WO1981000050A1 - Process for producing hepatitis b vaccine - Google Patents
Process for producing hepatitis b vaccine Download PDFInfo
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- WO1981000050A1 WO1981000050A1 PCT/US1980/000834 US8000834W WO8100050A1 WO 1981000050 A1 WO1981000050 A1 WO 1981000050A1 US 8000834 W US8000834 W US 8000834W WO 8100050 A1 WO8100050 A1 WO 8100050A1
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- Prior art keywords
- hbsag
- vaccine
- precipitate
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- particles
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- SPSXSWRZQFPVTJ-ZQQKUFEYSA-N hepatitis b vaccine Chemical compound C([C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)OC(=O)CNC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)C1=CC=CC=C1 SPSXSWRZQFPVTJ-ZQQKUFEYSA-N 0.000 title claims abstract description 7
- 229940124736 hepatitis-B vaccine Drugs 0.000 title claims abstract description 7
- 238000000034 method Methods 0.000 title claims description 20
- 229960005486 vaccine Drugs 0.000 claims abstract description 32
- 239000002245 particle Substances 0.000 claims abstract description 23
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 18
- 239000002244 precipitate Substances 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 7
- 238000005199 ultracentrifugation Methods 0.000 claims abstract description 7
- 102000004506 Blood Proteins Human genes 0.000 claims abstract description 6
- 108010017384 Blood Proteins Proteins 0.000 claims abstract description 6
- 241000700605 Viruses Species 0.000 claims abstract description 6
- 238000002523 gelfiltration Methods 0.000 claims abstract description 6
- 239000007900 aqueous suspension Substances 0.000 claims abstract description 4
- 230000007935 neutral effect Effects 0.000 claims abstract description 4
- 235000004252 protein component Nutrition 0.000 claims abstract description 4
- 230000005484 gravity Effects 0.000 claims abstract description 3
- 208000002672 hepatitis B Diseases 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 230000000415 inactivating effect Effects 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 5
- 239000000725 suspension Substances 0.000 claims description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 18
- 238000011282 treatment Methods 0.000 description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 9
- 241000282579 Pan Species 0.000 description 8
- 239000000427 antigen Substances 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 229910002012 Aerosil® Inorganic materials 0.000 description 5
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000005194 fractionation Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 241000282577 Pan troglodytes Species 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 229940009976 deoxycholate Drugs 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- KKEBXNMGHUCPEZ-UHFFFAOYSA-N 4-phenyl-1-(2-sulfanylethyl)imidazolidin-2-one Chemical compound N1C(=O)N(CCS)CC1C1=CC=CC=C1 KKEBXNMGHUCPEZ-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 206010019663 Hepatic failure Diseases 0.000 description 2
- 241000700721 Hepatitis B virus Species 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000008119 colloidal silica Substances 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 231100000835 liver failure Toxicity 0.000 description 2
- 208000007903 liver failure Diseases 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 102000007330 LDL Lipoproteins Human genes 0.000 description 1
- 108010007622 LDL Lipoproteins Proteins 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000003460 anti-nuclear Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 229960003964 deoxycholic acid Drugs 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
- 239000003480 eluent Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 238000012317 liver biopsy Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
-
- 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
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- This invention relates to a process for producing a hepatitis B vaccine containing HBsAg particles as main component thereof, and more particularly, to a process for producing said vaccine which com- prises removing human plasma components possibly causing adverse effects and components capable of causing hepatitis B infection by in ⁇ corporating a step of fractionation using a polyethylene glycol into the purification, process with success, and further carrying out a treatment for inactivating the infectious virus without causing any impairment of the antigenicity.
- HBsAg (HBs antigen) is known to be a constituent of a virus capable of causing hepatitis B (HBV). However, most of it is present as minute particles lacking infectivity and it can be found in the human or chimpanzee plasma. Electrophoretically, the HBsAg belongs to the class of human plasma globulin proteins.
- an HBs antibody-containing globulin preparation is administered so as to produce passive immunity, but the effect is of short duration. It goes without saying that the active immunization with a vaccine is the most preferable, as can be seen in the prevention of usual viral infections.
- a vaccine against hepatitis B is provided by isolating a hepatitis B virus substance and inactivating the same by an appropriate method or by separating the unchanged virus from an HBsAg followed by supplementary inactivation.
- This vaccine when ad ⁇ ministered to man or animal, induces production of antibodies against the hepatitis B virus and thus can prevent the infection with hepatitis B.
- Many attempts have been made to produce HBsAg vaccines.
- One method of inactivating the viral infectivity consists in heat treatment at 60 C for 10 hours or at 100°C for 2 minutes. The heat treatment, however, cannot assure sufficient decomposition of nucleic acids (DNA) which the HBV contains. Therefore, the technique now coming into wide use is the inactivation of HBV by formalin treatment.
- the most generally employed methods of isolating and puri ⁇ fying the HBsAg are ultracentrifugation and affinity chromatography. By using these techniques, almost pure HBsAg particles can be obtained.
- the conventional methods of isolation are generally complica ⁇ ted and present difficulties in industrial utilization thereof. More ⁇ over, a slight amount of concomitant human plasma proteins, once modi ⁇ fied by the formalin treatment, may, on administration to man, cause adverse effects, and therefore such proteins should be removed as co - pletely as possible. This trace amount of accompanying human plasma components cannot be removed even by repeated runs of ultracentrifuga ⁇ tion or affinity chromatography, so that some researchers took the view that such components should be present in the form bound to the HBsAg.
- the present inventors have paid their attention to the fact that polyethylene glycols exhibit excellent performance in fractiona- tion of human plasma proteins, and, as a result of investigations on the conditions of incorporation of the step of fractionation with polyethylene glycols into the process for producing HBsAg particles, have succeeded in obtaining highly pure HBsAg particles in high yields.
- the present invention consists in a process for producing a hepatitis B vaccine containing as main component thereof HBsAg (HBs antigen) particles free from human plasma components capable of causing hepatitis B infection, which comprises the steps of adjusting to approximately neutral the pH of an aqueous suspension of a crude HBsAg prepared from an HBsAg-containing human serum or plasma by
- OMPI removing therefrom most of plasma protein components, adding to said suspension a polyehtylene glycol to a concentration of 5-6% and thereby removing Dane particles capable of causing hepatitis B infection and immune complexes both as a precipitate, adding a further amount of a polyethylene glycol to the supernatant and thereby collecting HBsAg particles as a precipitate, recovering from said precipitate a specific fraction of HBsAg particles having a particle size of 22 nm and a
- the HBs antigen is recovered from the HBsAg-positive serum or plasma provided by healthy donors, and purified.
- a serum having an HBsAg titer of 1:16 or more as measured by the counter electrophoresis technique (CEP) may selectively be used for effective vaccine production.
- the two roughly classifiable subtypes "ad” and "ay” may be treated separately.
- the method of recovering the crude HBsAg is not limited, pro ⁇ vided that it is suited for large-scale purification.
- adsorption and elution on a colloid gel such as colloidal silica, for example, may preferably be employed.
- the crude HBsAg is the one from which most of the plasma protein components has been removed.
- the HBsAg is recovered as a precipitate by increasing the PEG concentration in the HBsAg-containing supernatant.
- the final PEG con ⁇ centration is 10-20% (w/v).
- the HBsAg-containing precipitate so obtained is then sub ⁇ jected to equalization or uniformization of the HBsAg particles and to removal of salting out reagents and other additives added during the production process.
- conventional techniques may be used, such as dialysis, gel filtration and ultracentrifugation.
- molecular sieve carriers applicable to substances having molecular weights between several hundred thousand and several million, such as Agarose (Sepharose 4B, 6B), cross-linked dextran and other high molecular polysaccharide granules, are employed.
- the recovery of the HBsAg fraction is carried out while assaying the HBsAg-positive fractions by immunological methods.
- the zonal centrifugation is pre ⁇ ferred, with a linear gradient of the concentration of cesium chloride
- the so-obtained HBsAg particles medicinally as a vaccine are desalted by dialysis, further sterilized by filtra ⁇ tion, and moreover inactivated so as to eliminate the possibility of remaining infectivity of the HBV.
- the inactivation is effected by adjusting the antigen purified in the manner mentioned above to a CEP titer of 1:20 and treating the antigen with 0.1-0.02% (w v) formalin, preferably 0.04% (w/v) formalin. After the formalin treatment, the final titer is adjusted to 1:4 as measured by the CEP. Finally, sterilization by filtration is carried out by using a 0.22 ⁇ ⁇ millipore filter.
- the vaccine may be administered at any dosage level and by any method of administration that will bring the vaccine into body fluids and make the same effectively act on the immunization mechanisms within the body so as to produce antibodies.
- the activity of the vaccine may be increased by admixing aluminum hydroxide or some other adjuvant known to those skilled in the art.
- the vaccine of the present invention is administered intra ⁇ muscularly or subcutaneously or via some other appropriate route. How ⁇ ever, intramuscular administration is preferred.
- intramuscular administration is preferred.
- the HBsAg vaccine so obtained, it was administered to the chimpanzee known to be the only animal species other than the human that could be infected with the HBV. Actually, however, no chimpanzee was infected. This fact revealed the safety of the vaccine and at the same time strongly suggested that the HBsAg vaccine might be effective in the prophylaxis of HBV, because the chimpanzees given the vaccine showed the production of HBs anti ⁇ bodies in the blood.
- this vaccine was then administered to actual patients under dialytic treatment. No adverse effects were observed, and the incidence rate of hepatitis B in the group of patients given the vaccine was significantly lower than that in the control group of patients. Thus the effectiveness of the vaccine has been established.
- the HBs antigen was obtained from an HBsAg-positive plasma from healthy blood donors by purifying said plasma.
- the plasma employed for the production of a vaccine had an HBsAg titer of not less than 1:16 when measured by the CEP (on a HYLAND Electrophoresis apparatus).
- the HBsAg subtypes "ad” and "ay” were purified separately.
- fibrin was removed together with ⁇ - lipoproteins by Mann's precipitation technique.
- the HBsAg was eluted from the Aerosil with one liter of a deoxycholate-containing borate buffer (0.01M sodium borate, 0.5% (w/v) sodium deoxycholate, pH 9).
- the elution was conducted at 37°C for 2 hours under stirring with a magnetic sti rrer.
- the Aerosil was separated by centrifugation on a Beckman TJ6 centrifuge at 1,600 g for 10 minutes.
- the supernatant ob- tained in the centrifugation contained the HBsAg.
- the supernatant was adjusted to pH 7,2 with 1N-HC1. (At a pH below 7, the deoxycholate becomes insoluble and precipitates out.)
- PEG 6,000 was added to a concentration of 5.5% (w/v). Stirring was effected with a magnetic sti rrer for 10 minutes to complete dissolu ⁇ tion. The mixture was then allowed to stand at 4°C for 5-6 hours. The resultant precipitate was removed by centrifugation at 1,600 g for 30 minutes. To the HBsAg-containing supernatant was added a further amount (12.5%) of PEG 6,000 to a final concentration of 18% (w/v). After stirring with a magnetic stirrer for 10 minutes to complete dissolution, the mixture was allowed to stand at 4°C overnight. There ⁇ after the mixture was centrifuged at 25,000 g for 15 minutes (Beckman J 21). The pellet contained the HBsAg. 55. Dissolution of PEG 6,000 pellet
- the gel filtration was carried out on a Sephadex 4B column ( 50/100, 50 mm in diameter, 1 in length, Pharmacia Fine Chemical, Uppsala, Sweden).
- the chro atographic gel was used after precoating with a normal human serum (50 ml). 15
- the equilibration of the column was performed with a PBS buffer having the following composition and the pH:
- the rate of eluent flow was 120 ml/h.
- the HBsAg-positive fractions were detected by the CEP, pooled, and concentrated on an Ami con Cell (XM 100 membrane DIAFL0) to a final 5 vol ume of 50 ml .
- the zonal ultracentrifugation at a linear cesium chloride concentra ⁇ tion gradient (density: 1.10-1.40) was performed on a zonal rotor (Type 0 Ti 14 Beckman L 5.65) at 43,000 rpm for 20 hours.
- the HBsAg-positive fractions (1.18-1.22) was pooled, and dialyzed against 9% sodium chloride.
- the antigen so purified was adjusted to a CEP titer of 1:20.
- the 35 subtypes "ad” and “ay”, after adjustment to equal titers and equal volumes, were pooled, and then subjected to formalin (30%) treatment.
- the formalin treatment was carried out by allowing the mixture at a formalin concentration of 1/2500 (i.e. 0.04%) to stand at 37°C for 48
- a sterilized aluminum hydroxide suspension was added to a final concentration of 0.1%.
- Test Example a Safety evaluation in chimpanzees.
- the vaccine prepared on a large scale was tested for its safety in 5 chimpanzees, there could not be obtained any chemical or serological evidence that they had been in ⁇ fected with the HBV.
- liver biopsy any histological sign of hepatic failure could not be found, either.
- 5 chimpanzees were injected intravenously with 5.0 ml of a concentrated HBsAg vaccine (CEP titer 1:8) twice at a one month interval. Three to four months later, 1.0 ml of the same vaccine preparation was administered to each animal by intramuscular injection.
- any of the five chimpanzees did not show any hepatic failure from both the laboratory and histological stand- points. Neither HBsAg nor anti HBs could be detected.
- the latex test was made, in the chimpanzees given the vaccine, to detect the possible existence of antiglobul n antibodies, antinuclear antibodies, antimitochondria anti ⁇ bodies and antismooth muscle antibodies. As a result, any autoantibodies could not be detected in any of the chimpanzees.
- the same vaccine preparation was administered to 353 men.
- the efficacy of the vaccine was evaluated by the appearance of humoral immune response to the HBsAg (anti HBs antibodies) and by an epide iological survey of the subjects given ⁇ the vaccine and the subjects not given the vaccine.
- the results obtained during the two year period after the immunization have proved that the vaccine preparation of the present invention can produce a prophylactic effect even in the circumstances where the risk of HB infection is high. No subjects given the vaccine preparation of the present invention showed any clinical or biological signs suggestive of autoimmune diseases.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
A hepatitis B vaccine is prepared by adjusting the pH of an aqueous suspension of a crude HBsAg to neutral by removing plasma protein components. Polyethylene glycol is added to a 5-6% concentration to thereby remove Dane particles and immune complexes as a precipitate. A further amount of a polyethylene glycol is added to thereby collect HBsAg particles as a precipitate. A specific fraction of HBsAg particles having a particle size of 22 nm and a specific gravity of 1.19-1.21 g/cm3 is recovered from the precipitate by gel filtration and ultracentrifugation. The virus contained in the fraction is inactivated and the fraction is made into a vaccine preparation.
Description
PROCESS FOR PRODUCING HEPATITIS B VACCINE
This invention relates to a process for producing a hepatitis B vaccine containing HBsAg particles as main component thereof, and more particularly, to a process for producing said vaccine which com- prises removing human plasma components possibly causing adverse effects and components capable of causing hepatitis B infection by in¬ corporating a step of fractionation using a polyethylene glycol into the purification, process with success, and further carrying out a treatment for inactivating the infectious virus without causing any impairment of the antigenicity.
HBsAg (HBs antigen) is known to be a constituent of a virus capable of causing hepatitis B (HBV). However, most of it is present as minute particles lacking infectivity and it can be found in the human or chimpanzee plasma. Electrophoretically, the HBsAg belongs to the class of human plasma globulin proteins.
According to one method of protection against infection with HBV, an HBs antibody-containing globulin preparation is administered so as to produce passive immunity, but the effect is of short duration. It goes without saying that the active immunization with a vaccine is the most preferable, as can be seen in the prevention of usual viral infections.
Theoretically, a vaccine against hepatitis B is provided by isolating a hepatitis B virus substance and inactivating the same by an appropriate method or by separating the unchanged virus from an HBsAg followed by supplementary inactivation. This vaccine, when ad¬ ministered to man or animal, induces production of antibodies against the hepatitis B virus and thus can prevent the infection with hepatitis B.
Many attempts have been made to produce HBsAg vaccines. One method of inactivating the viral infectivity consists in heat treatment at 60 C for 10 hours or at 100°C for 2 minutes. The heat treatment, however, cannot assure sufficient decomposition of nucleic acids (DNA) which the HBV contains. Therefore, the technique now coming into wide use is the inactivation of HBV by formalin treatment.
The most generally employed methods of isolating and puri¬ fying the HBsAg are ultracentrifugation and affinity chromatography. By using these techniques, almost pure HBsAg particles can be obtained. However, the conventional methods of isolation are generally complica¬ ted and present difficulties in industrial utilization thereof. More¬ over, a slight amount of concomitant human plasma proteins, once modi¬ fied by the formalin treatment, may, on administration to man, cause adverse effects, and therefore such proteins should be removed as co - pletely as possible. This trace amount of accompanying human plasma components cannot be removed even by repeated runs of ultracentrifuga¬ tion or affinity chromatography, so that some researchers took the view that such components should be present in the form bound to the HBsAg. It is an object of the present invention to provide a commer¬ cial process for producing a hepatitis B vaccine containing HBsAg par¬ ticles as main component thereof, which comprises removing plasma pro¬ tein components which may possibly become a cause for adverse effects, while maintaining the antigenicity of the HBsAg particles, then re- covering the HBsAg, and further inactivating the possibly accompanying HBV, that is Dane particles.
The present inventors have paid their attention to the fact that polyethylene glycols exhibit excellent performance in fractiona- tion of human plasma proteins, and, as a result of investigations on the conditions of incorporation of the step of fractionation with polyethylene glycols into the process for producing HBsAg particles, have succeeded in obtaining highly pure HBsAg particles in high yields.
The present invention consists in a process for producing a hepatitis B vaccine containing as main component thereof HBsAg (HBs antigen) particles free from human plasma components capable of causing hepatitis B infection, which comprises the steps of adjusting to approximately neutral the pH of an aqueous suspension of a crude HBsAg prepared from an HBsAg-containing human serum or plasma by
OMPI
removing therefrom most of plasma protein components, adding to said suspension a polyehtylene glycol to a concentration of 5-6% and thereby removing Dane particles capable of causing hepatitis B infection and immune complexes both as a precipitate, adding a further amount of a polyethylene glycol to the supernatant and thereby collecting HBsAg particles as a precipitate, recovering from said precipitate a specific fraction of HBsAg particles having a particle size of 22 nm and a
3 specific gravity of 1.19-1.21 g/cm by gel filtration and ultracentri¬ fugation, inactivating the virus contained in said fraction and making the fraction into the form of a vaccine preparation.
In the practice of the present invention, the HBs antigen is recovered from the HBsAg-positive serum or plasma provided by healthy donors, and purified. A serum having an HBsAg titer of 1:16 or more as measured by the counter electrophoresis technique (CEP) may selectively be used for effective vaccine production. The two roughly classifiable subtypes "ad" and "ay" may be treated separately.
The method of recovering the crude HBsAg is not limited, pro¬ vided that it is suited for large-scale purification. Thus, adsorption and elution on a colloid gel such as colloidal silica, for example, may preferably be employed. Preferably, the crude HBsAg is the one from which most of the plasma protein components has been removed.
From the resulting crude HBsAg, trace amounts of contaminants such as the infectious component of the HBV and the immune complexes are removed as a precipitate by fractionation using a polyethylene glycol. Polyethylene glycols (hereinafter PEG) having molecular weights of 2,000-10,000 are used. The treatment conditions are such that the aqueous solution is kept neutral (pH 6-8) and the temperature is maintained at 2-10 C (lower temperatures being preferable). The PEG concentration is 5-6% (w/v), preferably 5.3-5.7% (w/v). The time re- quired for the treatment is generally 3-7 hours. The resulting .precipi¬ tate is removed as a foreign matter.
The HBsAg is recovered as a precipitate by increasing the PEG concentration in the HBsAg-containing supernatant. The final PEG con¬ centration is 10-20% (w/v). The HBsAg-containing precipitate so obtained is then sub¬ jected to equalization or uniformization of the HBsAg particles and to removal of salting out reagents and other additives added during the production process. For these purposes, conventional techniques may be used, such as dialysis, gel filtration and ultracentrifugation.
- - EXζj-
OMFI
In the gel filtration, molecular sieve carriers applicable to substances having molecular weights between several hundred thousand and several million, such as Agarose (Sepharose 4B, 6B), cross-linked dextran and other high molecular polysaccharide granules, are employed. The recovery of the HBsAg fraction is carried out while assaying the HBsAg-positive fractions by immunological methods.
In the ultracentrifugation, the zonal centrifugation is pre¬ ferred, with a linear gradient of the concentration of cesium chloride
3 (CsCl), sodium bromide, etc. (1.10-1.40 g/cm ). Those HBsAg particles
3 that fall under the density range of 1.19-1.21 g/cm are recovered.
In using the so-obtained HBsAg particles medicinally as a vaccine, they are desalted by dialysis, further sterilized by filtra¬ tion, and moreover inactivated so as to eliminate the possibility of remaining infectivity of the HBV. The inactivation is effected by adjusting the antigen purified in the manner mentioned above to a CEP titer of 1:20 and treating the antigen with 0.1-0.02% (w v) formalin, preferably 0.04% (w/v) formalin. After the formalin treatment, the final titer is adjusted to 1:4 as measured by the CEP. Finally, sterilization by filtration is carried out by using a 0.22 ~ι millipore filter.
The vaccine may be administered at any dosage level and by any method of administration that will bring the vaccine into body fluids and make the same effectively act on the immunization mechanisms within the body so as to produce antibodies. The activity of the vaccine may be increased by admixing aluminum hydroxide or some other adjuvant known to those skilled in the art.
The vaccine of the present invention is administered intra¬ muscularly or subcutaneously or via some other appropriate route. How¬ ever, intramuscular administration is preferred. * For the confirmation of the safety of the HBsAg vaccine so obtained, it was administered to the chimpanzee known to be the only animal species other than the human that could be infected with the HBV. Actually, however, no chimpanzee was infected. This fact revealed the safety of the vaccine and at the same time strongly suggested that the HBsAg vaccine might be effective in the prophylaxis of HBV, because the chimpanzees given the vaccine showed the production of HBs anti¬ bodies in the blood.
Therefore, this vaccine was then administered to actual patients under dialytic treatment. No adverse effects were observed,
and the incidence rate of hepatitis B in the group of patients given the vaccine was significantly lower than that in the control group of patients. Thus the effectiveness of the vaccine has been established.
Example 1. Ant gen material
The HBs antigen was obtained from an HBsAg-positive plasma from healthy blood donors by purifying said plasma.
The plasma employed for the production of a vaccine had an HBsAg titer of not less than 1:16 when measured by the CEP (on a HYLAND Electrophoresis apparatus). The HBsAg subtypes "ad" and "ay" were purified separately.
As the material was a plasma, fibrin was removed together with β- lipoproteins by Mann's precipitation technique.
2. Adsorption of HBsAg on colloid silica To the supernatant resulting from Mann's precipitation, there was added Aerosil R380 (colloidal silica; Degussa, West Germany) to a con¬ centration of 2.5% (w/v). The mixture was then stirred gently by means of a magnetic sti rrer at 37°C for 3 hours. The reaction suspension was centrifuged on a Beckman TJ6 centrifuge at 1,600 g for 10 minutes. The HBsAg-containing Aerosil pellet was washed four times with one liter of a buffered EDTA (0.15M) -NaCl (0.15M) solution {pH 7.6).
3. Elution of HBsAg from Aerosil with deoxycholate-containing borate buffer
After the final washing, the HBsAg was eluted from the Aerosil with one liter of a deoxycholate-containing borate buffer (0.01M sodium borate, 0.5% (w/v) sodium deoxycholate, pH 9).
The elution was conducted at 37°C for 2 hours under stirring with a magnetic sti rrer. The Aerosil was separated by centrifugation on a Beckman TJ6 centrifuge at 1,600 g for 10 minutes. The supernatant ob- tained in the centrifugation contained the HBsAg.
4. Fractionation and concentration with PEG 6,000
The supernatant was adjusted to pH 7,2 with 1N-HC1. (At a pH below 7, the deoxycholate becomes insoluble and precipitates out.)
Then PEG 6,000 was added to a concentration of 5.5% (w/v). Stirring was effected with a magnetic sti rrer for 10 minutes to complete dissolu¬ tion. The mixture was then allowed to stand at 4°C for 5-6 hours. The resultant precipitate was removed by centrifugation at 1,600 g for 30 minutes. To the HBsAg-containing supernatant was added a further amount (12.5%) of PEG 6,000 to a final concentration of 18% (w/v).
After stirring with a magnetic stirrer for 10 minutes to complete dissolution, the mixture was allowed to stand at 4°C overnight. There¬ after the mixture was centrifuged at 25,000 g for 15 minutes (Beckman J 21). The pellet contained the HBsAg. 55. Dissolution of PEG 6,000 pellet
To the pellet was added 50 ml of 0.0175M sodium phosphate buffer (pH 6.8), and the mixture was homogenized by stirring with a magnetic stirrer for 4 hours, and then centrifuged at 1,600 g for 30 minutes. The supernatant (50 ml) contained the HBsAg. 106. Gel filtration chromatography
The gel filtration was carried out on a Sephadex 4B column ( 50/100, 50 mm in diameter, 1 in length, Pharmacia Fine Chemical, Uppsala, Sweden). The chro atographic gel was used after precoating with a normal human serum (50 ml). 15 The equilibration of the column was performed with a PBS buffer having the following composition and the pH:
NaCl (sodium chloride) 0.14M
Na2HP0» (d sodium hydrogen phosphate) 0.0113M
NaH2P0, (sodium dihydrogen phosphate) 0.0017M 0 NaN3 (sodium azide) 0.02% pH 7.2
The rate of eluent flow was 120 ml/h.
The HBsAg-positive fractions were detected by the CEP, pooled, and concentrated on an Ami con Cell (XM 100 membrane DIAFL0) to a final 5 vol ume of 50 ml .
7. Zonal ultracentrifugation with linear cesium chloride concentration gradient
The zonal ultracentrifugation at a linear cesium chloride concentra¬ tion gradient (density: 1.10-1.40) was performed on a zonal rotor (Type 0 Ti 14 Beckman L 5.65) at 43,000 rpm for 20 hours. The HBsAg-positive fractions (1.18-1.22) was pooled, and dialyzed against 9% sodium chloride.
8. Inactivation
The antigen so purified was adjusted to a CEP titer of 1:20. The 35 subtypes "ad" and "ay", after adjustment to equal titers and equal volumes, were pooled, and then subjected to formalin (30%) treatment. The formalin treatment was carried out by allowing the mixture at a formalin concentration of 1/2500 (i.e. 0.04%) to stand at 37°C for 48
~
hours and then at 4 C for 8 days. After the formalin treatment, the preparation was adjusted to a final antigen titer of 1:4 as measured by the CEP. Finally, the preparation was sterilized by filtration through a 0.22 u millipore filter. 9. Addition of adjuvant
A sterilized aluminum hydroxide suspension was added to a final concentration of 0.1%.
Test Example a) Safety evaluation in chimpanzees. When the vaccine prepared on a large scale was tested for its safety in 5 chimpanzees, there could not be obtained any chemical or serological evidence that they had been in¬ fected with the HBV. On liver biopsy, any histological sign of hepatic failure could not be found, either. More particularly, 5 chimpanzees were injected intravenously with 5.0 ml of a concentrated HBsAg vaccine (CEP titer 1:8) twice at a one month interval. Three to four months later, 1.0 ml of the same vaccine preparation was administered to each animal by intramuscular injection. During the six month period follow¬ ing the first administration, any of the five chimpanzees did not show any hepatic failure from both the laboratory and histological stand- points. Neither HBsAg nor anti HBs could be detected. Considering the possibility of autoimmune response, the latex test was made, in the chimpanzees given the vaccine, to detect the possible existence of antiglobul n antibodies, antinuclear antibodies, antimitochondria anti¬ bodies and antismooth muscle antibodies. As a result, any autoantibodies could not be detected in any of the chimpanzees. b) After the safety test and efficacy test in the five chimpanzees, the same vaccine preparation was administered to 353 men. The efficacy of the vaccine was evaluated by the appearance of humoral immune response to the HBsAg (anti HBs antibodies) and by an epide iological survey of the subjects given^the vaccine and the subjects not given the vaccine. The results obtained during the two year period after the immunization have proved that the vaccine preparation of the present invention can produce a prophylactic effect even in the circumstances where the risk of HB infection is high. No subjects given the vaccine preparation of the present invention showed any clinical or biological signs suggestive of autoimmune diseases.
OMPI
Claims
WHAT IS CLAIMED IS:
A process for producing a hepatitis B vaccine from an aqueous suspension of a crude HBsAg prepared from an HBsAg-containing human serum or plasma by the steps of adjusting the pH of the aqueous suspension and removing most of the plasma protein components approx¬ imately to neutral, characterized in the further steps of adding to the suspension a polyethylene glycol to a concentration of 5-6% and thereby removing Dane particles capable of causing hepatitis B in¬ fection and immune complexes, both as a precipitate, adding a further amount of polyethylene glycol to the supernatant and thereby collect¬ ing HBsAg particles as a precipitate, recovering from said precipitate a specific fraction of HBsAg particles having a particle size of 22 nm
3 and a specific gravity of 1.19-1.21 g/cm by gel filtration and ultracentrifugation, inactivating the virus contained in said fraction and making the fraction into a vaccine preparation.
U .
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19803049690 DE3049690A1 (en) | 1979-07-05 | 1980-07-03 | PROCESS FOR PRODUCING HEPATITIS B VACCINE |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP79/85493 | 1979-07-05 | ||
JP8549379A JPS5610119A (en) | 1979-07-05 | 1979-07-05 | Manufacture of b type hepatitis infection preventive vaccine |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1981000050A1 true WO1981000050A1 (en) | 1981-01-22 |
Family
ID=13860454
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1980/000834 WO1981000050A1 (en) | 1979-07-05 | 1980-07-03 | Process for producing hepatitis b vaccine |
Country Status (3)
Country | Link |
---|---|
JP (1) | JPS5610119A (en) |
GB (1) | GB2065473B (en) |
WO (1) | WO1981000050A1 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0112506A2 (en) * | 1982-11-29 | 1984-07-04 | Green Cross Corporation | A process for producing a hepatitis B infection preventing vaccine |
EP0155007A2 (en) * | 1984-03-16 | 1985-09-18 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Method for purification of HBs antigen |
FR2561256A1 (en) * | 1984-03-16 | 1985-09-20 | Pasteur Institut | Process for purifying HBs antigen type biological particles by flotation ultracentrifugation in a density gradient |
EP0159749A1 (en) * | 1984-04-04 | 1985-10-30 | Stichting Centraal Laboratorium van de Bloedtransfusiedienst van het Nederlandse Rode Kruis | Activated hepatitis B surface antigen product |
EP0291586A3 (en) * | 1981-08-31 | 1988-12-07 | Genentech, Inc. | Recombinant hepatitis b surface antigen and vaccine containing it |
EP0294071A2 (en) * | 1987-06-03 | 1988-12-07 | Merck & Co. Inc. | Hepatitis B vaccines made with pre-formed aluminum hydroxide gels, and processes thereof |
EP0384058A1 (en) * | 1989-02-09 | 1990-08-29 | Development Center For Biotechnology | Isolation of hepatitis B surface antigen from transformed yeast cells |
EP0533492A2 (en) * | 1991-09-18 | 1993-03-24 | Amgen Inc. | A hepatitis B vaccine formulation incorporating a bile acid salt |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0625069B2 (en) * | 1981-01-29 | 1994-04-06 | ブリティッシュ・テクノロジー・グループ・リミテッド | Method for producing hepatitis B vaccine |
FR2560890B1 (en) * | 1984-03-07 | 1987-10-16 | Grp Genie Genetique | COMPOSITION USEFUL FOR THE MANUFACTURE OF VACCINES CONTAINING PARTICLES CARRYING THE SURFACE ANTIGEN OF HEPATITIS B VIRUS AND THE POLYMERIZED HUMAN SERUM ALBUMIN RECEPTOR, ANIMAL CELLS CAPABLE OF PRODUCING SUCH PARTICLES |
JPS60258127A (en) * | 1984-06-04 | 1985-12-20 | Green Cross Corp:The | Preparation of hepatitis b vaccine |
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Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0291586A3 (en) * | 1981-08-31 | 1988-12-07 | Genentech, Inc. | Recombinant hepatitis b surface antigen and vaccine containing it |
US4565697A (en) * | 1982-11-29 | 1986-01-21 | Green Cross Corporation | Process for producing a hepatitis B infection preventing vaccine |
EP0112506A3 (en) * | 1982-11-29 | 1985-05-22 | The Green Cross Corporation | A process for producing a hepatitis b infection preventing vaccine |
EP0112506A2 (en) * | 1982-11-29 | 1984-07-04 | Green Cross Corporation | A process for producing a hepatitis B infection preventing vaccine |
FR2561256A1 (en) * | 1984-03-16 | 1985-09-20 | Pasteur Institut | Process for purifying HBs antigen type biological particles by flotation ultracentrifugation in a density gradient |
EP0155007A2 (en) * | 1984-03-16 | 1985-09-18 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Method for purification of HBs antigen |
EP0155007A3 (en) * | 1984-03-16 | 1988-12-07 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Method for purification of hbs antigen |
EP0159749A1 (en) * | 1984-04-04 | 1985-10-30 | Stichting Centraal Laboratorium van de Bloedtransfusiedienst van het Nederlandse Rode Kruis | Activated hepatitis B surface antigen product |
EP0294071A2 (en) * | 1987-06-03 | 1988-12-07 | Merck & Co. Inc. | Hepatitis B vaccines made with pre-formed aluminum hydroxide gels, and processes thereof |
EP0294071A3 (en) * | 1987-06-03 | 1990-08-01 | Merck & Co. Inc. | Hepatitis b vaccines made with pre-formed aluminum hydroxide gels, and processes thereof |
EP0384058A1 (en) * | 1989-02-09 | 1990-08-29 | Development Center For Biotechnology | Isolation of hepatitis B surface antigen from transformed yeast cells |
EP0533492A2 (en) * | 1991-09-18 | 1993-03-24 | Amgen Inc. | A hepatitis B vaccine formulation incorporating a bile acid salt |
EP0533492A3 (en) * | 1991-09-18 | 1994-08-10 | Amgen Inc | A hepatitis b vaccine formulation incorporating a bile acid salt |
Also Published As
Publication number | Publication date |
---|---|
GB2065473B (en) | 1984-02-29 |
GB2065473A (en) | 1981-07-01 |
JPS5610119A (en) | 1981-02-02 |
JPS5721246B2 (en) | 1982-05-06 |
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