CA2025263A1 - Complex suitable for carrying out a method of purifying pre-s hepatitis b surface antigen - Google Patents
Complex suitable for carrying out a method of purifying pre-s hepatitis b surface antigenInfo
- Publication number
- CA2025263A1 CA2025263A1 CA002025263A CA2025263A CA2025263A1 CA 2025263 A1 CA2025263 A1 CA 2025263A1 CA 002025263 A CA002025263 A CA 002025263A CA 2025263 A CA2025263 A CA 2025263A CA 2025263 A1 CA2025263 A1 CA 2025263A1
- Authority
- CA
- Canada
- Prior art keywords
- set forth
- hepatitis
- complex
- region
- surface antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000000427 antigen Substances 0.000 title claims abstract description 23
- 102000036639 antigens Human genes 0.000 title claims abstract description 22
- 108091007433 antigens Proteins 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims description 26
- 208000006454 hepatitis Diseases 0.000 title description 4
- 231100000283 hepatitis Toxicity 0.000 title description 4
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 33
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 33
- 208000002672 hepatitis B Diseases 0.000 claims abstract description 16
- 229920000642 polymer Polymers 0.000 claims abstract description 16
- 238000000746 purification Methods 0.000 claims abstract description 12
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 5
- 239000003599 detergent Substances 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 8
- 239000004202 carbamide Substances 0.000 claims description 8
- 229960005486 vaccine Drugs 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 6
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 6
- 229920000936 Agarose Polymers 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 230000003196 chaotropic effect Effects 0.000 claims description 3
- 238000002523 gelfiltration Methods 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 108060003951 Immunoglobulin Proteins 0.000 claims description 2
- 108090001090 Lectins Proteins 0.000 claims description 2
- 102000004856 Lectins Human genes 0.000 claims description 2
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 claims description 2
- 230000002209 hydrophobic effect Effects 0.000 claims description 2
- 230000003053 immunization Effects 0.000 claims description 2
- 238000002649 immunization Methods 0.000 claims description 2
- 102000018358 immunoglobulin Human genes 0.000 claims description 2
- 238000004255 ion exchange chromatography Methods 0.000 claims description 2
- 239000002523 lectin Substances 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].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 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 claims description 2
- AWDRATDZQPNJFN-VAYUFCLWSA-N taurodeoxycholic 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)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 AWDRATDZQPNJFN-VAYUFCLWSA-N 0.000 claims description 2
- 238000005199 ultracentrifugation Methods 0.000 claims description 2
- HEGSGKPQLMEBJL-RQICVUQASA-N (2r,3s,4s,5r)-2-(hydroxymethyl)-6-octoxyoxane-3,4,5-triol Chemical group CCCCCCCCOC1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RQICVUQASA-N 0.000 claims 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 claims 1
- GUQQBLRVXOUDTN-XOHPMCGNSA-N 3-[dimethyl-[3-[[(4r)-4-[(3r,5s,7r,8r,9s,10s,12s,13r,14s,17r)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl]azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CC(O)CS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 GUQQBLRVXOUDTN-XOHPMCGNSA-N 0.000 claims 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 1
- 101000872838 Hepatitis B virus genotype C subtype adr (isolate China/NC-1/1988) Small envelope protein Proteins 0.000 abstract description 14
- 230000001225 therapeutic effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 18
- 239000000243 solution Substances 0.000 description 16
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 239000002609 medium Substances 0.000 description 14
- 239000000872 buffer Substances 0.000 description 13
- 239000007983 Tris buffer Substances 0.000 description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 10
- 229920002684 Sepharose Polymers 0.000 description 9
- 239000000499 gel Substances 0.000 description 8
- 239000012528 membrane Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 210000003501 vero cell Anatomy 0.000 description 7
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 6
- 102000009027 Albumins Human genes 0.000 description 6
- 108010088751 Albumins Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 238000003119 immunoblot Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 6
- 229920002401 polyacrylamide Polymers 0.000 description 6
- 108010045897 polyalbumin Proteins 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 208000002109 Argyria Diseases 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 241000700618 Vaccinia virus Species 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000700721 Hepatitis B virus Species 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 241000220317 Rosa Species 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000004847 absorption spectroscopy Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000003431 cross linking reagent Substances 0.000 description 2
- -1 dextrane Polymers 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 2
- VGTPCRGMBIAPIM-UHFFFAOYSA-M sodium thiocyanate Chemical compound [Na+].[S-]C#N VGTPCRGMBIAPIM-UHFFFAOYSA-M 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- VHJLVAABSRFDPM-UHFFFAOYSA-N 1,4-dithiothreitol Chemical compound SCC(O)C(O)CS VHJLVAABSRFDPM-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes 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
- 239000004471 Glycine Substances 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 101710137302 Surface antigen S Proteins 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000005289 controlled pore glass Substances 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229920000592 inorganic polymer Polymers 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- 108010034897 lentil lectin Proteins 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910000000 metal hydroxide Inorganic materials 0.000 description 1
- 150000004692 metal hydroxides Chemical class 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001308 poly(aminoacid) Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 238000005245 sintering Methods 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Detergent Compositions (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Chemical Or Physical Treatment Of Fibers (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE:
There is disclosed a complex comprised of an insoluble polymer carrier to which monomeric human albumin is covalently bound and of a pre-S hepatitis B surface antigen bound in an elutable form to the monomeric human albumin by its pre-S(2)- and/or pre-S(1)-region. This complex may be used for therapeutic and diagnostic purposes and enables the rapid and efficient purification of pre-S-HBsAg by affinity chromatography.
There is disclosed a complex comprised of an insoluble polymer carrier to which monomeric human albumin is covalently bound and of a pre-S hepatitis B surface antigen bound in an elutable form to the monomeric human albumin by its pre-S(2)- and/or pre-S(1)-region. This complex may be used for therapeutic and diagnostic purposes and enables the rapid and efficient purification of pre-S-HBsAg by affinity chromatography.
Description
2 ~ 3 The invention relates to a complex comprised o an insoluble polymer earrier to which human albumin is covalently bound and of a pre-S hepatitis 33 surface antigen (pre-S-HBsAg) as well as to a method for the purification of pre-S-HBsAg.
From EP-A2- 0 243 103, a method of purifying pre-S-HBsAg is known, which consists in disrupting yeast cells expressing recombinant pre-S-HBsAg and separating pre-S-HBsAg from ~:he cell contents by affinity chromatography.
10 Polymerized human serum albumin (polyalbumin) covalently bound to a matrix serves as the adsorbent for pre-S-HBsAg.
~his polyalbumin i5 a product synthetically prepared in a high-molecular form by cross-linking agents, e.g., glutaraldehyde. The pre-S-HBsAg is adsorbed on the polyalbumin by its pre-S(2)-region and, after having washed off interfering substances, is eluted from the matrix and subjected to a secondary purification step.
It was evidenced that an efficient polyalbumin receptor existed in the pre-S region of HBsAg, which is 20 composed of a polypeptide including 55 amino acids and encoded by a section of the hepatitis virus DNA immediately preceding the S-region ("pre-S2") (Valenzuela et al., Bio/~echnoloqy Vol. 3: 317-320, 1985). Furthermore, it was demonstrated that both this immediately preceding region (pre-S2) and the total pre-S-region, in connection with the S-reyion (pre-Sl), eneode surface proteins that constitute reeeptors to hepatitis B virus (HBV) for the binding to eell membranes of liver eells.
HBsAg that contains a pre-S region for the first time 30 was obtained from infected human plasma in 1979 (Neurath 2 ~ 3 and Strick, Arch. Virol., 60: 79-81, 1979) and later on was produced also by way of genetic engineering by Valenzuela et al. (Bio/Technology 3: 317, 1985, and Nature 311: 67, 1984) and Paoletti et al. (PNAS 81: 193, 1984).
Subsequently, various recombinantly expressed pre-S-containing surface antigens were suggested as vaccines to induce the formation of antibodies against HBV. These antibodies are directed against the receptor of the virus, thus preventing its binding to the liver cell and the infection involved.
The purification of the antigens by affinity chromatography through polyalbumin has the disadvantage that impurities, yet even toxic substances, such as glutaraldehyde, are introduced into the eluate due to the use of cross-linking agents in the production of polymerization products.
It is the object of the present invention to eliminate this difficulty and to provide a complex which, in addition to the opportunity of a rapid and efficient affinity chromatographic purification of pre-S-HBsAg, offers new applications for therapeutic and diagnostic purposes.
The complex according to the invention is comprised of an insoluble polymer carrier based, in particular, on agarose or dextrane, to which monomeric human albumin is covalently bound, and of a pre-S hepatitis B surface antigen bound in an elutable form to the monomeric human albumin by its pre-S(2)- and/or pre-S(l)-region.
It was found according to the invention that pre-S-HBsAg would be complexed to the monomeric human albumin to an extent sufficient for affinity chromatographic $ ~
puriEication only if the latter were covalently bound to the carrier. If the albumin merely binds by way of adsorption, the pre-S-~lBsAg can be complexed to the albumin not at all or only extremely slightly such that an aEfinity chromatographic plJrification will not be possible. Another advantage of the complex according to the invention consists in that pre-S-H~sAg may be readily eluted, the protein thus being recoverable in a more gentle manner and a greater yield.
An insoluble polymer carrier to which monomeric human albumin is covalently bound is required for the Eormation of the complex of the invention. This insoluble polymer carrier, thus, also falls within the scope of the invention. To produce the insoluble polymer carrier according to the invention, any polymer capable of covalently binding proteins upon suitable activation may be used. The following carrier substances may be used:
- organic polymers, such as polyamides and vinyl polymers (polyacrylamide, polystyrene and polyvinyl aLcohols and derivatives thereof), as well as - natural polymers, such as cellulose, dextrane, agarose, chitin and polyamino acids, and - inorganic polymers, such as silica gel, glass and metal hydroxides.
These carrier substances may be used in the form of particles, e.g., as molecular sieves, in the form of membranes or oE plates, e.g., as microtiter plates.
Preferably, the insoluble polymer carrier is based on agarose or dextrane.
The complex according to the invention is storable over extended periods of time, i.e., both in the aqueous phase in which the carrier is present in the swollen state (e.g., as an affinity resin) and in the lyophilized state (e.g., as a membrane or a microtiter plate).
Lyophilization, preferably, is carried out in a volatile buffer containing glycine or glucose.
Pre-S-~IBsAg can be isolated from the complex according to the invention in high purity. The invention also relates to the use of the complex according to the invention for the preparation of diagnostics and vaccines, the vaccines being applicable both for active immunization against hepatitis B and for obtaining specific immunoglobulin of donors immunized with such vaccines.
The carrier according to the invention may be loaded with pre-S hepatitis B surface antigen in a simple manner by contacting an aqueous solution of hepatitis B surface antigen with the carrier, the pre-S-containing fractions of the hepatitis surface antigens being selectively adsorbed on the albumin molecules.
~he method according to the invention Eor the purification of pre-S hepatitis B surface antigen by using a complex according to the invention is characterized in that - the complex is washed with a buEfer solution to remove possibly present impurities, and - the pre-S hepatitis B surface antigen selectively adsorbed on the monomeric human albumin is cleaved and recovered either by treating the complex with an eluting agent containing chaotropic substances, such as urea, guanidine hydrochloride, thiocyanate, potassium chloride, ~2~
magnesium chloride or potassium iodide; or by treating the complex with detergents; or by treating the eomplex with a pH modifying agent;
whereupon a secondary purification step i8 carried out, if necessary.
A preferred embodiment of the method according to the invention is characterized in that ionic detergents, in particular sodium desoxycholate and taurodesoxycholic acid, or ~witterionic detergents, in particular 3 ~(3-cholamidopropyl)-dimethyl-ammonio~-l-propane sulfonate and 3~t3-cholamidopropyl)-dimethyl-ammonio~-2-hydroxy-1-propane sulfonate, or non-ionic detergents, in particular octyl glucopyranosides, may be used as the detergents.
It has proved that the quarternary structure of the eluted pre-S-~BsAg is not affected when using these detergents.
Preferably, the secondary purification step after elution comprises gel filtration, ultracentrifugation, hydrophobic chromatography, lectin affinity chromatography or ion exchange chromatography. Any of these purification steps yields pre-S-FlBsAg in a purity greater than 90 ~.
The invention wi:Ll be explained in more detail in the following:
The pre-S-HBsAg can be expressed from recombinant vaccinia virus that contains the genetic information of the pre-S-HBsAg. This technique has been described in the literature (Moss et al., Nature 311: 67 (19a4)).
At first, A high-titer virus stock of recombinant vaccinia virus was prepared by infecting vero celLs with 1 PFU/cell. After 2 to 3 days of incubation at 37C the ~ .J ~ r~ ~ ~
infected cells are shaken into the medium and pelleted by centrifugation at 5,000 g for 20 min. The supernatant is poured off and stored at ~C. The cells are washed three times in PBS, whereupon a trypsin solution is added to the cell suspension until a final concentration of trypsin of 0.025 ~ by mass is reached. After this, the suspension is gently stirred at 37C Eor 30 min, pooled with the supernatant, aliquoted into ampoules and deepfrozen at -80C. By this method, the virus titer is increased by approximately ten times as compared to that usually obtained in cell medium.
A vero cell inoculum is prepared by passaging cells in plastic Roux and Roller bottles (Nunc) in order to produce sufficient cells to be able to inoculate a 6-liter fermenter, which, in turn, serves as the inoculum for a ~0-liter vessel. To this end, a single ampoule of vero cells of a defined passage number at first is thawed and passaged to provide 12 confluent Roller bottles.
The cells are trypsinized, resuspended in medium 199 with 5 ~ by mass of foetal calf serum and mixed with a suspension of microcarriers (Cytodex 3, Pharmacia) and pumped into the fermenter to give a final concentration of 2 x 108 cells and 5 9 microcarrier per liter. At this stage, additional medium is added in an amount of one third of the final working volume.
The cells are allowed to adhere to the microcarriers for a period of three hours while slowly stirring the suspension. After this, further medium (DMEM containing 5 ~
by mass of foetal calf serum) is added in order to obtain the final working volume. As soon as a cell density of 6 to J ~ ~
8 x 108/liter has been achieved, a continuous perfusion with DMEM (containing S ~ by mass of foetal calf serum) is started. Upon achievement of a cell density of approximately 5 x 109/liter, the microcarriers are trypsinized and the cells with the microcarriers are pumped into the ~0-liter fermenter, which contains additional 5 9 microcarrier per liter. Upon adsorption, the fermenter is filled to a volume of ~0 liters and cultivation proceeds as described above.
When a cell density of 5 x 109/liter has been reached, the medium is pumped off after the microcarriers have settled. Five liters of the medium containing the recombinant microorganism are pumped into the fermenter so as to give a m.o.i.-value of about 2 PFU per recombinant cell. After adsorption of the virus, the fermenter is filled with medium 199 tcontaining 5 ~ by mass of foetal calf serum) to a volume of ~0 liters and is perfused with ~0 liters of the same medium Eor ~0 hours. After this period of time, about 80 ~ of the cells has detached from the microcarriers and is pumped off together with the medium.
The remaining cells still adhering to thè
microcarriers are detached thereErom by washing with medium under rapid stirring and are pumped off and pooled with the first fraction. The microcarriers are removed by means of a 70 mym s;eve. By centrifugation in a Beckmann JFC-Z
continuous flow rotor, the cells are pelleti~ed at 16,000 g. The medium is concentrated by ultrafiltration in the Pellilcon system (Millipore-Waters). Urea is added to the concentrated medium until an 8 M solution adjusts. This ~2~3 solution is dialyzed.
irhe conrentrated vero cell supernatant is added to a column packed with an insoluble polymer carrier substance to which human serum albumin is covalently bound. The coupling method is known to depend on the chemical nature of the carrier. When using Sepharose, activation is -~effected with CNBr, for instance, whereupon the monomeric human serum albumin is coupled this activated Sepharose.
After washing of the column with buffer 1 consisting of 0.2 M sodium acetate (pH 4.0) and 0.5 M NaCl, with bufEer 2 consisting oE 0.1 M Tris (pH 8.0) and 0.5 M NaCl, with a solution 3 consisting of 8 M urea, and finally with buffer 3 consisting of 0.02 M Tris (pH 7.4), the vero cell supernatant is applied to the column. As soon as the supernatant has been adsorbed by the column, the non-adsorbed proteins are washed from the column with buffer 4 consisting of 0.02 M Tris (pH 7.4) and 0.5 M NaCl. The adsorbed pre-S(2)-HBsAg is eluted with sodium thiocyanate in buffer 4 (l to about 4 M, preferably 3 M) at a pH of~
from 6 to about 8, preferably at pE~ 7 (buffer 5) or with 8 M urea in buffer 4, preferably with 4 M urea (buffer 6).
This separation according to the invention with monomeric human albumin results in a 145-fold enrichment of pre-S-HBsAg. Its identity is revealed by the immunoblotting technique (Burnette, Anal. Biochem., 112, 195, 1981) and by silver stained polyacrylamide gels (silver staining;
Morrissey, Anal. Biochem., 117, 307, 1981), the purity amounting to at least 80 %.
The pre-S(2)-HBsAg may be further purified, i.a., by gel filtration on Sepharose (Pharmacia). In doing so, the ~r3~
fraction obtained above is dialyzed against a buffer eonsisting of 0.02 M Tris (pH 7) and applied to the Sepharose column activated with 0,02 M Tris (pH 7). The purity of the pre-S-HBsAg can be shown to be greater than 90 % by silver staining and immunoblotting.
The realization of the method aecording to the invention is further illustrated by the following exemplary embodiments.
Example 1 Purifieation of pre-S(2)-HBsAg by monomerie human albumin affinity ehromatography The recombinant vaccinia virus with the genetic information for the pre-S(2)-HBsAg was obtained using the teehniques deseribed by Moss et al. (Nature 311: 67, 1984).
The high-titer stoek of the recombinant vaccinia virus was prepared as described above. The vero cells for infeetion with the recombinant virus were grown under the same conditions as described above. The concentrated dialyzed medium with the pre-S(2)-~BsAg was obtained in the previously described manner.
Monomeric human albumin was covalently bound to CNBr-aetivated Sepharose 4B (Pharmaeia) following the manufaeturer's instruetions. After this, a eolumn was paeked with a volume of 50 ml matrix and washed with 500 ml of buffer solution 1, 500 ml of buffer solution 2, 500 ml of solution 3 and, finally, 500 ml buffer solution 4.
The elariEied vero cell supernatant contained 8500 mg protein and 95 mg pre-S(2)-HBsAg and was pumped onto the prepared eolumn at a flow rate oE 100 ml/hour. When the total amount had entered the eolumn, the non-adsorbed 2~2~'~
proteins were washed from the column with buffer solution 4. The pre-S(2)-HBsAg was eluted with 3 M sodium thiocyanate, pH 7 (buffer solution 5). Identification and purity analysis were effected by silver staining and immunoblott ing f ollow ing polyacrylam ide gel electrophoresis.
Duplicate aliquots of the fractions eluted from the column were incubated for 15 min at 100C in a buffer consisting of 2 % by mass of sodium dodecyl sulfate (SDS), 10 0.125 M Tris-HCl (pH 6.8) and 100 mM dithiotreitol. The samples were electrophoresed through 12.5 3 by mass polyacrylamide separating gel for 2.5 hours at 65 mA/gel (Laemmli, Nature 227: 680, 1971). One set of the gel samples was stained with silver nitrate to visualize the polypeptides. The other set of the samples was assayed by immunoblotting, using rabbit antiserum.
The eluted fractions contained 42 mg protein and 68 mg pre-S(2)-HBsAg. Thus, a 145-~old enrichment of the pre-S(2)-HBsAg had been achieved.
The following Examples 2 and 3 illustrate the secondary purification of the fractions obtained in Example 1.
Ex~_ple 2:
A pre-S(2)-HBsAg-containing solution obtained according to Example 1 was further purified by column chromatography on Sepharose ~B (Pharmacia). At first, the column was prepared and washed with 500 ml 0.02 M Tris (pH
7.0) at a flow rate of 30 ml/hour. The pre-S(2)-ElBsAg-containing solution was dialyzed against this buErer, and 30 the dialyzed antigen present at a concentration of 10 mg/12 ml in addition to 6.2 mg protein/12 ml was added to the column. The column was activated with 0.02 M Tris (pH 7.0) and the eluted pre-S(2)-HBsAg-containing fraction contained 4.8 mg protein and 8.2 mg pre-S(2)-HBsAg. Silver-staining and immunoblotting oE the polyacrylamide gels carried out in the manner described in Example 1 revealed a purity of the pre-S(2)-HBsAg greater than 90 %.
_xa_~le 3.
A pre--S(2)-HBsAg-containing solution obtained according to Example 1 was further purified by column chromatography on lentil-lectin Sepharose 4B tPharmacia).
At first, the column was prepared and washed with 100 ml 0.02 M Tris (pH 7.0) at a flow rate of 50 ml/hour. The pre-S(2)-HBsAg-containing solution was dialyzed against this buffer, and the dialy~ed antigen present at a concentration of 21 mg/25 ml in addition to 13 mg protein/25 ml was applied onto the column at a flow rate of 25 ml/hour. After this, the non-adsorbed material was washed off with Tris buffer solution and the pre-S(2)-HBsAg was eluted with Tris buffer solution containing 5 ~ by mass of alpha-methylmannoside. The eluate contained 9.1 mg protein and 15.6 mg pre-S(2)-HBsAg. Silver staining and immunoblotting of the polyacrylamide gels were carried out as described in Example 1 and revealed a purity of the pre-S(2)-HBsAg greater than 90 %.
The purification of hepatitis B surface antigens bearing the pre-S(l)-region instead of the pre-S(2)-region or pre-S(2)- and pre-S(l)-regions is accomplished in a manner analogous to the exemplary embodimentæ described.
The method according to the invention may, indeed, be used to purify any protein that carries a pre-S(l)- and/or a 2~25263 pre-S~2)-region of HBsAg.
The following Examples ~ to 6 describe the use of various carrier substances and forms as well as coupling methods that serve the required purpose of use.
Exa_~le ~:
Coupling of monomeric human albumin to silicate carriers Possible carriers are silica gels or glass microbeads (controlled pore glass beads = CPG).
lO g of an aminated glass carrier (aminopropyl CPG, Pierce) are shaken with lO0 ml of a 2.5 ~ aqueous glutaraldehyde solution for four hours at room temperature.
Upon activation, the carrier is thoroughly washed free from aldehyde with deionized water. The thus activated carrier is incubated with 200 mg human serum albumin in 0.1 M
phosphate buffer (pH 8.0) for one hour. After blocking with l M ethanolamine and thorough washing with phosphate buffer, the carriPr is ready for use.
200 ml of a pre-S(2)-~lBsAg-containing cell culture supernataDt (ll mg pre-S(2)--HBsAg, 950 mg protein) are incubated with lO ml of the affinity carrier for two hours at 14C. Unbound protein is removed by washing with a buffer on a sintering suction filter and the specifically bound pre-$(2)-HBsAg is eluted by incubation of the packed carrier in lO ml 8 M urea solution. This assay yielded 6.8 mg highly pure pre-S(2)-HBsAg at an overall protein amount of 5.~ mg (determination according to Bradford).
Exa_ele 5:
Coupling of monomeric human albumin to membranes Membranes, e.g., of nylon, polyvinylidene difluoride or cellulose polyacrylamide mixed polymers are possible ~2~3 carriers. Nylon membrane (e.g., Zetabind, CUNO~ is partially hydrolyzed by incubation in HCl/E12O. The released amino groups are reacted with 1 M oxaldialdehyde in the presence of 0.1 M sodium cyanoborohydride at pH 7 for two hours. After washing with deionized water, human serum albumin (10 mg/mi in 0.1 M phosphate buffer pEI 7) is coupled to this membrane activated with reactive aldehyde groups in the presence of 0.1 M sodium cyanoborohydride.
Excess reactive groups are blocked by the addition of 1 M
ethanolamine. After washing with phosphate buffer (0.1 M;
pH ~3), the membrane may be used to selectively bind pre-S(2)-containing proteins.
The binding capacity oE a thus produced membrane is 5 to 10 myg pre-S(2)-HssAg/cm2.
Exam~le 6:
Coupling of a monomeric human albumin to microtiter plates To this end, functionalized polystyrene plates, such as, e.y., Amino-Plate (including primary amino groups) or Carbo-Plate (including carboxyl groups) (Nissho Iwai Corp., Tokyo) or similar materials may be used.
200 myl of a solution of N-ethoxycarbonyl-2-ethoxy-1,2-dlhydro-quinoline (~0 mM in 50 % aqueous ethanol) are pipetted into each well of a 96-well microtiter plate and incubated for two hours at ~0C. After careful washing with ethanol and deionized water, 200 myl of a 1 % aqueous solution of human serum albumin are each pipetted into a well and incubated at -~4C over night. After blocking with 1 M ethanolamine or 1 M acetate buffer pEI~ and subesequent washing, the plate is ready for use.
100 myl of a pre-S(2)-HBsAg-containing cell culture e3 2 ~ ~
supernatant are pipetted into the prepared microtiter plate in increasing dilutions and incubated at ~4C for two hours. After washing with buffer and incubation with enzyme-labeled anti-pre-S(2)-HBsAg ~monoclonal antibodies) following the known ELISA technique, the bound portion of pre-S(2)-protein is determined by substrate addition according to known methods of absorption spectroscopy.
If the microtiter plate is loaded with SM-denaturized pre-S(2)-HBsAg or synthetic pre-S(2)-peptide, a thus I0 treated plate may be used for assaying anti-pre-S(2)-antibodies (e.g., from patients' sera).
10 mg pre-S(2)-MBsAg in 10 ml phosphate buffer (0.1 M, pH 7.0~ are incubated with 100 myl mercaptoethanol in 10 ~
sodium dodecyl sulfate (SDS) for 5 min at 100C. In doing so, the immunologically active S-antigen of HBsAg is destroyed (Milich D.R. et al., Enhanced Immunogenicity of the Pre-S-Region of Mepatitis B Surface Antigen, Science 227: 1195-1199 (1985)). After blocking of the SM-groups with 500 mg iodoacetamide for one hour, excess reaction products are eliminated by dialysis and the pre-S(2)-antigen is diluted to 100 myg protein/ml with phosphate buffer pM 7.~.
A prepared HSA microtiter plate is coated with this pre-S(2) solution and subsequently washed. lrhus prepared plates may be stored even in the dry state.
100 myl of an anti-pre-S(2)-containing rabbit serum are each pipetted into the wells in increasing dilutions.
After incubation at ~4C over night, bound pre-S(2)-antibodies are assayed by means of absorption spectroscopy 30 by incubation with enzyme--labeled anti-rabbit serum and subsequent substrate addition.
Exa_ple 7:
Binding capacity and yield oE pre-S(2)-HBsAg when using various albumin carrier complexes Human serum albumin (HSA) and polymerized human serum albumin (poly-HSA) were covalently and adsorptively bound to various carrier substances in comparable amounts. The binding capacity and the yield of pre-S(2)-H~sAg after elution were determined by means of a HBsAg-specific ELISA
method a~d are summarized in the following Table:
2 ~ 3 Albumin Carrier Type of Capacity % yield after elution binding mg/g gel with 2M U ~M U 8M U 3M
KSCN
____________________________________________________________ mono CNBr- covalent 7.6 45 85 60 50 meric Sepha- via rose NH2 mono anti- adsorp- 0 meric HSA tive Sepha-rose poly- anti- adsorp- 5.8 - - - -meric HSA tive Sepha-rose poly- CNBr- covalent 6.~ 12 ~0 55 ~5 meric Sepha- via rose NH2 ____________________________________________________________ U = Urea It is apparent from the Table that hig'ner yields of pre-S(2)-HBsAg are obtained with the insoluble polymer carrier according to the invention, with which the monomeric human albumin is covalently bound to Sepharose in the present exemplary embodiment, than with Sepharose to which polyalbumin is covalently bound.
These remarkably higher yields are due to the more yentle eluting conditions made possible by employing the carrier of the invention.In addition, it can be seen that it is only the covalent binding of HSA to the carrier that results in the inventive characteristic oE the pre-S(2)-affinity and that a molecular sieve to which albumin is bound merely by adsorption is not suitable for affinity chromatographic purification.
From EP-A2- 0 243 103, a method of purifying pre-S-HBsAg is known, which consists in disrupting yeast cells expressing recombinant pre-S-HBsAg and separating pre-S-HBsAg from ~:he cell contents by affinity chromatography.
10 Polymerized human serum albumin (polyalbumin) covalently bound to a matrix serves as the adsorbent for pre-S-HBsAg.
~his polyalbumin i5 a product synthetically prepared in a high-molecular form by cross-linking agents, e.g., glutaraldehyde. The pre-S-HBsAg is adsorbed on the polyalbumin by its pre-S(2)-region and, after having washed off interfering substances, is eluted from the matrix and subjected to a secondary purification step.
It was evidenced that an efficient polyalbumin receptor existed in the pre-S region of HBsAg, which is 20 composed of a polypeptide including 55 amino acids and encoded by a section of the hepatitis virus DNA immediately preceding the S-region ("pre-S2") (Valenzuela et al., Bio/~echnoloqy Vol. 3: 317-320, 1985). Furthermore, it was demonstrated that both this immediately preceding region (pre-S2) and the total pre-S-region, in connection with the S-reyion (pre-Sl), eneode surface proteins that constitute reeeptors to hepatitis B virus (HBV) for the binding to eell membranes of liver eells.
HBsAg that contains a pre-S region for the first time 30 was obtained from infected human plasma in 1979 (Neurath 2 ~ 3 and Strick, Arch. Virol., 60: 79-81, 1979) and later on was produced also by way of genetic engineering by Valenzuela et al. (Bio/Technology 3: 317, 1985, and Nature 311: 67, 1984) and Paoletti et al. (PNAS 81: 193, 1984).
Subsequently, various recombinantly expressed pre-S-containing surface antigens were suggested as vaccines to induce the formation of antibodies against HBV. These antibodies are directed against the receptor of the virus, thus preventing its binding to the liver cell and the infection involved.
The purification of the antigens by affinity chromatography through polyalbumin has the disadvantage that impurities, yet even toxic substances, such as glutaraldehyde, are introduced into the eluate due to the use of cross-linking agents in the production of polymerization products.
It is the object of the present invention to eliminate this difficulty and to provide a complex which, in addition to the opportunity of a rapid and efficient affinity chromatographic purification of pre-S-HBsAg, offers new applications for therapeutic and diagnostic purposes.
The complex according to the invention is comprised of an insoluble polymer carrier based, in particular, on agarose or dextrane, to which monomeric human albumin is covalently bound, and of a pre-S hepatitis B surface antigen bound in an elutable form to the monomeric human albumin by its pre-S(2)- and/or pre-S(l)-region.
It was found according to the invention that pre-S-HBsAg would be complexed to the monomeric human albumin to an extent sufficient for affinity chromatographic $ ~
puriEication only if the latter were covalently bound to the carrier. If the albumin merely binds by way of adsorption, the pre-S-~lBsAg can be complexed to the albumin not at all or only extremely slightly such that an aEfinity chromatographic plJrification will not be possible. Another advantage of the complex according to the invention consists in that pre-S-H~sAg may be readily eluted, the protein thus being recoverable in a more gentle manner and a greater yield.
An insoluble polymer carrier to which monomeric human albumin is covalently bound is required for the Eormation of the complex of the invention. This insoluble polymer carrier, thus, also falls within the scope of the invention. To produce the insoluble polymer carrier according to the invention, any polymer capable of covalently binding proteins upon suitable activation may be used. The following carrier substances may be used:
- organic polymers, such as polyamides and vinyl polymers (polyacrylamide, polystyrene and polyvinyl aLcohols and derivatives thereof), as well as - natural polymers, such as cellulose, dextrane, agarose, chitin and polyamino acids, and - inorganic polymers, such as silica gel, glass and metal hydroxides.
These carrier substances may be used in the form of particles, e.g., as molecular sieves, in the form of membranes or oE plates, e.g., as microtiter plates.
Preferably, the insoluble polymer carrier is based on agarose or dextrane.
The complex according to the invention is storable over extended periods of time, i.e., both in the aqueous phase in which the carrier is present in the swollen state (e.g., as an affinity resin) and in the lyophilized state (e.g., as a membrane or a microtiter plate).
Lyophilization, preferably, is carried out in a volatile buffer containing glycine or glucose.
Pre-S-~IBsAg can be isolated from the complex according to the invention in high purity. The invention also relates to the use of the complex according to the invention for the preparation of diagnostics and vaccines, the vaccines being applicable both for active immunization against hepatitis B and for obtaining specific immunoglobulin of donors immunized with such vaccines.
The carrier according to the invention may be loaded with pre-S hepatitis B surface antigen in a simple manner by contacting an aqueous solution of hepatitis B surface antigen with the carrier, the pre-S-containing fractions of the hepatitis surface antigens being selectively adsorbed on the albumin molecules.
~he method according to the invention Eor the purification of pre-S hepatitis B surface antigen by using a complex according to the invention is characterized in that - the complex is washed with a buEfer solution to remove possibly present impurities, and - the pre-S hepatitis B surface antigen selectively adsorbed on the monomeric human albumin is cleaved and recovered either by treating the complex with an eluting agent containing chaotropic substances, such as urea, guanidine hydrochloride, thiocyanate, potassium chloride, ~2~
magnesium chloride or potassium iodide; or by treating the complex with detergents; or by treating the eomplex with a pH modifying agent;
whereupon a secondary purification step i8 carried out, if necessary.
A preferred embodiment of the method according to the invention is characterized in that ionic detergents, in particular sodium desoxycholate and taurodesoxycholic acid, or ~witterionic detergents, in particular 3 ~(3-cholamidopropyl)-dimethyl-ammonio~-l-propane sulfonate and 3~t3-cholamidopropyl)-dimethyl-ammonio~-2-hydroxy-1-propane sulfonate, or non-ionic detergents, in particular octyl glucopyranosides, may be used as the detergents.
It has proved that the quarternary structure of the eluted pre-S-~BsAg is not affected when using these detergents.
Preferably, the secondary purification step after elution comprises gel filtration, ultracentrifugation, hydrophobic chromatography, lectin affinity chromatography or ion exchange chromatography. Any of these purification steps yields pre-S-FlBsAg in a purity greater than 90 ~.
The invention wi:Ll be explained in more detail in the following:
The pre-S-HBsAg can be expressed from recombinant vaccinia virus that contains the genetic information of the pre-S-HBsAg. This technique has been described in the literature (Moss et al., Nature 311: 67 (19a4)).
At first, A high-titer virus stock of recombinant vaccinia virus was prepared by infecting vero celLs with 1 PFU/cell. After 2 to 3 days of incubation at 37C the ~ .J ~ r~ ~ ~
infected cells are shaken into the medium and pelleted by centrifugation at 5,000 g for 20 min. The supernatant is poured off and stored at ~C. The cells are washed three times in PBS, whereupon a trypsin solution is added to the cell suspension until a final concentration of trypsin of 0.025 ~ by mass is reached. After this, the suspension is gently stirred at 37C Eor 30 min, pooled with the supernatant, aliquoted into ampoules and deepfrozen at -80C. By this method, the virus titer is increased by approximately ten times as compared to that usually obtained in cell medium.
A vero cell inoculum is prepared by passaging cells in plastic Roux and Roller bottles (Nunc) in order to produce sufficient cells to be able to inoculate a 6-liter fermenter, which, in turn, serves as the inoculum for a ~0-liter vessel. To this end, a single ampoule of vero cells of a defined passage number at first is thawed and passaged to provide 12 confluent Roller bottles.
The cells are trypsinized, resuspended in medium 199 with 5 ~ by mass of foetal calf serum and mixed with a suspension of microcarriers (Cytodex 3, Pharmacia) and pumped into the fermenter to give a final concentration of 2 x 108 cells and 5 9 microcarrier per liter. At this stage, additional medium is added in an amount of one third of the final working volume.
The cells are allowed to adhere to the microcarriers for a period of three hours while slowly stirring the suspension. After this, further medium (DMEM containing 5 ~
by mass of foetal calf serum) is added in order to obtain the final working volume. As soon as a cell density of 6 to J ~ ~
8 x 108/liter has been achieved, a continuous perfusion with DMEM (containing S ~ by mass of foetal calf serum) is started. Upon achievement of a cell density of approximately 5 x 109/liter, the microcarriers are trypsinized and the cells with the microcarriers are pumped into the ~0-liter fermenter, which contains additional 5 9 microcarrier per liter. Upon adsorption, the fermenter is filled to a volume of ~0 liters and cultivation proceeds as described above.
When a cell density of 5 x 109/liter has been reached, the medium is pumped off after the microcarriers have settled. Five liters of the medium containing the recombinant microorganism are pumped into the fermenter so as to give a m.o.i.-value of about 2 PFU per recombinant cell. After adsorption of the virus, the fermenter is filled with medium 199 tcontaining 5 ~ by mass of foetal calf serum) to a volume of ~0 liters and is perfused with ~0 liters of the same medium Eor ~0 hours. After this period of time, about 80 ~ of the cells has detached from the microcarriers and is pumped off together with the medium.
The remaining cells still adhering to thè
microcarriers are detached thereErom by washing with medium under rapid stirring and are pumped off and pooled with the first fraction. The microcarriers are removed by means of a 70 mym s;eve. By centrifugation in a Beckmann JFC-Z
continuous flow rotor, the cells are pelleti~ed at 16,000 g. The medium is concentrated by ultrafiltration in the Pellilcon system (Millipore-Waters). Urea is added to the concentrated medium until an 8 M solution adjusts. This ~2~3 solution is dialyzed.
irhe conrentrated vero cell supernatant is added to a column packed with an insoluble polymer carrier substance to which human serum albumin is covalently bound. The coupling method is known to depend on the chemical nature of the carrier. When using Sepharose, activation is -~effected with CNBr, for instance, whereupon the monomeric human serum albumin is coupled this activated Sepharose.
After washing of the column with buffer 1 consisting of 0.2 M sodium acetate (pH 4.0) and 0.5 M NaCl, with bufEer 2 consisting oE 0.1 M Tris (pH 8.0) and 0.5 M NaCl, with a solution 3 consisting of 8 M urea, and finally with buffer 3 consisting of 0.02 M Tris (pH 7.4), the vero cell supernatant is applied to the column. As soon as the supernatant has been adsorbed by the column, the non-adsorbed proteins are washed from the column with buffer 4 consisting of 0.02 M Tris (pH 7.4) and 0.5 M NaCl. The adsorbed pre-S(2)-HBsAg is eluted with sodium thiocyanate in buffer 4 (l to about 4 M, preferably 3 M) at a pH of~
from 6 to about 8, preferably at pE~ 7 (buffer 5) or with 8 M urea in buffer 4, preferably with 4 M urea (buffer 6).
This separation according to the invention with monomeric human albumin results in a 145-fold enrichment of pre-S-HBsAg. Its identity is revealed by the immunoblotting technique (Burnette, Anal. Biochem., 112, 195, 1981) and by silver stained polyacrylamide gels (silver staining;
Morrissey, Anal. Biochem., 117, 307, 1981), the purity amounting to at least 80 %.
The pre-S(2)-HBsAg may be further purified, i.a., by gel filtration on Sepharose (Pharmacia). In doing so, the ~r3~
fraction obtained above is dialyzed against a buffer eonsisting of 0.02 M Tris (pH 7) and applied to the Sepharose column activated with 0,02 M Tris (pH 7). The purity of the pre-S-HBsAg can be shown to be greater than 90 % by silver staining and immunoblotting.
The realization of the method aecording to the invention is further illustrated by the following exemplary embodiments.
Example 1 Purifieation of pre-S(2)-HBsAg by monomerie human albumin affinity ehromatography The recombinant vaccinia virus with the genetic information for the pre-S(2)-HBsAg was obtained using the teehniques deseribed by Moss et al. (Nature 311: 67, 1984).
The high-titer stoek of the recombinant vaccinia virus was prepared as described above. The vero cells for infeetion with the recombinant virus were grown under the same conditions as described above. The concentrated dialyzed medium with the pre-S(2)-~BsAg was obtained in the previously described manner.
Monomeric human albumin was covalently bound to CNBr-aetivated Sepharose 4B (Pharmaeia) following the manufaeturer's instruetions. After this, a eolumn was paeked with a volume of 50 ml matrix and washed with 500 ml of buffer solution 1, 500 ml of buffer solution 2, 500 ml of solution 3 and, finally, 500 ml buffer solution 4.
The elariEied vero cell supernatant contained 8500 mg protein and 95 mg pre-S(2)-HBsAg and was pumped onto the prepared eolumn at a flow rate oE 100 ml/hour. When the total amount had entered the eolumn, the non-adsorbed 2~2~'~
proteins were washed from the column with buffer solution 4. The pre-S(2)-HBsAg was eluted with 3 M sodium thiocyanate, pH 7 (buffer solution 5). Identification and purity analysis were effected by silver staining and immunoblott ing f ollow ing polyacrylam ide gel electrophoresis.
Duplicate aliquots of the fractions eluted from the column were incubated for 15 min at 100C in a buffer consisting of 2 % by mass of sodium dodecyl sulfate (SDS), 10 0.125 M Tris-HCl (pH 6.8) and 100 mM dithiotreitol. The samples were electrophoresed through 12.5 3 by mass polyacrylamide separating gel for 2.5 hours at 65 mA/gel (Laemmli, Nature 227: 680, 1971). One set of the gel samples was stained with silver nitrate to visualize the polypeptides. The other set of the samples was assayed by immunoblotting, using rabbit antiserum.
The eluted fractions contained 42 mg protein and 68 mg pre-S(2)-HBsAg. Thus, a 145-~old enrichment of the pre-S(2)-HBsAg had been achieved.
The following Examples 2 and 3 illustrate the secondary purification of the fractions obtained in Example 1.
Ex~_ple 2:
A pre-S(2)-HBsAg-containing solution obtained according to Example 1 was further purified by column chromatography on Sepharose ~B (Pharmacia). At first, the column was prepared and washed with 500 ml 0.02 M Tris (pH
7.0) at a flow rate of 30 ml/hour. The pre-S(2)-ElBsAg-containing solution was dialyzed against this buErer, and 30 the dialyzed antigen present at a concentration of 10 mg/12 ml in addition to 6.2 mg protein/12 ml was added to the column. The column was activated with 0.02 M Tris (pH 7.0) and the eluted pre-S(2)-HBsAg-containing fraction contained 4.8 mg protein and 8.2 mg pre-S(2)-HBsAg. Silver-staining and immunoblotting oE the polyacrylamide gels carried out in the manner described in Example 1 revealed a purity of the pre-S(2)-HBsAg greater than 90 %.
_xa_~le 3.
A pre--S(2)-HBsAg-containing solution obtained according to Example 1 was further purified by column chromatography on lentil-lectin Sepharose 4B tPharmacia).
At first, the column was prepared and washed with 100 ml 0.02 M Tris (pH 7.0) at a flow rate of 50 ml/hour. The pre-S(2)-HBsAg-containing solution was dialyzed against this buffer, and the dialy~ed antigen present at a concentration of 21 mg/25 ml in addition to 13 mg protein/25 ml was applied onto the column at a flow rate of 25 ml/hour. After this, the non-adsorbed material was washed off with Tris buffer solution and the pre-S(2)-HBsAg was eluted with Tris buffer solution containing 5 ~ by mass of alpha-methylmannoside. The eluate contained 9.1 mg protein and 15.6 mg pre-S(2)-HBsAg. Silver staining and immunoblotting of the polyacrylamide gels were carried out as described in Example 1 and revealed a purity of the pre-S(2)-HBsAg greater than 90 %.
The purification of hepatitis B surface antigens bearing the pre-S(l)-region instead of the pre-S(2)-region or pre-S(2)- and pre-S(l)-regions is accomplished in a manner analogous to the exemplary embodimentæ described.
The method according to the invention may, indeed, be used to purify any protein that carries a pre-S(l)- and/or a 2~25263 pre-S~2)-region of HBsAg.
The following Examples ~ to 6 describe the use of various carrier substances and forms as well as coupling methods that serve the required purpose of use.
Exa_~le ~:
Coupling of monomeric human albumin to silicate carriers Possible carriers are silica gels or glass microbeads (controlled pore glass beads = CPG).
lO g of an aminated glass carrier (aminopropyl CPG, Pierce) are shaken with lO0 ml of a 2.5 ~ aqueous glutaraldehyde solution for four hours at room temperature.
Upon activation, the carrier is thoroughly washed free from aldehyde with deionized water. The thus activated carrier is incubated with 200 mg human serum albumin in 0.1 M
phosphate buffer (pH 8.0) for one hour. After blocking with l M ethanolamine and thorough washing with phosphate buffer, the carriPr is ready for use.
200 ml of a pre-S(2)-~lBsAg-containing cell culture supernataDt (ll mg pre-S(2)--HBsAg, 950 mg protein) are incubated with lO ml of the affinity carrier for two hours at 14C. Unbound protein is removed by washing with a buffer on a sintering suction filter and the specifically bound pre-$(2)-HBsAg is eluted by incubation of the packed carrier in lO ml 8 M urea solution. This assay yielded 6.8 mg highly pure pre-S(2)-HBsAg at an overall protein amount of 5.~ mg (determination according to Bradford).
Exa_ele 5:
Coupling of monomeric human albumin to membranes Membranes, e.g., of nylon, polyvinylidene difluoride or cellulose polyacrylamide mixed polymers are possible ~2~3 carriers. Nylon membrane (e.g., Zetabind, CUNO~ is partially hydrolyzed by incubation in HCl/E12O. The released amino groups are reacted with 1 M oxaldialdehyde in the presence of 0.1 M sodium cyanoborohydride at pH 7 for two hours. After washing with deionized water, human serum albumin (10 mg/mi in 0.1 M phosphate buffer pEI 7) is coupled to this membrane activated with reactive aldehyde groups in the presence of 0.1 M sodium cyanoborohydride.
Excess reactive groups are blocked by the addition of 1 M
ethanolamine. After washing with phosphate buffer (0.1 M;
pH ~3), the membrane may be used to selectively bind pre-S(2)-containing proteins.
The binding capacity oE a thus produced membrane is 5 to 10 myg pre-S(2)-HssAg/cm2.
Exam~le 6:
Coupling of a monomeric human albumin to microtiter plates To this end, functionalized polystyrene plates, such as, e.y., Amino-Plate (including primary amino groups) or Carbo-Plate (including carboxyl groups) (Nissho Iwai Corp., Tokyo) or similar materials may be used.
200 myl of a solution of N-ethoxycarbonyl-2-ethoxy-1,2-dlhydro-quinoline (~0 mM in 50 % aqueous ethanol) are pipetted into each well of a 96-well microtiter plate and incubated for two hours at ~0C. After careful washing with ethanol and deionized water, 200 myl of a 1 % aqueous solution of human serum albumin are each pipetted into a well and incubated at -~4C over night. After blocking with 1 M ethanolamine or 1 M acetate buffer pEI~ and subesequent washing, the plate is ready for use.
100 myl of a pre-S(2)-HBsAg-containing cell culture e3 2 ~ ~
supernatant are pipetted into the prepared microtiter plate in increasing dilutions and incubated at ~4C for two hours. After washing with buffer and incubation with enzyme-labeled anti-pre-S(2)-HBsAg ~monoclonal antibodies) following the known ELISA technique, the bound portion of pre-S(2)-protein is determined by substrate addition according to known methods of absorption spectroscopy.
If the microtiter plate is loaded with SM-denaturized pre-S(2)-HBsAg or synthetic pre-S(2)-peptide, a thus I0 treated plate may be used for assaying anti-pre-S(2)-antibodies (e.g., from patients' sera).
10 mg pre-S(2)-MBsAg in 10 ml phosphate buffer (0.1 M, pH 7.0~ are incubated with 100 myl mercaptoethanol in 10 ~
sodium dodecyl sulfate (SDS) for 5 min at 100C. In doing so, the immunologically active S-antigen of HBsAg is destroyed (Milich D.R. et al., Enhanced Immunogenicity of the Pre-S-Region of Mepatitis B Surface Antigen, Science 227: 1195-1199 (1985)). After blocking of the SM-groups with 500 mg iodoacetamide for one hour, excess reaction products are eliminated by dialysis and the pre-S(2)-antigen is diluted to 100 myg protein/ml with phosphate buffer pM 7.~.
A prepared HSA microtiter plate is coated with this pre-S(2) solution and subsequently washed. lrhus prepared plates may be stored even in the dry state.
100 myl of an anti-pre-S(2)-containing rabbit serum are each pipetted into the wells in increasing dilutions.
After incubation at ~4C over night, bound pre-S(2)-antibodies are assayed by means of absorption spectroscopy 30 by incubation with enzyme--labeled anti-rabbit serum and subsequent substrate addition.
Exa_ple 7:
Binding capacity and yield oE pre-S(2)-HBsAg when using various albumin carrier complexes Human serum albumin (HSA) and polymerized human serum albumin (poly-HSA) were covalently and adsorptively bound to various carrier substances in comparable amounts. The binding capacity and the yield of pre-S(2)-H~sAg after elution were determined by means of a HBsAg-specific ELISA
method a~d are summarized in the following Table:
2 ~ 3 Albumin Carrier Type of Capacity % yield after elution binding mg/g gel with 2M U ~M U 8M U 3M
KSCN
____________________________________________________________ mono CNBr- covalent 7.6 45 85 60 50 meric Sepha- via rose NH2 mono anti- adsorp- 0 meric HSA tive Sepha-rose poly- anti- adsorp- 5.8 - - - -meric HSA tive Sepha-rose poly- CNBr- covalent 6.~ 12 ~0 55 ~5 meric Sepha- via rose NH2 ____________________________________________________________ U = Urea It is apparent from the Table that hig'ner yields of pre-S(2)-HBsAg are obtained with the insoluble polymer carrier according to the invention, with which the monomeric human albumin is covalently bound to Sepharose in the present exemplary embodiment, than with Sepharose to which polyalbumin is covalently bound.
These remarkably higher yields are due to the more yentle eluting conditions made possible by employing the carrier of the invention.In addition, it can be seen that it is only the covalent binding of HSA to the carrier that results in the inventive characteristic oE the pre-S(2)-affinity and that a molecular sieve to which albumin is bound merely by adsorption is not suitable for affinity chromatographic purification.
Claims (16)
1. A complex comprised of an insoluble polymer carrier having monomeric human albumin covalently bound thereto and of a pre-S hepatitis B surface antigen having a pre-S(2)-region and a pre-S(1)-region and bound in an elutable form to said monomeric human albumin by at least one of said pre-S(2)-region and said pre-S(1)-region.
2. A complex as set forth in claim 1, wherein said insoluble polymer carrier is based on a substance selected from the group consisting of agarose and dextrane.
3. An insoluble polymer carrier to which monomeric human albumin is covalently bound.
4. An insoluble polymer carrier as set forth in claim 3 and based on a substance selected from the group consisting of agarose and dextrane.
5. A method of purifying pre-S hepatitis B surface antigen by using a complex comprised of an insoluble polymer carrier having monomeric human albumin covalently bound thereto and of a pre-S hepatitis B surface antigen having a pre-S(2)-region and a pre-S(1)-region and bound in an elutable form to said monomeric human albumin by at least one of said pre-S(2)-region and said pre-S(1)-region, which method comprises washing said complex with a buffer solution to remove possibly present impurities, and cleaving said complex and recovering said pre-S
hepatitis B surface antigen.
hepatitis B surface antigen.
6. A method as set forth in claim 5, wherein said pre-S
hepatitis B surface antigen is recovered by treating said complex with an eluting agent containing chaotropic substances.
hepatitis B surface antigen is recovered by treating said complex with an eluting agent containing chaotropic substances.
7. A method as set forth in claim 6, wherein said chaotropic substances are selected from the group consisting of urea, guanuidine hydrochloride, thiocyanate, potassium chloride, magnesium chloride and potassium iodide.
8. A method as set forth in claim 5, wherein said pre-S
hepatitis B surface antigen is recovered by treating said complex with a detergent.
hepatitis B surface antigen is recovered by treating said complex with a detergent.
9. A method as set forth in claim 5, wherein said pre-S
hepatitis B surface antigen is recovered by treating said complex with a pH modifying agent.
hepatitis B surface antigen is recovered by treating said complex with a pH modifying agent.
10. A method as set forth in claim 5, further comprising a secondary purification step.
11. A method as set forth in claim 8, wherein said detergent is selected from the group consisting of an ionic detergent, a zwitterionic detergent and a non-ionic detergent.
12. A method as set forth in claim 11, wherein said ionic detergent is selected from the group consisting of sodium desoxycholate and taurodesoxycholic acid.
13. A method as set forth in claim 11, wherein said zwitterionic detergent is selected form the group consisting of 3[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate and 3[(3-cholamidopropyl)-dimethyl-ammonio]-2-hydroxy-1-propane sulfonate.
14. A method as set forth in claim 11, wherein said non-ionic detergent is octyl glucopyranoside.
15. A method as set forth in claim 10, wherein said secondary purification step comprises a process selected from the group consisting of gel filtration, ultracentrifugation, hydrophobic chromatography, lectin affinity chromatography and ion exchange chromatography.
16. The use of a complex set forth in claim 1 for preparing diagnostics and vaccines, which vaccines are apt both for active immunization against hepatitis B and for obtaining specific immunoglobulin of donors immunized with such vaccines.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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ATA2198/89 | 1989-09-20 | ||
AT2198/89A AT392417B (en) | 1989-09-20 | 1989-09-20 | COMPLEXLY SUITABLE FOR PERFORMING A METHOD FOR CLEANING PRE-S-HEPATITIS-B-VIRUS SURFACE ANTIGENS |
Publications (1)
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CA2025263A1 true CA2025263A1 (en) | 1991-03-21 |
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CA002025263A Abandoned CA2025263A1 (en) | 1989-09-20 | 1990-09-13 | Complex suitable for carrying out a method of purifying pre-s hepatitis b surface antigen |
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EP (1) | EP0419446B1 (en) |
JP (1) | JP2721033B2 (en) |
AT (2) | AT392417B (en) |
CA (1) | CA2025263A1 (en) |
DE (1) | DE59008927D1 (en) |
DK (1) | DK0419446T3 (en) |
ES (1) | ES2074558T3 (en) |
FI (1) | FI101625B (en) |
NO (1) | NO300421B1 (en) |
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CN111659355B (en) * | 2020-06-23 | 2022-12-13 | 武汉瑞法医疗器械有限公司 | Alkylation modified hepatitis B virus immunoadsorbent and preparation method thereof |
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IL45247A0 (en) * | 1974-07-11 | 1974-10-22 | Yeda Res & Dev | Water insoluble protein preparations |
US4515714A (en) * | 1983-03-09 | 1985-05-07 | Juridicial Foundation, The Chemo-Semo-Sero-Therapeutic Research Institute | Method for purification of hepatitis B virus surface antigen |
ATE73349T1 (en) * | 1984-06-18 | 1992-03-15 | Chiron Corp | HEPATITIS SURFACE ANTIGEN PARTICLE VACCINATION. |
JPS6137738A (en) * | 1984-07-31 | 1986-02-22 | Tetsuo Nakamura | Vaccine for hepatitis b |
EP0243103B1 (en) * | 1986-04-25 | 1992-12-30 | Merck & Co. Inc. | Purification of pre-s hbsag by polymerized serum albumin affinity binding |
US4855055A (en) * | 1988-09-28 | 1989-08-08 | National Science Council | Isolation and purification pre-S2 containing hepatitis B virus surface antigen by chemical affinity chromatography |
-
1989
- 1989-09-20 AT AT2198/89A patent/AT392417B/en not_active IP Right Cessation
-
1990
- 1990-09-07 FI FI904412A patent/FI101625B/en not_active IP Right Cessation
- 1990-09-13 AT AT90890262T patent/ATE121417T1/en not_active IP Right Cessation
- 1990-09-13 CA CA002025263A patent/CA2025263A1/en not_active Abandoned
- 1990-09-13 DE DE59008927T patent/DE59008927D1/en not_active Expired - Fee Related
- 1990-09-13 ES ES90890262T patent/ES2074558T3/en not_active Expired - Lifetime
- 1990-09-13 EP EP90890262A patent/EP0419446B1/en not_active Expired - Lifetime
- 1990-09-13 DK DK90890262.0T patent/DK0419446T3/en active
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ES2074558T3 (en) | 1995-09-16 |
DE59008927D1 (en) | 1995-05-24 |
NO904085D0 (en) | 1990-09-19 |
FI904412A0 (en) | 1990-09-07 |
FI101625B1 (en) | 1998-07-31 |
FI101625B (en) | 1998-07-31 |
JP2721033B2 (en) | 1998-03-04 |
EP0419446B1 (en) | 1995-04-19 |
ATA219889A (en) | 1990-09-15 |
DK0419446T3 (en) | 1995-08-28 |
JPH03120300A (en) | 1991-05-22 |
NO904085L (en) | 1991-03-21 |
NO300421B1 (en) | 1997-05-26 |
ATE121417T1 (en) | 1995-05-15 |
AT392417B (en) | 1991-03-25 |
EP0419446A1 (en) | 1991-03-27 |
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