヒマシ油誘導体、例えばクレマフォー イーエル3 5(Cremaphor EL35)などの、リン脂質から選択できる。 他の概念によると、本発明は、以下を提供するものである: i) 上記方法のいずれかによって得られる微生物組成物、特にウィルス粒子 、例えばポリオウィルス粒子からなる微生物組成物;および ii)ウィルス粒子の貯蔵を目的とする本発明の組成物の使用。 本発明の各概念の好ましい態様は、必要であれば変更を加えて(mutatis mutan dis)、各他の概念と同様である。 本発明を下記実施例を参照しながら説明するが、本発明は下記実施例によって 何等制限されるものではない。実施例1 1mlの培養液当たり109個のポリオウィルス粒子(セービン株(Sabin stra in)、タイプ1、2、3)の懸濁液を蒸留水で1000倍に希釈した。この希釈 された懸濁液1mlを1mlの蒸留水における超音波処理された大豆のリン脂質 の分散液(100mg/mlの濃度)と混合した。リン脂質は加えずにウィルス のみを含ませた、コントロールのバイアル瓶を調製した。 双方のバイアル瓶の内容物を液体窒素中でシェルフリーズし(shell-freeze)、 一晩凍結乾燥した。翌日、1mlのオレイン酸をウィルス及びリン脂質を含むバ イアル瓶に添加した後、このバイアル瓶の内容物を数時間ローラーミキサーで混 合した。透明な溶液が得られた。 ポリオウィルスのコントロールのバイアル瓶を上記と同様にして調製した。ウ ィルスのみを含む、このコントロールのバイアル瓶に、1mlの培養液を添加し た。 10μlの油/ウィルス調製物を新たなバイアル瓶に移し、1mlの2%ウシ 胆汁抽出物(主にタウロコール酸ナトリウムを含む)溶液を添加した。この混合 物を、水相中に粒子を放出する目的で、よく振盪して、水中に油を分散させた。 一連の10倍希釈液を培養液で作製し、0.5 mlの各希釈液をビロ細胞(Viro cell)の密集した単層に添加し、4日間インキ ュベートし、無傷のウィルスの存在について試験した。同様の方法をコントロー ルのバイアル瓶の内容物にも行った。成長を、各単層におけるウィルスにより誘 導された細胞の溶解を目視で観察することによって評価した。成長は、以下のよ うにして2連の希釈液で記録した: これらの結果から、本発明の方法は、凍結乾燥単独に比べて、明らかに貯蔵さ れるウィルス調製物の生育可能性を向上することが示される。実施例2 5×108個粒子/mlを含むウィルス懸濁液(遠心して混入するタンパク質 を除去)(セービン株(Sabin strain)、タイプ1、2、3)を、200μlの懸 濁液を9.9mlの蒸留水に添加することにより50倍に希釈して、5×107 個粒子/mlの濃度を得た。この懸濁液を2.5mlの4個の等容のアリコート に分け、7mlのねじ込キャップ付きのガラス製バイアル瓶中に分散した。1ア リコートを本実験に使用し、2アリコートを実施例3に記載される実験に使用し た。 2.5mlの超音波処理されたリン脂質の分散液(100mg/m1)を、緩 やかに混合しながら希釈ウィルス粒子のアリコートに加えた。この混合液200 μlを20個の凍結乾燥用バイアル瓶中に分散し、残 りを、100μlのアリコートとして、「乾燥前」コントロールとして他のチュ ーブに移した。コントロールは、+4℃で一晩貯蔵された。凍結乾燥用バイアル 瓶を凍結乾燥器の遠心ローター中に置き、一晩凍結乾燥した。 翌日、100μlの培養液を、一晩凍結乾燥されたバイアル瓶10本の内容物 に加え、他の10本には100μlのオレイン酸(イギリス薬局方(B.P.))を添 加した。それぞれの群を「M」及び「O]と記した。「M」と記した2本のチュ ーブのサンプル10μlを新たな1mlのバイアル瓶に移して、25mg/ml タウロコール酸ナトリウムを含む0.1M重炭酸塩溶液1mlを添加し、よく混 合した。上記条件下で、油をよく分散させて、透明な溶液を得た。 サンプルの4×20μlのアリコートを、+4℃で一晩貯蔵された乾燥前コン トロール群から新たな1mlのバイアル瓶に移した。これらのバイアル瓶のうち の2本に1mlの培地を加え、他の2本には25mg/mlタウロコール酸ナト リウムを含む0.1M重炭酸塩溶液1mlを添加した。各バイアル瓶の内容物を よく混合した。 上記のようにして調製された懸濁液を用いて、存在するポリオウィルスの生育 可能性を測定するために、べロ細胞(Vero cell)の単層培養液で10倍希釈を行 った。結果は、50%の細胞障害効果が観察される最大の希釈率として表した。 実施例3 2.5mlの蒸留水を実施例2で調製されたウィルス粒子の1アリコートに添 加し、この群を「W」と記した。2.5mlのソルランC24(Solulan C24) (100mg/ml)を他のアリコートに加え、ゆるやかに混合した。この群を 「S」と記した。 200μlの各調製物を10本の凍結乾燥用バイアル瓶中に分散し、残りを、 100μlのアリコートとして、「乾燥前」コントロールとして他のチューブに 移した。コントロールは、+4℃で一晩貯蔵された。凍結乾燥用バイアル瓶を凍 結乾燥器の遠心ローター中に置き、一晩凍結乾燥した。 翌日、100μの培養液を、「W」群の各バイアル瓶に添加し、ゆるやかに混 合した。「S」群のバイアル瓶は、密閉し、5秒間、湯浴中で60℃に加熱して ソルランC24(Solulan C24)を溶融したところ、透明な溶液が得られた。室温 に冷却すると、この材料は固化した。90μlの培地を「S」群のバイアル瓶に 添加し、全容積を100μlとした。次に、10μlのサンプルを「S」及び「 W」群の各バイアル瓶から新たな1mlのバイアル瓶に移して、1mlの培地を それぞれに加えて、よく混合した。 新たな1mlのバイアル瓶に、乾燥前群のそれぞれのバイアル瓶のサンプルの 4×20μlを添加し、1mlの培地をそれぞれに加えた。各バイアル瓶の内容 物をよく混合した。 上記のようにして調製された懸濁液を用いて、べ口細胞(Vero cell)培養液で 10倍希釈を行い、存在するポリオウィルスの生育可能性を測定した。結果は、 50%の細胞障害効果が観察される最大の希釈率として表した。 DETAILED DESCRIPTION OF THE INVENTION How to store microorganisms The present invention also relates to a method for preserving a microorganism such that the microorganism maintains its infectivity. It is. In particular, the present invention relates to a method for storing virus particles. Storage / viability issues arise in connection with microbial storage. In particular, virus particles The child has problems related to the storage of viruses used for the following purposes: Perfume: (A) viral vectors used, for example, for gene therapy; (B) For the purpose of general research development, for example in a culture bank Storage of the virus (C) Wipes used to release into the environment for pest control in agriculture Luss; and (D) vaccine. Vaccines composed of viral particles have been used for many years. However, Such vaccines can be used in some cases without the viral components losing their infectivity. It is essential that they can be stored for a long time. Common storage methods include freezing or Frozen livers are mentioned, the latter generally involving reconstitution with water at a later stage. Remaining Remember, depending on the virus, the above process may lead to viability / infectivity. Some show a decrease. Viruses that are not properly stored as described above include poliovirus . This virus decomposes easily in aqueous suspension at room temperature and is stable for only 2 weeks at 0 ° C Not destroyed by freeze drying. For the above specific virus, preferred storage method Methods include freezing at -70 ° C or Cooling at 4 ° C. However, such storage conditions are Especially suitable for use in countries where there is a shortage of required equipment and supplies. Not right. International Application No. PCT / GB94 / 02495 states that it is solubilized in a hydrophobic phase. A composition comprising a hydrophilic substance and a method for preparing the same are disclosed. Also filed in the UK No. 9424901.8 teaches the retention of hydrophilic substances in the hydrophobic phase The same as described in PCT / GB94 / 02495, which further comprises components A composition is disclosed. In addition, UK Application No. 9424902.6 discloses compositions PCT / GB94 / 02495 which introduces a moiety that assists in the formation of Similar compositions are disclosed. In addition, UK Application No. 9422990.3 discloses that solubilized in a hydrophobic phase, Disclosed is an immunogenic composition comprising an immunogen suspended or dispersed. This exemption The epidermis may be a virus, and the composition is useful as a vaccine. Microorganisms, especially virus particles such as poliovirus particles, are reconstituted in aqueous media. Is converted to a form suitable for long-term storage at ambient temperature while maintaining infectivity. Was found to be. Therefore, such a composition has a common storage method. Very preferably used in countries where it is not very suitable and extremely freezing such viruses Or effective means that can be transported and stored without the need for long-term cooling. provide. Therefore, according to the first concept, the present invention has an infectious potential comprising the following steps: It provides methods for storing microorganisms that are maintained: (I) binding the microorganism to an amphiphile; and (Ii) solubilize, suspend or disperse the microorganisms in the hydrophobic phase. According to a preferred embodiment, the microorganism is a virus particle, in particular polio. Virus particles. Suitable methods for performing the above method include PCT / GB94 / 02495. , UK 9424901.8, UK 9424902.6 and UK 942 No. 2990.3. Hydrophobic solvents include, for example, long-chain fatty acids, medium-chain fatty acids, and branched-chain alcohols. , Monoglycerides, diglycerides, medium-chain triglycerides, long-chain triglycerides , Their halogenated (eg, fluorinated) analogs, or polyoxyethylene It is a lipid containing lipid. According to a preferred embodiment, the hydrophobic solvent is a mono-, di- or tri-glycol. Cerides or oleic acid. According to a preferred embodiment, the method comprises: (I) dispersing the microorganism together with the amphiphile in a liquid medium; (Ii) A hydrophilic head group (head group) that is oriented to microorganisms by removing the liquid medium. leaving an array of amphipathic molecules having p); and (Iii) providing a non-aqueous solvent around the microbial / amphiphile array; The liquid medium may be water, for example, lyophilization, centrifugal vacuum drying or other Can be removed by a suitable method. Preferably, in the above method, the amphiphile is a phospholipid, for example, Phosphatidylcholine (PC), lysophosphatidylcholine (lyso-PC ), Sphingomyelin or hexadecylphosphocholine Phosphatidyl derivatives, such as derivatives of It has a rucholine head group. Contains bile acids, glycolipids, polyoxyethylene Surfactants, lipophilic sulfates, betaines, sarcosine-containing surfactants, Solulan C24, polyoxyethylene 40 stearate (polyoxye thylene 40 stearate), a kind of surfactant of the Tween series, Span Shi Leeds surfactant or pegolated (polyethylene glycol) Castor oil derivatives (modified by the addition of a recall moiety), such as cremaphore EL35 (Cremaphor EL35). Although not intended to be limited to the following, the method For example, viral particles are thought to first form an array with amphipathic molecules . Next, the array is sequentially coated with a hydrophobic solvent. In this way, the water Access to the microorganisms is limited, which is when the microbial preparation is reconstituted from a lyophilized state. This explains that the storage characteristics when being built are improved. According to a second concept, the present invention provides a method for maintaining infectivity comprising the following steps: It provides a method for storing various microorganisms: (I) binding microorganisms to an amphiphile in the aqueous phase; and (Ii) removing water. Preferably, the water is removed by lyophilization. The amphiphile may be a phospholipid, for example, phosphatidylcholine ( PC), lysophosphatidylcholine (lyso-PC), sphingomyelin Or one derivative thereof such as hexadecylphosphocholine or phosphoryl Has a phosphatidylcholine head group, such as an amphiphilic polymer containing choline Things. Bile acids, glycolipids, polyoxyethylene-containing surfactants, lipophilic sulf Fate, betaine, sarcosine containing surfactant, Solulan C24 ), Polyoxyethylene 40 stearate, One of the surfactants of the Span series and one of the surfactants of the Span series Or pegolated (the addition of a polyethylene glycol moiety) (Modified) castor oil derivatives such as cremaphore IE 35 (Cremaphor EL35). According to a particularly preferred embodiment of the above concept, the amphiphile is sorbane C24 (Solulan C24), polyoxyethylene 40 stearate (polyoxyethylene 40 st earate), a type of surfactant of the Tween series, an interface of the Span series One type of activator or pegolated: polyethylene glycol moiety Castor oil derivatives, such as Cremaphor Y3 5 (Cremaphor EL35). According to a particularly preferred embodiment, the amphiphile comprises Solulan C24 or polyoxyethylene 40 stearate (p olyoxyethylene 40 stearate). Upon removal of water, the amphiphile / microbe array has an "open" form It is possible to Therefore, during rebuilding, water is still accessible to microorganisms And this will result in a loss of infectivity. Therefore, the above summary of the present invention According to another preferred embodiment, the method also comprises heating the mixture after removal of the water. Includes increasing the degree. This ensures an amphiphile / microbe array The structure thus taken is more condensed, and this allows access to water during reconstruction Is more restricted. When using a heating step, the amphiphile should remain solid after the removal of water May be, for example, lecithin, glycolipid, polyoxyethylene-containing surfactant Agent, lipophilic sulfate, betaine, sarcosine-containing surfactant, Sorran C2 4 (Solulan C24), polyoxyethylene 40 stearate (polyoxyethylene 40 s) tearate), a type of surfactant in the Tween series, and an interface in the Span series One type of activator or pegolated: polyethylene glycol moiety Castor oil derivatives, such as Cremaphor Y3 5 (Cremaphor EL35) and other phospholipids. According to another concept, the present invention provides: i) a microbial composition obtainable by any of the above methods, in particular virus particles A microbial composition comprising, for example, poliovirus particles; and ii) Use of the composition of the present invention for storage of virus particles. Preferred embodiments of each concept of the present invention are mutatis mutan dis), similar to each other concept. The present invention will be described with reference to the following examples. There is no restriction.Example 1 10 per 1 ml of culture9Poliovirus particles (Sabin strain in), suspensions of types 1, 2, 3) were diluted 1000-fold with distilled water. This dilution Sonicated soybean phospholipid in 1 ml of distilled water in 1 ml of distilled water (Concentration of 100 mg / ml). Virus without adding phospholipids A control vial containing only one was prepared. Shell-freeze the contents of both vials in liquid nitrogen, Lyophilized overnight. The next day, 1 ml of oleic acid was added to the virus and phospholipid containing After adding to the vial, mix the contents of the vial with a roller mixer for several hours. I combined. A clear solution was obtained. A control vial of poliovirus was prepared as described above. C Add 1 ml of culture to this control vial containing only virus. Was. Transfer 10 μl of the oil / virus preparation to a new vial and add 1 ml of 2% bovine A bile extract (primarily containing sodium taurocholate) solution was added. This mixture The material was shaken well to disperse the oil into the water in order to release the particles into the aqueous phase. A series of 10-fold dilutions were made in culture and 0.5 ml of each dilution was added to the dense monolayer of Viro cells and allowed to incubate for 4 days. And tested for the presence of intact virus. Controlling a similar method I went to the contents of Le vial. Growth driven by virus in each monolayer The induced cell lysis was assessed by visual observation. Growth is below And recorded in duplicate dilutions: From these results, it is clear that the method of the present invention can be stored more clearly than lyophilized alone. It is shown to improve the viability of the resulting virus preparation.Example 2 5 × 108Virus suspension containing individual particles / ml (protein contaminated by centrifugation) (Sabin strain, type 1, 2, 3) was added to a 200 μl suspension. The suspension was diluted 50-fold by adding to 9.9 ml of distilled water and7 A concentration of individual particles / ml was obtained. Aliquot 2.5 ml aliquots of this suspension into four And dispersed in 7 ml glass vials with screw caps. 1 a Recoat was used for this experiment and two aliquots were used for the experiment described in Example 3. Was. 2.5 ml of the sonicated phospholipid dispersion (100 mg / ml) Add to aliquots of diluted virus particles with gentle mixing. This mixture 200 μl was dispersed in 20 lyophilization vials and the remaining Aliquots as 100 μl aliquots and the other tubes as “before drying” controls. Moved to the lab. Controls were stored overnight at + 4 ° C. Lyophilization vial The jar was placed in the centrifugal rotor of a lyophilizer and lyophilized overnight. The next day, 100 μl of the culture was lyophilized overnight and the contents of 10 vials were removed. In addition, add 10 μl of oleic acid (British Pharmacopoeia (B.P.)) to the other 10 Added. Each group was marked “M” and “O.” Two tubes marked “M” Transfer 10 μl of the sample to a new 1 ml vial and add 25 mg / ml Add 1 ml of a 0.1 M bicarbonate solution containing sodium taurocholate and mix well. I combined. Under the above conditions, the oil was well dispersed to give a clear solution. An aliquot of 4 x 20 μl of the sample was placed in a pre-dried concentrator stored at + 4 ° C overnight. The troll group was transferred to a new 1 ml vial. Out of these vials 1 ml of medium was added to two of them, and 25 mg / ml of sodium taurocholate was added to the other two. 1 ml of a 0.1 M bicarbonate solution containing ium was added. The contents of each vial Mix well. Using the suspension prepared as described above, the growth of existing poliovirus To determine the potential, perform a 10-fold dilution in a monolayer culture of Vero cells. Was. The results were expressed as the highest dilution at which a 50% cytotoxic effect was observed. Example 3 Add 2.5 ml of distilled water to one aliquot of virus particles prepared in Example 2. In addition, this group was marked "W". 2.5 ml of Solulan C24 (100 mg / ml) was added to another aliquot and mixed gently. This group Marked "S". 200 μl of each preparation was dispersed in 10 lyophilization vials and the rest was 100 μl aliquots in other tubes as “before drying” controls Moved. Controls were stored overnight at + 4 ° C. Freeze vial for freeze drying Placed in centrifuge rotor of freeze dryer and lyophilized overnight. The next day, add 100 μl of the culture solution to each vial of the “W” group, and mix gently. I combined. The vials of the “S” group were sealed and heated to 60 ° C. in a hot water bath for 5 seconds. Melting Solulan C24 gave a clear solution. room temperature Upon cooling, the material solidified. 90 μl of medium into vials of “S” group Was added to bring the total volume to 100 μl. Next, 10 μl of the sample was added to the “S” and “ Transfer each vial from group "W" to a new 1 ml vial and transfer 1 ml of medium In addition to each, mixed well. In a new 1 ml vial, place a sample of each vial from the group before drying. 4 × 20 μl were added and 1 ml of medium was added to each. Contents of each vial Stir well. Using the suspension prepared as described above, in a Vero cell culture solution A 10-fold dilution was performed to determine the viability of the poliovirus present. Result is, Expressed as the highest dilution at which a 50% cytotoxic effect was observed.
───────────────────────────────────────────────────── フロントページの続き (81)指定国 EP(AT,BE,CH,DE, DK,ES,FI,FR,GB,GR,IE,IT,L U,MC,NL,PT,SE),OA(BF,BJ,CF ,CG,CI,CM,GA,GN,ML,MR,NE, SN,TD,TG),AP(KE,LS,MW,SD,S Z,UG),UA(AM,AZ,BY,KG,KZ,MD ,RU,TJ,TM),AL,AM,AT,AU,AZ ,BA,BB,BG,BR,BY,CA,CH,CN, CU,CZ,DE,DK,EE,ES,FI,GB,G E,HU,IL,IS,JP,KE,KG,KP,KR ,KZ,LC,LK,LR,LS,LT,LU,LV, MD,MG,MK,MN,MW,MX,NO,NZ,P L,PT,RO,RU,SD,SE,SG,SI,SK ,TJ,TM,TR,TT,UA,UG,US,UZ, VN (72)発明者 ハート,チャールズ,アンソニー イギリス国,リバプール エル69 3ビー エックス,デパートメント オブ メディ カル ミクロバイオロジー アンド ゲニ ト−ウリナリー メディシン,ザ ユニバ ーシティ オブ リバプール────────────────────────────────────────────────── ─── Continuation of front page (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, G E, HU, IL, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, P L, PT, RO, RU, SD, SE, SG, SI, SK , TJ, TM, TR, TT, UA, UG, US, UZ, VN (72) Inventor Hart, Charles, Anthony Liverpool El 69 3-Bee, UK X, Department of Medi Cal microbiology and Geni Toulinary Medicine, The Univer City of Liverpool
Claims (1)
Applications Claiming Priority (3)
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GB9521806.1 | 1995-10-25 | ||
GBGB9521806.1A GB9521806D0 (en) | 1995-10-25 | 1995-10-25 | Preservation methods |
PCT/GB1996/002615 WO1997015331A1 (en) | 1995-10-25 | 1996-10-25 | Methods of preserving microorganisms |
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JP2000501282A true JP2000501282A (en) | 2000-02-08 |
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JP9516410A Pending JP2000501282A (en) | 1995-10-25 | 1996-10-25 | How to store microorganisms |
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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 (1)
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JP2013523704A (en) * | 2010-03-31 | 2013-06-17 | スタビリテック リミテッド | Additives that stabilize virus particles, polypeptides or biomaterials |
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US7645608B2 (en) | 2004-08-17 | 2010-01-12 | Pml Microbiologicals, Inc. | Microorganism specimen storage, hydrating, transfer and applicator device |
GB2499480A (en) | 2010-03-31 | 2013-08-21 | Stabilitech Ltd | Method for preserving alum adjuvants and alum-adjuvanted vaccines |
JP5960120B2 (en) | 2010-03-31 | 2016-08-02 | スタビリテック リミテッド | Stabilization of virus particles |
WO2012075379A2 (en) | 2010-12-02 | 2012-06-07 | Oncolytics Biotech Inc. | Liquid viral formulations |
AU2011336410B2 (en) | 2010-12-02 | 2015-01-22 | Oncolytics Biotech Inc. | Lyophilized viral formulations |
GB201117233D0 (en) | 2011-10-05 | 2011-11-16 | Stabilitech Ltd | Stabilisation of polypeptides |
GB201406569D0 (en) | 2014-04-11 | 2014-05-28 | Stabilitech Ltd | Vaccine compositions |
GB2562241B (en) | 2017-05-08 | 2022-04-06 | Stabilitech Biopharma Ltd | Vaccine compositions |
EP4182473A1 (en) | 2020-07-20 | 2023-05-24 | Stratix Labs Corporation | Devices and methods for inoculating a target |
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AU7971194A (en) * | 1993-10-12 | 1995-05-04 | Chiron Corporation | Methods for preserving recombinant viruses |
GB9323588D0 (en) * | 1993-11-16 | 1994-01-05 | Cortecs Ltd | Hydrophobic preparation |
GB9424901D0 (en) * | 1994-12-09 | 1995-02-08 | Cortecs Ltd | Sequestration Agents |
GB9424902D0 (en) * | 1994-12-09 | 1995-02-08 | Cortecs Ltd | Solubilisation Aids |
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- 1996-10-25 ZA ZA9609015A patent/ZA969015B/en unknown
- 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 JP JP9516410A patent/JP2000501282A/en active Pending
- 1996-10-25 CN CN96198528A patent/CN1202829A/en active Pending
- 1996-10-25 BR BR9610932-7A patent/BR9610932A/en not_active Application Discontinuation
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013523704A (en) * | 2010-03-31 | 2013-06-17 | スタビリテック リミテッド | Additives that stabilize virus particles, polypeptides or biomaterials |
JP2016121155A (en) * | 2010-03-31 | 2016-07-07 | スタビリテック リミテッド | Excipients for stabilizing viral particles, polypeptides or biological material |
Also Published As
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ZA969015B (en) | 1998-04-28 |
BR9610932A (en) | 1999-12-21 |
WO1997015331A1 (en) | 1997-05-01 |
CN1202829A (en) | 1998-12-23 |
NZ320446A (en) | 1999-05-28 |
GB9521806D0 (en) | 1996-01-03 |
AU714485B2 (en) | 2000-01-06 |
NO981865D0 (en) | 1998-04-24 |
NO981865L (en) | 1998-06-24 |
EP0857069A1 (en) | 1998-08-12 |
KR19990067029A (en) | 1999-08-16 |
CA2235495A1 (en) | 1997-05-01 |
AU7318396A (en) | 1997-05-15 |
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