NO149611B - PROCEDURE FOR THE ISOLATION AND CLEANING OF IMMUNOLOGICALLY ACTIVE POLYRIBOSYL-RIBITOL PHOSPHATE (PRP), THE CAPSULAR POLYSACCARIDE OF HAEMOPHILUS INFLUENZAE - Google Patents
PROCEDURE FOR THE ISOLATION AND CLEANING OF IMMUNOLOGICALLY ACTIVE POLYRIBOSYL-RIBITOL PHOSPHATE (PRP), THE CAPSULAR POLYSACCARIDE OF HAEMOPHILUS INFLUENZAE Download PDFInfo
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- NO149611B NO149611B NO783635A NO783635A NO149611B NO 149611 B NO149611 B NO 149611B NO 783635 A NO783635 A NO 783635A NO 783635 A NO783635 A NO 783635A NO 149611 B NO149611 B NO 149611B
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- prp
- isolation
- haemophilus influenzae
- hydroxylapatite
- phosphate buffer
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- 238000000034 method Methods 0.000 title claims description 19
- 229920001282 polysaccharide Polymers 0.000 title claims description 12
- 241000606768 Haemophilus influenzae Species 0.000 title claims description 10
- 238000002955 isolation Methods 0.000 title claims description 8
- 229940047650 haemophilus influenzae Drugs 0.000 title claims 2
- 238000004140 cleaning Methods 0.000 title 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 12
- 239000012064 sodium phosphate buffer Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 150000004676 glycans Chemical class 0.000 claims description 11
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 11
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 11
- 239000005017 polysaccharide Substances 0.000 claims description 11
- 229910019142 PO4 Inorganic materials 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 8
- 229940045808 haemophilus influenzae type b Drugs 0.000 claims description 6
- 238000011282 treatment Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 claims description 2
- 230000004151 fermentation Effects 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000012869 ethanol precipitation Methods 0.000 claims 2
- 238000004108 freeze drying Methods 0.000 claims 2
- 238000004458 analytical method Methods 0.000 claims 1
- 238000004440 column chromatography Methods 0.000 claims 1
- 238000000502 dialysis Methods 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 17
- 239000011550 stock solution Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 14
- 229960005486 vaccine Drugs 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 10
- 108020004707 nucleic acids Proteins 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- 239000002158 endotoxin Substances 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 8
- 239000010452 phosphate Substances 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 241000588832 Bordetella pertussis Species 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 201000005702 Pertussis Diseases 0.000 description 7
- 239000000356 contaminant Substances 0.000 description 7
- 108091005461 Nucleic proteins Proteins 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 239000003463 adsorbent Substances 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 150000002972 pentoses Chemical class 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 229940001442 combination vaccine Drugs 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229940025294 hemin Drugs 0.000 description 2
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 229950006238 nadide Drugs 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- DLYUQMMRRRQYAE-UHFFFAOYSA-N tetraphosphorus decaoxide Chemical compound O1P(O2)(=O)OP3(=O)OP1(=O)OP2(=O)O3 DLYUQMMRRRQYAE-UHFFFAOYSA-N 0.000 description 2
- 229940071127 thioglycolate Drugs 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-M thioglycolate(1-) Chemical compound [O-]C(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- -1 Bio. gel HTP Chemical compound 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229920002274 Nalgene Polymers 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 239000007621 bhi medium Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229940075894 denatured ethanol Drugs 0.000 description 1
- CGMRCMMOCQYHAD-UHFFFAOYSA-J dicalcium hydroxide phosphate Chemical compound [OH-].[Ca++].[Ca++].[O-]P([O-])([O-])=O CGMRCMMOCQYHAD-UHFFFAOYSA-J 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000001698 pyrogenic effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- MKWYFZFMAMBPQK-UHFFFAOYSA-J sodium feredetate Chemical compound [Na+].[Fe+3].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O MKWYFZFMAMBPQK-UHFFFAOYSA-J 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/099—Bordetella
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
-
- 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
- A61K2039/70—Multivalent vaccine
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
Denne oppfinnelse angår en metode for isolering og rensning av det antigene polysakkarid polyribosylribitol-fosfat (PRP) fra Haemophilus influenzae type b-kulturer. This invention relates to a method for isolating and purifying the antigenic polysaccharide polyribosylribitol phosphate (PRP) from Haemophilus influenzae type b cultures.
Det rensede polyribitolfosfat (PRP) fra Haemophilus influenzae type b undersøkes for tiden som et beskyttelses-immunogen. Et aktivt antigent svar hos unge dyr og barn er imidlertid ikke oppnådd. Ifølge den tidligere kjente metode for rensning anvendes etanol og "Cetavolon" (heksadecyltrimetyl-ammoniumbromid). Forurensningene, endotoksiner, og pyrogene stoffer ble fjernet ved anvendelse av kald fenol eller kloroform og t-butanol, som kan resultere i tap av den antigene karakter hos dette polysakkarid. The purified polyribitol phosphate (PRP) from Haemophilus influenzae type b is currently being investigated as a protective immunogen. However, an active antigenic response in young animals and children has not been achieved. According to the previously known method of purification, ethanol and "Cetavolon" (hexadecyltrimethylammonium bromide) are used. The contaminants, endotoxins and pyrogenic substances were removed using cold phenol or chloroform and t-butanol, which may result in the loss of the antigenic character of this polysaccharide.
En ny fremgangsmåte for isolering og rensning av immunologisk aktivt PRP fra Haemophilus influenzae type b er ut-viklet . A new method for isolating and purifying immunologically active PRP from Haemophilus influenzae type b has been developed.
Foreliggende fremgangsmåte oppviser klare fordeler i forhold til tidligere kjente metoder, ved at alle forurensninger (nukleinsyrer, proteiner og endotoksiner) fjernes til minst mulige nivå ved behandling med hydroksylapatitt. PRP fremstilt ved den her beskrevne fremgangsmåte, gir et mer høymolekylært polysakkarid enn hva som tidligere er rapportert. The present method shows clear advantages compared to previously known methods, in that all contaminants (nucleic acids, proteins and endotoxins) are removed to the lowest possible level by treatment with hydroxylapatite. PRP produced by the method described here gives a higher molecular weight polysaccharide than previously reported.
Det er videre av betydning at PRP fremstilt i henhold til denne nye metode i høy grad er immunogent hos varmblodige dyr i motsetning til det PRP som fremstilles i henhold til tidligere kjente metoder. It is also important that PRP produced according to this new method is highly immunogenic in warm-blooded animals in contrast to the PRP produced according to previously known methods.
En fremgangsmåte for fjernelse av forurensninger (proteiner, nukleinsyrer og endotoksiner) i polysakkaridet PRP oppnås ved behandling av det delvis rensede polysakkarid med et fosfatholdig adsorpsjonsmiddel som ikke absorberer polysakkaridet under de angitte betingelser. A method for removing contaminants (proteins, nucleic acids and endotoxins) in the polysaccharide PRP is achieved by treating the partially purified polysaccharide with a phosphate-containing adsorbent which does not absorb the polysaccharide under the specified conditions.
(I) Isolering og rensning av polyribosylribitol- fosfat, (I) Isolation and purification of polyribosylribitol phosphate,
det kapsulære polysakkarid av Haemophilus influenzae type b. the capsular polysaccharide of Haemophilus influenzae type b.
Organismer, dyrkningsmedium og kultur Organisms, culture medium and culture
To stammer av Haemophilus influenzae type b anvendes. Two strains of Haemophilus influenzae type b are used.
Rab-stammen erholdes fra Grace Leidy, Babies Hospital, Columbia University, New York City, New York. CK-stammen ble isolert fra en pasient ved Waterbury Hospital Waterbury, Conn. Organismene får passere gjennom mus flere ganger for å sikre deres virulens. The Rab strain was obtained from Grace Leidy, Babies Hospital, Columbia University, New York City, New York. The CK strain was isolated from a patient at Waterbury Hospital Waterbury, Conn. The organisms are passed through mice several times to ensure their virulence.
Organismene isoleres ved autopsi fra hjernevev hos mus, sub-kultivert på enten 3,7%ig hjerne-hjerte-infusjonsmedium (BHI) The organisms are isolated at autopsy from mouse brain tissue, sub-cultured on either 3.7% brain-heart infusion medium (BHI)
(Difco Lab., Detroit, Mich.) eller på 5%ig BHI-agar komplettert med 0,01%ig nikotinamid-adenin-dinukleotid (NAD) (P-L Biochemicals, Milwaukee, Wis.) og l%ig (volum/volum) defibrinert hesteblod (Animal Blood Center, Syracuse, N.Y.), og fordeles derefter i porsjoner på 1 ml på ampuller, lyofiliseres og oppbevares ved -70°C. (Difco Lab., Detroit, Mich.) or on 5% BHI agar supplemented with 0.01% nicotinamide adenine dinucleotide (NAD) (P-L Biochemicals, Milwaukee, Wis.) and 1% (vol/vol ) defibrinated horse blood (Animal Blood Center, Syracuse, N.Y.), and then dispensed in 1-ml portions into vials, lyophilized, and stored at -70°C.
Grunnmedium anvendt for dyrkning av organismene er Basic medium used for cultivation of the organisms is
3,7%ig BHI. Grunnmediet suppleres med 10 mg NAD og 20 mg hemin (Eastman Kodak, Rochester, N.Y.) pr. liter. 1% (volum/volum) defibrinert hesteblod tilsettes pr. 50 ml podekultur. Supple-mentene nyfremstilles og filtreres gjennom en 0,4 5 p Nalgene filterenhet (Nalge Sybron Corp., Rochester, N.Y.) før anvendelse. For dyrkning av organismen i en 14 liters fermentor suppleres mediet ytterligere med 0,5% glukose.' 3.7% BHI. The basic medium is supplemented with 10 mg NAD and 20 mg hemin (Eastman Kodak, Rochester, N.Y.) per litres. 1% (volume/volume) defibrinated horse blood is added per 50 ml inoculum. The supplements are freshly prepared and filtered through a 0.45 µm Nalgene filter unit (Nalge Sybron Corp., Rochester, N.Y.) prior to use. For cultivation of the organism in a 14 liter fermenter, the medium is further supplemented with 0.5% glucose.'
For å tilberede en kolbe med podeorganismer opptines To prepare a flask of inoculum organisms, optines
1 ml frosset forrådskultur og overføres til 50 ml av det anrikede BHI-mediet supplert med defibrinert hesteblod og NAD. Kulturen dyrkes i 8 timer ved 37°C på en gyroskakeanordning med en hastighet av 150 omdr./minutt. Organismene settes i form av et l%ig podestoff til 500 ml porsjoner av anriket BHI-dyrkningsvæske i 2 liters kolber, som derefter inkuberes ved 37°C med moderat skakning på en gyroskakeanordning i 8 timer (for isolering av PRP) eller 14 timer (som podekulturer). 1 ml of frozen stock culture and transfer to 50 ml of the enriched BHI medium supplemented with defibrinated horse blood and NAD. The culture is grown for 8 hours at 37°C on a gyro device at a speed of 150 rpm. The organisms are added as a 1% inoculum to 500 ml portions of enriched BHI culture medium in 2 liter flasks, which are then incubated at 37°C with moderate shaking on a gyro shaker for 8 hours (for isolation of PRP) or 14 hours ( as graft cultures).
Fermenteringen av hver sats i en 14 liters fermentor initieres ved aseptisk overføring av 700 ml av podekulturen til 7 liter av den anrikede BHI-dyrkningsvæsken supplert med hemin istedenfor defibrinert hesteblod. Kulturen opprettholdes kontinu-erlig ved 37 - 1°C. Tanken omrøres med en hastighet på 150 omdr./ minutt, og en luftstrøm på 0,25 liter luft pr. liter dyrkningsvæske pr. minutt opprettholdes. Under dyrkningen tilsettes 0,001% av et silikon-antiskummiddel (FD-82, Hodag Chemical Corp.) efter behov. Kulturer dyrkes til bare eksponentiell tilvekst (8-10 x 10 9 levedyktige celler pr. ml), vanligvis ca. 8 timer, hvorefter dyrkningen avsluttes ved at man tilsetter 0,4%ig formaldehyd og oppbevarer kulturene natten over ved 4°C. The fermentation of each batch in a 14 liter fermenter is initiated by aseptically transferring 700 ml of the inoculum to 7 liters of the enriched BHI culture fluid supplemented with hemin instead of defibrinated horse blood. The culture is maintained continuously at 37 - 1°C. The tank is stirred at a speed of 150 rpm, and an air flow of 0.25 liters of air per liter of culture liquid per minute is maintained. During cultivation, 0.001% of a silicone antifoam agent (FD-82, Hodag Chemical Corp.) is added as needed. Cultures are grown to only exponential growth (8-10 x 10 9 viable cells per ml), usually approx. 8 hours, after which the cultivation is terminated by adding 0.4% formaldehyde and storing the cultures overnight at 4°C.
Isolering av polyribosylribitol- fosfat ( PRP) Isolation of polyribosylribitol phosphate (PRP)
Dyrkningsvæske fremstilt i henhold til det ovenstående sentrifugeres ved 27.000 x g i 30 minutter ved 4°C. Den celle-frie væsken på toppen oppbevares og behandles på følgende måte: Culture fluid prepared according to the above is centrifuged at 27,000 x g for 30 minutes at 4°C. The cell-free liquid on top is stored and processed as follows:
Trinn 1, etanolutfeining Step 1, ethanol sweep
Væsken på toppen i kulturen tilsettes natriumacetat (sluttkonsentrasjon 4%). Oppløsningen innstilles på pH 6,0-6,2, og 44 liter med metanol denaturert etanol tilsettes langsomt under kraftig omrøring ved 4°C. Blandingen innstilles på pH 6,8 med iseddik og oppbevares derefter i 12 timer ved 3°C. Den dannede felningen oppsamles ved dekantering og sentrifugeres derefter for dannelse av urent PRP. Sodium acetate is added to the liquid at the top of the culture (final concentration 4%). The solution is adjusted to pH 6.0-6.2, and 44 liters of methanol denatured ethanol are added slowly with vigorous stirring at 4°C. The mixture is adjusted to pH 6.8 with glacial acetic acid and then stored for 12 hours at 3°C. The resulting precipitate is collected by decantation and then centrifuged to form impure PRP.
Trinn 2, " Cetavlon" ( heksadecyltrimetylammoniumbromid)- behandling Step 2, "Cetavlon" (hexadecyltrimethylammonium bromide) treatment
Felningen fra trinn 1 løses i pyrogenfritt destillert vann og sentrifugeres for å fjerne residuet. Den klare, brune opp-løsning settes derefter langsomt til 100 ml 10%ig vann-oppløsning av "Cetavlon" under blanding (sluttkonsentrasjon 0,5%). Blandingen omrøres i 1 time og sentrifugeres derefter. Felningen av nukleinsyrer og PRP-"Cetavlon"-kompleks blandes med 2 liter 0,3M natriumklorid. Den uklare oppløsningen sentrifugeres for å fjerne uoppløselig materiale så som imklein-"Cetavlon"-salt. The precipitate from step 1 is dissolved in pyrogen-free distilled water and centrifuged to remove the residue. The clear, brown solution is then slowly added to 100 ml of a 10% water solution of "Cetavlon" while mixing (final concentration 0.5%). The mixture is stirred for 1 hour and then centrifuged. The precipitation of nucleic acids and PRP "Cetavlon" complex is mixed with 2 liters of 0.3M sodium chloride. The cloudy solution is centrifuged to remove insoluble material such as Imklein "Cetavlon" salt.
Væsken på toppen fortynnes med en lik volummengde vann, hvorved PRP-"Cetavlon"-saltet utfelles. Blandingen omrøres i 1 time, felningen oppsamles ved sentrifugering og oppløses derefter i 2 liter 0,3M natriumkloridoppløsning. The liquid on top is diluted with an equal volume of water, whereby the PRP "Cetavlon" salt is precipitated. The mixture is stirred for 1 hour, the precipitate is collected by centrifugation and then dissolved in 2 liters of 0.3M sodium chloride solution.
Trinn 3, etanolutfeining Step 3, ethanol sweep
"Cetavlon" og forurensningene av nukleinsyrer og proteiner fjernes ytterligere ved etanolutfeining (minst to ganger). PRP utfelles i henhold til trinn 1, mens "Cetavlon" oppløses i alkoholoppløsningen. PRP oppsamles ved sentrifugering, løses i 2 liter pyrogenfritt, destillert vann og utfelles derefter igjen som beskrevet ovenfor. Den endelige PRP-feiningen solubiliseres i 20 mM natriumfosfat, pH 6,8. "Cetavlon" and the contaminants of nucleic acids and proteins are further removed by ethanol purification (at least twice). PRP is precipitated according to step 1, while "Cetavlon" is dissolved in the alcohol solution. The PRP is collected by centrifugation, dissolved in 2 liters of pyrogen-free, distilled water and then precipitated again as described above. The final PRP sweep is solubilized in 20 mM sodium phosphate, pH 6.8.
Trinn 4, hydroksylapatittbehandling Step 4, hydroxylapatite treatment
Forurensninger (så som nukleinsyrer, proteiner og endotoksiner) i de delvis rensede PRP-preparater fjernes selektivt ved adsorpsjon på hydroksylapatitt [3-Ca^(P04) 2Ca(OH) ^\ . Impurities (such as nucleic acids, proteins and endotoxins) in the partially purified PRP preparations are selectively removed by adsorption on hydroxylapatite [3-Ca^(PO4) 2Ca(OH)^\ .
Oppfinnelsen bygger på den oppdagelse at polysakkaridet PRP ikke adsorberes av den hydroksylapatittholdige fosfatbuffer (0,02M) med en pH på 6,7-6,9; eller en fosfatbuffer- (0,02M) med The invention is based on the discovery that the polysaccharide PRP is not adsorbed by the hydroxylapatite-containing phosphate buffer (0.02M) with a pH of 6.7-6.9; or a phosphate buffer (0.02M) med
en pH på ca. 5,8; mens derimot forurensningene (så som nukleinsyrer, proteiner og endotoksiner) adsorberes under disse betingelser. Fremgangsmåten ifølge oppfinnelsen kan utføres satsvis eller i kolonnedrift. Ved satsvis utførelse settes hydroksyl-apatitten til det delvis rensede PRP-preparatet (i 0,02M natriumfosfatbuffer; pH 6,7-6,9). Blandingen blandes omhyggelig ved 2-10°C, og sentrifugeres for å fjerne uønskede faste substanser (adsorpsjonsmiddel og de forurensninger som er adsorbert av adsorpsjonsmidlet). Væsken på toppen underkastes nevnte prosedyre minst ytterligere to ganger. Den dannede oppløsningen filtreres gjennom "Millipore"-filter, dialyseres mot pyrogenfritt destillert vann og lyofiliseres. a pH of approx. 5.8; while, on the other hand, the contaminants (such as nucleic acids, proteins and endotoxins) are adsorbed under these conditions. The method according to the invention can be carried out in batches or in column operation. In batch execution, the hydroxyl apatite is added to the partially purified PRP preparation (in 0.02M sodium phosphate buffer; pH 6.7-6.9). The mixture is carefully mixed at 2-10°C, and centrifuged to remove unwanted solids (adsorbent and the contaminants adsorbed by the adsorbent). The liquid on top is subjected to the aforementioned procedure at least two more times. The resulting solution is filtered through a "Millipore" filter, dialyzed against pyrogen-free distilled water and lyophilized.
Ved kolonnedrift påføres delvis renset PRP i 0,02M natriumfosfatbuffer (pH ca. 5,8) i en kolonne inneholdende adsorpsjonsmidlet hydroksylapatitt, som er bragt til likevekt med 0,02M natriumfosfatbuffer, pH ca. 5,8, og elueres med en trinnvis gradient av natriumfosfatbuffer, pH ca. 5,8 fra 0,02M til 0,05M. Fraksjoner oppsamles og analyseres med hensyn til pentose (og dermed med hensyn til polyribosylribitolfosfat). De fraksjoner som er positive med hensyn til pentose, dialyseres mot pyrogenfritt, destillert vann og lyofiliseres. During column operation, partially purified PRP is applied in 0.02M sodium phosphate buffer (pH approx. 5.8) in a column containing the adsorbent hydroxylapatite, which has been brought to equilibrium with 0.02M sodium phosphate buffer, pH approx. 5.8, and eluted with a stepwise gradient of sodium phosphate buffer, pH approx. 5.8 from 0.02M to 0.05M. Fractions are collected and analyzed for pentose (and thus for polyribosylribitol phosphate). The fractions that are positive with respect to pentose are dialyzed against pyrogen-free, distilled water and lyophilized.
Oppfinnelsen skal illustreres nærmere ved hjelp av de følgende eksempler hvor de angitte temperaturer er i °C. The invention shall be illustrated in more detail by means of the following examples where the indicated temperatures are in °C.
Eksempel 1 Example 1
2,5 g hydroksylapatitt (f.eks. Bio. gel HTP, Bio-Rad Laboratories, Richmond, California) settes til 250 ml delvis rensede PRP-preparater (efter "Cetavlon"- og etanolbehandlinger) 2.5 g of hydroxylapatite (e.g. Bio. gel HTP, Bio-Rad Laboratories, Richmond, California) is added to 250 ml of partially purified PRP preparations (after "Cetavlon" and ethanol treatments)
(inneholdende ca. 1,0 mg PRP/ml) i 20 mM natriumfosfatbuffer, (containing approx. 1.0 mg PRP/ml) in 20 mM sodium phosphate buffer,
pH 6,9, og blandes i et iskaldt vannbad (1-4°C) i 1 time. Blandingen sentrifugeres i en Sorvall RC2-B i 30 minutter ved 16.000 x g. Væsken på toppen føres derefter gjennom et 0,65 m milliporefilter og underkastes nevnte prosedyre (hver gang behandlet med 2,5 g hydroksylapatitt) ytterligere to ganger. Den erholdte oppløsning filtreres gjennom 0,65 u og 0,45 m milliporefilter, pH 6.9, and mixed in an ice-cold water bath (1-4°C) for 1 hour. The mixture is centrifuged in a Sorvall RC2-B for 30 minutes at 16,000 x g. The supernatant is then passed through a 0.65 m millipore filter and subjected to the aforementioned procedure (each time treated with 2.5 g of hydroxylapatite) twice more. The solution obtained is filtered through 0.65 u and 0.45 m millipore filters,
dialyseres mot pyrogenfritt, destillert vann og lyofiliseres. dialyzed against pyrogen-free, distilled water and lyophilized.
Det lyofiliserte produkt oppviser kraftig immunogen aktivitet The lyophilized product exhibits strong immunogenic activity
(se nedenfor med hensyn til dyreforsøk med kombinert vaksine) (see below regarding animal testing with combined vaccine)
og inneholder en meget liten mengde forurensninger (så som nukleinsyrer, proteiner og endotoksiner) (se tabell I nedenfor). Ca. 170 mg lyofilisert PRP erholdes. Gjenvinningen av PRP ved denne fremgangsmåte er ca. 70%, beregnet på utgangspolysakkaridet. PRP bør oppbevares under egnede betingelser, f.eks. ved 4° i en eksikator over fosforpentoksyd og silikagel. and contains a very small amount of contaminants (such as nucleic acids, proteins and endotoxins) (see Table I below). About. 170 mg of lyophilized PRP is obtained. The recovery of PRP by this method is approx. 70%, calculated on the starting polysaccharide. PRP should be stored under suitable conditions, e.g. at 4° in a desiccator over phosphorus pentoxide and silica gel.
E ksempel 2 Example 2
Delvis renset PRP (300 mg PRP) oppløses i 100 ml 20 mM natriumfosfatbuffer, pH 5,8 (dvs. 3 mg PRP/ml). Denne oppløsning påføres en kolonne ved romtemperatur (5,0 x 45 cm) av hydroksylapatitt (ca. 250 ml lagvolum), som er bragt til likevekt med 20 mM natriumfos fatbuffer, pH 5,8, og elueres med en trinnvis gradient av 20 mM, 50 mM og 100 mM natriumf osf atbuf fer, pH 5,8. Partially purified PRP (300 mg PRP) is dissolved in 100 ml 20 mM sodium phosphate buffer, pH 5.8 (ie 3 mg PRP/ml). This solution is applied to a column at room temperature (5.0 x 45 cm) of hydroxylapatite (approx. 250 ml layer volume), which has been brought to equilibrium with 20 mM sodium phosphate buffer, pH 5.8, and eluted with a stepwise gradient of 20 mM , 50 mM and 100 mM sodium phosphate buffer, pH 5.8.
De individuelle fraksjoner (200 ml), eluert med 20 mM og 50 mM natriumfosfatbuffer (pH 5,8), som er positive med hensyn til pentose (pentosebestemmelse i henhold til originalmetoden), oppbevares, dialyseres mot pyrogenfritt, destillert vann og lyofiliseres. Man får ca. 206 mg av det lyofiliserte produkt, The individual fractions (200 ml), eluted with 20 mM and 50 mM sodium phosphate buffer (pH 5.8), which are positive for pentose (pentose determination according to the original method), are stored, dialyzed against pyrogen-free distilled water and lyophilized. You get approx. 206 mg of the lyophilized product,
PRP. PRP.
Renheten av polysakkaridet, PRP, analyseres ved at man bedømmer i hvilken utstrekning de er forurenset med nukleinsyrer, proteiner og endotoksiner. Proteinkonsentrasjonen bestemmes ifølge Lowry et al. i J. Biol. Chem. 193:265 (1951) med bovinserum-albumin som standard. Nukleinsyren bestemmes ved absorpsjon av PRP-oppløsningen ved 2 60 nm. Absorbansen av 50 yg nukleinsyre The purity of the polysaccharide, PRP, is analyzed by assessing the extent to which they are contaminated with nucleic acids, proteins and endotoxins. The protein concentration is determined according to Lowry et al. in J. Biol. Chem. 193:265 (1951) with bovine serum albumin as standard. The nucleic acid is determined by absorption of the PRP solution at 2 60 nm. The absorbance of 50 ug nucleic acid
i 1 ml vann i en celle med lysvei 1 cm ansees å være lik 1,0. Molekylstørrelsen bedømmes ved hjelp av "Sepharose" 4B- eller 2B-gelfiltrering på kolonner 1,5 x 90 cm (Pharmacia-Fine Chemicals, Pissataway, N.J.). Fordelingskoeffisienten (Kd) bestemmes fra elueringsmønsteret fremkalt ved originalreaksjonen (Herbert et al, Methods in Microbiology, vol. 5B, 285-291). in 1 ml of water in a cell with a light path of 1 cm is considered to be equal to 1.0. Molecular size is assessed by "Sepharose" 4B or 2B gel filtration on 1.5 x 90 cm columns (Pharmacia-Fine Chemicals, Pissataway, N.J.). The partition coefficient (Kd) is determined from the elution pattern produced by the original reaction (Herbert et al, Methods in Microbiology, vol. 5B, 285-291).
For undersøkelse av virkningen av en vaksine som inneholder PRP fra H. influenzae ble fremstilt en kombinasjonsvaksine med B.pertussis-antigener: A. En kombinasjonsvaksine inneholdende PRP fra H. influenzae type b og B. pertussis-antigener fremstilles i henhold til følgende: 140 mg PRP (fremstilt ifølge eksempel 2) oppløses i 350 ml steril,, fosfatbufret natriumkloridoppløsning (PBS) (0,113 g kaliumfosfat, 0,83 g dinatriumfosfat og 8,5 g natriumklorid pr. liter, pH 7,0, inneholdende 0,01% timerosal) og filtreres gjennom et 0,45 y millipore-filter. Denne konsentrerte PRP-oppløsning (400 yg/ml) anvendes for fremstilling av en kombinasjonsvaksine, som består av PRP og B. pertussis-antigener. To investigate the effect of a vaccine containing PRP from H. influenzae, a combination vaccine with B. pertussis antigens was prepared: A. A combination vaccine containing PRP from H. influenzae type b and B. pertussis antigens is prepared according to the following: 140 mg of PRP (prepared according to example 2) is dissolved in 350 ml of sterile, phosphate-buffered sodium chloride solution (PBS) (0.113 g of potassium phosphate, 0.83 g of disodium phosphate and 8.5 g of sodium chloride per liter, pH 7.0, containing 0.01% thimerosal) and filtered through a 0.45 µm millipore filter. This concentrated PRP solution (400 µg/ml) is used for the production of a combination vaccine, which consists of PRP and B. pertussis antigens.
150 ml av den konsentrerte PRP-oppløsningen (400 yg/ml) tilsettes 150 ml frisk B. pertussis (stamme 138)-cellesuspensjon i PBS, pH 7,0 (med opacitet 149; op, enhetsceller/ml) for fremstilling av forrådsoppløsningen av den kombinerte vaksine. Denne forrådsoppløsning (300 ml) holdes ved 4° inntil endotoksinnivået av pertussis har sunket (minst 90 dager). Steriliteten av forråds-oppløsningen undersøkes med tioglykolatmedier (ved 32-33°). pH i forrådsoppløsningen efter 90 dagers inkubering kontrolleres og innstilles på pH 7,0 i 1. Sluttproduktet (vaksine) anvendt for dyreforsøk, fremstilles ved fortynning av den kombinerte forråds-oppløsning med PBS, og den inneholder 10 yg PRP og 3,5-op-enheter av pertussisceller/O,5 ml (dose). 150 ml of the concentrated PRP solution (400 µg/ml) is added to 150 ml of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (with opacity 149; op, unit cells/ml) to prepare the stock solution of the combined vaccine. This stock solution (300 ml) is kept at 4° until the pertussis endotoxin level has decreased (at least 90 days). The sterility of the stock solution is checked with thioglycolate media (at 32-33°). The pH of the stock solution after 90 days of incubation is checked and adjusted to pH 7.0 in 1. The final product (vaccine) used for animal experiments is prepared by diluting the combined stock solution with PBS, and it contains 10 yg of PRP and 3.5-op -units of pertussis cells/0.5 ml (dose).
B. Forrådsoppløsningen av PRP (400 yg/ml) fremstilt ved at man oppløser 30 mg PRP (fremstilt ifølge eksempel 1) i 75 ml steril, fosfatbufret natriumkloridoppløsning (PBS) og filtreres gjennom et 0,45 y milliporefilter. Til 25 ml av dette konsentrerte PRP settes en like stor volummengde (dvs. 25 ml) frisk B. pertussis (stamme 138)-cellesuspensjon i PBS, pH 7,0 (inneholdende 149 op-enheter celler/ml) for fremstilling av forrådsoppløsningen av den kombinerte vaksine. Denne forrådsoppløsning (50 ml) holdes i minst 90 dager ved 4°. Steriliteten av forrådsoppløsningen undersøkes som angitt ovenfor med tioglykolatmedier. pH i forråds-oppløsningen er 7,0 ± 1. Sluttproduktet (vaksine) anvendt for dyreforsøk fremstilles ved fortynning av den kombinerte forråds-oppløsningen med PBS, og den inneholder 10 yg PRP og 3,7 op-enheter B. The stock solution of PRP (400 ug/ml) prepared by dissolving 30 mg of PRP (prepared according to Example 1) in 75 ml of sterile, phosphate buffered sodium chloride solution (PBS) and filtering through a 0.45 um millipore filter. To 25 ml of this concentrated PRP is added an equal volume (ie 25 ml) of fresh B. pertussis (strain 138) cell suspension in PBS, pH 7.0 (containing 149 op-units cells/ml) to prepare the stock solution of the combined vaccine. This stock solution (50 ml) is kept for at least 90 days at 4°. The sterility of the stock solution is tested as indicated above with thioglycolate media. The pH of the stock solution is 7.0 ± 1. The final product (vaccine) used for animal experiments is prepared by diluting the combined stock solution with PBS, and it contains 10 µg of PRP and 3.7 op units
av pertussisceller/O,5 ml (dose). of pertussis cells/O.5 ml (dose).
C. PRP-forrådsoppløsningen (200 ug/ml) i PBS (pH 7,0) fremstilles som i B. Pertussis-forrådsoppløsningen (inneholdende 75 op-enheter/ml) fremstilles ved fortynning av 25 ml frisk B. pertussis-cellesuspensjon (149 op-enheter/ml) med en like stor volummengde PBS. Begge /forrådsoppløsninger inkuberes separat i 90 dager eller noe lenger ved 4°. Den kombinerte vaksine fremstilles ved at man tar 14 ml PRP-forråds-oppløsning (200 yq/ ml) og 14 ml (avgiftet) forrådsoppløsning av pertussis-antigener (75 op-enheter/ml) og fortynner den med 112 ml PBS (sluttvolum 140 ml). Den ferdige vaksine for dyre-forsøk inneholder 10 Mg PRP og 3,7 op-enneter pertussis/0,5 ml (dose). C. The PRP stock solution (200 ug/ml) in PBS (pH 7.0) is prepared as in B. The pertussis stock solution (containing 75 op units/ml) is prepared by diluting 25 ml of fresh B. pertussis cell suspension (149 op-units/ml) with an equal volume of PBS. Both stock solutions are incubated separately for 90 days or slightly longer at 4°. The combined vaccine is prepared by taking 14 ml of PRP stock solution (200 yq/ml) and 14 ml (detoxified) stock solution of pertussis antigens (75 op units/ml) and diluting it with 112 ml of PBS (final volume 140 ml). The finished vaccine for animal experiments contains 10 Mg PRP and 3.7 pertussis pertussis units/0.5 ml (dose).
Resultatene med hensyn til antistoffsvaret på denne kombinerte vaksine hos unge rotter angis i tabell II. The results regarding the antibody response to this combined vaccine in young rats are shown in Table II.
Sammenligningsforsøk Comparison experiment
. I den følgende tabell er det vist at PRP fremstilt . In the following table it is shown that PRP is produced
i henhold til oppfinnelsen. Jean kombineres med B. pertussis celler og at den resulterende kombinerte vaksine fremkaller en betydelig større antistoff-reaksjon hos unge individer enn hva tilfellet er med vaksiner inneholdende PRP fremstilt ved en fenol-ekstraksjonsmetode. according to the invention. Jean is combined with B. pertussis cells and that the resulting combined vaccine elicits a significantly greater antibody reaction in young individuals than is the case with vaccines containing PRP prepared by a phenol extraction method.
Sammenligning av antistoff-reaksjon fremkalt med vaksiner inneholdende PRP fremstilt ved forskjellige fremgangsmåter Comparison of antibody response elicited by vaccines containing PRP prepared by different methods
Antistoff-speil - 0,15 ug/ml ansees å være beskyttende Antibody mirror - 0.15 ug/ml is considered protective
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US05/846,466 US4196192A (en) | 1977-10-28 | 1977-10-28 | Combined Haemophilus influenzae type b and pertussis vaccine |
US05/846,488 US4220717A (en) | 1977-12-22 | 1977-12-22 | Isolation and purification of polyribosyl ribitol phosphate from Haemophilus influenzae type b. |
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CA1209036A (en) * | 1982-08-20 | 1986-08-05 | Joseph S.C. Kuo | Combined haemophilus influenzae and diphtheria, pertussis, tetanus vaccine |
US4744982A (en) * | 1982-08-24 | 1988-05-17 | Hunter Kenneth W | Human monoclonal antibody reactive with polyribosylribitol phosphate |
EP0471177B1 (en) * | 1990-08-13 | 1995-10-04 | American Cyanamid Company | Filamentous hemagglutinin of bordetella pertussis as a carrier molecule for conjugate vaccines |
WO1993004183A1 (en) * | 1991-08-16 | 1993-03-04 | Merck & Co., Inc. | Process for converting lipid-containing bacterial capsular polysaccharide into lipid-free polysaccharide |
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US3395219A (en) * | 1964-12-11 | 1968-07-30 | Merck & Co Inc | Process for production of pertussis antigen |
NO116869B (en) * | 1965-01-19 | 1969-06-02 | Merck & Co Inc | |
US3465078A (en) * | 1965-10-21 | 1969-09-02 | Sydney Z Spiesel | Method of recovering antigens from bordetella pertussis cells |
NL174267B (en) * | 1969-05-20 | Roussel Uclaf | IMPROVEMENT OF THE PROCESS FOR THE PREPARATION OF A SOMATIC ANTIGEN ACCORDING TO DUTCH PATENT 169754 AND PROCESS FOR PREPARING A PHARMACEUTICAL PREPARATION. | |
US3636192A (en) * | 1970-01-13 | 1972-01-18 | Us Army | Meningococcal polysaccharide vaccines |
FR2253499B1 (en) * | 1973-12-10 | 1977-11-04 | Fabre Sa Pierre | |
DE2461439C3 (en) * | 1974-12-24 | 1980-03-20 | Behringwerke Ag, 3550 Marburg | Process for the preparation of a protective antigen from Bordetella pertussis and agent containing the same |
US3978209A (en) * | 1975-03-24 | 1976-08-31 | Merck & Co., Inc. | Endotoxin free meningococcus polysaccharides |
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