CA2359307A1 - Pertussis outer membrane protein - Google Patents
Pertussis outer membrane protein Download PDFInfo
- Publication number
- CA2359307A1 CA2359307A1 CA002359307A CA2359307A CA2359307A1 CA 2359307 A1 CA2359307 A1 CA 2359307A1 CA 002359307 A CA002359307 A CA 002359307A CA 2359307 A CA2359307 A CA 2359307A CA 2359307 A1 CA2359307 A1 CA 2359307A1
- Authority
- CA
- Canada
- Prior art keywords
- pertactin
- purified
- solution
- pertussis
- fha
- 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
- 201000005702 Pertussis Diseases 0.000 title claims description 25
- 101710116435 Outer membrane protein Proteins 0.000 title description 6
- 108010021711 pertactin Proteins 0.000 claims abstract description 117
- 241000588832 Bordetella pertussis Species 0.000 claims abstract description 27
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims abstract description 17
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims abstract description 17
- 239000012614 Q-Sepharose Substances 0.000 claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 229960005486 vaccine Drugs 0.000 claims description 41
- 102000036639 antigens Human genes 0.000 claims description 33
- 108091007433 antigens Proteins 0.000 claims description 33
- 239000000427 antigen Substances 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 22
- 201000010099 disease Diseases 0.000 claims description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 9
- 230000002480 immunoprotective effect Effects 0.000 claims description 9
- 208000015181 infectious disease Diseases 0.000 claims description 8
- 238000005342 ion exchange Methods 0.000 claims description 8
- 239000011159 matrix material Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 230000003053 immunization Effects 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims 3
- 108010081690 Pertussis Toxin Proteins 0.000 abstract description 34
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 abstract description 18
- 229910052921 ammonium sulfate Inorganic materials 0.000 abstract description 17
- 239000001166 ammonium sulphate Substances 0.000 abstract description 17
- 235000011130 ammonium sulphate Nutrition 0.000 abstract description 17
- 102000030621 adenylate cyclase Human genes 0.000 abstract description 14
- 108060000200 adenylate cyclase Proteins 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 13
- 239000002244 precipitate Substances 0.000 abstract description 9
- 239000000872 buffer Substances 0.000 abstract description 8
- 238000000108 ultra-filtration Methods 0.000 abstract description 8
- 239000000284 extract Substances 0.000 abstract description 7
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 abstract description 4
- 239000004202 carbamide Substances 0.000 abstract description 4
- 101710118506 69 kDa protein Proteins 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 26
- 210000004027 cell Anatomy 0.000 description 20
- 235000010633 broth Nutrition 0.000 description 19
- 239000012528 membrane Substances 0.000 description 19
- 235000018102 proteins Nutrition 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 18
- 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 14
- 239000000203 mixture Substances 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 9
- 238000000746 purification Methods 0.000 description 9
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 8
- 229940001007 aluminium phosphate Drugs 0.000 description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 8
- 239000010451 perlite Substances 0.000 description 8
- 235000019362 perlite Nutrition 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 239000012141 concentrate Substances 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000000706 filtrate Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000003755 preservative agent Substances 0.000 description 5
- 241000588807 Bordetella Species 0.000 description 4
- 230000002335 preservative effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000004224 protection Effects 0.000 description 4
- 235000019624 protein content Nutrition 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- QCDWFXQBSFUVSP-UHFFFAOYSA-N 2-phenoxyethanol Chemical compound OCCOC1=CC=CC=C1 QCDWFXQBSFUVSP-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 239000012736 aqueous medium Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 238000011031 large-scale manufacturing process Methods 0.000 description 3
- 229940066827 pertussis vaccine Drugs 0.000 description 3
- 229960005323 phenoxyethanol Drugs 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 229910021653 sulphate ion Inorganic materials 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000588779 Bordetella bronchiseptica Species 0.000 description 2
- 241000700198 Cavia Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000011403 purification operation Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 239000012465 retentate Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 229960000814 tetanus toxoid Drugs 0.000 description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 2
- 229940033663 thimerosal Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 241000588780 Bordetella parapertussis Species 0.000 description 1
- 101100114362 Caenorhabditis elegans col-7 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 206010011469 Crying Diseases 0.000 description 1
- 206010011878 Deafness Diseases 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 206010024769 Local reaction Diseases 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000282320 Panthera leo Species 0.000 description 1
- 101710194807 Protective antigen Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 229910052586 apatite Inorganic materials 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229960002713 calcium chloride Drugs 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229940030156 cell vaccine Drugs 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000036461 convulsion Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 229960002433 cysteine Drugs 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229960003983 diphtheria toxoid Drugs 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 235000003969 glutathione Nutrition 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000001698 pyrogenic effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Pertactin (formerly 69 kDa protein) is recovered in stable biologically pure form having no detectable adenylate cyclase activity from fermentation broth from the fermentation of Bordetella pertussis as well as from the cells. The broth is processed to selectively remove pertussis toxin (PT) and filamentous haemagglutinin (FHA), the pertactin is precipitated by ammonium sulphate and the precipitate is dissolved in buffer at pH 6.0 to 8.5, the solution then is passed through hydroxyapatite and Q-Sepharose chromatograph columns before final ultrafiltration. Cells are extracted with urea and the extract ultrafiltered and diafiltered. The pertactin is precipitated from the extract and the precipitate processed as above: In a variation, the broth is contacted with ammonium sulphate to precipitate pertactin, PT and FHA, the precipitate is dissolved and the PT and FHA selectively removed, before the solution is passed to the chromatograph columns.
Description
PERTUSSIS OUTER MEMBRANE PROTEIN
The present invention relates to an outer membrane protein of Bardetella pertus~~is, having a molecular weight' of approximately 69,000 Daltons, formerly called s the 69kDa protein and now called pertactin, provided in a form which has no detectable adenylate cyclase activity. The pertactin obtained from the fermentation broth and cellular extracts of B. pertussis by a specific purification procedure. The protein is used in to a "component" vaccine to protect against the disease of whooping cough.
This application is a division of copending Canadian Patent Application No. 2,224,052 filed April 3, 1991, which in turn is a division of Canadian Patent No.
15 2,079,887 filed April 3, 1991, Such applications claim pertactin having no detectable adenylate cyclase activity, the preparation thereof and compositions comprising the same.
The disease of whooping cough or pertussis is a 2o result of infection by Bordetella pertussis, and is a serious and debilitating human disease particularly in young children. For the last fifty years the disease has been controlled through large-scale immunization programmes. The current licensed vaccine in North 25 America is a "whole cell" vaccine prepared by growing the organism in fermentors and then treating the resulting B. pertussis cells with chemical agents, such as formaldehyde, to kill the organism and inactivate toxic proteins. The cells are resuspended and then used 30 directly or in combination with other antigens. This vaccine, although highly efficacious, has been associated with clinical symptoms that include fever, local reactions, high-pitched crying and convulsions.
Despite the fact that there is no proven relation 35 between these symptoms and the vaccine, there has been decreased public acceptance of this vaccine and in a number of countries, e.g. Japan, Sweden and the U.K., decreased immunization has led to outbreaks of the disease. The need for a more defined vaccine has been recognized and considerable effort has been directed by several manufacturers and researchers towards the s development of an efficacious pertussis vaccine that consists of a small number of highly purified proteins.
This vaccine has been termed a component vaccine.
This search has been hampered by a lack of information on the mechanism of pathogenesis of B.
to pertussis. Many virulence associated factors, such as pertussis toxin (PT), also known as lymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA), adenylate cyclase, lipopolysaccharide, agglutinogens and other outer membrane proteins have 15 been suggested for inclusion in an "acellular" vaccine, which is less defined than the component vaccine. Much of the work on acellular vaccines has concentrated on a PT-based vaccine. Results of a recent clinical trial have indicated that a vaccine consisting entirely of 2o PT-toxoid only partially protected children from the infection. A PT/FHA combination showed slightly higher efficacy but this was still lowE:r than that obtained for the whole-cell vaccine.
One potential protective antigen is an outer 2s membrane protein, with a molecular weight of approximately 69,000 daltons (pertactin) found in all virulent strains of B. pertussis. This protein is produced in relatively large amounts during the culture of the organism and can be purified from either the 3o fermentation broth or from cell extracts.
The potential importance of this outer membrane protein for inclusion in a human vaccine against whooping cough was suggested from attempts to prepare a vaccine to protect pigs against B. bronchiseptica 3s infection. Cell-surface extracts of B. bronchiseptica were used to immunize sows. Levels of antibody to a cell surface antigen with a molecular weight of 68,000 daltons correlated with protection of newborn piglets against infection. Similar antigens, with similar molecular weights, were detected in B. pertussis (approximately 69,000 daltons) and in B.parapertussis s (approximately 70,000 daltons). Immunisation with the protein obtained from B. pertussis protected. mice against intracerebral challenges with live organisms and antibodies to the protein conferred passive protection to mice in this test. Both active and passive to protection of mice in an aerosol challenge model have also been described.
The published procedures for purification of pertactin do not allow for the large-scale production of a highly purified, non-pyrogenic and stable antigen. One 15 reported method (Canadian Patent No. 1,253,073) involves acid-glycine extraction of th.e cells, anion-exchange chromatography and preparative iso-electric focussing.
However, the pertactin obtained has been reported to degrade into smaller fragments, to be sensitive to low 2o pH and to have adenylate cyclase activity. For these reasons, this extraction procedure is considered undesirable for large-scale production. In addition, iso-electric focussing is not amenable to large-scale production. A second procedure (U.S. Patent No.
25 5,101,014) involved the extraction of the outer membrane protein from the cells of an afimbriated strain of B.
pertussis. The protein was purified by a combination of DEAF-SepharoseTM and Affigel-blueT~ chromatographies. The potential of leaching the blue dye into the product 3o would be a possible safety concern. Neither method addresses the purification of pertactin directly from fermentation broths.
In one aspect, the present invention provides a purified pertactin produced by a method which comprises 35 contacting an impure solution of pertactin with hydroxyapatite. .
The present invention relates to an outer membrane protein of Bardetella pertus~~is, having a molecular weight' of approximately 69,000 Daltons, formerly called s the 69kDa protein and now called pertactin, provided in a form which has no detectable adenylate cyclase activity. The pertactin obtained from the fermentation broth and cellular extracts of B. pertussis by a specific purification procedure. The protein is used in to a "component" vaccine to protect against the disease of whooping cough.
This application is a division of copending Canadian Patent Application No. 2,224,052 filed April 3, 1991, which in turn is a division of Canadian Patent No.
15 2,079,887 filed April 3, 1991, Such applications claim pertactin having no detectable adenylate cyclase activity, the preparation thereof and compositions comprising the same.
The disease of whooping cough or pertussis is a 2o result of infection by Bordetella pertussis, and is a serious and debilitating human disease particularly in young children. For the last fifty years the disease has been controlled through large-scale immunization programmes. The current licensed vaccine in North 25 America is a "whole cell" vaccine prepared by growing the organism in fermentors and then treating the resulting B. pertussis cells with chemical agents, such as formaldehyde, to kill the organism and inactivate toxic proteins. The cells are resuspended and then used 30 directly or in combination with other antigens. This vaccine, although highly efficacious, has been associated with clinical symptoms that include fever, local reactions, high-pitched crying and convulsions.
Despite the fact that there is no proven relation 35 between these symptoms and the vaccine, there has been decreased public acceptance of this vaccine and in a number of countries, e.g. Japan, Sweden and the U.K., decreased immunization has led to outbreaks of the disease. The need for a more defined vaccine has been recognized and considerable effort has been directed by several manufacturers and researchers towards the s development of an efficacious pertussis vaccine that consists of a small number of highly purified proteins.
This vaccine has been termed a component vaccine.
This search has been hampered by a lack of information on the mechanism of pathogenesis of B.
to pertussis. Many virulence associated factors, such as pertussis toxin (PT), also known as lymphocytosis promoting factor (LPF), filamentous haemagglutinin (FHA), adenylate cyclase, lipopolysaccharide, agglutinogens and other outer membrane proteins have 15 been suggested for inclusion in an "acellular" vaccine, which is less defined than the component vaccine. Much of the work on acellular vaccines has concentrated on a PT-based vaccine. Results of a recent clinical trial have indicated that a vaccine consisting entirely of 2o PT-toxoid only partially protected children from the infection. A PT/FHA combination showed slightly higher efficacy but this was still lowE:r than that obtained for the whole-cell vaccine.
One potential protective antigen is an outer 2s membrane protein, with a molecular weight of approximately 69,000 daltons (pertactin) found in all virulent strains of B. pertussis. This protein is produced in relatively large amounts during the culture of the organism and can be purified from either the 3o fermentation broth or from cell extracts.
The potential importance of this outer membrane protein for inclusion in a human vaccine against whooping cough was suggested from attempts to prepare a vaccine to protect pigs against B. bronchiseptica 3s infection. Cell-surface extracts of B. bronchiseptica were used to immunize sows. Levels of antibody to a cell surface antigen with a molecular weight of 68,000 daltons correlated with protection of newborn piglets against infection. Similar antigens, with similar molecular weights, were detected in B. pertussis (approximately 69,000 daltons) and in B.parapertussis s (approximately 70,000 daltons). Immunisation with the protein obtained from B. pertussis protected. mice against intracerebral challenges with live organisms and antibodies to the protein conferred passive protection to mice in this test. Both active and passive to protection of mice in an aerosol challenge model have also been described.
The published procedures for purification of pertactin do not allow for the large-scale production of a highly purified, non-pyrogenic and stable antigen. One 15 reported method (Canadian Patent No. 1,253,073) involves acid-glycine extraction of th.e cells, anion-exchange chromatography and preparative iso-electric focussing.
However, the pertactin obtained has been reported to degrade into smaller fragments, to be sensitive to low 2o pH and to have adenylate cyclase activity. For these reasons, this extraction procedure is considered undesirable for large-scale production. In addition, iso-electric focussing is not amenable to large-scale production. A second procedure (U.S. Patent No.
25 5,101,014) involved the extraction of the outer membrane protein from the cells of an afimbriated strain of B.
pertussis. The protein was purified by a combination of DEAF-SepharoseTM and Affigel-blueT~ chromatographies. The potential of leaching the blue dye into the product 3o would be a possible safety concern. Neither method addresses the purification of pertactin directly from fermentation broths.
In one aspect, the present invention provides a purified pertactin produced by a method which comprises 35 contacting an impure solution of pertactin with hydroxyapatite. .
In the present invention, pertactin is obtained in large quantities from fermentor broth, which is the preferred source, and in a purified form, by using the processes described herein. The protein can be included in a product to be used for the widespread vaccination of children against whooping cough.
After growing the organi;~m in a fermentor, the cells are removed by centrifugation and filtration and the supernatant reduced in volume and sterilised. The to broth is diluted to a low ionic strength and, after removal of other antigens, the pertactin is isolated by chromatography on various substrates and may be further purified by ultrafiltration. The protein can also be isolated from the cells after extraction with urea, centrifugation and further processing to give a solution that can be treated as described above.
A method for the production of pertactin may comprise providing an impure aqueous solution of pertactin substantially free from other Bordetella 2o antigens, purifying pertactin in said aqueous solution by passing said aqueous solution sequentially in contact with a hydroxyapatite and an ion-exchange medium, and subjecting the resulting purified solution to ultrafiltration. Such method is claimed in CA
2;224,052.
The resulting product is very stable when purified by this method and has no detectable adenylate cyclase activity. This material is a biologically pure and stable pertactin having no detectable adenylate cyclase 3o activity.
The biologically pure and st=able pertactin provided herein is particularly useful as a component of a component vaccine. A component vaccine against disease caused by infection by Bordetella pertussis comprises immunoprotective biologically pure and stable pertactin having no detectable adenylate cyclase activity, as an active component thereof, and a physiologically-acceptable carrier therefor.
The component vaccine may also comprise at least one purified other immunoprotective pertussis antigen, 5 including pertussis toxin. (PT), filamentous haemagglutinin (FHA) and/or agglutinogens. The pertactin, on the one hand, and the at least one purified other immunoprotectivc= pertussis antigen, on another hand; may be separately isolated and purified io from a single fermentation of Bardetella pertussis.
A component vaccine against disease caused by infection by Bordetella pertuss.is comprises, as a first component, purified. pertactin having no detectable adenylate cyclase activity, as at least one additional component thereof, at least one purified other B.
pertussis antigen which is pertussis toxin (PT), filamentous- haemagglutinin (FHA) and/or agglutinogens;
and a pharmaceutically-acceptable carrier therefor.
Such component vaccine may be combined with 2o diphetheria toxoid (D) and tetanus toxoid (T) to provide a multivalent component vaccine which comprises (a) as a first component thereof, biologically pure and stable pertactin of Bordetella pertussis having no detectable adenylate cyclase activity; (b) as at least one 25 additional component thereof, at least one other purified B. pertussis antigen which is selected from the group consisting of pertussis toxin (PT), filamentous haemagglutinin (FHA) and agglutinogens; (c) as two further components thereof, two purified non-Bordetella 3o antigens which are diphtheria toxoid (D) tetanus toxoid (T); and (d) a pharmaceutically-acceptable carrier therefor. Such component vaccine is claimed in CA
2,224,052.
A method of making a component vaccine containing a 35 pertactin component may comprises separating from the pertussis component from an impure solution thereof which is substantially free from pertussis toxin and fila:entous haemaglutinin by a purification operation including contacting the impure solution with hydrcr~-apatite to remove contaminants substantially complet~i.y from the impure solution; and formulating the s pertactin component as a component of the component vaccine. Such procedure is claimed in CA 2,224,052.
The impure solution of pe:rtactin component may be provided by growing a strain of B. pertuss.is in a single fermentation to provide from the strain a composition to containing the B. pertussis ant_Lgens which are pertussis toxin (PT?, filamentous haE~mogglutinin (FHA) and pertactin and separating from the composition the PT and FHA.
The composition may be separated as a supernatant 15 from the growing of the strain of B. pertussis, agglutinogens may be purified from the remaining cellular material, and the purified agglutinogens may be formulated into the component vaccine along with purified and toxoided PT and FHA.
2o The biologically pure and stable pertactin having no detectable adenylate cyclase activity may be used for immunizing humans against disease caused by infection by Bordetella pertussis, preferably in combination with at least one purified other immunoprotective pertussis 25 antigen, including PT, FHA and/or agglutinogens and such uses constitute.aspects of the invention claimed in CA
2,079,887.
The biologically pure and stable pertactin having no detectable adenylate cyclase activity may be used in 3o the manufacture of a component vaccine against Bordetella pertussis, which may include at least one purified other immunoprotective pertussis antigen, as a component of the vaccine.
The purification operation. may further comprise, 35 for the pertactin which is obtained in a purified form having no detectable adenylate cyclase activity, contacting the impure solution of pertactin with a matrix composed of an ion-exchange medium, particularly Q-Sepharose~. In a preferred aspect of the invention, there is provided a method for the production of pertactin which comprises purifying an impure aqueous s solution of pertactin substantially free from non-ertactin components by contacting the aqueous solution sequentially with hydroxyapatite and a matrix composed of an ion-exchange medium to provide a purified solution of pertactin and recovering the purified solution of io pertactin. In such procedure, the matrix is particularly composed of Q-Sepharose~.
Broadly speaking, in an additional aspect of the invention claimed in CA 2, 224, 052, there is provided an improvement in a process for purifying an aqueous 15 solutino of pertactin. The improvement comprises contacting the impure solution of pertactin with hydroxyapatite.
The process described herein allows for the purification of several protein antigens for possible 2o inclusion in a component pertussis vaccine from a single fermentation of B. pertussis.
In the present invention, B. pertussis is grown in a fermentor under controlled conditions. Carbon sources and growth factors are supplemented either continuously 2s or in batches at various intervals during the fermentation until the pertussi.s proteins (PT, FHA and pertactin) are at the desired levels as determined by a specific enzyme-linked immunosorbent assay (ELISA) for each antigen. The fermentor broth is harvested, the 3o majority of the cells removed by centrifugation and the broth sterilised by microfiltration, preferably using known membrane filters of about 0.2 micron pore size:
The broth is concentrated, sa.y 10-fold, by membrane ultrafiltration and used for the purification of 3s pertussis toxin and FHA (see European Patent No.
0,336,736; U.S. Patent No. 4,997,915). The cells~are the source of material for the purification of the agglutinogens. Pertactin can be purified from both the broth or cells. The former is preferred as the majority of the protein is found in the broth.
s The first stage in the purification of the pertactin requires dilution of the broth to a low ionic strength and chromatography on Perlite* or other suitable solid particulate adsorbent material to remove the PT and FHA antigens, as moz-e fully described in the 1o aforementioned European Patent No. 0,336,736, U.S.
Patent No. 4,997,915). The PT and FHA antigens can be removed from the adsorbent material by treatment with an aqueous medium of high ionic strength for use in a component pertussis vaccine.
15 As used herein, the term "low ionic strength"
refers to an aqueous medium having a conductivity of about 11 mS/cm or less, preferably about 4 mS/cm or less. The unit of measurement mS/cm is millisiemen per centimeter. A Siemen (S) is a unit of conductivity and 2o is the equivalent of the inverse of resistance (ohm) and is sometimes designated mho. The term "high ionic strength" as used herein refers to an aqueous medium having a conductivity of greater than about 11 mS/cm and preferably at least about 50 mS/cm.
2s The remaining mixture then is concentrated by membrane filtration, subjected to ammonium sulphate precipitation and the resulting pellet dissolved in low ionic strength buffer, such as T'ris-.HCl, at a pH of 6.0 to 8.5 to yield a solution with a final conductivity of 3o generally less than about 4 mS/cm, typically approximately 3.4 mS/cm. The solution is chromatographed sequentially on an hydroxyapatite, and an ion-exchange medium, such as Q-Sepharose~. Pertactin elutes in the unbound fraction of both columns under the specified 35 buffer conductivity. However, if the conductivity is bower than 1.S mS/cm for hydroxy<~patite or 2.8 mS/cm for * - Trademark Q-Sepharose~, pertactin binds to both columns and can be eluted with a buffer having conductivities 1.5 mS/cm or greater for hydroxyapatite and 2.8 mS/cm for or greater Q-Sepharose~.
s The pertactin is further purified by ultrafiltration through about 100 to 300 kDa Nominal Molecular Weight Limit (NMWL) -membranes where it is collected in the filtrate, concentrated using membranes with a NMWL of about 30 kDa or less and sterile filtered io for use in combination with other pertussis antigens in a vaccine.
In an alternative procedure, pertactin along with FHA and PT are precipitated from fermentor broth, by addition of ammonium sulphate. The precipitate is 15 removed from .the residual broth and redissolved to provide an aqueous solution suitable for processing as described above first to remove the PT and FHA and then to purify the pertactin.
Pertactin also can be purified from B. pertussis 20 cells. The cells are extracted in a solution containing a high concentration (for example, 4 M) of urea for, say 1.5 hr. at room temperature. Cell debris is removed by centrifugation and the supernatant, which contains the protein, is subjected to ultra--filtration using 100 to 25 1000 kDa NMWL membranes. High molecular weight proteins, such as the agglutinogens, are retained while the majority of the pertactin is filtered through. The filtrate is concentrated, diafi7_tered using a 30 kDa or less NMWL membrane, and then precipitated by ammonium 3o sulphate and centrifuged to give a pellet for processing as described above.
Pertactin, although reportedly susceptible to proteolytic cleavage, is very stable when purified by the method of the invention.. SDS-PAGE analysis 35 indicates that the purified pert=actin is homogenous and essentially intact with traces of degradation products of molecular weights between 30 and 40 kDa. No evidence for further degradation or change in immunogenicity was found after several months of storage at 2 to 8°C or at higher temperatures (24°C, 37°C) . In contrast to a s previous publication (Canadian Patent No. 1,253,073), the finding that the pertactin prepared by this method does not show any detectable adenylate cyclase activity and has enhanced stability makes it an ideal candidate for inclusion into a component. vaccine for protection to against B. pertussis.
EXAMPLES.
Methods of protein biochemistry, immunochemistry used but not explicitly described in this disclosure and these Examples, are amply reported in the scientific literature and are well within the ability of those skilled in the art.
Example 1 This Example illustrates the growth of B. pertussis in fermentors.
B. pertussis was seeded into a fermentor containing 250 L of broth (modified Staines-Scholte medium). During the period of fermentation, monosodium glutamate and the growth factors, glutathione, ferrous sulphate, calcium chloride, ascorbic acid, niacin and cysteine were added 2s during the fermentation process to increase antigen yields. At the end of a 48 hr. fermentation period, the broth was centrifuged to remove the majority of cells and the supernatant, which contains PT, FHA and most of the pertactin, was further clarified by ultrafiltration 3o through cellulose acetate membranes (0.22~.m,pore size).
The sterilised filtrate was concentrated approximately 10-fold using a 20 kDa NMWL membrane and assayed for protein content and for antigen by antigen-specific ELISAs.
After growing the organi;~m in a fermentor, the cells are removed by centrifugation and filtration and the supernatant reduced in volume and sterilised. The to broth is diluted to a low ionic strength and, after removal of other antigens, the pertactin is isolated by chromatography on various substrates and may be further purified by ultrafiltration. The protein can also be isolated from the cells after extraction with urea, centrifugation and further processing to give a solution that can be treated as described above.
A method for the production of pertactin may comprise providing an impure aqueous solution of pertactin substantially free from other Bordetella 2o antigens, purifying pertactin in said aqueous solution by passing said aqueous solution sequentially in contact with a hydroxyapatite and an ion-exchange medium, and subjecting the resulting purified solution to ultrafiltration. Such method is claimed in CA
2;224,052.
The resulting product is very stable when purified by this method and has no detectable adenylate cyclase activity. This material is a biologically pure and stable pertactin having no detectable adenylate cyclase 3o activity.
The biologically pure and st=able pertactin provided herein is particularly useful as a component of a component vaccine. A component vaccine against disease caused by infection by Bordetella pertussis comprises immunoprotective biologically pure and stable pertactin having no detectable adenylate cyclase activity, as an active component thereof, and a physiologically-acceptable carrier therefor.
The component vaccine may also comprise at least one purified other immunoprotective pertussis antigen, 5 including pertussis toxin. (PT), filamentous haemagglutinin (FHA) and/or agglutinogens. The pertactin, on the one hand, and the at least one purified other immunoprotectivc= pertussis antigen, on another hand; may be separately isolated and purified io from a single fermentation of Bardetella pertussis.
A component vaccine against disease caused by infection by Bordetella pertuss.is comprises, as a first component, purified. pertactin having no detectable adenylate cyclase activity, as at least one additional component thereof, at least one purified other B.
pertussis antigen which is pertussis toxin (PT), filamentous- haemagglutinin (FHA) and/or agglutinogens;
and a pharmaceutically-acceptable carrier therefor.
Such component vaccine may be combined with 2o diphetheria toxoid (D) and tetanus toxoid (T) to provide a multivalent component vaccine which comprises (a) as a first component thereof, biologically pure and stable pertactin of Bordetella pertussis having no detectable adenylate cyclase activity; (b) as at least one 25 additional component thereof, at least one other purified B. pertussis antigen which is selected from the group consisting of pertussis toxin (PT), filamentous haemagglutinin (FHA) and agglutinogens; (c) as two further components thereof, two purified non-Bordetella 3o antigens which are diphtheria toxoid (D) tetanus toxoid (T); and (d) a pharmaceutically-acceptable carrier therefor. Such component vaccine is claimed in CA
2,224,052.
A method of making a component vaccine containing a 35 pertactin component may comprises separating from the pertussis component from an impure solution thereof which is substantially free from pertussis toxin and fila:entous haemaglutinin by a purification operation including contacting the impure solution with hydrcr~-apatite to remove contaminants substantially complet~i.y from the impure solution; and formulating the s pertactin component as a component of the component vaccine. Such procedure is claimed in CA 2,224,052.
The impure solution of pe:rtactin component may be provided by growing a strain of B. pertuss.is in a single fermentation to provide from the strain a composition to containing the B. pertussis ant_Lgens which are pertussis toxin (PT?, filamentous haE~mogglutinin (FHA) and pertactin and separating from the composition the PT and FHA.
The composition may be separated as a supernatant 15 from the growing of the strain of B. pertussis, agglutinogens may be purified from the remaining cellular material, and the purified agglutinogens may be formulated into the component vaccine along with purified and toxoided PT and FHA.
2o The biologically pure and stable pertactin having no detectable adenylate cyclase activity may be used for immunizing humans against disease caused by infection by Bordetella pertussis, preferably in combination with at least one purified other immunoprotective pertussis 25 antigen, including PT, FHA and/or agglutinogens and such uses constitute.aspects of the invention claimed in CA
2,079,887.
The biologically pure and stable pertactin having no detectable adenylate cyclase activity may be used in 3o the manufacture of a component vaccine against Bordetella pertussis, which may include at least one purified other immunoprotective pertussis antigen, as a component of the vaccine.
The purification operation. may further comprise, 35 for the pertactin which is obtained in a purified form having no detectable adenylate cyclase activity, contacting the impure solution of pertactin with a matrix composed of an ion-exchange medium, particularly Q-Sepharose~. In a preferred aspect of the invention, there is provided a method for the production of pertactin which comprises purifying an impure aqueous s solution of pertactin substantially free from non-ertactin components by contacting the aqueous solution sequentially with hydroxyapatite and a matrix composed of an ion-exchange medium to provide a purified solution of pertactin and recovering the purified solution of io pertactin. In such procedure, the matrix is particularly composed of Q-Sepharose~.
Broadly speaking, in an additional aspect of the invention claimed in CA 2, 224, 052, there is provided an improvement in a process for purifying an aqueous 15 solutino of pertactin. The improvement comprises contacting the impure solution of pertactin with hydroxyapatite.
The process described herein allows for the purification of several protein antigens for possible 2o inclusion in a component pertussis vaccine from a single fermentation of B. pertussis.
In the present invention, B. pertussis is grown in a fermentor under controlled conditions. Carbon sources and growth factors are supplemented either continuously 2s or in batches at various intervals during the fermentation until the pertussi.s proteins (PT, FHA and pertactin) are at the desired levels as determined by a specific enzyme-linked immunosorbent assay (ELISA) for each antigen. The fermentor broth is harvested, the 3o majority of the cells removed by centrifugation and the broth sterilised by microfiltration, preferably using known membrane filters of about 0.2 micron pore size:
The broth is concentrated, sa.y 10-fold, by membrane ultrafiltration and used for the purification of 3s pertussis toxin and FHA (see European Patent No.
0,336,736; U.S. Patent No. 4,997,915). The cells~are the source of material for the purification of the agglutinogens. Pertactin can be purified from both the broth or cells. The former is preferred as the majority of the protein is found in the broth.
s The first stage in the purification of the pertactin requires dilution of the broth to a low ionic strength and chromatography on Perlite* or other suitable solid particulate adsorbent material to remove the PT and FHA antigens, as moz-e fully described in the 1o aforementioned European Patent No. 0,336,736, U.S.
Patent No. 4,997,915). The PT and FHA antigens can be removed from the adsorbent material by treatment with an aqueous medium of high ionic strength for use in a component pertussis vaccine.
15 As used herein, the term "low ionic strength"
refers to an aqueous medium having a conductivity of about 11 mS/cm or less, preferably about 4 mS/cm or less. The unit of measurement mS/cm is millisiemen per centimeter. A Siemen (S) is a unit of conductivity and 2o is the equivalent of the inverse of resistance (ohm) and is sometimes designated mho. The term "high ionic strength" as used herein refers to an aqueous medium having a conductivity of greater than about 11 mS/cm and preferably at least about 50 mS/cm.
2s The remaining mixture then is concentrated by membrane filtration, subjected to ammonium sulphate precipitation and the resulting pellet dissolved in low ionic strength buffer, such as T'ris-.HCl, at a pH of 6.0 to 8.5 to yield a solution with a final conductivity of 3o generally less than about 4 mS/cm, typically approximately 3.4 mS/cm. The solution is chromatographed sequentially on an hydroxyapatite, and an ion-exchange medium, such as Q-Sepharose~. Pertactin elutes in the unbound fraction of both columns under the specified 35 buffer conductivity. However, if the conductivity is bower than 1.S mS/cm for hydroxy<~patite or 2.8 mS/cm for * - Trademark Q-Sepharose~, pertactin binds to both columns and can be eluted with a buffer having conductivities 1.5 mS/cm or greater for hydroxyapatite and 2.8 mS/cm for or greater Q-Sepharose~.
s The pertactin is further purified by ultrafiltration through about 100 to 300 kDa Nominal Molecular Weight Limit (NMWL) -membranes where it is collected in the filtrate, concentrated using membranes with a NMWL of about 30 kDa or less and sterile filtered io for use in combination with other pertussis antigens in a vaccine.
In an alternative procedure, pertactin along with FHA and PT are precipitated from fermentor broth, by addition of ammonium sulphate. The precipitate is 15 removed from .the residual broth and redissolved to provide an aqueous solution suitable for processing as described above first to remove the PT and FHA and then to purify the pertactin.
Pertactin also can be purified from B. pertussis 20 cells. The cells are extracted in a solution containing a high concentration (for example, 4 M) of urea for, say 1.5 hr. at room temperature. Cell debris is removed by centrifugation and the supernatant, which contains the protein, is subjected to ultra--filtration using 100 to 25 1000 kDa NMWL membranes. High molecular weight proteins, such as the agglutinogens, are retained while the majority of the pertactin is filtered through. The filtrate is concentrated, diafi7_tered using a 30 kDa or less NMWL membrane, and then precipitated by ammonium 3o sulphate and centrifuged to give a pellet for processing as described above.
Pertactin, although reportedly susceptible to proteolytic cleavage, is very stable when purified by the method of the invention.. SDS-PAGE analysis 35 indicates that the purified pert=actin is homogenous and essentially intact with traces of degradation products of molecular weights between 30 and 40 kDa. No evidence for further degradation or change in immunogenicity was found after several months of storage at 2 to 8°C or at higher temperatures (24°C, 37°C) . In contrast to a s previous publication (Canadian Patent No. 1,253,073), the finding that the pertactin prepared by this method does not show any detectable adenylate cyclase activity and has enhanced stability makes it an ideal candidate for inclusion into a component. vaccine for protection to against B. pertussis.
EXAMPLES.
Methods of protein biochemistry, immunochemistry used but not explicitly described in this disclosure and these Examples, are amply reported in the scientific literature and are well within the ability of those skilled in the art.
Example 1 This Example illustrates the growth of B. pertussis in fermentors.
B. pertussis was seeded into a fermentor containing 250 L of broth (modified Staines-Scholte medium). During the period of fermentation, monosodium glutamate and the growth factors, glutathione, ferrous sulphate, calcium chloride, ascorbic acid, niacin and cysteine were added 2s during the fermentation process to increase antigen yields. At the end of a 48 hr. fermentation period, the broth was centrifuged to remove the majority of cells and the supernatant, which contains PT, FHA and most of the pertactin, was further clarified by ultrafiltration 3o through cellulose acetate membranes (0.22~.m,pore size).
The sterilised filtrate was concentrated approximately 10-fold using a 20 kDa NMWL membrane and assayed for protein content and for antigen by antigen-specific ELISAs.
Example 2 This Example illustrates the large-scale removal of PT and FHA using a chromatographic column of Perlite.
The broth concentrate, prepared as described in s Example 1, was diluted with water to a conductivity of <4 mS/cm and subjected to chromatography on a Perlite column (12 cm[H] x 37 cm[D]), previously equilibrated with water, at a protein to Perlite ratio of approximately 3 mg per millilitre and a linear flow rate to of approximately 100 cm/hr. Proteins bound to the Perlite were almost exclusively PT and FHA while pertactin was found in the flow-through.
Example 3 This Example illustrates the precipitation of 15 pertactin using ammonium sulphate fractionation.
The flow-through fraction from Example 2 was concentrated to a volume of approximately 10 litres by ultrafiltration using 10 kDa . NMWL membranes. The resultant solution usually had a protein concentration 20 of 1 to 2 mg/ml. While stirring at room temperature, ammonium sulphate (3.5 Kg/10 L of concentrate or 350 w/v) was slowly added, and the mixture left to dissolve before transferring to a refrigerator at 2 to 8°C and stirred for an additional two hours, preferably 2s overnight. The precipitate was collected by centrifugation and dissolved :in 2 litres of 10 mM
Tris.HCl, pH 6.0 to 8.5. A second ammonium sulphate fractionation (25% w/v) was effected by slowly adding ammonium sulphate (500 g) to the 2 litres of solution 3o and stirring for at least 2 hours after the solution was cooled to 2 to 8°C, usually overnight. Finally the precipitate was collected by centrifugation, dissolved in 2 L of Tris.HCl; pH 7.5; and the conductivity adjusted to approximately 3.4 mS/cm by adding either 35 ammonium sulphate (if below 3.4 mS/cm) or 10 mM
Tris.HCl, pH 7.5 (if higher than 3.4 mS/cm).
The broth concentrate, prepared as described in s Example 1, was diluted with water to a conductivity of <4 mS/cm and subjected to chromatography on a Perlite column (12 cm[H] x 37 cm[D]), previously equilibrated with water, at a protein to Perlite ratio of approximately 3 mg per millilitre and a linear flow rate to of approximately 100 cm/hr. Proteins bound to the Perlite were almost exclusively PT and FHA while pertactin was found in the flow-through.
Example 3 This Example illustrates the precipitation of 15 pertactin using ammonium sulphate fractionation.
The flow-through fraction from Example 2 was concentrated to a volume of approximately 10 litres by ultrafiltration using 10 kDa . NMWL membranes. The resultant solution usually had a protein concentration 20 of 1 to 2 mg/ml. While stirring at room temperature, ammonium sulphate (3.5 Kg/10 L of concentrate or 350 w/v) was slowly added, and the mixture left to dissolve before transferring to a refrigerator at 2 to 8°C and stirred for an additional two hours, preferably 2s overnight. The precipitate was collected by centrifugation and dissolved :in 2 litres of 10 mM
Tris.HCl, pH 6.0 to 8.5. A second ammonium sulphate fractionation (25% w/v) was effected by slowly adding ammonium sulphate (500 g) to the 2 litres of solution 3o and stirring for at least 2 hours after the solution was cooled to 2 to 8°C, usually overnight. Finally the precipitate was collected by centrifugation, dissolved in 2 L of Tris.HCl; pH 7.5; and the conductivity adjusted to approximately 3.4 mS/cm by adding either 35 ammonium sulphate (if below 3.4 mS/cm) or 10 mM
Tris.HCl, pH 7.5 (if higher than 3.4 mS/cm).
Example 4 This Example illustrates the precipitation of pertactin from pertussis fermentation broth concentrates.
Ammonium sulphate (250 g/L of broth) was added to fermentor broth concentrates and the mixture stirred for more than two hours after the mixture had reached 2 to 8°C. The precipitate was col7.ected by centrifugation, dissolved in 10 mM Tris.HCl, pH 7.5, and the same buffer to added until the conductivity was < 4.0 mS/cm. This solution contained all the PT, FHA and pertactin. The solution was subjected to Perlite chromatography (as described in Example 2) to remove PT and FHA and the Perlite flow-through was subjected to hydroxyapatite and is Q-Sepharose~ chromatography (sef=_ Example 5) .
Example 5 This Example illustrates the chromatography of pertactin on hydroxyapatite and Q-Sepharose~.
Hydroxyapatite was packed into a suitable size 2o column, preferably 5 cm [D] x 10 cm [H] and equilibrated with 10 mM Tris.HCl buffer, pH 7.5, containing 15 mM
ammonium sulphate (conductivity approximately 3.4 mS/cm). Q-Sepharose~ was packed into a similar column and equilibrated with the same :buffer. The two columns 2s were connected in series with the hydroxyapatite column upstream of the Q-Sepharose~ column. The resolubilised pertactin (from Example 3) when subjected to chromatography on the two columns in series did not bind to the matrices and the flow-through fraction was 3o collected. After filtration through a 300 kDa NMWL
membrane, the filtrate containing the pertactin was concentrated and diafiltered using <30 kDa NMWL
membranes and finally sterile filtered using a 0.22 ~.m membrane.
Ammonium sulphate (250 g/L of broth) was added to fermentor broth concentrates and the mixture stirred for more than two hours after the mixture had reached 2 to 8°C. The precipitate was col7.ected by centrifugation, dissolved in 10 mM Tris.HCl, pH 7.5, and the same buffer to added until the conductivity was < 4.0 mS/cm. This solution contained all the PT, FHA and pertactin. The solution was subjected to Perlite chromatography (as described in Example 2) to remove PT and FHA and the Perlite flow-through was subjected to hydroxyapatite and is Q-Sepharose~ chromatography (sef=_ Example 5) .
Example 5 This Example illustrates the chromatography of pertactin on hydroxyapatite and Q-Sepharose~.
Hydroxyapatite was packed into a suitable size 2o column, preferably 5 cm [D] x 10 cm [H] and equilibrated with 10 mM Tris.HCl buffer, pH 7.5, containing 15 mM
ammonium sulphate (conductivity approximately 3.4 mS/cm). Q-Sepharose~ was packed into a similar column and equilibrated with the same :buffer. The two columns 2s were connected in series with the hydroxyapatite column upstream of the Q-Sepharose~ column. The resolubilised pertactin (from Example 3) when subjected to chromatography on the two columns in series did not bind to the matrices and the flow-through fraction was 3o collected. After filtration through a 300 kDa NMWL
membrane, the filtrate containing the pertactin was concentrated and diafiltered using <30 kDa NMWL
membranes and finally sterile filtered using a 0.22 ~.m membrane.
Example 6:
This Example illustrates the purification of pertactin by blinding to Q-Sepha:rose~.
The Perlite flow-through fraction from Example 2, s was concentrated to approximately 7 L, having a protein concentration usually between :1.5 to 3.0 mg/ml. Solid ammonium sulphate was added t:o the concentrate at a ratio of approximately 35% (w/v). The mixture was stirred for 2 or more hours at 2 to 8°C. The collected to precipitate was dissolved in approximately 500 rnl of 10 mM Tris.HCl, pH 8.0, and then precipitated with ammonium sulphate (100 g/L). After a rr~inimum of 2 hr. stirring at 2 to 8°C, the supernatant was isolated by centrifugation. An additional aliquot of ammonium 15 sulphate (100 g/L) was added to the supernatant to precipitate the pertactin. The precipitate was dissolved and adjusted with 10 mM Tris.HCl, pH8.0, to a conductivity of approximately 3.4 mS/cm. The pertactin is purified according to Example 5 by passing through 2o tandem hydroxyapatite/Q-Sepharose~ columns (each 11 cm [D) X 8 cm [H]), ultrafiltered through a 300 kDa membrane and concentrated with a 10 to 30 kDa membrane.
The pertactin then was solvent-exchanged by either dialysis, diafiltration or using a solvent exchange 25 column into a solution in 10 mM Tris.HCl, pH 8.0 (conductivity approximately 0.6 mS/cm), then bound to a Q-Sepharose~ column (11 cm [D] x 8 cm [H) ) equilibrated in 10 mM Tris.HCl, pH 8Ø Ths= column was washed with mM Tris.HCl, pH 8.0, containing 5 mM ammonium 3o sulphate (conductivity approximately 1.7 mS/cm) and the pertactin was eluted with 10 to 100 mM (preferably 50 mM) phosphate buffer at pH 8Ø The purified pertactin is solvent exchanged into PBS and sterile filtered.
Alternatively, after the ammonium sulphate 35 precipitation steps, the pertactin solution with an ionic strength adjusted to approximately 3.4 mS/cm is passed through the hydroxyapatite column alone. The run-through fraction is concentrated and solvent exchanged into 10 mM Tris.HCl, pH 8.0 and bound directly onto the Q-Sepharose~ column. After washing with 10 mM
Tris.HCl, pH 8.0, containing 5 mM ammonium sulphate, the pertactin is eluted from the Q-Sepharose~ column with 10-100 mM (preferably 50 mM) phosphate buffer at pH 8Ø
The purified pertactin is ult:rafiltered through a 300 kDa membrane, concentrated and diafiltered with a 10 to to 30 kDa membrane and sterile filtered.
Example 7 This Example illustrates the extraction of pertactin from B. pertussis cells.
B. pertussis cells (5o w/v) were suspended in phosphate buffered saline (10 mM sodium phosphate, pH
7.5, 0.15 M sodium chloride ~PBS~) containing 4 M urea and the suspension was dispersed for 15 seconds to 60 minutes, and stirred for 1.5 hr. at room temperature.
Cell debris was removed by centrifugation at 5 to 6,OOOxg. The cellular extract was filtered through 100 kDa or 300 kDa membranes and the retentate diafiltered with 2 to 3 volumes of PBS. The filtrate from the membranes and the diafiltrate were combined and concentrated to one-fifth the original volume using <_30 kDa NWML membranes and further diafiltered with 5 volumes of PBS.
Example 8 This Example illustrates the preparation of pertactin from extracts of B. pe.rtussis cells.
3o The retentate from Example 7 was precipitated by the slow addition of ammonium sulphate (25% w/w) at room temperature and the mixture allowed to stir at 2 to 8°C
for an additional 2 hrs, preferably overnight. The precipitate was dissolved in 10 mM Tris.HCl buffer, pH
7.5, and saturated ammonium sulphate solution added to achieve a conductivity corresponding to that of 10 mM
Tris.HCl, pH 7.5, containing 15 mM ammonium sulphate (3.5 mS/cm). The pertactin was then in a form for purification by hydroxyapatite/Q-Sepharose~
chromatography as described in Example 5.
s Example 9 This Example illustrates the immunogenicity of pertactin combined with other antigens.
A solution of purified pertactin was mixed with aluminium phosphate (3 mg/ml) and varying amounts of to pertactin (1 to 20 ~.g) were combined with constant amounts of PT toxoid, FHA toxoid and agglutinogens, as well as dipheria toxoid (D) and teterum toxoid (T).
Guinea pigs (10 per group and with a 400 to 450 g weight range) were injected at days 0 and 21 and were bled at 15 day 28. A good antibody response to pertactin was observed with doses as low as 1 fig. No significant difference in antibody responses was observed between doses of 1 to 10 ~g and consistency was obtained between various lots of pertactin (See Table I below).
2o Example 10 This Example illustrates the stability of purified pertactin.
The stability of the .pertactin antigen was monitored with and without combination with aluminium 2s phosphate and after combination with other pertussis antigens in a candidate vaccine formulation. Samples of pertactin (without aluminium phosphate) were stored for various times at -20°C, 2 to 8°C, 24°C and 37°C.
Two lots were studied with different preservatives (thimerosal 3o and phenoxy-ethanol). It was noticeable that whichever preservative was used there was no reduction in the pertactin-specific ELISA value up to 3 months. A
reduction was observed with phenoxyethanol as a preservative in the 6 and 12 month values. The results 35 are reproduced shown in Table II below.
This Example illustrates the purification of pertactin by blinding to Q-Sepha:rose~.
The Perlite flow-through fraction from Example 2, s was concentrated to approximately 7 L, having a protein concentration usually between :1.5 to 3.0 mg/ml. Solid ammonium sulphate was added t:o the concentrate at a ratio of approximately 35% (w/v). The mixture was stirred for 2 or more hours at 2 to 8°C. The collected to precipitate was dissolved in approximately 500 rnl of 10 mM Tris.HCl, pH 8.0, and then precipitated with ammonium sulphate (100 g/L). After a rr~inimum of 2 hr. stirring at 2 to 8°C, the supernatant was isolated by centrifugation. An additional aliquot of ammonium 15 sulphate (100 g/L) was added to the supernatant to precipitate the pertactin. The precipitate was dissolved and adjusted with 10 mM Tris.HCl, pH8.0, to a conductivity of approximately 3.4 mS/cm. The pertactin is purified according to Example 5 by passing through 2o tandem hydroxyapatite/Q-Sepharose~ columns (each 11 cm [D) X 8 cm [H]), ultrafiltered through a 300 kDa membrane and concentrated with a 10 to 30 kDa membrane.
The pertactin then was solvent-exchanged by either dialysis, diafiltration or using a solvent exchange 25 column into a solution in 10 mM Tris.HCl, pH 8.0 (conductivity approximately 0.6 mS/cm), then bound to a Q-Sepharose~ column (11 cm [D] x 8 cm [H) ) equilibrated in 10 mM Tris.HCl, pH 8Ø Ths= column was washed with mM Tris.HCl, pH 8.0, containing 5 mM ammonium 3o sulphate (conductivity approximately 1.7 mS/cm) and the pertactin was eluted with 10 to 100 mM (preferably 50 mM) phosphate buffer at pH 8Ø The purified pertactin is solvent exchanged into PBS and sterile filtered.
Alternatively, after the ammonium sulphate 35 precipitation steps, the pertactin solution with an ionic strength adjusted to approximately 3.4 mS/cm is passed through the hydroxyapatite column alone. The run-through fraction is concentrated and solvent exchanged into 10 mM Tris.HCl, pH 8.0 and bound directly onto the Q-Sepharose~ column. After washing with 10 mM
Tris.HCl, pH 8.0, containing 5 mM ammonium sulphate, the pertactin is eluted from the Q-Sepharose~ column with 10-100 mM (preferably 50 mM) phosphate buffer at pH 8Ø
The purified pertactin is ult:rafiltered through a 300 kDa membrane, concentrated and diafiltered with a 10 to to 30 kDa membrane and sterile filtered.
Example 7 This Example illustrates the extraction of pertactin from B. pertussis cells.
B. pertussis cells (5o w/v) were suspended in phosphate buffered saline (10 mM sodium phosphate, pH
7.5, 0.15 M sodium chloride ~PBS~) containing 4 M urea and the suspension was dispersed for 15 seconds to 60 minutes, and stirred for 1.5 hr. at room temperature.
Cell debris was removed by centrifugation at 5 to 6,OOOxg. The cellular extract was filtered through 100 kDa or 300 kDa membranes and the retentate diafiltered with 2 to 3 volumes of PBS. The filtrate from the membranes and the diafiltrate were combined and concentrated to one-fifth the original volume using <_30 kDa NWML membranes and further diafiltered with 5 volumes of PBS.
Example 8 This Example illustrates the preparation of pertactin from extracts of B. pe.rtussis cells.
3o The retentate from Example 7 was precipitated by the slow addition of ammonium sulphate (25% w/w) at room temperature and the mixture allowed to stir at 2 to 8°C
for an additional 2 hrs, preferably overnight. The precipitate was dissolved in 10 mM Tris.HCl buffer, pH
7.5, and saturated ammonium sulphate solution added to achieve a conductivity corresponding to that of 10 mM
Tris.HCl, pH 7.5, containing 15 mM ammonium sulphate (3.5 mS/cm). The pertactin was then in a form for purification by hydroxyapatite/Q-Sepharose~
chromatography as described in Example 5.
s Example 9 This Example illustrates the immunogenicity of pertactin combined with other antigens.
A solution of purified pertactin was mixed with aluminium phosphate (3 mg/ml) and varying amounts of to pertactin (1 to 20 ~.g) were combined with constant amounts of PT toxoid, FHA toxoid and agglutinogens, as well as dipheria toxoid (D) and teterum toxoid (T).
Guinea pigs (10 per group and with a 400 to 450 g weight range) were injected at days 0 and 21 and were bled at 15 day 28. A good antibody response to pertactin was observed with doses as low as 1 fig. No significant difference in antibody responses was observed between doses of 1 to 10 ~g and consistency was obtained between various lots of pertactin (See Table I below).
2o Example 10 This Example illustrates the stability of purified pertactin.
The stability of the .pertactin antigen was monitored with and without combination with aluminium 2s phosphate and after combination with other pertussis antigens in a candidate vaccine formulation. Samples of pertactin (without aluminium phosphate) were stored for various times at -20°C, 2 to 8°C, 24°C and 37°C.
Two lots were studied with different preservatives (thimerosal 3o and phenoxy-ethanol). It was noticeable that whichever preservative was used there was no reduction in the pertactin-specific ELISA value up to 3 months. A
reduction was observed with phenoxyethanol as a preservative in the 6 and 12 month values. The results 35 are reproduced shown in Table II below.
Potential vaccine combinations in aluminium phosphate were stored at 2 to 8°C, 24°C and 37°C.
Stability of the antigen was monitored by general appearance, pertactin-specific ELISA, protein content, s SDS-PAGE, Western blot analysis with monospecific anti-pertactin antisera and immunogenicity studies in guinea pigs. The pertactin :has shown no changes in stability for any storage time whether alone or in combination with either adjuvant or other antigens, as to shown in Table III below. Ths= materials used for the experiment were pertactin alone with aluminium phosphate adjuvant and vaccine combinations with aluminium phosphate.
SUMMARY OF DISCLOSURE
is In summary of this disclosure, the present invention provides novel procedures for recovery of pertactin in stable form suitab_Le for incorporation as a component in a component vaccine, from fermentation products of Bordetella species, using column 2o chromatography and ultrafiltrat:ion. Modifications are possible within the scope of this invention.
Stability of the antigen was monitored by general appearance, pertactin-specific ELISA, protein content, s SDS-PAGE, Western blot analysis with monospecific anti-pertactin antisera and immunogenicity studies in guinea pigs. The pertactin :has shown no changes in stability for any storage time whether alone or in combination with either adjuvant or other antigens, as to shown in Table III below. Ths= materials used for the experiment were pertactin alone with aluminium phosphate adjuvant and vaccine combinations with aluminium phosphate.
SUMMARY OF DISCLOSURE
is In summary of this disclosure, the present invention provides novel procedures for recovery of pertactin in stable form suitab_Le for incorporation as a component in a component vaccine, from fermentation products of Bordetella species, using column 2o chromatography and ultrafiltrat:ion. Modifications are possible within the scope of this invention.
TABLE I:
DOSE RESPONSE AND CONSISTENCY
OF PRODUCTION OF PERTACTIN
ANTIGEN . a ~g ELISA ~g/mL
a . - 9.90 0.8 69kDa , - 10.80 0.7 . ''.~" 9.00 1.20 C:P4DTOD3A*6. ~ 9.00 0.58 ' ' 9.00 1.07 a) - Log2(reactive titres/100: eight animals/group.
* - These materials were vaccine preparations.
Dose: The antigens were dissolved in 1 ml and the dose/animal was 0.5 ml on. Day 0 and 0.5 ml on Day 21. Animals were bled on Day 28.
DOSE RESPONSE AND CONSISTENCY
OF PRODUCTION OF PERTACTIN
ANTIGEN . a ~g ELISA ~g/mL
a . - 9.90 0.8 69kDa , - 10.80 0.7 . ''.~" 9.00 1.20 C:P4DTOD3A*6. ~ 9.00 0.58 ' ' 9.00 1.07 a) - Log2(reactive titres/100: eight animals/group.
* - These materials were vaccine preparations.
Dose: The antigens were dissolved in 1 ml and the dose/animal was 0.5 ml on. Day 0 and 0.5 ml on Day 21. Animals were bled on Day 28.
TABLE II
STABILITY OF UNADSORBED PERTACTIN
M01VTHS TEMP (°C) ~g/~, ~,g/~, Cz~.i61-'fH ~ -G ~ 14.i 6 1.~
J
r n p p -G V ~ ',j Gb v Gb 151 -GV ly ~G~v1-r - 49' -'~ 14 __ G 5 L~
1 ~ '~
~4 3~
w v LS y '-' ~ 1 J
~~
62361.-TH = Pertactin preparation that contains 0.01% thimerosal as the preservative.
62361-P - Pertactin preparation that contains 0.5%
2-phenoxyethanol as the preservative.
a) = Protein contents of the samples were determined by BCA assay (Pierce) after TCA precipitation of the sample.
b) = Protein content of sample a.t zero time was determined by Kjeldahl.
STABILITY OF UNADSORBED PERTACTIN
M01VTHS TEMP (°C) ~g/~, ~,g/~, Cz~.i61-'fH ~ -G ~ 14.i 6 1.~
J
r n p p -G V ~ ',j Gb v Gb 151 -GV ly ~G~v1-r - 49' -'~ 14 __ G 5 L~
1 ~ '~
~4 3~
w v LS y '-' ~ 1 J
~~
62361.-TH = Pertactin preparation that contains 0.01% thimerosal as the preservative.
62361-P - Pertactin preparation that contains 0.5%
2-phenoxyethanol as the preservative.
a) = Protein contents of the samples were determined by BCA assay (Pierce) after TCA precipitation of the sample.
b) = Protein content of sample a.t zero time was determined by Kjeldahl.
TABLE III
STABILITY OF PERTACTIN
IN VACCINE COMBINATIONS
-_- .. -____ ....~......'vy.a t11V11-O71 L
MONTHS TEMP. (C) %~ c a Eu.g/mL TITRES~af A69K0~
" 10.7 1.28 9.9 0.83 11.2 1.28 b ~ 9.9 1.0 "'r'wvJy ~ 10.9 0.64 9.5 1.0 _ .- ~ . ~~ 10 . 1 0 . 74 3 _. 9.9 0.00 ' .z-' 9.0 0.89 .JJ 9.2 0.84 - " '.~" 10.5 0.85 ~. 7.5 0.93 "
C:1'4DT001 . . 9.0 1.20 "' -'-' 10 . 6 0 . 92 _ ~, 9.1 1.13 Note: For samples b, c and d the sample was diluted to 6 ~g/ml of pertactin prior to injection and the animals were given 0.5 ml on Day 0 and 0.5 ml on Day 28.
Note: For samples a and f the antigens were in 1 ml and the dose was 0.5 ml on Day 0 and 0.5 ml on Day 28.
a) Log2(reactive titres/100): eight animals/group.
b) Pertactin solution along adsorbed with aluminium phosphate.
Contained 54 ~g/ml of pertactin.
c) Pertactin solution along adsorbed with aluminium phosphate.
Contained 134 ~g/ml of pertactin.
d) Pertactin solution along adsorbed with aluminium phosphate.
Contained 77 ug/ml of pertactin.
e) Vaccine combination containing 10 ~g/ml of pertactin.
f) Vaccine combination containing 6 ~.tg/ml of pertactin.
STABILITY OF PERTACTIN
IN VACCINE COMBINATIONS
-_- .. -____ ....~......'vy.a t11V11-O71 L
MONTHS TEMP. (C) %~ c a Eu.g/mL TITRES~af A69K0~
" 10.7 1.28 9.9 0.83 11.2 1.28 b ~ 9.9 1.0 "'r'wvJy ~ 10.9 0.64 9.5 1.0 _ .- ~ . ~~ 10 . 1 0 . 74 3 _. 9.9 0.00 ' .z-' 9.0 0.89 .JJ 9.2 0.84 - " '.~" 10.5 0.85 ~. 7.5 0.93 "
C:1'4DT001 . . 9.0 1.20 "' -'-' 10 . 6 0 . 92 _ ~, 9.1 1.13 Note: For samples b, c and d the sample was diluted to 6 ~g/ml of pertactin prior to injection and the animals were given 0.5 ml on Day 0 and 0.5 ml on Day 28.
Note: For samples a and f the antigens were in 1 ml and the dose was 0.5 ml on Day 0 and 0.5 ml on Day 28.
a) Log2(reactive titres/100): eight animals/group.
b) Pertactin solution along adsorbed with aluminium phosphate.
Contained 54 ~g/ml of pertactin.
c) Pertactin solution along adsorbed with aluminium phosphate.
Contained 134 ~g/ml of pertactin.
d) Pertactin solution along adsorbed with aluminium phosphate.
Contained 77 ug/ml of pertactin.
e) Vaccine combination containing 10 ~g/ml of pertactin.
f) Vaccine combination containing 6 ~.tg/ml of pertactin.
Claims (14)
1. Purified pertactin produced by a method which comprises contacting an impure solution of pertactin with hydroxyapatite.
2. The purified pertactin of claim 1 wherein said impure solution is passed in contact with said hydroxyapatite.
3. The purified pertactin of claim 1 or 2 wherein, following contact with hydroxyapatite, the purified pertactin solution is passed in contact with a matrix compound of an ion-exchange medium to provide a further purified pertactin.
4. The purified pertactin of claim 3 wherein said purified pertactin solution is passed in contact with said matrix composed of an ion-exchange medium and a further purified solution of pertactin elutes in an unbound fraction.
5. The purified pertactin of either of claim 3 or 4 wherein said further purified solution of pertactin is passed in contact with said matrix composed of ion-exchange medium to bind pertactin thereto and the bound pertactin is eluted to provide an additionally purified solution of pertactin.
6. The purified pertactin of any one of claims 3 to 5 wherein said matrix compound of an ion-exchange medium is Q-Sepharose®.
7. The purified pertactin of any one of claims 1 to 6 wherein said method is followed by formulating the purified pertactin as a compound of a component vaccine.
8. The use of the purified pertactin claimed in any one of claims 1 to 7 for immunizing humans against disease caused by infection by Bordetella pertussis.
9. The use of claim 8 wherein said purified pertactin is used in combination with at least one purified other immunoprotective pertussis antigen.
10. The use of claim 9 wherein said at least one other immunoprotective pertussis antigen is selected from the group consisting of PT, FHA and agglutinogens.
11. The use of the purified pertactin claimed in any one of claims 1 to 7 in the manufacture of a component vaccine against Bordetella pertussis.
12. The use of claim 11 wherein said at least one purified other immunoprotective pertussis antigen is employed as a component of the vaccine.
13. The use of claim 12 wherein said at least one purified other immunoprotective pertussis antigen is selected from the group consisting of PT, FHA and agglutinogens.
14. The use of claims 12 or 13 wherein the pertactin and said at least one purified other immunoprotective pertussis antigen have been purified from a single fermentation of Bordetella pertussis.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9007656.1 | 1990-04-04 | ||
GB909007656A GB9007656D0 (en) | 1989-04-04 | 1990-04-04 | Control system |
CA002224052A CA2224052C (en) | 1990-04-04 | 1991-04-03 | Pertussis outer membrane protein |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002224052A Division CA2224052C (en) | 1990-04-04 | 1991-04-03 | Pertussis outer membrane protein |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2359307A1 true CA2359307A1 (en) | 1991-10-05 |
Family
ID=25679910
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002359307A Abandoned CA2359307A1 (en) | 1990-04-04 | 1991-04-03 | Pertussis outer membrane protein |
Country Status (1)
Country | Link |
---|---|
CA (1) | CA2359307A1 (en) |
-
1991
- 1991-04-03 CA CA002359307A patent/CA2359307A1/en not_active Abandoned
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2079887C (en) | Purification of a pertussis outer membrane protein | |
EP0336736B1 (en) | Purification of pertussis toxins and production of vaccine | |
AU714493B2 (en) | Multivalent DTP-polio vaccines | |
JP4589852B2 (en) | Non-cellular pertussis vaccine and method for producing the same | |
JPH10504717A (en) | Pneumococcal polysaccharide-recombinant pneumolysin conjugate vaccines for immunization against pneumococcal infection | |
US4705686A (en) | Process for the preparation of acellular Bordetalla pertussis vaccine | |
CA1280693C (en) | Purification of pertussis antigens | |
AU641711B2 (en) | Process for purification of a 69 000 dalton antigenetic protein from bordetella pertussis | |
Novotny et al. | Biologic and protective properties of the 69-kDa outer membrane protein of Bordetella pertussis: a novel formulation for an acellular pertussis vaccine | |
US4788058A (en) | B. pertussis vaccine production using carbodiimides | |
US4774086A (en) | Process for the purification, solubilization and/or detoxification of protein antigens of bacteria of the Bordetella genus using a carbonate buffer and an acellular anti-whooping cough vaccine | |
JPH0834742B2 (en) | Pertussis endotoxin removal method, pertussis toxoid and its manufacturing method | |
US6444211B2 (en) | Purification of a pertussis outer membrane protein | |
WO1995029934A1 (en) | Method of separating protective components of bordetella pertussis | |
CA2224052C (en) | Pertussis outer membrane protein | |
CA2359307A1 (en) | Pertussis outer membrane protein | |
US20010051163A1 (en) | Purification of a pertussis outer membrane protein | |
WO2009016651A1 (en) | Simplified means for obtaining prn from bordetella pertussis | |
CA2192165A1 (en) | Process for purifying necrotoxin produced by bordetella and toxoid |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Dead |