WO2008134830A1 - Method to obtain monophosphoryl lipid a from bordetella pertussis as a by-product of the cellular pertussis vaccine production - Google Patents
Method to obtain monophosphoryl lipid a from bordetella pertussis as a by-product of the cellular pertussis vaccine production Download PDFInfo
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- WO2008134830A1 WO2008134830A1 PCT/BR2007/000073 BR2007000073W WO2008134830A1 WO 2008134830 A1 WO2008134830 A1 WO 2008134830A1 BR 2007000073 W BR2007000073 W BR 2007000073W WO 2008134830 A1 WO2008134830 A1 WO 2008134830A1
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- Prior art keywords
- vaccine
- bordetella pertussis
- bpmpla
- final bulk
- product
- Prior art date
Links
- 241000588832 Bordetella pertussis Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 25
- 229940066827 pertussis vaccine Drugs 0.000 title claims abstract description 22
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- 239000006227 byproduct Substances 0.000 title claims abstract description 12
- 229940035032 monophosphoryl lipid a Drugs 0.000 title claims abstract description 11
- 230000001413 cellular effect Effects 0.000 title claims abstract description 9
- 239000003599 detergent Substances 0.000 claims abstract description 11
- 239000000706 filtrate Substances 0.000 claims abstract description 11
- 239000012675 alcoholic extract Substances 0.000 claims abstract description 9
- 239000012646 vaccine adjuvant Substances 0.000 claims abstract description 8
- 229940124931 vaccine adjuvant Drugs 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000004108 freeze drying Methods 0.000 claims abstract description 7
- 230000003308 immunostimulating effect Effects 0.000 claims abstract description 7
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 229960005486 vaccine Drugs 0.000 claims abstract description 7
- 239000008215 water for injection Substances 0.000 claims abstract description 7
- 238000005903 acid hydrolysis reaction Methods 0.000 claims abstract description 6
- 239000002671 adjuvant Substances 0.000 claims abstract description 6
- 229960003971 influenza vaccine Drugs 0.000 claims abstract description 4
- 238000006386 neutralization reaction Methods 0.000 claims abstract description 4
- 239000002158 endotoxin Substances 0.000 claims description 32
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 8
- 230000001476 alcoholic effect Effects 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 229960003964 deoxycholic acid Drugs 0.000 claims description 3
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims description 3
- 238000001704 evaporation Methods 0.000 claims description 3
- 230000008020 evaporation Effects 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000011146 sterile filtration Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 239000003446 ligand Substances 0.000 claims description 2
- 229920006008 lipopolysaccharide Polymers 0.000 description 16
- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical group O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 description 11
- KYQCXUMVJGMDNG-UHFFFAOYSA-N 4,5,6,7,8-pentahydroxy-2-oxooctanoic acid Chemical compound OCC(O)C(O)C(O)C(O)CC(=O)C(O)=O KYQCXUMVJGMDNG-UHFFFAOYSA-N 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 239000011574 phosphorus Substances 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 241000894007 species Species 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 150000004665 fatty acids Chemical group 0.000 description 3
- 150000004676 glycans Polymers 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 150000004804 polysaccharides Polymers 0.000 description 3
- RVBUGGBMJDPOST-UHFFFAOYSA-N 2-thiobarbituric acid Chemical compound O=C1CC(=O)NC(=S)N1 RVBUGGBMJDPOST-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000007398 colorimetric assay Methods 0.000 description 2
- 238000009295 crossflow filtration Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000132 electrospray ionisation Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- KHOITXIGCFIULA-UHFFFAOYSA-N Alophen Chemical compound C1=CC(OC(=O)C)=CC=C1C(C=1N=CC=CC=1)C1=CC=C(OC(C)=O)C=C1 KHOITXIGCFIULA-UHFFFAOYSA-N 0.000 description 1
- 241000588807 Bordetella Species 0.000 description 1
- 241000759568 Corixa Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101000986989 Naja kaouthia Acidic phospholipase A2 CM-II Proteins 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 108010073443 Ribi adjuvant Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000973887 Takayama Species 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002346 endotoxic effect Effects 0.000 description 1
- 108010072542 endotoxin binding proteins Proteins 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- -1 glycosyl phosphate Chemical compound 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 150000002386 heptoses Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000000752 ionisation method Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000012859 sterile filling Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
- C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
- C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
- C07H13/06—Fatty acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the present invention relates to the production of monophosphoryl Lipid A from Bordetella pertussis (BpMPLA) as a by-product of cellular pertussis vaccine production, more specifically as a by-product of cellular pertussis vaccine obtained through the process developed by Raw et al, in their patent pending application PCT/BR2004/000172. More particularly, it concerns a method of purifying BpMPLA from processing a usually rejected alcoholic extract from the final bulk of Bordetella pertussis vaccine, subjecting it to an affinity column and submitting the detergent eluted fraction to acid hydrolysis followed by neutralization, freeze-drying and resuspension with water for injection plus mild detergent.
- the new ⁇ pMPLA is used in immunostimulatory applications, as a vaccine adjuvant. More particularly, said SpMPLA is used as adjuvant for influenza vaccine with promissory results.
- lipopolysaccharide LPS
- endotoxin LPS
- LPS lipoprotein
- Lipid A hydrophobic lipid
- the hydrophilic core domain consists of KDO (2-keto-3-deoxyoctonate), heptoses, and neutral sugars such as galactose.
- Lipid A comprises a number of species that have the same overall Lipid A structure (two acylated GIcNAc-P residues) but differ in the number of fatty acid moieties. Removal by hydrolysis of the polysaccharide chains from LPS produces Lipid A, either as the naturally occurring, cytotoxic diphosphoryl form or the less toxic monophosphoryl form (Rietschel et al, 1987, Caroff et al, 1988; Erridge et al, 2002).
- Lipid A Biological diversity of detoxified Lipid A is exemplified by its multifunctional activities, including acting as an adjuvant against vaccine antigens.
- Monophosphoryl Lipid A is a component of the Ribi Adjuvant System (Oureshi et al, 1982; Takayama & Oureshi 1991, Gupta & Siber, 1995, Hunter, 2002).
- the approximate (or average) molecular weight is 1.7-1.8 kDa, depending on the number and identity of fatty acid chains present.
- the fatty acid composition will vary depending upon the method of production.
- the present invention is an alternative method to obtain monophosphoryl Lipid A from Bordetella pertussis (SpMPLA) as a by-product of cellular pertussis vaccine production, for use in immunostimulatory applications such as a vaccine adjuvant.
- SpMPLA Bordetella pertussis
- FIG. 1 is a flowchart of the BpMPLA production as a by-product of Bordetella pertussis final bulk
- FIG. 2 shows Assignment of main ionic specie of MPLA from Bordetella pertussis
- FIG. 3 are diagrams showing Electrospray Mass Spectrometry, positive ionization (a and b) and negative ionization (c and d) (ESP -/+ Esquire 3000 plus Bruker) - MPLA from Bordetella pertussis;
- FIG. 4 is a Table showing some Analytical Results of MPLA obtained through the present method.
- the present invention relates to a method to obtain monophosphoryl Lipid A from Bordetella pertussis (SpMPLA) as a by-product of cellular pertussis vaccine production, for use in immunostimulatory applications such as a vaccine adjuvant. More particularly, the present invention relates to a method of purifying BpMPLA from processing a usually rejected alcoholic extract, supernatant or filtrate from the final bulk of Bordetella pertussis vaccine (by-product from main vaccine production).
- SpMPLA Bordetella pertussis
- the present method presents the following steps: a) Treatment of the final bulk alcoholic of Bordetella pertussis vaccine (1) (1g of the wet mass to 20 ml of the solvent solution -1g/20ml); b) Centrifugation or tangencial flow filtration (2) of said treated final bulk alcoholic of Bordetella pertussis vaccine, resulting in a alcoholic extract, supernatant or filtrate (3); c) Subjecting said alcoholic extract, supernatant or filtrate (3) from the final bulk of Bordetella pertussis vaccine to an affinity column (4), more particularly to an endotoxin affinity column, which uses the immobilized ligand polymixin B, resulting in an enriched endotoxin fraction; d) Elution of said enriched endotoxin fraction with sodium deoxycholate (1%DOC) (5); e) Submitting said eluted fraction to acid hydrolysis, with acetic acid (2N Hac) at 100 0 C during 60 minutes (6); f)
- Said resuspended BpMPLA is used in immunostimulatory applications, as a vaccine adjuvant. More particularly, said resuspended BpMPLA is used as an adjuvant for influenza vaccine.
- Example 1 General Methods:
- the inactivated single harvest of the Bordetella pertussis vaccine (final bulk) was concentrated by tangential flow filtration before alcoholic solution treatment for decreasing the LPS contamination by lipids and proteins. To perform this step, it must be considered knowing the wet mass/ml of the Bp Final Bulk and then calculate to each 1g of the wet mass to 20 ml of the solvent solution (1g/20ml). After the alcoholic treatment, a new tangential flow filtration was done to remove the solvent and the rejected filtrate was used as the starting material to purify the SpMPLA. The KDO determination was performed at that step. Obtention of the endotoxin rich fraction:
- the rejected filtrate was then passed through an endotoxin affinity column (polymixin column) and eluted with 1% solution of sodium deoxycholate (1% DOC), maximum with 2 volumes of the column.
- the endotoxin binding capacity per ml of gel was previously determined for inactivated single harvest of Bordetella pertussis to avoid overloading.
- the gel capacity to bind up to 2 mg LPS/ml was determined.
- the affinity chromatography was performed under refrigerator temperature (2-8 0 C). The sample was slowly pumping over the gel for extended periods of time (8-16hr) instead of using a single gravity filtration through the column.
- the volume of the eluted rich endotoxin fraction was determined and also the KDO content.
- the endotoxin rich fraction (s) was then subjected to an acid hydrolysis with 2N acetic acid (v/v) at 100 0 C during one hour.
- the detergent precipitate was removed by paper filtration before acid evaporation under vacuum followed by pH adjustment to pH 5-6 with 3N NaOH.
- the recovered neutralized filtrate yielded the BpMPLA and it must be a negative material for KDO determination.
- the phosphorus assay was performed to determine the Lipid A concentration. Sterile filtration, filling and freeze-drying of SpMPLA:
- a 0.22 ⁇ m PVDF membrane was used to sterile filtrated the SpMPLA before the filling and freeze-drying without any stabilizer or cryoprotector.
- the freeze-drying BpMPLA was analysed by mass spectrometry. Resuspension of the BpMPLA:
- Spectrophotometer was used for all colorimetric assays.
- KDO determination The thiobarbituric acid (TBA) assay by Karkhanis et al (1978) was used for
- Mass spectrometry was the analytical tool used to measure the molecular mass of BpMPLA.
- the sample was inserted directly into the ionization source, or undergone high-pressure liquid chromatography (HPLC) en route to the ionization source.
- HPLC high-pressure liquid chromatography
- the ionization method used was Electrospray Ionization (ESI). To detect the negatively charged ions of the sample the analysis was done using negative ionization mode.
- the used equipment was the Esquire 3000 plus, Bruker.
- Figure 2 shows Assignment of main ionic specie of MPLA from Bordetella pertussis.
- Figure 3 are diagrams showing Electrospray Mass Spectrometry, positive ionization (a and b) and negative ionization (c and d) (ESP -/+ Esquire 3000 plus Bruker) - MPLA from Bordetella pertussis. The results of the ESP analysis of the MPLA from Bordetella pertussis showed that the main observed ionic specie was 1291.7 m/z.
- Figure 4 is a Table showing some Analytical Results of MPLA obtained through the present method. References:
- Vaccine 20 S7-S12.
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Abstract
A method to obtain monophosphoryl Lipid A from Bordetella pertussis (BpMPLA) as a by-product of cellular pertussis vaccine production, for use in immunostimulatory applications such as a vaccine adjuvant, and more particularly, a method of purifying BpMPLA from processing a usually rejected alcoholic extract, supernatant or filtrate from the final bulk of Bordetella pertussis vaccine (by-product from main vaccine production). This method of purifying BpMPLA comprises the processing of a usually rejected alcoholic extract from the final bulk of Bordetella pertussis vaccine, the subjecting of this final bulk to an affinity column and the submission of the detergent eluted fraction to acid hydrolysis followed by neutralization, freeze-drying and resuspension with water for injection plus mild detergent. The new BpMPLA is used in immunostimulatory applications, more particularly, as a vaccine adjuvant, and still more particularly, as an adjuvant for influenza vaccine, with promissory results.
Description
TITLE OF THE INVENTION
METHOD TO OBTAIN MONOPHOSPHORYL LIPID A FROM BORDETELLA PERTUSSISAS A BY-PRODUCT OF THE CELLULAR PERTUSSIS VACCINE PRODUCTION
FIELD OF THE INVENTION
The present invention relates to the production of monophosphoryl Lipid A from Bordetella pertussis (BpMPLA) as a by-product of cellular pertussis vaccine production, more specifically as a by-product of cellular pertussis vaccine obtained through the process developed by Raw et al, in their patent pending application PCT/BR2004/000172. More particularly, it concerns a method of purifying BpMPLA from processing a usually rejected alcoholic extract from the final bulk of Bordetella pertussis vaccine, subjecting it to an affinity column and submitting the detergent eluted fraction to acid hydrolysis followed by neutralization, freeze-drying and resuspension with water for injection plus mild detergent. The new βpMPLA is used in immunostimulatory applications, as a vaccine adjuvant. More particularly, said SpMPLA is used as adjuvant for influenza vaccine with promissory results.
BACKGROUND OF THE INVENTION
The lysis of gram-negative bacteria causes them to release lipopolysaccharide (LPS) from the outer membrane of their cell wall. For most purposes the terms lipopolysaccharide or LPS and endotoxin are synonymous.
The induction of bactericidal antibodies by LPS is well known, but severe adverse reactions after injection in animals or humans contraindicates the use of LPS as such in vaccines (Luderitz et al, 1982; Morrison & Ryan, 1987, Rietschel et al, 1994). It has been observed that the component of LPS most
responsible for endotoxic activity is Lipid A. LPS is composed of a hydrophobic lipid (Lipid A), a hydrophilic core polysaccharide chain, and a hydrophilic O- antigenic polysaccharide side chain. The hydrophilic core domain consists of KDO (2-keto-3-deoxyoctonate), heptoses, and neutral sugars such as galactose. The outer O-antigen consists of units of two to eight sugars repeated many times. Lipid A comprises a number of species that have the same overall Lipid A structure (two acylated GIcNAc-P residues) but differ in the number of fatty acid moieties. Removal by hydrolysis of the polysaccharide chains from LPS produces Lipid A, either as the naturally occurring, cytotoxic diphosphoryl form or the less toxic monophosphoryl form (Rietschel et al, 1987, Caroff et al, 1988; Erridge et al, 2002).
Biological diversity of detoxified Lipid A is exemplified by its multifunctional activities, including acting as an adjuvant against vaccine antigens. Monophosphoryl Lipid A is a component of the Ribi Adjuvant System (Oureshi et al, 1982; Takayama & Oureshi 1991, Gupta & Siber, 1995, Hunter, 2002). The approximate (or average) molecular weight is 1.7-1.8 kDa, depending on the number and identity of fatty acid chains present. The fatty acid composition will vary depending upon the method of production.
Several methods of production are described and they took in account efforts towards attenuating the toxic attributes of LPS without diminishing the imrnunostimulatory benefits of these compounds. Myers et al described methods for production of 3D-MLA (2001 ,2003). In these methods the LPS obtained from a deep rough strain of gram-negative bacterium could suffer dephosphorylation and decarbohydration using mineral acid solutions of moderate strength (e.g. 0.1 N HCI) followed base hydrolysis (for instance, CM 2:1 (v/v) saturated with 0.5M Na2CO3 pH 10.5) under controlled conditions and after flash evaporated the solvent. Or the cells suffered an extraction with an aliphatic alcoholic solution (75wt % of alcohol having from 1 to 4 carbon atoms and the balance water) producing cells poor of phospholipid content who after suffered extraction with chloroform and methanol (CM).
SUMMARY AND OBJECTS OF THE INVENTION
The present invention is an alternative method to obtain monophosphoryl Lipid A from Bordetella pertussis (SpMPLA) as a by-product of cellular pertussis vaccine production, for use in immunostimulatory applications such as a vaccine adjuvant.
BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation of the present invention and many of the attendant advantages thereof will be readily understood by reference of the following detailed description when taken in conjunction with the accompanying drawings, in which:
- Figure 1 is a flowchart of the BpMPLA production as a by-product of Bordetella pertussis final bulk;
- Figure 2 shows Assignment of main ionic specie of MPLA from Bordetella pertussis;
- Figure 3 are diagrams showing Electrospray Mass Spectrometry, positive ionization (a and b) and negative ionization (c and d) (ESP -/+ Esquire 3000 plus Bruker) - MPLA from Bordetella pertussis;
- Figure 4 is a Table showing some Analytical Results of MPLA obtained through the present method.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method to obtain monophosphoryl Lipid A from Bordetella pertussis (SpMPLA) as a by-product of cellular pertussis vaccine production, for use in immunostimulatory applications such as a vaccine adjuvant.
More particularly, the present invention relates to a method of purifying BpMPLA from processing a usually rejected alcoholic extract, supernatant or filtrate from the final bulk of Bordetella pertussis vaccine (by-product from main vaccine production). According to Figure 1, the present method presents the following steps: a) Treatment of the final bulk alcoholic of Bordetella pertussis vaccine (1) (1g of the wet mass to 20 ml of the solvent solution -1g/20ml); b) Centrifugation or tangencial flow filtration (2) of said treated final bulk alcoholic of Bordetella pertussis vaccine, resulting in a alcoholic extract, supernatant or filtrate (3); c) Subjecting said alcoholic extract, supernatant or filtrate (3) from the final bulk of Bordetella pertussis vaccine to an affinity column (4), more particularly to an endotoxin affinity column, which uses the immobilized ligand polymixin B, resulting in an enriched endotoxin fraction; d) Elution of said enriched endotoxin fraction with sodium deoxycholate (1%DOC) (5); e) Submitting said eluted fraction to acid hydrolysis, with acetic acid (2N Hac) at 1000C during 60 minutes (6); f) Filtration, more particularly simple paper filtration (7), to separate the detergent precipitate; g) Acid evaporation under vacuum (8); h) Neutralization to pH 5-6 (9), followed by sterile filtration (10), resulting in the new BpMPLA (11); i) Filling and freeze-drying (12) of said new BpMPLA; j) Resuspension (13) of said new BpMPLA before use with water for injection (WFI) plus detergent.
Said resuspended BpMPLA is used in immunostimulatory applications, as a vaccine adjuvant. More particularly, said resuspended BpMPLA is used as an adjuvant for influenza vaccine.
Example 1 : General Methods:
Preparation of monophosphoryl Lipid A from the final bulk of Bordetella pertussis vaccine (SpMPLA) Obtention of the rejected filtrate:
The inactivated single harvest of the Bordetella pertussis vaccine (final bulk) was concentrated by tangential flow filtration before alcoholic solution treatment for decreasing the LPS contamination by lipids and proteins. To perform this step, it must be considered knowing the wet mass/ml of the Bp Final Bulk and then calculate to each 1g of the wet mass to 20 ml of the solvent solution (1g/20ml). After the alcoholic treatment, a new tangential flow filtration was done to remove the solvent and the rejected filtrate was used as the starting material to purify the SpMPLA. The KDO determination was performed at that step. Obtention of the endotoxin rich fraction:
The rejected filtrate was then passed through an endotoxin affinity column (polymixin column) and eluted with 1% solution of sodium deoxycholate (1% DOC), maximum with 2 volumes of the column. The endotoxin binding capacity per ml of gel was previously determined for inactivated single harvest of Bordetella pertussis to avoid overloading. The gel capacity to bind up to 2 mg LPS/ml was determined. The affinity chromatography was performed under refrigerator temperature (2-80C). The sample was slowly pumping over the gel for extended periods of time (8-16hr) instead of using a single gravity filtration through the column. The volume of the eluted rich endotoxin fraction was determined and also the KDO content.
Acid hydrolysis of the endotoxin rich fraction:
The endotoxin rich fraction (s) was then subjected to an acid hydrolysis with 2N acetic acid (v/v) at 1000C during one hour. The detergent precipitate was removed by paper filtration before acid evaporation under vacuum followed by pH adjustment to pH 5-6 with 3N NaOH. The recovered neutralized filtrate
yielded the BpMPLA and it must be a negative material for KDO determination. The phosphorus assay was performed to determine the Lipid A concentration. Sterile filtration, filling and freeze-drying of SpMPLA:
A 0.22 μm PVDF membrane was used to sterile filtrated the SpMPLA before the filling and freeze-drying without any stabilizer or cryoprotector. The freeze-drying BpMPLA was analysed by mass spectrometry. Resuspension of the BpMPLA:
The resuspension of the vials with BpMPLA was done with water for injection, which contained a mild detergent to avoid aggregation. Example 2:
Analytical Methods: Colorimetric assays:
Spectrophotometer was used for all colorimetric assays. KDO determination: The thiobarbituric acid (TBA) assay by Karkhanis et al (1978) was used for
KDO determination. Phosphorus assay:
In this procedure, phosphate in samples containing Lipid A is satisfactorily estimated by phosphorus determination (Rouser et al, 1970). BpMPLA analysis by mass spectrometry:
Mass spectrometry was the analytical tool used to measure the molecular mass of BpMPLA. The sample was inserted directly into the ionization source, or undergone high-pressure liquid chromatography (HPLC) en route to the ionization source. The ionization method used was Electrospray Ionization (ESI). To detect the negatively charged ions of the sample the analysis was done using negative ionization mode. The used equipment was the Esquire 3000 plus, Bruker.
Figure 2 shows Assignment of main ionic specie of MPLA from Bordetella pertussis. Figure 3 are diagrams showing Electrospray Mass Spectrometry, positive
ionization (a and b) and negative ionization (c and d) (ESP -/+ Esquire 3000 plus Bruker) - MPLA from Bordetella pertussis. The results of the ESP analysis of the MPLA from Bordetella pertussis showed that the main observed ionic specie was 1291.7 m/z. Figure 4 is a Table showing some Analytical Results of MPLA obtained through the present method. References:
- Caroff M, Tacken A, Szabό L (1988) - Detergent-accelerated hydrolysis of bacterial endotoxins and determination of the anomeric configuration of glycosyl phosphate present in the "isolated lipid A" fragment of the Bordetella pertussis endotoxin. Carbohydr Res 2:273-82.
- Erridge C, Bennett-Guerrero E, Poxton IR (2002) - Structure and function of lipopolysaccharides. Microbes I nfec 4:837-51.
- Gupta RK & Siber GR (1995) - Adjuvants for human bacines - current status, problems and future prospects. Vaccine 13: 1263-76.
- Hunter, RL (2002) - Overview of vaccine adjuvants: present and future. Vaccine 20: S7-S12.
- Karkhanis YD Zeltner JA, Jackson JJ, Carlo DJ (1978) - A new and improved microassay to determine 2-keto-3- deoxyoctonate in lipopolysaccharide of gram-negative bacteria. Anal. Biochem. 85:595-601.
- Luderitz O, Galanos C, Rietschel ET (1982) - Endotoxins og Gram-negative bacteria. Pharm Ther 15: 383-402.
- Morrison DC & Ryan JL (1987) - Endotoxins and disease mechanisms. Ann Rev Med. 38: 417-32. - Myers KR, Snyder DS (2003) - Methods for the production of 3-O-deactivated- 4'-monophosphoryl lipid a (3D-MLA) - United States Patent Application 20030031684 -Kind Code A1 February 13, 2003, Corixa Corporation, Seattle WA.
- Myers KR, Snyder DS1 Blackman C, Whitaker - K 3-O - deacylated-4'- monophosphoryl Lipid A (3D-MLA) - (2001 ) USPTO 60280089.033001.
- Oureshi N, Takayama K, Ribi E (1982) - Purification and structural determination of non-toxic lipid A obtained from the lipopolysaccharide of Salmonella thyphimurium. - J Biol Chem 257: 11808-15.
- Raw I, Kubrusly FS, Dias WO, Esteves Ml, Furuyama N, Horton DSPQ, Leme E, Quintilio W, Sakauchi MA, Prado SMA, Higashi HG - Process for obtention of new cellular pertussis vaccine - PCT/BR2004/000172 - WO/2006/002502.
- Rietschel ET, Brade H, Brade L, Brandenburg K, Schade FU, Seydel U, Zahringer U, Galanos C, Luderitz O, Westphal O, Labischinski H, Kusumoto S, Shiba T (1987) - Lipid A, the endotoxic center of bacterial lipopolysacharides: relation of chemical structure to biological activity. Prog Clin Biol Res 231: 25- 34.
- Rietschel ET, Kirikae T, Schade FU, Mamat U, Schmidt G, Loppnow H, Ulmer AJ, Zahringer U, Seydel U, Di Padova F, Schreier M, Brade H (1994) - Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J 8: 217-225.
- Rouser G, Fkeischer S, Yamamoto - A Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970 5(5): 494-6.
- Takayama KK, Oureshi N (1991) - Novel lipid A derivatives and uses therefore. WO 91/01134- PCT/US90/04145.
Claims
What is claimed is:
1st) A method to obtain monophosphoryl Lipid A from Bordetella pertussis (SpMPLA) as a by-product of cellular pertussis vaccine production, and more particularly, a method of purifying BpMPLA from processing a usually rejected alcoholic extract, supernatant or filtrate from the final bulk of Bordetella pertussis vaccine (by-product from main vaccine production), comprising the following steps: a) Treatment of the final bulk alcoholic of Bordetella pertussis vaccine (1) (1g of the wet mass to 20 ml of the solvent solution -1g/20ml); b) Centrifugation or tangencial flow filtration (2) of said treated final bulk alcoholic of Bordetella pertussis vaccine, resulting in a alcoholic extract, supernatant or filtrate (3); c) Subjecting said alcoholic extract, supernatant or filtrate (3) from the final bulk of Bordetella pertussis vaccine to an affinity column (4), more particularly to an endotoxin affinity column, which uses the immobilized ligand polymixin B, resulting in an enriched endotoxin fraction; d) Elution of said enriched endotoxin fraction with sodium deoxycholate (1%DOC) (5); e) Submitting said eluted fraction to acid hydrolysis, with acetic acid (2N Hac) at 1000C during 60 minutes (6); f) Filtration, more particularly simple paper filtration (7), to separate the detergent precipitate; g) Acid evaporation under vacuum (8); h) Neutralization to pH 5-6 (9), followed by sterile filtration (10), resulting in the new βpMPLA (11); i) Filling and freeze-drying (12) of said new SpMPLA; j) Resuspension of said new SpMPLA before use with water for injection (WFI) plus detergent (12).
2nd) The method according to claim 1 , wherein said resuspended βpMPLA is used in immunostimulatory applications.
3rd) The method according to claim 1 , wherein said resuspended SpMPLA is used as a vaccine adjuvant.
4th) The method according to claim 1, wherein said resuspended BpMPLA is used as an adjuvant for influenza vaccine.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/BR2007/000073 WO2008134830A1 (en) | 2007-03-22 | 2007-03-22 | Method to obtain monophosphoryl lipid a from bordetella pertussis as a by-product of the cellular pertussis vaccine production |
| BRPI0721393A BRPI0721393B8 (en) | 2007-03-22 | 2007-03-22 | method to obtain monophosphorylated lipid from bordetella pertussis as a by-product of cellular pertussis vaccine production |
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| PCT/BR2007/000073 WO2008134830A1 (en) | 2007-03-22 | 2007-03-22 | Method to obtain monophosphoryl lipid a from bordetella pertussis as a by-product of the cellular pertussis vaccine production |
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| PCT/BR2007/000073 WO2008134830A1 (en) | 2007-03-22 | 2007-03-22 | Method to obtain monophosphoryl lipid a from bordetella pertussis as a by-product of the cellular pertussis vaccine production |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2220211A (en) * | 1988-06-29 | 1990-01-04 | Ribi Immunochem Research Inc | Modified lipopolysaccharides |
| WO1994019013A1 (en) * | 1993-02-19 | 1994-09-01 | Smithkline Beecham Corporation | Influenza vaccine compositions containing 3-o-deacylated monophosphoryl lipid a |
| US6013640A (en) * | 1998-08-21 | 2000-01-11 | Ribi Immunochem Research, Inc. | Phosphoglycolipid and methods for its use |
| WO2002078637A2 (en) * | 2001-03-30 | 2002-10-10 | Corixa Corporation | Methods for the production of 3-o-deactivated-4'-monophosphoryl lipid a (3d-mla) |
| BR0303762A (en) * | 2003-08-29 | 2005-04-12 | Xirlene Santos Salgueiro | Processes for obtaining bordetella pertussis lipopolysaccharide and for obtaining and preparing lipid for bordetella pertussis monophosphorylate, and for using lipid for bordetella pertussus monophosphorylate as an adjuvant in immunizations and vaccines |
| BR0303416A (en) * | 2003-08-21 | 2005-05-10 | Univ Fed Sao Paulo Unifesp | Processes for obtaining bordetella pertussis lipopolysaccharide and for obtaining and preparing bordetella pertussis monophosphorylated lipid-a, use of bordetella pertussis monophosphorylated lipid-a and vaccines |
-
2007
- 2007-03-22 WO PCT/BR2007/000073 patent/WO2008134830A1/en active Application Filing
- 2007-03-22 BR BRPI0721393A patent/BRPI0721393B8/en active IP Right Grant
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2220211A (en) * | 1988-06-29 | 1990-01-04 | Ribi Immunochem Research Inc | Modified lipopolysaccharides |
| WO1994019013A1 (en) * | 1993-02-19 | 1994-09-01 | Smithkline Beecham Corporation | Influenza vaccine compositions containing 3-o-deacylated monophosphoryl lipid a |
| US6013640A (en) * | 1998-08-21 | 2000-01-11 | Ribi Immunochem Research, Inc. | Phosphoglycolipid and methods for its use |
| WO2002078637A2 (en) * | 2001-03-30 | 2002-10-10 | Corixa Corporation | Methods for the production of 3-o-deactivated-4'-monophosphoryl lipid a (3d-mla) |
| BR0303416A (en) * | 2003-08-21 | 2005-05-10 | Univ Fed Sao Paulo Unifesp | Processes for obtaining bordetella pertussis lipopolysaccharide and for obtaining and preparing bordetella pertussis monophosphorylated lipid-a, use of bordetella pertussis monophosphorylated lipid-a and vaccines |
| BR0303762A (en) * | 2003-08-29 | 2005-04-12 | Xirlene Santos Salgueiro | Processes for obtaining bordetella pertussis lipopolysaccharide and for obtaining and preparing lipid for bordetella pertussis monophosphorylate, and for using lipid for bordetella pertussus monophosphorylate as an adjuvant in immunizations and vaccines |
Non-Patent Citations (6)
| Title |
|---|
| DATABASE CAPLUS [online] accession no. STN Database accession no. (2005:1197878) * |
| DATABASE CAPLUS [online] accession no. STN Database accession no. (2006:1266855) * |
| DATABASE CAPLUS [online] QURESHI N. ET AL.: "Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595. Purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination", accession no. STN Database accession no. (1985:419515) * |
| DATABASE CAPLUS [online] QURESHI N. ET AL.: "Purification and structural determination of nontoxic lipid A obtained from the lipopolysaccharide of Salmonella typhimurium", accession no. STN Database accession no. (1982:576580) * |
| JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 257, no. 19, 1982, pages 11808 - 11815 * |
| JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 260, no. 9, 1985, pages 5271 - 5278 * |
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| BRPI0721393B1 (en) | 2020-08-18 |
| BRPI0721393A2 (en) | 2013-01-01 |
| BRPI0721393B8 (en) | 2022-09-06 |
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