Purification method

Abstract

A purification method is provided for removing impurities, such as GroEL, from a sample of outer membrane vesicles (OMV). For example, the method is applicable to OMV derived from Bordetella or Neisseria, such as Neisseria gonorrhoeae. Also provided are OMV purified by the methods described herein and immunogenic compositions, such as vaccines, containing the same.

Description

Technical Field

[0001] Sequence Listing This application contains an electronically submitted sequence listing (VB66853WO01.xml, size: 6,434 bytes, and creation date: July 18, 2023) which is hereby incorporated by reference in its entirety.

[0002] Field of the Invention There is provided a method in the field of purification methods, for example, a method for purifying outer membrane vesicles (OMVs). Also provided is a method for removing one or more impurities from a sample of OMVs. Also provided are OMVs purified by such methods and immunogenic compositions, such as vaccines, containing such OMVs.

Background Art

[0003] Background of the Invention Gram-negative bacteria can spontaneously release OMVs from their outer membranes during growth, and these are referred to as natural OMVs (nOMVs). The formation of nOMVs can be promoted by modification of certain bacterial components (International Publication No. 2006 / 046143, Berlanda Scorza et al. (2008) Mol Cell Proteomics 7:473-85), whereby the bacteria are genetically engineered to exhibit a hyperblebbing phenotype and produce nOMVs, and thus they are also referred to as generalized modules of membrane antigens (GMMA). OMVs can also be produced by disruption of the whole bacteria. Examples of OMV production methods include the use of detergent treatment to produce OMVs referred to as detergent-extracted OMVs (dOMVs) (European Patent No. 0011243, Fredriksen et al. (1991) NIPH Ann. 14(2):67-80), detergent-free methods (International Publication No. 2004 / 019977), sonication (Hozbor et al. (1999) Curr Microbiol 38:273-8), and the like.

[0004] OMVs are rich in immunogenic cell surface - associated antigens, periplasmic antigens, and secreted antigens and are used, for example, in vaccines against Neisseria meningitidis serotype B (Tan et al. (2010) N Engl J Med. 362(16):1511 - 20). OMVs contain components that act as adjuvants and are suitable for this use because they induce a strong immune response against antigens. Thus, OMVs can mimic natural bacteria more closely than purified protein antigens or other bacterial components. However, because OMVs have a more complex structure derived from bacteria, they are usually more difficult to prepare than recombinant protein antigens.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, there is a need to provide an improved method for preparing OMVs, particularly for use in immunogenic compositions and vaccines.

Means for Solving the Problems

[0006] Summary of the Invention In one aspect, a method for substantially removing or reducing the concentration or amount of one or more impurities from a sample containing outer membrane vesicles (OMVs), comprising: (a) concentrating and washing the sample by tangential flow filtration (TFF) through a TFF membrane to obtain a residual concentrate containing OMVs; and (b) filtering the residual concentrate by flow - through chromatography using a chromatographic matrix comprising a porous outer layer, an internal core containing octylamine ligand, and beads having a molecular weight cut - off of about 700 kDa to obtain a flow - through of purified OMVs, thereby obtaining purified OMVs.

[0007] In a second aspect, a method for preparing OMV from bacteria of the genus Bordetella, comprising: (a) homogenizing a fermentation harvest of bacteria of the genus Bordetella to thereby obtain a homogenized fermentation harvest containing bacteria of the genus Bordetella; (b) treating the homogenized fermentation harvest with DOC (i.e., for disrupting the outer membrane of bacteria of the genus Bordetella) and optionally benzonase to produce a crude preparation of DOC-extracted OMV; (c) centrifuging and / or filtering the crude preparation of DOC-extracted OMV to produce a sample containing DOC-extracted OMV and one or more impurities; (d) (i) concentrating and washing a sample containing DOC-extracted OMV and one or more impurities by tangential flow filtration (TFF) through a membrane to obtain a retentate concentrate containing OMV and (ii) removing one or more impurities by filtering the retentate concentrate by flow-through chromatography using a chromatography matrix comprising a porous outer layer, an internal core containing octylamine ligand, and beads having a molecular weight cut-off of about 700 kDa to obtain a flow-through of purified OMV; and (e) sterile filtering the flow-through of purified OMV using a 0.22 μm filter or consisting essentially of such steps is provided.

[0008] In a third aspect, a method for preparing OMV from bacteria of the genus Neisseria, comprising the steps of: (i) recovering a fermentation broth 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; (v) filtering the crude preparation to produce a sample containing OMV and one or more impurities; (vi) removing or reducing the concentration of one or more impurities by the following: (vii) concentrating and washing a sample containing OMV and one or more impurities by TFF through a membrane to obtain a residual concentrate containing OMV; and (viii) filtering the residual concentrate by flow-through chromatography using a chromatographic matrix comprising a porous outer layer, an internal core containing an octylamine ligand, and beads having a molecular weight cut-off of about 700 kDa to obtain a flow-through of purified OMV; and (ix) sterile filtering the flow-through of purified OMV using a 0.22 μm filter. A continuous process is provided.

[0009] In a fourth aspect, a method for optionally reducing the concentration of the 60 kDa chaperonin GroEL in a sample of OMV derived from the genus Bordetella or Neisseria, comprising filtering a sample of OMV by flow-through chromatography using a chromatographic matrix comprising a porous outer layer, an internal core containing an octylamine ligand, and beads having a molecular weight cut-off of about 700 kDa to obtain a flow-through of purified OMV, wherein the 60 kDa chaperonin GroEL is present in an amount of less than 1.5% of the total protein. A method is provided.

[0010] In a fifth aspect, a population of purified OMV produced by the method according to any one of the first, second, third, or fourth aspects is provided.

[0011] In a sixth aspect, a population of purified OMVs that is at least 85% pure or about 85% to about 100% pure, optionally with a purity measured, for example, by size exclusion ultra performance liquid chromatography (UPLC) such as size exclusion UPLC, and determining the ratio between the major peak and the low molecular size peak, is provided.

[0012] In a seventh aspect, a population of purified OMVs for use in medicine is provided.

Brief Description of the Drawings

[0013]

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Mode for Carrying Out the Invention

[0014] Isolated bacterial OMVs have been proposed as components for use, for example, as immunogenic components in vaccines. However, it has been found that the reproducible production of a sufficient amount of sufficiently pure OMVs suitable for large-scale vaccine manufacture is difficult.

[0015] The inventors have developed a process suitable for purifying OMVs, such as a large-scale process or an industrial process. Accordingly, a method for substantially removing or reducing the concentration or amount of one or more impurities from a sample containing OMVs, comprising: (a) concentrating and washing the sample by tangential flow filtration (TFF) through a TFF membrane to obtain a residual concentrate containing OMVs; and (b) filtering the residual concentrate by flow-through chromatography using a chromatography matrix comprising a porous outer layer, an internal core containing an octylamine ligand, and beads having a molecular weight cut-off of about 700 kDa to obtain a flow-through of purified OMVs, thereby providing a method for obtaining purified OMVs. In some embodiments, the method is suitable for purifying OMVs by, for example, substantially removing or reducing the concentration or amount of one or more impurities from a sample containing OMVs. In some embodiments, the one or more impurities include protein impurities. In some embodiments, the one or more impurities include, or are, the chaperonin GroEL (GroEL) of 60 kDa. In one embodiment, the chaperonin GroEL of 60 kDa 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 any one of SEQ ID NOs: 1-4. In one embodiment, the chaperonin GroEL of 60 kDa comprises, or has, the sequence of any one of SEQ ID NOs: 1-4.

[0016] In some embodiments, the methods provided herein are methods of 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 in which the amount or concentration of one or more impurities in the purified OMVs is less than 10%, such as 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 one or more impurities in the sample comprising OMVs, as measured as a percentage of the total protein in the sample. In one embodiment, the methods provided herein remove at least 80% of one or more impurities from a sample comprising OMVs, such as 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 one or more impurities are removed, as measured as a percentage of the total protein content. In some embodiments, the methods provided herein reduce the amount of one or more impurities in a sample comprising OMVs to about one tenth, about one twentieth, about one thirtieth, about one fortieth, about one fiftieth, such as about one thirty to about one sixtieth, about one forty to about one sixtieth, about one fifty to about one sixtieth or less, as measured by the total protein content in the sample comprising OMVs. These values can be determined by comparing the concentration or amount of one or more impurities in the sample comprising OMVs after step (a), i.e., in the residual fraction concentrate, to that remaining in the sample comprising OMVs after step (b), i.e., in the flow-through of the purified OMVs. The terms "purifying" and "purification" are intended to refer to procedures in which the concentration or amount of at least one undesired compound(s), i.e., each "impurity(ies)", is reduced, such as removed, relative to the desired compound. In one embodiment, the impurity comprises GroEL, such as the impurity is GroEL, such as SEQ ID NOs: 1-4, and the desired compound is an OMV or a population of OMVs or a preparation of OMVs. In one embodiment, the concentration or amount of GroEL and / or other impurities can be determined using techniques known to those of skill in the art, such as by liquid chromatography-mass spectrometry (LC-MS), or by SDS-PAGE.In some embodiments, the methods provided herein are methods of reducing the amount or concentration of GroEL in a sample comprising OMV. In some embodiments, the methods provided herein are methods of substantially removing GroEL from a sample comprising OMV, e.g., as compared to the amount or concentration of GroEL in a sample comprising OMV prior to use of the method. In one embodiment, the methods provided herein are suitable for substantially reducing the amount or concentration of GroEL in a sample of OMV, e.g., as compared to the amount or concentration of GroEL in a sample comprising OMV prior to use of the method. In some embodiments, the amount or concentration of GroEL in the purified OMV 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 a sample comprising OMV when measured as a percentage of the total protein in the sample. In one embodiment, the methods provided herein remove at least 80% of GroEL from a sample comprising OMV when measured as a percentage of the total protein content, e.g., 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 GroEL is removed. In some embodiments, the methods provided herein reduce the amount of GroEL in a sample comprising OMV to about one-tenth, about one-twentieth, about one-thirtieth, about one-fortieth, about one-fiftieth, e.g., about one-thirtieth to about one-sixtieth, about one-fortieth to about one-sixtieth, about one-fiftieth to about one-sixtieth or less when measured by the total protein content in the sample comprising OMV. These values can be determined by comparing the concentration or amount of GroEL in the sample comprising OMV after step (a), i.e., in the residual fraction concentrate, to that remaining in the sample comprising OMV after step (b), i.e., in the flow-through of the purified OMV.

[0017] The methods provided herein reproducibly produce, batch-to-batch, preparations or populations of OMVs obtained by disruption of the outer membrane of Bordetella bacteria that have high purity (e.g., at least 95%) and / or a yield consistently higher than 40 mg / L (when measured against either protein and / or lipid content) and / or a narrow particle size distribution (polydispersity index or PDI) of less than 0.3. The methods provided herein consistently produce preparations or populations of OMVs released into the culture medium by, for example, bacteria of the genus Neisseria that have high purity (e.g., at least 90%, e.g., at least 91%, 92%, 93%, 94%, or 95%) and / or a yield higher than 40 mg / L (when measured against protein content) and / or a narrow particle size distribution (polydispersity index or PDI) of less than 0.3.

[0018] OMVs produced by the methods provided herein are sufficiently pure and suitable for use as an active ingredient of an immunogenic composition.

[0019] OMVs produced by processes known in the art generally contain a protein referred to as chaperonin or GroEL (bp3495, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4) with a molecular weight of 60 kDa. However, GroEL is not incorporated within the OMVs; rather, GroEL appears to be loosely associated with the OMV surface. 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 strong protective antigen, for example, in a murine aerosol model for pertussis. Thus, while purification processes known in the art appear to produce suitably pure OMVs with an acceptably low level of background contaminants / impurities, GroEL can nevertheless be present in an amount sufficient to influence the antibody response to other OMV-related antigens. Surprisingly, in addition to the advantages described above, the methods provided herein substantially reduce or substantially remove GroEL from samples of OMVs. Accordingly, the methods provided herein can be methods for reducing the amount and / or concentration of GroEL from samples of OMVs, for example, methods for substantially removing GroEL from samples of OMVs.

[0020] Preferably, the above method replaces the downstream processing steps for OMV purification that rely on ultracentrifugation, including density gradient centrifugation, sucrose cushion centrifugation, or others that may be difficult to perform on a larger scale. Thus, the method provided herein is suitable for the industrial-scale production of OMVs. Thus, in some embodiments, the method described herein is performed without or does not include the use of ultracentrifugation, such as ultracentrifugation having a centrifugal force greater than about 30,000×g or about 30,000×g, and optionally the method is performed without or does not include the use of ultracentrifugation after step (a). With respect to the industrial-scale production of OMVs, the inventors mean that the sample containing OMVs is prepared from a fermentation culture having a volume of about 20 liters to about 10,000 liters, about 25 liters to about 5,000 liters, about 25 liters to about 2,000 liters, about 50 liters to about 1,000 liters, greater than or equal to 20 liters, greater than or equal to 25 liters, greater than or equal to 50 liters, or greater than or equal to 100 liters, such as 20 liters, 25 liters, 50 liters, 100 liters, 500 liters, or 1,000 liters.

[0021] Outer membrane vesicles (OMV) OMVs are known in the art and can be produced by natural / spontaneous "blebbing" from the outer membrane of bacteria. Also, OMVs can be artificially prepared by mechanical and / or chemical disruption of bacterial cells to form vesicles.

[0022] In some embodiments, the OMVs are obtained from Gram-negative bacteria. In some embodiments, the OMVs are obtained from any genus of Gram-negative bacteria, such as any species from the genera Escherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia, Brucella, Chlamydia, Haemophilus, Legionella, Porphyromonas, Pseudomonas, Yersinia, Helicobacter, Salmonella, Vibrio, etc.

[0023] For example, the bacteria can 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-encapsulated strains), Legionella pneumophila, Neisseria gonorrhoeae, meningococcus, Neisseria lactamica, Porphyromonas gingivalis, Pseudomonas aeruginosa, Yersinia enterocolitica, Helicobacter pylori, Salmonella enterica (including serotypes typhi and typhimurium, and serotypes paratyphi and enteritidis), Shigella spp. (e.g., S. dysenteriae, S. flexneri, S. boydii or S. sonnei), Vibrio cholerae, and the like.

[0024] In one embodiment, the OMV is obtained from bacteria of the genus Bordetella, optionally from Bordetella pertussis. In one embodiment, the OMV is obtained from bacteria of the genus Neisseria, optionally from Neisseria gonorrhoeae. In one embodiment, the OMV is obtained from bacteria of the genus Porphyromonas, optionally from Porphyromonas gingivalis. In one embodiment, the OMV is obtained from bacteria of the genus Escherichia, optionally from Escherichia coli. In one embodiment, the OMV is obtained from bacteria of strain K or strain B of E. coli, and optionally the E. coli bacteria are selected from any one of strain BL21(DE3), BLR(DE3), and Escherichia coli HMS174(DE3). In one embodiment, the OMV is obtained from bacteria of the genus Salmonella, optionally from Salmonella typhi or Salmonella typhimurium.

[0025] In some embodiments, the bacteria are wild-type bacteria. In some embodiments, the bacteria are recombinant bacteria. For example, the bacteria are genetically modified to inactivate genes leading to a toxic phenotype, such as modification of natural lipid A structure, oligosaccharide core, or natural lipopolysaccharide (LPS) to disrupt the outer O antigen. The absence of the O antigen in LPS is as useful as the absence of hexaacylated lipid A. In some embodiments, the bacteria are genetically modified by mutation to reduce the pyrogenicity of the bacterial lipopolysaccharide (LPS). Specific mutations include, by way of non-limiting example, mutations in IpxLl, synX, IgtA, htrA, msbBl, msbB2, virG, lpxA and their homologs. Mutations suitable for down-regulating or abolishing expression include point mutations, gene deletions, gene insertions, and any modification of the genomic sequence that results in a change in gene expression, such as reduction, inactivation or silencing. In one embodiment, the mutation is a deletion.

[0026] Inactivation of toxins, for example to prevent the expression of Shiga toxin or pertussis toxin, is also suitable.

[0027] In some embodiments, the bacterium is a high bleb-forming bacterium. As used herein, the term "high bleb-forming" refers to a mutant strain of a bacterium that spontaneously releases more outer membrane vesicles (OMVs) (e.g., per unit time) than the wild-type or parental strain from which it is derived. Generally, a high bleb-forming variant releases more OMVs than the wild-type or parental strain from which it is derived, for example, more than 10%, more than 20%, more than 30%, or more than 40%. The high bleb-forming bacterium may be a naturally occurring mutant strain or may be genetically modified to exhibit a high bleb-forming phenotype. The term "wild-type" in relation to a bacterium refers to a bacterium that has not been chemically or genetically modified in any way (other than growth in a culture medium). Neisseria strains, such as Neisseria meningitidis and Neisseria gonorrhoeae, are non-limiting examples and may be genetically modified to exhibit a high bleb-forming phenotype by down-regulating or eliminating the expression of GNA33. Similar mutations are known in other bacteria, for example, Haemophilus influenzae, Moraxella catarrhalis, and Escherichia coli strains may be genetically modified to exhibit a high bleb-forming phenotype by down-regulating or eliminating the 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 may be genetically modified to exhibit a high bleb-forming phenotype by down-regulating or eliminating the expression of one or more tolR or OmpA. Suitable mutations for down-regulating or eliminating expression include point mutations, gene deletions, gene insertions, and any modification of the genomic sequence 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.

[0028] The high-burden-forming bacteria can be further genetically engineered by one or more processes selected from the following groups: (a) a process of down-regulating the expression of immunodominant variable antigens or non-protective antigens; (b) a process of up-regulating the expression of protective outer membrane protein (OMP) antigens; (c) a process of down-regulating genes involved in making the lipid A portion of LPS toxic; (d) a process of up-regulating genes involved in making the lipid A portion of LPS less toxic; and (e) a process of genetically modifying the bacteria to express heterologous antigens.

[0029] In some embodiments, a sample containing OMVs is artificially prepared from bacteria at a pH high enough so as not to precipitate the detergent, using a detergent treatment that includes treating the bacteria with a bile salt detergent (e.g., salts such as lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, ursolic acid, sodium deoxycholate). Other techniques can be carried out substantially in the absence of detergent, using techniques such as sonication, homogenization, microfluidization, cavitation, osmotic shock, grinding, French press, mixing, and the like.

[0030] The starting material for the methods provided herein is a sample containing OMVs. In one embodiment, the sample is substantially free of whole bacteria, whether alive or dead.

[0031] In some embodiments, the sample containing OMV is obtained from bacteria of the genus Bordetella. In some embodiments, the bacteria of the genus Bordetella include, or are, one or more of Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. In some embodiments, the bacteria include, or are, Bordetella pertussis. In some embodiments, Bordetella pertussis includes, or is, recombinant bacteria. In some embodiments, the recombinant Bordetella pertussis includes recombinant bacteria that contain an S1 gene containing mutations R9K and E129G and optionally express a genetically detoxified pertussis toxoid PT-9K / 129G. In some embodiments, the recombinant Bordetella pertussis bacteria do not express the dermonecrotic toxin (DNT) gene. In some embodiments, the recombinant Bordetella pertussis bacteria express a genetically detoxified pertussis toxoid PT-9K / 129G and do not express the dermonecrotic toxin (DNT) gene. Suitable recombinant Bordetella pertussis bacteria are disclosed in International Publication No. WO 2020 / 094580, which is incorporated herein by reference. In some embodiments, the recombinant Bordetella pertussis bacteria include at least one genomic LpxA gene encoding a mutant LpxA protein and / or at least a genomic insertion of a heterologous LpxD gene, as disclosed, for example, in International Publication No. WO 2021 / 064050, which is incorporated herein by reference. In some embodiments, the recombinant Bordetella pertussis includes recombinant bacteria that produce lipid A having (i) a C3' acyl chain length of about 10 carbons (C10), (ii) a C2' acyl chain length of about 10 carbons (C10), and / or (iii) a C2 acyl chain length of about 10 carbons (C10). Suitable recombinant Bordetella pertussis bacteria are disclosed in International Publication No. WO 2021 / 064050, which is incorporated herein by reference. In some embodiments, the recombinant Bordetella pertussis includes recombinant bacteria that contain an S1 gene containing mutations R9K and E129G and optionally express a genetically detoxified pertussis toxoid PT-9K / 129G, and includes recombinant bacteria that produce lipid A having (i) a C3' acyl chain length of about 10 carbons (C10), (ii) a C2' acyl chain length of about 10 carbons (C10), and / or (iii) a C2 acyl chain length of about 10 carbons (C10).

[0032] In some embodiments, the OMV is obtained from bacteria of the genus Neisseria. In some embodiments, the bacteria of the genus Neisseria include or are one or more selected from Neisseria meningitidis and Neisseria gonorrhoeae. In some embodiments, the bacteria are Neisseria meningitidis. In some embodiments, the bacteria are Neisseria meningitidis serogroup B (MenB). In some embodiments, the bacteria are Neisseria meningitidis serogroup B (MenB) strains selected from the group consisting of NZ98 / 254, NG H36, BZ 232, DK 353, B6116 / 77, BZ 163, 0085 / 00, NG P20, 0046 / 02, M11 40123, M12 240069, N5 / 99, 99M, or M07240677. In some embodiments, the OMV is obtained from the Neisseria meningitidis strain NZ98 / 254 or 99M. In one embodiment, the Neisseria meningitidis is the NZ98 / 254 strain. In one embodiment, the Neisseria meningitidis is the 99M strain. In some embodiments, the bacteria are Neisseria gonorrhoeae, which is also referred to as gonococcal bacteria or gonococcus.

[0033] In one embodiment, the Neisseria gonorrhoeae is a Neisseria gonorrhoeae bacterium comprising a genetic modification(s). In some embodiments, the Neisseria gonorrhoeae bacterium into which the genetic modification(s) is introduced (i.e., the unmodified Neisseria gonorrhoeae) is any strain of Neisseria gonorrhoeae bacterium. In some embodiments, the unmodified Neisseria gonorrhoeae is strain FA1090, F62, WHO-G, WHO-M, WHO-N, GC14, BG7, BG8, BG27, SK92, or MS11. In one embodiment, the unmodified Neisseria gonorrhoeae is strain FA1090. The Neisseria gonorrhoeae bacterium of strain FA1090 is known in the art and is commercially available from the American Type Culture Collection (see ATCC, e.g., accession number #700825, 1081 University Blvd, Manassas, Virginia 20110, US). In certain embodiments, the genetic modification(s) reduces the endotoxin activity of the OMV as compared to the wild-type or unmodified Neisseria gonorrhoeae. In some embodiments, the genetic modification(s) that reduces the endotoxin activity results in a Neisseria gonorrhoeae bacterium that produces OMVs having an increased level of penta-acylated lipid A, a decreased level of hexa-acylated lipid A, or a higher ratio of penta-acylated lipid A to hexa-acylated lipid A, as compared to the lipid A present in the OMVs in each wild-type or unmodified Neisseria gonorrhoeae (i.e., the wild-type or unmodified Neisseria gonorrhoeae for comparison before genetic modification that produces OMVs having an increased level of penta-acylated lipid A, a decreased level of hexa-acylated lipid A, or a higher ratio of penta-acylated lipid A to hexa-acylated lipid A).

[0034] In some embodiments, the Neisseria gonorrhoeae is a genetically modified Neisseria gonorrhoeae bacterium comprising a genetic modification(s) that decreases or abolishes the expression and / or function of the lipid A biosynthetic lauroyl acyltransferase (lpxl1) gene, mRNA, and / or polypeptide.

[0035] In a further embodiment, the Neisseria gonorrhoeae bacterium comprises a further genetic modification that decreases or abolishes the expression and / or function of the reduction-modifiable protein (rmp) gene, mRNA, and / or polypeptide.

[0036] In one embodiment, the Neisseria gonorrhoeae bacterium is derived from the FA1090 strain. In one embodiment, the Neisseria gonorrhoeae bacterium comprises a genetic modification(s) that (I) reduces or eliminates the expression and / or function of the lipid A biosynthetic lauroyl acyltransferase (lpxl1) gene, mRNA, and / or polypeptide, and (II) reduces or eliminates the expression and / or function of the reducible modification protein (rmp) gene, mRNA, and / or polypeptide. In one embodiment, the Neisseria gonorrhoeae bacterium is derived from the FA1090 strain and comprises a genetic modification(s) that (I) reduces or eliminates the expression and / or function of the lipid A biosynthetic lauroyl acyltransferase (lpxl1) gene, mRNA, and / or polypeptide, and (II) reduces or eliminates the expression and / or function of the reducible modification protein (rmp) gene, mRNA, and / or polypeptide.

[0037] The lpxl1 gene (also referred to as msbB) encodes the polypeptide lipid A biosynthetic lauroyl acyltransferase (Lpxl1). Lpxl1 plays a role in lipid A biosynthesis. A Neisseria gonorrhoeae organism genetically modified to provide a reduced or undetectable functional lpxl1-encoded protein produces OMVs with reduced endotoxin. This is because the amount and nature of lipid A acylation are major factors affecting 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.

[0038] The rmp gene encodes the polypeptide reducible modification protein (Rmp).

[0039] In the context of the present disclosure, "decreased expression" means that the Neisseria gonorrhoeae bacteria provided herein express less lpxl1 and rmp mRNA and / or Lpxl1 and Rmp protein as compared to an unmodified (wild-type) Neisseria gonorrhoeae strain or a Neisseria gonorrhoeae strain containing the wild-type lpxl1 / rmp gene. Expression can be considered decreased if any reduction in mRNA and / or protein expression is observed as compared to an unmodified (wild-type) Neisseria gonorrhoeae strain or a Neisseria gonorrhoeae strain containing the wild-type lpxl1 / rmp gene. Expression is decreased if a reduction of greater than 5%, greater than 10%, greater than 25%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95% in mRNA and / or protein expression is observed as compared to mRNA and / or protein expression, respectively, in an unmodified (wild-type) Neisseria gonorrhoeae strain or a Neisseria gonorrhoeae strain containing the wild-type lpxl1 / rmp gene. In the context of the present disclosure, "abolished expression" means that Lpxl1 mRNA and / or protein, and Rmp mRNA and / or protein cannot be detected in the Neisseria gonorrhoeae bacteria provided herein using techniques that would be used by one of ordinary skill in the art to measure expression.

[0040] The function of Lpxl1 can be determined, for example, by examining the extent to which the lipid A component of outer membrane vesicle lipooligosaccharide is penta-acylated relative to hexa-acylation (e.g., by analyzing the acylation state of lipid A using mass spectrometry as described in van der Ley et al. Infection and immunity vol. 69, 10(2001): 5981-90). When the genetically modified gonococcal bacterium contains a genetic modification that reduces or abolishes the function of the Lpxl1 protein, lipid A is penta-acylated despite evidence suggesting the presence of lpxl1 mRNA and / or protein (e.g., lipid A is 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 100% penta-acylated).

[0041] In one embodiment, a decrease or elimination of the expression and / or function of the Lpxl1 polypeptide results in pentaacylation of lipid A, and optionally the acylation of lipid A is determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectrometry. In one embodiment, the genetically modified Neisseria gonorrhoeae provided herein contains lipooligosaccharide (LOS) together with pentaacylated lipid A. The acylation of lipid A can be determined, for example, by extracting lipid A followed by analysis by MADI-TOF spectrometry. Specifically, a decrease or elimination of the expression and / or function of the Lpxl1 polypeptide results in a lipooligosaccharide (LOS) containing lipid A lacking lauric acid that would have been added if LpxL1 was functionally expressed. A decrease or elimination of the expression and / or function of the Lpxl1 polypeptide results in a LOS containing lipid A lacking the secondary lauroyl chain from the non-reducing end of the GlcN disaccharide of lipid A. A decrease or elimination of the expression and / or function of the Lpxl1 polypeptide results in a LOS containing lipid A lacking the C12 acyloxyacyl chain (from the non-reducing end). A decrease or elimination of the expression and / or function of the Lpxl1 polypeptide results in a LOS containing lipid A lacking lauric acid at the secondary 2'-O-position of the distal non-reducing end glucosamine of the β-(1-->6)D-glucosamine dimer (thus the isolated 3-hydroxymyristyl moiety is present as an amide bond on the distal glucosamine of lipid A).

[0042] In one embodiment, a decrease or elimination of the expression and / or function of the lpxl1 polypeptide results in pentaacylation of lipid A that is greater than 50%, such as greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95% or greater than 99% of lipid A. In one embodiment, a decrease or elimination of the expression and / or function of the Lpxl1 polypeptide results in 100% pentaacylation of lipid A. In one embodiment, the genetically modified Neisseria gonorrhoeae provided herein has a reduced ability to activate Toll-like receptor 4 (TLR4) compared to Neisseria gonorrhoeae containing the wild-type lpxl1 gene.

[0043] In some embodiments, a sample containing OMVs comprises OMVs and one or more impurities, such as GroEL.

[0044] In one embodiment, a sample containing OMVs is obtained by disrupting the outer membrane of bacteria, such as bacteria of the genus Bordetella, and comprises DOC-extracted OMVs and one or more impurities, such as GroEL. In one embodiment, a sample containing OMVs obtained by disrupting the outer membrane of bacteria of the genus Bordetella comprises DOC-extracted OMVs and GroEL. In one embodiment, a sample of OMVs obtained by disrupting the outer membrane of bacteria of the genus Bordetella comprises DOC-extracted OMVs and GroEL bound to the surface of the OMVs.

[0045] In some embodiments, a sample containing OMVs is obtained by detergent disruption of the outer membrane of bacteria, such as bacteria of the genus Bordetella or Neisseria. In one embodiment, the detergent is DOC (e.g., CAS number 302-95-4) optionally containing benzonase (e.g., CAS number 9025-65-4).

[0046] In certain embodiments, the detergent is DOC at a concentration of about 0.1% to about 0.5%, optionally including benzonase, for example DOC having a concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4% or about 0.5%. In some embodiments, the sample containing OMVs is obtained by detergent disruption of the outer membrane of bacteria, such as bacteria of the genus Bordetella, at a temperature of about 30°C to about 45°C, for example 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 can be used at a concentration of 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 containing OMVs is obtained by detergent disruption of the outer membrane of bacteria, such as bacteria of the genus Bordetella, at a temperature of about 30°C to about 45°C, for example about 30°C to about 42°C, for example about 30°C to about 40°C, for example about 30°C, about 35°C or about 40°C, using DOC at a concentration of about 0.1% to about 0.5%, for example 0.1% or 0.5%. In one embodiment, the sample containing OMVs is obtained by detergent disruption of the outer membrane of bacteria of the genus Bordetella at a temperature of about 40°C, using DOC at a concentration of about 0.5%, optionally including benzonase.

[0047] In some embodiments, a sample comprising OMVs is obtained by (i) homogenizing a fermentation harvest of bacteria, such as bacteria of the genus Bordetella, thereby obtaining a homogenized fermentation harvest; (ii) treating the homogenized fermentation harvest with DOC, for disrupting the outer membrane of bacteria, such as bacteria of the genus Bordetella, 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. In some embodiments, a sample of OMVs is obtained by (i) homogenizing a fermentation harvest of bacteria of the genus Bordetella, thereby obtaining a homogenized fermentation harvest; (ii) treating the homogenized fermentation harvest with DOC at a concentration of about 0.1% to about 0.5% and optionally benzonase at a temperature of about 35°C to about 40°C for disrupting the outer membrane of bacteria of the genus Bordetella, 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. The homogenization and / or treatment (steps (i) and / or (ii), respectively) can be carried out in the presence of a suitable buffer. By way of non-limiting example, suitable buffers include Tris buffers, such as Tris-HCl buffer. In one embodiment, the buffer is Tris-HCl buffer, such as 20 mM Tris-HCl buffer. Suitable buffers have a pH of about 8 to about 9, such as a pH of 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 homogenization and / or treatment (steps (i) and / or (ii), respectively) is carried out in the presence of at least one protease inhibitor. Suitable protease inhibitors are known in the art and, by way of non-limiting example, include cOmplete EDTA-free protease inhibitor cocktail (Roche).

[0048] In one embodiment, a sample containing OMVs comprises, or consists of, for example, OMVs released into the culture medium by bacteria of the genus Neisseria. In one embodiment, a sample containing OMVs comprises, for example, OMVs released into the culture medium by bacteria of the genus Neisseria, and one or more impurities, such as GroEL. In one embodiment, a sample containing OMVs comprises, for example, OMVs released into the culture medium by bacteria of the genus Neisseria, and GroEL. In one embodiment, a sample containing OMVs comprises, for example, OMVs released into the culture medium by bacteria of the genus Neisseria, and GroEL bound to the surface of the OMVs.

[0049] In some embodiments, a sample of OMVs is obtained by (i) recovering the fermentation broth 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 of OMVs. In some embodiments, in step (iv), MgCl2 is added to the intermediate to obtain an MgCl2 concentration of about 0.1 mM to about 10 mM, such as about 0.5 mM to about 5 mM, such as 1 mM. The intermediate is treated with benzonase treatment using a benzonase concentration of about 10 kU / L to about 100 kU / L, such as about 10 kU / L, about 20 kU / L, about 30 kU / L, about 40 kU / L, 50 kU / L, about 60 kU / L, about 70 kU / L, about 80 kU / L, about 90 kU / L, or about 100 kU / L in step (iv). In one embodiment, the benzonase concentration is 50 kU / L. In one embodiment, the intermediate is treated with benzonase at a temperature of about 0 °C to about 10 °C, such as about 2 °C to about 8 °C. In one embodiment, the intermediate is treated with benzonase for a period of about 1 day to about 3 months, such as about 3 days to 2 months. In one embodiment, MgCl2 is added to the intermediate to obtain an MgCl2 concentration of about 1 mM, and the intermediate is treated with 50 kU / L benzonase at a temperature of about 2 °C to about 8 °C for a period of about 3 days to 2 months.

[0050] Step (a) Tangential Flow Filtration (TFF) The first step of the method provided herein involves (a) concentrating and washing a sample containing OMVs by TFF using a TFF membrane to obtain a residual concentrate containing OMVs.

[0051] In TFF, the sample solution from the supply reservoir passes tangentially along the surface of the filter membrane and returns to the supply reservoir where the sample solution can be recycled. Components larger than the pores of the filter membrane, such as OMVs, are retained and pass along the membrane surface, while components smaller than the pores of the filter membrane pass through the filter. The sample solution that passes along the membrane surface and returns to the supply reservoir is referred to as the retentate. The sample solution that has passed through the membrane is generally referred to as the permeate or filtrate.

[0052] The membrane used in TFF has a molecular weight cut-off. The molecular weight cut-off (MWCO) or nominal molecular weight cut-off (NMWCO) is defined as the minimum molecular weight of the solute that is 90% retained by the membrane (e.g., as described by the manufacturer).

[0053] In some embodiments, the TFF is performed using a TFF membrane having a molecular weight cut-off of from about 200 kDa to about 800 kDa, such as from about 200 kDa to about 400 kDa or from about 500 kDa to about 750 kDa, such as, about 200 kDa, about 250 kDa, about 300 kDa, about 350 kDa, about 400 kDa, about 500 kDa, about 550 kDa, about 600 kDa, about 650 kDa, about 700 kDa or about 750 kDa MWCO. In certain embodiments, the TFF membrane has a MWCO of about 500 kDa. In other embodiments, the TFF membrane has a MWCO of about 750 kDa. In other embodiments, for example when the bacterium is a bacterium of the genus Neisseria, the TFF membrane has a MWCO of about 300 kDa.

[0054] In some embodiments, the TFF membrane has a pore size of from about 0.1 mm to about 0.75 mm, such as from about 0.4 mm to about 0.6 mm, such as about 0.4 mm, about 0.5 mm or about 0.6 mm. To avoid doubt, the pore size (sometimes referred to as the lumen diameter) is calculated using standard methods known in the art and is typically determined by the manufacturer. The pore size is the average diameter of the pores in the membrane based on the assumption that all the pores in the membrane are circular.

[0055] In some embodiments, the TFF membrane is a hollow fiber membrane. In one embodiment, the TFF membrane is a hollow fiber membrane having an MWCO of about 750 kDa or about 300 kDa. In one embodiment, the TFF membrane is a hollow fiber membrane having an MWCO of about 750 kDa or about 300 kDa and a pore size of about 0.5 mm. In one embodiment, the membrane is a hollow fiber membrane having an MWCO of about 750 kDa or about 300 kDa, a pore size of about 0.5 mm and an area of about 290 cm 2 In one embodiment, the membrane is a hollow fiber membrane having an MWCO of about 750 kDa, a pore size of about 0.5 mm, an area of about 290 cm 2 and a nominal flow path length of 60 cm. In some embodiments, the TFF membrane is, for example, the GE Healthcare UFP-750-C-3X2MA hollow fiber membrane or an equivalent having the following properties: a cut-off of 750 kDa, a pore size of 0.5 mm and a working surface area of 290 cm 2 . In some embodiments, the TFF membrane is a Millipore 500 kDa cassette or equivalent, or a Sartorius 300 kDa cassette or equivalent.

[0056] The TFF in step (a) includes concentrating and washing the sample. In one embodiment, the TFF in step (a) includes at least one cycle including ultrafiltration and diafiltration. In one embodiment, the TFF in step (a) includes at least one ultrafiltration cycle and at least one cycle of diafiltration. To avoid doubt, in the TFF process, concentration (by ultrafiltration) and washing (by diafiltration) are filtration steps that use the same TFF membrane but are carried out using various operating parameters further discussed below.

[0057] "Ultrafiltration" is generally used to refer to a TFF membrane filtration method in which a liquid is pressed against a semipermeable membrane by pressure, such as liquid pressure, such as water pressure. Suspended high molecular weight solids, such as OMVs, are retained by the membrane ("retentate"), while water and low molecular weight solutes pass through the membrane ("filtrate"). Thus, the term retentate is used to refer to the components or parts of a solution that are retained by the TFF membrane and do not pass through (in a limited sense, the term retentate may also include parts of the sample that are small enough to pass through the membrane but have not yet passed through, as will be understood by those skilled in the art).

[0058] In the methods provided herein, ultrafiltration is mainly used to concentrate the sample, but to avoid doubt, it will be understood by those skilled in the art that ultrafiltration also, by its nature, removes / excludes some impurities. As used herein, the concentration of a sample, such as a reference to "concentrating the sample", generally refers to increasing the amount of a particular target material relative to the volume of the fluid in which the target material is disposed, in the ordinary meaning of the term. 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) with respect to the sample containing OMVs. The sample obtained after the concentration step may be referred to as the concentrate.

[0059] "Diafiltration" refers to the use of a TFF membrane to remove, replace, or reduce the concentration of impurities in a solution based on molecular size. Similar to ultrafiltration, the TFF membrane retains molecules larger than the pores of the membrane, such as OMVs (the "retentate"), while smaller molecules, such as salts, solvents, and water, pass freely through the membrane (the "filtrate"). However, generally during diafiltration, a solution, such as a buffer, is introduced into the recirculation tank at a rate substantially the same as the rate at which the filtrate is removed, thereby replacing the buffer and / or washing the retentate while maintaining a constant volume of liquid in the sample. In one embodiment, the retentate is diluted with a solvent and refiltered to reduce the concentration of soluble permeate components. During diafiltration, OMVs remain in the retentate and components, such as impurities, are "washed away" and pass through the membrane into the filtrate, thereby removing the impurities and / or replacing the buffer.

[0060] Concentration of OMVs in the sample is performed using at least one cycle of ultrafiltration. In some embodiments, the ultrafiltration includes at least one cycle of ultrafiltration, for example, two or more, three or more, four or more, five or more cycles of ultrafiltration. In one embodiment, at least one ultrafiltration cycle concentrates the OMVs in the sample by at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or at least about 15-fold, such as about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, or about 15-fold. In some embodiments, at least one ultrafiltration cycle concentrates the OMVs in the sample by about 2.5-fold to about 20-fold, about 5-fold to about 20-fold, about 6-fold to about 20-fold, about 7-fold to about 20-fold, about 8-fold to about 20-fold, about 9-fold to about 20-fold, about 10-fold to about 20-fold, about 11-fold to about 20-fold, about 12-fold to about 20-fold, about 13-fold to about 20-fold, about 14-fold to about 20-fold, or about 15-fold to about 20-fold. The OMVs in the sample can be concentrated to a protein concentration of 2 - 5 mg / ml ± 1% by ultrafiltration. In some embodiments, the OMVs can be concentrated to a protein concentration of about 2% to about 10% (weight / volume) by ultrafiltration.

[0061] The washing of the sample is carried out using at least one cycle of diafiltration. In some embodiments, the diafiltration includes at least one cycle of diafiltration, for example, includes two or more, three or more, four or more, five or more cycles of diafiltration. In one embodiment, the diafiltration includes two cycles of diafiltration. In one embodiment, at least one cycle of diafiltration washes the sample with a washing solution having a volume of at least about five times the sample volume, for example, a washing solution having a volume of about ten times the sample volume, about fifteen times the sample volume or about twenty times the sample volume.

[0062] In some embodiments, at least one diafiltration cycle washes the sample with a wash solution that is at least about 5 times the sample volume, at least about 10 times the sample volume, or at least about 15 times the sample volume, for example, about 5 times the sample volume, about 6 times the sample volume, about 7 times the sample volume, about 8 times the sample volume, about 9 times the sample volume, about 10 times the sample volume, about 11 times the sample volume, about 12 times the sample volume, about 13 times the sample volume, about 14 times the sample volume, about 15 times the sample volume, about 16 times the sample volume, about 17 times the sample volume, about 18 times the sample volume, about 19 times the sample volume, or about 20 times the sample volume. In some embodiments, at least one diafiltration cycle washes the sample with a wash solution that is from about 5 times the sample volume to about 20 times the sample volume, from about 5 times the sample volume to about 20 times the sample volume, from about 6 times the sample volume to about 20 times the sample volume, from about 7 times the sample volume to about 20 times the sample volume, from about 8 times the sample volume to about 20 times the sample volume, from about 9 times the sample volume to about 20 times the sample volume, from about 10 times the sample volume to about 20 times the sample volume, from about 11 times the sample volume to about 20 times the sample volume, from about 12 times the sample volume to about 20 times the sample volume, from about 13 times the sample volume to about 20 times the sample volume, from about 14 times the sample volume to about 20 times the sample volume, or from about 15 times the sample volume to about 20 times 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 buffer, and the like. In one embodiment, the wash solution optionally contains other components, such as EDTA or sucrose. In some embodiments, for example, when the bacterium is of the genus Bordetella, the wash buffer is DPBS. In some embodiments, the wash solution is DPBS that optionally contains EDTA, such as about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM EDTA, such as about 5 mM EDTA. In some embodiments, the wash solution is DPBS that contains EDTA, such as about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, or 8 mM EDTA, such as about 5 mM EDTA. In some embodiments, for example, when the bacterium is of the genus Bordetella, the wash solution is Tris buffer, such as 20 mM Tris buffer having an appropriate pH. The appropriate pH is from about 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 washing solution contains sucrose, for example, 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 washing solution is a 20 mM Tris buffer having a pH of about 8.6 and optionally containing about 3% sucrose. In certain embodiments, the washing solution is DPBS containing about 5 mM EDTA. In some embodiments, for example, when the bacteria are bacteria of the genus Neisseria, the washing solution is a sodium phosphate buffer, such as a Na2HPO4 / NaH2PO4 buffer, such as 10 mM Na2HPO4 / NaH2PO4 having an appropriate pH. The appropriate 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, 6.8, 6.9, or 7.0. In some embodiments, the washing solution contains, for example, about 50 mM to about 250 mM sodium chloride, such as 50 mM, 100 mM, 150 mM, 200 mM or 250 mM. In certain embodiments, the washing solution is 10 mM Na2HPO4 / NaH2PO4 pH 6.8 containing 150 mM NaCl. In some embodiments, for example, when the bacteria are bacteria of the genus Neisseria, the washing solution is phosphate buffered saline (PBS) pH 7.4, such as a buffer containing 8.1 mM Na2HPO4, 1.5 mM KHPO4, 2.7 mM KCl and 136.9 mM NaCl.

[0063] 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.

[0064] In some embodiments, when using TFF, the operating parameters can be set such that concentration and washing are carried out substantially simultaneously. Since the same TFF membrane and equipment are used, the diafiltration parameters can be such that they result in an increase in the concentration of the components they retain, such as OMV. In continuous diafiltration, the solvent is continuously added to the retentate at the same rate at which the filtrate is produced, in which case the retentate volume and the concentration of the retained components do not change during the process. On the other hand, in discontinuous or sequential dilution diafiltration, following the ultrafiltration step, solvent is added to the retentate side, and when the volume of solvent added to the retentate side is less than the volume of the filtrate produced, the concentration of the components in the retentate increases (because the volume of liquid in the retentate decreases).

[0065] Thus, the TFF step (a) may optionally include a dilution step prior to concentration and washing, where a certain volume of liquid is added to the sample containing OMV, thereby increasing the sample volume. The increase in the initial working volume can be suitable as it can facilitate subsequent processing steps, such as washing, for example by reducing the likelihood of membrane fouling. If the dilution step is used prior to concentration and washing, the volume of liquid used for dilution is removed during washing and / or concentration.

[0066] 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 OMV. Flow-through chromatography is a separation technique in which the sample interacts with a chromatography substrate. The "chromatography substrate" is any type of solid phase that separates the target of interest from other molecules present in the sample as a result of differences in, for example, size and / or binding affinity. The chromatography substrate is generally contained in a suitable housing, such as a chromatography column.

[0067] Potential mixtures or impurities in the sample bind to and are retained by the chromatographic matrix, while the target in the sample that does not bind or is not retained can be recovered. Without wishing to be bound by theory, for example, in the "flow-through" mode of operation in step (b) of the process, the residual concentrate containing OMV is applied to the chromatographic matrix in, for example, a chromatography column and flows through it. As the residual concentrate flows through the column, the residual concentrate interacts with the chromatographic matrix contained in the column. During the interaction, OMV does not bind to the chromatographic matrix, while impurities (including GroEL) can bind to the chromatographic matrix and / or be trapped by the chromatographic matrix. In this process, a flow-through of purified OMV containing lower levels of impurities, lower concentrations of impurities, fewer amounts of impurities, or no impurities is obtained. The flow-through of purified OMV flows out of the chromatography column and is recovered. The flow-through of purified OMV contains lower levels or amounts of impurities (e.g., GroEL) compared to the residual concentrate before it interacts with the chromatographic matrix.

[0068] Suitable methods for flow-through chromatography include gel filtration, mixed-mode resin column chromatography, ion exchange column chromatography, affinity matrix chromatography, and hydrophobic interaction chromatography.

[0069] In some embodiments, the chromatographic matrix is a size exclusion resin suitable for gel filtration, such as Sephacryl S-500 resin.

[0070] In some embodiments, the chromatographic substrate is a mixed-mode chromatography resin suitable for mixed-mode resin column chromatography. The mixed-mode chromatography resin is functionalized with ligands, thereby enabling various types of interactions, such as, by way of non-limiting example, ion exchange, affinity binding, and / or size exclusion. In one embodiment, the chromatographic substrate includes, for example, a porous outer layer and an internal core, such as beads having an internal core activated with a ligand, such as a plurality of beads. In one embodiment, the chromatographic substrate has a porous outer layer (preventing large molecules from entering the core) and an internal core, such as beads having an internal core activated with a ligand including an octylamine ligand, with a molecular weight cut-off of about 600 kDa to about 800 kDa, such as 600 kDa to 800 kDa, about 700 kDa, such as 700 kDa. 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 known in the art include determining the dynamic binding capacity of the chromatographic substrate using proteins of various sizes, such as ovalbumin (Mr 45000), apoferritin (Mr 475000), thyroglobulin (Mr 660000), and bovine IgM (approx. Mr 900000), and evaluating up to which protein size the sample protein can penetrate into the bead core and bind to the substrate. In one embodiment, the beads have a non-functionalized outer layer (without ligand) and a functionalized core to which a ligand, such as an octylamine ligand, is bound. The term "octylamine ligand" refers to a ligand having the functional group CH3CH2CH2CH2CH2CH2CH2CH2NH-, where the pKa of the protonated octylamine before optional binding to the medium is about 10.65. In one embodiment, the matrix of the chromatographic substrate is high-flow agarose having a particle size of, for example, about 75 μm to about 95 μm, such as about 85 μm (i.e., the average particle size of the cumulative volume distribution).In one embodiment, the chromatographic substrate is the mixed-mode chromatography resin CaptoCore 700 (GE Lifesciences) or an equivalent, and the mixed-mode chromatography resin CaptoCore 700 comprises a porous outer layer, an inner core containing octylamine ligands, and a matrix of highly cross-linked agarose beads having a particle size of about 85 μm and a molecular weight cut-off of about 700 kDa. Other suitable chromatographic substrates are known in the art.

[0071] In one embodiment, steps (a) and (b) are sequentially performed without using intermediate processing steps other than collection / storage, and the output of step (a) (the residual concentrate containing OMVs) is the direct input of step (b). In some embodiments, process step (a) and process step (b) are discontinuous process steps, where step (a) is completed with the residual concentrate containing OMVs collected and optionally stored for a certain period, and then step (b) is initiated, and the residual concentrate containing OMVs is applied to a flow-through chromatographic substrate, such as a chromatography column. In some embodiments, process step (a) and process step (b) are continuous process steps. As used herein, the reference to "continuous process" refers to a process having a series of two or more processing steps, where the output from an upstream step (e.g., step (a)) is continuously transferred to a downstream step (e.g., step (b)) without waiting for the upstream processing step to complete, and then the downstream processing step is initiated. For example, the residual concentrate containing OMVs continuously flows directly from the TFF device to a flow-through type chromatography column, i.e., the TFF device is in fluid communication with the flow-through chromatography column.

[0072] In one embodiment, the process further includes or consists of step (a') of filtering the concentrate residue to remove the precipitate. In one embodiment, steps (a) and (a') are sequentially performed without using intermediate processing steps other than collection / storage, and the output of step (a) is the direct input for step (a'). In some embodiments, process step (a) and process step (a') are discontinuous process steps, where after step (a) is completed, step (a') is initiated. In some embodiments, steps (a) and (a') are continuous process steps.

[0073] Step (c) Sterile filtration OMVs prepared using the methods provided herein are generally used as components in immunogenic compositions, such as pharmaceutical compositions, such as vaccine compositions. Thus, such components are desirably sterile and free of bacterial and / or viral contaminants. Accordingly, the methods provided herein may further include the step of sterile filtering the purified OMVs. "Sterile filtering" or "sterile filtering" refers to the removal of viruses and / or bacteria from a solution of viruses and / or bacteria by passing the solution through a filter having pores small enough that viruses and / or bacteria cannot pass through. Thus, a "sterile preparation of purified OMVs" refers to a preparation or population of purified OMVs that has passed through a filter having a pore size small enough to prevent the passage of viruses and / or bacteria and that results in a preparation that is free of, or substantially free of, bacterial contaminants. The size of bacteria generally ranges from about 0.2 μm to about 600 μm, and a filter having a pore size of about 0.22 μm or less is small enough to produce a sterile filtrate and result in a sterile preparation of purified OMVs. Accordingly, step (c) includes sterile filtering the flow-through of the purified OMVs using a 0.22 μm filter to produce a sterile preparation of the purified OMVs. In one embodiment, steps (b) and (c) are performed sequentially without using intermediate processing steps other than collection / storage, and the output of step (b) is the direct input of step (c). In some embodiments, process step (b) and process step (c) are discontinuous process steps, where step (c) is initiated after step (b) is completed. In some embodiments, steps (b) and (c) are continuous process steps.

[0074] Preferably, the methods provided herein enable a recovery or yield of at least 25 mg of OMVs (based on protein content), such as at least 40 mg of protein / L of fermentation harvest or culture used in the preparation of a sample of OMVs, such as at least 70 mg of protein / L of culture or more.

[0075] The term "polydispersity" (or "dispersity" as recommended by IUPAC) is used to describe the degree of non-uniformity of the size distribution of a population of particles. The "polydispersity index" (PDI) is a dimensionless parameter used in the art to define the size distribution of lipid nanoparticles. The higher the value of the polydispersity index, the broader the spread of the individual particle sizes that make up the population. The lower the value of the polydispersity index, the more uniformly and tightly the particle sizes of the individual nanoparticles that make up the population are classified. The PDI can be determined using the dynamic light scattering (DLS) technique described herein, which is known in the art. Preparations or populations of purified OMVs produced by the methods provided herein have a polydispersity index of about 0.3 or less, such as about 0.1 to about 0.3, about 0.2 to about 0.3, such as about 0.2 to about 0.27.

[0076] The term "z-average radius" means the average radius of OMVs in a preparation or population of purified OMVs as measured by dynamic light scattering (DLS) as described herein. In one embodiment, the DLS measurement is performed using a Malvern Zetasizer Nano ZS (Malvern, Herrenberg, Germany) equipped with a 633 nm He-Ne laser and operating at an angle of 173°. The scattered light detected at 173° is automatically adjusted by a laser attenuation filter. For data analysis, the viscosity and refractive index (RI) of a 3% sucrose solution (at 25 °C) are used. The software used to collect and analyze the data is Zetasizer software version 7.13. The temperature is set at 25 °C. Each sample (80 μL) is characterized in a disposable polystyrene microcuvette (ZEN0040, Alfatest) having a path length of 10 mm. The hydrodynamic diameter of the OMVs is represented by the Z average value (general purpose algorithm) of three measurements out of eleven runs for each sample, and the PDI of the calculated size values is also provided.

[0077] In one embodiment, a preparation or population of purified OMVs provided herein, e.g., from bacteria of the genus Bordetella, such as a preparation or population of purified OMVs obtained from bacteria of Bordetella pertussis, has a Z-average radius of from about 50 nm to about 150 nm, such as from about 50 nm to about 140 nm, from 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.

[0078] In one embodiment, a preparation or population of purified OMVs provided herein has a z-average radius of from about 10 nm to about 125 nm, such as from about 20 nm to about 110 nm, from about 30 nm to about 100 nm, from about 40 nm to about 90 nm, from about 50 nm to about 80 nm, from about 60 nm to about 70 nm. In one embodiment, a preparation or population of purified OMVs provided herein, e.g., from bacteria of the genus Escherichia, such as from bacteria of Escherichia coli strain K or strain B (wherein optionally, the bacteria of Escherichia coli are selected from any one of strains BL21(DE3), BLR(DE3), and Escherichia coli HMS174(DE3)), has a Z-average radius of from about 15 nm to about 75 nm, such as from about 20 nm to about 70 nm, from about 30 nm to about 60 nm, from about 40 nm to about 50 nm. In one embodiment, a preparation or population of purified OMVs provided herein, e.g., from bacteria of the genus Neisseria, such as from purified OMVs obtained from Neisseria gonorrhoeae, has a z-average radius of from about 30 nm to about 110 nm, such as from about 40 nm to about 100 nm, from about 50 nm to about 90 nm, from about 60 nm to about 80 nm.

[0079] In some embodiments, the preparation or population of purified OMVs is at least 85% pure, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, such as about 85%, about 90%, about 92%, about 93%, about 94% or about 95% pure. In one embodiment, the population of purified OMVs is about 85% to about 100% pure, about 90% to about 100% pure, about 95% to about 100% pure. In one embodiment, the population of purified OMVs has a purity of about 85% to about 99%, about 90% to about 99%, about 91% to about 99%, about 92% to about 99%, about 93% to about 99%, about 94% to about 99%, about 95% to about 99%, about 96% to about 99%, about 97% to about 99% or about 98% to about 99%. In one embodiment, the purity is measured using size exclusion UPLC, such as by size exclusion UPLC (fluorescence detection), and the ratio between the major peak (due to OMVs) and the smaller, low molecular size peaks (due to free proteins) can be determined. It will be understood by those skilled in the art that purity can also be defined based on the amount or level of residual total impurities. Thus, the impurities can be 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% impurities, such as free impurities, such as the level or concentration of protein impurities, present in the preparation or population of purified OMVs. In one embodiment, the preparation or population of purified OMVs contains 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% impurities, such as free impurities, such as protein impurities.

[0080] Preparations or populations of purified OMVs produced using the methods provided herein may contain from about 0.01% to about 10% GroEL, such as from about 0.01% to about 9% GroEL, from about 0.01% to about 8% GroEL, from about 0.01% to about 7% GroEL, from about 0.01% to about 6% GroEL, from about 0.01% to about 5% GroEL, from about 0.01% to about 4% GroEL, from about 0.01% to about 3% GroEL, from about 0.01% to about 2% GroEL, or from about 0.01% to about 1.5%, such as about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 0.75%, about 1%, or about 1.05% GroEL, as a percentage of total protein. In one embodiment, preparations or populations of purified OMVs produced using the methods provided herein contain GroEL at about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1.5% or less, or about 1% or less, such as less than 5% GroEL, less than 4% GroEL, less than 3% GroEL, less than 2% GroEL, less than 1.5% GroEL, or less than 1.1% GroEL, as a percentage of total protein. In one embodiment, the amount of GroEL may be provided as a relative amount or an absolute amount. In one embodiment, the amount of GroEL is provided as a relative amount, such as as a percentage of total protein in a preparation or population of purified OMVs. In one embodiment, the amount of GroEL is a relative amount determined using mass spectrometry.

[0081] Furthermore, in addition to the even lower 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, such as 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. In the context of this 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, such as a pharmaceutical composition, such as a vaccine composition. Thus, as described below, the preparation or population of purified OMVs can be combined with one or more pharmaceutically acceptable carriers, excipients or other components to produce an immunogenic composition.

[0082] Immunogenic composition Preparations or populations of purified OMVs are capable of eliciting an immune response, including, for example, a primary and / or boosted immune response against, for example, Bordetella spp., such as Bordetella pertussis, or Neisseria spp., such as Neisseria gonorrhoeae, when administered to a suitable subject, such as a mammal. The immune response can be a cellular or 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 capable of neutralizing Bordetella pertussis or Neisseria gonorrhoeae infection and / or pathogenicity. The immune response elicited by the OMVs can be directed against or induced against one or more antigens present in the OMVs. Thus, compositions comprising the purified OMVs provided herein are immunogenic. The term "immunogenic composition" broadly refers to any composition provided herein that, when administered to a subject, can elicit an immune response, such as an antibody or cellular immune response, against the antigen(s) present in the OMVs. Immunogenic compositions may also be referred to in the art as "pharmaceuticals" and refer, for example, to preparations or populations 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 further described below, in addition to the antigens present in the OMVs, the immunogenic compositions may be formulated to contain other antigenic components from, for example, Bordetella spp., Neisseria spp. and / or other organisms capable of infecting a suitable subject and causing symptoms of a disease or disorder.

[0083] When an immunogenic composition prevents, alleviates, mitigates, or eliminates a disease resulting from a bacterial and / or viral infection from a subject, the immunogenic composition may be referred to as a vaccine. Thus, in certain embodiments, the immunogenic composition is a vaccine. The vaccines provided herein may be prophylactic (i.e., to prevent infection, for example, by inducing a primary and / or boosted immune response) or therapeutic (i.e., to treat infection, for example, by inducing a primary and / or boosted immune response), but are usually prophylactic. Even if a subject expresses antibodies, there may be a deviation or delay before the immune system can repel the infection, so a prophylactic vaccine does not guarantee complete protection from the disease. Thus, to avoid doubt, the term prophylactic vaccine includes vaccines used to reduce the impact of future infections, for example, by reducing the severity and / or duration of such infections.

[0084] The terms "defense against infection" and / or "providing protective immunity" mean that the immune system of a subject is primed (e.g., by vaccination), induces an immune response, and repels infection. In some embodiments, the induced immune response can repel infection against Bordetella, optionally Bordetella pertussis, or Neisseria, optionally Neisseria gonorrhoeae. Thus, a vaccinated subject may be susceptible to infection but can better control, fend off, or repel infection than a control subject. The immunogenic composition used as a vaccine comprises an immunologically effective amount of OMV. "Immunologically effective amount" means an amount sufficient to generate an immune response effective in the treatment or prevention of infection and disease, for either a single dose to a suitable subject or as part of a series of administrations. Generally, the desired result is to be able to defend a subject against a pathogen or to generate an antigen (e.g., a pathogen)-specific immune response that contributes thereto. This amount varies depending on the health and physical condition of the individual to be treated, age, taxonomic group of the individual to be treated (e.g., non-human primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the treating physician in the medical situation, and other relevant factors. The amount is expected to fall within a relatively broad range that can be determined by routine experimentation, e.g., by clinical trials.

[0085] The term "antigen" refers to a substance that, when administered to a subject, elicits an immune response induced against a certain substance. The OMVs purified by the methods provided herein are complex components containing a plurality of antigens, for example, those within the OMV lumen, or displayed on the surface of the OMV, or bound to the surface of the OMV. Unless otherwise noted, this plurality of antigens is taken together, and the OMVs are referred to as antigens or antigenic components. In some embodiments, when administered to a subject, the OMV component of the immunogenic composition, although not wishing to be limited by theory, elicits an immune response induced against Bordetella spp., such as Bordetella pertussis, or Neisseria spp., such as Neisseria gonorrhoeae. In some embodiments, the immune response induced against Bordetella spp. or Neisseria spp. is protective, i.e., it can prevent or reduce infection and / or colonization caused by Bordetella spp., optionally Bordetella pertussis, or Neisseria spp., optionally Neisseria gonorrhoeae. Thus, the composition can be pharmaceutically acceptable. Generally, the immunogenic composition contains components in addition to the OMVs. For example, the immunogenic composition usually contains one or more pharmaceutical carrier(s) and / or excipient(s) (a detailed discussion of such components is described in Gennaro (2000) Remington: The Science and Practice of Pharmacy. 20th edition, ISBN:0683306472).

[0086] A "pharmaceutically acceptable carrier" is a carrier that does not itself induce the production of antibodies. Such carriers are well known to those skilled 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 agents or emulsifiers, pH buffering substances, and the like.

[0087] The immunogenic composition, such as a vaccine, may also contain one or more adjuvants. The term "adjuvant" as used herein refers to a compound that, when administered together with an antigen(s), enhances the subject's immune response against the antigen(s).

[0088] The composition may contain a preservative, such as thimerosal or 2-phenoxyethanol. In one embodiment, the composition provided herein is substantially free of mercury material (i.e., less than 5 μg / ml), for example, free of thimerosal. Compositions that do not contain mercury are also provided. Compositions that do not contain thimerosal, such as thimerosal-free vaccines, are also provided.

[0089] To control tonicity, physiological salts, such as sodium salts, may be included. Sodium chloride (NaCl) may be used, which may be present at 1 - 20 mg / ml, for example, about 10 ± 2 mg / ml of NaCl.

[0090] The composition generally has an osmotic pressure in the range of 200 mOsm / kg to 400 mOsm / kg, such as 240 - 360 mOsm / kg, such as 290 - 310 mOsm / kg.

[0091] The composition may contain one or more buffers. Examples of common buffers include phosphate buffer, Tris buffer, borate buffer, succinate buffer, histidine buffer, or citrate buffer. The buffer is usually included in the range of 5 - 20 mM.

[0092] The pH of the composition is generally from about pH 5.0 to about pH 8.1, more typically from about pH 6.0 to about pH 8.0, such as from about pH 6.5 to about pH 7.5, or from about pH 7.0 to about pH 7.8.

[0093] In one embodiment, the immunogenic composition is sterile.

[0094] The composition may contain material for single immunization or may contain material for multiple immunizations (i.e., a "multi-dose" kit). The inclusion of a preservative is provided, for example, in a multi-dose configuration. As an alternative to (or in addition to) including a preservative in a multi-dose composition, the composition may be contained in a container having a sterile adapter for removal of the material.

[0095] Immunogenic compositions, such as human vaccines, are typically administered in a dosage of about 0.5 ml, although, for example, partial dosages, such as half dosages (i.e., about 0.25 ml) for children, may be administered.

[0096] Adjuvants that can be used in the compositions provided herein include mineral-containing compositions such as aluminum salts and calcium salts. Compositions provided herein can include mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates or mixtures of various mineral compounds. Non-limiting examples of aluminum adjuvants include aluminum hydroxide, aluminum oxyhydroxide salts, aluminum phosphate, aluminum hydroxyphosphate, aluminum hydroxyphosphate sulfate, and the like.

[0097] In some embodiments, the adjuvant comprises aluminum phosphate that can be obtained by precipitation. The reaction conditions and concentration during precipitation affect the degree of substitution of phosphate for hydroxyl in the salt. The PO4 / Al 3+ molar ratio of the aluminum phosphate adjuvant is 0.3 to 1.2, such as 0.8 to 1.2, such as 0.95 ± 0.1. Aluminum phosphate can be, for example, amorphous for hydroxyphosphates. The aluminum phosphate provided herein can have a point of zero charge (PZC) of 4.0 to 7.0, such as 5.0 to 6.5, such as about 5.7.

[0098] Al in the composition for administration to a patient +++The concentration can be from about 10 mg / ml to about 0.01 mg / ml, such as 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 5 mg / ml, about 4 mg / ml, about 3 mg / ml, about 2 mg / ml, about 1 mg / ml, about 0.3 mg / ml, about 0.05 mg / ml or about 0.01 mg / ml. In one embodiment, the range is from about 0.3 mg / ml to about 1 mg / ml. In some embodiments, a maximum of 0.85 mg / dose is provided, such as about 0.5 mg / dose, about 0.4 mg / dose, about 0.3 mg / dose, about 0.2 mg / dose or about 0.1 mg / dose.

[0099] The immunogenic compositions provided herein may include Toll-like receptor (TLR) agonists, such as Toll-like receptor 2 agonist (TLR2a), Toll-like receptor 3 agonist (TLR3a), Toll-like receptor 4 agonist (TLR4a), Toll-like receptor 7 agonist (TLR7a), Toll-like receptor 8 agonist (TLR8a), or Toll-like receptor 9 agonist (TLR9a). Examples of Toll-like receptor agonists that may be included in the compositions provided herein include TLR2 agonists (e.g., Pam3CSK4), TLR4 agonists (e.g., aminoalkyl glucosaminide phosphate, such as E6020), TLR7 agonists (e.g., imiquimod or benzonaphthyridine, such as SMIP7.10), TLR8 agonists (e.g., resiquimod (which is also a TLR7 agonist)), and / or TLR9 agonists (e.g., IC31 or CpG1018). The immunogenic compositions provided herein may include both a TLR agonist and at least one aluminum salt adjuvant, such as aluminum phosphate and / or aluminum hydroxide. In some embodiments, the immunogenic compositions provided herein substantially do not contain an aluminum adjuvant, i.e., they contain only residual or trace amounts of aluminum salt adjuvant(s), or contain no measurable amount of aluminum salt adjuvant(s). In one embodiment, when the immunogenic compositions provided herein include a TLR agonist, the TLR agonist is selected from the group consisting of IC31, E6020, CpG1018, and SMIP7.10. In one embodiment, the immunogenic compositions provided herein include a TLR agonist selected from the group consisting of IC31, E6020, CpG1018, and SMIP7.10, and an aluminum salt adjuvant selected from the group consisting of aluminum hydroxide and aluminum phosphate. In one embodiment, the immunogenic compositions provided herein include (i) a TLR agonist selected from the group consisting of TLR4 agonist E6020, TLR7 agonist SMIP7.10, and TLR9 agonist Cpg1018, and (ii) an aluminum salt selected from the group consisting of aluminum hydroxide and aluminum phosphate.TLR agonists can have a molecular weight of less than 2000 Da.

[0100] TLR7 agonists, such as SMIP7.10, can include at least one adsorbable moiety. Inclusion of such moieties in TLR agonists allows the TLR agonists to be adsorbed to insoluble aluminum salts (e.g., by ligand exchange or any other suitable mechanism), and their immunological behavior is improved. Phosphorus-containing adsorbable moieties are useful, and thus the adsorbable moiety can include phosphate, phosphonate, phosphinate, phosphonite, phosphinite, etc. TLR agonists can include at least one phosphonate group. In some embodiments, the compositions provided herein can include a TLR7 agonist that includes a phosphonate group. This phosphonate group can enable adsorption of the agonist to an insoluble aluminum salt. In some embodiments, the TLR7 agonist includes 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 as shown below and is optionally adsorbed to an aluminum adjuvant.

[0101] [Chemical formula]

[0102] In some embodiments, the TLR agonist is water-soluble. Thus, the TLR agonist can form a homogeneous solution when mixed with water in an aqueous buffer at pH 7 at 25 °C and 1 atmosphere pressure and provide a solution having a concentration of at least 50 μg / ml. Thus, the term "water-soluble" excludes substances that are only sparingly soluble under these conditions.

[0103] The composition is generally administered in an aqueous form to a suitable subject, such as a patient, such as a suitable mammal, such as a human. However, the composition may be in a non-aqueous form prior to administration. For example, some vaccines are manufactured in an aqueous form and subsequently filled, dispensed, and administered in an aqueous form as well, while other vaccines are lyophilized during manufacture and may be reconstituted into an aqueous form at the time of use, such as at the time of administration. Thus, the compositions provided herein may include, or be, dried compositions, such as lyophilized formulations.

[0104] The compositions provided herein can be prepared as an injectable, either as a liquid solution or as a suspension. Solid forms suitable for solution or suspension in a liquid vehicle prior to injection can also be prepared (e.g., lyophilized compositions or spray-lyophilized compositions). The composition can be prepared for pulmonary administration using, for example, a fine powder or a spray, such as an inhalant. The composition can be prepared, for example, as drops for administration to the nose, ear, or eye. The composition may be present in kit form designed such that a complex composition is reconstituted immediately prior to administration to the patient. Such a kit can include one or more lyophilized antigens and one or more antigens in liquid form for reconstitution of the lyophilized antigen. For example, if the composition is to be prepared immediately prior to use (e.g., when the components are presented in lyophilized form) and is presented as a kit, the kit may include two vials, or the kit may include one filled syringe and one vial, and the contents of the syringe are used to reactivate the contents of the vial prior to injection.

[0105] Additional antigenic components of the compositions of the invention The immunogenic compositions provided herein are generally combination vaccines and can include one or more additional antigen(s) in addition to OMVs as provided herein.

[0106] In some embodiments, the OMVs provided herein are obtained from bacteria of the genus Bordetella, optionally from Bordetella pertussis, and one or more additional antigen(s) is an antigen capable of stimulating or generating an immune response against viral and / or bacterial pathogens. Examples of common bacterial pathogens include, but are not limited to, Corynebacterium diphtheriae, Clostridium tetani, and Haemophilus influenzae type b. Examples of common viral pathogens include, but are not limited to, poliovirus and hepatitis B virus. Thus, in addition to the OMVs provided herein, the immunogenic compositions provided herein may further comprise one or more antigenic components capable of generating an immune response, such as a protective immune response, against Corynebacterium diphtheriae, Clostridium tetani, Bordetella pertussis, hepatitis B virus, Haemophilus influenzae type b, or poliovirus when administered to a subject. To avoid doubt, reference to additional or further antigens is intended to refer to antigenic components included in the immunogenic compositions provided herein beyond and in addition to the OMVs and their constituents. For example, small amounts of pertussis toxoid may be found as a constituent of the OMV, but reference to pertussis toxoid as a further antigen refers to an amount of pertussis toxoid that is clearly added in excess of the OMV, e.g., as an isolated protein antigen.

[0107] Suitable additional antigenic components known in the art from bacterial and viral pathogens, described in more detail below, include the following: - Hepatitis B virus: surface and / or core antigens. - Corynebacterium diphtheriae: diphtheria toxoid or CRM 197 (diphtheria toxoid variant). - Clostridium tetani: tetanus toxoid. - Bordetella pertussis: acellular pertussis antigens selected from the group consisting of pertussis toxoid (PT), filamentous hemagglutinin (FHA), pertactin, adenylate cyclase or immunogenic fragments thereof, FIM2, and FIM3. - Haemophilus influenzae type b: capsular polysaccharide, such as polyribosyl ribitol phosphate [PRP]-protein conjugate. - Poliovirus: inactivated poliovirus (IPV) containing, for example, type 1, type 2, and type 3 strains selected from Mahoney type 1, MEF-1 type 2, and Saukett type 3 strains, or any of type 1 and type 3 strains.

[0108] In some embodiments, the OMVs provided herein and obtained from Bordetella species, preferably Bordetella pertussis, are conjugated to capsular polysaccharide derived from Haemophilus influenzae type b to form an immunogenic conjugate. This conjugate can be combined with one or more additional antigens in the immunogenic composition as described above. In some embodiments, the immunogenic composition of the conjugate between Bordetella OMV and Hib polysaccharide is a fully liquid and stable composition further comprising an antigen selected from the group consisting of diphtheria 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 of about 5 to about 100 kDa. The Hib sugar moiety of the conjugate may be used in its full-length native form, including full-length PRP as prepared from Hib bacteria, or alternatively, the Hib sugar moiety of the conjugate may be fragmented from its native length according to various methods described in the art, and optionally, size fractions of these fragments may also be used. In some embodiments, the PRP can be a synthetic polysaccharide. Suitable conjugates of Bordetella OMV to Hib capsular polysaccharide are disclosed in International Publication No. WO 2020 / 043874, which is incorporated herein by reference.

[0109] Acellular pertussis antigen In addition to the OMVs provided herein, which are obtained from Bordetella bacteria and optionally from Bordetella pertussis bacteria, the immunogenic composition can further comprise one or more acellular pertussis (aP) antigens selected from, for example, the following well-known and well-characterized Bordetella 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 "69-kilodalton outer membrane protein"); (4) type 2 fimbriae ("FIM2"); (5) type 3 fimbriae ("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 treated as a single antigen (referred to as FIM2 / 3). PTc can be prepared by chemical treatment of pertussis toxin (pertussisistoxin) with formaldehyde and / or glutaraldehyde. In one embodiment, as an alternative to this chemical detoxification procedure, PT is a mutant PT with reduced enzymatic activity by mutagenesis, for example, genetically detoxified pertussis toxoid (PTg), for example, the genetically detoxified pertussis toxoid known in the art and called the PT-9K / 129G variant. The PT-9K / 129G variant is a double mutant with substitutions at residues 9 (Arg to Lys) and 129 (Glu to Gly) of the S1 subunit. In some embodiments, the immunogenic composition comprises the acellular pertussis antigens PT, FHA, and PRN, for example, PTg, FHA, and PRN. In some embodiments, the immunogenic composition also comprises the acellular pertussis antigen FIM2 / FIM3. In some embodiments, the immunogenic composition comprises the acellular pertussis antigens PTg, FHA, FIM2 / FIM3, but does not comprise PRN.

[0110] In one embodiment, PT, FHA and PRN are prepared by isolation from a Bordetella 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 to hydroxyapatite gel), while pertactin can be extracted from cells by heat treatment and aggregation (e.g., using barium chloride). The antigen can be purified by successive chromatography 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.

[0111] FHA and pertactin can be treated with formaldehyde before use.

[0112] The aP antigen(s) can be used in a non-adsorbed state, but can be adsorbed onto one or more aluminum salt adjuvant(s) before use. Typically, the aP antigen is substantially free of mercury-based preservatives, such as thimerosal.

[0113] Cell-free pertussis antigen can be present in the immunogenic compositions provided herein in an amount capable of inducing an immune response when administered. Ideally, the cell-free pertussis antigen can induce a protective immune response. The amounts of cell-free pertussis antigen are typically expressed in micrograms. The concentration of PT in the vaccine is usually between 5 and 50 μg / ml. Typical PT concentrations are 5 μg / ml, 16 μg / ml, 20 μg / ml or 50 μg / ml, for example, about 20 μg per dose or about 25 μg per dose. The concentration of FHA in the vaccine is usually between 10 and 50 μg / ml. Typical FHA concentrations are 10 μg / ml, 16 μg / ml or 50 μg / ml, for example, about 20 μg per dose or about 25 μg per dose. The concentration of pertactin in the vaccine is usually between 5 and 16 μg / ml. Typical pertactin concentrations are 5 μg / ml, 6 μg / ml or 16 μg / ml, for example, about 3 μg per dose or about 8 μg per dose. FIM2 and FIM3 can be present at concentrations of 5 μg / ml and 16 μg / ml. Typical concentrations of FIM2 and FIM3 are 5 μg / ml, 10 μg / ml or 20 μg / ml, for example, about 5 μg per dose or about 10 μg per dose. Booster vaccines for adolescents and adults typically contain between 2.5 and 8 μg of PT, between 4 and 8 μg of FHA, and between 2.5 and 8 μg of pertactin per 0.5 ml dose. Typically, the booster vaccine contains 4 μg of PT, 4 μg of FHA and 8 μg of pertactin, more preferably 5 μg of PT, 2.5 μg of FHA and 2.5 μg of pertactin per 0.5 ml dose. Pediatric vaccines usually contain 7 μg of PT, 10 μg of FHA and 10 μg of pertactin per 0.5 ml dose.

[0114] When the aqueous component contains each of PT, FHA and pertactin, their weight ratios can be different, for example, about 16:16:5, about 5:10:6, about 20:20:3, about 25:25:8 or about 10:5:3 (PT:FHA:PRN).

[0115] 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 antitoxin antibodies after injection. Diphtheria toxoid used in diphtheria vaccines is well known in the art. In one embodiment, the diphtheria toxoid is prepared by formaldehyde treatment. The diphtheria toxoid can be obtained by growing Corynebacterium diphtheriae in a growth medium supplemented with bovine extracts (e.g., Fenton medium, or Linggoud & Fenton medium), followed by formaldehyde treatment, ultrafiltration, and precipitation. In one embodiment, the growth medium for growing Corynebacterium diphtheriae does not contain animal-derived components. The toxoided material can then be treated by a process including sterile filtration and / or dialysis. Alternatively, genetically detoxified diphtheria toxin (e.g., CRM197) can be used, but it may be treated with formaldehyde to maintain long-term stability during storage. The diphtheria toxoid can be adsorbed onto an adjuvant, such as an aluminum salt adjuvant.

[0116] The amount of diphtheria toxin and / or toxoid in the composition is generally measured in "Lf" units ("agglutination unit" or "limiting agglutination dose" or "limit of agglutination"), which is defined as the amount of toxin / toxoid that results in an optimally agglutinated mixture when mixed with 1 international unit of antitoxin (Lyng (1990) Biologicals 18:11-17). For example, "Diphtheria Toxoid, Plain" (NIBSC code: 69 / 017) is supplied by NIBSC, which contains 300 LF per ampoule, and "The 2nd International Reference Reagent For Diphtheria Toxoid For Flocculation Test" (NIBSC Code: 02 / 176) is also supplied, which contains 900 Lf per ampoule. The concentration of diphtheria toxin or toxoid in the composition can be readily determined using an agglutination assay by comparison with a reference material calibrated against such reference reagents.

[0117] The immunogenicity of diphtheria toxoid in the composition is generally expressed in international units (IU). The potency can be evaluated by comparing the protection afforded by the composition in experimental animals (usually guinea pigs) with a reference vaccine calibrated in IU. "4th WHO International Standard for Diphtheria Toxoid (Adsorbed)" (NIBSC code: 07 / 216) is supplied by NIBSC, which contains 213 IU per ampoule and is suitable for calibrating such assays.

[0118] According to the IU measurement value, the composition generally contains at least 30 IU / dose. The composition typically contains diphtheria toxoid between 20 and 80 Lf / ml, typically about 50 Lf / ml. Booster vaccines for adolescents and adults typically contain between 4 Lf / ml and 8 Lf / ml, for example, 2.5 Lf, preferably 4 Lf of diphtheria toxoid per 0.5 ml dose. Pediatric vaccines typically contain between 20 and 50 Lf / ml, for example, 10 Lf or 25 Lf of diphtheria toxoid per 0.5 ml dose.

[0119] The purity of a protein preparation can be expressed by the ratio of a specific protein to the total protein mass. The purity of diphtheria toxoid in a composition is generally expressed in units of Lf of diphtheria toxoid per unit mass of protein (non-dialyzable) nitrogen. By way of example, a very high purity toxin / toxoid may have a purity exceeding 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).

[0120] Tetanus Clostridium tetani causes tetanus. Tetanus toxin can be processed to yield a protective toxoid. The toxoid is used in tetanus vaccines and is well known in the art. Thus, the combination vaccines provided herein can include tetanus toxoid. In one embodiment, the tetanus toxoid is prepared by formaldehyde treatment. Tetanus toxoid can be obtained by growing Clostridium tetani in a growth medium (e.g., Latham medium derived from bovine casein), followed by formaldehyde treatment, ultrafiltration and precipitation. The growth medium for growing Clostridium tetani may not contain animal-derived components. The material can then be processed by a process including sterile filtration and / or dialysis. Tetanus toxoid can be adsorbed onto an adjuvant, such as an aluminum salt adjuvant.

[0121] The amount of tetanus toxoid can be expressed in units of "Lf" (see below), which is defined as the amount of toxoid that produces an optimally aggregated mixture when mixed with 1 international unit of antitoxin. The "The 2nd International Reference Reagent for Tetanus Toxoid For Flocculation Test" (NIBSC Code: 04 / 150) is supplied by NIBSC and contains 690 LF per ampoule, by which the measured values can be calibrated.

[0122] Booster vaccines for adolescents and adults typically contain 5 Lf of tetanus toxoid per 0.5 ml dose. Pediatric vaccines typically contain between 5 and 10 Lf of tetanus toxoid per 0.5 ml dose.

[0123] The immunogenicity of tetanus toxoid is measured in international units (IU), and the protection afforded by the composition in experimental animals (usually guinea pigs) is evaluated by comparison with a reference vaccine, such as the NIBSC "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 the compositions provided herein is at least 35 IU per dose, for example at least 70 IU / ml. In one embodiment, the potency of tetanus toxoid in the compositions provided herein is at least 40 IU per dose. However, in booster vaccines for adults and adolescents, a reduced potency of 20 IU / dose can be tolerated since the antigen content is reduced compared to pediatric vaccines intended for primary immunization.

[0124] The purity of tetanus toxoid in the composition is generally expressed in units of Lf of tetanus toxoid per unit mass of protein (non-dialyzable) nitrogen. Tetanus toxoid should have a purity of at least 1000 Lf / mg N.

[0125] Hib Haemophilus influenzae type b ("Hib") causes bacterial meningitis. Hib vaccines are usually based on capsular polysaccharide antigens whose preparation 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). Bacteria of the Haemophilus influenzae species can be cultured without animal-derived components. Hib polysaccharides are conjugated to carrier proteins, especially to enhance their immunogenicity in children. Normal carrier proteins for these conjugates are tetanus toxoid, diphtheria toxoid, CRM 197 derivatives of diphtheria toxin, or outer membrane protein complexes (OMPC) derived from serogroup B meningococci. Thus, the combination vaccines provided herein can include Hib capsular polysaccharides conjugated to a carrier protein, such as tetanus toxoid, diphtheria toxoid or OMPC.

[0126] The sugar moiety of the conjugate can include full-length polyribosyl ribitol phosphate (PRP) prepared from Hib bacteria, and / or fragments of full-length PRP. A saccharide:protein ratio (w / w) between 1:5 (i.e., excess protein) and 5:1 (i.e., excess saccharide), For example, conjugates having ratios between 1:2 and 5:1 and between 1:1.25 and 1:2.5 can be used. However, in some vaccines, the weight ratio of saccharide to carrier protein is between 1:2.5 and 1:3.5.

[0127] The amount of Hib antigen is usually expressed in μg of carbohydrate. The concentration of carbohydrate in the vaccine is typically between 3 and 30 μg / ml, for example 20 μg / ml. In one embodiment, administration of the Hib conjugate results in an anti-PRP antibody concentration of ≧0.15 μg / ml, for example ≧1 μg / ml, which are standard response thresholds. In some embodiments, the Hib polyribosyl ribitol phosphate is a synthetic polysaccharide, for example as described in Verez-Bencomo et al. Science. 2004 Jul 23;305(5683):522-5.

[0128] As described above, in some embodiments, the Hib capsular polysaccharide antigen may be conjugated to the OMVs provided herein obtained from species of the genus Bordetella.

[0129] Hepatitis B virus Hepatitis B virus (HBV) is the cause of viral hepatitis. The HBV virion is composed of an inner core surrounded by an outer protein coat or capsid, and the core contains the viral DNA genome. The main component of the capsid is a protein known as the hepatitis B surface antigen, or more commonly as "HBsAg", which is usually a polypeptide of 226 amino acids with a molecular weight of approximately 24 kDa. All existing hepatitis B vaccines contain HBsAg, and this antigen stimulates the production of anti-HBsAg antibodies that protect against HBV infection when administered to normal patients. Accordingly, the immunogenic compositions provided herein can include HBsAg.

[0130] In the case of vaccine production, HBsAg can be produced in several ways. For example, by expressing the protein by recombinant DNA methods. The HBsAg for use in the methods provided herein needs to be recombinantly expressed, for example, in yeast cells. Suitable yeasts include hosts of the genus Saccharomyces (e.g., S. cerevisiae), the genus Hansenula (e.g., H. polymorpha), or the genus Pichia. Yeasts can be cultured without animal-derived components. HBsAg expressed in yeast is generally non-glycosylated, and in one embodiment, the non-glycosylated form of HBsAg is used in the immunogenic compositions provided herein. HBsAg expressed in yeast has high immunogenicity and can be prepared without the risk of blood product contamination. Many methods for purifying HBsAg from recombinant yeast are known in the art.

[0131] HBsAg generally assumes the form of substantially spherical particles (average diameter of about 20 nm), including a lipid matrix containing phospholipids. HBsAg particles expressed in yeast may contain phosphatidylinositol, which is not found in native HBV virions.

[0132] In one embodiment, HBsAg is derived from HBV subtype adw2.

[0133] The amount of HBsAg is usually expressed in micrograms. The combined vaccine containing HBsAg usually contains between 5 and 60 μg / ml. The concentration of HBsAg in the compositions provided herein is, for example, less than 60 μg / ml, for example, ≤55 μg / ml, ≤50 μg / ml, ≤45 μg / ml, ≤40 μg / ml, etc. A concentration of about 20 μg / ml is typical, for example 10 μg per dose. In some embodiments, the composition contains "low dose" HBsAg. This means that the HBsAg concentration in the composition is ≤5 μg / ml, for example, <4, <3, <2.5, <2, <1, etc. Thus, for a typical unit dose volume of 0.5 ml, the amount of HBsAg is less than 2.5 μg, for example <2, <1.5, <1, <0.5, etc.

[0134] Poliovirus Poliovirus causes polio. Inactivated poliovirus vaccine (IPV) has been known for many years. Thus, the combined vaccine provided herein can contain inactivated poliovirus antigen.

[0135] Poliovirus can be grown in cell culture. In one embodiment, Vero cell lines derived from monkey kidneys are used in the culture. Vero cells can conveniently be cultured microcarriers. After growth, the virions can be purified using techniques such as ultrafiltration, diafiltration, and chromatography. When materials from animals (optionally bovine) are used in cell culture, they need to be obtained from sources free of transmissible spongiform encephalopathy (TSE), for example, free of bovine spongiform encephalopathy (BSE). In one embodiment, poliovirus is grown in cells cultured in a medium free of animal-derived components.

[0136] Prior to administration to a patient, the poliovirus must be inactivated, which can be done by treatment with formaldehyde (or, for example, a non-aldehyde agent). Polio can be caused by one of three types of poliovirus. The three types are similar and cause the same symptoms, but are very different antigenically, and infection by one type does not protect against infection by the other types. In one embodiment, the compositions provided herein use three poliovirus antigens: 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 can also be used. These viruses are, for example, grown, purified, and inactivated individually and then combined to provide a bulk trivalent mixture for use in the compositions provided herein.

[0137] The IPV component is usually expressed in "DU" units ("D antigen units"; Module 6 of WHO's The immunological basis for immunization series (Robertson)). The combined vaccine usually contains between 1 and 100 DU per dose per poliovirus type, for example, about 40 DU of type 1 poliovirus, about 8 DU of type 2 poliovirus and about 32 DU of type 3 poliovirus, but lower doses (WO2008 / 028956; WO2008 / 028957) can be used, for example, 10 - 20 DU for type 1, 2 - 4 DU for type 2 and 8 - 20 DU for type 3. The combined vaccines provided herein can contain "low dose" polioviruses. In the case of type 1 poliovirus, "low dose" means that the virus concentration in the composition is ≤ 20 DU / ml, for example < 18, < 16, < 14, < 12, < 10, etc. In the case of type 2 poliovirus, "low dose" means that the virus concentration in the composition is ≤ 4 DU / ml, for example < 3, < 2, < 1, < 0.5, etc. In the case of type 3 poliovirus, "low dose" means that the virus concentration in the composition is ≤ 16 DU / ml, for example < 14, < 12, < 10, < 8, < 6, etc. When all three of types 1, 2 and 3 polioviruses are present, the three antigens can be present in a DU ratio of 5:1:4, respectively, or in any other suitable ratio, for example in a ratio of 15:32:45 when using the Sabin strain (Liao et al. (2012) J Infect Dis. 205:237 - 43). Low dose antigens from the Sabin strain are useful, in which case type 1 is ≤ 10 DU, type 2 is ≤ 20 DU, type 3 is ≤ 30 DU (per unit dose, usually per 0.5 ml).

[0138] When using the IPV component and growing its poliovirus in Vero cells, the vaccine composition contains, for example, less than 10 ng / ml of Vero cell DNA, for example ≤ 1 ng / ml, for example ≤ 500 pg / ml or ≤ 50 pg / ml, for example, less than 10 ng / ml of Vero cell DNA that is ≥ 50 base pairs in length.

[0139] Preparation of combined vaccine The antigen components derived from the above pathogens for use in vaccines are usually referred to by the following abbreviations: for diphtheria toxoid, "D"; for tetanus toxoid, "T"; for pertussis antigen, "P", where "aP" is acellular pertussis antigen (e.g., including at least OMV, PT, and FHA provided herein and optionally pertactin and / or FIM2 / FIM3), for hepatitis B surface antigen, HBsAg, for conjugate Haemophilus influenzae type b capsular polysaccharide, "Hib", and for trivalent inactivated poliovirus, "IPV".

[0140] The embodiments provided herein include, but are not limited to, mixed 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

[0141] In some embodiments, these mixed vaccines contain only the listed antigens as active ingredients, but may further contain excipients such as adjuvants, buffers, etc. In some embodiments, the aP component consists of OMV, PT (e.g., genetically detoxified PT), and FHA provided herein. In some embodiments, the aP component consists of OMV, PT (e.g., genetically detoxified PT), FHA, and PRN provided herein. In some embodiments, the aP component consists of OMV, PT (preferably genetically detoxified PT), FHA, and FIM2 / FIM3 provided herein. In some embodiments, the aP component consists of OMV, PT (e.g., genetically detoxified PT), FHA, PRN, and FIM2 / FIM3 provided herein.

[0142] In the case of pediatric combination vaccines, the D:T ratio is typically greater than 1 (i.e., pediatric vaccines usually have an excess of D in Lf units), generally between 2:1 and 3:1 (measured in Lf units), for example 2.5:1. In contrast, for booster vaccines administered to adolescents or adults (who have typically received at least one pediatric combination vaccine, usually containing D and T), the T:D ratio is typically greater than 1 (i.e., booster vaccines usually have an excess of T in Lf units), generally between 1.5:1 and 2.5:1, for example 2:1.

[0143] One useful vaccine comprises the OMVs provided herein, and per unit dose, 2 Lf D, 5 Lf T, 4 μg PT-9K / 129G, 4 μg FHA, and 8 μg pertactin. Another useful vaccine comprises the OMVs provided herein, and per unit dose, 25 Lf D, 10 Lf T, 25 μg PT-9K / 129G, 25 μg FHA, and 8 μg pertactin. Combinations of D and T are of interest.

[0144] Immunization In addition to providing immunogenic compositions as described above, the present specification also provides the use of OMVs and immunogenic compositions in methods for enhancing an immune response in a mammal, including administering an OMV or an immunogenic composition provided herein to a mammal. Typically, the immune response is an antibody response. In some embodiments, the antibody response is a protective antibody response. The present specification also provides compositions for use in such methods.

[0145] The present specification also provides a method for protecting a mammal from a bacterial infection and / or disease, including administering to the mammal an immunogenic composition provided herein.

[0146] Also provided herein are compositions for use as a medicament (e.g., as an immunogenic composition or as a vaccine). Also provided is a population of purified OMVs as disclosed herein for use in a medicament. Also provided is the use of the OMVs provided herein in the manufacture of a medicament for preventing bacterial infection in a mammal.

[0147] In one embodiment, the mammal is a human. The human can be an adult or, for example, a child. When the vaccine is for prophylactic use, the human is, for example, a child (e.g., an infant or a baby). When the vaccine is for therapeutic use, the human is, for example, an adult. A vaccine for children can also be administered to adults to evaluate, for example, safety, dosage, immunogenicity, etc.

[0148] The effectiveness of a therapeutic treatment can be tested by monitoring for bacterial infection after administration of the composition provided herein. The effectiveness of a prophylactic treatment can be tested by monitoring for an immune response to an immunogenic protein or other antigen in the vesicles after administration of the composition. The immunogenicity of the composition provided herein can be determined by administering it to a subject (e.g., a child between 12 and 16 months of age) and then determining standard serological parameters. These immune responses are generally determined around 4 weeks after administration of the composition and compared to the values determined prior to administration of the composition. When more than one dose of the composition is administered, determinations may be made after more than one administration.

[0149] The compositions provided herein will generally be administered directly to a patient. Direct delivery can be by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or into the interstitial space of a tissue), or by each of rectal, oral, vaginal, topical, transdermal, intranasal, intraocular, intra-aural, intralung, or other mucosal administrations. In one embodiment, intramuscular administration to the thigh or upper arm is provided. The injection can be via a needle (e.g., a subcutaneous needle), but alternatively, needle-free injection can be used. A typical intramuscular dose is about 0.5 ml.

[0150] The immunogenic compositions provided herein can be used to induce systemic and / or mucosal immunity.

[0151] The dosing regimen may be a single-dose schedule or a multiple-dose schedule. Multiple doses can be used in a primary immunization schedule and / or a booster immunization schedule. A booster dosing schedule can also be carried out after the primary dosing schedule. Appropriate timings between priming doses (e.g., 4 to 16 weeks), and appropriate timings between priming and boosting can be determined conventionally.

[0152] General The term "comprising" includes "including"; for example, a composition "comprising" X can further include additional elements, such as X+Y. The term "substantially" does not exclude "completely"; for example, a composition "substantially free of" Y may completely lack Y, or Y may be present as an incidental impurity at a level that does not affect the properties of the composition. In some embodiments, the term "comprising" refers to including the indicated active agent, such as the recited polypeptide, as well as other active agents and pharmaceutically acceptable carriers, excipients, emollients, stabilizers, etc. known in the pharmaceutical industry. In some embodiments, the term "consisting essentially of" refers to a composition where the only active ingredient of the composition is the specified active ingredient(s), e.g., an antigen, but other compounds can be included for purposes such as stabilizing or preserving the formulation that do not directly contribute to the therapeutic effect of the specified active ingredient. Use of the transitional phrase "consisting essentially of" is intended to define a claim scope that includes the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. See In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in original); see also MPEP§2111.03. Thus, the term "consisting essentially of" is not intended to be construed as equivalent to "comprising" when used in a claim. The term "consisting of" and variations thereof mean "limited to" unless specifically specified otherwise. In certain areas, the term "including active ingredients consisting of" can be used instead of "consisting essentially of".The term "about" in relation to the numerical value x means, for example, x ± 10%, x ± 5%, x ± 4%, x ± 3%, x ± 2%, x ± 1%. When a method refers to steps such as, for example, (a), (b), (c), etc., these are intended to be consecutive, i.e., step (a) precedes step (b) and step (c) follows step (b). However, unless otherwise specified, a process that includes a step of mixing two or more components does not require a specific mixing order. Therefore, the components can be mixed in any order. If there are three components, two of the components can be mixed with each other and then the mixture can be mixed with the third component.

[0153] Numbered embodiments Embodiment 1. A method for substantially removing or reducing the concentration or amount of one or more impurities from a sample containing outer membrane vesicles (OMV), comprising: (a) concentrating and washing the sample by tangential flow filtration (TFF) through a tangential flow filtration (TFF) membrane to obtain a residual concentrate containing OMV; and (b) filtering the residual concentrate by flow-through chromatography using a chromatographic matrix comprising a porous outer layer, an internal core containing an octylamine ligand, and beads having a molecular weight cut-off of about 700 kDa to obtain a flow-through of purified OMV, thereby obtaining purified OMV.

[0154] Embodiment 2. The method of Embodiment 1, wherein the OMV is obtained from Gram-negative bacteria.

[0155] Embodiment 3. The OMV is (i) bacteria of the genus Bordetella, optionally obtained from Bordetella pertussis, optionally the bacteria contain the S1 gene containing the mutations R9K and E129G, and optionally express the genetically detoxified pertussis toxoid PT-9K / 129G, and / or the bacteria are (I) recombinant bacteria producing lipid A having a C3' acyl chain length of about 10 carbon atoms (C10), and / or (II) a C2' acyl chain length of about 10 carbon atoms (C10), and / or (III) a C2 acyl chain length of about 10 carbon atoms (C10), or (ii) A method according to embodiment 2, comprising bacteria of the genus Neisseria, optionally obtained from Neisseria gonorrhoeae, optionally genetically modified Neisseria gonorrhoeae bacteria, for example, comprising genetic modification(s) that reduce endotoxin activity compared to wild-type and / or unmodified Neisseria gonorrhoeae bacteria.

[0156] Embodiment 4. A method according to any one of embodiments 1 to 3, wherein the TFF membrane has a molecular weight cut-off of about 500 kDa.

[0157] Embodiment 5. A method according to any one of embodiments 1 to 3, wherein the TFF membrane is a hollow fiber membrane having a molecular weight cut-off of about 750 kDa or about 300 kDa.

[0158] Embodiment 6. A method according to embodiment 4 or 5, wherein the TFF in step (a) comprises concentrating and washing the sample by at least one ultrafiltration cycle and at least one cycle of diafiltration.

[0159] Embodiment 7. A method according to embodiment 6, wherein at least one ultrafiltration cycle concentrates the sample by at least about 5-fold, for example about 10-fold.

[0160] Embodiment 8. A method according to embodiment 7, wherein at least one cycle of diafiltration washes the sample with at least 5 volumes of wash solution, for example 10 volumes of wash solution, optionally the bacteria are bacteria of the genus Bordetella, and the wash solution is selected from (1) Dulbecco's phosphate buffered saline (DPBS) optionally containing EDTA, for example about 5 mM EDTA, or (2) 20 mM Tris buffer pH 8.6 optionally containing sucrose, for example about 3% sucrose, or optionally the bacteria are bacteria of the genus Neisseria, and the wash solution is selected from (1) 10 mM Na2HPO4 / NaH2PO4 pH 6.8 containing 150 mM NaCl or (2) phosphate buffered saline (PBS) pH 7.4.

[0161] Embodiment 9. A method according to any one of embodiments 1 to 8, wherein the sample of OMVs is obtained by deoxycholate (DOC) disruption of the outer membrane of bacteria of the genus Bordetella.

[0162] Embodiment 10. The method of Embodiment 9, wherein a sample containing OMV is prepared by a method comprising: (i) homogenizing a fermentation broth of bacteria of the genus Bordetella to obtain a homogenized fermentation broth; (ii) treating the homogenized fermentation broth with DOC and optionally benzonase to produce a crude preparation of DOC-extracted OMV; and (iii) centrifuging and / or filtering the crude preparation of DOC-extracted OMV to produce a sample containing OMV.

[0163] Embodiment 11. The method of Embodiment 10, wherein the homogenized fermentation broth is treated with DOC and benzonase.

[0164] Embodiment 12. The method according to any one of Embodiments 1 to 8, wherein the sample containing OMV comprises or consists of OMV released into the culture medium.

[0165] Embodiment 13. The method of Embodiment 12, wherein a sample containing OMV is prepared by a method comprising: (i) optionally centrifuging a fermentation broth of bacteria of the genus Neisseria; (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 OMV.

[0166] Embodiment 14. The method according to any one of Embodiments 1 to 13, further comprising: (a') filtering the residue to remove the precipitate; and / or (c) passing the purified OMV through a 0.22 μm filter for sterile filtration to produce a sterile preparation containing the purified OMV.

[0167] Embodiment 15. A method for preparing OMV from Bordetella bacteria, comprising: (i) homogenizing a fermentation broth of Bordetella bacteria to obtain a homogenized fermentation broth containing Bordetella bacteria; (ii) treating the homogenized fermentation broth with DOC to disrupt the outer membrane of Bordetella bacteria, and optionally treating with benzonase to produce a crude preparation of DOC-extracted OMV; (ii) centrifuging and / or filtering the crude preparation of DOC-extracted OMV to produce a sample containing DOC-extracted OMV and one or more impurities; (iv) removing or reducing the concentration of one or more impurities by the following: (v) concentrating and washing the sample containing DOC-extracted OMV and one or more impurities by tangential flow filtration (TFF) through a TFF membrane to obtain a retentate concentrate containing OMV; and (vi) filtering the retentate concentrate by flow-through chromatography using a chromatography matrix comprising a porous outer layer, an internal core containing octylamine ligand, and beads having a molecular weight cut-off of about 700 kDa to obtain a flow-through of purified OMV; and (vii) sterile filtering the flow-through of purified OMV using a 0.22 μm filter.

[0168] Embodiment 16. The method of Embodiment 15, wherein the Bordetella bacteria is Bordetella pertussis.

[0169] Embodiment 17. The method of Embodiment 16, wherein the Bordetella pertussis is a recombinant bacterium that contains the S1 gene containing mutations R9K and E129G and optionally expresses the genetically detoxified pertussis toxoid PT-9K / 129G, and / or (i) has a C3' acyl chain length of about 10 carbons (C10), and / or (ii) has a C2' acyl chain length of about 10 carbons (C10), and / or (iii) is a recombinant bacterium that produces lipid A having a C2 acyl chain length of about 10 carbons (C10).

[0170] Embodiment 18. A method for preparing OMV from bacteria of the genus Neisseria, comprising: (i) centrifuging a fermentation broth; (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 OMV and one or more impurities; (vi) removing or reducing the concentration of one or more impurities by the following: (vii) concentrating and washing a sample containing OMV and one or more impurities by TFF through a membrane to obtain a residual concentrate containing OMV; and (viii) filtering the residual concentrate by flow-through chromatography using a chromatography matrix comprising a porous outer layer, an internal core containing an octylamine ligand, and beads having a molecular weight cut-off of about 700 kDa to obtain a flow-through of purified OMV; and (ix) sterile filtering the flow-through of purified OMV using a 0.22 μm filter.

[0171] Embodiment 19. The method of Embodiment 18, wherein the bacteria of the genus Neisseria is Neisseria gonorrhoeae.

[0172] Embodiment 20. The method of Embodiment 18 or 19, wherein the Neisseria gonorrhoeae is a Neisseria gonorrhoeae bacterium comprising a genetic modification(s), optionally wherein the genetic modification(s) reduces the endotoxin activity of the OMV as compared to wild-type or unmodified Neisseria gonorrhoeae.

[0173] Embodiment 21. The method of Embodiment 20, wherein the genetic modification(s) that reduces endotoxin activity results in an increase in the level of pentaacylated lipid A and a decrease in the level of hexaacylated lipid A as compared to lipid A present in wild-type and / or unmodified Neisseria gonorrhoeae.

[0174] Embodiment 22. The method of Embodiment 21, wherein the genetic modification(s) reduces or abolishes the expression and / or function of the lipid A biosynthetic lauroyl acyltransferase (lpxl1) gene, mRNA, and / or polypeptide.

[0175] Embodiment 23. The method of Embodiment 22, wherein the gonococcal bacterium comprises further genetic modification that reduces or eliminates the expression and / or function of the reducible-modifiable protein (rmp) gene, mRNA, and / or polypeptide.

[0176] Embodiment 24. The method of any one of Embodiments 1 to 23, wherein one or more impurities comprise the 60 kDa chaperonin GroEL or are the 60 kDa chaperonin GroEL.

[0177] Embodiment 25. A method for reducing the concentration of the 60 kDa chaperonin GroEL in a sample of OMVs optionally derived from Bordetella or Neisseria, comprising filtering a sample of OMVs by flow-through chromatography using a chromatographic matrix comprising a porous outer layer, an inner core comprising an octylamine ligand, and beads having a molecular weight cut-off of about 700 kDa to obtain a flow-through of purified OMVs, wherein the 60 kDa chaperonin GroEL is present in an amount of less than 1.5% of the total protein.

[0178] Embodiment 26. The method of Embodiment 24 or 25, wherein the 60 kDa 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 any one of SEQ ID NOs: 1 to 4, and optionally the 60 kDa chaperonin GroEL comprises or has the sequence of any one of SEQ ID NOs: 1 to 4.

[0179] Embodiment 27. A population of purified OMVs produced by the method of any one of Embodiments 1 to 26.

[0180] Embodiment 28. A population of purified OMVs that is at least 85% pure or about 85% to about 100% pure.

[0181] 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.

[0182] Population of purified OMVs of Embodiment 28 or 29, wherein the purity is measured using size exclusion ultra-high performance liquid chromatography (UPLC), for example by size exclusion UPLC, and the ratio between the major peak and the low molecular size peak is determined.

[0183] Population of purified OMVs of any one of Embodiments 28 to 30, wherein one or more impurities are present at an impurity 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 about 1%, for example free of impurities, for example protein impurities.

[0184] Population of purified OMVs of any one of Embodiments 28 to 31, wherein one or more impurities comprise the 60 kDa chaperonin GroEL or are the 60 kDa chaperonin GroEL.

[0185] Population of purified OMVs of Embodiment 32, wherein the 60 kDa 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 any one of SEQ ID NOs: 1-4, and optionally the 60 kDa chaperonin GroEL comprises or has the sequence of any one of SEQ ID NOs: 1-4.

[0186] Population of purified OMVs of Embodiment 32 or 33, comprising from about 0.01% to about 10% GroEL as a percentage of the total protein.

[0187] Population of purified OMVs of any one of Embodiments 32 to 34, comprising less than 5% GroEL.

[0188] Population of purified OMVs according to any one of embodiments 27 to 35, substantially free of process-related impurities.

[0189] Embodiment 37. A population of purified OMVs according to any one of embodiments 27 to 36, wherein the purified OMVs are obtained from Bordetella and conjugated to the capsular polysaccharide of Haemophilus influenzae type b (Hib).

[0190] Embodiment 38. The OMVs are from Gram-negative bacteria, optionally (i) from bacteria of the genus Bordetella, optionally from Bordetella pertussis, optionally the bacteria contain the S1 gene with mutations R9K and E129G, and optionally express the genetically detoxified pertussis toxoid PT-9K / 129G, and / or the bacteria are recombinant bacteria that produce lipid A having (I) a C3' acyl chain length of about 10 carbons (C10), and / or (II) a C2' acyl chain length of about 10 carbons (C10), and / or (III) a C2 acyl chain length of about 10 carbons (C10), or (ii) from bacteria of the genus Neisseria, optionally from Neisseria gonorrhoeae, optionally the bacteria are genetically modified Neisseria gonorrhoeae bacteria, for example containing one or more genetic modifications that reduce endotoxin activity compared to wild-type and / or unmodified Neisseria gonorrhoeae, a population of purified OMVs according to any one of embodiments 28 to 37.

[0191] Embodiment 39. A population of purified OMVs according to any one of embodiments 27 to 38 for use in medicine.

[0192] [Examples] [Example 1] Fermentation and deoxycholic acid (DOC) preparation of OMVs The frozen contents of an ampoule containing the working seed of Bordetella pertussis strain Tohama were thawed at room temperature for 10 minutes.

[0193] Addition of 1 g / L of dimethylcyclodextrin and 10 g / L of acid casein hydrolysate, replacement of 40 mg / L of L-cystine with 40 mg / L of L-cysteine, and use of higher concentrations of Na-L-glutamate (11.84 g / L), reduced glutathione (150 mg / L) and ascorbic acid (400 mg / L) in 30 ml of fresh medium (source Stainer and Scholte (J. Gen. Microbiol. 63:211-220 (1971))). Inoculate 400 μl of the working seed into each of two 250 ml shaking flasks, incubate at 35 °C (+ / -1 °C) and 150 rpm for 24 hours (+ / -1 hour) to produce the first preculture.

[0194] When the optical density (OD 650nm ) of the first preculture reached 1 to 1.5 at 650 nm, they were used to inoculate two 3 L shaking flasks containing 1 L of fresh medium. The second preculture flasks were incubated at 35 °C (+ / -1 °C) and 150 rpm for 24 hours (+ / -1 hour), and then they were pooled. 1500 ml of the pooled preculture was used to inoculate a 20 L fermenter (Biolafitte (trademark)) containing 10 L of fresh medium and 3 ml of antifoaming agent. Batch fermentation was carried out for 24 hours using the following parameters: temperature 35 °C, head pressure 0.4 bar, constant air flow rate of 20 NL / min, pH 7.2 (adjusted using 50% acetic acid), and maximum stirring speed of 50 rpm to 1000 rpm. During fermentation, the temperature (35 °C) and head pressure (0.4 bar) were maintained at constant levels. A mechanical foam breaker was used to control foaming during fermentation. The air flow rate was gradually increased during fermentation according to a predefined curve. The level of dissolved oxygen was set at 25%, and was adjusted by increasing stirring when DO fell below 25%. The minimum stirring speed was set at 50 rpm and the maximum stirring speed was set at 1000 rpm. The pH was adjusted to 7.2 by addition of 50% acetic acid (w / v or weight / volume).

[0195] During fermentation, the growth of the culture was monitored by OD 650nmIt was monitored as such. When the oxygen consumption decreased (as a result of glutamate consumption) and caused a decrease in the stirring speed, the fermentation was stopped. At the end of the fermentation, the cells were pelleted by centrifugation at 5000×g for 30 minutes (4 °C). The supernatant was filtered through a Sartobran-P (0.45 μm + 0.22 μm) filter and dispensed into 1 L flasks. The supernatant and cell pellet were stored at -20 °C.

[0196] For the detergent extraction of OMVs, the frozen bacterial pellet was thawed and resuspended and homogenized in 4 volumes of 20 mM Tris-HCl - 2 mM EDTA buffer (pH 8.6), 100 U / mL benzonase together with cOmplete™ EDTA-free protease inhibitor cocktail and incubated with stirring for 30 minutes at room temperature. cOmplete™ EDTA-free protease inhibitor cocktail was added to 1 volume of either extraction buffer 1 (20 mM TrisHCl, 0.5% DOC, 2 mM EDTA, pH 8.6 buffer) or extraction buffer 2 (20 mM TrisHCl, 0.1% DOC, 2 mM EDTA, pH 8.6 buffer), and the suspension was incubated with stirring for 30 minutes at either 30 °C or 40 °C. After 30 minutes, 100 U / mL benzonase was added. The suspension was incubated and stirred for a further 30 minutes. Cell debris was removed by centrifugation at 20,000 g for 30 minutes (4 °C), and the supernatant containing OMVs was taken out and sterile filtered (0.22 μm). Each 250 ml of the solution containing DOC-extracted OMVs was diluted to 500 ml with OMV buffer (Tris 20 mM pH 8.6 + 3% sucrose). The generalized DOC extraction procedure used is shown in Figure 1.

[0197] [Example 2] Preparation of OMVs using 0.1% DOC at 30 °C, purification by TFF and ultracentrifugation DOC-extracted outer membrane vesicles derived from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (extraction buffer 2) at 30 °C. The OMV preparation was purified using a combination of TFF and ultracentrifugation (Figure 2).

[0198] In the first purification step, TFF was performed as follows using a Millipore 500 kDa cassette: The preparation was concentrated 10-fold to 50 ml (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi). The concentrated preparation was diafiltered at a constant volume (50 ml) using 500 ml of OMV buffer (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi). 1550 U of benzonase (Merck 327 U / μl) was added to the residue containing OMV and stirred for 1 hour (pump flow rate 50 ml / min, permeate valve closed, no counterpressure). The concentrated preparation was further diafiltered with 500 ml of OMV buffer (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi), and 50 ml of the residue was recovered in reverse flow. The system was further washed with 50 ml of OMV buffer, and the washings were pooled with the residue to obtain a final volume of approximately 100 ml (residue + washings).

[0199] After TFF, the pooled residue was centrifuged twice at 150,000 g for 2 hours, the supernatant was removed, the pellet containing OMV was resuspended in 30 ml of DPBS, aliquoted into 1 ml and 15 ml, and stored at -70 °C.

[0200] The lipid concentration was measured using the fluorescent dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide] (also referred to as FM 4-64, Molecular Probes), and a standard calibration curve was created for a known amount of DOPC. FM 4-64 is a lipophilic styryl dye that selectively intercalates into lipid membranes and stains lipid membranes with red fluorescence (excitation / emission maxima approximately 515 / 640 nm). The protein content was measured using the Lowry method known in the art.

[0201]

Table 1

[0202] TFF followed by ultracentrifugation removed free proteins and aggregates (reducing the protein content by approximately 50%), while generally retaining OMVs (the lipid content in the final sample was reduced by about 12%). However, both the purity (85%) and yield (25 mg / L of the fermentation culture based on protein content) were low, and the 60 kDa chaperonin GroEL was not removed (Figure 3). Furthermore, after ultracentrifugation, the pellet was difficult to resuspend, and as a result, sterile filtration was impossible.

[0203] [Example 3] Preparation of OMVs using 0.1% DOC at 40 °C, TFF, and purification by 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 (Figure 2).

[0204] Increasing the temperature during DOC extraction did not affect subsequent purification using TFF followed by ultracentrifugation. The resulting pellet was very difficult to resuspend, contained a high level of precipitate, and as a result, sterile filtration was impossible. The resulting yield was also low, at 17 mg / L of the fermentation culture (based on protein content), and the 60 kDa chaperonin was not removed. Considering the low recovery and purity obtained, ultracentrifugation as the second step of the two-step purification process following TFF seems infeasible for the production of OMVs on a manufacturing scale.

[0205] [Example 4] Preparation of OMVs using 0.1% DOC at 40 °C, TFF, and purification by flow-through chromatography (Sephacryl S-500 gel filtration) The DOC-extracted outer membrane vesicles derived from Bordetella pertussis were prepared according to Example 1 using 0.1% DOC (extraction buffer 2) at 40°C. The OMV preparation was purified using a combination of TFF and flow-through chromatography (Sephacryl S-500 gel filtration) (Figure 4). TFF was performed as follows using a Millipore 500 kDa cassette:

[0206] The DOC-extracted OMV was concentrated 10-fold to 50 ml (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi). The concentrated preparation was diafiltered to a constant volume (50 ml) using 500 ml of OMV buffer (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi). 1550 U of benzonase (Merck 327 U / μl) was added to the residue containing OMV and stirred for 1 hour (pump flow rate 50 ml / min, permeate valve closed, no counter pressure). The concentrated preparation was further diafiltered with 500 ml of OMV buffer (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi), and 50 ml of the residue was recovered in reverse flow. The system was further washed with 50 ml of OMV buffer, and the washings were pooled with the residue to obtain a final volume of approximately 100 ml (residue + washings).

[0207] After TFF, further purification was performed by gel filtration using Sephacryl S-500. The pooled residue was applied to a Sephacryl S-500 High Resolution chromatography column (10 mm × 450 mm × 2 = 70.6 ml). Washing was performed using OMV buffer, and OMV was eluted in the flow-through (flow-through + washings = 115 ml). The eluate was pooled and sterile filtered under laminar flow using a Sterivex GV 0.22 μm filter (surface 10 cm 2 , Millipore) and aliquoted into 1 ml and 15 ml portions. The aliquots were stored at -70°C.

[0208] The OMV yield was improved by replacing ultracentrifugation with Sephacryl S-500 gel filtration (yield 35.7 mg / L). However, the 60 kDa chaperonin was not completely removed (data not shown). Furthermore, a 3 L column was required to process 150 ml of the OMV preparation, suggesting a volume constraint for scaling up the process to an industrial level.

[0209] [Example 5] Preparation of OMV using 0.1% DOC at 40°C, TFF, and purification by flow-through chromatography (CaptoCore 700 gel filtration) The 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 OMV preparation was purified using a combination of TFF and flow-through chromatography (CaptoCore 700 gel filtration) (Figure 5). TFF was performed using a Millipore 500 kDa cassette as follows:

[0210] The DOC-extracted OMV was concentrated 10-fold to 50 ml (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi). The concentrated preparation was diafiltered to a constant volume (50 ml) using 500 ml of OMV buffer (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi). 1550 U of benzonase (Merck 327 U / ul) was added to the residue containing OMV and stirred for 1 hour (pump flow rate 50 ml / min, permeate valve closed, no counterpressure). The concentrated preparation was further diafiltered with 500 ml of OMV buffer (pump flow rate 125 ml / min, TMP 5 + / - 0.5 psi), and 50 ml of the residue was recovered in reverse flow. The system was further washed with 50 ml of OMV buffer, and the washings were pooled with the residue to obtain a final volume of approximately 100 ml (residue + washings).

[0211] After TFF, further purification was performed by gel filtration using CaptoCore 700. The pooled retentate was applied to a CaptoCore 700 (GE Healthcare) column (109 ml column volume, 1.3 cm radius, pre-equilibrated in OMV buffer) at 11.5 ml / min (130 cm / hour). Washing was performed with OMV buffer at the same flow rate. The solution containing OMV was eluted in the flow-through (flow-through + wash = 115 ml). The eluate was pooled and sterile filtered under laminar flow using a Sterivex GV 0.22 μm filter (10 cm surface 2 , Millipore) and aliquoted into 1 ml and 15 ml aliquots. The aliquots were stored at -70 °C.

[0212] The use of CaptoCore 700 improved both the yield (44 mg / L) and purity (greater than 95%) of the OMV preparation. The process also significantly reduced the amount of 60 kDa chaperonin (GroEL) (Figure 6). Furthermore, a 130 ml column (or smaller) is required to process 150 ml of the OMV preparation, a typical TFF retentate, meaning that the process is industrially scalable.

[0213] [Example 6] Preparation of OMV using 0.5% DOC at 30 °C, TFF and purification by 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 OMV preparation was purified using a combination of TFF and CaptoCore as before (Figure 5).

[0214] Increasing the DOC concentration to 0.5% and reducing the temperature to 30 °C during extraction had a significant effect on the yield, which increased to 203 mg / L of culture. Furthermore, the process yielded a preparation that almost completely removed free proteins not bound to GroEL and OMV (purity greater than 95%) (Figures 7(a) and (b)).

[0215] [Example 7] Comparison of OMVs purified using various processes / conditions OMVs were prepared and purified using the conditions described below according to the method provided above:

[0216]

Table 2

[0217] More stringent extraction conditions led to an increase in the protein / lipid ratio and an increase in the DLS measured size. However, the polydispersity remained low (Pdi < 0.3).

[0218] [Example 8] Preparation of OMVs using 0.5% DOC at 30 °C, purification by TFF (750 kDa) and CaptoCore 700 In this experiment, the purification process of Example 5 was repeated 4 times, but the 500 kDa filter used in the TFF step was replaced with a 750 kDa hollow fiber filter (Figure 8) (GE Healthcare, UFP-750-C-H24LA, surface 0.0042 m 2 , dead volume 12 ml, buffer Tris 20 mM - sucrose 3% (P / V) pH 8.6, final recovery volume approximately 100 ml):

[0219]

Table 3

[0220] Compared to the process using a standard cassette for TFF, the yield obtained using the hollow fiber was generally higher (purity > 95%) and a further reduction in impurities was observed (Figure 9). The SE-UPLC profile showed excellent reproducibility while DLS showed good product homogeneity.

[0221]

Table 4

[0222] Despite the pore size, the 750 kDa hollow fiber membrane alone was unable to remove the 60 kDa chaperonin from the OMV preparation, and the CaptoCore700 step was still necessary (Figure 10). The SDS PAGE of OMV purified using the hollow fiber membrane showed a pattern similar but not identical to that of OMV purified using the 500 kDa cassette (Figure 11).

[0223] Replacing the 500 kDa cassette with a 750 kDa hollow fiber membrane for TFF resulted in increased OMV yield, purity, and homogeneity. Reproducibility was also excellent with respect to both protein yield and product quality.

[0224] [Example 9] Evaluation of the removal of 60 kDa chaperonin regarding the CaptoCore 700 column The removal of the 60 kDa chaperonin GroEL by CaptoCore 700 was determined using mass spectrometry. The relative amount of GroEL in each sample was measured using mass spectrometry (MS) as 9.4% of the total protein in the ultrafiltration residue (i.e., before filtration) and 1.06% of the total protein in the ultrafiltration permeate (i.e., after filtration). Using the Lowry assay, the total protein in the ultrafiltration residue was 230.6 mg, while the total protein in the ultrafiltration permeate was 36.5 mg.

[0225] Therefore, 21.7 mg of the 60 kDa chaperonin GroEL was present in the ultrafiltration residue, while 0.39 mg was present in the ultrafiltration permeate after filtration. Approximately 98.3% of the 60 kDa chaperonin GroEL was removed from the sample by the filtration step of CaptoCore 700 (the clearance was approximately 56-fold) (Figure 12).

[0226] [Example 10] Manufacturing process of Neisseria gonorrhoeae OMV The upstream process included an inoculation step, a fermentation step, a recovery step, and a filtration step. During the pre-culture step, the flask was inoculated with a Neisseria gonorrhoeae liquid seed vial. The number of growth steps (number and volume of flasks and / or pre-fermenters) depended on the final scale of fermentation. Subcultures (passages) of seed growth and inoculation of the fermenter were carried out when the culture was in complete exponential growth. Biomass was formed during batch fermentation, and the product, i.e., GMMA, was released into the medium. When the carbon source in the medium was depleted, the fermented product was harvested and primary recovery was carried out by centrifugation: the pellet containing biomass was discarded, and the supernatant containing the product was filtered through 0.2 μm (filtration 1) to reduce the bioburden (complete removal of pathogenic cells). This intermediate was treated by adding Benzonase to hydrolyze genomic DNA. The material was filtered (filtration 2a), pathogenic cells were removed from the supernatant, and a holding time at 2 - 8 °C was applied to perform DNA hydrolysis before the purification process. Before purification, the supernatant was clarified by filtration through 0.2 μm (filtration 2) to remove possible precipitates, and the resulting intermediate was named SNF-BF. Tangential flow filtration (TFF) was carried out to achieve an improvement in product purity by reducing soluble proteins (intended to be Neisseria gonorrhoeae proteins not belonging to GMMA), nucleic acids, and residues of the fermentation medium. The diafiltration retentate was collected and filtered to remove precipitates (filtration 3). The resulting intermediate was named DR and used as the starting material for the next chromatographic step. CaptoCore 700 was used as the resin for the chromatographic step operating in flow-through mode to remove soluble proteins and DNA bound to the resin. The resulting intermediate was the CaptoCore 700 eluate (CC700EL2). Before performing 0.2 μm orthogonal filtration, CC700EL2 was diluted to a total protein concentration targeting 1.2 ± 0.1 mg / ml as needed to maximize the volume of the DS. After filtration 4, the resulting intermediate was named concentrated bulk (CB), and this solution was filled into a suitable container and frozen at -70 °C as a low bioburden material. The description of the manufacturing process for DS is outlined in Figure 13.

[0227] [Example 11] Technical details of CaptoCore 700 chromatography The purpose of this CaptoCore chromatography step is to increase the purity with respect to DNA and other protein removal (benzonase and soluble proteins).

[0228] The DR intermediate was applied to two CaptoCore 700 columns (column volume (CV approximately 1 L)) connected in series and pre-equilibrated with 20 CV of PBS pH 7.4 at 300 - 500 cm / hour. Washing was carried out using the same buffer at the same flow rate. Chromatography was carried out in flow-through mode, and thus, the start of peak collection was in the loading block and the end was in the washing block. Peak collection was carried out manually after the UV signal at 280 nm (without method instructions).

[0229] [Example 12] Implementation of CaptoCore 700 in the production process of Neisseria gonorrhoeae OMV The production process of the GMMA-FA1090-2KO drug substance was improved by replacing the ultracentrifugation step between filtration 3 and filtration 4 with CaptoCore 700 (multimodal chromatography). The GMMA-FA1090-2KO drug substance contains Neisseria gonorrhoeae bacteria derived from strain FA1090 and also contains (I) genetic modification(s) that reduce or eliminate the expression and / or function of the lipid A biosynthetic lauroyl acyltransferase (lpxl1) gene, mRNA, and / or polypeptide, and (II) genetic modification(s) that reduce or eliminate the expression and / or function of the reduced-modified protein (rmp) gene, mRNA, and / or polypeptide.

[0230] When using CaptoCore 700 chromatography (yield = 55%) instead of the ultracentrifugation step (yield = 68%), a slight decrease in yield was observed. The yield was evaluated by measuring the OMV content by SEC-Dye HPLC method based on the quantification of FM4-64 for samples after isocratic SEC chromatography and standard GMMA preparations with known protein concentrations.

[0231] As shown in Fig. 14, when using a CaptoCore 700 column instead of the ultracentrifugation step, an increase in purity was observed, and as shown in Fig. 15, GroEL protein impurities were completely removed.

[0232] It should be understood that the present invention has been described by way of example only, and that modifications can be made while remaining within the scope and spirit of the present invention.

[0233] Sequence SEQ ID NO:1: Chaperonin "GroEL" of 60 kDa; Bordetella pertussis (strain Tohama I / ATCC BAA-589 / NCTC 13251) TIFF2025524990000006.tif42167

[0234] SEQ ID NO:2: Molecular chaperone GroEL [Neisseria gonorrhoeae FA1090] GenBank: AAW90696.1 TIFF2025524990000007.tif42167

[0235] SEQ ID NO:3: Molecular chaperone GroEL [Neisseria meningitidis NZ98 / 254] TIFF2025524990000008.tif42167

[0236] SEQ ID NO:4: Molecular chaperone GroEL [Neisseria meningitidis 99M] TIFF2025524990000009.tif42167

Claims

1. A method for substantially removing or reducing the concentration or amount of one or more impurities from a sample containing outer membrane vesicles (OMVs), comprising: (a) concentrating and washing the sample by tangential flow filtration (TFF) through a tangential flow filtration (TFF) membrane to obtain a residual concentrate containing OMVs; and (b) filtering the residual concentrate by flow-through chromatography using a chromatographic substrate comprising a porous outer layer, an inner core containing an octylamine ligand, and beads having a molecular weight cutoff of about 700 kDa to obtain a flow-through of purified OMVs, thereby obtaining purified OMVs.

2. The method according to claim 1, wherein the TFF film has a molecular weight cutoff of approximately 500 kDa.

3. The method according to claim 1, wherein the TFF membrane is a hollow fiber membrane having a molecular weight cutoff of about 750 kDa or about 300 kDa.

4. Step (a) of TFF comprises concentrating and washing the sample by at least one ultrafiltration cycle and at least one diafiltration cycle, optionally concentrating the sample at least about 5 times, e.g., about 10 times, optionally washing the sample with at least 5 times the volume of washing solution, e.g., 10 times the volume of washing solution, optionally if the bacteria are of the genus Bordetella and the washing solution is (1) optionally Dulbecco's phosphate-buffered saline (DPBS) containing EDTA, e.g., about 5 mM EDTA, or (2) optionally 20 mM Tris buffer pH 8.6 containing sucrose, e.g., about 3% sucrose, or optionally if the bacteria are of the genus Neisseria and the washing solution is (1) 10 mM NaCl containing 150 mM NaCl 2 Hpo 4 / NaH 2 PO 4 The method according to claim 2, wherein pH 6.8 or (2) phosphate-buffered saline (PBS) pH 7.4 is selected.

5. The method according to claim 1, wherein a sample of OMV is obtained by deoxycholic acid (DOC) disruption of the outer membrane of a bacterium of the genus Bordetella, and an optional sample containing OMV is prepared by a method comprising: (i) homogenizing a fermentation sample of a bacterium of the genus Bordetella to obtain a homogenized fermentation sample; (ii) treating the homogenized fermentation sample with DOC and optionally benzonase to produce a crude preparation of DOC-extracted OMV; and (iii) centrifuging and / or filtering the crude preparation of DOC-extracted OMV to produce a sample containing OMV.

6. The method according to claim 1, wherein the OMV-containing sample contains or consists of OMV released into a culture medium, and optionally the OMV-containing sample is prepared by a method comprising: (i) optionally the step of centrifuging a fermentation sample of a bacterium of the genus Neisseria; (ii) the step of collecting the supernatant; (iii) the step of filtering the supernatant to obtain an intermediate; (iv) the step of treating the intermediate with benzonase to produce a crude preparation; and (v) the step of filtering the crude preparation to produce an OMV-containing sample.

7. The method according to claim 1, further comprising (a') filtering out the residue to remove the precipitate, and / or (c) sterilizing the purified OMV by passing it through a 0.22 μm filter to produce a sterile preparation containing the purified OMV.

8. A method for preparing OMV from bacteria of the genus Bordetella, comprising: (i) homogenizing a fermentation sample of bacteria of the genus Bordetella to obtain a homogenized fermentation sample containing bacteria of the genus Bordetella; (ii) treating the homogenized fermentation sample with DOC to disrupt the outer membrane of bacteria of the genus Bordetella, and optionally treating it with benzonase to produce a crude preparation of DOC-extracted OMV; (ii) centrifuging and / or filtering the crude preparation of DOC-extracted OMV to produce a sample containing DOC-extracted OMV and one or more impurities; and (iv) removing or reducing the concentration of one or more impurities by the method of claim 1.

9. A method for preparing OMV from bacteria of the genus Neisseria, comprising the following steps in sequence: (i) centrifuging a fermentation sample; (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 OMV and one or more impurities; and (vi) removing or reducing the concentration of one or more impurities by the method of claim 1.

10. The method according to claim 1, wherein one or more impurities contain or are 60 kDa chaperonin GroEL.

11. A method according to claim 10 for optionally reducing the concentration of 60 kDa chaperonin GroEL in a sample of OMV from the genus Bordetella or Neisseria, comprising filtering the OMV sample by flow-through chromatography using a chromatographic substrate comprising a porous outer layer, an inner core containing an octylamine ligand, and beads having a molecular weight cutoff of about 700 kDa, to obtain a flow-through of purified OMV, wherein 60 kDa chaperonin GroEL is present in an amount less than 1.5% of the total protein.

12. A population of purified OMV produced by the method described in claim 1.

13. A population of purified OMVs that is at least 85% pure, or approximately 85% to approximately 100% pure, wherein the purity is optionally measured using size exclusion ultrafast liquid chromatography (UPLC), for example by size exclusion ULC, and the ratio between major peaks and small molecular size peaks is determined.

14. A population of purified OMV according to claim 13, wherein one or more impurities are present at levels or concentrations 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%, and is free from impurities, such as protein impurities.

15. A population of purified OMV according to claim 14, wherein one or more impurities constitute about 0.01% to about 10% of GroEL as a percentage of total protein.

16. A group of purified OMV according to claim 13, wherein purified OMV is obtained from the genus Bordetella and conjugated to the capsular polysaccharide of Haemophilus influenzae type b (Hib).

17. OMV is obtained from Gram-negative bacteria, or from bacteria: (i) Recombinant bacteria of the genus Bordetella, optionally Bordetella pertussis, optionally containing the S1 gene with mutants R9K and E129G, optionally expressing the genetically detoxified pertussis toxoid PT-9K / 129G, and / or recombinant bacteria that produce lipid A having (I) a C3' acyl chain length of approximately 10 carbon atoms (C10), and / or (II) a C2' acyl chain length of approximately 10 carbon atoms (C10), and / or (III) a C2 acyl chain length of approximately 10 carbon atoms (C10), or (ii) A bacterium of the genus Neisseria, optionally Neisseria gonorrhoeae, optionally the bacterium is a genetically modified gonococcal bacterium, for example, comprising a gene modification (may include more) that reduces endotoxin activity compared to wild-type and / or unmodified Neisseria gonorrhoeae, optionally the gene modification (may include more) that reduces endotoxin activity results in an increased level of pentaacylated lipid A and a decreased level of hexaacylated lipid A compared to lipid A present in wild-type and / or unmodified Neisseria gonorrhoeae, optionally the gene modification (may include more) that reduces or eliminates the expression and / or function of the lipid A biosynthesis lauroyl acyltransferase (lpxl1) gene, mRNA, and / or polypeptide, optionally the Neisseria gonorrhoeae bacterium comprises further gene modifications that reduce or eliminate the expression and / or function of reductively modifiable protein (rmp) gene, mRNA, and / or polypeptide. The method according to claim 1, or the group of purified OMVs according to claim 13.

18. A composition comprising a population of purified OMV according to claim 13 for use in pharmaceuticals.

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