WO2024023265A2 - Purification process - Google Patents

Purification process Download PDF

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WO2024023265A2
WO2024023265A2 PCT/EP2023/070922 EP2023070922W WO2024023265A2 WO 2024023265 A2 WO2024023265 A2 WO 2024023265A2 EP 2023070922 W EP2023070922 W EP 2023070922W WO 2024023265 A2 WO2024023265 A2 WO 2024023265A2
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Prior art keywords
omvs
optionally
sample
purified
impurities
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PCT/EP2023/070922
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French (fr)
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WO2024023265A3 (en
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Anne Renée J. VANDERCAMMEN
Vincenzo NARDI DEI DA FILICAIA DOTTI
Wael CHAHIM
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Glaxosmithkline Biologicals Sa
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Publication of WO2024023265A2 publication Critical patent/WO2024023265A2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/099—Bordetella
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3847—Multimodal interactions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/235—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bordetella (G)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/08—Specific process operations in the concentrate stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2676—Centrifugal separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/26—Further operations combined with membrane separation processes
    • B01D2311/2697—Chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/10—Cross-flow filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/16—Diafiltration
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/36—Neisseria

Definitions

  • nOMVs native OMVs
  • GMMA generalized modules for membrane antigens
  • OMV production methods include the use of detergent treatments to produce OMVs referred to as detergent extracted OMVs (dOMVs) (EP0011243; Fredriksen et al. (1991) NIPH Ann.14(2):67-80), detergent-free methods (WO2004/019977), sonication (Hozbor et al. (1999) Curr Microbiol 38:273-8), etc.
  • OMVs are rich in immunogenic cell surface-associated, periplasmic, and secreted antigens and have been used in vaccines, for example, against Neisseria meningitidis serogroup B (Tan et al. (2010) N Engl J Med. 362(16):1511-20).
  • OMVs contain components that act as adjuvants, eliciting strong immune responses against the antigens.
  • OMVs may more closely mimic the native bacterium than purified protein antigens or other bacterial components.
  • OMVs are more complex structures derived from bacteria, they are typically more difficult to prepare than recombinant protein antigens. Therefore, there is the need to provide improved processes for preparing OMVs in particular for use in immunogenic compositions and vaccines.
  • TFF tangential flow filtration
  • a process for preparing OMVs from Bordetella bacteria comprising or consisting essentially of: (a) homogenising a fermentation harvest of the Bordetella bacteria, thereby obtaining a homogenised fermentation harvest comprising the Bordetella bacteria; (b) treating the homogenised fermentation harvest with DOC (i.e.
  • a process for preparing OMVs from Neisseria bacteria comprising the sequential steps of: (i) recovering the fermentation harvest by centrifugation; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce a sample containing OMVs and one or more impurities; (vi) removing or reducing the concentration of the one or more impurities by (vii) concentrating and washing the sample containing OMVs and one or more impurities by TFF through a membrane to obtain a retentate concentrate comprising OMVs and (viii) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa
  • a process for reducing the concentration of the 60kDa chaperonin GroEL in a sample of OMVs comprising: filtering the sample of OMVs by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs wherein the 60kDa chaperonin GroEL is present in an amount less than 1.5% of total protein.
  • a population of purified OMVs produced by a process according to any one of the first, second, third or fourth aspect.
  • a population of purified OMVs that is at least 85% pure or from about 85% to about 100% pure, optionally wherein purity is measured using size exclusion Ultra-performance liquid chromatography (UPLC), for example by size exclusion UPLC to determine the ratio between the main peak and the low molecular size peak.
  • UPLC size exclusion Ultra-performance liquid chromatography
  • FIG.2 General schematic of the two-stage OMV purification process using TFF (500kDa) and Ultracentrifugation (UC).
  • FIG.3 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS PAGE) Gel of samples taken at each stage of OMV purification: (1) Molecular weight marker, (2) TFF Retentate, (3) UC Supernatant, and (4) UC Pellet. Arrow indicates presence of the 60kDa chaperonin (GroEL) throughout.
  • FIG.4 General schematic of the two-stage OMV purification process using TFF (500kDa) and Sephacryl S-500 Gel filtration.
  • FIG.5 General schematic of the two-stage OMV purification process using TFF (500kDa) and CaptoCore 700 gel filtration.
  • FIG.6 SDS PAGE Gel of samples taken before (2: Load) and after (4-7: Pool) gel filtration using a CaptoCore 700 column (1: Molecular weight marker). Arrow indicates 60kDa chaperonin (GroEL) which is significantly removed from the OMV sample using CaptoCore 700.
  • FIG. 7(a) SDS Page Gel of OMV samples taken at each stage of OMV purification: (MW) Molecular weight marker, (TFF) TFF Retentate, (CaptoCore) Eluate from CaptoCore column.
  • FIG. 6 SDS PAGE Gel of samples taken before (2: Load) and after (4-7: Pool) gel filtration using a CaptoCore 700 column (1: Molecular weight marker). Arrow indicates 60kDa chaperonin (GroEL) which is significantly removed from the OMV sample using CaptoCore 700.
  • FIG. 7(b) Analytical gel filtration Size Exclusion Chromatography (SEC) analysis of OMV samples follow TFF and CaptoCore gel filtration. The process resulted in a preparation that almost completely removed GroEL and free-proteins not associated with the OMVs.
  • FIG.8 General schematic of the two-stage OMV purification process using TFF (750kDa hollow fibre filter) and CaptoCore 700 gel filtration.
  • FIG. 9 Size Exclusion Ultra Performance Liquid Chromatographic (SE-UPLC) profiles indicated excellent reproducibility of processes using a 750kDa Hollow Fibre TFF step compared to processes using a standard cassette for TFF. Yield obtained with Hollow Fibre was generally higher (purity >95%) and a further reduction in impurities was seen.
  • SE-UPLC Size Exclusion Ultra Performance Liquid Chromatographic
  • FIG. 10 Despite the pore size, the 750 kDa hollow fibre membrane again could not remove the 60kDa chaperonin from OMV preparations - a CaptoCore700 step was still employed.
  • FIG. 13 Flow chart of gonococcal OMV process production, purpose of the steps and nomenclature of relative intermediates.
  • FIG. 14 Purity by Size exclusion-high-performance liquid chromatography (SEC-HPLC) method of downstream process intermediate generated with two different processes.
  • DR is starting Material of second purification step
  • CB is a Concentrated Bulk of Drug substance (DS).
  • R10 is a batch and its CB is produced using Ultracentrifugation
  • R11 is a batch with the implementation of Capto Core 700 in the DS production.
  • FIG.15 Purity by SDS-PAGE of CB. The table shows the purity increase using Capto Core 700 (92%) instead of ultracentrifugation (83%). The SDS-Page image highlights the specific effect of Capto Core 700 on GroEL removal.
  • DETAILED DESCRIPTION OF THE INVENTION Isolated bacterial OMVs have been proposed as components for use in vaccines, for example as immunogenic components. However, the reproducible production of sufficiently pure OMVs in an amount suitable for large-scale vaccine manufacture has proven challenging.
  • Inventors have developed processes, for example large-scale or industrial processes, suitable for purifying OMVs.
  • a process for substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising OMVs comprising: (a) concentrating and washing the sample by TFF through a TFF membrane to obtain a retentate concentrate comprising the OMVs; (b) filtering the retentate concentrate by flow-through chromatography to obtain a flow-through of purified OMVs using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa, thereby obtaining purified OMVs.
  • the processes are suitable for purifying OMVs, for example by substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising OMVs.
  • the one or more impurities comprise protein impurities.
  • the one or more impurities comprise or is the 60kDa chaperonin GroEL (GroEL).
  • the 60kDa chaperonin GroEL comprises a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of SEQ ID NO: 1-4.
  • the 60kDa chaperonin GroEL comprises or has the sequence of anyone of SEQ ID NO: 1-4).
  • the process provided herein is a process for substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising OMVs.
  • substantially removing or reducing the concentration or amount of one or more impurities is intended to refer to embodiments wherein the amount or contentration of the one or more impurities in the purified OMVs is reduced to less than 10%, for example less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1.5% of the amount or concentration of the one or more impurities in the sample comprising OMVs, when measured as a percentage of total protein in the sample.
  • the processes provided herein remove at least 80% of the one or more impurities from a sample comprising OMVs, as measured by percentage of total protein content, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% of the one or more impurities is removed.
  • the processes provided herein reduce the amount of one or more impurities in a sample comprising OMVs by at least about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, such as about 30 to about 60 times, about 40 to about 60 times, about 50 to about 60 times, as measured by total protein content in the sample comprising OMVs.
  • These values may be determined by comparing the concentration or amount of the one or more impurities in the sample comprising OMVs after step (a), i.e. in the retentate concentrate, with that remaining in the sample comprising OMVs after step (b), i.e. in the flow-through of purified OMVs.
  • purify and “purifying” are intended to refer to procedures by which the concentration or amount of at least one undesired compound or compounds, an “impurity” or “impurities” respectively, is/are reduced, for example removed, relative to the desired compound.
  • the impurities comprise GroEL, for example the impurity is GroEL, for example SEQ ID NO: 1-4, and the desired compound is an OMV or population of OMVs or preparations of OMVs.
  • the concentration or amount of GroEL and/or other impurities may be determined using techniques known to the skilled person in the art, for example by Liquid chromatography–mass spectrometry (LC-MS) or by SDS-PAGE.
  • the process provided herein is a process for reducing the amount or concentration of GroEL in a sample comprising OMVs.
  • the process provided herein is a process for substantially removing GroEL from a sample comprising OMVs, for example, in comparison to the amount or concentration of GroEL in a sample comprising OMVs prior to use of the process.
  • the processes provided herein are suitable for substantially reducing the amount or concentration of GroEL in a sample of OMVs, for example, in comparison to the amount or concentration of GroEL in a sample comprising OMVs prior to use of the process.
  • the amount or contentration of GroEL in the purified OMVs is reduced to less than 5%, less than 4%, less than 3%, less than 2% or less than 1.5% of the amount or concentration of GroEL in the sample comprising OMVs, when measured as a percentage of total protein in the sample.
  • the processes provided herein remove at least 80% of the GroEL from a sample comprising OMVs, as measured by percentage of total protein content, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% of the GroEL is removed.
  • the processes provided herein reduce the amount of GroEL in a sample comprising OMVs by at least about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, such as about 30 to about 60 times, about 40 to about 60 times, about 50 to about 60 times, as measured by total protein content in the sample comprising OMVs.
  • These values may be determined by comparing the concentration or amount of GroEL in the sample comprising OMVs after step (a), i.e. in the retentate concentrate, with that remaining in the sample comprising OMVs after step (b), i.e. in the flow-through of purified OMVs.
  • the processes provided herein are reproducible between batches consistently producing preparations or populations of OMVs obtained by disruption of the outer membrane of Bordetella bacteria having high purity (for example, of at least 95%) and/or with a yield consistently greater than 40mg/L (as measured by reference to either protein and/or lipid content) and/or having a narrow particle size distribution (polydispersity index or PDI) of ⁇ 0.3.
  • the processes of provided herein consistently produce preparations or populations of OMVs released in the medium, for example by Neisseria bacteria, having high purity (for example, of at least 90%, such as at least 91%, 92%, 93%, 94%, or 95%) and/or with a yield greater than 40mg/L (as measured by reference to protein content) and/or having a narrow particle size distribution (polydispersity index or PDI) of ⁇ 0.3.
  • OMVs produced by the processes provided herein are sufficiently pure and suitable for use as an active principle in an immunogenic composition.
  • OMVs produced by processes known in the art generally comprise a protein referred to as the 60kDa chaperonin or GroEL (bp3495, SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4).
  • GroEL is not integrated within the OMVs, rather it appears to be loosely associated with the OMV surface.
  • the Bordetella GroEL protein is immunodominant and capable of inducing an antibody response but, in contrast to other protein antigens, GroEL does not appear to be a potent protective antigen, for example, in the mouse aerosol model for pertussis.
  • the process provided herein may be a process for reducing the amount and/or concentration of GroEL from a sample of OMVs such as a process for substantially removing GroEL from a sample of OMVs.
  • the above-noted processes replace downstream processing steps for OMV purification relying on ultracentrifugation, including density gradient centrifugation, sucrose cushion centrifugation, or others that may be difficult to implement on a larger scale. Therefore, the processes provided herein are suitable for industrial, large-scale production of OMVs. Therefore, in some embodiments the processes described herein are performed without, or do not comprise, an ultracentrifugation, for example, an ultracentrifugation that has a centrifugal force of greater than or about 30,000 x g is applied, optionally wherein the processes are performed without, or do not comprise, an ultracentrifugation after step (a).
  • the sample comprising OMVs is prepared from a fermentation culture having a volume of from about 20 litres to about 10000 litres, from about 25 litres to about 5000 litres, from about 25 litres to about 2000 litres, from about 50 litres to about 1000 litres, greater than or equal to 20 litres, greater than or equal to 25 litres, greater than or equal to 50 litres or greater than or equal to 100 litres, such as 20 litres, 25 litres, 50 litres, 100 litres, 500 litres or 1000 litres.
  • OMVs Outer Membrane Vesicles
  • OMVs are known in the art and may be produced by natural/spontaneous ‘blebbing’ from the outer membrane of the bacterium. They may also be artificially prepared by mechanical and/or chemical disruption of the bacterial cell to form vesicles. In some embodiments, the OMVs are obtained from Gram negative bacteria.
  • the OMVs are obtained from any Gram negative bacteria genera, such as from species in any of genera Escherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia, Brucella, Chlamydia Haemophilus, Legionella, Porphyromonas, Pseudomonas, Yersinia, Helicobacter, Salmonella, Vibrio, etc.
  • the bacterium may be Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Moraxella catarrhalis, Escherichia coli, Haemophilus influenzae (including non-typeable stains), Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, Porphyromonas gingivalis, Pseudomonas aeruginosa, Yersinia enterocolitica, Helicobacter pylori, Salmonella enterica (including serovars typhi and typhimurium, as well as serovars paratyphi and enteritidis), Shigella (such as SIG,
  • the OMVs are obtained from Bordetella bacteria, optionally from Bordetella pertussis.
  • the OMVs are obtained from Neisseria bacteria, optionally from Neisseria gonorroheae.
  • the OMVs are obtained from Porphyromonas bacteria, optionally from Porphyromonas gingivalis.
  • the OMVs are obtained from Escherichia bacteria, optionally from Escherichia coli.
  • the OMVs are obtained from Escherichia coli bacteria of strain K or strain B strains, optionally wherein the Escherichia coli bacteria are selected from any one of strains BL21(DE3), BLR(DE3), and E. coli HMS174(DE3).
  • the OMVs are obtained from Salmonella bacteria, optionally from Salmonella typhi or Salmonella typhimurium.
  • the bacterium is a wild-type bacterium. In some embodiments, the bacterium is a recombinant bacterium.
  • the bacterium is genetically modified to inactivate genes which lead to a toxic phenotype, such as modifications of native lipopolysaccharide (LPS), for example to disrupt the native lipid A structure, the oligosaccharide core, or the outer O antigen. Absence of O antigen in the LPS is useful, as is absence of hexa-acylated lipid A.
  • the bacterium is genetically modified by mutation to reduce the pyrogenic potential of the lipopolysaccharide (LPS) of the bacteria.
  • mutations include, by way of non-limiting example, mutations in IpxLl, synX, IgtA, htrA, msbBl, msbB2, virG, lpxA and homologues thereof.
  • Suitable mutations for down-regulating or abolishing expression include point mutations, gene deletions, gene insertions, and any modification of genomic sequences that results in a change in gene expression, such as a reduction, inactivation or silencing.
  • the mutation is a deletion.
  • Inactivation of toxins for example to prevent expression of Shiga toxin or pertussis toxin, is also advantageous.
  • the bacterium is an hyper-blebbing bacterium.
  • hyper- blebbing refers to a mutant strain of bacteria that spontaneously releases OMVs in greater quantities than a wild-type or parent strain from which it was derived (e.g., per unit of time). In general, hyperblebbing mutants release greater quantities of OMVs than the wild-type or parent strain from which it was derived, for example, greater than 10%, greater than 20%, greater than 30% or greater than 40%.
  • the hyper-blebbing bacterium may be a naturally occurring mutant strain or may be genetically modified to exhibit a hyper-blebbing phenotype.
  • wild-type with reference to bacteria refers to a bacterium that has not been modified either chemically or genetically in any way whatsoever (other than growth in culture medium).
  • Neisserial strains such as Neisseria meningitidis or Neisseria gonorrhoeae, may be genetically modified to exhibit a hyper-blebbing phenotype by down-regulating or abolishing expression of, by way of non-limiting example, GNA33.
  • Similar mutations are known in other bacteria, for example, Haemophilus influenza, Moraxella catarrhalis and Escherichia coli strains may be genetically modified to exhibit a hyper-blebbing phenotype by down-regulating or abolishing expression of one or more genes selected from the group consisting of tolQ, tolR, tolX, tolA and tolB.
  • Strains of Shigella flexneri, Shigella dysenteriae, Shigella boydii and Shigella sonnei can be genetically modified to exhibit a hyper-blebbing phenotype by down- regulating or abolishing expression of one or more tolR or OmpA.
  • Suitable mutations for down- regulating or abolishing expression include point mutations, gene deletions, gene insertions, and any modification of genomic sequences that results in a change in gene expression, for example a reduction, for example inactivation or silencing. Further suitable mutations are known in the art.
  • the hyper-blebbing bacterium may be further genetically engineered by one or more processes selected from the following group: (a) a process of down-regulating expression of immunodominant variable or non-protective antigens, (b) a process of up-regulating expression of protective outer membrane protein (OMP) antigens, (c) a process of down-regulating a gene involved in rendering the lipid A portion of LPS toxic, (d) a process of up-regulating a gene involved in rendering the lipid A portion of LPS less toxic, and (e) a process of genetically modifying the bacterium to express a heterologous antigen.
  • the sample comprising OMVs are be prepared artificially from bacteria using detergent treatment (e.g.
  • deoxycholate or sarkosyl
  • DOC deoxycholate
  • sarkosyl a bile acid salt detergent
  • Other techniques may be performed substantially in the absence of detergent using techniques such as sonication, homogenisation, microfluidisation, cavitation, osmotic shock, grinding, French press, blending, etc.
  • the starting material for the process provided herein is a sample comprising OMVs. In one embodiment, the sample is substantially free from whole bacteria, whether living or dead.
  • the sample comprising OMVs is obtained from Bordetella bacteria.
  • the Bordetella bacterium comprises or is one or more of Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica.
  • the bacterium comprises or is Bordetella pertussis.
  • the Bordetella pertussis comprises or is a recombinant bacterium.
  • the recombinant Bordetella pertussis comprises or is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G. In some embodiments the recombinant Bordetella pertussis bacterium does not express the dermonecrotic toxin (DNT) gene. In some embodiments the recombinant Bordetella pertussis bacterium expresses the genetically detoxified pertussis toxoid PT-9K/129G and does not express the dermonecrotic toxin (DNT) gene.
  • Suitable recombinant Bordetella pertussis bacteria are disclosed in WO2020/094580, which is incorporated by reference herein.
  • the recombinant Bordetella pertussis bacterium comprises at least one genomic LpxA gene encoding a mutated LpxA protein, and/or at least a genomic insertion of a heterologous LpxD gene, as disclosed for instance in WO2021/064050, which is incorporated herewith by reference.
  • the recombinant Bordetella pertussis comprises or is a recombinant bacterium that produces lipid A comprising (i) C3’ acyl chains having a length of about 10 carbon atoms (C10); (ii) C2’ acyl chains having a length of about 10 carbon atoms (C10); and/or (iii) C2 acyl chains having a length of about 10 carbon atoms (C10).
  • Suitable recombinant Bordetella pertussis bacteria are disclosed in WO2021/064050, which is incorporated by reference herein.
  • the recombinant Bordetella pertussis comprises or is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G and comprises or is a recombinant bacterium that produces lipid A comprising (i) C3’ acyl chains having a length of about 10 carbon atoms (C10); (ii) C2’ acyl chains having a length of about 10 carbon atoms (C10); and/or (iii) C2 acyl chains having a length of about 10 carbon atoms (C10).
  • the OMVs are obtained from Neisseria bacteria.
  • the Neisseria bacterium comprises or is one or more selected from Neisseria meningitidis and Neisseria gonorrhoeae.
  • the bacterium is Neisseria meningitidis.
  • the bacterium is Neisseria meningitidis serogroup B (MenB).
  • the bacterium is a Neisseria meningitidis serogroup B (MenB) strain selected from the group consisting of: NZ98/254, NG H36, BZ 232, DK 353, B6116/77, BZ 163, 0085/00, NG P20, 0046/02, M1140123, M12 240069, N5/99, 99M, or M07240677.
  • the OMVs are obtained from Neisseria meningitidis strain NZ98/254 or 99M.
  • the Neisseria meningitidis is of strain NZ98/254.
  • the Neisseria meningitidis is of strain 99M.
  • the bacterium is Neisseria gonorrhoeae, which is also referred to as gonococcal bacteria or gonococcus.
  • the Neisseria gonorrhoeae is a Neisseria gonorrhoeae bacterium that comprises genetic modification(s).
  • the Neisseria gonorrhoeae bacterium to which the genetic modification(s) is introduced i.e the unmodified Neisseria gonorrhoeae
  • the Neisseria gonorrhoeae bacterium to which the genetic modification(s) is introduced is a gonococcal bacterium of any strain.
  • the unmodified Neisseria gonorrhoeae is of strain FA1090, F62, WHO-G, WHO-M, WHO-N, GC14, BG7, BG8, BG27, SK92, or MS11.
  • the unmodified Neisseria gonorrhoeae is of strain FA1090.
  • Gonococcal bacteria of strain FA1090 are known in the and are commercially available from the American Type Culture Collection (ATCC, see for example Deposit Number #700825, 1081 University Boulevard, Manassas, Virginia 20110, US).
  • said genetic modification(s) reduce the endotoxin activity of the OMVs compared to a wild-type or unmodified Neisseria gonorrhoeae.
  • said genetic modification(s) that reduces the endotoxin activity results a genetically modified Neisseria gonorrhoeae that produces OMVs having increased levels of pentaacylated lipid A, decreased levels of hexaacylated lipid A, or a higher proportion of pentaacylated lipid A to hexaacylated lipid A compared to the lipid A that is in the OMVs in the respective a wild-type or unmodified Neisseria gonorrhoeae (i.e.
  • the comparative wild-type or unmodified Neisseria gonorrhoeae prior to the genetic modifications that produces OMV having increased levels of pentaacylated lipid A, decreased levels of hexaacylated lipid A, or a higher proportion of pentaacylated lipid A to hexaacylated lipid A).
  • the Neisseria gonorrhoeae is a genetically modified gonococcal bacterium, comprising genetic modification(s) that (I) decreases or abolishes expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide.
  • the Neisseria gonorrhoeae bacterium comprises a further genetic modification that decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide.
  • the Neisseria gonorrhoeae bacterium is from strain FA1090.
  • the Neisseria gonorrhoeae bacterium comprises genetic modification(s) that (I) decreases or abolishes expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide; and (II) decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide.
  • lpxl1 lipid A biosynthesis lauroyl acyltransferase
  • rmp reduction modifiable protein
  • the Neisseria gonorrhoeae bacterium is from strain FA1090 and comprises genetic modification(s) that (I) decreases or abolishes expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide; and (II) decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide.
  • the lpxl1 gene also referred to as msbB
  • Lpxl1 plays a role in lipid A biosynthesis.
  • Lpxl1 polypeptide
  • the rmp gene encodes the polypeptide reduction modifiable protein (Rmp).
  • “decreased expression” means that the gonococcal bacterium provided herein expresses less lpxl1 and rmp mRNA and/or Lpxl1 and Rmp protein compared to an unmodified (wild type) gonococcal strain or a gonococcal strain comprising the wild type lpxl1 / rmp genes. Expression may be considered decreased when any reduction in mRNA and/or protein expression is observed compared to an unmodified (wild type) gonococcal strain or a gonococcal strain comprising the wild type lpxl1 / rmp genes.
  • Expression may be considered decreased when an over 5%, over 10%, over 25%, over 50%, over 60%, over 70%, over 80% over 90% or over 95% reduction in mRNA and/or protein expression is observed compared to the mRNA and/or protein expression, respectively, in an unmodified (wild-type) gonococcal strain or a gonococcal strain comprising the wild type lpxl1 / rmp genes.
  • “abolished expression” means that no Lpxl1 mRNA and/or protein and no Rmp mRNA and/or protein can be detected in the gonococcal bacterium provided herein using the technique used by the skilled person to measure expression.
  • Lpxl1 can be determined for example by examining the extent to which the Lipid A component of the outer membrane vesicle lipooligosaccharide is penta-acylated as opposed to being hexa-acylated (for example by analysing the acylation state of lipid A using mass spectrometry e.g. as described in van der Ley et al. Infection and immunity vol.69,10 (2001): 5981-90).
  • the Lipid A will be penta-acylated (for example it will be at least 80%; at least 90%; at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%; or is 100% penta-acylated), despite evidence to suggest presence of lpxl1 mRNA and/or protein.
  • acylation of lipid A is determined by Matrix- Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) spectrometry.
  • MALDI-TOF Matrix- Assisted Laser Desorption/Ionization-Time of Flight
  • the genetically modified gonococcal bacterium provided herein comprises lipooligosaccharide (LOS) with penta-acylated lipid A.
  • the acylation of Lipid A can be determined for example by extracting lipid A followed by analysis by MADI-TOF spectrometry.
  • Lpxl1 polypeptide results in lipooligosaccharide (LOS) comprising a lipid A lacking the lauric acid that LpxL1 would have added, had it been functionally expressed.
  • LOS lipooligosaccharide
  • Decreased or abolished expression and/or function of the Lpxl1 polypeptide results in a LOS comprising a lipid A lacking the secondary lauroyl chain from the nonreducing end of the GlcN disaccharide of lipid A.
  • Decreased or abolished expression and/or function of the Lpxl1 polypeptide results in a LOS comprising a lipid A lacking the C12 acyloxyacyl chain (from the non-reducing end).
  • Lpxl1 polypeptide results in a LOS comprising a lipid A lacking the lauric acid in the secondary 2’-O-position of the distal nonreducing terminal glucosamine of the ⁇ -(1--> 6) D-glucosamine dimer (consequently a lone 3-hydroxymyristyl moiety exists in amide linkage on the distal glucosamine of the lipid A).
  • decreased or abolished expression and/or function of the lpxl1 polypeptide results in above 50% penta-acylation of lipid A, for example above 60%, above 70%, above 80%, above 90%, above 95% or above 99%.
  • the genetically modified gonococcal bacterium provided herein has a reduced capacity for activating Toll- like receptor 4 (TLR4) compared to a gonococcus comprising the wild-type lpxl1 gene.
  • TLR4 Toll- like receptor 4
  • the sample comprising OMVs comprises OMVs and one or more impurities, for example GroEL.
  • the sample comprising OMVs is obtained by disruption of the outer membrane of bacteria, for example Bordetella bacteria, and comprises DOC-extracted OMVs and one or more impurities, for example GroEL.
  • the sample comprising OMVs obtained by disruption of the outer membrane of Bordetella bacteria comprises DOC-extracted OMVs and GroEL.
  • the sample of OMVs obtained by disruption of the outer membrane of Bordetella bacteria comprises DOC-extracted OMVs and GroEL associated with the surface of the OMVs.
  • the sample comprising OMVs is obtained by detergent disruption of the outer membrane of bacteria, for example Bordetella or Neisseria bacteria.
  • the detergent is DOC (for example, CAS No. 302-95-4) optionally comprising benzonase (for example, CAS No. 9025-65-4).
  • the detergent is DOC having a concentration of from about 0.1% to about 0.5% DOC, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4% or about 0.5%, optionally comprising benzonase.
  • the sample comprising OMVs is obtained by detergent disruption of the outer membrane of bacteria, for example Bordetella bacteria, at a temperature of from about 30°C to about 45°C, for example, from about 30°C to about 40°C, for example, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C or about 40°C.
  • benzonase may be used at a concentration of from about 50 U/ml to about 2000 U/ml, for example about 50 U/ml, about 100 U/ml, about 150 U/ml or about 1500 U/ml.
  • the sample comprising OMVs is obtained by detergent disruption of the outer membrane of bacteria, for example Bordetella bacteria, using DOC at a concentration of about 0.1% to about 0.5%, such as 0.1% or 0.5%, at a temperature of from about 30°C to about 45°C, for example, from about 30°C to about 42°C, from about 30°C to about 40°C for example, about 30°C, about 35°C or about 40°C.
  • the sample comprising OMVs is obtained by detergent disruption of the outer membrane of Bordetella bacteria using DOC at a concentration of about 0.5%, optionally comprising benzonase, at a temperature of about 40°C.
  • the sample comprising OMVs is obtained by: (i) homogenising a fermentation harvest of bacteria, for example Bordetella bacteria, thereby obtaining a homogenised fermentation harvest; (ii) treating the homogenised fermentation harvest with DOC to disrupt the outer membrane of the bacteria, for example the Bordetella bacteria, and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; (iii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce the sample.
  • the sample of OMVs is obtained by: (i) homogenising a fermentation harvest of Bordetella bacteria, thereby obtaining a homogenised fermentation harvest; (ii) treating the homogenised fermentation harvest with DOC at a concentration of from about 0.1% to about 0.5% at a temperature of from about 35°C to about 40°C to disrupt the outer membrane of the Bordetella bacteria and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; (iii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce the sample.
  • the homogenisation and/or treating may be performed in the presence of a suitable buffer.
  • suitable buffers include Tris buffers such as a Tris-HCl buffer.
  • the buffer is a Tris-HCl buffer, for example, a 20mM Tris-HCl buffer.
  • Suitable buffers will have a pH of from about 8 to 9, such as about 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or about 9.
  • the homogenisation and/or treating are performed in the presence of at least one protease inhibitor.
  • Suitable protease inhibitors are known in the art, and include, by way of non-limiting example, the cOmplete EDTA-free protease inhibitor cocktail (Roche).
  • the sample comprising OMVs comprises or consists of OMVs released in the medium, for example by Neisseria bateria.
  • the sample comprising OMVs comprises OMVs released in the medium, for example by Neisseria bateria, and one or more impurities, for example GroEL.
  • the sample comprising OMVs comprises OMVs released in the medium, for example by Neisseria bateria, and GroEL.
  • the sample comprising OMVs comprises OMVs that were released in the medium, for example by Neisseria bateria, and GroEL associated with the surface of the OMVs.
  • the sample of OMVs is obtained by: (i) recovering the fermentation harvest by centrifugation; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce the sample of OMVs.
  • MgCl2 is added to the intermediate to obtain a MgCl2 concentration of from about 0.1mM to about 10mM, for example from about 0.5mM to about 5mM, for example of 1mM.
  • the intermediate is treated with benzonase treatment in step (iv) using a benzonase concentration of from about 10kU/L to about 100kU/L, for example about 10kU/L, about 20kU/L, about 30kU/L, about 40kU/L, 50kU/L, about 60kU/L, about 70kU/L, about 80kU/L, about 90kU/L, or about 100kU/L.
  • the benzonase concentration is 50kU/L.
  • the intermediate is treated with benzonase at a temperature of from about 0°C to about 10°C, for example, from about 2°C to about 8°C.
  • the intermediate is treated with benzonase for a period of from around 1 day to around 3 months, for example for a period of from around 3 days to 2 months.
  • MgCl2 is added to the intermediate to obtain a MgCl2 concentration of about 1mM and the intermediate is treated with 50kU/L benzonase at a temperature of from about 2°C to about 8°C for a period of from around 3 days to 2 months.
  • Step (a) Tangential Flow Filtration (TFF) A first step of the process provided herein comprises (a) concentrating and washing a sample comprising OMVs by TFF using a TFF membrane to obtain a retentate concentrate comprising the OMVs.
  • TFF a sample solution from a feed reservoir passes tangentially along the surface of a filter membrane and back to the feed reservoir, where it may be recirculated. Components larger than the pores of the filter membrane — such as OMVs - are retained, passing along the membrane surface, whilst components that are smaller than the pores of the filter membrane pass through the filter. Sample solution that passes along the membrane surface and back to the feed reservoir, is referred to as the retentate. Sample solution that passes through the membrane is generally referred to as the permeate or filtrate.
  • Membranes used in TFF will have a molecular weight cutoff.
  • Molecular weight cutoff or nominal molecular weight cutoff (NMWCO) is defined as the minimum molecular weight of a solute that is 90% retained by the membrane (e.g., as stated by the manufacturer).
  • the TFF is performed using a TFF membrane having a molecular weight cutoff of from about 200 kDa to about 800kDa, for example, a MWCO of from about 200kDa to about 400kDa or from about 500kDa to about 750kDa, for example, about 200kDa, about 250kDa, about 300kDa, about 350kDa, about 400kDa, about 500kDa, about 550kDa, about 600kDa, about 650kDa, about 700kDa or about 750kDa.
  • the TFF membrane has a MWCOof about 500kDa. In other embodiments, the TFF membrane has a MWCOof about 750kDa. In other embodiments, for example when the bacterium is a Neisseria bacterium, the TFF membrane has a MWCOof about 300kDa. In some embodiments, the TFF membrane has a pore size of from about 0.1mm to about 0.75mm, for example from about 0.4mm to about 0.6mm, for example, about 0.4mm, about 0.5mm or about 0.6mm. For the avoidance of doubt, the pore size (sometimes referred to as lumen diameter) is calculated using standard methods known in the art, usually determined by the manufacturer.
  • the pore size is a mean diameter of the pores in the membrane based on the assumption that all pores in the membrane are circular.
  • the TFF membrane is a hollow fibre membrane.
  • the TFF membrane is a hollow fibre membrane having a MWCO of about 750kDa or about 300kDa.
  • the TFF membrane is a hollow fibre membrane having a MWCO of about 750kDa or about 300kDa and a pore size of about 0.5mm.
  • the membrane is a hollow fibre membrane having a MWCO of about 750kDa or about 300kDa, a pore size of about 0.5mm and an area of about 290cm 2 .
  • the membrane is a hollow fibre membrane having a MWCO of about 750kDa, a pore size of about 0.5mm, an area of about 290cm 2 and a nominal flow path length of 60cm.
  • the TFF membrane is a GE Healthcare UFP-750-C-3X2MA hollow fibre membrane or equivalent, for example, having the following characteristics: 750 kDa cutoff, pore size 0.5 mm and working surface area of 290 cm 2 .
  • the TFF membrane is a Millipore 500kDa Cassette or equivalent, or a Sartorius 300kDa cassette or equivalent.
  • the TFF of step (a) comprises concentrating and washing the sample.
  • the TFF step (a) comprises at least one cycle that includes ultrafiltration and diafiltration.
  • the TFF of step (a) comprises at least one ultrafiltration cycle and at least one cycle of diafiltration.
  • concentration (by ultrafiltration) and washing (by diafiltration) are filtration steps performed using the same TFF membrane but with varying operating parameters, discussed further below.
  • Ultrafiltration is generally used to refer to TFF membrane filtration methods in which pressure, for example liquid pressure such as water pressure, forces the liquid against a semi- permeable membrane.
  • retentate Suspended high molecular weight solids, such as OMVs, are retained by the membrane (the “retentate”) while water and low molecular weight solutes pass through the membrane (the “filtrate”).
  • the term retentate is used to refer to those components or portion of a solution retained by, and which do not cross, the TFF membrane (the skilled person will appreciate that, to a limited extent, it may also includes some portions of the sample that are small enough to cross the membrane but have not yet done so).
  • ultrafiltration is used primarily to concentrate the sample but for the avoidance of doubt, the skilled person will appreciate that by its nature, ultrafiltration also removes/eliminates some impurities.
  • concentration of a sample for example “concentrates the sample” generally refers to the ordinary meaning of that term, namely increasing the amount of a particular subject material relative to the volume of fluid in which the subject material is disposed.
  • ultrafiltration liquid is removed from the sample (but not replaced) thereby reducing the aqueous content of the sample.
  • OMVs are retained in the sample by the TFF membrane thereby increasing the concentration of OMVs per unit volume of fluid in the retentate concentrate of step (a) respective to the sample comprising OMVs.
  • a sample obtained following a concentration step may be called a concentrate.
  • Diafiltration refers to the use of the TFF membrane to remove, replace or lower the concentration of impurities in a solution on the basis of molecular size.
  • the TFF membrane retains molecules, such as OMVs, that are larger than the pores of the membrane (the “retentate”) while smaller molecules such as salts, solvents and water pass freely through the membrane (the “filtrate”).
  • a solution for example a buffer
  • the retentate is diluted with solvent and re-filtered to reduce the concentration of soluble permeate components.
  • the ultrafiltration comprises at least one cycle of ultrafiltration, for example comprises two or more, three or more, four or more, five or more cycles of ultrafiltration.
  • the at least one ultrafiltration cycle concentrates the OMVs in sample by at least about 2.5 times, by at least about 5 times, by at least about 10 times or by at least about 15 times, such as about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 time, about 13 times, about 14 times or about 15 times.
  • the at least one ultrafiltration cycle concentrates the OMVs in the sample by: from about 2.5 times to about 20 times, from about 5 times to about 20 times, from about 6 times to about 20 times, from about 7 times to about 20 times, from about 8 times to about 20 times, from about 9 times to about 20 times, from about 10 times to about 20 times, from about 11 times to about 20 times, from about 12 times to about 20 times, from about 13 times to about 20 times, from about 14 times to about 20 times, or from about 15 times to about 20 times.
  • the OMVs in the sample may be concentrated to a protein concentration of 2-5 mg/ml ⁇ 1% by ultrafiltration.
  • the OMVs may be concentrated by ultrafiltration to a protein concentration of about 2% to about 10% (weight / volume). Washing of the sample is performed using at least one cycle of diafiltration.
  • the diafiltration comprises at least one cycle of diafiltration, for example comprises two or more, three or more, four or more, five or more cycles of diafiltration.
  • the diafiltration comprises two cycles of diafiltration.
  • the at least one cycle of diafiltration washes the sample with at least about 5 volumes of a wash solution relative to the sample volume, for example about 10 volumes of a wash solution, about 15 volumes of a wash solution or about 20 volumes of a wash solution relative to the sample volume.
  • the at least one diafiltration cycle washes the sample with at least about 5 volumes of a wash solution relative to the sample volume, with at least about 10 volumes, or with at least about 15 volumes of a wash solution relative to the sample volume, such as about 5 volumes, about 6 volumes, about 7 volumes, about 8 volumes, about 9 volumes, about 10 volumes, about 11 volumes, about 12 volumes, about 13 volumes, about 14 volumes, about 15 volumes, about 16 volumes, about 17 volumes, about 18 volumes, about 19 volumes, or about 20 volumes of a wash solution relative to the sample volume.
  • the at least one diafiltration cycle washes the sample with: from about 5 volumes to about 20 volumes, from about 5 volumes to about 20 volumes, from about 6 volumes to about 20 volumes, from about 7 volumes to about 20 volumes, from about 8 volumes to about 20 volumes, from about 9 volumes to about 20 volumes, from about 10 volumes to about 20 volumes, from about 11 volumes to about 20 volumes, from about 12 volumes to about 20 volumes, from about 13 volumes to about 20 volumes, from about 14 volumes to about 20 volumes, or from about 15 volumes to about 20 volumes of a wash solution relative to the sample volume.
  • Suitable wash solutions are known in the art and include, by way of non-limiting example, Dulbecco's phosphate-buffered saline (DPBS), Tris buffers and the like.
  • the wash solutions optionally comprise other components such as EDTA or sucrose.
  • the wash buffer is DPBS.
  • the wash solution is DPBS optionally comprising EDTA for example, about 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM or 8mM EDTA, for example about 5mM EDTA.
  • the wash solution is DPBS comprising EDTA, for example, about 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM or 8mM EDTA, for example about 5mM EDTA.
  • the wash solution is a Tris buffer, for example a 20mM Tris buffer having a suitable pH.
  • a suitable pH is from about pH 8.0 to about 9.0, such as pH 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.
  • the wash solution comprises sucrose, for example from about 1% to about 10% sucrose, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, for example about 3% sucrose.
  • the wash solution is a 20mM Tris buffer having a pH of about 8.6 optionally comprising about 3% sucrose.
  • the wash solution is DPBS comprising about 5mM EDTA.
  • the wash solution is a sodium phosphate buffer, for example a Na 2 HPO 4 /NaH 2 PO 4 buffer, for example a 10mM Na 2 HPO 4 /NaH 2 PO 4 having a suitable pH.
  • a suitable pH is from about pH 6.0 to about pH 7.0, such as pH 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6., 6.7, 6.7, 6.8, 6.9, or 7.0.
  • the wash solution comprises sodium chloride, for example from about 50mM to about 250mM, such as 50mM, 100mM, 150mM, 200mM or 250mM.
  • the wash solution is 10mM Na 2 HPO 4 /NaH 2 PO 4 pH 6.8 comprising 150mM NaCl.
  • the wash solution is phosphate-buffer saline (PBS) pH 7.4, such as a buffer comprising 8.1mM Na 2 HPO 4 , 1.5 mM KHPO 4 , 2.7 mM KCl and 136.9 mM NaCl.
  • PBS phosphate-buffer saline
  • concentration and washing are performed sequentially, one after the other, for example, in some embodiments the sample may first be concentrated and then washed and in other embodiments, the sample may first be washed and then concentrated.
  • the operating parameters may be set such that concentration and washing are performed at substantially the same time. Since the same TFF membrane and equipment is used, the diafiltration parameters may be such that they lead to an increase in the concentration of retained components, such as OMVs.
  • OMVs concentration of retained components
  • continuous diafiltration a solvent is continuously added to the retentate at the same rate as the filtrate is generated and in this case, the retentate volume and the concentration of retained components do not change during the process.
  • the TFF step (a) may optionally comprise a step of dilution prior to concentration and washing wherein a volume of a liquid is added to the sample comprising OMVs, thereby increasing the sample volume.
  • An increase in initial working volume may be advantageous since it can make the following processing steps, such as washing, easier for example, by reducing the likelihood of clogging of the membrane.
  • Step (b) Flow-Through Chromatography
  • the retentate concentrate is filtered by flow-through chromatography to obtain a flow-through of purified OMVs.
  • Flow-through chromatography is a separation technique in which sample interacts with a chromatographic substrate.
  • a “chromatographic substrate” is any kind of solid phase which separates a target of interest from other molecules present in a sample as a result of, for example, differences in size and/or binding affinity. Chromatographic substrates will generally be contained in a suitable housing, for example a chromatography column.
  • the retentate concentrate that comprises OMVs is applied to and flows through a chromatographic substrate, for example in a chromatography column, and as the retentate concentrate flows through the column, it interacts with a chromatographic substrate contained in the column. During the interaction, OMVs do not bind to the chromatographic substrate while impurities (including GroEL) are able to bind to and/or are trapped in the chromatographic substrate.
  • a flow-through of purified OMVs are obtained, which comprises lower levels of impurities, lower concentrations of impurities, lower amounts of impurities, fewer impurities, or no impurities.
  • the flow-through of purified OMVs flows out of the chromatography column and is recovered.
  • the flow-through of purified OMVs comprises lower levels or amounts of impurities (such as GroEL) compared to the retentate concentrate prior to interacting with the chromatographic substrate.
  • Suitable methods of flow-through chromatography include gel filtration, mixed mode resin column chromatography, ion exchange column chromatography, affinity matrix chromatography and hydrophobic interaction chromatography.
  • the chromatographic substrate is a size-exclusion resin, suitable for gel filtration, such as Sephacryl S-500 resin.
  • the chromatographic substrate is a mixed mode chromatography resin, suitable for mixed mode resin column chromatography. Mixed-mode chromatography resins are functionalised with ligands thereby being capable of different types of interaction such as, by way of non-limiting example, ion exchange, affinity binding and/or size exclusion.
  • the chromatographic substrate comprises beads, for example a plurality of beads, having a porous outer layer and an inner core, for example a ligand activated inner core.
  • the chromatographic substrate comprises beads having a porous outer layer having a molecular weight cut-off from about 600kDa to about 800kDa, for example from 600kDa to 800kDa, such of about 700kDa, for example of 700kDa (that prevents large molecules from entering the core) and an inner core, for example a ligand activated inner core, comprising octylamine ligands.
  • the term “molecular weight cut-off” refers to the size cut-off of the outer layer.
  • methods for measuring the molecular weight cut-off comprise determining the dynamic binding capacity of the chromatographic substrate using proteins of different sizes, such as ovalbumin (Mr 45 000), apoferritin (Mr 475 000), thyroglobulin (Mr 660 000), and bovine IgM (approx. Mr 900 000), and evaluating up to which protein size the sample protein is able to enter the bead core and bind to the substrate.
  • the beads have an nonfunctionalized outer layer (without ligand) and a functionalized core with an attached ligand, such as an octylamine ligand.
  • octylamine ligand refers to a ligand having the fuctional group CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 NH-, optionally wherein the pKa of the protonated octlymanime before attachment to the maxim, is about 10.65.
  • the matrix of the chromatographic substrate is high-flow agarose, for example having a particle size (i.e. an average particle size of the cumulative volume distribution) from about 75 ⁇ m to aout 95 ⁇ m, such as of about 85 ⁇ m.
  • the chromatographic substrate is the mixed-mode chromatography resin Capto Core 700 (GE Lifesciences) or equivalent, which comprises a matrix of highly cross-linked garaose beads with particle size of about 85 ⁇ m having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa.
  • Capto Core 700 GE Lifesciences
  • Other suitable chromatographic substrates are known in the art.
  • steps (a) and (b) are performed sequentially without intermediate processing steps, other than collection/storage, and the output of step (a) (the retentate concentrate comprising OMVs) is the direct input for step (b).
  • process steps (a) and (b) are discontinuous process steps wherein step (a) is completed with the retentate concentrate comprising OMVs being collected, optionally stored for a period of time, before step (b) is started and the retentate concentrate comprising OMVs is applied to the flow-through chromatographic substrate, such as to the chromatography column.
  • process steps (a) and (b) are continuous process steps.
  • a “continuous process” refers to a process having two or more processing steps in a series, wherein the output from an upstream step (for example, step (a)) is transferred to a downstream step (for example, step (b)) continuously without waiting for the upstream processing step to run to completion before the downstream processing step is started.
  • the retentate concentrate comprising OMVs continuously flows from the TFF equipment directly to the flow-through chromatography column, i.e. the TFF equipment is in fluid communication with the flow-through chromatography column.
  • the process further comprises a step (a’) comprising or consisting of filtering the concentrate retentate to remove precipitate.
  • steps (a) and (a’) are performed sequentially without intermediate processing steps, other than collection/storage, and the output of step (a) is the direct input for step (a’).
  • process steps (a) and (a’) are discontinuous process steps wherein step (a) is completed before step (a’) is started.
  • steps (a) and (a’) are continuous process steps.
  • Step (c) Sterile filtration OMVs prepared using the processes provided herein will generally be for use as components in immunogenic compositions such as pharmaceutical compositions, for example vaccine compositions. As such, it is desirable that such components are sterile and free of bacterial and/or viral contamination.
  • the processes provided herein may further comprise the step of sterile filtering the purified OMVs.
  • Step filtration or “sterile filtering” refers to the removal of virus and/or bacteria from a solution by passing the solution through a filter having pores of a sufficiently small diameter that virus and/or bacteria cannot pass through.
  • a “sterile preparation of purified OMVs” refers to a preparation or population of purified OMVs that has been passed through a filter having a pore size that is small enough to prevent the passage of virus and/or bacteria and result in a preparation free or substantially free of bacterial contaminants.
  • step (c) comprises sterile filtering the flow-through of purified OMVs using a 0.22 ⁇ m filter to produce a sterile preparation of purified OMVs.
  • steps (b) and (c) are performed sequentially without intermediate processing steps, other than collection/storage, and the output of step (b) is the direct input for step (c).
  • process steps (b) and (c) are discontinuous process steps wherein step (b) is completed before step (c) is started.
  • steps (b) and (c) are continuous process steps.
  • processes provided herein enable a recovery or yield of at least 25 mg of OMVs (based on protein content) per litre of fermentation harvest or culture used in the preparation of the sample of OMVs, for example at least 40mg protein/L culture, such as at least 70mg protein/L culture or more.
  • polydispersity (or “dispersity” as recommended by IUPAC) is used to describe the degree of non-uniformity of a size distribution of a population of particles.
  • the “Polydispersity Index” (PDI) is a dimensionless parameter used in the art to to define the size distribution of the lipid nanoparticles.
  • the PDI may be determined using dynamic light scattering (DLS) techniques known in the art and descried herein. Preparations or populations of purified OMVs produced by the processes provided herein have a polydispersity index of about 0.3 or less, for example, about 0.1 to about 0.3, about 0.2 to about 0.3, such as about 0.2 to about 0.27.
  • z-average radius means the average radius of OMVs in the preparation or population of purified OMVs as measured by Dynamic Light Scattering (DLS) as described herein.
  • DLS measurements are performed with a Malvern Zetasizer Nano ZS (Malvern, Herremberg, Germany) equipped with a 633 nm He–Ne laser and operating at an angle of 173°. Scattering light detected at 173° is automatically adjusted by laser attenuation filters.
  • the viscosity and refractive index (RI) of 3% sucrose solution (at 25 °C) are used.
  • the software used to collect and analyze the data is the Zetasizer software version 7.13. Temperature is set at 25 °C.
  • Each sample (80 ⁇ L) is characterized in single-use polystyrene microcuvette (ZEN0040, Alfatest) with a path length of 10 mm.
  • the hydrodynamic diameter of OMV is expressed by a Z- average value (general purpose algorithm) of three measurements of eleven runs for each sample, providing also a PDI of the size values calculated.
  • preparations or populations of purified OMVs provided herein for example purified OMV obtained from Bordetella bacteria, for example B.
  • pertussis bacteria will have a z-average radius of from about 50 nm to about 150 nm, for example, about 50 nm to about 140 nm, about 50 nm to about 80 nm, such as about 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm or about 76 nm.
  • preparations or populations of purified OMVs provided herein will have a z-average radius of from about 10 nm to about 125nm, for example, about 20 nm to about 110 nm, about 30 nm to about 100 nm, about 40 nm to about 90 nm, about 50 nm to about 80 nm, about 60 nm to about 70 nm.
  • preparations or populations of purified OMVs provided herein for example purified OMV obtained from Escherichia coli bacteria, for example Escherichia coli bacteria of strain K or strain B strains, optionally wherein the Escherichia coli bacteria are selected from any one of strains BL21(DE3), BLR(DE3), and E. coli HMS174(DE3), will have a z-average radius of from about 15 nm to about 75 nm, for example, about 20 nm to about 70 nm, about 30 nm to about 60 nm, about 40 nm to about 50 nm.
  • preparations or populations of purified OMVs provided herein for example purified OMV obtained from Neisseria bacteria, for example Neisseria gonorrhoeae, will have a z-average radius of from about 30 nm to about 110 nm, for example, about 40 nm to about 100 nm, about 50 nm to about 90 nm, about 60 nm to about 80 nm.
  • preparations or a population of purified OMVs will be least 85% pure, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, for example, about 85%, about 90%, about 92%, about 93%, about 94% or about 95%.
  • populations of purified OMVs will be from about 85% to about 100% pure, from about 90% to about 100% pure, from about 95% to about 100% pure. In one embodiment, populations of purified OMVs will have a purity of from about 85% to about 99%, from about 90% to about 99%, from about 91% to about 99%, from about 92% to about 99%, from about 93% to about 99%, from about 94% to about 99%, from about 95% to about 99%, from about 96% to about 99%, from about 97% to about 99% or from about 98% to about 99%.
  • purity may be measured using size exclusion UPLC, for example by size exclusion UPLC (fluorescence detection) to determine the ratio between the main peak (attributed to OMVs) and the smaller, low molecular size peak (attributed to free proteins).
  • size exclusion UPLC fluorescence detection
  • purity may be defined by reference to the amount or level of total impurities remaining.
  • impurities may be present in preparations or populations of purified OMVs at a level or concentration of less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than about 1% of impurities, for example of free impurities, such as protein impurities.
  • preparations or populations of purified OMVs comprise no more than 20%, no more than 15%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2% or no more than about 1% of impurities, for example of free impurities, such as protein impurities.
  • Preparations or populations of purified OMVs produced using the processes provided herein may comprise from about 0.01% to about 10% of GroEL, such as from about 0.01% to about 9% of GroEL, from about 0.01% to about 8% of GroEL, from about 0.01% to about 7% of GroEL, from about 0.01% to about 6% of GroEL, from about 0.01% to about 5% of GroEL, from about 0.01% to about 4% of GroEL, from about 0.01% to about 3% of GroEL, from about 0.01% to about 2% of GroEL or from about 0.01% to about 1.5% as a percentage of total protein, for example, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 0.75%, about 1% or about 1.05% GroEL.
  • GroEL such as from about 0.01% to about 9% of GroEL, from about 0.01% to about 8% of GroEL, from about 0.01% to about 7% of GroEL, from about 0.01% to about 6% of GroEL, from about 0.0
  • preparations or populations of purified OMVs produced using the processes provided herein will comprise no more than about 5% GroEL, no more than about 4% GroEL, no more than about 3% GroEL, no more than about 2% GroEL, no more than about 1.5% GroEL or no more than about 1% GroEL as a percentage of total protein, for example, less than 5% of GroEL, less than 4% of GroEL, less than 3% of GroEL, less than 2% of GroEL, less than 1.5% of GroEL or less than 1.1% of GroEL.
  • the amount of GroEL may be provided as a relative or absolute amount.
  • the amount of GroEL is provided as a relative amount for example, as a percentage of total protein in the preparation or population of purified OMVs. In one embodiment, the amount of GroEL is a relative amount determined using mass analysis. Further, and in addition to the reduced amount of GroEL, the preparation or population of purified OMVs is substantially free of process related impurities, such as those associated with upstream production or purification processes, for example media components used in fermentation or reagents used in cell disruption. Thus, the preparation or population of purified OMVs has the purity required for formulation and pharmaceutical use as a drug substance.
  • drug substance is used to refer to a product suitable for use as an active ingredient in an immunogenic composition such as a medicament, for example a pharmaceutical composition such as a vaccine composition.
  • a pharmaceutical composition such as a vaccine composition.
  • the preparation or population of purified OMVs may be combined with one or more pharmaceutically acceptable carriers, excipients or other components to produce an immunogenic composition.
  • Immunogenic compositions The preparation or population of purified OMVs is capable of eliciting an immune response, for example comprising eliciting a primary immune response and/or a boosted immune response, for example to Bordetella, for example Bordetella pertussis, or Neisseria, for example Neisseria gonorrhoeae when administered to a suitable subject, for example a mammal.
  • the immune response may be a cellular or a humoral immune response.
  • the immune response is an antibody response.
  • the immune response is a T-cell immune response that can neutralise the infection and/or virulence of Bordetella pertussis or Neisseria gonorrhoeae.
  • compositions comprising purified OMVs provided herein are immunogenic.
  • immunogenic composition broadly refers to any composition comprising OMVs provided herein that may be administered to a subject to elicit an immune response, such as an antibody or cellular immune response, against an antigen or antigens present in the OMVs.
  • An immunogenic composition may also be referred to as a “drug product” in the art and refers to the preparation or population of purified OMVs formulated in a form suitable for administration to a subject, such as a human, for example as a pharmaceutical composition, for example, as a vaccine.
  • immunogenic compositions may be formulated to contain other antigenic components, for example from Bordetella, Neisseria and/or other organisms capable of infecting the suitable subject and causing illness or symptoms of disease.
  • the immunogenic compositions prevent, ameliorate, palliate, or eliminate disease from the subject arising as a result of bacterial and/or viral infection, they may be referred to as a vaccine.
  • the immunogenic composition is a vaccine.
  • Vaccines provided herein may either be prophylactic (i.e. to prevent infection, for example by eliciting a primary and/or boosted immune response) or therapeutic (i.e.
  • prophylactic vaccines do not guarantee complete protection from disease because even if a subject develops antibodies, there may be a lag or delay before the immune system is able to fight off the infection. Therefore, and for the avoidance of doubt, the term prophylactic vaccine encompasses vaccines that are used to ameliorate the effects of a future infection, for example by reducing the severity and/or duration of such an infection.
  • protection against infection and/or “provide protective immunity” means that the immune system of a subject has been primed (e.g by vaccination) to trigger an immune response and repel infection.
  • the immune response triggered is capable of repelling infection against Bordetella, optionally Bordetella pertussis, or Neisseria, optionally Neisseria gonorrhoeae.
  • a vaccinated subject may thus get infected, but is better able to control and fight-off or repel the infection than a control subject.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of OMVs.
  • immunologically effective amount it is meant that the administration of that amount to a suitable subject, either in a single dose or as part of a series, sufficient to generate an immune response effective for treatment or prevention of infection and disease.
  • the desired result is the production of an antigen (e.g., pathogen)-specific immune response that is capable of or contributes to protecting the subject against the pathogen.
  • an antigen e.g., pathogen
  • This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials, for example by clinical trials.
  • antigen refers to a substance that, when administered to a subject, elicits an immune response directed against said substance.
  • OMVs purified by the processes provided herein are complex components comprising a plurality of antigens, for example, within the OMV lumen or displayed on or associated with the surface of the OMVs. Unless indicated otherwise, this plurality of antigens will be considered collectively and the OMVs will be referred to as an antigen or antigenic component.
  • the immunogenic composition when administered to a subject, the immunogenic composition— and without wishing to be limited by theory the OMV component of the immunogenic composition— will elicit an immune response directed against Bordetella, for example, Bordetella pertussis, or Neisseria, for example Neisseria gonorrhoeae.
  • the immune response directed against Bordetella or Neisseria is protective, that is, it can prevent or reduce infection and/or colonisation caused by Bordetella, optionally Bordetella pertussis, or Neisseria, optionally Neisseria gonorrhoeae.
  • Compositions may thus be pharmaceutically acceptable.
  • immunogenic compositions include components in addition to the OMVs, for example, they typically include one or more pharmaceutical carrier(s) and/or excipient(s) (a thorough discussion of such components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472).
  • a “pharmaceutically acceptable carrier” is a carrier that does not itself induce the production of antibodies.
  • Such carriers include, by way of non-limiting example, polysaccharides, sucrose, trehalose, lactose, diluents, such as water, saline including phosphate buffered saline, glycerol and auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, etc.
  • Immunogenic compositions may also comprise one or more adjuvants.
  • adjuvant refers to a compound that enhances a subject's immune response to antigen(s) when administered conjointly with the antigen(s).
  • the composition may include a preservative such as thiomersal or 2-phenoxyethanol.
  • compositions provided herein are substantially free from (i.e. less than 5 ⁇ g/ml) mercurial material e.g. thiomersal-free.
  • Compositions containing no mercury are also provided.
  • Thiomersal-free compositions, for example thiomersal-free vaccines, are also provided.
  • a physiological salt such as a sodium salt may be included.
  • Sodium chloride (NaCl) may be used, which may be present at between 1 and 20 mg/ml, for example about 10+2mg/ml NaCl.
  • Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, for example between 240-360 mOsm/kg, and for example within the range of 290-310 mOsm/kg.
  • Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-20mM range.
  • the pH of a composition will generally be between from about pH 5.0 to about pH 8.1, and more typically between from about pH 6.0 and about pH 8.0, such as between about pH 6.5 and about pH 7.5, or between about pH 7.0 and about pH 7.8.
  • immunogenic compositions are sterile.
  • the composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a ‘multidose’ kit).
  • the inclusion of a preservative is provided, for example in multidose arrangements.
  • the compositions may be contained in a container having an aseptic adaptor for removal of material.
  • Immunogenic compositions for example human vaccines, are typically administered in a dosage volume of about 0.5ml, although fractional doses, such as a half dose (i.e. about 0.25ml) may be administered, for example, to children.
  • Adjuvants which may be used in compositions provided herein include mineral containing compositions such as aluminium salts and calcium salts.
  • the compositions provided herein may include mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates or mixtures of different mineral compounds.
  • Aluminium adjuvants include, by way of non-limiting example, aluminium hydroxide, aluminium oxyhydroxide salts, aluminium phosphate, aluminium hydroxyphosphates, aluminium hydroxyphosphate sulfate and the like.
  • the adjuvant comprises Aluminium phosphate which may be obtained by precipitation.
  • the reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt.
  • the PO 4 /Al 3+ molar ratio of an aluminium phosphate adjuvant may be between 0.3 and 1.2, for example between 0.8 and 1.2, and for example 0.95+0.1.
  • the aluminium phosphate may be amorphous, for example for hydroxyphosphate salts.
  • Aluminium phosphates provided herein may have a point of zero charge (PZC) of between 4.0 and 7.0, for example between 5.0 and 6.5 e.g. about 5.7.
  • concentration of Al +++ in a composition for administration to a patient may be between from about 10mg/ml to about 0.01mg/ml, for example, about 5 mg/ml or less, about 4 mg/ml or less, about 3 mg/ml or less, about 2 mg/ml or less, about 1 mg/ml or less, for example, about 5mg/ml, about 4mg/ml, about 3mg/ml, about 2mg/ml, about 1mg/ml, about 0.3mg/ml, about 0.05mg/ml or about 0.01mg/ml.
  • the range is between from about 0.3mg/ml to about 1mg/ml. In some embodiments, a maximum of 0.85mg/dose is provided, for example about 0.5mg/dose, about 0.4mg/dose, about 0.3mg/dose, about 0.2mg/dose or about 0.1mg/dose.
  • compositions provided herein may comprise a Toll like receptor (TLR) agonist such as a Toll like receptor 2 agonist (TLR2a), a Toll like receptor 3 agonist (TLR3a), a Toll like receptor 4 agonist (TLR4a), a Toll like receptor 7 agonist (TLR7a), a Toll like receptor 8 agonist (TLR8a) or a Toll like receptor 9 agonist TLR9a).
  • TLR Toll like receptor
  • Compositions provided herein may include a Toll like receptor agonist selected from the group consisting of a TLR2 agonist (e.g. Pam3CSK4), a TLR4 agonist (e.g.
  • an aminoalkyl glucosaminide phosphate such as E6020
  • a TLR7 agonist e.g. imiquimod or a benzonaphthyridine, for example, SMIP7.10
  • a TLR8 agonist e.g. resiquimod (also a TLR7 agonist)
  • a TLR9 agonist e.g. IC31 or CpG1018.
  • Immunogenic compositions provided herein may comprise both a TLR agonist and at least one aluminium salt adjuvant such as aluminium phosphate and/or aluminium hydroxide.
  • immunogenic compositions provided herein are substantially aluminium adjuvant free, i.e., they comprise only a residual or trace amount of aluminium salt adjuvant(s) or do not comprise a measurable amount of aluminium salt adjuvant(s).
  • the TLR agonist is selected from the group consisting of IC31, E6020, CpG1018 and SMIP7.10.
  • immunogenic compositions provided herein comprise a TLR agonist selected from the group consisting of IC31, E6020, CpG1018 and SMIP7.10; and an aluminium salt adjuvant selected from the group consisting of an aluminium hydroxide and aluminium phosphate.
  • immunogenic compositions provided herein comprise (i) a TLR agonist selected from the group consisting of the TLR4 agonist E6020, the TLR7 agonist SMIP7.10 and the TLR9 agonist Cpg1018 and (ii) an aluminium salt selected from the group consisting of an aluminium hydroxide and aluminium phosphate.
  • TLR agonists may have a molecular weight of ⁇ 2000Da.
  • TLR7 agonists, such as SMIP7.10 may include at least one adsorptive moiety. The inclusion of such moieties in TLR agonists allows them to adsorb to insoluble aluminium salts (e.g. by ligand exchange or any other suitable mechanism) and improves their immunological behaviour.
  • Phosphorus- containing adsorptive moieties are useful, and so an adsorptive moiety may comprise a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, etc.
  • the TLR agonist may include at least one phosphonate group.
  • a composition provided herein may include a TLR7 agonist which includes a phosphonate group. This phosphonate group can allow adsorption of the agonist to an insoluble aluminium salt.
  • the TLR7 agonist comprises a benzonaphthyridine chemical scaffold for example, in some embodiments, the TLR agonist is 3-(5- amino-2-(2-methyl-4-(2-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo [f]- [1,7]naphthyridin-8-yl)propanoic acid, shown below, optionally adsorbed to an aluminum adjuvant.
  • TLR agonists are water-soluble. Thus, they can form a homogenous solution when mixed in an aqueous buffer with water at pH 7 at 25°C and 1 atmosphere pressure to give a solution which has a concentration of at least 50 ⁇ g/ml.
  • compositions will generally be administered to a suitable subject, for example a patient, for example a suitable mammal, such as a human, in aqueous form. Prior to administration, however, the composition may have been in a non-aqueous form. For instance, some vaccines are manufactured in aqueous form, then filled and distributed and administered also in aqueous form whereas other vaccines may be lyophilised during manufacture and reconstituted into an aqueous form at the time of use, for example at the time of administration. Thus, a composition provided herein may comprise or be a dried composition, such as a lyophilised formulation.
  • compositions provided herein may be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition).
  • the composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray.
  • the composition may be prepared for nasal, aural or ocular administration e.g. as drops.
  • the composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient.
  • Such kits may comprise one or more lyophilised antigens and one or more antigens in liquid form for reconstitution of the lyophilised antigens.
  • kits may comprise two vials, or it may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection.
  • kits may comprise two vials, or it may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection.
  • Further antigenic components of compositions of the invention Immunogenic compositions provided herein will generally be combination vaccines and in addition to OMVs as provided herein, may include one or more further antigen(s).
  • the OMVs provided herein are obtained from Bordetella bacteria, optionally from Bordetella pertussis and the one or more further antigen(s) are antigens capable of stimulating or generating an immune response against viral and/or bacterial pathogens.
  • Typical bacterial pathogens include, but are not limited to, Corynebacterium diphtheriae; Clostridium tetani and Haemophilus influenzae type b.
  • Typical viral pathogens include, but are not limited to, poliovirus and hepatitis B virus.
  • immunogenic compositions provided herein may further comprise one or more antigenic components that, when administered to a subject, is/are capable of generating an immune response, for example a protective immune response, against Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, hepatitis B virus, Haemophilus influenzae type B or polio virus.
  • an immune response for example a protective immune response, against Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, hepatitis B virus, Haemophilus influenzae type B or polio virus.
  • additional or further antigens is intended to refer to antigenic components included in the immunogenic compositions provided herein beyond, i.e. in addition to, the OMVs and constituents thereof.
  • pertussis toxoid may be found as a constituent of OMVs but reference to pertussis toxoid as a further antigen refers to an amount of pertussis toxoid that is specifically added over and above the OMVs, for example, as an isolated protein antigen.
  • suitable further antigenic components known in the art from the bacterial and viral pathogens described in more detail below include: – Hepatitis B virus: surface and/or core antigens.
  • Corynebacterium diphtheriae diphtheria toxoid or CRM197 (a diphtheria toxoid mutant).
  • Clostridium tetani tetanus toxoid.
  • Bordetella pertussis acellular pertussis antigens selected from the group consisting of pertussis toxoid (PT), filamentous haemagglutinin (FHA), pertactin, adenylate cyclase or an iimmunogenic fragment thereof, FIM2 and FIM3.
  • PT pertussis toxoid
  • FHA filamentous haemagglutinin
  • FIM2 and FIM3 adenylate cyclase or an iimmunogenic fragment thereof
  • Haemophilus influenzae B capsular polysaccharide, for example a polyribosyl ribitol phosphate [PRP]-protein conjugate.
  • Polio virus inactivated poliovirus (IPV) comprising either type 1, type 2 and type 3 strains or type 1 and type 3 strains, for example, selected from Mahoney type 1, MEF-1 type 2 and.
  • the OMVs provided herein and obtained from Bordetella species are conjugated to a capsular polysaccharide from Haemophilus influenzae type b to form an immunogenic conjugate.
  • This conjugate may be combined with one or more further antigens in an immunogenic composition as described above.
  • an immunogenic composition of the conjugates between Bordetella OMVs and Hib polysaccharide is a fully liquid, stable composition further comprising an antigen selected from the group consisting of Diphteria Toxoid (DT), Tetanus Toxoid (TT), Hepatitis B (HepB) antigen, inactivated Poliovirus (IPV) and one or more acellular pertussis (aP) antigens.
  • the Hib capsular polysaccharide has a molecular weight between from about 5 to about 100 KDa.
  • the Hib saccharide moiety of the conjugate may be used in its full-length native form, comprising full-length PRP as prepared from Hib bacteria, or, as an alternative, it may be fragmented from its natural length according to different methods described in the art; optionally, a size fraction of these fragments can also be used.
  • the PRP may be a synthetic polysaccharide. Suitable conjugates of Bordetella OMVs to Hib capsular polysaccharide are disclosed in WO2020/043874, which is incorporated herein by reference.
  • immunogenic compositions may further comprise one or more acellular pertussis (aP) antigens, for example selected from the following well-known and well-characterized B.pertussis antigens: (1) detoxified pertussis toxin (pertussis toxoid, or ‘PT’), for example as chemically detoxified pertussis toxoid (PTc) or genetically detoxified pertussis toxoid (PTg); (2) filamentous hemagglutinin (‘FHA’); (3) pertactin (also known as ‘PRN’ or the ‘69 kiloDalton outer membrane protein’); (4) fimbriae type 2 (‘FIM2’); (5) fimbriae type 3 (‘FIM3’) and (6) adenylate cyclase (AC) or an immunogenic fragment thereof.
  • PT detoxified pertussis toxin
  • PTc chemically detoxified pertussis toxoid
  • PTg genetically detoxified pert
  • both detoxified PT and FHA are used.
  • FIM2 and FIM3 are typically co-purified and processed as a single antigen (referred to as FIM2/3).
  • PTc may be prepared by chemical treatment of pertussis toxin with formaldehyde and/or glutaraldehyde.
  • the PT is a mutant PT in which enzymatic activity has been reduced by mutagenesis, for example, genetically detoxified pertussis toxoid (PTg), for example the genetically detoxified pertussis toxoid is the genetically detoxified pertussis toxoid known in the art and referred to as the PT-9K/129G mutant.
  • immunogenic compositions comprise the acellular pertussis antigens PT, FHA and PRN, for example PTg, FHA and PRN. In some embodiments the immunogenic compositions also comprise the acellular pertussis antigens FIM2/FIM3. In some embodiments, immunogenic compositions comprise the acellular pertussis antigens PTg, FHA, FIM2/FIM3 but do not contain PRN.
  • PT, FHA and PRN are prepared by isolation from B.pertussis culture grown in a suitable medium, such as modified Stainer-Scholte liquid medium.
  • PT and FHA can be isolated from the fermentation broth (e.g. by adsorption on hydroxyapatite gel), whereas pertactin can be extracted from the cells by heat treatment and flocculation (e.g. using barium chloride).
  • the antigens can be purified in successive chromatographic and/or precipitation steps.
  • PT and FHA can be purified by hydrophobic chromatography, affinity chromatography and size exclusion chromatography.
  • Pertactin can be purified by ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography.
  • FHA and pertactin may be treated with formaldehyde prior to use.
  • the aP antigen(s) may be used in an unadsorbed state, but may be adsorbed onto one or more aluminium salt adjuvant(s) before being used.
  • the aP antigens are substantially free from mercurial preservatives such as thimerosal.
  • the acellular pertussis antigens may be present in the immunogenic compositions provided herein in an amount that is capable of eliciting an immune response when administered. Ideally, the acellular pertussis antigens can elicit a protective immune response. Quantities of acellular pertussis antigens are typically expressed in micrograms.
  • the concentration of PT in a vaccine is usually between 5 and 50 ⁇ g/ml. Typical PT concentrations are 5 ⁇ g/ml, 16 ⁇ g/ml, 20 ⁇ g/ml or 50 ⁇ g/ml, for example, about 20 ⁇ g per dose or about 25 ⁇ g per dose.
  • the concentration of FHA in a vaccine is usually between 10 and 50 ⁇ g/ml. Typical FHA concentrations are 10 ⁇ g/ml, 16 ⁇ g/ml or 50 ⁇ g/ml, for example, about 20 ⁇ g per dose or about 25 ⁇ g per dose.
  • the concentration of pertactin in a vaccine is usually between 5 and 16 ⁇ g/ml.
  • Typical pertactin concentrations are 5 ⁇ g/ml, 6 ⁇ g/ml or 16 ⁇ g/ml, for example, about 3 ⁇ g per dose or about 8 ⁇ g per dose.
  • FIM2 and FIM3 may be present at a concentration 5 and 16 ⁇ g/ml.
  • Typical concentrations of FIM2 and FIM3 are 5 ⁇ g/ml, 10 ⁇ g/ml or 20 ⁇ g/ml, for example, for example, about 5 ⁇ g per dose or about 10 ⁇ g per dose.
  • Booster vaccines for adolescents and adults typically contain 2.5 to 8 ⁇ g PT, between 4 and 8 ⁇ g FHA and between 2.5 and 8 ⁇ g pertactin per 0.5 ml dose.
  • a booster vaccine comprises 4 ⁇ g PT, 4 ⁇ g FHA and 8 ⁇ g pertactin, for example 5 ⁇ g PT, 2.5 ⁇ g FHA and 2.5 ⁇ g pertactin, per 0.5 ml dose.
  • a paediatric vaccine usually comprises 7 ⁇ g PT, 10 ⁇ g FHA and 10 ⁇ g pertactin, per 0.5 ml dose.
  • their weight ratios can vary, but may be e.g. about 16:16:5, about 5:10:6, about 20:20:3, about 25:25:8, or about 10:5:3 (PT:FHA:PRN).
  • Diphtheria Corynebacterium diphtheriae causes diphtheria.
  • Diphtheria toxin can be treated (e.g. using formalin or formaldehyde) to remove toxicity while retaining the ability to induce specific anti-toxin antibodies after injection.
  • the diphtheria toxoids used in diphtheria vaccines are well known in the art.
  • diphtheria toxoids are those prepared by formaldehyde treatment.
  • the diphtheria toxoid can be obtained by growing C. diphtheriae in growth medium (e.g. Fenton medium, or Linggoud & Fenton medium), which may be supplemented with bovine extract, followed by formaldehyde treatment, ultrafiltration and precipitation.
  • growth medium e.g. Fenton medium, or Linggoud & Fenton medium
  • the growth medium for growing C. diphtheriae is free from animal-derived components.
  • the toxoided material may then be treated by a process comprising sterile filtration and/or dialysis.
  • genetically detoxified diphtheria toxin e.g., CRM197
  • CRM197 genetically detoxified diphtheria toxin
  • the diphtheria toxoid may be adsorbed onto an adjuvant such as an aluminium salt adjuvant.
  • Quantities of diphtheria toxin and/or toxoid in a composition are generally measured in the ‘Lf’ unit (“flocculating units”, or the “limes flocculating dose”, or the “limit of flocculation”), defined as the amount of toxin/toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture (Lyng (1990) Biologicals 18:11-17).
  • the NIBSC supplies ‘Diphtheria Toxoid, Plain’ (NIBSC code: 69/017), which contains 300 LF per ampoule, and also supplies ‘The 2nd International Reference Reagent For Diphtheria Toxoid For Flocculation Test’ (NIBSC Code: 02/176) which contains 900 Lf per ampoule.
  • the concentration of diphtheria toxin or toxoid in a composition can readily be determined using a flocculation assay by comparison with a reference material calibrated against such reference reagents.
  • the immunizing potency of diphtheria toxoid in a composition is generally expressed in international units (IU).
  • compositions generally include at least 30 IU/dose.
  • Compositions typically include between 20 and 80 Lf/ml of diphtheria toxoid, typically about 50 Lf/ml.
  • Booster vaccines for adolescents and adults typically contain between 4 Lf/ml and 8 Lf/ml of diphtheria toxoid, e.g., 2.5 Lf, for example 4 Lf, per 0.5 ml dose.
  • Paediatric vaccines typically contain between 20 and 50 Lf/ml of diphtheria toxoid, e.g. 10 Lf or 25 Lf per 0.5 ml dose.
  • Purity of a protein preparation can be expressed by the ratio of specific protein to total protein.
  • the purity of diphtheria toxoid in a composition is generally expressed in units of Lf diphtheria toxoid per unit mass of protein (nondialysable) nitrogen.
  • a very pure toxin/toxoid might have a purity of more than 1700 Lf/mg N, indicating that most or all of the protein in the composition is diphtheria toxin/toxoid (Kuhmlann & Rieger (1995) Immunol Infect Dis 5:10-4 ).
  • Tetanus Clostridium tetani causes tetanus.
  • Tetanus toxin can be treated to give a protective toxoid.
  • the toxoids are used in tetanus vaccines and are well known in the art.
  • a combination vaccine provided herein can include a tetanus toxoid.
  • tetanus toxoids are those prepared by formaldehyde treatment.
  • the tetanus toxoid can be obtained by growing C.tetani in growth medium (e.g. a Latham medium derived from bovine casein), followed by formaldehyde treatment, ultrafiltration and precipitation.
  • the growth medium for growing C.tetani may be free from animal- derived components.
  • the material may then be treated by a process comprising sterile filtration and/or dialysis.
  • the tetanus toxoid may be adsorbed onto an adjuvant, for example, an aluminium salt adjuvant.
  • Quantities of tetanus toxoid can be expressed in ‘Lf’ units (see below), defined as the amount of toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture.
  • the NIBSC supplies ‘The 2nd International Reference Reagent for Tetanus Toxoid For Flocculation Test’ (NIBSC Code: 04/150) which contains 690 LF per ampoule, by which measurements can be calibrated.
  • Booster vaccines for adolescents and adults typically contain 5 Lf of tetanus toxoid per 0.5 ml dose.
  • Paediatric vaccines typically contain between 5 and 10 Lf of tetanus toxoid per 0.5 ml dose.
  • the immunizing potency of tetanus toxoid is measured in international units (IU), assessed by comparing the protection afforded by a composition in laboratory animals (typically guinea pigs) with a reference vaccine e.g. using NIBSC’s ‘Tetanus Toxoid Adsorbed Third International Standard 2000’ (Sesardic et al. (2002) Biologicals 30:49-68; NIBSC code: 98/552), which contains 469 IU per ampoule.
  • the potency of tetanus toxoid in a composition provided herein should be at least 35 IU per dose e.g. at least 70 IU/ml.
  • the potency of tetanus toxoid in a composition provided herein is at least 40 IU per dose. However, in booster vaccines for adults and adolescents, a reduced potency of 20 IU/dose may be acceptable because of the reduced antigen content in comparison to paediatric vaccine intended for primary immunization.
  • the purity of tetanus toxoid in a composition is generally expressed in units of Lf tetanus toxoid per unit mass of protein (non-dialyzable) nitrogen.
  • the tetanus toxoid should have a purity of at least 1000 Lf/mg N.
  • Hib Haemophilus influenzae type b (‘Hib’) causes bacterial meningitis.
  • Hib vaccines are typically based on the capsular saccharide antigen the preparation of which is well documented in the art (Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Buttery & Moxon (2000) J R Coll Physicians Lond 34:163- 168, etc).
  • the H.influenzae bacteria can be cultured in the absence of animal-derived components.
  • the Hib saccharide is conjugated to a carrier protein in order to enhance its immunogenicity, especially in children.
  • Typical carrier proteins in these conjugates are tetanus toxoid, diphtheria toxoid, the CRM197 derivative of diphtheria toxin, or an outer membrane protein complex (OMPC) from serogroup B meningococcus.
  • OMPC outer membrane protein complex
  • a combination vaccine provided herein can include a Hib capsular saccharide conjugated to a carrier protein such as tetanus toxoid, diphtheria toxoid or OMPC.
  • the saccharide moiety of the conjugate may comprise full-length polyribosylribitol phosphate (PRP) as prepared from Hib bacteria, and/or fragments of full-length PRP.
  • Conjugates with a saccharide:protein ratio (w/w) of between 1:5 (i.e. excess protein) and 5:1 (i.e. excess saccharide) may be used e.g. ratios between 1:2 and 5:1 and ratios between 1:1.25 and 1:2.5.
  • the weight ratio of saccharide to carrier protein is between 1:2.5 and 1:3.5.
  • Quantities of Hib antigens are typically expressed in ⁇ g of saccharide.
  • the concentration of saccharide in a vaccine is typically between from 3 to 30 ⁇ g/ml e.g. 20 ⁇ g/ml.
  • administration of the Hib conjugate results in an anti-PRP antibody concentration of >0.15 ⁇ g/ml, and for example >1 ⁇ g/ml, and these are the standard response thresholds.
  • the Hib polyribosylribitol phosphate is a synthetic polysaccharide (for example, as described in Verez- Bencomo et al. Science.
  • the Hib capsular polysaccharide antigen may be conjugated to the OMVs provided herein obtained from Bordetella species.
  • Hepatitis B virus Hepatitis B virus (HBV) is a cause of viral hepatitis.
  • the HBV virion consists of an inner core surrounded by an outer protein coat or capsid, and the core contains the viral DNA genome.
  • the major component of the capsid is a protein known as HBV surface antigen or, more commonly, ‘HBsAg’, which is typically a 226-amino acid polypeptide with a molecular weight of ⁇ 24 kDa.
  • immunogenic compositions provided herein can include HBsAg.
  • HBsAg can be made in a number of ways. For example, by expressing the protein by recombinant DNA methods. HBsAg for use with the method provided herein should be recombinantly expressed, e.g. in yeast cells. Suitable yeasts include Saccharomyces (such as S.cerevisiae), Hanensula (such as H.polymorpha) or Pichia hosts.
  • the yeasts can be cultured in the absence of animal-derived components.
  • Yeast-expressed HBsAg is generally non-glycosylated; in one embodiment the non-glycosylated form of HBsAg is used in the immunogenic compositions provided herein.
  • Yeast-expressed HBsAg is highly immunogenic and can be prepared without the risk of blood product contamination. Many methods for purifying HBsAg from recombinant yeast are known in the art.
  • the HBsAg will generally be in the form of substantially-spherical particles (average diameter of about 20nm), including a lipid matrix comprising phospholipids.
  • Yeast-expressed HBsAg particles may include phosphatidylinositol, which is not found in natural HBV virions.
  • the HBsAg is from HBV subtype adw2.
  • Quantities of HBsAg are typically expressed in micrograms.
  • Combination vaccines containing HBsAg usually include between 5 and 60 ⁇ g/ml.
  • the concentration of HBsAg in a composition provided herein is for example less than 60 ⁇ g/ml e.g. ⁇ 55 ⁇ g/ml, ⁇ 50 ⁇ g/ml, ⁇ 45 ⁇ g/ml, ⁇ 40 ⁇ g/ml, etc.
  • a concentration of about 20 ⁇ g/ml is typical e.g.
  • a composition includes a ‘low dose’ of HBsAg.
  • concentration of HBsAg in the composition is ⁇ 5 ⁇ g/ml e.g. ⁇ 4, ⁇ 3, ⁇ 2.5, ⁇ 2, ⁇ 1, etc.
  • the amount of HBsAg is less than 2.5 ⁇ g e.g. ⁇ 2, ⁇ 1.5, ⁇ 1, ⁇ 0.5, etc.
  • Poliovirus Poliovirus causes poliomyelitis.
  • IPV Inactivated polio virus vaccine
  • a combination vaccine provided herein can include an inactivated poliovirus antigen. Polioviruses may be grown in cell culture.
  • a culture uses a Vero cell line, derived from monkey kidney.
  • Vero cells can conveniently be cultured microcarriers. After growth, virions may be purified using techniques such as ultrafiltration, diafiltration, and chromatography. Where animal (and optionally bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encephalopathies (TSEs), and for example free from bovine spongiform encephalopathy (BSE).
  • TSEs transmissible spongiform encephalopathies
  • BSE bovine spongiform encephalopathy
  • polioviruses are grown in cells cultured in medium free of animal-derived components.
  • Polioviruses Prior to administration to patients, polioviruses must be inactivated, and this can be achieved by treatment with formaldehyde (or, for example, a non-aldehyde agent). Poliomyelitis can be caused by one of three types of poliovirus. The three types are similar and cause identical symptoms, but they are antigenically very different and infection by one type does not protect against infection by others. In one embodiment, three poliovirus antigens are used with the compositions provided herein: poliovirus Type 1 (e.g. Mahoney strain), poliovirus Type 2 (e.g. MEF-1 strain), and poliovirus Type 3 (e.g. Saukett strain).
  • poliovirus Type 1 e.g. Mahoney strain
  • poliovirus Type 2 e.g. MEF-1 strain
  • poliovirus Type 3 e.g. Saukett strain
  • strains of poliovirus Type 1, Type 2 and Type 3 are known in the art and may also be used.
  • the viruses are for example grown, purified and inactivated individually, and are then combined to give a bulk trivalent mixture for use with the composition provided herein.
  • Quantities of IPV are typically expressed in the ‘DU’ unit (the “D-antigen unit”; Module 6 of WHO’s The immunological basis for immunization series (Robertson)).
  • Combination vaccine usually comprise between 1-100 DU per polioviral type per dose e.g., about 40 DU of type 1 poliovirus, about 8 DU of type 2 poliovirus, and about 32 DU of type 3 poliovirus, but it is possible to use lower doses than these (WO2008/028956; WO2008/028957) e.g.10-20 DU for type 1, 2-4 DU for type 2, and 8-20 DU for type 3.
  • a combination vaccine provided herein can include a ‘low dose’ of a poliovirus. For a Type 1 poliovirus this means that the concentration of the virus in the composition is ⁇ 20 DU/ml e.g.
  • Type 2 poliovirus this means that the concentration of the virus in the composition is ⁇ 4 DU/ml e.g. ⁇ 3, ⁇ 2, ⁇ 1, ⁇ 0.5, etc.
  • a Type 3 poliovirus this means that the concentration of the virus in the composition is ⁇ 16 DU/ml e.g. ⁇ 14, ⁇ 12, ⁇ 10, ⁇ 8, ⁇ 6, etc.
  • the three antigens can be present at a DU ratio of 5:1:4 respectively, or at any other suitable ratio e.g.
  • a vaccine composition for example contains less than 10ng/ml, for example ⁇ 1ng/ml e.g. ⁇ 500pg/ml or ⁇ 50 pg/ml of Vero cell DNA e.g. less than 10ng/ml of Vero cell DNA that is >50 base pairs long.
  • Antigenic components from these pathogens for use in vaccines are commonly referred to by abbreviated names: ‘D’ for diphtheria toxoid; ‘T’ for tetanus toxoid; ‘P’ for pertussis antigens, with ‘aP’ being acellular pertussis antigens (e.g. including at least OMVs provided herein, PT and FHA and optionally pertactin and/or FIM2/FIM3); HBsAg for hepatitis B surface antigen; ‘Hib’ for conjugated H.influenzae b capsular saccharide; and ‘IPV’ for 3-valent inactivated poliovirus.
  • D diphtheria toxoid
  • T for tetanus toxoid
  • P pertussis antigens
  • HBsAg for hepatitis B surface antigen
  • Hib for conjugated H.influenzae b capsular saccharide
  • IPV
  • Embodiments provided herein include, but are not limited to combination vaccines comprising the following components: – D, T, aP – D, T, aP, IPV – D, T, aP, HBsAg – D, T, aP, Hib – D, T, aP, Hib, IPV – D, T, aP, HBsAg, Hib – D, T, aP, HBsAg, IPV – D, T, aP, HBsAg, IPV – D, T, aP, HBsAg, IPV, Hib
  • these combination vaccines contain only the antigens listed as active ingredients but may further comprise excipients such as adjuvants, buffers and the like.
  • the aP component consists of OMVs provided herein, PT (for example genetically detoxified PT) and FHA.
  • the aP component consists of OMVs provided herein, PT (for example genetically detoxified PT), FHA and PRN.
  • the aP component consists of OMVs provided herein, PT (preferably genetically detoxified PT), FHA and FIM2/FIM3.
  • the aP component consists of OMVs provided herein, PT (for example genetically detoxified PT), FHA, PRN and FIM2/FIM3.
  • the ratio of D:T is typically greater than 1 (i.e.
  • paediatric vaccines usually have excess D in Lf units) and generally between 2:1 and 3:1 (measured in Lf units), e.g.2.5:1.
  • the ratio of T:D is typically greater than 1 (i.e. booster vaccines usually have excess T in Lf units) and generally between 1.5:1 and 2.5:1, e.g. 2:1.
  • One useful vaccine includes OMVs provided herein and, per unit dose, 2Lf D, 5Lf T, 4 ⁇ g PT- 9K/129G, 4 ⁇ g FHA and 8 ⁇ g pertactin.
  • OMVs provided herein and, per unit dose, 25Lf D, 10Lf T, 25 ⁇ g PT-9K/129G, 25 ⁇ g FHA and 8 ⁇ g pertactin. Combinations with D and T are of interest.
  • Immunisation In addition to providing immunogenic compositions as described above, also it is provided herein the use of OMVs and immunogenic compositions in a method for raising an immune response in a mammal, comprising administering OMVs or an immunogenic composition provided herein to the mammal.
  • the immune response is an antibody response.
  • the antibody response is a protective antibody response. It is also provided herein compositions for use in such methods.
  • compositions for use as medicaments e.g. as immunogenic compositions or as vaccines.
  • a population of purified OMVs as disclosed herein for use in medicine. It also provides the use of OMVs provided herein in the manufacture of a medicament for preventing a bacterial infection in a mammal.
  • the mammal is a human.
  • the human may be an adult or, for example, a child.
  • the vaccine is for prophylactic use, the human is for example a child (e.g.
  • Efficacy of therapeutic treatment can be tested by monitoring bacterial infection after administration of the composition provided herein.
  • Efficacy of prophylactic treatment can be tested by monitoring immune responses against immunogenic proteins in the vesicles or other antigens after administration of the composition. Immunogenicity of compositions provided herein can be determined by administering them to test subjects (e.g. children 12-16 months age) and then determining standard serological parameters. These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition.
  • compositions provided herein will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • parenteral injection e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue
  • rectal oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration.
  • intramuscular administration to the thigh or the upper arm is provided. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used.
  • a typical intramuscular dose is about 0.5 ml.
  • Immunogenic compositions may be used to elicit systemic and/or mucosal immunity.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined.
  • General The term “comprising” encompasses “including” e.g. a composition “comprising” X may include something additional e.g. X + Y. The word “substantially” does not exclude “completely” e.g.
  • composition which is “substantially free” from Y may be completely free from Y or it may mean that Y is present in the composition, if at all, as an incidental impurity at a level that does not effect the properties of the composition.
  • the term “comprising” refers to the inclusion of the indicated active agent, such as recited polypeptides, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry.
  • the term “consisting essentially of” refers to a composition, whose only active ingredient is the indicated active ingredient(s), for example antigens, however, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient.
  • Use of the transitional phrase “consisting essentially” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, and those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
  • step (c) follows step (b) which is preceded by step (a).
  • a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • Embodiment 3 The process of embodiment 2, wherein the OMVs are obtained from: (i) Bordetella bacteria, optionally from Bordetella pertussis, optionally wherein the bacterium is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G and/or wherein the bacterium is a recombinant bacterium that produces lipid A having (I) C3’ acyl chains of about 10 carbon atoms (C10) in length; and/or (II) C2’ acyl chains of about 10 carbon atoms (C10) in length; and/or (III) C2 acyl chains of about 10 carbon atoms (C10) in length; or (ii) Neisseria bacteria, optionally from Neisseria gonorrhoeae, optionally where
  • Embodiment 4 The process of any one of embodiments 1 to 3 wherein the TFF membrane has a molecular weight cut off of about 500 kDa.
  • Embodiment 5. The process of any one of embodiments 1 to 3 wherein the TFF membrane is a hollow fibre membrane having a molecular weight cut off of about 750 kDa or about 300 kDa.
  • Embodiment 6. The process of embodiment 4 or 5 wherein the TFF of step (a) comprises concentrating and washing the sample by at least one ultrafiltration cycle and at least one cycle of diafiltration.
  • Embodiment 7. The process of embodiment 6 wherein the at least one ultrafiltration cycle concentrates the sample by at least about 5 times, for example about 10 times.
  • a wash solution for example 10 volumes of a wash solution
  • the bacterium is a Bordetella bacterium
  • the wash solution is selected from (1) Dulbecco's phosphate-buffered saline (DPBS) optionally comprising EDTA, for example about 5mM EDTA, or (2) 20mM Tris buffer pH 8.6 optionally comprising sucrose, for example about 3% sucrose; or, optionally wherein the bacterium is a Neisseria bacterium, the wash solution is selected from (1) 10mM Na 2 HPO 4 /NaH 2 PO 4 pH 6.8 comprising 150mM NaCl or (2) phosphate-buffer saline (PBS) pH 7.4.
  • DPBS Dulbecco's phosphate-buffered saline
  • PBS phosphate-buffer saline
  • Embodiment 9 The process of any one of the preceding embodiments, wherein the sample of OMVs is obtained by deoxycholate (DOC) disruption of the outer membrane of Bordetella bacteria.
  • Embodiment 10 The process of embodiment 9, wherein the sample comprising OMVs is prepared by a process comprising the steps of (i) homogenising a fermentation harvest of Bordetella bacteria, thereby obtaining a homogenised fermentation harvest; (ii) treating the homogenised fermentation harvest with DOC and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; and (iii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce the sample comprising OMVs.
  • Embodiment 11 The process of any one of the preceding embodiments, wherein the sample of OMVs is obtained by deoxycholate (DOC) disruption of the outer membrane of Bordetella bacteria.
  • Embodiment 10 The process of embodiment 9, wherein the sample comprising OMVs
  • Embodiment 12 The process of any one of embodiments 1 to 8, wherein the sample comprising OMVs comprises or consists of OMVs released in the medium.
  • Embodiment 13 The process of embodiment 12, wherein the sample comprising OMVs is prepared by a process comprising the steps of (i) centrifuging a fermentation harvest, optionally of Neisseria bacteria; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce the sample comprising OMVs.
  • Embodiment 14 The process of embodiment 10, wherein the homogenised fermentation harvest is treated with DOC and benzonase.
  • a process for preparing OMVs from Bordetella bacteria comprising: (i) homogenising a fermentation harvest of Bordetella bacteria, thereby obtaining a homogenised fermentation harvest comprising the Bordetella bacteria; (ii) treating the homogenised fermentation harvest with DOC to disrupt the outer membrane of the Bordetella bacteria and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; (ii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce a sample containing DOC-extracted OMVs and one or more impurities; (iv) removing or reducing the concentration of the one or more impurities by (v) concentrating and washing the sample containing DOC-extracted OMVs and one or more impurities by TFF through a TFF membrane to obtain a retentate concentrate comprising OMVs and (vi) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having
  • Embodiment 16 The process of embodiment 15 wherein the Bordetella bacteria is Bordetella pertussis.
  • Embodiment 17 The process of embodiment 16, wherein the Bordetella pertussis is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G and/or is a recombinant bacterium that produces lipid A having (i) C3’ acyl chains of about 10 carbon atoms (C10) in length; and/or (ii) C2’ acyl chains of about 10 carbon atoms (C10) in length; and/or (iii) C2 acyl chains of about 10 carbon atoms (C10) in length.
  • Embodiment 18 A process for preparing OMVs from Neisseria bacteria, the process comprising the sequential steps of: (i) centrifuging a fermentation harvest ; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce a sample containing OMVs and one or more impurities; (vi) removing or reducing the concentration of the one or more impurities by (vii) concentrating and washing the sample containing OMVs and one or more impurities by TFF through a membrane to obtain a retentate concentrate comprising OMVs and (viii) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a
  • Embodiment 19 The process of embodiment 18, wherein the Neisseria bacteria is Neisseria gonorrhoeae.
  • Embodiment 20 The process of embodiment 18 or 19, wherein the Neisseria gonorrhoeae is a Neisseria gonorrhoeae bacterium that comprises genetic modification(s), optionally wherein said genetic modification(s) reduce the endotoxin activity of the OMVs compared to a wild-type or unmodified Neisseria gonorrhoeae.
  • Embodiment 21 Embodiment 21.
  • Neisseria gonorrhoeae bacterium comprises a further genetic modification that decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide.
  • rmp reduction modifiable protein
  • Embodiment 24 The process of any preceding embodiments wherein the one or more impurities comprise or is the 60kDa chaperonin GroEL.
  • Embodiment 25 Embodiment 25.
  • a process for reducing the concentration of the 60kDa chaperonin GroEL in a sample of OMVs, optionally from Bordetella or Neisseria comprising: filtering the sample of OMVs by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs wherein the 60kDa chaperonin GroEL is present in an amount less than 1.5% of total protein.
  • Embodiment 26 Embodiment 26.
  • the 60kDa chaperonin GroEL comprises a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of SEQ ID NO: 1-4, optionally wherein the 60kDa chaperonin GroEL comprises or has the sequence of anyone of SEQ ID NO: 1-4.
  • Embodiment 27 A population of purified OMVs produced by a process according to any one of the preceding embodiments.
  • Embodiment 28 A population of purified OMVs that is at least 85% pure or from about 85% to about 100% pure.
  • Embodiment 29 A population of purified OMVs produced by a process according to any one of the preceding embodiments.
  • Embodiment 30. The population of purified OMVs of embodiment 28 or 29, wherein purity is measured using size exclusion Ultra-performance liquid chromatography (UPLC), for example by size exclusion UPLC to determine the ratio between the main peak and the low molecular size peak.
  • UPLC size exclusion Ultra-performance liquid chromatography
  • Embodiment 32 The population of purified OMVs of any one of embodiments 28 to 31, wherein the one or more impurities comprise or is the 60kDa chaperonin GroEL.
  • Embodiment 33 Embodiment 33.
  • the population of purified OMVs of embodiment 32 wherein the 60kDa chaperonin GroEL comprise a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of SEQ ID NO: 1-4, optionally wherein the 60kDa chaperonin GroEL comprises or has the sequence of anyone of SEQ ID NO: 1-4.
  • Embodiment 34 The population of purified OMVs of embodiments 32 or 33, comprising from about 0.01% to about 10% of GroEL as a percentage of total protein.
  • Embodiment 35 The population of purified OMVs of embodiments 32 or 33, comprising from about 0.01% to about 10% of GroEL as a percentage of total protein.
  • Embodiment 36 The population of purified OMVs of any one of embodiments 32 to 34, comprising less that 5% GroEL.
  • Embodiment 36 The population of purified OMVs of any one of embodiments 27 to 35, wherein the population is substantially free of process related impurities.
  • Embodiment 37 The population of purified OMVs of any one of embodiments 27 to 36, wherein the purified OMVs are obtained from Bordetella and are conjugated to a capsular polysaccharide of Haemophilus influenzae type b (Hib).
  • Embodiment 38 Embodiment 38.
  • Embodiment 39 The population of purified OMVs of any one of embodiments 27 to 38 for use in medicine.
  • EXAMPLES Example 1 – Fermentation and Deoxycholate (DOC) preparation of OMVs The frozen contents of an ampoule containing a working seed of Bordetella pertussis Tohama strain was thawed at room temperature for 10 minutes. Two 250 ml shake-flasks containing 30 ml fresh medium (adapted from Stainer and Scholte (J. Gen. Microbiol.
  • DOC Fermentation and Deoxycholate
  • the first pre-cultures reached an optical density at 650 nm (OD650nm) of 1-1.5, they were used to inoculate two 3L shake-flasks containing 1L of fresh medium.
  • the second pre-culture flasks were incubated at 35° C. (+/ ⁇ 1° C.) and 150 rpm for 24 h (+/ ⁇ 1 h), after which they were pooled. 1500ml of the pooled pre-culture was used to inoculate a 20 L-fermentor (BiolafitteTM ) containing 10L of fresh medium and 3ml antifoam agent.
  • Batch fermentation was performed for 24h using the following parameters: temperature 35° C., head pressure 0.4 bar, air flow rate 20NL/min constant, pH 7.2 (regulated using acetic acid 50%) and stirring speed 50 rpm to 1000rpm max.
  • temperature 35° C.
  • head pressure 0.4 bar
  • a mechanical foam breaker was used to control foaming during the fermentation.
  • the air flow rate was progressively increased during the fermentation, according to a pre-defined curve.
  • the level of dissolved oxygen was set at 25% and regulated by increasing stirring when the DO fell below 25%.
  • the minimum stirring speed was set at 50 rpm; the maximum stirring speed was set at 1000 rpm.
  • the pH was regulated at 7.2 by addition of acetic acid 50% (w/v or weight/volume).
  • growth of the culture was monitored as OD650nm. Fermentation was stopped once the oxygen consumption decreased (as a consequence of glutamate exhaustion) resulting in a decrease in stirring speed.
  • cells were pelleted by centrifugation at 5000xg for 30 minutes (4°C). The supernatant was filtered with a Sartobran-P (0.45 ⁇ m + 0.22 ⁇ m) filter and dispensed into 1L flasks. The supernatant and cell pellet was stored at -20°C.
  • the frozen bacterial pellets were thawed, resuspended and homogenised in four volumes of 20mM Tris-HCl - 2mM EDTA buffer (pH 8,6), 100 U/mL benzonase with cOmpleteTM EDTA-free Protease Inhibitor Cocktail and incubated with stirring for 30 min at room temperature.
  • extraction buffer 1 (20 mM TrisHCl, 0.5% DOC, 2mM EDTA, pH 8.6 buffer) or extraction buffer 2 (20 mM TrisHCl, 0.1% DOC, 2mM EDTA, pH 8.6 buffer) was added with cOmpleteTM EDTA-free Protease Inhibitor Cocktail and the suspension was incubated with stirring for 30 min at a temperature of either 30°C or 40°C. After 30 minutes, 100U/mL Benzonase was added. The suspension was incubated and stirred for a further 30 minutes. Cellular debris was removed by centrifugation at 20,000g for 30 min (4°C), the supernatant containing the OMVs was removed and sterile filtered (0.22 ⁇ m).
  • Each 250 ml of solution comprising DOC extracted OMVs was diluted to 500 ml with OMV buffer (Tris 20mM pH 8.6 + 3% sucrose).
  • OMV buffer Tris 20mM pH 8.6 + 3% sucrose.
  • the generalised DOC extraction procedure used is shown in Fig. 1.
  • Example 2 Preparation of OMVs using 0.1% DOC at 30oC, Purification by TFF and Ultracentrifugation DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (Extraction Buffer 2) at 30°C. The preparation of OMVs was purified using a combination of TFF and Ultracentrifugation (Fig. 2).
  • TFF was performed using a Millipore 500kDa Cassette as follows: The preparations were concentrated 10-fold to 50 ml (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). The concentrated preparation was diafiltered at constant volume (50 ml) with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). 1550 U of benzonase (Merck 327 U/ul) was added to the retentate containing the OMVs and stirred for 1h (Pump flow 50ml/min, permeate valve closed, no counterpressure).
  • the concentrated preparation was further diafiltered with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi) and 50 ml of retentate was recovered in reverse flow.
  • the system was washed with a further 50 ml of OMV buffer and the wash was pooled with the retentate to yield a final volume of about 100ml (retentate + wash).
  • the pooled retentate was centrifuged twice for 2 hours at 150,000g, supernatant was removed and the pellet comprising OMVs was resuspended in 30ml of DPBS, aliquoted in 1ml and 15 ml aliquots and stored at -70°C.
  • Lipid concentration was measured using the fluorescent dye FM 4-64 [N-(3- Triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide] (also referred to as FM 4-64, Molecular Probes) to generate a standard calibration curve versus known amounts of DOPC.
  • FM 4-64 is a lipophilic styryl dye that selectively intercalates into the lipid membrane staining them with red fluorescence (excitation/emission maxima ⁇ 515/640 nm). Protein content was determined using the Lowry method known in the art.
  • TFF TFF followed by ultracentrifugation eliminated free proteins and aggregates (reducing protein content by around 50%) whilst generally retaining OMVs (lipid content in the final sample was reduced by about 12%).
  • purity (85%) and yield (25mg/L of fermentation culture based on protein content) were both low and the 60kDa chaperonin, GroEL, was not removed (Fig. 3).
  • the pellet was difficult to resuspend such that sterile filtration was not possible.
  • Example 3 Preparation of OMVs using 0.1% DOC at 40oC, Purification by TFF and Ultracentrifugation DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (Extraction Buffer 2) at 40°C. The preparation of OMVs was purified using a combination of TFF and Ultracentrifugation as before (Fig. 2). Increasing the temperature during DOC extraction had no impact on subsequent purification using TFF followed by ultracentrifugation. The resulting pellet was very hard to resuspend and contained a high level of precipitate such that sterile filtration was not possible.
  • Example 4 Preparation of OMVs using 0.1% DOC at 40oC, Purification by TFF and flow- through chromatography (Sephacryl S-500 gel filtration) DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (Extraction Buffer 2) at 40°C.
  • the preparation of OMVs was purified using a combination of TFF and flow-through chromatography (Sephacryl S-500 gel filtration) (Fig.4).
  • TFF was performed using a Millipore 500kDa Cassette as follows: The DOC extracted OMVs were concentrated 10-fold to 50 ml (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). The concentrated preparation was diafiltered at constant volume (50 ml) with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi).
  • Example 5 Preparation of OMVs using 0.1% DOC at 40oC, Purification by TFF and flow- through chromatography (CaptoCore 700 gel filtration) DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (Extraction Buffer 2) at 40°C. The preparation of OMVs was purified using a combination of TFF and flow-through chromatography (CaptoCore 700 gel filtration) (Fig. 5).
  • TFF was performed using a Millipore 500kDa Cassette as follows:
  • the DOC extracted OMVs were concentrated 10-fold to 50 ml (Pump flow 125ml /min, TMP 5 +/- 0.5 psi).
  • the concentrated preparation was diafiltered at constant volume (50 ml) with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi).
  • 1550 U of benzonase Merck 327 U/ul was added to the retentate containing the OMVs and stirred for 1h (Pump flow 50ml/min, permeate valve closed, no counterpressure).
  • the concentrated preparation was further diafiltered with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi) and 50 ml of retentate was recovered in reverse flow.
  • the system was washed with a further 50 ml of OMV buffer and the wash was pooled with the retentate to yield a final volume of about 100ml (retentate + wash).
  • further purification was performed by gel filtration using CaptoCore 700.
  • the pooled retentate was applied to a CaptoCore700 (GE Healthcare) column (109 ml column volume, radius 1,3 cm, pre-equilibrated in OMV buffer) at 11,5 ml/min (130 cm/h).
  • Example 6 Preparation of OMVs using 0.5% DOC at 30oC, Purification by TFF and CaptoCore 700 DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.5% DOC (Extraction Buffer 1) at 30°C. The preparation of OMVs was purified using a combination of TFF and CaptoCore as before (Fig. 5). Increasing the concentration of DOC to 0.5% and reducing the temperature to 30°C during extraction had a significant impact on yield which increased to 203 mg/L of culture.
  • Example 8 Preparation of OMVs using 0.5% DOC at 30oC, Purification by TFF (750kDa) and CaptoCore 700
  • the purification process of Example 5 was repeated four times but the 500kDa filter used in the TFF step was substituted with a 750kDa hollow fibre filter (Fig. 8), (GE Healthcare, UFP-750-C-H24LA, Surface 0,0042 m 2 , Dead volume 12 ml, Buffer Tris 20mM – Sucrose 3% (P/V) pH 8,6, Final recovered volume ca. 100ml):
  • the yield obtained with Hollow Fiber was generally higher (purity >95%) and a further reduction in impurities was seen (Fig. 9).
  • Example 9 Evaluation of Chaperonin 60kDa removal on CaptoCore 700 column The removal of the 60kDa chaperonin GroEL by CaptoCore 700 was determined using mass analysis. The relative amount of GroEL in each sample was measured using mass spectrometry (MS) as 9.4% of total protein in the ultrafiltration retentate (i.e. before filtration) and and 1.06% of total protein in the ultrafiltration permeate (i.e. after filtration). Using the Lowry assay, total protein in the ultrafiltration retentate was 230.6 mg whilst total protein in the ultrafiltration permeate was 36.5 mg.
  • MS mass spectrometry
  • Example 10 manufacturing process of Gonococcal OMVs Upstream process comprised the steps of inoculum, fermentation, recovery & filtration steps. During the preculture step a flask was inoculated with Neisseria gonorrhoeae liquid seed vial.
  • the number of expansion steps depended from the final scale of fermentation.
  • the passages of seed expansions and fermenter inoculum were performed when the culture was in full exponential growth.
  • the product i.e. GMMA
  • the fermentation was harvested and primary recovery was performed by centrifugation: pellet containing biomass was discarded and the supernatant, containing the product, was filtered at 0,2 ⁇ m (Filtration 1) to reduce bioburden (complete pathogen cell removal).
  • This intermediate was processed by adding benzonase in order to hydrolyze genomic DNA.
  • the material was filtered (filtration 2a) to remove pathogen cells from the supernatant and a holding time at 2-8°C was applied in order to execute the DNA hydrolysis before the purification process.
  • the supernatant was clarified by 0,2 ⁇ m filtration (Filtration 2) removing possible precipitate and the resulting intermediate was named SNF-BF.
  • the TFF was performed achieving a product purity improvement by reduction of soluble proteins (intended as gonococcus proteins not belonging to GMMA), nucleic acids and residual of fermentation medium.
  • the diaretentate was collected and filtered to remove precipitate (Filtration 3).
  • the resulting intermediate was named DR, starting material for the next chromatographic steps.
  • Capto Core 700 was used as resin for chromatographic step operated in flow-through mode to remove soluble proteins and DNA which are bound to the resin.
  • the resulting intermediate was the Capto Core 700 eluate (CC700EL2).
  • CC700EL2 was diluted, if needed, to a total protein concentration targeted at 1,2 ⁇ 0,1 mg/ml in order to maximize the volume of DS.
  • Filtration 4 the intermediate obtained was named Concentrated Bulk (CB), this solution was filled into suitable containers and frozen at -70°C as low bioburden material.
  • CB Concentrated Bulk
  • Example 11 Technical details of Capto Core 700 chromatography
  • the purpose of this Capto Core chromatography step is to increase the purity in tems of DNA and other proteins removal (benzonase and soluble proteins).
  • the DR intermediate was applied to two CaptoCore700 columns connected in series (column volume (CV ⁇ 1 L) previously equilibrated with 20 CV of PBS pH 7.4 at 300-500 cm/h. Washing was performed with the same buffer at the same flow rate. Chromatography was performed as flow- throught mode, so the start of peak collection is in the load block and the end in rinse block. The peak collection was performed manually (without method instruction) following the UV signal at 280 nm.
  • Example 12 Implementation of Capto Core 700 in the manufacturing process of Gonococcal OMVs
  • the manufacturing process of the GMMA-FA1090-2KO drug substance was improved by the substitution of an untracentrifugation step between Filtration 3 and Filtration 4 with Capto Core 700 (multimodal chromatography).
  • the GMMA-FA1090-2KO drug substance comprises a Neisseria gonorrhoeae bacterium is from strain FA1090 and comprises genetic modification(s) that (I) decreases or abolishes expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide; and (II) decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide.
  • lpxl1 lauroyl acyltransferase
  • rmp reduction modifiable protein
  • SEQ ID NO:1 60 kDa chaperonin “GroEL”; Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251)
  • SEQ ID NO: 2 molecular chaperone GroEL [Neisseria gonorrhoeae FA 1090] GenBank: AAW90696.1
  • SEQ ID NO: 3 molecular chaperone GroEL [Neisseria meningitidis NZ98/254]
  • SEQ ID NO: 4 molecular chaperone GroEL [Neisseria meningitidis 99M]

Abstract

It is provided a purification processe for removing impurities, such as GroEL, from samples of outer membrane vesicles (OMVs). For example, the process is applied to OMVs derived from Bordetella or from Neisseria, for example Neisseria gonorrhoeae. Also provided are OMVs purified by the processes described herein and immunogenic compositions, such as vaccines, comprising the same.

Description

PURIFICATION PROCESS SEQUENCE LISTING The instant application contains an electronically submitted Sequence Listing (VB66853WO01.xml; Size: (6.434 bytes; and Date of Creation: 2023-07-18) which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION That which is provided is in the field of purification processes, for example processes for purifying outer membrane vesicles (OMVs). Processes are also provided for for removing one or more impurities from a sample of OMVs. What is also provided is in the field of OMVs purified by such processes and immunogenic compositions, such as vaccines, comprising said OMVs. BACKGROUND TO THE INVENTION Gram-negative bacteria can spontaneously release OMVs from their outer membranes during growth, which are referred to as native OMVs (nOMVs). The formation of nOMVs can be facilitated by modification of certain bacterial components (WO2006/046143; Berlanda Scorza et al. (2008) Mol Cell Proteomics 7:473-85) whereby bacteria are genetically engineered to exhibit a hyperblebbing phenotype to produce nOMVs, which are thus also referred to as generalized modules for membrane antigens (GMMA). OMVs can also be produced by disruption of whole bacteria. OMV production methods include the use of detergent treatments to produce OMVs referred to as detergent extracted OMVs (dOMVs) (EP0011243; Fredriksen et al. (1991) NIPH Ann.14(2):67-80), detergent-free methods (WO2004/019977), sonication (Hozbor et al. (1999) Curr Microbiol 38:273-8), etc. OMVs are rich in immunogenic cell surface-associated, periplasmic, and secreted antigens and have been used in vaccines, for example, against Neisseria meningitidis serogroup B (Tan et al. (2010) N Engl J Med. 362(16):1511-20). They are suited for this use because OMVs contain components that act as adjuvants, eliciting strong immune responses against the antigens. Thus, OMVs may more closely mimic the native bacterium than purified protein antigens or other bacterial components. However, as OMVs are more complex structures derived from bacteria, they are typically more difficult to prepare than recombinant protein antigens. Therefore, there is the need to provide improved processes for preparing OMVs in particular for use in immunogenic compositions and vaccines. SUMMARY OF THE INVENTION In one aspect, it is provided a process for substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising outer membrane vesicles (OMVs), the process comprising: (a) concentrating and washing the sample by tangential flow filtration (TFF) through a TFF membrane to obtain a retentate concentrate comprising the OMVs; (b) filtering the retentate concentrate by flow-through chromatography to obtain a flow-through of purified OMVs using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa, thereby obtaining purified OMVs. In a second aspect, there is provided a process for preparing OMVs from Bordetella bacteria, the process comprising or consisting essentially of: (a) homogenising a fermentation harvest of the Bordetella bacteria, thereby obtaining a homogenised fermentation harvest comprising the Bordetella bacteria; (b) treating the homogenised fermentation harvest with DOC (i.e. to disrupt the outer membrane of the Bordetella bacteria) and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; (c) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce a sample containing DOC extracted OMVs and one or more impurities; (d) removing the one or more impurities by (i) concentrating and washing the sample containing DOC extracted OMVs and one or more impurities by TFF through a membrane to obtain a retentate concentrate comprising OMVs and (ii) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs; and (e) sterile filtering the flow-through of purified OMVs using a 0.22µm filter. In a third aspect, there is provided a process for preparing OMVs from Neisseria bacteria, the process comprising the sequential steps of: (i) recovering the fermentation harvest by centrifugation; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce a sample containing OMVs and one or more impurities; (vi) removing or reducing the concentration of the one or more impurities by (vii) concentrating and washing the sample containing OMVs and one or more impurities by TFF through a membrane to obtain a retentate concentrate comprising OMVs and (viii) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs; and (ix) sterile filtering the flow-through of purified OMVs using a 0.22µm filter. In a fourth aspect, there is provided a process for reducing the concentration of the 60kDa chaperonin GroEL in a sample of OMVs, optionally from Bordetella or Neisseria, the process comprising: filtering the sample of OMVs by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs wherein the 60kDa chaperonin GroEL is present in an amount less than 1.5% of total protein. In a fifth aspect, there is provided a population of purified OMVs produced by a process according to any one of the first, second, third or fourth aspect. In a sixth aspect, there is provided a population of purified OMVs that is at least 85% pure or from about 85% to about 100% pure, optionally wherein purity is measured using size exclusion Ultra-performance liquid chromatography (UPLC), for example by size exclusion UPLC to determine the ratio between the main peak and the low molecular size peak. In a seventh aspect, there is provided a population of purified OMVs for use in medicine. DESCRIPTION OF DRAWINGS/FIGURES FIG.1: Generalised schematic of the DOC extraction procedure used in the Examples. FIG.2: General schematic of the two-stage OMV purification process using TFF (500kDa) and Ultracentrifugation (UC). FIG.3: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS PAGE) Gel of samples taken at each stage of OMV purification: (1) Molecular weight marker, (2) TFF Retentate, (3) UC Supernatant, and (4) UC Pellet. Arrow indicates presence of the 60kDa chaperonin (GroEL) throughout. FIG.4: General schematic of the two-stage OMV purification process using TFF (500kDa) and Sephacryl S-500 Gel filtration. FIG.5: General schematic of the two-stage OMV purification process using TFF (500kDa) and CaptoCore 700 gel filtration. FIG.6: SDS PAGE Gel of samples taken before (2: Load) and after (4-7: Pool) gel filtration using a CaptoCore 700 column (1: Molecular weight marker). Arrow indicates 60kDa chaperonin (GroEL) which is significantly removed from the OMV sample using CaptoCore 700. FIG. 7(a): SDS Page Gel of OMV samples taken at each stage of OMV purification: (MW) Molecular weight marker, (TFF) TFF Retentate, (CaptoCore) Eluate from CaptoCore column. FIG. 7(b): Analytical gel filtration Size Exclusion Chromatography (SEC) analysis of OMV samples follow TFF and CaptoCore gel filtration. The process resulted in a preparation that almost completely removed GroEL and free-proteins not associated with the OMVs. FIG.8: General schematic of the two-stage OMV purification process using TFF (750kDa hollow fibre filter) and CaptoCore 700 gel filtration. FIG. 9: Size Exclusion Ultra Performance Liquid Chromatographic (SE-UPLC) profiles indicated excellent reproducibility of processes using a 750kDa Hollow Fibre TFF step compared to processes using a standard cassette for TFF. Yield obtained with Hollow Fibre was generally higher (purity >95%) and a further reduction in impurities was seen. Note that the different retention times seen were due to use of different UPLC systems. FIG. 10: Despite the pore size, the 750 kDa hollow fibre membrane again could not remove the 60kDa chaperonin from OMV preparations - a CaptoCore700 step was still employed. FIG. 11: SDS PAGE of OMVs purified using a 750kDa hollow fibre membrane followed by CaptoCore 700 gel filtration showed similar but not identical profiles to OMVs purified with the 500kDa cassette followed by CaptoCore 700 gel filtration (1=MW, 2=750kDa Hollow Fibre, 3=500kDa Cassette, 4=MW). FIG.12: Around 98,3% of the 60kDa chaperonin GroEL is removed from the sample by the CaptoCore 700 filtration step (clearance is around 56 times) (1=UF Retentate, 2=Drug Substance following CaptoCore 700 filtration, 3=MW marker, arrow indicates 60kDa Chaperonin GroEl). FIG. 13: Flow chart of gonococcal OMV process production, purpose of the steps and nomenclature of relative intermediates. FIG. 14: Purity by Size exclusion-high-performance liquid chromatography (SEC-HPLC) method of downstream process intermediate generated with two different processes. DR is starting Material of second purification step, CB is a Concentrated Bulk of Drug substance (DS). R10 is a batch and its CB is produced using Ultracentrifugation, R11 is a batch with the implementation of Capto Core 700 in the DS production. FIG.15: Purity by SDS-PAGE of CB. The table shows the purity increase using Capto Core 700 (92%) instead of ultracentrifugation (83%). The SDS-Page image highlights the specific effect of Capto Core 700 on GroEL removal. DETAILED DESCRIPTION OF THE INVENTION Isolated bacterial OMVs have been proposed as components for use in vaccines, for example as immunogenic components. However, the reproducible production of sufficiently pure OMVs in an amount suitable for large-scale vaccine manufacture has proven challenging. The Inventors have developed processes, for example large-scale or industrial processes, suitable for purifying OMVs. Thus, it is provided a process for substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising OMVs, the process comprising: (a) concentrating and washing the sample by TFF through a TFF membrane to obtain a retentate concentrate comprising the OMVs; (b) filtering the retentate concentrate by flow-through chromatography to obtain a flow-through of purified OMVs using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa, thereby obtaining purified OMVs. In some embodiments, the processes are suitable for purifying OMVs, for example by substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising OMVs. In some embodiments, the one or more impurities comprise protein impurities. In some embodiments, the one or more impurities comprise or is the 60kDa chaperonin GroEL (GroEL). In one embodiment, the 60kDa chaperonin GroEL comprises a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of SEQ ID NO: 1-4. In one embodiment, the 60kDa chaperonin GroEL comprises or has the sequence of anyone of SEQ ID NO: 1-4). In some embodiments, the process provided herein is a process for substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising OMVs. The term “substantially removing or reducing the concentration or amount of one or more impurities” is intended to refer to embodiments wherein the amount or contentration of the one or more impurities in the purified OMVs is reduced to less than 10%, for example less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1.5% of the amount or concentration of the one or more impurities in the sample comprising OMVs, when measured as a percentage of total protein in the sample. In one embodiment, the processes provided herein remove at least 80% of the one or more impurities from a sample comprising OMVs, as measured by percentage of total protein content, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% of the one or more impurities is removed. In some embodiments, the processes provided herein reduce the amount of one or more impurities in a sample comprising OMVs by at least about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, such as about 30 to about 60 times, about 40 to about 60 times, about 50 to about 60 times, as measured by total protein content in the sample comprising OMVs. These values may be determined by comparing the concentration or amount of the one or more impurities in the sample comprising OMVs after step (a), i.e. in the retentate concentrate, with that remaining in the sample comprising OMVs after step (b), i.e. in the flow-through of purified OMVs. The terms “purify” and “purifying” are intended to refer to procedures by which the concentration or amount of at least one undesired compound or compounds, an “impurity” or “impurities” respectively, is/are reduced, for example removed, relative to the desired compound. In one embodiment, the impurities comprise GroEL, for example the impurity is GroEL, for example SEQ ID NO: 1-4, and the desired compound is an OMV or population of OMVs or preparations of OMVs. In one embodiment, the concentration or amount of GroEL and/or other impurities may be determined using techniques known to the skilled person in the art, for example by Liquid chromatography–mass spectrometry (LC-MS) or by SDS-PAGE. In some embodiments, the process provided herein is a process for reducing the amount or concentration of GroEL in a sample comprising OMVs. In some embodiments, the process provided herein is a process for substantially removing GroEL from a sample comprising OMVs, for example, in comparison to the amount or concentration of GroEL in a sample comprising OMVs prior to use of the process. In one embodiment, the processes provided herein are suitable for substantially reducing the amount or concentration of GroEL in a sample of OMVs, for example, in comparison to the amount or concentration of GroEL in a sample comprising OMVs prior to use of the process. In some embodiments, the amount or contentration of GroEL in the purified OMVs is reduced to less than 5%, less than 4%, less than 3%, less than 2% or less than 1.5% of the amount or concentration of GroEL in the sample comprising OMVs, when measured as a percentage of total protein in the sample. In one embodiment, the processes provided herein remove at least 80% of the GroEL from a sample comprising OMVs, as measured by percentage of total protein content, for example, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% of the GroEL is removed. In some embodiments, the processes provided herein reduce the amount of GroEL in a sample comprising OMVs by at least about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, such as about 30 to about 60 times, about 40 to about 60 times, about 50 to about 60 times, as measured by total protein content in the sample comprising OMVs. These values may be determined by comparing the concentration or amount of GroEL in the sample comprising OMVs after step (a), i.e. in the retentate concentrate, with that remaining in the sample comprising OMVs after step (b), i.e. in the flow-through of purified OMVs. The processes provided herein are reproducible between batches consistently producing preparations or populations of OMVs obtained by disruption of the outer membrane of Bordetella bacteria having high purity (for example, of at least 95%) and/or with a yield consistently greater than 40mg/L (as measured by reference to either protein and/or lipid content) and/or having a narrow particle size distribution (polydispersity index or PDI) of <0.3. The processes of provided herein consistently produce preparations or populations of OMVs released in the medium, for example by Neisseria bacteria, having high purity (for example, of at least 90%, such as at least 91%, 92%, 93%, 94%, or 95%) and/or with a yield greater than 40mg/L (as measured by reference to protein content) and/or having a narrow particle size distribution (polydispersity index or PDI) of <0.3. OMVs produced by the processes provided herein are sufficiently pure and suitable for use as an active principle in an immunogenic composition. OMVs produced by processes known in the art generally comprise a protein referred to as the 60kDa chaperonin or GroEL (bp3495, SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4). However, GroEL is not integrated within the OMVs, rather it appears to be loosely associated with the OMV surface. The Bordetella GroEL protein is immunodominant and capable of inducing an antibody response but, in contrast to other protein antigens, GroEL does not appear to be a potent protective antigen, for example, in the mouse aerosol model for pertussis. Thus, whilst purification processes known in the art appear to produce suitably pure OMVs having an acceptably low level of background contaminants/impurities, GroEL may nonetheless be present in an amount sufficient to impact the antibody response to other OMV associated antigens. Surprisingly, and in addition to the above described advantages, processes provided herein significantly reduce or substantially remove GroEL from a sample of OMVs. Thus, the process provided herein may be a process for reducing the amount and/or concentration of GroEL from a sample of OMVs such as a process for substantially removing GroEL from a sample of OMVs. Advantageously, the above-noted processes replace downstream processing steps for OMV purification relying on ultracentrifugation, including density gradient centrifugation, sucrose cushion centrifugation, or others that may be difficult to implement on a larger scale. Therefore, the processes provided herein are suitable for industrial, large-scale production of OMVs. Therefore, in some embodiments the processes described herein are performed without, or do not comprise, an ultracentrifugation, for example, an ultracentrifugation that has a centrifugal force of greater than or about 30,000 x g is applied, optionally wherein the processes are performed without, or do not comprise, an ultracentrifugation after step (a). With regard to industrial, large-scale production of OMVs the Inventors mean that the sample comprising OMVs is prepared from a fermentation culture having a volume of from about 20 litres to about 10000 litres, from about 25 litres to about 5000 litres, from about 25 litres to about 2000 litres, from about 50 litres to about 1000 litres, greater than or equal to 20 litres, greater than or equal to 25 litres, greater than or equal to 50 litres or greater than or equal to 100 litres, such as 20 litres, 25 litres, 50 litres, 100 litres, 500 litres or 1000 litres. Outer Membrane Vesicles (OMVs) OMVs are known in the art and may be produced by natural/spontaneous ‘blebbing’ from the outer membrane of the bacterium. They may also be artificially prepared by mechanical and/or chemical disruption of the bacterial cell to form vesicles. In some embodiments, the OMVs are obtained from Gram negative bacteria. In some embodiments, the OMVs are obtained from any Gram negative bacteria genera, such as from species in any of genera Escherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia, Brucella, Chlamydia Haemophilus, Legionella, Porphyromonas, Pseudomonas, Yersinia, Helicobacter, Salmonella, Vibrio, etc. For example, the bacterium may be Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Moraxella catarrhalis, Escherichia coli, Haemophilus influenzae (including non-typeable stains), Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, Porphyromonas gingivalis, Pseudomonas aeruginosa, Yersinia enterocolitica, Helicobacter pylori, Salmonella enterica (including serovars typhi and typhimurium, as well as serovars paratyphi and enteritidis), Shigella (such as S. dysenteriae, S. flexneri, S. boydii or S. sonnei), Vibrio cholerae, etc. In one embodiment, the OMVs are obtained from Bordetella bacteria, optionally from Bordetella pertussis. In one embodiment, the OMVs are obtained from Neisseria bacteria, optionally from Neisseria gonorroheae. In one embodiment, the OMVs are obtained from Porphyromonas bacteria, optionally from Porphyromonas gingivalis. In one embodiment, the OMVs are obtained from Escherichia bacteria, optionally from Escherichia coli. In one embodiment, the OMVs are obtained from Escherichia coli bacteria of strain K or strain B strains, optionally wherein the Escherichia coli bacteria are selected from any one of strains BL21(DE3), BLR(DE3), and E. coli HMS174(DE3). In one embodiment, the OMVs are obtained from Salmonella bacteria, optionally from Salmonella typhi or Salmonella typhimurium. In some embodiments, the bacterium is a wild-type bacterium. In some embodiments, the bacterium is a recombinant bacterium. For example the bacterium is genetically modified to inactivate genes which lead to a toxic phenotype, such as modifications of native lipopolysaccharide (LPS), for example to disrupt the native lipid A structure, the oligosaccharide core, or the outer O antigen. Absence of O antigen in the LPS is useful, as is absence of hexa-acylated lipid A. In some embodiments, the bacterium is genetically modified by mutation to reduce the pyrogenic potential of the lipopolysaccharide (LPS) of the bacteria. Particular mutations include, by way of non-limiting example, mutations in IpxLl, synX, IgtA, htrA, msbBl, msbB2, virG, lpxA and homologues thereof. Suitable mutations for down-regulating or abolishing expression include point mutations, gene deletions, gene insertions, and any modification of genomic sequences that results in a change in gene expression, such as a reduction, inactivation or silencing. In one embodiment, the mutation is a deletion. Inactivation of toxins, for example to prevent expression of Shiga toxin or pertussis toxin, is also advantageous. In some embodiments, the bacterium is an hyper-blebbing bacterium. The term hyper- blebbing, as used herein, refers to a mutant strain of bacteria that spontaneously releases OMVs in greater quantities than a wild-type or parent strain from which it was derived (e.g., per unit of time). In general, hyperblebbing mutants release greater quantities of OMVs than the wild-type or parent strain from which it was derived, for example, greater than 10%, greater than 20%, greater than 30% or greater than 40%. The hyper-blebbing bacterium may be a naturally occurring mutant strain or may be genetically modified to exhibit a hyper-blebbing phenotype. The term "wild-type" with reference to bacteria refers to a bacterium that has not been modified either chemically or genetically in any way whatsoever (other than growth in culture medium). Neisserial strains, such as Neisseria meningitidis or Neisseria gonorrhoeae, may be genetically modified to exhibit a hyper-blebbing phenotype by down-regulating or abolishing expression of, by way of non-limiting example, GNA33. Similar mutations are known in other bacteria, for example, Haemophilus influenza, Moraxella catarrhalis and Escherichia coli strains may be genetically modified to exhibit a hyper-blebbing phenotype by down-regulating or abolishing expression of one or more genes selected from the group consisting of tolQ, tolR, tolX, tolA and tolB. Strains of Shigella flexneri, Shigella dysenteriae, Shigella boydii and Shigella sonnei can be genetically modified to exhibit a hyper-blebbing phenotype by down- regulating or abolishing expression of one or more tolR or OmpA. Suitable mutations for down- regulating or abolishing expression include point mutations, gene deletions, gene insertions, and any modification of genomic sequences that results in a change in gene expression, for example a reduction, for example inactivation or silencing. Further suitable mutations are known in the art. The hyper-blebbing bacterium may be further genetically engineered by one or more processes selected from the following group: (a) a process of down-regulating expression of immunodominant variable or non-protective antigens, (b) a process of up-regulating expression of protective outer membrane protein (OMP) antigens, (c) a process of down-regulating a gene involved in rendering the lipid A portion of LPS toxic, (d) a process of up-regulating a gene involved in rendering the lipid A portion of LPS less toxic, and (e) a process of genetically modifying the bacterium to express a heterologous antigen. In some embodiments, the sample comprising OMVs are be prepared artificially from bacteria using detergent treatment (e.g. with deoxycholate (DOC) or sarkosyl) including treating bacteria with a bile acid salt detergent (e.g. salts of lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursocholic acid, etc., with sodium deoxycholate) at a pH sufficiently high not to precipitate the detergent. Other techniques may be performed substantially in the absence of detergent using techniques such as sonication, homogenisation, microfluidisation, cavitation, osmotic shock, grinding, French press, blending, etc. The starting material for the process provided herein is a sample comprising OMVs. In one embodiment, the sample is substantially free from whole bacteria, whether living or dead. In some embodiments, the sample comprising OMVs is obtained from Bordetella bacteria. In some embodiments, the Bordetella bacterium comprises or is one or more of Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. In some embodiments, the bacterium comprises or is Bordetella pertussis. In some embodiments, the Bordetella pertussis comprises or is a recombinant bacterium. In some embodiments, the recombinant Bordetella pertussis comprises or is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G. In some embodiments the recombinant Bordetella pertussis bacterium does not express the dermonecrotic toxin (DNT) gene. In some embodiments the recombinant Bordetella pertussis bacterium expresses the genetically detoxified pertussis toxoid PT-9K/129G and does not express the dermonecrotic toxin (DNT) gene. Suitable recombinant Bordetella pertussis bacteria are disclosed in WO2020/094580, which is incorporated by reference herein. In some embodiments, the recombinant Bordetella pertussis bacterium comprises at least one genomic LpxA gene encoding a mutated LpxA protein, and/or at least a genomic insertion of a heterologous LpxD gene, as disclosed for instance in WO2021/064050, which is incorporated herewith by reference. In some embodiments, the recombinant Bordetella pertussis comprises or is a recombinant bacterium that produces lipid A comprising (i) C3’ acyl chains having a length of about 10 carbon atoms (C10); (ii) C2’ acyl chains having a length of about 10 carbon atoms (C10); and/or (iii) C2 acyl chains having a length of about 10 carbon atoms (C10). Suitable recombinant Bordetella pertussis bacteria are disclosed in WO2021/064050, which is incorporated by reference herein. In some embodiments, the recombinant Bordetella pertussis comprises or is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G and comprises or is a recombinant bacterium that produces lipid A comprising (i) C3’ acyl chains having a length of about 10 carbon atoms (C10); (ii) C2’ acyl chains having a length of about 10 carbon atoms (C10); and/or (iii) C2 acyl chains having a length of about 10 carbon atoms (C10). In some embodiments, the OMVs are obtained from Neisseria bacteria. In some embodiments, the Neisseria bacterium comprises or is one or more selected from Neisseria meningitidis and Neisseria gonorrhoeae. In some embodiments, the bacterium is Neisseria meningitidis. In some emodiments, the bacterium is Neisseria meningitidis serogroup B (MenB). In some embodiments, the bacterium is a Neisseria meningitidis serogroup B (MenB) strain selected from the group consisting of: NZ98/254, NG H36, BZ 232, DK 353, B6116/77, BZ 163, 0085/00, NG P20, 0046/02, M1140123, M12 240069, N5/99, 99M, or M07240677. In some embodiments, the OMVs are obtained from Neisseria meningitidis strain NZ98/254 or 99M. In one embodiments, the Neisseria meningitidis is of strain NZ98/254. In one embodiment, the Neisseria meningitidis is of strain 99M. In some embodiments, the bacterium is Neisseria gonorrhoeae, which is also referred to as gonococcal bacteria or gonococcus. In one embodiment, the Neisseria gonorrhoeae is a Neisseria gonorrhoeae bacterium that comprises genetic modification(s). In some embodiments, the Neisseria gonorrhoeae bacterium to which the genetic modification(s) is introduced (i.e the unmodified Neisseria gonorrhoeae) is a gonococcal bacterium of any strain. In some embodiments the unmodified Neisseria gonorrhoeae is of strain FA1090, F62, WHO-G, WHO-M, WHO-N, GC14, BG7, BG8, BG27, SK92, or MS11. In one embodiment, the unmodified Neisseria gonorrhoeae is of strain FA1090. Gonococcal bacteria of strain FA1090 are known in the and are commercially available from the American Type Culture Collection (ATCC, see for example Deposit Number #700825, 1081 University Blvd, Manassas, Virginia 20110, US). In certain embodiments, said genetic modification(s) reduce the endotoxin activity of the OMVs compared to a wild-type or unmodified Neisseria gonorrhoeae. In some embodiments, said genetic modification(s) that reduces the endotoxin activity results a genetically modified Neisseria gonorrhoeae that produces OMVs having increased levels of pentaacylated lipid A, decreased levels of hexaacylated lipid A, or a higher proportion of pentaacylated lipid A to hexaacylated lipid A compared to the lipid A that is in the OMVs in the respective a wild-type or unmodified Neisseria gonorrhoeae (i.e. the comparative wild-type or unmodified Neisseria gonorrhoeae prior to the genetic modifications that produces OMV having increased levels of pentaacylated lipid A, decreased levels of hexaacylated lipid A, or a higher proportion of pentaacylated lipid A to hexaacylated lipid A). In some embodiments the Neisseria gonorrhoeae is a genetically modified gonococcal bacterium, comprising genetic modification(s) that (I) decreases or abolishes expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide. In a further embodiment, the Neisseria gonorrhoeae bacterium comprises a further genetic modification that decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide. In one embodiment, the Neisseria gonorrhoeae bacterium is from strain FA1090. In one embodiment the Neisseria gonorrhoeae bacterium comprises genetic modification(s) that (I) decreases or abolishes expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide; and (II) decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide. In one embodiment the Neisseria gonorrhoeae bacterium is from strain FA1090 and comprises genetic modification(s) that (I) decreases or abolishes expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide; and (II) decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide. The lpxl1 gene (also referred to as msbB) encodes the polypeptide Lipid A biosynthesis lauroyl acyltransferase (Lpxl1). Lpxl1 plays a role in lipid A biosynthesis. Neisserial organisms genetically modified to provide for decreased or no detectable functional lpxl1 encoded protein produce OMVs with reduced endotoxicity. This is because the amount of lipid A acylation and the nature of the acylation are major factors that affect LOS toxicity [Makda Fisseha et al. Infection and Immunity Jun 2005, 73 (7) 4070-4080]. Lpxl1 (polypeptide) may also be referred to as the Lpxl1 enzyme. The rmp gene encodes the polypeptide reduction modifiable protein (Rmp). In the context of the present disclosure, “decreased expression” means that the gonococcal bacterium provided herein expresses less lpxl1 and rmp mRNA and/or Lpxl1 and Rmp protein compared to an unmodified (wild type) gonococcal strain or a gonococcal strain comprising the wild type lpxl1 / rmp genes. Expression may be considered decreased when any reduction in mRNA and/or protein expression is observed compared to an unmodified (wild type) gonococcal strain or a gonococcal strain comprising the wild type lpxl1 / rmp genes. Expression may be considered decreased when an over 5%, over 10%, over 25%, over 50%, over 60%, over 70%, over 80% over 90% or over 95% reduction in mRNA and/or protein expression is observed compared to the mRNA and/or protein expression, respectively, in an unmodified (wild-type) gonococcal strain or a gonococcal strain comprising the wild type lpxl1 / rmp genes. In the context of the present disclosure, “abolished expression” means that no Lpxl1 mRNA and/or protein and no Rmp mRNA and/or protein can be detected in the gonococcal bacterium provided herein using the technique used by the skilled person to measure expression. The function of Lpxl1 can be determined for example by examining the extent to which the Lipid A component of the outer membrane vesicle lipooligosaccharide is penta-acylated as opposed to being hexa-acylated (for example by analysing the acylation state of lipid A using mass spectrometry e.g. as described in van der Ley et al. Infection and immunity vol.69,10 (2001): 5981-90). If the genetically modified gonococcal bacterium comprises a genetic modification that decreases or abolishes the function of the Lpxl1 protein, the Lipid A will be penta-acylated (for example it will be at least 80%; at least 90%; at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%; or is 100% penta-acylated), despite evidence to suggest presence of lpxl1 mRNA and/or protein. In an embodiment, decreased or abolished expression and/or function of the Lpxl1 polypeptide results in penta-acylation of lipid A, optionally wherein the acylation of lipid A is determined by Matrix- Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) spectrometry. In an embodiment, the genetically modified gonococcal bacterium provided herein comprises lipooligosaccharide (LOS) with penta-acylated lipid A. The acylation of Lipid A can be determined for example by extracting lipid A followed by analysis by MADI-TOF spectrometry. Specifically, decreased or abolished expression and/or function of the Lpxl1 polypeptide results in lipooligosaccharide (LOS) comprising a lipid A lacking the lauric acid that LpxL1 would have added, had it been functionally expressed. Decreased or abolished expression and/or function of the Lpxl1 polypeptide results in a LOS comprising a lipid A lacking the secondary lauroyl chain from the nonreducing end of the GlcN disaccharide of lipid A. Decreased or abolished expression and/or function of the Lpxl1 polypeptide results in a LOS comprising a lipid A lacking the C12 acyloxyacyl chain (from the non-reducing end). Decreased or abolished expression and/or function of the Lpxl1 polypeptide results in a LOS comprising a lipid A lacking the lauric acid in the secondary 2’-O-position of the distal nonreducing terminal glucosamine of the β-(1--> 6) D-glucosamine dimer (consequently a lone 3-hydroxymyristyl moiety exists in amide linkage on the distal glucosamine of the lipid A). In an embodiment, decreased or abolished expression and/or function of the lpxl1 polypeptide results in above 50% penta-acylation of lipid A, for example above 60%, above 70%, above 80%, above 90%, above 95% or above 99%. In an embodiment, decreased or abolished expression and/or function of the Lpxl1 polypeptide results in 100% penta-acylation of lipid A. In an embodiment, the genetically modified gonococcal bacterium provided herein has a reduced capacity for activating Toll- like receptor 4 (TLR4) compared to a gonococcus comprising the wild-type lpxl1 gene. In some embodiments, the sample comprising OMVs comprises OMVs and one or more impurities, for example GroEL. In one embodiment, the sample comprising OMVs is obtained by disruption of the outer membrane of bacteria, for example Bordetella bacteria, and comprises DOC-extracted OMVs and one or more impurities, for example GroEL. In one embodiment, the sample comprising OMVs obtained by disruption of the outer membrane of Bordetella bacteria comprises DOC-extracted OMVs and GroEL. In one embodiment, the sample of OMVs obtained by disruption of the outer membrane of Bordetella bacteria comprises DOC-extracted OMVs and GroEL associated with the surface of the OMVs. In some embodiments, the sample comprising OMVs is obtained by detergent disruption of the outer membrane of bacteria, for example Bordetella or Neisseria bacteria. In one embodiment, the detergent is DOC (for example, CAS No. 302-95-4) optionally comprising benzonase (for example, CAS No. 9025-65-4). In certain embodiments, the detergent is DOC having a concentration of from about 0.1% to about 0.5% DOC, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4% or about 0.5%, optionally comprising benzonase. In some embodiments, the sample comprising OMVs is obtained by detergent disruption of the outer membrane of bacteria, for example Bordetella bacteria, at a temperature of from about 30°C to about 45°C, for example, from about 30°C to about 40°C, for example, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C or about 40°C. When used, benzonase may be used at a concentration of from about 50 U/ml to about 2000 U/ml, for example about 50 U/ml, about 100 U/ml, about 150 U/ml or about 1500 U/ml. In some embodiments, the sample comprising OMVs is obtained by detergent disruption of the outer membrane of bacteria, for example Bordetella bacteria, using DOC at a concentration of about 0.1% to about 0.5%, such as 0.1% or 0.5%, at a temperature of from about 30°C to about 45°C, for example, from about 30°C to about 42°C, from about 30°C to about 40°C for example, about 30°C, about 35°C or about 40°C. In one embodiment, the sample comprising OMVs is obtained by detergent disruption of the outer membrane of Bordetella bacteria using DOC at a concentration of about 0.5%, optionally comprising benzonase, at a temperature of about 40°C. In some embodiments, the sample comprising OMVs is obtained by: (i) homogenising a fermentation harvest of bacteria, for example Bordetella bacteria, thereby obtaining a homogenised fermentation harvest; (ii) treating the homogenised fermentation harvest with DOC to disrupt the outer membrane of the bacteria, for example the Bordetella bacteria, and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; (iii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce the sample. In some embodiments, the sample of OMVs is obtained by: (i) homogenising a fermentation harvest of Bordetella bacteria, thereby obtaining a homogenised fermentation harvest; (ii) treating the homogenised fermentation harvest with DOC at a concentration of from about 0.1% to about 0.5% at a temperature of from about 35°C to about 40°C to disrupt the outer membrane of the Bordetella bacteria and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; (iii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce the sample. The homogenisation and/or treating (steps (i) and/or (ii) respectively) may be performed in the presence of a suitable buffer. By way of non-limiting example, suitable buffers include Tris buffers such as a Tris-HCl buffer. In one embodiment, the buffer is a Tris-HCl buffer, for example, a 20mM Tris-HCl buffer. Suitable buffers will have a pH of from about 8 to 9, such as about 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or about 9. In one embodiment, the homogenisation and/or treating (steps (i) and/or (ii) respectively) are performed in the presence of at least one protease inhibitor. Suitable protease inhibitors are known in the art, and include, by way of non-limiting example, the cOmplete EDTA-free protease inhibitor cocktail (Roche). In one embodiment, the sample comprising OMVs comprises or consists of OMVs released in the medium, for example by Neisseria bateria. In one embodiment, the sample comprising OMVs comprises OMVs released in the medium, for example by Neisseria bateria, and one or more impurities, for example GroEL. In one embodiment, the sample comprising OMVs comprises OMVs released in the medium, for example by Neisseria bateria, and GroEL. In one embodiment, the sample comprising OMVs comprises OMVs that were released in the medium, for example by Neisseria bateria, and GroEL associated with the surface of the OMVs. In some embodiment, the sample of OMVs is obtained by: (i) recovering the fermentation harvest by centrifugation; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce the sample of OMVs. In some embodiments, in step (iv) MgCl2 is added to the intermediate to obtain a MgCl2 concentration of from about 0.1mM to about 10mM, for example from about 0.5mM to about 5mM, for example of 1mM. The intermediate is treated with benzonase treatment in step (iv) using a benzonase concentration of from about 10kU/L to about 100kU/L, for example about 10kU/L, about 20kU/L, about 30kU/L, about 40kU/L, 50kU/L, about 60kU/L, about 70kU/L, about 80kU/L, about 90kU/L, or about 100kU/L. In one embodiment, the benzonase concentration is 50kU/L. In one embodiment, the intermediate is treated with benzonase at a temperature of from about 0°C to about 10°C, for example, from about 2°C to about 8°C. In one embodiment, the intermediate is treated with benzonase for a period of from around 1 day to around 3 months, for example for a period of from around 3 days to 2 months. In one embodiment, MgCl2 is added to the intermediate to obtain a MgCl2 concentration of about 1mM and the intermediate is treated with 50kU/L benzonase at a temperature of from about 2°C to about 8°C for a period of from around 3 days to 2 months. Step (a) Tangential Flow Filtration (TFF) A first step of the process provided herein comprises (a) concentrating and washing a sample comprising OMVs by TFF using a TFF membrane to obtain a retentate concentrate comprising the OMVs. In TFF a sample solution from a feed reservoir passes tangentially along the surface of a filter membrane and back to the feed reservoir, where it may be recirculated. Components larger than the pores of the filter membrane – such as OMVs - are retained, passing along the membrane surface, whilst components that are smaller than the pores of the filter membrane pass through the filter. Sample solution that passes along the membrane surface and back to the feed reservoir, is referred to as the retentate. Sample solution that passes through the membrane is generally referred to as the permeate or filtrate. Membranes used in TFF will have a molecular weight cutoff. Molecular weight cutoff (MWCO) or nominal molecular weight cutoff (NMWCO) is defined as the minimum molecular weight of a solute that is 90% retained by the membrane (e.g., as stated by the manufacturer). In some embodiments, the TFF is performed using a TFF membrane having a molecular weight cutoff of from about 200 kDa to about 800kDa, for example, a MWCO of from about 200kDa to about 400kDa or from about 500kDa to about 750kDa, for example, about 200kDa, about 250kDa, about 300kDa, about 350kDa, about 400kDa, about 500kDa, about 550kDa, about 600kDa, about 650kDa, about 700kDa or about 750kDa. In certain embodiments, the TFF membrane has a MWCOof about 500kDa. In other embodiments, the TFF membrane has a MWCOof about 750kDa. In other embodiments, for example when the bacterium is a Neisseria bacterium, the TFF membrane has a MWCOof about 300kDa. In some embodiments, the TFF membrane has a pore size of from about 0.1mm to about 0.75mm, for example from about 0.4mm to about 0.6mm, for example, about 0.4mm, about 0.5mm or about 0.6mm. For the avoidance of doubt, the pore size (sometimes referred to as lumen diameter) is calculated using standard methods known in the art, usually determined by the manufacturer. The pore size is a mean diameter of the pores in the membrane based on the assumption that all pores in the membrane are circular. In some embodiments, the TFF membrane is a hollow fibre membrane. In one embodiment, the TFF membrane is a hollow fibre membrane having a MWCO of about 750kDa or about 300kDa. In one embodiment, the TFF membrane is a hollow fibre membrane having a MWCO of about 750kDa or about 300kDa and a pore size of about 0.5mm. In one embodiment, the membrane is a hollow fibre membrane having a MWCO of about 750kDa or about 300kDa, a pore size of about 0.5mm and an area of about 290cm2. In one embodiment, the membrane is a hollow fibre membrane having a MWCO of about 750kDa, a pore size of about 0.5mm, an area of about 290cm2 and a nominal flow path length of 60cm. In some embodiments, the TFF membrane is a GE Healthcare UFP-750-C-3X2MA hollow fibre membrane or equivalent, for example, having the following characteristics: 750 kDa cutoff, pore size 0.5 mm and working surface area of 290 cm2. In some embodiments, the TFF membrane is a Millipore 500kDa Cassette or equivalent, or a Sartorius 300kDa cassette or equivalent. The TFF of step (a) comprises concentrating and washing the sample. In one embodiment, the TFF step (a) comprises at least one cycle that includes ultrafiltration and diafiltration. In one embodiment, the TFF of step (a) comprises at least one ultrafiltration cycle and at least one cycle of diafiltration. For the avoidance of doubt, in the TFF step, concentration (by ultrafiltration) and washing (by diafiltration) are filtration steps performed using the same TFF membrane but with varying operating parameters, discussed further below. “Ultrafiltration” is generally used to refer to TFF membrane filtration methods in which pressure, for example liquid pressure such as water pressure, forces the liquid against a semi- permeable membrane. Suspended high molecular weight solids, such as OMVs, are retained by the membrane (the “retentate”) while water and low molecular weight solutes pass through the membrane (the “filtrate”). Thus, the term retentate is used to refer to those components or portion of a solution retained by, and which do not cross, the TFF membrane (the skilled person will appreciate that, to a limited extent, it may also includes some portions of the sample that are small enough to cross the membrane but have not yet done so). In the processers provided herein, ultrafiltration is used primarily to concentrate the sample but for the avoidance of doubt, the skilled person will appreciate that by its nature, ultrafiltration also removes/eliminates some impurities. As used herein, reference to concentration of a sample, for example “concentrates the sample” generally refers to the ordinary meaning of that term, namely increasing the amount of a particular subject material relative to the volume of fluid in which the subject material is disposed. For example, during ultrafiltration liquid is removed from the sample (but not replaced) thereby reducing the aqueous content of the sample. OMVs are retained in the sample by the TFF membrane thereby increasing the concentration of OMVs per unit volume of fluid in the retentate concentrate of step (a) respective to the sample comprising OMVs. A sample obtained following a concentration step may be called a concentrate. “Diafiltration” refers to the use of the TFF membrane to remove, replace or lower the concentration of impurities in a solution on the basis of molecular size. As with ultrafiltration, the TFF membrane retains molecules, such as OMVs, that are larger than the pores of the membrane (the “retentate”) while smaller molecules such as salts, solvents and water pass freely through the membrane (the “filtrate”). However, generally during diafiltration, a solution, for example a buffer, is introduced into the recycle tank at substantially the same rate at which filtrate is removed, thereby exchanging buffer and/or washing the retentate whilst maintaining a constant volume of liquid in the sample. In one embodiment, the retentate is diluted with solvent and re-filtered to reduce the concentration of soluble permeate components. During diafiltration, OMVs remain in the retentate and components, such as impurities, are ‘washed’ out, passing through the membrane, and into the filtrate thereby removing the impurities and/or exchanging buffers. Concentration of OMVs in the sample is performed using at least one cycle of ultrafiltration. In some embodiments, the ultrafiltration comprises at least one cycle of ultrafiltration, for example comprises two or more, three or more, four or more, five or more cycles of ultrafiltration. In one embodiment, the at least one ultrafiltration cycle concentrates the OMVs in sample by at least about 2.5 times, by at least about 5 times, by at least about 10 times or by at least about 15 times, such as about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 time, about 13 times, about 14 times or about 15 times. In some embodiments, the at least one ultrafiltration cycle concentrates the OMVs in the sample by: from about 2.5 times to about 20 times, from about 5 times to about 20 times, from about 6 times to about 20 times, from about 7 times to about 20 times, from about 8 times to about 20 times, from about 9 times to about 20 times, from about 10 times to about 20 times, from about 11 times to about 20 times, from about 12 times to about 20 times, from about 13 times to about 20 times, from about 14 times to about 20 times, or from about 15 times to about 20 times. The OMVs in the sample may be concentrated to a protein concentration of 2-5 mg/ml ± 1% by ultrafiltration. In some embodiments, the OMVs may be concentrated by ultrafiltration to a protein concentration of about 2% to about 10% (weight / volume). Washing of the sample is performed using at least one cycle of diafiltration. In some embodiments, the diafiltration comprises at least one cycle of diafiltration, for example comprises two or more, three or more, four or more, five or more cycles of diafiltration. In one embodiment the diafiltration comprises two cycles of diafiltration. In one embodiment, the at least one cycle of diafiltration washes the sample with at least about 5 volumes of a wash solution relative to the sample volume, for example about 10 volumes of a wash solution, about 15 volumes of a wash solution or about 20 volumes of a wash solution relative to the sample volume. In some embodiments, the at least one diafiltration cycle washes the sample with at least about 5 volumes of a wash solution relative to the sample volume, with at least about 10 volumes, or with at least about 15 volumes of a wash solution relative to the sample volume, such as about 5 volumes, about 6 volumes, about 7 volumes, about 8 volumes, about 9 volumes, about 10 volumes, about 11 volumes, about 12 volumes, about 13 volumes, about 14 volumes, about 15 volumes, about 16 volumes, about 17 volumes, about 18 volumes, about 19 volumes, or about 20 volumes of a wash solution relative to the sample volume. In some embodiments, the at least one diafiltration cycle washes the sample with: from about 5 volumes to about 20 volumes, from about 5 volumes to about 20 volumes, from about 6 volumes to about 20 volumes, from about 7 volumes to about 20 volumes, from about 8 volumes to about 20 volumes, from about 9 volumes to about 20 volumes, from about 10 volumes to about 20 volumes, from about 11 volumes to about 20 volumes, from about 12 volumes to about 20 volumes, from about 13 volumes to about 20 volumes, from about 14 volumes to about 20 volumes, or from about 15 volumes to about 20 volumes of a wash solution relative to the sample volume. Suitable wash solutions are known in the art and include, by way of non-limiting example, Dulbecco's phosphate-buffered saline (DPBS), Tris buffers and the like. In one embodiment, the wash solutions optionally comprise other components such as EDTA or sucrose. In some embodiments, for example when the bacterium is Bordetella, the wash buffer is DPBS. In some embodiments, the wash solution is DPBS optionally comprising EDTA for example, about 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM or 8mM EDTA, for example about 5mM EDTA. In some embodiments, the wash solution is DPBS comprising EDTA, for example, about 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM or 8mM EDTA, for example about 5mM EDTA. In some embodiments, for example when the bacterium is Bordetella, the wash solution is a Tris buffer, for example a 20mM Tris buffer having a suitable pH. A suitable pH is from about pH 8.0 to about 9.0, such as pH 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0. In one embodiment, the wash solution comprises sucrose, for example from about 1% to about 10% sucrose, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, for example about 3% sucrose. In certain embodiments, the wash solution is a 20mM Tris buffer having a pH of about 8.6 optionally comprising about 3% sucrose. In certain embodiments, the wash solution is DPBS comprising about 5mM EDTA. In some embodiments, for example when the bacterium is a Neisseria bacterium, the wash solution is a sodium phosphate buffer, for example a Na2HPO4/NaH2PO4 buffer, for example a 10mM Na2HPO4/NaH2PO4 having a suitable pH. A suitable pH is from about pH 6.0 to about pH 7.0, such as pH 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6., 6.7, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the wash solution comprises sodium chloride, for example from about 50mM to about 250mM, such as 50mM, 100mM, 150mM, 200mM or 250mM. In certain embodiments, the wash solution is 10mM Na2HPO4/NaH2PO4 pH 6.8 comprising 150mM NaCl. In some embodiments, for example when the bacterium is a Neisseria bacterium, the wash solution is phosphate-buffer saline (PBS) pH 7.4, such as a buffer comprising 8.1mM Na2HPO4, 1.5 mM KHPO4, 2.7 mM KCl and 136.9 mM NaCl. In some embodiments, concentration and washing are performed sequentially, one after the other, for example, in some embodiments the sample may first be concentrated and then washed and in other embodiments, the sample may first be washed and then concentrated. In some embodiments, using TFF the operating parameters may be set such that concentration and washing are performed at substantially the same time. Since the same TFF membrane and equipment is used, the diafiltration parameters may be such that they lead to an increase in the concentration of retained components, such as OMVs. In continuous diafiltration, a solvent is continuously added to the retentate at the same rate as the filtrate is generated and in this case, the retentate volume and the concentration of retained components do not change during the process. On the other hand, in discontinuous or sequential dilution diafiltration, an ultrafiltration step is followed by the addition of solvent to the retentate side; if the volume of solvent added to the retentate side is less than the volume of filtrate generated, then the concentration of components in the retentate will increase (as the volume of liquid in the retentate decreases). Thus, the TFF step (a) may optionally comprise a step of dilution prior to concentration and washing wherein a volume of a liquid is added to the sample comprising OMVs, thereby increasing the sample volume. An increase in initial working volume may be advantageous since it can make the following processing steps, such as washing, easier for example, by reducing the likelihood of clogging of the membrane. Where a step of dilution is used prior to concentration and washing, the volume of liquid used for dilution is removed during washing and/or concentration. Step (b) Flow-Through Chromatography In the next step of the process, the retentate concentrate is filtered by flow-through chromatography to obtain a flow-through of purified OMVs. Flow-through chromatography is a separation technique in which sample interacts with a chromatographic substrate. A “chromatographic substrate” is any kind of solid phase which separates a target of interest from other molecules present in a sample as a result of, for example, differences in size and/or binding affinity. Chromatographic substrates will generally be contained in a suitable housing, for example a chromatography column. Potential contaminants or impurities in the sample bind to the chromatographic substrate and are retained whilst a target within the sample which is not bound or retained can be recovered. Without wishing to be bound by theory, in a “flow-through” mode of operation, such as in step (b) of the process, the retentate concentrate that comprises OMVs is applied to and flows through a chromatographic substrate, for example in a chromatography column, and as the retentate concentrate flows through the column, it interacts with a chromatographic substrate contained in the column. During the interaction, OMVs do not bind to the chromatographic substrate while impurities (including GroEL) are able to bind to and/or are trapped in the chromatographic substrate. In this process, a flow-through of purified OMVs are obtained, which comprises lower levels of impurities, lower concentrations of impurities, lower amounts of impurities, fewer impurities, or no impurities. The flow-through of purified OMVs flows out of the chromatography column and is recovered. The flow-through of purified OMVs comprises lower levels or amounts of impurities (such as GroEL) compared to the retentate concentrate prior to interacting with the chromatographic substrate. Suitable methods of flow-through chromatography include gel filtration, mixed mode resin column chromatography, ion exchange column chromatography, affinity matrix chromatography and hydrophobic interaction chromatography. In some embodiments, the chromatographic substrate is a size-exclusion resin, suitable for gel filtration, such as Sephacryl S-500 resin. In some embodiments, the chromatographic substrate is a mixed mode chromatography resin, suitable for mixed mode resin column chromatography. Mixed-mode chromatography resins are functionalised with ligands thereby being capable of different types of interaction such as, by way of non-limiting example, ion exchange, affinity binding and/or size exclusion. In one embodiment, the chromatographic substrate comprises beads, for example a plurality of beads, having a porous outer layer and an inner core, for example a ligand activated inner core. In one embodiment, the chromatographic substrate comprises beads having a porous outer layer having a molecular weight cut-off from about 600kDa to about 800kDa, for example from 600kDa to 800kDa, such of about 700kDa, for example of 700kDa (that prevents large molecules from entering the core) and an inner core, for example a ligand activated inner core, comprising octylamine ligands. The term “molecular weight cut-off” refers to the size cut-off of the outer layer. For example, methods for measuring the molecular weight cut-off, which are know in the art, comprise determining the dynamic binding capacity of the chromatographic substrate using proteins of different sizes, such as ovalbumin (Mr 45 000), apoferritin (Mr 475 000), thyroglobulin (Mr 660 000), and bovine IgM (approx. Mr 900 000), and evaluating up to which protein size the sample protein is able to enter the bead core and bind to the substrate. In one embodiment the beads have an nonfunctionalized outer layer (without ligand) and a functionalized core with an attached ligand, such as an octylamine ligand. The term “octylamine ligand” refers to a ligand having the fuctional group CH3CH2CH2CH2CH2CH2CH2CH2NH-, optionally wherein the pKa of the protonated octlymanime before attachment to the mediu, is about 10.65. In one embodiment, the matrix of the chromatographic substrate is high-flow agarose, for example having a particle size (i.e. an average particle size of the cumulative volume distribution) from about 75 µm to aout 95 µm, such as of about 85 µm. In one embodiment, the chromatographic substrate is the mixed-mode chromatography resin Capto Core 700 (GE Lifesciences) or equivalent, which comprises a matrix of highly cross-linked garaose beads with particle size of about 85 µm having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa. Other suitable chromatographic substrates are known in the art. In one embodiment, steps (a) and (b) are performed sequentially without intermediate processing steps, other than collection/storage, and the output of step (a) (the retentate concentrate comprising OMVs) is the direct input for step (b). In some embodiments, process steps (a) and (b) are discontinuous process steps wherein step (a) is completed with the retentate concentrate comprising OMVs being collected, optionally stored for a period of time, before step (b) is started and the retentate concentrate comprising OMVs is applied to the flow-through chromatographic substrate, such as to the chromatography column. In some embodiments, process steps (a) and (b) are continuous process steps. As used herein, reference to a “continuous process” refers to a process having two or more processing steps in a series, wherein the output from an upstream step (for example, step (a)) is transferred to a downstream step (for example, step (b)) continuously without waiting for the upstream processing step to run to completion before the downstream processing step is started. For example, the retentate concentrate comprising OMVs continuously flows from the TFF equipment directly to the flow-through chromatography column, i.e. the TFF equipment is in fluid communication with the flow-through chromatography column. In one embodiment, the process further comprises a step (a’) comprising or consisting of filtering the concentrate retentate to remove precipitate. In one embodiment, steps (a) and (a’) are performed sequentially without intermediate processing steps, other than collection/storage, and the output of step (a) is the direct input for step (a’). In some embodiments, process steps (a) and (a’) are discontinuous process steps wherein step (a) is completed before step (a’) is started. In some embodiments, steps (a) and (a’) are continuous process steps. Step (c) Sterile filtration OMVs prepared using the processes provided herein will generally be for use as components in immunogenic compositions such as pharmaceutical compositions, for example vaccine compositions. As such, it is desirable that such components are sterile and free of bacterial and/or viral contamination. Therefore, the processes provided herein may further comprise the step of sterile filtering the purified OMVs. “Sterile filtration” or “sterile filtering” refers to the removal of virus and/or bacteria from a solution by passing the solution through a filter having pores of a sufficiently small diameter that virus and/or bacteria cannot pass through. Thus, a “sterile preparation of purified OMVs” refers to a preparation or population of purified OMVs that has been passed through a filter having a pore size that is small enough to prevent the passage of virus and/or bacteria and result in a preparation free or substantially free of bacterial contaminants. Bacteria generally range in size from about 0.2 μm to about 600 μm and filters having a pore size of about 0.22 μm or less are sufficiently small to produce sterile filtrates and result in a sterile preparation of purified OMVs. Thus, step (c) comprises sterile filtering the flow-through of purified OMVs using a 0.22µm filter to produce a sterile preparation of purified OMVs. In one embodiment, steps (b) and (c) are performed sequentially without intermediate processing steps, other than collection/storage, and the output of step (b) is the direct input for step (c). In some embodiments, process steps (b) and (c) are discontinuous process steps wherein step (b) is completed before step (c) is started. In some embodiments, steps (b) and (c) are continuous process steps. Advantageously, processes provided herein enable a recovery or yield of at least 25 mg of OMVs (based on protein content) per litre of fermentation harvest or culture used in the preparation of the sample of OMVs, for example at least 40mg protein/L culture, such as at least 70mg protein/L culture or more. The term “polydispersity” (or “dispersity” as recommended by IUPAC) is used to describe the degree of non-uniformity of a size distribution of a population of particles. The “Polydispersity Index” (PDI) is a dimensionless parameter used in the art to to define the size distribution of the lipid nanoparticles. The higher the value of the polydispersity index, the broader the spread of individual particle sizes making up the population. The lower the value of the polydispersity index, the more uniform and tightly grouped the particle sizes of the individual nanoparticles making up the population will be. The PDI may be determined using dynamic light scattering (DLS) techniques known in the art and descried herein. Preparations or populations of purified OMVs produced by the processes provided herein have a polydispersity index of about 0.3 or less, for example, about 0.1 to about 0.3, about 0.2 to about 0.3, such as about 0.2 to about 0.27. The term “z-average radius” means the average radius of OMVs in the preparation or population of purified OMVs as measured by Dynamic Light Scattering (DLS) as described herein. In one embodiment, DLS measurements are performed with a Malvern Zetasizer Nano ZS (Malvern, Herremberg, Germany) equipped with a 633 nm He–Ne laser and operating at an angle of 173°. Scattering light detected at 173° is automatically adjusted by laser attenuation filters. For data analysis, the viscosity and refractive index (RI) of 3% sucrose solution (at 25 °C) are used. The software used to collect and analyze the data is the Zetasizer software version 7.13. Temperature is set at 25 °C. Each sample (80 μL) is characterized in single-use polystyrene microcuvette (ZEN0040, Alfatest) with a path length of 10 mm. The hydrodynamic diameter of OMV is expressed by a Z- average value (general purpose algorithm) of three measurements of eleven runs for each sample, providing also a PDI of the size values calculated. In one embodiment, preparations or populations of purified OMVs provided herein, for example purified OMV obtained from Bordetella bacteria, for example B. pertussis bacteria, will have a z-average radius of from about 50 nm to about 150 nm, for example, about 50 nm to about 140 nm, about 50 nm to about 80 nm, such as about 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 66 nm, 67 nm, 68 nm, 69 nm, 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm or about 76 nm. In one embodiment, preparations or populations of purified OMVs provided herein will have a z-average radius of from about 10 nm to about 125nm, for example, about 20 nm to about 110 nm, about 30 nm to about 100 nm, about 40 nm to about 90 nm, about 50 nm to about 80 nm, about 60 nm to about 70 nm. In one embodiment, preparations or populations of purified OMVs provided herein, for example purified OMV obtained from Escherichia coli bacteria, for example Escherichia coli bacteria of strain K or strain B strains, optionally wherein the Escherichia coli bacteria are selected from any one of strains BL21(DE3), BLR(DE3), and E. coli HMS174(DE3), will have a z-average radius of from about 15 nm to about 75 nm, for example, about 20 nm to about 70 nm, about 30 nm to about 60 nm, about 40 nm to about 50 nm. In one embodiment, preparations or populations of purified OMVs provided herein, for example purified OMV obtained from Neisseria bacteria, for example Neisseria gonorrhoeae, will have a z-average radius of from about 30 nm to about 110 nm, for example, about 40 nm to about 100 nm, about 50 nm to about 90 nm, about 60 nm to about 80 nm. In some embodiments, preparations or a population of purified OMVs will be least 85% pure, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, for example, about 85%, about 90%, about 92%, about 93%, about 94% or about 95%. In one embodiment, populations of purified OMVs will be from about 85% to about 100% pure, from about 90% to about 100% pure, from about 95% to about 100% pure. In one embodiment, populations of purified OMVs will have a purity of from about 85% to about 99%, from about 90% to about 99%, from about 91% to about 99%, from about 92% to about 99%, from about 93% to about 99%, from about 94% to about 99%, from about 95% to about 99%, from about 96% to about 99%, from about 97% to about 99% or from about 98% to about 99%. In one embodiment, purity may be measured using size exclusion UPLC, for example by size exclusion UPLC (fluorescence detection) to determine the ratio between the main peak (attributed to OMVs) and the smaller, low molecular size peak (attributed to free proteins). The skilled person will also recognise that purity may be defined by reference to the amount or level of total impurities remaining. Thus, impurities may be present in preparations or populations of purified OMVs at a level or concentration of less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than about 1% of impurities, for example of free impurities, such as protein impurities. In one embodiment, preparations or populations of purified OMVs comprise no more than 20%, no more than 15%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2% or no more than about 1% of impurities, for example of free impurities, such as protein impurities. Preparations or populations of purified OMVs produced using the processes provided herein may comprise from about 0.01% to about 10% of GroEL, such as from about 0.01% to about 9% of GroEL, from about 0.01% to about 8% of GroEL, from about 0.01% to about 7% of GroEL, from about 0.01% to about 6% of GroEL, from about 0.01% to about 5% of GroEL, from about 0.01% to about 4% of GroEL, from about 0.01% to about 3% of GroEL, from about 0.01% to about 2% of GroEL or from about 0.01% to about 1.5% as a percentage of total protein, for example, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 0.75%, about 1% or about 1.05% GroEL. In one embodiment, preparations or populations of purified OMVs produced using the processes provided herein will comprise no more than about 5% GroEL, no more than about 4% GroEL, no more than about 3% GroEL, no more than about 2% GroEL, no more than about 1.5% GroEL or no more than about 1% GroEL as a percentage of total protein, for example, less than 5% of GroEL, less than 4% of GroEL, less than 3% of GroEL, less than 2% of GroEL, less than 1.5% of GroEL or less than 1.1% of GroEL. In one embodiment, the amount of GroEL may be provided as a relative or absolute amount. In one embodiment, the amount of GroEL is provided as a relative amount for example, as a percentage of total protein in the preparation or population of purified OMVs. In one embodiment, the amount of GroEL is a relative amount determined using mass analysis. Further, and in addition to the reduced amount of GroEL, the preparation or population of purified OMVs is substantially free of process related impurities, such as those associated with upstream production or purification processes, for example media components used in fermentation or reagents used in cell disruption. Thus, the preparation or population of purified OMVs has the purity required for formulation and pharmaceutical use as a drug substance. Within the context of the present application, the term “drug substance” is used to refer to a product suitable for use as an active ingredient in an immunogenic composition such as a medicament, for example a pharmaceutical composition such as a vaccine composition. Thus, and as described below, the preparation or population of purified OMVs may be combined with one or more pharmaceutically acceptable carriers, excipients or other components to produce an immunogenic composition. Immunogenic compositions The preparation or population of purified OMVs is capable of eliciting an immune response, for example comprising eliciting a primary immune response and/or a boosted immune response, for example to Bordetella, for example Bordetella pertussis, or Neisseria, for example Neisseria gonorrhoeae when administered to a suitable subject, for example a mammal. The immune response may be a cellular or a humoral immune response. In some embodiments, the immune response is an antibody response. In some embodiments, the immune response is a T-cell immune response that can neutralise the infection and/or virulence of Bordetella pertussis or Neisseria gonorrhoeae. The immune response elicited by the OMV may be directed toward or against one or more antigens present in the OMVs. Thus, compositions comprising purified OMVs provided herein are immunogenic. The term “immunogenic composition” broadly refers to any composition comprising OMVs provided herein that may be administered to a subject to elicit an immune response, such as an antibody or cellular immune response, against an antigen or antigens present in the OMVs. An immunogenic composition may also be referred to as a “drug product” in the art and refers to the preparation or population of purified OMVs formulated in a form suitable for administration to a subject, such as a human, for example as a pharmaceutical composition, for example, as a vaccine. As described further below, in addition to antigens present in the OMVs, immunogenic compositions may be formulated to contain other antigenic components, for example from Bordetella, Neisseria and/or other organisms capable of infecting the suitable subject and causing illness or symptoms of disease. When the immunogenic compositions prevent, ameliorate, palliate, or eliminate disease from the subject arising as a result of bacterial and/or viral infection, they may be referred to as a vaccine. Hence, in certain embodiments, the immunogenic composition is a vaccine. Vaccines provided herein may either be prophylactic (i.e. to prevent infection, for example by eliciting a primary and/or boosted immune response) or therapeutic (i.e. to treat infection, for example by eliciting a primary and/or boosted immune response), but will typically be prophylactic. Prophylactic vaccines do not guarantee complete protection from disease because even if a subject develops antibodies, there may be a lag or delay before the immune system is able to fight off the infection. Therefore, and for the avoidance of doubt, the term prophylactic vaccine encompasses vaccines that are used to ameliorate the effects of a future infection, for example by reducing the severity and/or duration of such an infection. The terms “protection against infection” and/or “provide protective immunity” means that the immune system of a subject has been primed (e.g by vaccination) to trigger an immune response and repel infection. In some embodiments, the immune response triggered is capable of repelling infection against Bordetella, optionally Bordetella pertussis, or Neisseria, optionally Neisseria gonorrhoeae. A vaccinated subject may thus get infected, but is better able to control and fight-off or repel the infection than a control subject. Immunogenic compositions used as vaccines comprise an immunologically effective amount of OMVs. By “immunologically effective amount”, it is meant that the administration of that amount to a suitable subject, either in a single dose or as part of a series, sufficient to generate an immune response effective for treatment or prevention of infection and disease. Commonly, the desired result is the production of an antigen (e.g., pathogen)-specific immune response that is capable of or contributes to protecting the subject against the pathogen. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials, for example by clinical trials. The term “antigen” refers to a substance that, when administered to a subject, elicits an immune response directed against said substance. OMVs purified by the processes provided herein are complex components comprising a plurality of antigens, for example, within the OMV lumen or displayed on or associated with the surface of the OMVs. Unless indicated otherwise, this plurality of antigens will be considered collectively and the OMVs will be referred to as an antigen or antigenic component. In some embodiments, when administered to a subject, the immunogenic composition— and without wishing to be limited by theory the OMV component of the immunogenic composition— will elicit an immune response directed against Bordetella, for example, Bordetella pertussis, or Neisseria, for example Neisseria gonorrhoeae. In some embodiments, the immune response directed against Bordetella or Neisseria is protective, that is, it can prevent or reduce infection and/or colonisation caused by Bordetella, optionally Bordetella pertussis, or Neisseria, optionally Neisseria gonorrhoeae. Compositions may thus be pharmaceutically acceptable. Generally, immunogenic compositions include components in addition to the OMVs, for example, they typically include one or more pharmaceutical carrier(s) and/or excipient(s) (a thorough discussion of such components is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN: 0683306472). A “pharmaceutically acceptable carrier” is a carrier that does not itself induce the production of antibodies. Such carriers are well known to those of ordinary skill in the art and include, by way of non-limiting example, polysaccharides, sucrose, trehalose, lactose, diluents, such as water, saline including phosphate buffered saline, glycerol and auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, etc. Immunogenic compositions, for example vaccines, may also comprise one or more adjuvants. The term “adjuvant” as used herein refers to a compound that enhances a subject's immune response to antigen(s) when administered conjointly with the antigen(s). The composition may include a preservative such as thiomersal or 2-phenoxyethanol. In one embodiment, the compositions provided herein are substantially free from (i.e. less than 5µg/ml) mercurial material e.g. thiomersal-free. Compositions containing no mercury are also provided. Thiomersal-free compositions, for example thiomersal-free vaccines, are also provided. To control tonicity, a physiological salt, such as a sodium salt may be included. Sodium chloride (NaCl) may be used, which may be present at between 1 and 20 mg/ml, for example about 10+2mg/ml NaCl. Compositions will generally have an osmolality of between 200 mOsm/kg and 400 mOsm/kg, for example between 240-360 mOsm/kg, and for example within the range of 290-310 mOsm/kg. Compositions may include one or more buffers. Typical buffers include: a phosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer; or a citrate buffer. Buffers will typically be included in the 5-20mM range. The pH of a composition will generally be between from about pH 5.0 to about pH 8.1, and more typically between from about pH 6.0 and about pH 8.0, such as between about pH 6.5 and about pH 7.5, or between about pH 7.0 and about pH 7.8. In one embodiment, immunogenic compositions are sterile. The composition may include material for a single immunisation, or may include material for multiple immunisations (i.e. a ‘multidose’ kit). The inclusion of a preservative is provided, for example in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material. Immunogenic compositions, for example human vaccines, are typically administered in a dosage volume of about 0.5ml, although fractional doses, such as a half dose (i.e. about 0.25ml) may be administered, for example, to children. Adjuvants which may be used in compositions provided herein include mineral containing compositions such as aluminium salts and calcium salts. The compositions provided herein may include mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates or mixtures of different mineral compounds. Aluminium adjuvants include, by way of non-limiting example, aluminium hydroxide, aluminium oxyhydroxide salts, aluminium phosphate, aluminium hydroxyphosphates, aluminium hydroxyphosphate sulfate and the like. In some embodiments, the adjuvant comprises Aluminium phosphate which may be obtained by precipitation. The reaction conditions and concentrations during precipitation influence the degree of substitution of phosphate for hydroxyl in the salt. The PO4/Al3+ molar ratio of an aluminium phosphate adjuvant may be between 0.3 and 1.2, for example between 0.8 and 1.2, and for example 0.95+0.1. The aluminium phosphate may be amorphous, for example for hydroxyphosphate salts. Aluminium phosphates provided herein may have a point of zero charge (PZC) of between 4.0 and 7.0, for example between 5.0 and 6.5 e.g. about 5.7. The concentration of Al+++ in a composition for administration to a patient may be between from about 10mg/ml to about 0.01mg/ml, for example, about 5 mg/ml or less, about 4 mg/ml or less, about 3 mg/ml or less, about 2 mg/ml or less, about 1 mg/ml or less, for example, about 5mg/ml, about 4mg/ml, about 3mg/ml, about 2mg/ml, about 1mg/ml, about 0.3mg/ml, about 0.05mg/ml or about 0.01mg/ml. In one embodiment, the range is between from about 0.3mg/ml to about 1mg/ml. In some embodiments, a maximum of 0.85mg/dose is provided, for example about 0.5mg/dose, about 0.4mg/dose, about 0.3mg/dose, about 0.2mg/dose or about 0.1mg/dose. Immunogenic compositions provided herein may comprise a Toll like receptor (TLR) agonist such as a Toll like receptor 2 agonist (TLR2a), a Toll like receptor 3 agonist (TLR3a), a Toll like receptor 4 agonist (TLR4a), a Toll like receptor 7 agonist (TLR7a), a Toll like receptor 8 agonist (TLR8a) or a Toll like receptor 9 agonist TLR9a). Compositions provided herein may include a Toll like receptor agonist selected from the group consisting of a TLR2 agonist (e.g. Pam3CSK4), a TLR4 agonist (e.g. an aminoalkyl glucosaminide phosphate, such as E6020), a TLR7 agonist (e.g. imiquimod or a benzonaphthyridine, for example, SMIP7.10), a TLR8 agonist (e.g. resiquimod (also a TLR7 agonist)) and/or a TLR9 agonist (e.g. IC31 or CpG1018). Immunogenic compositions provided herein may comprise both a TLR agonist and at least one aluminium salt adjuvant such as aluminium phosphate and/or aluminium hydroxide. In some embodiments, immunogenic compositions provided herein are substantially aluminium adjuvant free, i.e., they comprise only a residual or trace amount of aluminium salt adjuvant(s) or do not comprise a measurable amount of aluminium salt adjuvant(s). In one embodiment, when immunogenic compositions provided herein comprise a TLR agonist, the TLR agonist is selected from the group consisting of IC31, E6020, CpG1018 and SMIP7.10. In one embodiment, immunogenic compositions provided herein comprise a TLR agonist selected from the group consisting of IC31, E6020, CpG1018 and SMIP7.10; and an aluminium salt adjuvant selected from the group consisting of an aluminium hydroxide and aluminium phosphate. In one menodiment, immunogenic compositions provided herein comprise (i) a TLR agonist selected from the group consisting of the TLR4 agonist E6020, the TLR7 agonist SMIP7.10 and the TLR9 agonist Cpg1018 and (ii) an aluminium salt selected from the group consisting of an aluminium hydroxide and aluminium phosphate. TLR agonists may have a molecular weight of <2000Da. TLR7 agonists, such as SMIP7.10, may include at least one adsorptive moiety. The inclusion of such moieties in TLR agonists allows them to adsorb to insoluble aluminium salts (e.g. by ligand exchange or any other suitable mechanism) and improves their immunological behaviour. Phosphorus- containing adsorptive moieties are useful, and so an adsorptive moiety may comprise a phosphate, a phosphonate, a phosphinate, a phosphonite, a phosphinite, etc. The TLR agonist may include at least one phosphonate group. In some embodiments, a composition provided herein may include a TLR7 agonist which includes a phosphonate group. This phosphonate group can allow adsorption of the agonist to an insoluble aluminium salt. In some embodiments, the TLR7 agonist comprises a benzonaphthyridine chemical scaffold for example, in some embodiments, the TLR agonist is 3-(5- amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)ethoxy)ethoxy)phenethyl)benzo [f]- [1,7]naphthyridin-8-yl)propanoic acid, shown below, optionally adsorbed to an aluminum adjuvant.
Figure imgf000028_0001
In some embodiments, TLR agonists are water-soluble. Thus, they can form a homogenous solution when mixed in an aqueous buffer with water at pH 7 at 25°C and 1 atmosphere pressure to give a solution which has a concentration of at least 50 μg/ml. The term “water-soluble” thus excludes substances that are only sparingly soluble under these conditions. Compositions will generally be administered to a suitable subject, for example a patient, for example a suitable mammal, such as a human, in aqueous form. Prior to administration, however, the composition may have been in a non-aqueous form. For instance, some vaccines are manufactured in aqueous form, then filled and distributed and administered also in aqueous form whereas other vaccines may be lyophilised during manufacture and reconstituted into an aqueous form at the time of use, for example at the time of administration. Thus, a composition provided herein may comprise or be a dried composition, such as a lyophilised formulation. Compositions provided herein may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition). The composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared for nasal, aural or ocular administration e.g. as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient. Such kits may comprise one or more lyophilised antigens and one or more antigens in liquid form for reconstitution of the lyophilised antigens. For example, where a composition is to be prepared extemporaneously prior to use (e.g. where a component is presented in lyophilised form) and is presented as a kit, the kit may comprise two vials, or it may comprise one ready-filled syringe and one vial, with the contents of the syringe being used to reactivate the contents of the vial prior to injection. Further antigenic components of compositions of the invention Immunogenic compositions provided herein will generally be combination vaccines and in addition to OMVs as provided herein, may include one or more further antigen(s). In some embodiments, the OMVs provided herein are obtained from Bordetella bacteria, optionally from Bordetella pertussis and the one or more further antigen(s) are antigens capable of stimulating or generating an immune response against viral and/or bacterial pathogens. Typical bacterial pathogens include, but are not limited to, Corynebacterium diphtheriae; Clostridium tetani and Haemophilus influenzae type b. Typical viral pathogens include, but are not limited to, poliovirus and hepatitis B virus. Thus, in addition to the OMVs provided herein, immunogenic compositions provided herein may further comprise one or more antigenic components that, when administered to a subject, is/are capable of generating an immune response, for example a protective immune response, against Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, hepatitis B virus, Haemophilus influenzae type B or polio virus. For the avoidance of doubt, reference to additional or further antigens is intended to refer to antigenic components included in the immunogenic compositions provided herein beyond, i.e. in addition to, the OMVs and constituents thereof. For example, small amounts of pertussis toxoid may be found as a constituent of OMVs but reference to pertussis toxoid as a further antigen refers to an amount of pertussis toxoid that is specifically added over and above the OMVs, for example, as an isolated protein antigen. Suitable further antigenic components known in the art from the bacterial and viral pathogens described in more detail below include: – Hepatitis B virus: surface and/or core antigens. – Corynebacterium diphtheriae: diphtheria toxoid or CRM197 (a diphtheria toxoid mutant). – Clostridium tetani: tetanus toxoid. – Bordetella pertussis: acellular pertussis antigens selected from the group consisting of pertussis toxoid (PT), filamentous haemagglutinin (FHA), pertactin, adenylate cyclase or an iimmunogenic fragment thereof, FIM2 and FIM3. – Haemophilus influenzae B: capsular polysaccharide, for example a polyribosyl ribitol phosphate [PRP]-protein conjugate. – Polio virus: inactivated poliovirus (IPV) comprising either type 1, type 2 and type 3 strains or type 1 and type 3 strains, for example, selected from Mahoney type 1, MEF-1 type 2 and. Saukett type 3 strains. In some embodiments the OMVs provided herein and obtained from Bordetella species, preferably from Bordetella pertussis, are conjugated to a capsular polysaccharide from Haemophilus influenzae type b to form an immunogenic conjugate. This conjugate may be combined with one or more further antigens in an immunogenic composition as described above. In some embodiments, an immunogenic composition of the conjugates between Bordetella OMVs and Hib polysaccharide is a fully liquid, stable composition further comprising an antigen selected from the group consisting of Diphteria Toxoid (DT), Tetanus Toxoid (TT), Hepatitis B (HepB) antigen, inactivated Poliovirus (IPV) and one or more acellular pertussis (aP) antigens. In some embodiments the Hib capsular polysaccharide has a molecular weight between from about 5 to about 100 KDa. The Hib saccharide moiety of the conjugate may be used in its full-length native form, comprising full-length PRP as prepared from Hib bacteria, or, as an alternative, it may be fragmented from its natural length according to different methods described in the art; optionally, a size fraction of these fragments can also be used. In some embodiments the PRP may be a synthetic polysaccharide. Suitable conjugates of Bordetella OMVs to Hib capsular polysaccharide are disclosed in WO2020/043874, which is incorporated herein by reference. Acellular pertussis antigens In addition to OMVs provided herein, obtained from Bordetella bacteria, optionally from Bordetella pertussis, immunogenic compositions may further comprise one or more acellular pertussis (aP) antigens, for example selected from the following well-known and well-characterized B.pertussis antigens: (1) detoxified pertussis toxin (pertussis toxoid, or ‘PT’), for example as chemically detoxified pertussis toxoid (PTc) or genetically detoxified pertussis toxoid (PTg); (2) filamentous hemagglutinin (‘FHA’); (3) pertactin (also known as ‘PRN’ or the ‘69 kiloDalton outer membrane protein’); (4) fimbriae type 2 (‘FIM2’); (5) fimbriae type 3 (‘FIM3’) and (6) adenylate cyclase (AC) or an immunogenic fragment thereof. In one embodiment, both detoxified PT and FHA are used. FIM2 and FIM3 are typically co-purified and processed as a single antigen (referred to as FIM2/3). PTc may be prepared by chemical treatment of pertussis toxin with formaldehyde and/or glutaraldehyde. In one embodiment, as an alternative to this chemical detoxification procedure the PT is a mutant PT in which enzymatic activity has been reduced by mutagenesis, for example, genetically detoxified pertussis toxoid (PTg), for example the genetically detoxified pertussis toxoid is the genetically detoxified pertussis toxoid known in the art and referred to as the PT-9K/129G mutant. The PT-9K/129G mutant is a double mutant with substitutions at residues 9 (Arg to Lys) and 129 (Glu to Gly) of the S1 subunit. In some embodiments immunogenic compositions comprise the acellular pertussis antigens PT, FHA and PRN, for example PTg, FHA and PRN. In some embodiments the immunogenic compositions also comprise the acellular pertussis antigens FIM2/FIM3. In some embodiments, immunogenic compositions comprise the acellular pertussis antigens PTg, FHA, FIM2/FIM3 but do not contain PRN. In one embodiment, PT, FHA and PRN are prepared by isolation from B.pertussis culture grown in a suitable medium, such as modified Stainer-Scholte liquid medium. PT and FHA can be isolated from the fermentation broth (e.g. by adsorption on hydroxyapatite gel), whereas pertactin can be extracted from the cells by heat treatment and flocculation (e.g. using barium chloride). The antigens can be purified in successive chromatographic and/or precipitation steps. PT and FHA can be purified by hydrophobic chromatography, affinity chromatography and size exclusion chromatography. Pertactin can be purified by ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography. FHA and pertactin may be treated with formaldehyde prior to use. The aP antigen(s) may be used in an unadsorbed state, but may be adsorbed onto one or more aluminium salt adjuvant(s) before being used. Typically, the aP antigens are substantially free from mercurial preservatives such as thimerosal. The acellular pertussis antigens may be present in the immunogenic compositions provided herein in an amount that is capable of eliciting an immune response when administered. Ideally, the acellular pertussis antigens can elicit a protective immune response. Quantities of acellular pertussis antigens are typically expressed in micrograms. The concentration of PT in a vaccine is usually between 5 and 50μg/ml. Typical PT concentrations are 5μg/ml, 16μg/ml, 20μg/ml or 50μg/ml, for example, about 20µg per dose or about 25µg per dose. The concentration of FHA in a vaccine is usually between 10 and 50μg/ml. Typical FHA concentrations are 10μg/ml, 16μg/ml or 50μg/ml, for example, about 20µg per dose or about 25µg per dose. The concentration of pertactin in a vaccine is usually between 5 and 16μg/ml. Typical pertactin concentrations are 5μg/ml, 6μg/ml or 16μg/ml, for example, about 3µg per dose or about 8µg per dose. FIM2 and FIM3 may be present at a concentration 5 and 16μg/ml. Typical concentrations of FIM2 and FIM3 are 5μg/ml, 10μg/ml or 20μg/ml, for example, for example, about 5µg per dose or about 10µg per dose. Booster vaccines for adolescents and adults typically contain 2.5 to 8 μg PT, between 4 and 8 μg FHA and between 2.5 and 8 μg pertactin per 0.5 ml dose. Typically, a booster vaccine comprises 4 μg PT, 4 μg FHA and 8 μg pertactin, for example 5 μg PT, 2.5 μg FHA and 2.5 μg pertactin, per 0.5 ml dose. A paediatric vaccine usually comprises 7 μg PT, 10 μg FHA and 10 μg pertactin, per 0.5 ml dose. Where the aqueous component includes each of PT, FHA and pertactin, their weight ratios can vary, but may be e.g. about 16:16:5, about 5:10:6, about 20:20:3, about 25:25:8, or about 10:5:3 (PT:FHA:PRN). Diphtheria Corynebacterium diphtheriae causes diphtheria. Diphtheria toxin can be treated (e.g. using formalin or formaldehyde) to remove toxicity while retaining the ability to induce specific anti-toxin antibodies after injection. The diphtheria toxoids used in diphtheria vaccines are well known in the art. In one embodiment, diphtheria toxoids are those prepared by formaldehyde treatment. The diphtheria toxoid can be obtained by growing C. diphtheriae in growth medium (e.g. Fenton medium, or Linggoud & Fenton medium), which may be supplemented with bovine extract, followed by formaldehyde treatment, ultrafiltration and precipitation. In one embodiment, the growth medium for growing C. diphtheriae is free from animal-derived components. The toxoided material may then be treated by a process comprising sterile filtration and/or dialysis. Alternatively, genetically detoxified diphtheria toxin (e.g., CRM197) may be used which may be formaldehyde-treated to maintain long- term stability during storage. The diphtheria toxoid may be adsorbed onto an adjuvant such as an aluminium salt adjuvant. Quantities of diphtheria toxin and/or toxoid in a composition are generally measured in the ‘Lf’ unit (“flocculating units”, or the “limes flocculating dose”, or the “limit of flocculation”), defined as the amount of toxin/toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture (Lyng (1990) Biologicals 18:11-17). For example, the NIBSC supplies ‘Diphtheria Toxoid, Plain’ (NIBSC code: 69/017), which contains 300 LF per ampoule, and also supplies ‘The 2nd International Reference Reagent For Diphtheria Toxoid For Flocculation Test’ (NIBSC Code: 02/176) which contains 900 Lf per ampoule. The concentration of diphtheria toxin or toxoid in a composition can readily be determined using a flocculation assay by comparison with a reference material calibrated against such reference reagents. The immunizing potency of diphtheria toxoid in a composition is generally expressed in international units (IU). The potency can be assessed by comparing the protection afforded by a composition in laboratory animals (typically guinea pigs) with a reference vaccine that has been calibrated in IUs. NIBSC supplies the ‘4th WHO International Standard for Diphtheria Toxoid (Adsorbed)’ (NIBSC code: 07/216) which contains 213 IU per ampoule, and is suitable for calibrating such assays. By IU measurements, compositions generally include at least 30 IU/dose. Compositions typically include between 20 and 80 Lf/ml of diphtheria toxoid, typically about 50 Lf/ml. Booster vaccines for adolescents and adults typically contain between 4 Lf/ml and 8 Lf/ml of diphtheria toxoid, e.g., 2.5 Lf, for example 4 Lf, per 0.5 ml dose. Paediatric vaccines typically contain between 20 and 50 Lf/ml of diphtheria toxoid, e.g. 10 Lf or 25 Lf per 0.5 ml dose. Purity of a protein preparation can be expressed by the ratio of specific protein to total protein. The purity of diphtheria toxoid in a composition is generally expressed in units of Lf diphtheria toxoid per unit mass of protein (nondialysable) nitrogen. For instance, a very pure toxin/toxoid might have a purity of more than 1700 Lf/mg N, indicating that most or all of the protein in the composition is diphtheria toxin/toxoid (Kuhmlann & Rieger (1995) Immunol Infect Dis 5:10-4 ). Tetanus Clostridium tetani causes tetanus. Tetanus toxin can be treated to give a protective toxoid. The toxoids are used in tetanus vaccines and are well known in the art. Thus, a combination vaccine provided herein can include a tetanus toxoid. In one embodiment, tetanus toxoids are those prepared by formaldehyde treatment. The tetanus toxoid can be obtained by growing C.tetani in growth medium (e.g. a Latham medium derived from bovine casein), followed by formaldehyde treatment, ultrafiltration and precipitation. The growth medium for growing C.tetani may be free from animal- derived components. The material may then be treated by a process comprising sterile filtration and/or dialysis. The tetanus toxoid may be adsorbed onto an adjuvant, for example, an aluminium salt adjuvant. Quantities of tetanus toxoid can be expressed in ‘Lf’ units (see below), defined as the amount of toxoid which, when mixed with one International Unit of antitoxin, produces an optimally flocculating mixture. The NIBSC supplies ‘The 2nd International Reference Reagent for Tetanus Toxoid For Flocculation Test’ (NIBSC Code: 04/150) which contains 690 LF per ampoule, by which measurements can be calibrated. Booster vaccines for adolescents and adults typically contain 5 Lf of tetanus toxoid per 0.5 ml dose. Paediatric vaccines typically contain between 5 and 10 Lf of tetanus toxoid per 0.5 ml dose. The immunizing potency of tetanus toxoid is measured in international units (IU), assessed by comparing the protection afforded by a composition in laboratory animals (typically guinea pigs) with a reference vaccine e.g. using NIBSC’s ‘Tetanus Toxoid Adsorbed Third International Standard 2000’ (Sesardic et al. (2002) Biologicals 30:49-68; NIBSC code: 98/552), which contains 469 IU per ampoule. The potency of tetanus toxoid in a composition provided herein should be at least 35 IU per dose e.g. at least 70 IU/ml. In one embodiment, the potency of tetanus toxoid in a composition provided herein is at least 40 IU per dose. However, in booster vaccines for adults and adolescents, a reduced potency of 20 IU/dose may be acceptable because of the reduced antigen content in comparison to paediatric vaccine intended for primary immunization. The purity of tetanus toxoid in a composition is generally expressed in units of Lf tetanus toxoid per unit mass of protein (non-dialyzable) nitrogen. The tetanus toxoid should have a purity of at least 1000 Lf/mg N. Hib Haemophilus influenzae type b (‘Hib’) causes bacterial meningitis. Hib vaccines are typically based on the capsular saccharide antigen the preparation of which is well documented in the art (Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Buttery & Moxon (2000) J R Coll Physicians Lond 34:163- 168, etc). The H.influenzae bacteria can be cultured in the absence of animal-derived components.The Hib saccharide is conjugated to a carrier protein in order to enhance its immunogenicity, especially in children. Typical carrier proteins in these conjugates are tetanus toxoid, diphtheria toxoid, the CRM197 derivative of diphtheria toxin, or an outer membrane protein complex (OMPC) from serogroup B meningococcus. Thus, a combination vaccine provided herein can include a Hib capsular saccharide conjugated to a carrier protein such as tetanus toxoid, diphtheria toxoid or OMPC. The saccharide moiety of the conjugate may comprise full-length polyribosylribitol phosphate (PRP) as prepared from Hib bacteria, and/or fragments of full-length PRP. Conjugates with a saccharide:protein ratio (w/w) of between 1:5 (i.e. excess protein) and 5:1 (i.e. excess saccharide) may be used e.g. ratios between 1:2 and 5:1 and ratios between 1:1.25 and 1:2.5. In some vaccines, however, the weight ratio of saccharide to carrier protein is between 1:2.5 and 1:3.5. Quantities of Hib antigens are typically expressed in μg of saccharide. The concentration of saccharide in a vaccine is typically between from 3 to 30μg/ml e.g. 20μg/ml. In one embodiment, administration of the Hib conjugate results in an anti-PRP antibody concentration of >0.15µg/ml, and for example >1µg/ml, and these are the standard response thresholds. In some embodiments, the Hib polyribosylribitol phosphate is a synthetic polysaccharide (for example, as described in Verez- Bencomo et al. Science. 2004 Jul 23;305(5683):522-5. As said above, in some embodiments the Hib capsular polysaccharide antigen may be conjugated to the OMVs provided herein obtained from Bordetella species. Hepatitis B virus Hepatitis B virus (HBV) is a cause of viral hepatitis. The HBV virion consists of an inner core surrounded by an outer protein coat or capsid, and the core contains the viral DNA genome. The major component of the capsid is a protein known as HBV surface antigen or, more commonly, ‘HBsAg’, which is typically a 226-amino acid polypeptide with a molecular weight of ~24 kDa. All existing hepatitis B vaccines contain HBsAg, and when this antigen is administered to a normal patient, it stimulates the production of anti-HBsAg antibodies which protect against HBV infection. Thus, immunogenic compositions provided herein can include HBsAg. For vaccine manufacture, HBsAg can be made in a number of ways. For example, by expressing the protein by recombinant DNA methods. HBsAg for use with the method provided herein should be recombinantly expressed, e.g. in yeast cells. Suitable yeasts include Saccharomyces (such as S.cerevisiae), Hanensula (such as H.polymorpha) or Pichia hosts. The yeasts can be cultured in the absence of animal-derived components. Yeast-expressed HBsAg is generally non-glycosylated; in one embodiment the non-glycosylated form of HBsAg is used in the immunogenic compositions provided herein. Yeast-expressed HBsAg is highly immunogenic and can be prepared without the risk of blood product contamination. Many methods for purifying HBsAg from recombinant yeast are known in the art. The HBsAg will generally be in the form of substantially-spherical particles (average diameter of about 20nm), including a lipid matrix comprising phospholipids. Yeast-expressed HBsAg particles may include phosphatidylinositol, which is not found in natural HBV virions. In one embodiment, the HBsAg is from HBV subtype adw2. Quantities of HBsAg are typically expressed in micrograms. Combination vaccines containing HBsAg usually include between 5 and 60 µg/ml. The concentration of HBsAg in a composition provided herein is for example less than 60 µg/ml e.g. ≤55 µg/ml, ≤50 µg/ml, ≤45 µg/ml, ≤40 µg/ml, etc. A concentration of about 20 µg/ml is typical e.g. 10µg per dose. In some embodiments, a composition includes a ‘low dose’ of HBsAg. This means that the concentration of HBsAg in the composition is ≤5 μg/ml e.g. <4, <3, <2.5, <2, <1, etc. In a typical 0.5ml unit dose volume, therefore, the amount of HBsAg is less than 2.5μg e.g. <2, <1.5, <1, <0.5, etc. Poliovirus Poliovirus causes poliomyelitis. Inactivated polio virus vaccine (IPV) has been known for many years. Thus, a combination vaccine provided herein can include an inactivated poliovirus antigen. Polioviruses may be grown in cell culture. In one embodiment, a culture uses a Vero cell line, derived from monkey kidney. Vero cells can conveniently be cultured microcarriers. After growth, virions may be purified using techniques such as ultrafiltration, diafiltration, and chromatography. Where animal (and optionally bovine) materials are used in the culture of cells, they should be obtained from sources that are free from transmissible spongiform encephalopathies (TSEs), and for example free from bovine spongiform encephalopathy (BSE). In one embodiment, polioviruses are grown in cells cultured in medium free of animal-derived components. Prior to administration to patients, polioviruses must be inactivated, and this can be achieved by treatment with formaldehyde (or, for example, a non-aldehyde agent). Poliomyelitis can be caused by one of three types of poliovirus. The three types are similar and cause identical symptoms, but they are antigenically very different and infection by one type does not protect against infection by others. In one embodiment, three poliovirus antigens are used with the compositions provided herein: poliovirus Type 1 (e.g. Mahoney strain), poliovirus Type 2 (e.g. MEF-1 strain), and poliovirus Type 3 (e.g. Saukett strain). Other strains of poliovirus Type 1, Type 2 and Type 3 are known in the art and may also be used. The viruses are for example grown, purified and inactivated individually, and are then combined to give a bulk trivalent mixture for use with the composition provided herein. Quantities of IPV are typically expressed in the ‘DU’ unit (the “D-antigen unit”; Module 6 of WHO’s The immunological basis for immunization series (Robertson)). Combination vaccine usually comprise between 1-100 DU per polioviral type per dose e.g., about 40 DU of type 1 poliovirus, about 8 DU of type 2 poliovirus, and about 32 DU of type 3 poliovirus, but it is possible to use lower doses than these (WO2008/028956; WO2008/028957) e.g.10-20 DU for type 1, 2-4 DU for type 2, and 8-20 DU for type 3. A combination vaccine provided herein can include a ‘low dose’ of a poliovirus. For a Type 1 poliovirus this means that the concentration of the virus in the composition is ≤20 DU/ml e.g. <18, <16, <14, <12, <10, etc. For a Type 2 poliovirus this means that the concentration of the virus in the composition is ≤4 DU/ml e.g. <3, <2, <1, <0.5, etc. For a Type 3 poliovirus this means that the concentration of the virus in the composition is ≤16 DU/ml e.g. <14, <12, <10, <8, <6, etc. Where all three of Types 1, 2 and 3 polio virus are present the three antigens can be present at a DU ratio of 5:1:4 respectively, or at any other suitable ratio e.g. a ratio of 15:32:45 when using Sabin strains (Liao et al. (2012) J Infect Dis. 205:237-43). A low dose of antigen from Sabin strains is useful, with ≤10 DU type 1, ≤20 DU type 2, and ≤30 DU type 3 (per unit dose, typically 0.5 ml). Where an IPV component is used, and the polioviruses were grown on Vero cells, a vaccine composition for example contains less than 10ng/ml, for example ≤1ng/ml e.g. ≤500pg/ml or ≤50 pg/ml of Vero cell DNA e.g. less than 10ng/ml of Vero cell DNA that is >50 base pairs long. Preparing a combination vaccine Antigenic components from these pathogens for use in vaccines are commonly referred to by abbreviated names: ‘D’ for diphtheria toxoid; ‘T’ for tetanus toxoid; ‘P’ for pertussis antigens, with ‘aP’ being acellular pertussis antigens (e.g. including at least OMVs provided herein, PT and FHA and optionally pertactin and/or FIM2/FIM3); HBsAg for hepatitis B surface antigen; ‘Hib’ for conjugated H.influenzae b capsular saccharide; and ‘IPV’ for 3-valent inactivated poliovirus. Embodiments provided herein include, but are not limited to combination vaccines comprising the following components: – D, T, aP – D, T, aP, IPV – D, T, aP, HBsAg – D, T, aP, Hib – D, T, aP, Hib, IPV – D, T, aP, HBsAg, Hib – D, T, aP, HBsAg, IPV – D, T, aP, HBsAg, IPV, Hib In some embodiments, these combination vaccines contain only the antigens listed as active ingredients but may further comprise excipients such as adjuvants, buffers and the like. In some embodiments the aP component consists of OMVs provided herein, PT (for example genetically detoxified PT) and FHA. In some embodiments the aP component consists of OMVs provided herein, PT (for example genetically detoxified PT), FHA and PRN. In some embodiments the aP component consists of OMVs provided herein, PT (preferably genetically detoxified PT), FHA and FIM2/FIM3. In some embodiments the aP component consists of OMVs provided herein, PT (for example genetically detoxified PT), FHA, PRN and FIM2/FIM3. For paediatric combination vaccines, the ratio of D:T is typically greater than 1 (i.e. paediatric vaccines usually have excess D in Lf units) and generally between 2:1 and 3:1 (measured in Lf units), e.g.2.5:1. In contrast, for booster vaccine that are administered to adolescents or adults (who usually have received at least one paediatric combination vaccine comprising D and T), the ratio of T:D is typically greater than 1 (i.e. booster vaccines usually have excess T in Lf units) and generally between 1.5:1 and 2.5:1, e.g. 2:1. One useful vaccine includes OMVs provided herein and, per unit dose, 2Lf D, 5Lf T, 4µg PT- 9K/129G, 4µg FHA and 8µg pertactin. Another useful vaccine includes OMVs provided herein and, per unit dose, 25Lf D, 10Lf T, 25µg PT-9K/129G, 25µg FHA and 8µg pertactin. Combinations with D and T are of interest. Immunisation In addition to providing immunogenic compositions as described above, also it is provided herein the use of OMVs and immunogenic compositions in a method for raising an immune response in a mammal, comprising administering OMVs or an immunogenic composition provided herein to the mammal. Typically, the immune response is an antibody response. In some embodiments, the antibody response is a protective antibody response. It is also provided herein compositions for use in such methods. It is also provided a method for protecting a mammal against a bacterial infection and/or disease, comprising administering to the mammal an immunogenic composition as provided herein. It is also provided herein compositions for use as medicaments (e.g. as immunogenic compositions or as vaccines). It is also provided a population of purified OMVs as disclosed herein for use in medicine. It also provides the use of OMVs provided herein in the manufacture of a medicament for preventing a bacterial infection in a mammal. In one embodiment, the mammal is a human. The human may be an adult or, for example, a child. Where the vaccine is for prophylactic use, the human is for example a child (e.g. a toddler or infant); where the vaccine is for therapeutic use, the human is for example an adult. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc. Efficacy of therapeutic treatment can be tested by monitoring bacterial infection after administration of the composition provided herein. Efficacy of prophylactic treatment can be tested by monitoring immune responses against immunogenic proteins in the vesicles or other antigens after administration of the composition. Immunogenicity of compositions provided herein can be determined by administering them to test subjects (e.g. children 12-16 months age) and then determining standard serological parameters. These immune responses will generally be determined around 4 weeks after administration of the composition, and compared to values determined before administration of the composition. Where more than one dose of the composition is administered, more than one post-administration determination may be made. Compositions provided herein will generally be administered directly to a patient. Direct delivery may be accomplished by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal administration. In one embodiment, intramuscular administration to the thigh or the upper arm is provided. Injection may be via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is about 0.5 ml. Immunogenic compositions provided herein may be used to elicit systemic and/or mucosal immunity. Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined. General The term “comprising” encompasses “including” e.g. a composition “comprising” X may include something additional e.g. X + Y. The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y or it may mean that Y is present in the composition, if at all, as an incidental impurity at a level that does not effect the properties of the composition. In some implementations, the term “comprising” refers to the inclusion of the indicated active agent, such as recited polypeptides, as well as inclusion of other active agents, and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc., as are known in the pharmaceutical industry. In some implementations, the term “consisting essentially of” refers to a composition, whose only active ingredient is the indicated active ingredient(s), for example antigens, however, other compounds may be included which are for stabilizing, preserving, etc. the formulation, but are not involved directly in the therapeutic effect of the indicated active ingredient. Use of the transitional phrase “consisting essentially” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, and those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term “consisting essentially of” when used in a claim is not intended to be interpreted to be equivalent to “comprising”. The term “consisting of” and variations thereof means “limited to” unless expressly specified otherwise. In certain territories, the term “comprising an active ingredient consisting of” may be used in place of “consisting essentially”. The term “about” in relation to a numerical value x means, for example, x±10%, x±5%, x±4%, x±3%, x±2%, x±1%. Where methods refer to steps, for example as (a), (b), (c), etc., these are intended to be sequential, i.e., step (c) follows step (b) which is preceded by step (a). Unless specifically stated however, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc. NUMBERED EMBODIMENTS Embodiment 1. A process for substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising outer membrane vesicles (OMVs), the process comprising: (a) concentrating and washing the sample by tangential flow filtration (TFF) through a TFF membrane to obtain a retentate concentrate comprising the OMVs; (b) filtering the retentate concentrate by flow-through chromatography to obtain a flow-through of purified OMVs using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa, thereby obtaining purified OMVs. Embodiment 2. The process of embodiment 1, wherein the OMVs are obtained from Gram negative bacteria. Embodiment 3. The process of embodiment 2, wherein the OMVs are obtained from: (i) Bordetella bacteria, optionally from Bordetella pertussis, optionally wherein the bacterium is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G and/or wherein the bacterium is a recombinant bacterium that produces lipid A having (I) C3’ acyl chains of about 10 carbon atoms (C10) in length; and/or (II) C2’ acyl chains of about 10 carbon atoms (C10) in length; and/or (III) C2 acyl chains of about 10 carbon atoms (C10) in length; or (ii) Neisseria bacteria, optionally from Neisseria gonorrhoeae, optionally wherein the bacterium is a genetically modified gonococcal bacterium, for example comprises genetic modification(s) that reduce the endotoxin activity compared to a wild-type and/or unmodified Neisseria gonorrhoeae. Embodiment 4. The process of any one of embodiments 1 to 3 wherein the TFF membrane has a molecular weight cut off of about 500 kDa. Embodiment 5. The process of any one of embodiments 1 to 3 wherein the TFF membrane is a hollow fibre membrane having a molecular weight cut off of about 750 kDa or about 300 kDa. Embodiment 6. The process of embodiment 4 or 5 wherein the TFF of step (a) comprises concentrating and washing the sample by at least one ultrafiltration cycle and at least one cycle of diafiltration. Embodiment 7. The process of embodiment 6 wherein the at least one ultrafiltration cycle concentrates the sample by at least about 5 times, for example about 10 times. Embodiment 8. The process of embodiment 7 wherein the at least one cycle of diafiltration washes the sample with at least 5 volumes of a wash solution, for example 10 volumes of a wash solution, optionally wherein the bacterium is a Bordetella bacterium, wherein the wash solution is selected from (1) Dulbecco's phosphate-buffered saline (DPBS) optionally comprising EDTA, for example about 5mM EDTA, or (2) 20mM Tris buffer pH 8.6 optionally comprising sucrose, for example about 3% sucrose; or, optionally wherein the bacterium is a Neisseria bacterium, the wash solution is selected from (1) 10mM Na2HPO4/NaH2PO4 pH 6.8 comprising 150mM NaCl or (2) phosphate-buffer saline (PBS) pH 7.4. Embodiment 9. The process of any one of the preceding embodiments, wherein the sample of OMVs is obtained by deoxycholate (DOC) disruption of the outer membrane of Bordetella bacteria. Embodiment 10. The process of embodiment 9, wherein the sample comprising OMVs is prepared by a process comprising the steps of (i) homogenising a fermentation harvest of Bordetella bacteria, thereby obtaining a homogenised fermentation harvest; (ii) treating the homogenised fermentation harvest with DOC and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; and (iii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce the sample comprising OMVs. Embodiment 11. The process of embodiment 10, wherein the homogenised fermentation harvest is treated with DOC and benzonase. Embodiment 12. The process of any one of embodiments 1 to 8, wherein the sample comprising OMVs comprises or consists of OMVs released in the medium. Embodiment 13. The process of embodiment 12, wherein the sample comprising OMVs is prepared by a process comprising the steps of (i) centrifuging a fermentation harvest, optionally of Neisseria bacteria; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce the sample comprising OMVs. Embodiment 14. The process of any one of the preceding embodiments, further comprising the steps of: (a’) filtering the retentate to remove precipitate, and/or (c) sterile filtering the purified OMVs through a 0.22µm filter to produce a sterile preparation comprising purified OMVs. Embodiment 15. A process for preparing OMVs from Bordetella bacteria, the process comprising: (i) homogenising a fermentation harvest of Bordetella bacteria, thereby obtaining a homogenised fermentation harvest comprising the Bordetella bacteria; (ii) treating the homogenised fermentation harvest with DOC to disrupt the outer membrane of the Bordetella bacteria and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; (ii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce a sample containing DOC-extracted OMVs and one or more impurities; (iv) removing or reducing the concentration of the one or more impurities by (v) concentrating and washing the sample containing DOC-extracted OMVs and one or more impurities by TFF through a TFF membrane to obtain a retentate concentrate comprising OMVs and (vi) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs; and (vii) sterile filtering the flow-through of purified OMVs using a 0.22µm filter. Embodiment 16. The process of embodiment 15 wherein the Bordetella bacteria is Bordetella pertussis. Embodiment 17. The process of embodiment 16, wherein the Bordetella pertussis is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G and/or is a recombinant bacterium that produces lipid A having (i) C3’ acyl chains of about 10 carbon atoms (C10) in length; and/or (ii) C2’ acyl chains of about 10 carbon atoms (C10) in length; and/or (iii) C2 acyl chains of about 10 carbon atoms (C10) in length. Embodiment 18. A process for preparing OMVs from Neisseria bacteria, the process comprising the sequential steps of: (i) centrifuging a fermentation harvest ; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce a sample containing OMVs and one or more impurities; (vi) removing or reducing the concentration of the one or more impurities by (vii) concentrating and washing the sample containing OMVs and one or more impurities by TFF through a membrane to obtain a retentate concentrate comprising OMVs and (viii) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs; and (ix) sterile filtering the flow-through of purified OMVs using a 0.22µm filter. Embodiment 19. The process of embodiment 18, wherein the Neisseria bacteria is Neisseria gonorrhoeae. Embodiment 20. The process of embodiment 18 or 19, wherein the Neisseria gonorrhoeae is a Neisseria gonorrhoeae bacterium that comprises genetic modification(s), optionally wherein said genetic modification(s) reduce the endotoxin activity of the OMVs compared to a wild-type or unmodified Neisseria gonorrhoeae. Embodiment 21. The process of embodiment 20 wherein said geneitic modification(s) that reduce the endotoxin activity results in increased levels of pentaacylated lipid A and decreased levels of hexaacylated lipid A compared to the lipid A present in a wild-type and/or unmodified Neisseria gonorrhoeae. Embodiment 22. The process of embodiment 21, wherein said genetic modification(s) decrease or abolish expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide. Embodiment 23. The process of embodiment 22 wherein the Neisseria gonorrhoeae bacterium comprises a further genetic modification that decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide. Embodiment 24. The process of any preceding embodiments wherein the one or more impurities comprise or is the 60kDa chaperonin GroEL. Embodiment 25. A process for reducing the concentration of the 60kDa chaperonin GroEL in a sample of OMVs, optionally from Bordetella or Neisseria, the process comprising: filtering the sample of OMVs by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs wherein the 60kDa chaperonin GroEL is present in an amount less than 1.5% of total protein. Embodiment 26. The process of embodiment 24 or 25, wherein the 60kDa chaperonin GroEL comprises a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of SEQ ID NO: 1-4, optionally wherein the 60kDa chaperonin GroEL comprises or has the sequence of anyone of SEQ ID NO: 1-4. Embodiment 27. A population of purified OMVs produced by a process according to any one of the preceding embodiments. Embodiment 28. A population of purified OMVs that is at least 85% pure or from about 85% to about 100% pure. Embodiment 29. The population of purified OMVs of embodiment 27 or 28, wherein the purified OMVs are at least 95% pure or from about 95% to about 100% pure. Embodiment 30. The population of purified OMVs of embodiment 28 or 29, wherein purity is measured using size exclusion Ultra-performance liquid chromatography (UPLC), for example by size exclusion UPLC to determine the ratio between the main peak and the low molecular size peak. Embodiment 31. The population of purified OMVs of any one of embodiments 28 to 30, wherein one or more impurities are present at a level or concentration of less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than about 1% of impurities, for example free of impurities, such as protein impurities. Embodiment 32. The population of purified OMVs of any one of embodiments 28 to 31, wherein the one or more impurities comprise or is the 60kDa chaperonin GroEL. Embodiment 33. The population of purified OMVs of embodiment 32, wherein the 60kDa chaperonin GroEL comprise a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of SEQ ID NO: 1-4, optionally wherein the 60kDa chaperonin GroEL comprises or has the sequence of anyone of SEQ ID NO: 1-4. Embodiment 34. The population of purified OMVs of embodiments 32 or 33, comprising from about 0.01% to about 10% of GroEL as a percentage of total protein. Embodiment 35. The population of purified OMVs of any one of embodiments 32 to 34, comprising less that 5% GroEL. Embodiment 36. The population of purified OMVs of any one of embodiments 27 to 35, wherein the population is substantially free of process related impurities. Embodiment 37. The population of purified OMVs of any one of embodiments 27 to 36, wherein the purified OMVs are obtained from Bordetella and are conjugated to a capsular polysaccharide of Haemophilus influenzae type b (Hib). Embodiment 38. The population of of purified OMVs of any one of embodiments 28 to 37, wherein the OMVs are obtained from Gram negative bacteria, optionally from: (i) Bordetella bacteria, optionally from Bordetella pertussis, optionally wherein the bacterium is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G and/or wherein the bacterium is a recombinant bacterium that produces lipid A having (I) C3’ acyl chains of about 10 carbon atoms (C10) in length; and/or (II) C2’ acyl chains of about 10 carbon atoms (C10) in length; and/or (III) C2 acyl chains of about 10 carbon atoms (C10) in length; or (ii) Neisseria bacteria, optionally from Neisseria gonorrhoeae, optionally wherein the bacterium is a genetically modified gonococcal bacterium, for example comprises genetic modification(s) that reduce the endotoxin activity compared to a wild-type and/or unmodified Neisseria gonorrhoeae. Embodiment 39. The population of purified OMVs of any one of embodiments 27 to 38 for use in medicine. EXAMPLES Example 1 – Fermentation and Deoxycholate (DOC) preparation of OMVs The frozen contents of an ampoule containing a working seed of Bordetella pertussis Tohama strain was thawed at room temperature for 10 minutes. Two 250 ml shake-flasks containing 30 ml fresh medium (adapted from Stainer and Scholte (J. Gen. Microbiol. 63:211-220 (1971)) by the addition of dimethylcyclodextrin 1 g/L and acid casein hydrolysate 10 g/L, the replacement of L-cystine 40 mg/L with L-cysteine 40 mg/L, and the use of higher concentrations of Na-L-Glutamate (11.84 g/L), reduced glutathione (150 mg/L) and ascorbic acid (400 mg/L)) were each inoculated with 400µl of working seed and incubated at 35° C. (+/−1° C.) and 150 rpm for 24 h (+/−1 h) to produce a first pre-culture. Once the first pre-cultures reached an optical density at 650 nm (OD650nm) of 1-1.5, they were used to inoculate two 3L shake-flasks containing 1L of fresh medium. The second pre-culture flasks were incubated at 35° C. (+/−1° C.) and 150 rpm for 24 h (+/−1 h), after which they were pooled. 1500ml of the pooled pre-culture was used to inoculate a 20 L-fermentor (Biolafitte™ ) containing 10L of fresh medium and 3ml antifoam agent. Batch fermentation was performed for 24h using the following parameters: temperature 35° C., head pressure 0.4 bar, air flow rate 20NL/min constant, pH 7.2 (regulated using acetic acid 50%) and stirring speed 50 rpm to 1000rpm max. During the fermentation, the temperature (35° C.) and head pressure (0.4 bar) were maintained at a constant level. A mechanical foam breaker was used to control foaming during the fermentation. The air flow rate was progressively increased during the fermentation, according to a pre-defined curve. The level of dissolved oxygen was set at 25% and regulated by increasing stirring when the DO fell below 25%. The minimum stirring speed was set at 50 rpm; the maximum stirring speed was set at 1000 rpm. The pH was regulated at 7.2 by addition of acetic acid 50% (w/v or weight/volume). During fermentation, growth of the culture was monitored as OD650nm. Fermentation was stopped once the oxygen consumption decreased (as a consequence of glutamate exhaustion) resulting in a decrease in stirring speed. At the end of fermentation, cells were pelleted by centrifugation at 5000xg for 30 minutes (4°C). The supernatant was filtered with a Sartobran-P (0.45µm + 0.22µm) filter and dispensed into 1L flasks. The supernatant and cell pellet was stored at -20°C. For detergent extraction of OMV, the frozen bacterial pellets were thawed, resuspended and homogenised in four volumes of 20mM Tris-HCl - 2mM EDTA buffer (pH 8,6), 100 U/mL benzonase with cOmplete™ EDTA-free Protease Inhibitor Cocktail and incubated with stirring for 30 min at room temperature. One volume of either extraction buffer 1 (20 mM TrisHCl, 0.5% DOC, 2mM EDTA, pH 8.6 buffer) or extraction buffer 2 (20 mM TrisHCl, 0.1% DOC, 2mM EDTA, pH 8.6 buffer) was added with cOmplete™ EDTA-free Protease Inhibitor Cocktail and the suspension was incubated with stirring for 30 min at a temperature of either 30°C or 40°C. After 30 minutes, 100U/mL Benzonase was added. The suspension was incubated and stirred for a further 30 minutes. Cellular debris was removed by centrifugation at 20,000g for 30 min (4°C), the supernatant containing the OMVs was removed and sterile filtered (0.22µm). Each 250 ml of solution comprising DOC extracted OMVs was diluted to 500 ml with OMV buffer (Tris 20mM pH 8.6 + 3% sucrose). The generalised DOC extraction procedure used is shown in Fig. 1. Example 2 Preparation of OMVs using 0.1% DOC at 30ºC, Purification by TFF and Ultracentrifugation DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (Extraction Buffer 2) at 30°C. The preparation of OMVs was purified using a combination of TFF and Ultracentrifugation (Fig. 2). In a first purification step, TFF was performed using a Millipore 500kDa Cassette as follows: The preparations were concentrated 10-fold to 50 ml (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). The concentrated preparation was diafiltered at constant volume (50 ml) with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). 1550 U of benzonase (Merck 327 U/ul) was added to the retentate containing the OMVs and stirred for 1h (Pump flow 50ml/min, permeate valve closed, no counterpressure). The concentrated preparation was further diafiltered with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi) and 50 ml of retentate was recovered in reverse flow. The system was washed with a further 50 ml of OMV buffer and the wash was pooled with the retentate to yield a final volume of about 100ml (retentate + wash). Following TFF, the pooled retentate was centrifuged twice for 2 hours at 150,000g, supernatant was removed and the pellet comprising OMVs was resuspended in 30ml of DPBS, aliquoted in 1ml and 15 ml aliquots and stored at -70°C. Lipid concentration was measured using the fluorescent dye FM 4-64 [N-(3- Triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide] (also referred to as FM 4-64, Molecular Probes) to generate a standard calibration curve versus known amounts of DOPC. FM 4-64 is a lipophilic styryl dye that selectively intercalates into the lipid membrane staining them with red fluorescence (excitation/emission maxima ~515/640 nm). Protein content was determined using the Lowry method known in the art.
Figure imgf000045_0001
TFF followed by ultracentrifugation eliminated free proteins and aggregates (reducing protein content by around 50%) whilst generally retaining OMVs (lipid content in the final sample was reduced by about 12%). However, purity (85%) and yield (25mg/L of fermentation culture based on protein content) were both low and the 60kDa chaperonin, GroEL, was not removed (Fig. 3). In addition, following ultracentrifugation, the pellet was difficult to resuspend such that sterile filtration was not possible. Example 3 Preparation of OMVs using 0.1% DOC at 40ºC, Purification by TFF and Ultracentrifugation DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (Extraction Buffer 2) at 40°C. The preparation of OMVs was purified using a combination of TFF and Ultracentrifugation as before (Fig. 2). Increasing the temperature during DOC extraction had no impact on subsequent purification using TFF followed by ultracentrifugation. The resulting pellet was very hard to resuspend and contained a high level of precipitate such that sterile filtration was not possible. The resulting yield was also low at 17mg/L of fermentation culture (based on protein content) and the 60kDa chaperonin was not removed. In view of the low recovery and purity obtained, ultracentrifugation as a second step of a two-step purification process following TFF does not appear to be viable for production of OMVs on a manufacturing scale. Example 4 Preparation of OMVs using 0.1% DOC at 40ºC, Purification by TFF and flow- through chromatography (Sephacryl S-500 gel filtration) DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (Extraction Buffer 2) at 40°C. The preparation of OMVs was purified using a combination of TFF and flow-through chromatography (Sephacryl S-500 gel filtration) (Fig.4). TFF was performed using a Millipore 500kDa Cassette as follows: The DOC extracted OMVs were concentrated 10-fold to 50 ml (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). The concentrated preparation was diafiltered at constant volume (50 ml) with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). 1550 U of benzonase (Merck 327 U/ul) was added to the retentate containing the OMVs and stirred for 1h (Pump flow 50ml/min, permeate valve closed, no counterpressure). The concentrated preparation was further diafiltered with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi) and 50 ml of retentate was recovered in reverse flow. The system was washed with a further 50 ml of OMV buffer and the wash was pooled with the retentate to yield a final volume of about 100ml (retentate + wash). Following TFF, further purification was performed by gel filtration using Sephacryl S-500. Pooled retentate was applied to a Sephacryl S-500 High Resolution chromatography column (10 mm x 450 mm x 2. = 70,6 ml). Washing was performed with OMV buffer and the OMVs were eluted in the flow- through (flow-through + Wash = 115 ml). Eluate was pooled and sterile-filtered under laminar flow using a Sterivex GV 0.22 um filter (10 cm2 surface, Millipore) and aliquoted in 1ml and 15 ml aliquots. Aliquots were stored at -70°C. The OMV yield was improved by replacing ultracentrifugation with Sephacryl S-500 gel filtration (yield 35.7 mg/L). However, the 60kDa chaperonin was not completely removed (data not shown). In addition, to process 150ml of OMV preparation, a 3L column is necessary implying that there are volume constraints for upscale of the process to an industrial level. Example 5 Preparation of OMVs using 0.1% DOC at 40ºC, Purification by TFF and flow- through chromatography (CaptoCore 700 gel filtration) DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (Extraction Buffer 2) at 40°C. The preparation of OMVs was purified using a combination of TFF and flow-through chromatography (CaptoCore 700 gel filtration) (Fig. 5). TFF was performed using a Millipore 500kDa Cassette as follows: The DOC extracted OMVs were concentrated 10-fold to 50 ml (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). The concentrated preparation was diafiltered at constant volume (50 ml) with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi). 1550 U of benzonase (Merck 327 U/ul) was added to the retentate containing the OMVs and stirred for 1h (Pump flow 50ml/min, permeate valve closed, no counterpressure). The concentrated preparation was further diafiltered with 500ml of OMV buffer (Pump flow 125ml /min, TMP 5 +/- 0.5 psi) and 50 ml of retentate was recovered in reverse flow. The system was washed with a further 50 ml of OMV buffer and the wash was pooled with the retentate to yield a final volume of about 100ml (retentate + wash). Following TFF, further purification was performed by gel filtration using CaptoCore 700. The pooled retentate was applied to a CaptoCore700 (GE Healthcare) column (109 ml column volume, radius 1,3 cm, pre-equilibrated in OMV buffer) at 11,5 ml/min (130 cm/h). Washing was performed with OMV buffer at the same flow rate. Solution containing the OMVs eluted in the flow-through (flow- through + Wash = 115 ml). Eluate was pooled and sterile-filtered under laminar flow using a Sterivex GV 0.22 um filter (10 cm2 surface, Millipore) and aliquoted in 1ml and 15 ml aliquots. Aliquots were stored at -70°C. The use of CaptoCore 700 improved both yield (44mg/L) and purity (>95%) of the OMV preparation. The process also significantly reduced the amount of 60kDa chaperonin (GroEL) (Fig. 6). Further, in order to process 150 ml of OMV preparation, a typical TFF retentate, a 130 ml column (or smaller) is required meaning that the process is industrially scaleable. Example 6 Preparation of OMVs using 0.5% DOC at 30ºC, Purification by TFF and CaptoCore 700 DOC extracted outer membrane vesicles from Bordetella pertussis were prepared according to Example 1 using 0.5% DOC (Extraction Buffer 1) at 30°C. The preparation of OMVs was purified using a combination of TFF and CaptoCore as before (Fig. 5). Increasing the concentration of DOC to 0.5% and reducing the temperature to 30°C during extraction had a significant impact on yield which increased to 203 mg/L of culture. In addition, the process resulted in a preparation that almost completely removed GroEL and free-proteins not associated with the OMVs (purity >95%) (Fig. 7(a) and (b)). Example 7 Comparison of OMVs purified using different processes/conditions OMVs were prepared and purified using the conditions described below following the methods provided above:
Figure imgf000048_0001
Harsher extraction conditions lead to increased ratio proteins/lipids and increased DLS measured size. However polydispersity remained low (Pdi<0,3). Example 8 Preparation of OMVs using 0.5% DOC at 30ºC, Purification by TFF (750kDa) and CaptoCore 700 In this experiment, the purification process of Example 5 was repeated four times but the 500kDa filter used in the TFF step was substituted with a 750kDa hollow fibre filter (Fig. 8), (GE Healthcare, UFP-750-C-H24LA, Surface 0,0042 m2, Dead volume 12 ml, Buffer Tris 20mM – Sucrose 3% (P/V) pH 8,6, Final recovered volume ca. 100ml):
Figure imgf000049_0001
Compared to processes using a standard cassette for TFF, the yield obtained with Hollow Fiber was generally higher (purity >95%) and a further reduction in impurities was seen (Fig. 9). SE-UPLC profiles indicated excellent reproducibility whilst DLS indicated good product homogeneity:
Figure imgf000049_0002
Despite the pore size, the 750 kDa hollow fibre membrane alone could not remove the 60kDa chaperonin from OMV preparations - a CaptoCore700 step was still necessary (Fig. 10). SDS PAGE of OMVs purified with a hollow fibre membrane showed a similar but not identical pattern to OMVs purified with the 500kDa cassette (Fig. 11). Substituting 500 kDa cassettes with 750kDa hollow fibre membranes for TFF resulted in an increase in yield, purity and homogeneity of OMVs. Reproducibility was also excellent, both in terms of protein yield and product quality. Example 9 Evaluation of Chaperonin 60kDa removal on CaptoCore 700 column The removal of the 60kDa chaperonin GroEL by CaptoCore 700 was determined using mass analysis. The relative amount of GroEL in each sample was measured using mass spectrometry (MS) as 9.4% of total protein in the ultrafiltration retentate (i.e. before filtration) and and 1.06% of total protein in the ultrafiltration permeate (i.e. after filtration). Using the Lowry assay, total protein in the ultrafiltration retentate was 230.6 mg whilst total protein in the ultrafiltration permeate was 36.5 mg. Thus, 21.7mg of the 60kDa chaperonin GroEL was present in the ultrafiltration retentate whilst 0.39mg was present after filtration in the ultrafiltration permeate. Around 98,3% of the 60kDa chaperonin GroEL is removed from the sample by the CaptoCore 700 filtration step (clearance is around 56 times) (Fig. 12). Example 10: manufacturing process of Gonococcal OMVs Upstream process comprised the steps of inoculum, fermentation, recovery & filtration steps. During the preculture step a flask was inoculated with Neisseria gonorrhoeae liquid seed vial. The number of expansion steps (number and volume of flasks and/or pre-fermenter) depended from the final scale of fermentation. The passages of seed expansions and fermenter inoculum were performed when the culture was in full exponential growth. During a batch fermentation biomass was formed and the product, i.e. GMMA, was released in the medium. When the carbon sources in the media were depleted, the fermentation was harvested and primary recovery was performed by centrifugation: pellet containing biomass was discarded and the supernatant, containing the product, was filtered at 0,2 μm (Filtration 1) to reduce bioburden (complete pathogen cell removal). This intermediate was processed by adding benzonase in order to hydrolyze genomic DNA. The material was filtered (filtration 2a) to remove pathogen cells from the supernatant and a holding time at 2-8°C was applied in order to execute the DNA hydrolysis before the purification process. Before purification, the supernatant was clarified by 0,2 μm filtration (Filtration 2) removing possible precipitate and the resulting intermediate was named SNF-BF. The TFF was performed achieving a product purity improvement by reduction of soluble proteins (intended as gonococcus proteins not belonging to GMMA), nucleic acids and residual of fermentation medium. The diaretentate was collected and filtered to remove precipitate (Filtration 3). The resulting intermediate was named DR, starting material for the next chromatographic steps. Capto Core 700 was used as resin for chromatographic step operated in flow-through mode to remove soluble proteins and DNA which are bound to the resin. The resulting intermediate was the Capto Core 700 eluate (CC700EL2). Before performing a 0,2 μm orthogonal filtration the CC700EL2 was diluted, if needed, to a total protein concentration targeted at 1,2 ± 0,1 mg/ml in order to maximize the volume of DS. After Filtration 4 the intermediate obtained was named Concentrated Bulk (CB), this solution was filled into suitable containers and frozen at -70°C as low bioburden material. The description of the manufacturing process for the DS is outlined in Figure 13. Example 11: Technical details of Capto Core 700 chromatography The purpose of this Capto Core chromatography step is to increase the purity in tems of DNA and other proteins removal (benzonase and soluble proteins). The DR intermediate was applied to two CaptoCore700 columns connected in series (column volume (CV ~ 1 L) previously equilibrated with 20 CV of PBS pH 7.4 at 300-500 cm/h. Washing was performed with the same buffer at the same flow rate. Chromatography was performed as flow- throught mode, so the start of peak collection is in the load block and the end in rinse block. The peak collection was performed manually (without method instruction) following the UV signal at 280 nm. Example 12: Implementation of Capto Core 700 in the manufacturing process of Gonococcal OMVs The manufacturing process of the GMMA-FA1090-2KO drug substance was improved by the substitution of an untracentrifugation step between Filtration 3 and Filtration 4 with Capto Core 700 (multimodal chromatography). The GMMA-FA1090-2KO drug substance comprises a Neisseria gonorrhoeae bacterium is from strain FA1090 and comprises genetic modification(s) that (I) decreases or abolishes expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide; and (II) decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide. A slight decrease of yield was observed using Capto Core 700 chromatography (yield = 55%) instead an ultracentrifugation steps (yield = 68 %). Yield was evaluated by measuring OMV content by SEC-Dye HPLC method, based on quantification of FM4-64 on samples and standard GMMA preparations with known protein concentration after isochratic SEC chromatography. An increase of purity was observed using Capto Core 700 column instead an ultracentrifugation steps as shown in Fig. 14, with the GroEL protein impurity being completely removed, as shown in Fig. 15. It will be understood that the subject matter has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the subject matter.
SEQUENCES SEQ ID NO:1: 60 kDa chaperonin “GroEL”; Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251)
Figure imgf000052_0001
SEQ ID NO: 2: molecular chaperone GroEL [Neisseria gonorrhoeae FA 1090] GenBank: AAW90696.1
Figure imgf000052_0002
SEQ ID NO: 3: molecular chaperone GroEL [Neisseria meningitidis NZ98/254]
Figure imgf000052_0003
SEQ ID NO: 4: molecular chaperone GroEL [Neisseria meningitidis 99M]
Figure imgf000052_0004

Claims

CLAIMS 1. A process for substantially removing or reducing the concentration or amount of one or more impurities from a sample comprising outer membrane vesicles (OMVs), the process comprising: (a) concentrating and washing the sample by tangential flow filtration (TFF) through a TFF membrane to obtain a retentate concentrate comprising the OMVs; (b) filtering the retentate concentrate by flow-through chromatography to obtain a flow-through of purified OMVs using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa, thereby obtaining purified OMVs.
2. The process of claim 1 wherein the TFF membrane has a molecular weight cut off of about 500 kDa.
3. The process of claim 1 wherein the TFF membrane is a hollow fibre membrane having a molecular weight cut off of about 750 kDa or about 300 kDa. 4. The process of claim 2 or 3 wherein the TFF of step (a) comprises concentrating and washing the sample by at least one ultrafiltration cycle and at least one cycle of diafiltration, optionally wherein the at least one ultrafiltration cycle concentrates the sample by at least about 5 times, for example about 10 times, optionally wherein the at least one cycle of diafiltration washes the sample with at least 5 volumes of a wash solution, for example 10 volumes of a wash solution, optionally wherein the bacterium is a Bordetella bacterium, wherein the wash solution is selected from (1) Dulbecco's phosphate-buffered saline (DPBS) optionally comprising EDTA, for example about 5mM EDTA, or (2) 20mM Tris buffer pH 8.6 optionally comprising sucrose, for example about 3% sucrose; or, optionally wherein the bacterium is a Neisseria bacterium, the wash solution is selected from (1) 10mM Na2HPO4/NaH2PO4 pH 6.8 comprising 150mM NaCl or (2) phosphate-buffer saline (PBS) pH 7.
4.
5. The process of any one of the preceding claims, wherein the sample of OMVs is obtained by deoxycholate (DOC) disruption of the outer membrane of Bordetella bacteria, optionally wherein the sample comprising OMVs is prepared by a process comprising the steps of (i) homogenising a fermentation harvest of Bordetella bacteria, thereby obtaining a homogenised fermentation harvest; (ii) treating the homogenised fermentation harvest with DOC and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; and (iii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce the sample comprising OMVs.
6. The process of any one of claims 1 to 5, wherein the sample comprising OMVs comprises or consists of OMVs released in the medium, optionally wherein the sample comprising OMVs is prepared by a process comprising the steps of (i) centrifuging a fermentation harvest, optionally of Neisseria bacteria; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce the sample comprising OMVs.
7. The process of any one of the preceding claims, further comprising the steps of: (a’) filtering the retentate to remove precipitate, and/or (c) sterile filtering the purified OMVs through a 0.22µm filter to produce a sterile preparation comprising purified OMVs.
8. A process for preparing OMVs from Bordetella bacteria, the process comprising: (i) homogenising a fermentation harvest of Bordetella bacteria, thereby obtaining a homogenised fermentation harvest comprising the Bordetella bacteria; (ii) treating the homogenised fermentation harvest with DOC to disrupt the outer membrane of the Bordetella bacteria and optionally benzonase to produce a crude preparation of DOC-extracted OMVs; (ii) centrifuging and/or filtering the crude preparation of DOC-extracted OMVs to produce a sample containing DOC-extracted OMVs and one or more impurities; (iv) removing or reducing the concentration of the one or more impurities by (v) concentrating and washing the sample containing DOC-extracted OMVs and one or more impurities by TFF through a TFF membrane to obtain a retentate concentrate comprising OMVs and (vi) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs; and (vii) sterile filtering the flow-through of purified OMVs using a 0.22µm filter.
9. A process for preparing OMVs from Neisseria bacteria, the process comprising the sequential steps of: (i) centrifuging a fermentation harvest ; (ii) collecting the supernatant; (iii) filtering the supernatant to obtain an intermediate; (iv) treating the intermediate with benzonase to produce a crude preparation; and (v) filtering the crude preparation to produce a sample containing OMVs and one or more impurities; (vi) removing or reducing the concentration of the one or more impurities by (vii) concentrating and washing the sample containing OMVs and one or more impurities by TFF through a membrane to obtain a retentate concentrate comprising OMVs and (viii) filtering the retentate concentrate by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs; and (ix) sterile filtering the flow-through of purified OMVs using a 0.22µm filter.
10. The process of any preceding claim wherein the one or more impurities comprise or is the 60kDa chaperonin GroEL.
11. A process for reducing the concentration of the 60kDa chaperonin GroEL in a sample of OMVs, optionally from Bordetella or Neisseria, the process comprising: filtering the sample of OMVs by flow-through chromatography using a chromatographic substrate comprising beads having a porous outer layer, an inner core comprising octylamine ligands, and a molecular weight cut off of about 700 kDa to obtain a flow-through of purified OMVs wherein the 60kDa chaperonin GroEL is present in an amount less than 1.5% of total protein.
12. The process of claim 10 or 11, wherein the 60kDa chaperonin GroEL comprises a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of SEQ ID NO: 1-4, optionally wherein the 60kDa chaperonin GroEL comprises or has the sequence of anyone of SEQ ID NO: 1-4.
13. A population of purified OMVs produced by a process according to any one of the preceding claims.
14. A population of purified OMVs that is at least 85% pure or from about 85% to about 100% pure, optionally wherein purity is measured using size exclusion Ultra-performance liquid chromatography (UPLC), for example by size exclusion UPLC to determine the ratio between the main peak and the low molecular size peak.
15. The population of purified OMVs of claims 13 or 14, wherein the purified OMVs are at least 95% pure or from about 95% to about 100% pure.
16. The population of purified OMVs of any one of claims 13 to 15, wherein one or more impurities are present at a level or concentration of less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than about 1% of impurities, for example free of impurities, such as protein impurities.
17. The population of purified OMVs of any one of claims 13 to 16, wherein the one or more impurities comprise or is the 60kDa chaperonin GroEL, optionally wherein the 60kDa chaperonin GroEL comprise a polypeptide having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to anyone of SEQ ID NO: 1-4, optionally wherein the 60kDa chaperonin GroEL comprises or has the sequence of anyone of SEQ ID NO: 1-4.
18. The population of purified OMVs of claim 17, comprising from about 0.01% to about 10% of GroEL as a percentage of total protein.
19. The population of purified OMVs of any one of claims 13 to 18, wherein the population is substantially free of process related impurities.
20. The population of purified OMVs of any one of claims 13 to 19, wherein the purified OMVs are obtained from Bordetella and are conjugated to a capsular polysaccharide of Haemophilus influenzae type b (Hib).
21. The process of any one of claims 1 to 12, the population of purified OMVs of any one of claims 13 to 20, wherein the OMVs are obtained from Gram negative bacteria or wherein the bacteria is: (i) Bordetella bacteria, optionally Bordetella pertussis, optionally wherein the bacterium is a recombinant bacterium comprising an S1 gene comprising the mutations R9K and E129G and optionally expressing the genetically detoxified pertussis toxoid PT- 9K/129G and/or wherein the bacterium is a recombinant bacterium that produces lipid A having (I) C3’ acyl chains of about 10 carbon atoms (C10) in length; and/or (II) C2’ acyl chains of about 10 carbon atoms (C10) in length; and/or (III) C2 acyl chains of about 10 carbon atoms (C10) in length; or (ii) Neisseria bacteria, optionally Neisseria gonorrhoeae, optionally wherein the bacterium is a genetically modified gonococcal bacterium, for example comprises genetic modification(s) that reduce the endotoxin activity compared to a wild-type and/or unmodified Neisseria gonorrhoeae, optionally wherein said geneitic modification(s) that reduce the endotoxin activity results in increased levels of pentaacylated lipid A and decreased levels of hexaacylated lipid A compared to the lipid A present in a wild-type and/or unmodified Neisseria gonorrhoeae, optionally wherein said genetic modification(s) decrease or abolish expression and/or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and/or polypeptide, optionally wherein the Neisseria gonorrhoeae bacterium comprises a further genetic modification that decreases or abolishes expression and/or function of the reduction modifiable protein (rmp) gene, mRNA, and/or polypeptide.
22. The population of purified OMVs of any one of claims 13 to 21 for use in medicine.
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