Bioconjugate compositions and methods for producing bioconjugates

Abstract

The present invention relates to improved pharmaceutical compositions comprising one or more bioconjugate compositions. Each bioconjugate composition comprises a mixture of monoglycosylated and polyglycosylated bioconjugates of a specific E. coli O-antigen polysaccharide covalently coupled to a Pseudomonas aeruginosa exoprotein A carrier protein. The invention also relates to a method for producing purified bioconjugates from gram-negative host cells and to bioconjugates obtained or obtainable by said method.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from European patent application EP 22 211 401.9 filed on December 5, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] References to electronically submitted sequence listings

[0004] The contents of the electronic sequence listing (CRU6090EPEPA1.xml; size: 4,785 bytes; creation date: November 28, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Compared to commensal Escherichia coli, extraintestinal pathogenic Escherichia coli (ExPEC) strains express a diverse array of virulence factors that enable them to colonize the gastrointestinal tract and cause a variety of extraintestinal infections, resulting in a substantial healthcare cost burden due to hospitalizations and mortality. Neonates, the elderly, and immunocompromised patients are particularly susceptible to ExPEC infections, including invasive ExPEC disease (IED).

[0006] O-antigens comprise the immunodominant component of the cell wall lipopolysaccharide (LPS) of Gram-negative bacteria, including Escherichia coli. Currently, >180 serologically distinct E. coli O-antigens have been identified, with the vast majority of ExPEC isolates classified into fewer than 20 O-antigen serotypes. The full-length E. coli O-antigen polysaccharide (O-PS) typically consists of approximately 10 to 25 repeating sugar units. In wild-type E. coli, this O-PS is linked to a highly conserved LPS core structure. Each of these components, the O-PS components and the LPS core structure, are synthesized separately by enzymes primarily encoded in the rfb and rfa gene clusters. Following O-antigen polymerization, the O-PS backbone can typically be modified by the addition of acetyl or glucose residues.

[0007] Efforts to develop vaccines to prevent ExPEC infections have focused on multivalent compositions comprising bioconjugates of E. coli O-antigen polysaccharides covalently linked to a carrier protein [see, e.g., Poolman and Wacker, J. Infect. Dis. (2016) v. 213(1), pp. 6-13; WO 2015 / 124769; WO 2017 / 035181; WO 2020 / 191082A1].

[0008] In general, glycoconjugate vaccines have been shown to be effective against CD4 +Efficient activation of T cells is driven by endosome-generated polysaccharide antigen fragments covalently linked to peptides derived from carrier proteins [see, eg, Avci et al., Nat. Med. (2011), 17: 1602-1609].

[0009] Although it appears to be a promising class of vaccines, the production of complex bioconjugates is generally a challenging task, especially the production of bioconjugates comprising bacterial O-antigen polysaccharides covalently coupled to a carrier protein. However, several production methods have been described, including methods for purifying such bioconjugates [see, for example, WO 2009 / 104074; WO 2020 / 191082; WO 2022 / 214620; van den Dobbelsteen et al., Vacine (2016), 34: 4152-4160].

[0010] Typically, O-EPA bioconjugates can be produced by enzymatic conjugation of the O-antigen polysaccharide (O-PS) component to a carrier protein in a Gram-negative bacterial host cell (such as E. coli), using, for example, the PglB oligosaccharyltransferase system [see, for example, WO 2015 / 124769; WO 2020 / 191082; Poolman and Wacker, J. Infect. Dis. (2016) v. 213(1), pp. 6-13 and references therein]. In this method, the coupling of the O-PS component to the carrier protein occurs in the periplasmic space, i.e., the space between the inner plasma membrane and the outer membrane of Gram-negative bacteria (such as E. coli).

[0011] Specifically, WO 2022 / 214620 describes a method for commercial-scale production of O-EPA bioconjugates. Thus, this method represents a significant improvement over previous methods for producing O-EPA bioconjugates, which included a size exclusion chromatography step (SEC), making these methods less favorable for large-scale production [e.g., van den Dobbelsteen et al., Vaccine (2016), 34:4152-4160; Burckhardt et al., Vaccine (2019), 37(38):5762–5769; Ravenscroft et al., Glycobiology (2016), 26(1):51–62; WO 2009 / 104074; or WO 2015 / 124769]. Thus, WO 2022 / 214620 describes for the first time a method suitable for large-scale production of O-EPA bioconjugates, such as production in bioreactors with volumes ranging from 100 L to 20,000 L. However, despite the significant advantages over previous approaches, this approach can be further improved, for example, to provide bioconjugate compositions having a relatively high proportion of polyglycosylated forms of O-EPA bioconjugates, i.e., EPA carrier proteins in which more than one glycosylation site is occupied, such as EPA carrier proteins linked to 2, 3, or 4 O-antigen polysaccharides. In turn, a higher proportion of polyglycosylated forms of O-EPA bioconjugates results in a reduced amount of carrier protein relative to the amount of O-antigen polysaccharide. This is particularly important because minimizing the amount of carrier protein mitigates the hypothesized risk of adverse events caused by carrier protein or carrier protein-induced immunosuppression [see, e.g., Juergens et al., Hum Vaccin Immunother. 2018; 14(8): 1948–1956; Knuf et al. Vaccine 2011; 29(31): 4881–4890].

[0012] Although generally well tolerated, it is important to develop pharmaceutical compositions that minimize the risk of adverse events, even if the risk is hypothetical. This is particularly important for the development of vaccines, which are typically administered to healthy people.

[0013] In view of the above-mentioned T cell mechanism of action (Avci et al., Nat. Med. (2011), 17: 1602–1609) and the goal of developing vaccines with minimized risk of adverse events, the development of bioconjugate compositions with a relatively high degree of polyglycosylation and methods for producing such bioconjugates is considered to be an important improvement over currently known bioconjugate compositions and production methods.

[0014] Thus, there is an unmet medical need to provide improved O-EPA bioconjugate compositions and, accordingly, improved pharmaceutical compositions comprising one or more O-EPA bioconjugate compositions.

[0015] Furthermore, those skilled in the art understand that efficient production methods are needed to obtain sufficient safe products for vaccination of large populations in an economically viable manner. Therefore, there is always a need for improved methods of manufacturing and / or purifying such complex bioconjugates.

[0016] For example, in addition to improvements related to certain properties of the bioconjugates themselves obtained therefrom (e.g., improved glycosylation), the methods described in WO 2022 / 214620 can also provide further improvements in the efficient large-scale production of bioconjugates (e.g., culturing host cell cultures in bioreactors with volumes of at least 100 L to 20,000 L). Such improvements may particularly relate to yield, purity, and buffer consumption. For example, reduced buffer consumption is desirable for both economic (lower costs) and environmental reasons (less waste).

[0017] Therefore, there is still a need for improved methods of producing purified bioconjugates comprising bacterial O-antigen polysaccharides covalently coupled to a carrier protein, in particular E. coli O-antigen-EPA bioconjugates, to address the above-mentioned deficiencies. Summary of the Invention

[0018] The present invention relates to improved pharmaceutical compositions comprising one or more bioconjugate compositions. Each bioconjugate composition comprises a mixture of bioconjugates, each bioconjugate comprising a bacterial O-antigen polysaccharide (EPA) of a specific Escherichia coli (E. coli) serotype covalently coupled to a Pseudomonas aeruginosa ExoProtein A carrier protein. Each O-EPA bioconjugate can be monoglycosylated, with one O-antigen polysaccharide linked to EPA, or polyglycosylated, with two, three, or four O-antigen polysaccharides linked to EPA, such that the bioconjugate composition is a mixture of monoglycosylated and polyglycosylated forms of O-EPA bioconjugates. In certain embodiments, the EPA carrier protein has the amino acid sequence of SEQ ID NO: 1. The present invention also relates to methods for producing purified O-EPA bioconjugates from Gram-negative host cells. The methods comprise providing the bioconjugates in a filtered periplasmic fraction (FPF) of the host cells and purifying the bioconjugates. The purification comprises the following steps: (i) a first anion exchange chromatography step followed by (ii) a mixed mode chromatography (MMC) step, followed by (iii) a hydrophobic interaction chromatography (HIC) step and (iv) a second anion exchange chromatography step. The present invention also relates to an O-EPA bioconjugate composition obtainable by or obtained by the method according to the present invention. DETAILED DESCRIPTION

[0019] The above objects are achieved by a pharmaceutical composition as defined in claim 1, a method for producing an O-EPA bioconjugate as defined in claim 5, and an O-EPA bioconjugate composition obtained or obtainable by said method as defined in claim 16. Further aspects of the invention are disclosed in the description and the independent claims, and preferred embodiments are disclosed in the description and the dependent claims.

[0020] The present invention will be described in more detail below. It should be understood that the various embodiments, preferred embodiments and ranges provided / disclosed in this specification may be combined in any manner. In addition, depending on the specific embodiment, the selected definition, embodiment or range may not apply.

[0021] Unless otherwise stated, the following definitions apply to this specification:

[0022] As used herein, the terms "a," "an," "the" and similar referents used in the context of the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0023] As used herein, the terms "including," "comprising," and "comprising" are used herein in their open, non-limiting sense. It should be understood that the various embodiments, preferences, and ranges can be combined in any combination.

[0024] As used herein, when the term "about" is used in conjunction with a numerical value, it refers to any numerical value that is within ±10% (preferably ±5% or ±1%) of the recited numerical value.

[0025] Several abbreviations are used in this specification, including:

[0026] AEX1 First Anion Exchange Chromatography

[0027] AEX2 Secondary Anion Exchange Chromatography

[0028] cGE capillary gel electrophoresis

[0029] cHA ceramic hydroxyapatite

[0030] Periplasmic fraction from CPF centrifugation

[0031] CV column volume

[0032] DOGY Glycosylation degree

[0033] EPA Pseudomonas aeruginosa exoprotein A (also known as exotoxin A)

[0034] ExPEC extraintestinal pathogenic Escherichia coli

[0035] FPF filtered host cell periplasmic fraction

[0036] HA Hydroxyapatite

[0037] HCP host cell protein

[0038] HIC Hydrophobic Interaction Chromatography

[0039] HPLC high-performance liquid chromatography

[0040] IC-PAD Ion Chromatography with Pulsed Amperometric Detection

[0041] IED Invasive ExPEC disease

[0042] MMC mixed mode chromatography

[0043] MMR Multimodal Chromatography Resins

[0044] O-EPA Bioconjugate of EPA carrier protein covalently coupled to bacterial O-antigen polysaccharide

[0045] PF periplasmic fraction

[0046] RP-HPLC reversed-phase high-performance liquid chromatography

[0047] SEC size exclusion chromatography

[0048] SE-HPLC Size Exclusion High Performance Liquid Chromatography

[0049] sEPA Short-chain glycosylated EPA

[0050] TFF Tangential Flow Filtration

[0051] WFI Water for Injection

[0052] The term "glycoconjugate" is known in the art and describes, in particular, a chemical entity covalently bound to one or more polysaccharides. Such glycoconjugates can be obtained by bioconjugation in living cells ("bioconjugates" or "bioconjugates") or by chemical conjugation of polysaccharides ("chemical" or "synthetic" glycoconjugates). Particularly suitable chemical entities are proteins, and the corresponding glycoconjugates are glycoproteins. In particular, the term glycoconjugate relates to conjugated products in which a polysaccharide (i.e., a glycan) is covalently coupled to a carrier protein. If the carrier protein contains more than one glycosylation site, a mixture of glycoconjugates, in particular bioconjugates, is obtained, for example a mixture of mono-, di-, tri- and tetra-glycosylated bioconjugates. An example of a carrier protein containing four glycosylation sites is the EPA carrier protein having the amino acid sequence of SEQ ID NO: 1.

[0053] A "bioconjugate composition" encompasses such a mixture and may include a bioconjugate of a single polysaccharide (e.g., as in a drug substance that is a glycoconjugate of an O-antigen polysaccharide of a specific bacterial serotype coupled to a carrier protein; sometimes referred to as a "monovalent composition") or may also include a bioconjugate of more than one polysaccharide (e.g., as in a pharmaceutical product in which O-antigen polysaccharides of multiple bacterial serotypes, each separately coupled to a carrier protein, are contained in the same composition; sometimes referred to as a "multivalent composition").

[0054] The term glycoprotein includes “glycoconjugate vaccines.” In glycoconjugate vaccines, the emphasis is on the glycan portion, as the glycan is the relevant antigen against which an immune response is desired, and the protein portion serves merely as a carrier to elicit the desired T cell memory immune response.

[0055] The term "polysaccharide" is known in the art and describes in particular polymeric carbohydrates composed of monosaccharide units linked together by glycosidic bonds, which may be linear or branched. Such polysaccharides are characterized by their repeating units, each of which is described by its respective monosaccharide composition. The repeating unit comprises one or more monosaccharides, which may also be chemically modified (e.g., amidated, sulfonated, acetylated, phosphorylated, etc.). Common monosaccharides in the repeating unit are cyclic or linear monosaccharides containing 3-7 carbon atoms. In the specific case of glycoconjugate vaccines, the conjugated polysaccharide is derived from a pathogenic species (e.g., Escherichia coli), and the repeating unit is defined by the genetics of the specific pathogen. Therefore, the repeating unit can be a specific marker / identifier for the pathogen.

[0056] The term "polysaccharide component" therefore refers to one or more polysaccharide chains of a glycoconjugate. A polysaccharide can be a monomer or polymer of a sugar residue, but typically contains at least three sugars and can be linear or branched. Polysaccharides can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, mannose phosphate, 6'-sulfo N-acetyl-glucosamine, etc.). The term "polysaccharide" includes homopolymers and heteropolymers of sugar residues. The term "polysaccharide" also encompasses the polysaccharide components of glycoconjugates (e.g., glycoproteins, glycopeptides).

[0057] As used herein, the term "O-acetylated polysaccharide" refers to a polysaccharide in which one or more monosaccharides of a repeating unit are modified by acetylation. One or more existing hydroxyl groups of the monosaccharide are acetylated. For pathogen-derived repeating units used in glycoconjugate vaccines, O-acetylation of certain monosaccharides can affect the induction of an immune response to the pathogen. Examples of pathogen-derived polysaccharide components are shown in Table 1.

[0058] The term "glycan" / "glycan chain" is a synonym for "polysaccharide" as defined below. Accordingly, in the context of the present invention, "glycan" and the prefix "glyco-" also refer to the carbohydrate portion of a glycoconjugate (e.g., a glycoprotein). Thus, "glycosylation" refers to the covalent attachment of one or more polysaccharides to a protein, such that the protein is "monoglycosylated" or "polyglycosylated," respectively.

[0059] As used herein, the term "serotype" refers to glycoconjugates with different polysaccharide chains derived from different bacterial serotypes. Examples of identified glycans from several E. coli serotypes are listed in Table 1 below.

[0060] As used herein, the term "adjusting the load" refers to adjusting the load of a process intermediate (e.g., a host cell periplasmic component containing an O-EPA conjugate, or a fraction containing O-EPA after a first, second, or third purification step) to conditions suitable for applying the process intermediate to a chromatography resin for further purification. Unless otherwise indicated or clearly contradicted by the context, "adjusting the load" refers to adjusting the conductivity of the load to a target conductivity suitable for a subsequent purification step and / or adjusting the pH of the load to a target pH suitable for a subsequent purification step. Additionally, "adjusting the load" includes adjusting the concentration of the process intermediate, i.e., reducing the processing volume, particularly by TFF.

[0061] The terms "resin" and "medium" are used synonymously herein and relate to a chromatographic resin or chromatographic medium for separating a target protein (i.e., an O-EPA bioconjugate) from impurities. Resins useful in the present invention can be in different forms, such as microbeads, filters (membranes), cartridges, etc., all of which are considered resins according to the present invention. In certain embodiments, the resin is in the form of microbeads that can be used for a column. In certain embodiments, the resin is in the form of a membrane having functional groups. In certain embodiments, the resin is in the form of a directly usable cartridge. Resins that can be used according to the present invention can be commercially available from suppliers such as Cytiva (formerly GE Healthcare), Bio-Rad, and / or other suppliers.

[0062] The term "capture step" is known in the art and refers to the first chromatography step, the purpose of which is to bind the protein of interest from the crude sample and separate it from key contaminants (such as proteases and glycosidases). The target protein (e.g., O-EPA bioconjugate) is concentrated and transferred to a buffer that maintains the functional and structural integrity of the O-EPA bioconjugate. Removal of other key contaminants can also be achieved by carefully optimizing the binding conditions.

[0063] The focus of optimizing the capture step is on capacity and speed. Therefore, in order to maximize the capacity and / or speed of the separation in this first step, it is acceptable to compromise on resolution.

[0064] The term "polishing step" is known in the art and relates to a chromatography step performed as a final chromatography step in order to further increase the purity of the target protein (eg O-EPA bioconjugate).

[0065] The term "bind-elute mode" is known in the art and refers to a separation mode that works by first binding the sample components (especially the protein / bioconjugate of interest) to the chromatographic resin. Once the sample components are bound, the resin is washed with a buffer, thereby removing unbound material. The bound material is then eluted. This separation mode is in contrast to flow-through mode, in which the pH / ionic strength of the sample and buffer are selected so that the protein does not bind and flows through the column, leaving most or specific impurities bound to the column.

[0066] In an embodiment of the present invention, in steps ii) to v), conditions are first adjusted to allow binding of the O-EPA bioconjugate to the chromatography medium, and subsequently adjusted to allow elution of the O-EPA bioconjugate from said medium, i.e. each chromatography step is performed in bind-elute mode.

[0067] Active and inactive particles: Active particles are particles that contain one or more living microorganisms (such as bacteria). These can affect the sterility of the drug product.

[0068] Non-viable particles are particles that do not contain living microorganisms but can serve as a transport vehicle for active particles.

[0069] The size of the active and inactive particles is generally from about 0.2 μm to 30 μm, typically from about 0.2 μm to 5 μm.

[0070] As used herein, the term "drug substance" refers to a bulk product of an individual bioconjugate (e.g., an E. coli O-antigen polysaccharide covalently coupled to an EPA carrier protein, e.g., an E. coli O25B O-antigen covalently coupled to EPA) at a concentration higher than that of the product ultimately administered to a subject in need thereof. The drug substance can be produced after purification of the bioconjugate. The drug substance can be stored, for example, in a more concentrated form in a suitable formulation buffer (see, e.g., WO2018 / 077853, WO2020 / 191082), for example, under frozen conditions, e.g., at -70°C.

[0071] As used herein, the term "drug product" refers to a formulation of a bioconjugate, in particular an E. coli O-antigen polysaccharide, each conjugated to an EPA carrier protein, in its final form for administration to a subject in need thereof. As used herein, the term "drug product" particularly relates to a multivalent vaccine composition, such as a tetravalent ExPEC saccharide conjugate vaccine composition, comprising E. coli O-antigen polysaccharides O25B, O1A, O2, and O6A, each conjugated to an EPA carrier protein. Other non-limiting examples of multivalent saccharide conjugate vaccine compositions are, for example, nine-valent saccharide conjugate vaccine compositions comprising E. coli O-antigen polysaccharides O1A, O2, O4, O6A, O15, O16, O18A, O25B and O75, and decavalent saccharide conjugate vaccine compositions comprising, for example, E. coli O-antigen polysaccharides O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B and O75, each of which is coupled to an EPA carrier protein (see, for example, WO2020 / 191082 and WO2022 / 058945 for examples of decavalent and nine-valent saccharide conjugate vaccine compositions, respectively). The drug product can generally be prepared by mixing the drug substances of the respective glycoconjugates and diluting as needed with a suitable formulation buffer (see, for example, WO2018 / 077853, WO2020 / 191082, WO2022 / 058945) to produce the target dose of vaccine.

[0072] exist First aspect The present invention relates to a method for producing a purified bioconjugate from Gram-negative bacterial host cells. The bioconjugate specifically comprises a bacterial O-antigen polysaccharide covalently coupled to a carrier protein, the carrier protein being Pseudomonas aeruginosa exoprotein A (O-EPA bioconjugate). It should be understood that the Pseudomonas aeruginosa exoprotein A (EPA) carrier protein is a detoxified or recombinant version thereof.

[0073] The method comprises providing a filtered periplasmic fraction (FPF) of a host cell expressing a bioconjugate (step i). The FPF comprises the bioconjugate. The method further comprises several chromatographic purification steps (described herein as steps ii to v).

[0074] Step ii comprises "subjecting the optionally loaded FPF to a first anion exchange chromatography (AEX 1) step to obtain a first AEX eluate (AEX 1) comprising the bioconjugate." This step is a capture step.

[0075] Step iii comprises "subjecting the optionally loaded AEX1 eluate to a mixed mode chromatography (MMC) step on a multimodal resin (MMR) to obtain an MMR eluate, wherein the MMR comprises an anion exchange function and a hydrophobic function to obtain an MMR eluate comprising the bioconjugate."

[0076] Step iv comprises "subjecting the loaded MMR eluate to a hydrophobic interaction chromatography (HIC) step to obtain a HIC eluate comprising the bioconjugate."

[0077] Step v includes "subjecting the loaded HIC eluate to a second anion exchange chromatography (AEX 2) step to obtain an AEX2 eluate containing the bioconjugate as a product." This step is a polishing step.

[0078] In purification steps ii to v, ie in all chromatography steps, conditions are first adjusted to allow binding of the bioconjugate to the chromatography medium and subsequently adjusted to allow elution of the bioconjugate from the chromatography medium (bind-elute mode).

[0079] It should be understood that step iv is performed in the order indicated. When following this purification scheme, the eluate after each chromatography step is more enriched in O-EPA bioconjugate than the eluate from the previous step, i.e., the purity of the O-EPA bioconjugate increases after each chromatography step compared to the previous step.

[0080] Each of the above-mentioned production steps is known per se individually. However, it was found that a specific combination of step iv above is particularly suitable for the production of a variety of different O-EPA bioconjugates and is amenable to large-scale production.

[0081] The O-EPA bioconjugates produced by the methods of the present invention typically have a purity of ≥90% as measured by SE-HPLC, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100%, or a purity of ≥95% as measured by RP-HPLC, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100%. Typically, the purity of the O-EPA bioconjugates as measured by SE-HPLC or RP-HPLC is ≥95%, preferably ≥98%, e.g., 98%, 99%, 99.5%, 99.7%, 99.9%, or 100%. A purity of at least 90% or greater (e.g., at least 95% or at least 98%) as measured by SE-HPLC is advantageous for components of pharmaceutical products intended for administration to humans, such as multivalent bioconjugate vaccines comprising multiple O-EPA conjugates.

[0082] Furthermore, when following the above protocol, the overall yield of O-EPA bioconjugate is acceptable for a large-scale process for preparing a pharmaceutical formulation of the O-EPA bioconjugate (e.g., at least 5% overall yield, preferably at least 10% overall yield, such as about 5-35% overall yield [relative to the O-EPA bioconjugate present in the filtered periplasmic fraction], from a bioreactor of at least 150 L, with a purity of at least 90% as measured by SE-HPLC. Typically, the purity is at least 95% as measured by SE-HPLC or RP-HPLC).

[0083] Importantly and surprisingly, specific combinations of step iv generally result in O-EPA bioconjugate compositions having a relatively high proportion of polyglycosylated O-EPA bioconjugates.

[0084] Further details on each of the steps in step iv are provided below:

[0085] Step i In the first step, FPF of a Gram-negative bacterial host cell expressing an O-EPA bioconjugate is provided. As described above, the FPF contains the O-EPA bioconjugate.

[0086] The provision of filtered periplasmic host cell fractions comprising O-EPA bioconjugates is known per se (see, for example, WO 2009 / 104074, WO 2015 / 124769 or WO 2020 / 191082).

[0087] To obtain the periplasmic fraction by osmotic shock treatment, cells are first incubated in a buffer having a relatively high volume molar osmotic pressure concentration (osmolarity) (hypertonic) and then incubated in a buffer having a relatively low volume molar osmotic pressure concentration (hypotonic). This osmotic shock treatment results in at least partial removal of the cell wall and the production of spheroplasts (i.e., cells, particularly Gram-negative cells, from which the cell wall has been at least partially removed). As a result, most host cell proteins are retained in the spheroplasts, while periplasmic proteins are released into the suspension medium.

[0088] Typically, E. coli host cells encoding PglB, EPA, and enzymes for biosynthesis of the corresponding O-antigen polysaccharide are cultured in a bioreactor having a volume of about 100 L to 20,000 L, such as 150 L to 5,000 L, such as 200 L or 800 L or 2,000 L, wherein the cells produce the O-EPA bioconjugate. Typically, the cells are harvested when they are in a stationary phase by centrifugation, such as by continuous centrifugation, such as using a disc centrifuge, after the culture is cooled to below 20° C. The cells are resuspended in a suitable liquid, such as a 0.9% NaCl solution or Tris-buffered saline (TBS), and subjected to an osmotic shock, preferably using a solution at about 2-15° C., preferably about 6-10° C. Osmotic shock can be, for example, by adding a sucrose solution (e.g., 60% sucrose, pH 8, 480mM Tris-HCl, 24mM EDTA) to a sucrose target concentration of about 20-30%, for example, 25%, to the cells, and the mixture is incubated at about 2-15°C, preferably at about 6-10°C for about 15 minutes to 4 hours, for example, about 1 hour, while mixing. After incubation with sucrose, the cells / sucrose solution is mixed with a solution of low osmotic pressure value, for example, by mixing at 6-10°C with about 4x volumes of 10mM Tris-HCl (pH 8.0). Mixing can be, for example, performed by a static mixer. The O-EPA product is released from the periplasmic space into the supernatant (referred to as the periplasmic fraction PF), and the PF is collected. The material can then be clarified, for example, by separating and removing the cell debris in the PF using a disc centrifuge, thereby collecting the supernatant (referred to as the centrifuged periplasmic fraction CPF). The CPF is preferably further filtered to remove remaining cell debris, for example, by filtration through a depth and bioburden reduction filter (e.g., a membrane with a pore size of about 0.2 μm), and the resulting material is collected as a filtered periplasmic fraction (FPF). This FPF can then be used in the chromatographic purification method of the invention as described herein [starting from step ii].

[0089] Therefore, in one embodiment, step i further comprises (i-1) incubating the Gram-negative bacterial host cells in a bioreactor having a volume of 100 L to 20,000 L (e.g., 150 L to 5,000 L, such as 200 L or 800 L or 2,000 L) at a temperature of 34° C. to 36° C. (e.g., 35° C.) and growing them to a stationary phase before harvesting;

[0090] This is followed by (i-2) harvesting the Gram-negative bacterial host cells, wherein the harvesting comprises a continuous flow centrifugation step to obtain harvested Gram-negative bacterial host cells comprising the O-EPA bioconjugate in the periplasm.

[0091] Therefore, in another embodiment, step i further comprises (i-3) subjecting the host cells to an osmotic shock treatment to obtain a periplasmic fraction of the Gram-negative bacterial host cells containing the O-EPA bioconjugate. It will be clear to those skilled in the art that step (i-3) is after step (i-2). In certain embodiments, the osmotic shock treatment in step (i-3) comprises adding a sucrose solution, preferably further comprising EDTA, to the cells to a target concentration of about 25% sucrose, incubating the mixture at about 6-10°C for about 15 minutes to 2 hours while mixing, and then adding a solution having a low volume molar osmotic pressure concentration (e.g., 10 mM Tris-HCl pH 8) to the cell / sucrose solution at about 6-10°C to reduce the osmotic pressure value by at least four times (compared to a composition comprising 25% sucrose), thereby releasing the O-EPA bioconjugate from the periplasm into the supernatant, collecting the supernatant (periplasmic fraction), and then filtering the periplasmic fraction to obtain a filtered periplasmic fraction.

[0092] In a further embodiment, step i further comprises a step (i-4) of filtering to obtain a filtered periplasmic fraction (FPF) comprising the O-EPA bioconjugate. As described above, the filtration step is used to remove remaining cell debris, for example, by filtering through a depth and bioburden reduction filter (e.g., a membrane with a pore size of about 0.2 μm).

[0093] In one embodiment, step (i) comprises:

[0094] (i-1) incubating the host cells in a bioreactor with a volume of 100 L to 20,000 L (e.g., 150 L to 5,000 L) at a temperature of 34° C. to 36° C. and growing to a stationary phase before harvesting;

[0095] (i-2) harvesting the host cells by a continuous flow centrifugation step to obtain harvested host cells containing the bioconjugate; and

[0096] (i-3) subjecting the host cells to osmotic shock to obtain a periplasmic fraction of the host cells, wherein the periplasmic fraction contains the bioconjugate; and

[0097] (i-4) Filtration to obtain a filtered periplasmic fraction (FPF) containing the O-EPA bioconjugate.

[0098] Host Cell: In a preferred embodiment, the Gram-negative bacterial host cell from which the O-EPA bioconjugate is obtained comprises genetic information encoding (a) a bacterial O-antigen polysaccharide and (b) a recombinant Pseudomonas aeruginosa exoprotein A (EPA) comprising at least one glycosylation site and (c) a metabolic apparatus for N-glycosylation of EPA with the O-antigen polysaccharide, thereby producing the O-EPA conjugate in vivo in the periplasm of the Gram-negative bacterial host cell.

[0099] In a preferred embodiment, the Gram-negative bacterial host cell is an E. coli host cell, more preferably an E. coli K-12 host cell, such as E. coli K-12 strain W3110.

[0100] Details in this regard are described in more detail below and in the prior art, for example in WO 2009 / 104074, WO 2020 / 191082 and WO 2020 / 191088 (incorporated herein in their entirety).

[0101] In a preferred embodiment, the waaL gene is deleted or functionally inactivated from the genome of the host cell of the present invention. The terms "waaL" and "waaL gene" refer to an O-antigen ligase gene encoding a membrane-bound enzyme whose active site is located in the periplasm. The enzyme encoded by the waaL gene transfers undecaprenyl phosphate (UPP)-bound O-antigen to the lipid A core to form lipopolysaccharide. Deletion or disruption of the endogenous waaL gene (e.g., ΔwaaL strains) disrupts the transfer of O-antigen to lipid A and can instead enhance the transfer of O-antigen to another available biomolecule (e.g., a carrier protein expressed in the host cell of the present invention).

[0102] In one embodiment of the host cell of the present invention, the E. coli gtrABS gene responsible for glycosylation of the O16 O-antigen is deleted from the host cell genome or functionally inactivated. In a preferred embodiment, the E. coli gtrABS gene is deleted from the genome of the E. coli W3110 host cell or functionally inactivated. While the gtrA and gtrB genes are highly homologous and interchangeable among different serotypes, the gtrS gene encodes a serotype-specific O-antigen glycosyltransferase. In E. coli W3110, GtrS can transfer a glucose (Glc) residue to the GlcNAc sugar in the αL-Rha-(1→3)-D-GlcNAc motif of the E. coli O16 O-antigen.

[0103] O-antigen polysaccharide: In a preferred embodiment, the O-antigen polysaccharide is specific for a Gram-negative bacterium (preferably Escherichia coli) selected from the list consisting of Escherichia and Shigella.

[0104] In one embodiment, the O-antigen polysaccharide is selected from the group consisting of E. coli O-antigen polysaccharides O1A, O2, O4, O8, O6A, O15, O16, O18A, O25B and O75.

[0105] In one embodiment, the O-antigen polysaccharide is selected from the group consisting of E. coli O-antigen polysaccharides O1A, O2, O4, O6A, O15, O16, O18A, O25B and O75.

[0106] In one embodiment, the O-antigen polysaccharide is selected from the group consisting of E. coli O-antigen polysaccharides O1A, O2, O6A, O16, O18A, O25B and O75.

[0107] In one embodiment, the O-antigen polysaccharide is selected from the group consisting of E. coli O-antigen polysaccharides O1A, O2 and O25B.

[0108] In one embodiment, the O-antigen polysaccharide is E. coli O-antigen polysaccharide O1A.

[0109] In one embodiment, the O-antigen polysaccharide is E. coli O-antigen polysaccharide O2.

[0110] In one embodiment, the O-antigen polysaccharide is E. coli O-antigen polysaccharide O25B.

[0111] In E. coli, the gene products involved in the biosynthesis of the O-antigen polysaccharide are encoded by the rfb locus. Therefore, the host cells provided herein preferably also comprise a nucleotide sequence corresponding to the rfb locus of an E. coli serotype selected from the list consisting of O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B and O75.

[0112] As used herein, "O-antigen rfb locus" and "O-antigen rfb gene cluster" refer to a locus in the genome of Gram-negative bacteria that contains a cluster of genes that together encode the enzymatic machinery capable of synthesizing the O-antigen polysaccharide structure. The term rfb locus preferably refers to a genomic locus from the genus Escherichia, particularly Escherichia coli.

[0113] In certain embodiments, the O-antigen rfb locus is heterologous to the host cell, for example, introduced into a precursor cell of the host cell and preferably integrated into its genome. Preferably, if the original rfb gene cluster is present in the precursor cell, it has been replaced by the O-antigen rfb gene cluster in the host cell to enable the production of a bioconjugate of an O-antigen polysaccharide, preferably selected from the list consisting of E. coli O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B and O75-antigen polysaccharides.

[0114] An exemplary sequence of the rfb gene cluster (rfb locus) that can be used in a production strain of a bioconjugate with the above-mentioned E. coli serotype O-antigen polysaccharide is as follows:

[0115] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O1A has the sequence of SEQ ID NO: 11 of WO 2020 / 191082.

[0116] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O2 has the sequence of SEQ ID NO: 12 of WO 2020 / 191082.

[0117] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigenic polysaccharide of E. coli serotype O4 has the sequence of SEQ ID NO: 9 of WO 2020 / 191082. In an exemplary and non-limiting embodiment, the production strain comprising the rfb locus encoding the O-antigenic polysaccharide of E. coli serotype O4 further comprises a nucleic acid encoding an E. coli O4-specific GtrS protein, such as SEQ ID NO: 4 of WO 2020 / 191082.

[0118] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O6A has the sequence of SEQ ID NO: 13 of WO 2020 / 191082.

[0119] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O8 has the sequence of SEQ ID NO: 14 of WO 2020 / 191082.

[0120] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O15 has the sequence of SEQ ID NO: 15 of WO 2020 / 191082.

[0121] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O16 has the sequence of SEQ ID NO: 16 of WO 2020 / 191082.

[0122] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O18A has the sequence of SEQ ID NO: 5 of WO 2022 / 208430, wherein the Wzy O-antigen polymerase encoded in the rfb locus preferably has the amino acid sequence of SEQ ID NO: 1 of WO 2022 / 208430, except that the amino acid sequence contains isoleucine at a position corresponding to position 199 in the amino acid sequence, lysine at a position corresponding to position 377 in the amino acid sequence, and alanine at a position corresponding to position 395 in the amino acid sequence.

[0123] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O25B has the sequence of SEQ ID NO: 18 of WO 2020 / 191082.

[0124] In an exemplary and non-limiting embodiment, the rfb locus encoding the O-antigen polysaccharide of Escherichia coli serotype O75 has the sequence of SEQ ID NO: 19 of WO 2020 / 191082.

[0125] The repeating unit structures of Escherichia coli O-antigen polysaccharides O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B and O75 are shown in Table 1.

[0126] Optionally, the chain length of the O-antigen can be manipulated by manipulating the natural Wzz O-antigen chain length regulator mechanism, for example, by overexpressing or supplementing the Wzz O-antigen chain regulator, for example, by replacing the E. coli wzzB gene (see, for example, US2018 / 0099038, WO 2020 / 039359) with species from the genus Salmonella and Shigella or one of its counterparts from Pseudomonas aeruginosa (e.g., Salmonella enterica counterparts, fepE or other wzz homologs), thereby, for example, increasing the number of repeat units of the O-antigen, whether or not the Wzy protein is additionally overexpressed. The effects of these species wzz-like genes have been described in the literature. However, these are not required to obtain bioconjugates with good immunogenicity, and in a preferred embodiment, the bioconjugates according to the present invention are prepared without manipulating the Wzz chain length regulator mechanism.

[0127] Carrier protein: The carrier protein is detoxified exoprotein A (EPA) of Pseudomonas aeruginosa, also known as recombinant EPA.

[0128] Various detoxified protein variants of EPA have been described in the literature and can be used as carrier proteins. In certain embodiments, the EPA carrier protein used in the bioconjugates of the present invention is modified to render the protein less toxic and / or more susceptible to glycosylation. For example, detoxification can be achieved by mutation and deletion of the catalytically essential residues L552V and ΔE553 (see, e.g., Lukac et al., 1988, Infect Immun, 56:3095-3098; Ho et al., 2006, Hum Vaccin, 2:89-98). In a specific embodiment, the carrier protein used to generate the bioconjugates of the present invention is modified to optimize the number of glycosylation sites in the carrier protein, thereby reducing the concentration of the protein administered, for example, in an immunogenic composition in the form of a bioconjugate. In a specific embodiment, the host cell encodes EPA comprising 1-10, preferably 2-4, more preferably 4 glycosylation sites, wherein the glycosylation sites comprise the glycosylation consensus sequence Asn-X-Ser (Thr), wherein X can be any amino acid except Pro, more preferably the glycosylation consensus sequence Asp (Glu) -X-Asn-Z-Ser (Thr), wherein X and Z are independently selected from any amino acid except Pro. Therefore, in a preferred embodiment, the carrier protein is a recombinant EPA comprising 4 N-glycosylation sites, wherein the N-glycosylation sites comprise the glycosylation consensus sequence Asn-X-Ser (Thr), preferably the glycosylation consensus sequence Asp (Glu) -X-Asn-Z-Ser (Thr). In one embodiment, the carrier protein comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 and comprises 1-10, preferably 2-4, preferably 4 glycosylation sites comprising the glycosylation consensus sequence Asn-X-Ser(Thr), more preferably the glycosylation consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr). In a preferred embodiment, the carrier protein is recombinant EPA comprising the amino acid sequence of SEQ ID NO: 1. In another preferred embodiment, the host cell comprises a nucleotide sequence encoding the carrier protein comprising SEQ ID NO: 1.

[0129] Metabolic apparatus for N-glycosylation of EPA: Gram-negative host cells preferably contain an oligosaccharyltransferase (OST) that transfers oligosaccharides to N-glycosylation sites on carrier proteins. Thus, preferably, the host cells as provided herein contain a nucleotide sequence encoding an oligosaccharyltransferase (OST). As used herein, oligosaccharyltransferases are enzymes that transfer lipid-linked oligosaccharides to residues in a nascent polypeptide chain that contain a glycosylation consensus motif, such as an asparagine (Asn, N) residue in a nascent polypeptide chain that contains an N-glycosylation consensus motif, an example of which is a motif having the amino acid sequence Asn-X-Ser(Thr) or (Asp(Glu)-X-Asn-Z-Ser(Thr)). Throughout this application, it is understood that for the N-glycosylation motif Asn-X-Ser (Thr), X can be any amino acid except proline, and for the N-glycosylation motif Asp (Glu) -X-Asn-Z-Ser (Thr), both X and Z can be any amino acid except proline. Preferably, such oligosaccharyltransferases transfer oligosaccharides to the asparagine residue of the glycosylation consensus sequence Asp (Glu) -X-Asn-Z-Ser (Thr) in the polypeptide chain of the carrier protein as described herein. The nucleic acid encoding the oligosaccharyltransferase can be native to the host cell or can be introduced into the host cell using genetic methods. In a preferred embodiment, the oligosaccharyltransferase is heterologous to the host cell. E. coli does not naturally contain oligosaccharyltransferases, so if E. coli is used as a host cell for the production of bioconjugates, a heterologous oligosaccharyltransferase is contained in such a host cell, for example, by genetic engineering. In view of the present disclosure, the oligosaccharyltransferase can be from any source known in the art.

[0130] In certain preferred embodiments, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter. For example, in one embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al., 2002, Science 298: 1790-1793; see also, e.g., NCBI gene ID: 3231775, UniProt accession number 086154). In another embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter lari (see, e.g., NCBI gene ID: 7410986).

[0131] In a specific embodiment, the oligosaccharyltransferase is PglB from Campylobacter jejuni, including the native (wild-type) protein or any variant thereof, such as those described in WO 2016 / 107818 and WO 2016 / 107819. PglB can transfer lipid-linked oligosaccharides to asparagine residues in the consensus sequences Asn-X-Ser (Thr) and Asp (Glu) -X-Asn-Z-Ser (Thr). In certain embodiments, the PglB oligosaccharyltransferase is a polypeptide having oligosaccharyltransferase activity as defined herein, comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2. In a preferred embodiment, the PglB oligosaccharyltransferase comprises an amino acid sequence identical to SEQ ID NO: 2 or an amino acid sequence identical to SEQ ID NO: 2 but having specific amino acid changes that specifically optimize expression of bioconjugates of specific E. coli O-antigen serotypes (see, for example, WO 2020 / 191088, and further examples of the present application below). Thus, in a preferred embodiment, the host cell comprises a nucleotide sequence encoding an oligosaccharyltransferase comprising SEQ ID NO: 2 or a variant thereof as described herein. In certain embodiments, one or more endogenous glycosylation consensus sequences in wild-type PglB have been mutated to prevent PglB autoglycosylation, such as SEQ ID NO: 2 comprising the mutation N534Q. Examples of variant PglBs suitable for use in host cells provided herein include PglB of SEQ ID NO: 2 (e.g., for bioconjugates of EPA with the O-antigen of E. coli serotypes O2, O8, O18A, or O25B); PglB of SEQ ID NO: 2 comprising the mutation N311V; PglB of SEQ ID NO: 2 comprising the mutations N311V, K482R, D384H, and A669V (e.g., for bioconjugates of EPA with the O-antigen of E. coli serotypes O1A or O15); PglB of SEQ ID NO: 2 comprising the mutations Y77H and N311V (e.g., for bioconjugates of EPA with the O-antigen of E. coli serotypes O4); PglB of SEQ ID NO: 2 comprising the mutations Y77H, S80R, Q287P, K289R, and N311V. PglB of SEQ ID NO: 2 (e.g., for bioconjugates of EPA with the O-antigen of E. coli serotype O6A or O16); and PglB of SEQ ID NO: 2 comprising mutations Y77R and N311V (e.g., for bioconjugates of EPA with the O-antigen of E. coli serotype O75).

[0132] As will be appreciated by those skilled in the art, the polypeptide of the present invention (such as a carrier protein, an oligosaccharyl transferase, and an enzyme of the rfb gene cluster, including a polypeptide having, for example, the Wzy O-antigen polymerase activity provided in WO 2020 / 191088 or WO 2022 / 208430) contained in the host cell of the present invention is conveniently provided to the host cell in the form of a nucleic acid encoding the polypeptide of the present invention. The nucleic acid encoding the polypeptide of the present invention is preferably a nucleic acid construct, specifically an expression construct comprising one or more expression cassettes for expressing the polypeptide of the present invention, wherein the nucleotide sequence encoding the polypeptide of the present invention is operably connected to an expression control sequence for expressing the polypeptide in the host cell of the present invention. Suitable expression control sequences generally include at least a promoter and may include other sequence elements, such as, in particular, a Shine-Dalgarno sequence and a transcription termination sequence. The promoter may be a constitutive promoter, or it may be a promoter whose activity can be regulated, for example, by being suppressed or induced under certain conditions (such as temperature changes or the presence of certain chemicals or proteins in the cell), all of which are also well known in the art. The limiting examples of suitable promoters are ara, phoA, tac, tet, trc, trp, PBAD, λPL, T5 or T7 promoters. The nucleic acid construct can be extrachromosomal, such as a plasmid or other vector, or the nucleic acid construct can be integrated into the genome of the host cell of the present invention. The construction and / or synthetic molecular biological methods for expressing the nucleic acid construct of the polypeptide in the host cell of the present invention are generally well known in the art. In certain embodiments, the rfb locus is integrated into the genome of the host cell. In certain embodiments, the nucleic acid encoding oligosaccharyl transferase and the nucleic acid encoding the EPA carrier protein are present on a plasmid in the host cell.

[0133] Step ii In the second step, the O-EPA bioconjugate is purified by a first anion exchange chromatography step (AEX1; step ii). This step serves to reduce the processing volume and process-related impurities originating from the fermentation process, i.e., the incubation of the host cells and the extraction of the periplasmic fraction containing the O-EPA bioconjugate. The main task of AEX1 is to remove most of the DNA, endotoxins and host cell proteins (HCPs). In particular, impurities that can lead to degradation of the O-EPA conjugate, such as proteases, peptidases and glycosidases, are removed. Therefore, in particular, the AEX1 step is a capture step.

[0134] In certain embodiments, AEX1 is performed using a strong anion exchange resin. In certain embodiments, the resin is a ceramic resin, e.g., having rigid, incompressible properties and a high dynamic binding capacity at high flow rates. A non-limiting example of a resin particularly suitable for the AEX1 step is Q Ceramic HyperD F resin. In certain embodiments, the AEX1 step is performed in bind-elute mode.

[0135] Step ii comprises subjecting the optionally loaded FPF to a first anion exchange chromatography step (AEX 1) to obtain a first AEX eluate (AEX 1). This step is known per se. Generally, it is not necessary to adjust the load of the filtered periplasmic fraction before AEX 1. Instead, the filtered periplasmic fraction is usually directly subjected to AEX 1 unless the conditions (particularly conductivity) of the filtered periplasmic fraction are not suitable for direct AEX 1. Therefore, adjusting the load of the FPF before AEX 1 is arbitrary.

[0136] In certain embodiments, step ii further comprises one or more of the following steps (ii-1) to (ii-3), preferably performed in the order shown:

[0137] In one embodiment, this comprises (ii-1) contacting the filtered periplasmic fraction, optionally adjusted for loading, with an AEX1 medium and washing the medium containing the bound O-EPA bioconjugate with a wash buffer. Typically, the wash buffer has a relatively low salt concentration and a relatively low conductivity. An example of a suitable low-salt, low-conductivity wash buffer is a buffer comprising 10 mM Tris and 50 mM NaCl at pH 8.5.

[0138] In one embodiment, this includes (ii-2) eluting the O-EPA bioconjugate with an elution buffer, which typically comprises a relatively high salt concentration and a relatively high conductivity. An example of a suitable high-salt, high-conductivity buffer is a buffer comprising 10 mM or 50 mM Tris and 1 M NaCl at pH 8.5. Typically, elution is performed using a step gradient.

[0139] For each of these steps, it is apparent that the identity of the AEX1 resin, the AEX1 column or membrane or cassette format, the exact buffer composition, pH and / or salt concentration or conductivity, and the gradient can be varied as known to those skilled in the art based on the current disclosure.

[0140] In one embodiment, this includes (ii-3) optionally combining fractions enriched in the O-EPA bioconjugate to obtain an AEX1 eluate. Typically, fractions enriched in the O-EPA conjugate are identified by performing SDS-PAGE analysis or any suitable analysis to determine the amount or relative purity of the desired O-EPA conjugate. Performing SDS-PAGE analysis to identify fractions containing the desired product, i.e., the O-EPA bioconjugate, is well known to those skilled in the art. It will be apparent to those skilled in the art that, in addition to combining fractions based on analysis of the contents of these fractions, it is also possible to simply collect a larger range of fractions known to contain product during elution, especially once the process steps have been established and experience with the product elution profile has been gained.

[0141] Step iii In a third step, the O-EPA bioconjugate is further purified by subjecting the optionally loaded AEX1 eluate to a mixed-mode chromatography (MMC) step on a multimodal resin (MMR) to obtain an MMR eluate, the MMR comprising both anion exchange and hydrophobic functionalities. This chromatography step further removes process-related impurities, particularly endotoxins and host cell proteins (HCPs). Surprisingly, it has been found that MMRs comprising both anion exchange and hydrophobic functionalities are particularly suitable for separating bioconjugates as described herein. As shown herein, the use of this step significantly allows for the acquisition of O-EPA bioconjugate compositions with increased polyglycosylation levels compared to previously preferred methods. Furthermore, the use of an MMR reduces the overall buffer consumption of the overall purification process, which is believed to be beneficial due to reduced costs and reduced environmental burden (less waste). This is particularly important for large-scale production processes, such as incubating host cells in bioreactors with volumes ranging from 100 L to 20,000 L, e.g., 150 L to 5,000 L, such as 200 L, 800 L, or 2,000 L. Various MMRs comprising anion exchange functionality and hydrophobic functionality (ie comprising cationic and hydrophobic groups) are commercially available, non-limiting examples include PPA HyperCel, HEA HyperCel, MEP HyperCel, and Capto adhere. In certain embodiments, the MMR is Capto adhere.

[0142] In a preferred embodiment, the MMR comprises a ligand of formula (I),

[0143]

[0144] in

[0145] RES stands for the resin of the stationary phase;

[0146] R 1represents a C1-C4 alkyl group, preferably a methyl group;

[0147] R 2 represents a C1-C4 alkyl group substituted by a phenyl, tolyl or xylyl group, preferably a benzyl group;

[0148] R 3 represents a C1-C4 alkyl group substituted by a hydroxyl group or a mercapto group, preferably a 2-hydroxyethyl group.

[0149] An example of a suitable ligand of formula (I) is N-benzyl-N-methylethanolamine.

[0150] In a preferred embodiment, the MMR is a compressible resin. Compared to rigid resins, compressible resins as described herein have the advantage of relatively high wear resistance during slurry preparation (mixing the resin with the eluent or wash buffer). Higher wear resistance leads to higher reusability of the resin. Therefore, compressible MMR as described herein is economically advantageous.

[0151] Suitable compressible MMRs comprising anion exchange functionality and hydrophobic functionality are commercially available, for example Capto ad-here.

[0152] Adjusting the load of the AEX1 eluate before MMC is optional. Typically, there is no need to adjust the load of the AEX1 eluate before the MMC step. Instead, the AEX1 eluate is typically subjected to MMC directly. In alternative embodiments, the load of the AEX1 eluate is adjusted. For example, the pH and conductivity are adjusted to predetermined values ​​by adding a suitable buffer, for example, the pH is adjusted to 7.2±0.2 using a buffer containing 50 mM BisTris, pH 6.0, and in a subsequent step, the conductivity is adjusted to a target conductivity of 7.5-9 mS / cm using a buffer containing 10 mM BisTris, pH 7.0. In a preferred embodiment, the MMC step is performed in bind-elute mode. Step iii is performed after step ii.

[0153] In certain embodiments, step iii further comprises one or more of the following steps (iii-1) to (iii-4), preferably performed in the order shown:

[0154] In one embodiment, this comprises (iii-1) performing a particle reduction filtration 1. This step results in the removal of both active and inactive particles.

[0155] In one embodiment, this includes (iii-2), contacting the optionally adjusted AEX1 eluate with the MMR, and subjecting the MMR comprising the bound O-EPA bioconjugate to several washing steps. Typically, in the first washing step, the MMR is washed with a buffer having a relatively low conductivity. An example of a suitable low conductivity wash buffer is a buffer comprising 50mM BisTris, 50mM NaCl, pH 6.0. Typically, in the second washing step, the MMR is washed with a buffer having a higher conductivity and a lower pH than the first wash buffer. An example of a suitable second wash buffer is a buffer comprising 50mM acetate, 250mM NaCl, pH 4.7. Typically, in the third washing step, the MMR is washed again with a buffer having a relatively low conductivity (such as the low conductivity wash buffer used for the first washing step, for example, a buffer comprising 50mM BisTris, 50mM NaCl, pH 6.0).

[0156] In one embodiment, this includes (iii-3), eluting the O-EPA bioconjugate with a buffer having a relatively high conductivity. An example of a suitable high conductivity elution buffer is an elution buffer comprising 50 mM BisTris, 2 M NaCl, pH 6.0.

[0157] In one embodiment, this includes (iii-4), combining fractions enriched in the O-EPA bioconjugate to obtain an MMR eluate. In a preferred embodiment, fixed-volume fractions are combined to obtain an MMR eluate enriched in the O-EPA bioconjugate, rather than combining fractions based on SDS-PAGE analysis. Of course, in addition to combining fractions, one can simply collect a fraction known to contain product during elution, especially once the process steps have been established and experience with the product elution profile has been gained.

[0158] For each of these steps, it will be apparent that the identity of the MMR, column format, exact buffer composition, pH and / or salt concentration or conductivity, and gradient may be varied as known to those skilled in the art based on the current disclosure.

[0159] Step iv In the fourth step, the O-EPA bioconjugate is further purified by subjecting the loaded MMR eluate to a hydrophobic interaction chromatography (HIC) step to obtain a HIC eluate containing the bioconjugate. The HIC step is used to further remove HCPs, especially non-glycosylated EPA and various medium-sized HCPs (approximately 40-60 kDa).

[0160] In certain embodiments, step iv, "subjecting the MMR eluate obtained in step (iii) of adjusting the load to a hydrophobic interaction chromatography (HIC) step to obtain a HIC eluate" further comprises one or more of the following steps (iv-1) to (iv-5), preferably in the order shown.

[0161] In one embodiment, this includes (iv-1), adjusting the conductivity of the load of the MMR eluate. The conductivity is adjusted using a loading buffer suitable for binding to the HIC medium, which loading buffer typically has a relatively high conductivity. An example of a suitable loading buffer is a buffer comprising 2M potassium phosphate, pH 7.0. Typically, the load of the MMR eluate is adjusted by adding a loading buffer whose weight is three times that of the MMR eluate. A non-limiting example of a hydrophobic interaction medium that can be used for the HIC of the present invention is a Sartobind Phenyl adsorber, such as a Sartobind Phenyl Jumbo 5L capsule.

[0162] In one embodiment, this includes (iv-2), performing a particle reduction filtration 2 to remove both active and inactive particles.

[0163] In one embodiment, this comprises (iv-3) contacting the adjusted MMR eluate with a HIC medium and washing the medium containing the bound O-EPA bioconjugate with a suitable buffer (typically a buffer with a relatively high conductivity, such as a buffer comprising 2 M potassium phosphate, pH 7.0), and optionally followed by a second washing step with a buffer with reduced conductivity (e.g. a mixture comprising 70% of a buffer comprising 2 M potassium phosphate, pH 7.0 and 30% of WFI water).

[0164] In one embodiment, this includes (iv-4) eluting the O-EPA bioconjugate using a suitable elution buffer (typically an elution buffer with relatively low conductivity). Preferably, elution is performed using a step gradient using a suitable elution buffer. An example of a suitable buffer for eluting the O-EPA bioconjugate is a mixture containing 30% of a buffer comprising 2 M potassium phosphate, pH 7.0 and 70% WFI water.

[0165] For each of these steps, it will be apparent that the identity of the HIC resin, column or membrane or cartridge format, exact buffer composition, pH and / or salt concentration or conductivity, and gradient may vary as known to those skilled in the art based on the current disclosure.

[0166] In one embodiment, this includes (iv-5), combining fractions enriched in O-EPA bioconjugate to obtain a HIC eluate. It will be clear to those skilled in the art that, in addition to combining fractions, one can simply collect a wider range of fractions known to contain product during elution, especially once the process steps have been established and experience with the product elution profile has been gained.

[0167] Step v : In the fifth step, the O-EPA bioconjugate is further purified by "subjecting the HIC eluate obtained in the load adjustment step (iv) to a second anion exchange chromatography (AEX2) step to obtain a second AEX eluate as a product". The AEX2 step is used to further remove process-related protein impurities, including non-glycosylated EPA, short-chain glycosylated EPA (sEPA; i.e., EPA carrier protein, which has a polysaccharide chain of only about 1-3 repeating units), and in particular E. coli transaldolase B (~37 kDa). Therefore, in one aspect, the present invention provides a polishing step using anion exchange chromatography to reduce the amount of transaldolase B in a preparation containing the O-EPA bioconjugate. In certain embodiments, the O-EPA bioconjugate has undergone previous purification steps, including anion exchange, MMR, and hydrophobic interaction chromatography steps in sequence.

[0168] Suitable AEX media for this AEX2 step are commercially available, and a non-limiting example is Source Q resin.

[0169] In certain embodiments, step (v) further comprises one or more of the following steps (v-1) to (v-4), preferably performed in the order shown.

[0170] In one embodiment, this includes (v-1), performing tangential flow filtration 1 (TFF1) of the load of the HIC eluate to reduce conductivity and reduce pH. The TFF1 step is a diafiltration step. In this step, a new buffer is added to the feed at a rate identical to the permeate flow rate (i.e., the rate at which the feed passes through the membrane). Thus, the HIC eluate in a suitable buffer is obtained, and the volume of the HIC eluate remains constant (buffer exchange). Typical suitable diafiltration volumes are, for example, 5-6 times the volume of the HIC eluate, but if necessary, this can be adjusted (for example, the conductivity and pH of the retentate) according to conventional tests of those skilled in the art. A suitable buffer for this buffer exchange is, for example, pH 6.0, a buffer comprising 10mM BisTris and 50mM NaCl.

[0171] In one embodiment, this includes (v-2), performing a particle reduction filtration 3 to remove both active and inactive particles.

[0172] In one embodiment, this includes (v-3), contacting the loaded filtered HIC eluate with the AEX2 medium and eluting the O-EPA bioconjugate with an elution buffer. In certain embodiments, elution is performed, in particular, by a step gradient followed by a linear gradient of increasing salt concentration. Typically, the elution buffer has a relatively high salt concentration and high conductivity. A non-limiting example of a suitable method for eluting the O-EPA bioconjugate is applying 7.5 CV of 21% buffer containing 10 mM BisTris, 200 mM NaCl, pH 6.0 (buffer V) in buffer U (buffer U containing 10 mM BisTris, 50 mM NaCl, pH 6.0) (step gradient), followed by a linear gradient of 21-56% buffer V in buffer U over 7-8 CV, for example 7.5 CV.

[0173] In one embodiment, this includes (v-4), combining fractions enriched in O-EPA bioconjugate to obtain the AEX2 eluate as product.

[0174] In one embodiment, during step (v), the O-EPA bioconjugate is bound to an AEX2 matrix and eluted by a step gradient as described above, followed by a linear gradient of increasing salt concentration, to obtain an O-EPA bioconjugate with a purity of at least 90%, preferably at least 95%, more preferably at least 98% or at least 99%.

[0175] For each of these steps, it is clear that the characteristics of the AEX2 resin, the AEX2 column or membrane or cartridge format, the exact buffer composition, pH and / or salt concentration or conductivity, the gradient, and the strategy for fraction pooling can vary, as known to those skilled in the art based on the current disclosure.

[0176] As mentioned above:

[0177] In certain embodiments, AEX1 is performed in bind-elute mode.

[0178] In certain embodiments, MMC is performed in bind-elute mode.

[0179] In certain embodiments, HIC is performed in bind-elute mode.

[0180] In certain embodiments, AEX2 is performed in bind-elute mode.

[0181] In certain embodiments, AEX1, MMC, HIC, and AEX2 are performed in bind-elute mode.

[0182] When following the above protocol, in each of the second, third, fourth and fifth steps, i.e. in each chromatography step (steps ii-v), the relative amount of O-EPA conjugate to total protein in the collected eluate is higher than that in the load.

[0183] In certain embodiments, the production method further comprises an additional step (vi), wherein the load of the AEX2 eluate is adjusted to a pharmaceutically acceptable buffer and concentration, thereby obtaining the purified O-EPA conjugate as a drug substance.

[0184] In certain embodiments, step (vi) further comprises one or more of the following steps (vi-1) to (vi-3), preferably performed in the order shown:

[0185] In one embodiment, this includes (vi-1), performing tangential flow filtration 2 (TFF2) of the AEX2 eluate to convert to a pharmaceutically acceptable buffer and concentration of O-EPA bioconjugate. In one embodiment, TFF2 is performed with an excipient buffer comprising 6.19 mM KH2PO4, 3.81 mM Na2HPO4, 5% (w / w) sorbitol, 10 mM methionine, pH 7.0. In one embodiment, after TFF2, polysorbate-80 is added to a concentration of 0.02% (w / w) to obtain the purified O-EPA bioconjugate in a pharmaceutically acceptable buffer. Thus, in one embodiment, the pharmaceutically acceptable buffer comprises 6.19 mM KH2PO4, 3.81 mM Na2HPO4, 5% (w / w) sorbitol, 10 mM methionine, 0.02% (w / w) polysorbate-80, pH 7.0. Typical suitable diafiltration volumes for TFF2 are, for example, 5-6 times the volume of the AEX2 eluate, but this can be adjusted if necessary based on routine testing by a person skilled in the art (e.g. conductivity and pH of the retentate).

[0186] Optionally, the pH of the load of the AEX2 eluate is adjusted before TFF2. A suitable pH for TFF2 is, for example, 6.5 ± 0.2, and an example of a suitable buffer is 100 mM Na2HPO4 buffer (buffer W). Typically and preferably, the pH of the load of the AEX2 eluate is not adjusted before TFF2 (in which case the pH is typically about 6.0 ± 0.2).

[0187] In one embodiment, this includes (vi-2) subjecting the purified O-EPA bioconjugate to bioburden filtration to obtain the purified O-EPA bioconjugate drug substance. This bioburden filtration can be performed, for example, using a filter having a PES membrane with a cutoff of 0.45 + 0.2 μm, such as a Sartopore 2 Capsule Size 9 filter.

[0188] In one embodiment, this includes (vi-3) aliquoting and freezing the purified O-EPA bioconjugate drug substance to obtain a drug substance.

[0189] In certain embodiments, the production method further comprises an additional step (vii) wherein several purified O-EPA bioconjugate drug substances are combined, thereby obtaining a multivalent drug product.

[0190] In certain embodiments, the multivalent pharmaceutical product comprises at least four, five, six, seven, eight, nine, or ten purified O-EPA bioconjugates comprising an O-antigen polysaccharide selected from the list consisting of E. coli O-serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75. In certain embodiments, the multivalent pharmaceutical product comprises a purified O-EPA bioconjugate comprising an O-antigen polysaccharide selected from the list consisting of E. coli O-serotypes O1A, O2, and O25B.

[0191] In certain embodiments, the multivalent drug product comprises at least a purified O-EPA bioconjugate comprising the O-antigen polysaccharide of Escherichia coli O-serotype O1A.

[0192] In certain embodiments, the multivalent drug product comprises at least a purified O-EPA bioconjugate comprising the O-antigen polysaccharide of Escherichia coli O-serotype O2.

[0193] In certain embodiments, the multivalent drug product comprises at least a purified O-EPA bioconjugate comprising the O-antigen polysaccharide of Escherichia coli O-serotype O25B.

[0194] In certain embodiments, the multivalent drug product comprises at least a purified O-EPA bioconjugate comprising O-antigen polysaccharides of E. coli O-serotypes O1A, O2, O6A, and O25B.

[0195] In certain embodiments, the multivalent pharmaceutical product comprises at least four, preferably at least eight, and more preferably nine purified O-EPA bioconjugates comprising an O-antigen polysaccharide selected from the list consisting of E. coli O-serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, preferably E. coli O-serotypes O1A, O2, O4, O6A, O15, O16, O18A, O25B, and O75. In further embodiments, conjugates of different E. coli serotypes (i.e., O-antigen polysaccharides covalently coupled to a carrier protein) can be added, for example, to obtain a multivalent pharmaceutical product comprising 10-20 conjugates (e.g., O-EPA conjugates). Such conjugates of different E. coli serotypes can also be bioconjugates and can also be purified according to the methods described herein. In a specific embodiment, a nine-valent drug product is provided, comprising no more than nine purified O-EPA bioconjugates according to the present invention, wherein the O-antigen polysaccharide in the nine-valent drug product consists of E. coli O-serotypes O1A, O2, O4, O6A, O15, O16, O18A, O25B, and O75. In a specific embodiment, a ten-valent drug product is provided, comprising no more than ten purified O-EPA bioconjugates according to the present invention, wherein the O-antigen polysaccharide in the ten-valent drug product consists of E. coli O-serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75.

[0196] In alternative embodiments, additional filtration steps, such as tangential flow filtration, ultrafiltration / diafiltration (a variant of tangential flow filtration), dead-end filtration, and / or sterile filtration, as well as optional concentration and / or dilution and / or buffer exchange steps, may be added at certain points in the process, but preferably no additional chromatography steps are included, and thus the process preferably does not include more than four chromatography steps. Preferably, the process according to the present invention does not include either a size exclusion chromatography step or a hydroxyapatite chromatography step for purifying the bioconjugate. Of course, further purification steps, such as chromatography steps, can be added to the process of the present invention to obtain even higher purities, but this will inevitably lead to a decrease in overall yield and an increase in process complexity and material consumption, and is therefore undesirable because it may make the process economically unfeasible for large-scale production of biopharmaceutical products, whereas the process of the present invention already results in the desired purity level of the drug product suitable for administration to humans, at a yield that makes the process of the present invention economically and practically feasible.

[0197] Further details regarding the method steps and terminology used are provided below:

[0198] Anion Exchange Chromatography (AEX): Generally speaking, in ion exchange chromatography, binding is based on electrostatic charge. In the case of AEX, the resin has positively charged functional groups, so sample components with negatively charged functional groups will bind to it. As known to those skilled in the art, AEX can be performed using either weak or strong anion exchangers. In certain embodiments of the present invention, strong anion exchange resins are used in the AEX steps (AEX1 and AEX2). Non-limiting examples of functional groups suitable for AEX resins are quaternary ammonium groups. Such resins are commercially available and include, for example, Q Ceramic HyperD F resin and Source 15Q resin. Based on this disclosure and common sense, one skilled in the art will know how to adapt the different available AEX resins for use in the methods of the present invention. As a non-limiting example, AEX1 can be suitably performed using Q Ceramic HyperD F resin, and AEX2 can be suitably performed using Source 15Q resin. As the salt concentration increases, salt ions in the elution buffer compete with the resin-bound species for binding, and the bound species are displaced and eluted. Alternatively, when the pH is changed, the bound protein is titrated and eventually becomes uncharged or has the same charge as the functional groups of the resin, resulting in rejection and elution of the bound protein.

[0199] Mixed mode chromatography: The term mixed mode chromatography (MMC) is known in the art and is used synonymously with multimodal chromatography herein. The abbreviation MMC is not to be confused with the commercially available product "Capto MMC multimodal chromatography resin" (Cytiva). The term relates to a chromatographic method that utilizes more than one form of interaction between the stationary phase and the target protein (e.g., a bioconjugate) to achieve separation from impurities. Therefore, in MMC, the binding to the multimodal resin (MMR) and the separation of the protein rely on at least two different types of interactions. In some cases, binding is determined by a combination of electrostatic and hydrophobic interactions, such as in the case of an MMR comprising an anion exchange function and a hydrophobic function. A non-limiting example of such a resin is an MMR comprising a ligand of Formula I (e.g., N-benzyl-N-methylethanolamine). Such resins can be, in particular, compressible resins and are commercially available, such as Capto Adhere multimodal resins. In addition, hydrogen bonds may contribute to binding.

[0200] However, this type of MMC is distinct from hydroxyapatite chromatography (HA), such as ceramic HA (cHA), which is sometimes considered a specific form of mixed-mode chromatography. In HA (or cHA), separation is achieved, in particular, through a combination of ionic and metal-affinity interactions (e.g., CHT ceramic hydroxyapatite XT resin). Typically, proteins bind to HA via HA-phosphate (cation exchange) or HA-calcium interactions (metal affinity). Small basic proteins typically bind to HA via phosphate-cation exchange, while acidic proteins typically bind primarily via calcium affinity interactions. Large proteins typically bind to HA using both mechanisms. However, while HA (or cHA) is a suitable stationary phase for a wide range of applications, including the purification of bioconjugates, it does not achieve the superior results of the MMR of the present invention in current bioconjugate purification applications. For example, the use of HA carries a higher cost. Furthermore, the MMR of the present invention significantly and unexpectedly results in an increase in the polysaccharide yield in the bioconjugate compositions prepared according to the methods of the present invention, compared to similar methods using cHA instead of MMR [see WO 2022 / 214620].

[0201] Hydrophobic Interaction Chromatography (HIC): In HIC, the target protein (e.g., O-EPA bioconjugate) is separated from impurities based on its hydrophobicity. Target proteins containing hydrophobic and hydrophilic regions are typically applied to an HIC column in a high salt buffer. The salt in the buffer reduces the solvation of the target protein. As the solvation decreases, the exposed hydrophobic regions are adsorbed by the HIC resin. The more hydrophobic the molecule, the less salt is required to promote binding. Typically, a decreasing salt gradient is used to elute the sample from the column in order of increasing hydrophobicity. Sample elution can also be aided by adding other components (such as detergents) to the elution buffer. A non-limiting example of a HIC capsule suitable for performing the above-mentioned HIC step is the Sartobind Phenyl Jumbo 5L capsule. Based on this disclosure and various commercially available HIC resins, one skilled in the art will be able to modify and use the HIC resin.

[0202] Bioconjugate: This term is as discussed above. Specifically, bioconjugates are glycoconjugates produced in host cells, wherein the host cell machinery produces the glycan and the carrier protein and links the glycan to the carrier protein, for example, via N-linkage to asparagine or arginine. A particularly preferred host cell for producing bioconjugates is Escherichia coli, which preferably contains nucleic acids encoding: (i) the carrier protein, (ii) an oligosaccharyltransferase, such as Campylobacter jejuni PglB, which is capable of covalently linking the O-antigen polysaccharide to an asparagine (Asn) residue in the glycosylation consensus sequence (Asn-X-Ser (Thr), where X can be any amino acid except Pro) in the carrier protein via N-linked glycosylation, and (iii) an rfb gene cluster encoding enzymes responsible for producing the O-antigen polysaccharide of the desired serotype. By creating host cells with different rfb gene loci, different bioconjugates can be prepared, for example, containing O-antigen polysaccharides from different E. coli or Shigella serotypes. Cultivation of such host cells produces bioconjugates in the periplasm of the host cells containing a carrier protein covalently linked to an O-antigen polysaccharide encoded by the rfb locus. A more detailed description of the production of bioconjugates in such host cells can be found, for example, in WO 2009 / 104074, WO 2015 / 124769, WO 2017 / 035181, or WO 2020 / 191082. Optimized variants of the PglB oligosaccharyltransferase for producing bioconjugates of specific E. coli O-antigens are described in WO 2020 / 191088. The present invention relates to new and improved methods for purifying the bioconjugates produced from such host cells and bioconjugate compositions that can be obtained by or from such host cells. The host cells used to produce bioconjugates are typically bacterial cells, preferably Gram-negative bacterial cells. In a preferred embodiment, the host cell is E. coli. O-EPA bioconjugates therefore need to be purified from E. coli host cell proteins. An example of a host cell protein is transaldolase B, particularly E. coli transaldolase B, and the methods described in the present invention enable preparations of O-EPA to be obtained with very low amounts of host cell proteins, including very low amounts of transaldolase B. Surprisingly, transaldolase B appears to be one of the most abundant residual host cell proteins that is difficult to remove from O-EPA bioconjugates. Host cells are often engineered to express bioconjugates in the periplasm, so a good starting point for purifying O-EPA bioconjugates is from the periplasmic fraction of host cells (e.g., from Gram-negative host cells, such as E. coli host cells).

[0203] Particularly useful bioconjugates include carrier proteins to which one or more polysaccharides are attached. Such bioconjugates are used, for example, as active ingredients in vaccines designed to induce a functional immune response against the polysaccharides of the bioconjugate. In embodiments of the present invention, the bioconjugate comprises a carrier protein and one or more polysaccharides covalently bound to the carrier protein, preferably 1-4 polysaccharides covalently bound to the carrier protein.

[0204] In an embodiment of the present invention, the bioconjugate is a conjugate product containing an E. coli O-antigen polysaccharide covalently bound to a carrier protein. In an embodiment of the present invention, the bioconjugate is a conjugate product containing an Shigella O-antigen polysaccharide covalently bound to a carrier protein. The term O-antigen is known in the art and follows its conventional meaning, which is not to be confused with O-linked. In a typical embodiment, the O-antigen polysaccharide is N-linked to the carrier protein. The term O-antigen polysaccharide generally refers to a repeating polysaccharide polymer contained in the LPS of bacteria (e.g., E. coli). The O-antigen of E. coli is a polymer of immunogenic repeating oligosaccharides (typically 1-40 repeating units, e.g., 5-30 repeating units) and is commonly used for serotyping and glycoconjugate vaccine production.

[0205] Carrier protein: This term is as discussed above. A particularly suitable carrier protein in the context of the present invention is the detoxified exotoxin A of Pseudomonas aeruginosa (EPA) (the terms exotoxin A of Pseudomonas aeruginosa and exoprotein A of Pseudomonas aeruginosa or EPA are used interchangeably). In a specific embodiment, the carrier protein is the detoxified exotoxin A of Pseudomonas aeruginosa. For EPA, various detoxified protein variants have been described in the literature and can be used as carrier proteins. For example, detoxification can be achieved by mutation and deletion of catalytically essential residues L552V and ΔE553.

[0206] Preferably, the EPA carrier protein comprises 1-20, preferably 1-10, preferably 2-4 glycosylation sites.

[0207] In certain embodiments, EPA comprises four glycosylation sites having the amino acid sequence Asn-X-Ser(Thr), preferably Asp(Glu)-X-Asn-Z-Ser(Thr). See, for example, WO 2015 / 124769, WO 2017 / 035181, or WO 2020 / 191082 for descriptions of examples of bioconjugation of E. coli O-antigen polysaccharides with EPA carrier proteins, or see, for example, WO 2009 / 104074 for descriptions of examples of bioconjugation of Shigella O-antigen polysaccharides with EPA carrier proteins. In a non-limiting preferred embodiment, the carrier protein of the bioconjugate of the present invention comprises SEQ ID NO: 1. In certain embodiments, during expression in the host cell, the carrier protein comprises a signal sequence that targets the carrier protein to the periplasmic space. Various signal sequences can be used. In a non-limiting embodiment, the signal sequence comprises SEQ ID NO: 3. The signal sequence may be cleaved after the protein is translocated to the periplasm and, therefore, may no longer be present in the final carrier protein of the bioconjugate.

[0208] Polysaccharide: This term is as discussed above. Suitable polysaccharides include 1-100, such as 1-50, 1-40, 1-30, 1-20 and 1-10, 3-50, 3-40, for example at least 5, such as 5-40, for example 7-30, for example 7-25, for example 5-20, for example 10-20 repeating units n. Such repeating units contain (i.e., comprise or consist of) (i) unmodified monosaccharides and / or (ii) modified monosaccharides. In non-limiting embodiments, the term "modified monosaccharide" includes N-acetylation, O-acetylation, amidation and / or amination of monosaccharides. Such modified monosaccharides may include zero, one or more modifications, for example, zero, one, two or three of the above modifications, on the same monosaccharide.

[0209] In a particular embodiment, the modified monosaccharide is an O-acetylated and / or N-acetylated monosaccharide, in particular comprises one O-acetylated or N-acetylated monosaccharide.

[0210] In an embodiment of the present invention, suitable repeating units comprise a monosaccharide selected from the group consisting of mannose, rhamnose, glucose, fucose, galactose, modified mannose, modified rhamnose, modified glucose, modified fucose and modified galactose.

[0211] In an embodiment of the present invention, the O-polysaccharide is specific for a Gram-negative bacterium, preferably Escherichia coli, selected from the list of Escherichia and Shigella.

[0212] Non-limiting and exemplary structures of E. coli O-antigen polysaccharides are shown in Table 1 below. A single repeating unit of each E. coli O-antigen polysaccharide is shown. In this table, each n is independently an integer from 1 to 100, such as 1 to 50, 1 to 40, 1 to 30, 1 to 20, and 1 to 10, 3 to 50, 3 to 40, 5 to 30, for example, at least 5, such as 5 to 40, for example, 7 to 30, for example, 7 to 25, for example, 10 to 20, but in some cases can be 1 to 2. In certain preferred embodiments of the bioconjugate composition of E. coli O-antigen polysaccharides purified according to the methods of the present invention, the average value of n is between 5 and 30, preferably 10 to 25, and more preferably 10 to 20.

[0213] Table 1. Examples of structures of Escherichia coli O-antigen polysaccharides

[0214]

[0215]

[0216] The structures of other O-antigen polysaccharides of different serotypes of E. coli or Shigella or other bacteria are known and can be found in the art.

[0217] Bioconjugate composition: This term is known in the art and is discussed above. In the case of a carrier protein carrying multiple glycosylation sites (e.g., the EPA carrier protein having SEQ ID NO: 1), the production of bioconjugates typically results in a mixture of bioconjugates (herein, a bioconjugate composition) rather than a single bioconjugate product.

[0218] As used herein, in certain embodiments, the term "bioconjugate composition" thus refers to a mixture of bioconjugates, wherein each bioconjugate comprises the same carrier protein and the same O-antigen polysaccharide, but the individual bioconjugates differ in the number of occupied glycosylation sites (e.g., a "monovalent" composition). In the case of an EPA carrier protein comprising four N-glycosylation sites, for example, an EPA carrier protein comprising a four-fold glycosylation consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), such as the EPA carrier protein having SEQ ID NO: 1, a monovalent bioconjugate composition typically comprises several individual bioconjugates, each bioconjugate having 1-4 glycans attached to the EPA carrier protein, i.e., a monotetraglycosylated, ditetraglycosylated, tritetraglycosylated, or tetraglycosylated bioconjugate.

[0219] Depending on the individual bioconjugate, in particular the O-EPA bioconjugate as defined above, not all individual bioconjugates must be present, for example, an O-EPA bioconjugate composition may comprise mono-, di-, and tri-glycosylated O-EPA bioconjugates, but not tetra-glycosylated O-EPA bioconjugates. However, an O-EPA bioconjugate composition as defined above may also comprise all four glycosylation forms, i.e., mono-, di-, tri-, and tetra-glycosylated O-EPA bioconjugates.

[0220] In other embodiments, the bioconjugate composition can be a mixture of several monovalent compositions, i.e., a mixture comprising multiple drug substances, each comprising a carrier protein covalently bound to an O-antigen polysaccharide of a specific bacterial serotype via bioconjugation, wherein the total composition (a "multivalent" composition) comprises different monovalent compositions, such that the multivalent composition comprises different O-antigen polysaccharides of different bacterial serotypes, each covalently coupled to a carrier protein. In certain embodiments, such a multivalent composition is also referred to as a "drug product" because it can be used as a vaccine composition for administration to a subject to induce an immune response against the bacterial serotype of the O-antigen polysaccharide contained in the composition. Each individual serotype of bioconjugate drug substance present in such a multivalent composition can also be considered part of the monovalent composition as described above. For example, in the case of an EPA carrier protein comprising four N-linked glycosylation sites, mono-, di-, tri-, and / or tetra-glycosylated bioconjugates of each serotype may be present in the multivalent composition.

[0221] Thus, a bioconjugate composition comprising a carrier protein (EPA) having SEQ ID NO: 1 covalently linked to an O-antigen polysaccharide of a specific E. coli serotype (O-EPA bioconjugate) comprises a mixture of bioconjugates, wherein the O-EPA bioconjugate can be monoglycosylated with one O-antigen polysaccharide linked to EPA, or polyglycosylated with two, three, or four O-antigen polysaccharides linked to EPA, such that the bioconjugate composition is a mixture of monoglycosylated and polyglycosylated forms of O-EPA bioconjugate.

[0222] Polyglycosylated bioconjugate forms: As used herein, the term "polyglycosylated form of a bioconjugate" relates to a bioconjugate comprising a carrier protein covalently linked to more than one O-antigen polysaccharide of the same serotype, i.e., more than one N-glycosylation site is occupied. As described above, an O-EPA bioconjugate comprising an EPA carrier protein having four N-glycosylation sites (e.g., an EPA carrier protein having SEQ ID NO: 1) can be monoglycosylated, diglycosylated, triglycosylated, or tetraglycosylated. In this case, the term "polyglycosylated form" therefore relates to the sum of the diglycosylated, triglycosylated, and tetraglycosylated forms of the bioconjugate.

[0223] Comparison with Previous Methods for Producing O-EPA Bioconjugates: As described above, WO 2022 / 214620 describes methods for producing O-EPA bioconjugates suitable for large-scale production of bioconjugates. However, the previously described methods for producing O-EPA bioconjugates comprising an EPA carrier protein covalently linked to an O-antigen polysaccharide of a specific E. coli serotype yield bioconjugate compositions containing a relatively low proportion of the polyglycosylated form of the O-EPA bioconjugate (i.e., EPA carrier protein linked to two, three, or four O-antigen polysaccharides). Consequently, pharmaceutical compositions containing such specific O-EPA bioconjugates necessarily contain compositions of such specific O-EPA bioconjugates having a relatively low proportion of the polyglycosylated form of the O-EPA bioconjugate.

[0224] The degree of glycosylation (DOGY) of O-EPA bioconjugates can be measured by different methods. An established and particularly suitable method is capillary gel electrophoresis (cGE). In cGE, charged molecules are separated based on their size in a capillary tube filled with a porous gel matrix. cGE is commonly used to separate large biomolecules, such as proteins or glycoproteins. For example, in the case of O-EPA bioconjugates, the presence of covalently bound E. coli O-antigen polysaccharide causes the mobility of the EPA carrier protein in the capillary tube to change.

[0225] Advantageously, DOGY is measured according to the following protocol:

[0226] The O-EPA bioconjugate was concentrated to a target concentration of 3 mg / mL and separated in a bare fused silica capillary (total capillary length 30.2 cm, inner diameter 50 μm) containing SDS-gel buffer (pH 8.0, 0.2% SDS, 1% PEG6000) using a voltage of -15 kV. Advantageously, the absorbance of the bioconjugate was measured at 220 nm for cGE. The relative amounts (peak area percentage) of the mono-, di-, tri-, and tetra-glycosylated O-EPA bioconjugate forms were determined by integrating the corresponding peak segments in the electropherogram to determine the percentage of glycosylated forms. Based on the percentage of mono-, di-, tri-, and tetra-glycosylated O-EPA bioconjugate forms, a comparison between the abundance of mono- and poly-glycosylated forms of the O-EPA bioconjugate can be established.

[0227] Comparison of the DOGY of several exemplary O-EPA bioconjugate compositions produced according to the methods of WO 2022 / 214620 (Example 2) or the methods of the present invention described herein (Example 1), as measured by cGE, shows that the methods of the present invention result in a higher proportion of polyglycosylated forms of the O-EPA bioconjugates. This is shown in Example 3 (Table 4).

[0228] therefore, In the second aspect The present invention relates to O-EPA bioconjugate compositions obtainable or obtainable by the production methods of the invention described herein. In particular, the present invention relates to O-EPA bioconjugate compositions comprising a carrier protein having SEQ ID NO: 1 covalently linked to an O-antigen polysaccharide of Escherichia coli (particularly selected from serotypes O1A, O2, or O25B), obtainable or obtainable by the methods of the invention. These bioconjugate compositions are particularly suitable for use in the manufacture of pharmaceutical compositions as described herein.

[0229] In certain embodiments, the present invention relates to an O-EPA bioconjugate composition obtainable or obtained by the methods of the invention described herein, wherein each O-EPA bioconjugate comprises an EPA carrier protein comprising four N-glycosylation sites having the amino acid sequence Asn-X-Ser(Thr), preferably having the amino acid sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro, such as an EPA carrier protein comprising the amino acid sequence of SEQ ID NO: 1; and

[0230] The carrier protein is covalently linked to an E. coli O-antigen polysaccharide selected from the group consisting of O1A, O2, O4, O8, O6A, O15, O16, O18A, O25B, and O75. In certain embodiments, the E. coli O-antigen polysaccharide is selected from the group consisting of O1A, O2, O6A, O16, O18A, O25B, and O75. In certain embodiments, the E. coli O-antigen polysaccharide is selected from the group consisting of O1A, O2, and O25B.

[0231] In certain embodiments, the present invention relates to an O-EPA bioconjugate composition obtainable by or obtained by the methods of the invention described herein, selected from:

[0232] (a) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O1A, at least 50% of which is polyglycosylated;

[0233] (b) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O2, at least 44% of which is polyglycosylated;

[0234] (c) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O6A, at least 64% of which is polyglycosylated;

[0235] (d) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O16, at least 65% of which is polyglycosylated;

[0236] (e) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O18A, at least 70% of which is polyglycosylated;

[0237] (f) a bioconjugate composition of an O-EPA bioconjugate of E. coli serotype O25B, at least 50% of which is polyglycosylated; and / or

[0238] (g) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O75, at least 50% of which is polyglycosylated,

[0239] The percentages of polyglycosylated and monoglycosylated O-EPA were each determined by capillary gel electrophoresis.

[0240] It should be understood that the definitions, terms, characteristics, parameters disclosed herein (particularly for the first aspect of the present invention) are also applicable to the second aspect.

[0241] exist The third aspectThe present invention relates to specific pharmaceutical compositions comprising one or more bioconjugate compositions as defined herein. This aspect particularly provides pharmaceutical compositions comprising bioconjugate compositions wherein the EPA carrier protein is covalently linked to an O-antigen polysaccharide of an E. coli serotype selected from O1A, O2, O6A, O15, O16, O18A, O25B, or O75. These pharmaceutical compositions exhibit particularly beneficial amounts of the EPA carrier protein in a polyglycosylated form. This aspect is explained in further detail below. It should be understood that the definitions, terms, characteristics, and parameters disclosed herein (particularly with respect to the first aspect of the invention) also apply to this third aspect.

[0242] In a preferred embodiment, the pharmaceutical composition comprises one or more bioconjugate compositions. Each of these bioconjugate compositions comprises the Pseudomonas aeruginosa exoprotein A carrier protein (EPA) having SEQ ID NO: 1 covalently linked to an O-antigen polysaccharide of a specific Escherichia coli serotype (O-EPA bioconjugate). Each of the O-EPA bioconjugates can be monoglycosylated, with one O-antigen polysaccharide linked to EPA, or polyglycosylated, with two, three, or four O-antigen polysaccharides linked to EPA, such that the bioconjugate composition is a mixture of monoglycosylated and polyglycosylated forms of O-EPA bioconjugates. In particular, the pharmaceutical composition comprises one or more of the following:

[0243] (a) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O1A, at least 50% of which is polyglycosylated;

[0244] (b) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O2, at least 44% of which is polyglycosylated;

[0245] (c) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O6A, at least 64% of which is polyglycosylated;

[0246] (d) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O16, at least 65% of which is polyglycosylated;

[0247] (e) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O18A, at least 70% of which is polyglycosylated;

[0248] (f) a bioconjugate composition of an O-EPA bioconjugate of E. coli serotype O25B, at least 50% of which is polyglycosylated; and / or

[0249] (g) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O75, at least 50% of which is polyglycosylated,

[0250] The percentages of polyglycosylated and monoglycosylated O-EPA were each determined by cGE.

[0251] Further details regarding bioconjugate compositions (a)-(g) are provided below (DOGY measurements by cGE as described above):

[0252] Regarding composition (a): In one embodiment, the bioconjugate composition of the O-EPA bioconjugate of E. coli serotype O1A comprises at least 50% polyglycosylated O-EPA and no more than 80% polyglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (a) comprises 50-80% polyglycosylated O-EPA, such as 50%, 55%, 60%, 65%, 67%, 70%, 75%, or 80% polyglycosylated O-EPA. In one embodiment, bioconjugate composition (a) comprises at least 55% polyglycosylated O-EPA. Furthermore, in certain embodiments, bioconjugate composition (a) comprises at least 20% monoglycosylated O-EPA and no more than 50% monoglycosylated O-EPA. Thus, in certain embodiments, the bioconjugate composition (a) comprises 20-50% monoglycosylated O-EPA, such as 20%, 25%, 30%, 35%, 40%, 45%, or 50% monoglycosylated O-EPA.

[0253] Regarding composition (b): In one embodiment, the bioconjugate composition of the O-EPA bioconjugate of E. coli serotype O2 comprises at least 44% polyglycosylated O-EPA and no more than 90% polyglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (b) comprises 44-90% polyglycosylated O-EPA, such as 44%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 75%, 80%, 85%, or 90% polyglycosylated O-EPA. In certain embodiments, bioconjugate composition (b) comprises at least 50% polyglycosylated O-EPA. Furthermore, in certain embodiments, bioconjugate composition (b) comprises at least 10% monoglycosylated O-EPA and no more than 56% monoglycosylated O-EPA. Thus, in certain embodiments, the bioconjugate composition (b) comprises 10-56% monoglycosylated O-EPA, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 56% monoglycosylated O-EPA.

[0254] Regarding composition (c): In one embodiment, the bioconjugate composition of the O-EPA bioconjugate of E. coli serotype O6A comprises at least 64% polyglycosylated O-EPA and no more than 90% polyglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (c) comprises 64-90% polyglycosylated O-EPA, such as 64%, 65%, 70%, 72%, 75%, 80%, 85%, or 90% polyglycosylated O-EPA. In certain embodiments, bioconjugate composition (c) comprises at least 70% polyglycosylated O-EPA. Furthermore, in certain embodiments, bioconjugate composition (c) comprises at least 10% monoglycosylated O-EPA and no more than 36% monoglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (c) comprises 10-36% monoglycosylated O-EPA, e.g., 10%, 15%, 20%, 25%, 30%, 35%, or 36% monoglycosylated O-EPA.

[0255] Regarding composition (d): In one embodiment, the bioconjugate composition of the O-EPA bioconjugate of E. coli serotype O16 comprises at least 65% polyglycosylated O-EPA and no more than 90% polyglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (d) comprises 65-90% polyglycosylated O-EPA, for example, 65%, 70%, 75%, 80%, 81%, 85%, or 90% polyglycosylated O-EPA. In certain embodiments, bioconjugate composition (d) comprises at least 70% polyglycosylated O-EPA. Furthermore, in certain embodiments, bioconjugate composition (d) comprises at least 10% monoglycosylated O-EPA and no more than 35% monoglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (d) comprises 10-35% monoglycosylated O-EPA, for example, 10%, 15%, 20%, 25%, 30%, or 35% monoglycosylated O-EPA.

[0256] Regarding composition (e): In one embodiment, the bioconjugate composition of the O-EPA bioconjugate of E. coli serotype O18A comprises at least 70% polyglycosylated O-EPA and no more than 95% polyglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (e) comprises 70-95% polyglycosylated O-EPA, for example, 70%, 75%, 76%, 80%, 85%, 90%, or 95% polyglycosylated O-EPA. In certain embodiments, bioconjugate composition (e) comprises at least 80% polyglycosylated O-EPA. Furthermore, in certain embodiments, bioconjugate composition (e) comprises at least 5% monoglycosylated O-EPA and no more than 30% monoglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (e) comprises 5-30% monoglycosylated O-EPA, for example, 5%, 10%, 15%, 20%, 25%, or 30% monoglycosylated O-EPA.

[0257] Regarding composition (f): In one embodiment, the bioconjugate composition of the O-EPA bioconjugate of E. coli serotype O25B comprises at least 50% polyglycosylated O-EPA and no more than 90% polyglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (f) comprises 50-90% polyglycosylated O-EPA, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 88%, or 90% polyglycosylated O-EPA. In certain embodiments, bioconjugate composition (f) comprises at least 60% polyglycosylated O-EPA. Furthermore, in certain embodiments, bioconjugate composition (f) comprises at least 10% monoglycosylated O-EPA and no more than 50% monoglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (f) comprises 10-50% monoglycosylated O-EPA, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% monoglycosylated O-EPA.

[0258] Regarding composition (g): In one embodiment, the bioconjugate composition of the O-EPA bioconjugate of E. coli serotype O75 comprises at least 50% polyglycosylated O-EPA and no more than 90% polyglycosylated O-EPA. Thus, in certain embodiments, bioconjugate composition (g) comprises 50-90% polyglycosylated O-EPA, such as 50%, 55%, 60%, 65%, 66%, 70%, 75%, 80%, 85%, or 90% polyglycosylated O-EPA. In certain embodiments, bioconjugate composition (g) comprises at least 60% polyglycosylated O-EPA. Furthermore, in certain embodiments, bioconjugate composition (h) comprises at least 10% monoglycosylated O-EPA and no more than 50% monoglycosylated O-EPA. Thus, in certain embodiments, the bioconjugate composition (g) comprises 10-50% monoglycosylated O-EPA, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% monoglycosylated O-EPA.

[0259] Preferably, each of the bioconjugate compositions (a)-(g) has a purity of at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100%, as measured by SE-HPLC, or a purity of at least 95%, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100%, as measured by RP-HPLC. Typically, each of the bioconjugate compositions (a)-(g) has a purity of at least 95%, e.g., at least 96%, 97%, 98%, 99%, as measured by SE-HPLC or RP-HPLC. In certain embodiments, one or more of the bioconjugate compositions (a)-(g) are in a container and have a volume of about 0.5 mL to about 100 L, e.g., about 1 mL to about 10 L, and the concentration of the bioconjugate is about 4 μg / mL to about 5000 μg / mL, e.g., about 15 μg / mL to about 1000 μg / mL, e.g., about 16 or 32 μg / mL (based on the concentration measured based on the amount of polysaccharide, as conventionally known in the art; typically, these bioconjugates have a polysaccharide / protein (w / w) ratio of about 0.1 to about 0.5, e.g., about 0.15 to about 0.45, such as about 0.2 to about 0.4).

[0260] In one embodiment, the pharmaceutical composition comprises the bioconjugate composition (a).

[0261] In one embodiment, the pharmaceutical composition comprises a bioconjugate composition (b).

[0262] In one embodiment, the pharmaceutical composition comprises the bioconjugate composition (f).

[0263] In one embodiment, the pharmaceutical composition comprises at least four bioconjugate compositions selected from (a)-(g) as defined above, preferably at least (a), (b), (c) and (f).

[0264] In another embodiment, the pharmaceutical composition comprises all seven bioconjugate compositions (a)-(g) as defined above.

[0265] In another embodiment, the pharmaceutical composition as defined above further comprises a bioconjugate composition (h) of an O-EPA bioconjugate of E. coli serotype O15. In certain embodiments, the O15-EPA bioconjugate composition (h) comprises a carrier protein (EPA) having SEQ ID NO: 1 covalently linked to an O-antigen polysaccharide of E. coli serotype O15.

[0266] The O15-EPA bioconjugate can be monoglycosylated with one O-antigen polysaccharide linked to EPA, or polyglycosylated with two, three, or four O-antigen polysaccharides linked to EPA, such that the bioconjugate composition is a mixture of monoglycosylated and polyglycosylated forms of the O15-EPA bioconjugate, at least 50% of which are polyglycosylated. In one embodiment, the bioconjugate composition (h) of the O-EPA bioconjugate of E. coli serotype O15 comprises at least 50% polyglycosylated O-EPA and no more than 95% polyglycosylated O-EPA. Thus, in certain embodiments, the bioconjugate composition (h) comprises 50-95% polyglycosylated O-EPA, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 85%, 90%, or 95% polyglycosylated O-EPA. Furthermore, in certain embodiments, the bioconjugate composition (h) comprises at least 5% monoglycosylated O-EPA and no more than 50% monoglycosylated O-EPA. Thus, in certain embodiments, the bioconjugate composition (h) comprises 5-50% monoglycosylated O-EPA, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% monoglycosylated O-EPA.

[0267] The percentages of polyglycosylated and monoglycosylated O-EPA were determined by cGE as described above.

[0268] Preferably, the bioconjugate composition (h) is at least 90% pure, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% pure, as measured by SE-HPLC, or at least 95% pure, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% pure, as measured by RP-HPLC. Typically, the bioconjugate composition (h) is at least 95% pure, e.g., at least 96%, 97%, 98%, 99% pure, as measured by SE-HPLC or RP-HPLC.

[0269] In certain embodiments, the pharmaceutical composition described above comprises each of the bioconjugate compositions (a), (b), (c), (d), (e), (f), (g), and (h) and has a volume of about 0.5 mL to about 10 L, e.g., about 1 mL to about 1 L, and a total concentration of the bioconjugate of about 90 μg / mL to about 600 μg / mL, e.g., about 170 μg / mL to about 300 μg / mL (based on the amount of polysaccharide measured, as conventionally known in the art; typically the polysaccharide / protein (w / w) ratio of the bioconjugate in the composition is about 0.1 to about 0.5, e.g., about 0.15 to about 0.45, e.g., about 0.2 to about 0.4).

[0270] In another embodiment, the pharmaceutical composition as defined above further comprises a bioconjugate composition (i) of an O-EPA bioconjugate of E. coli serotype O4. In certain embodiments, the O4-EPA bioconjugate composition (i) comprises a carrier protein (EPA) having SEQ ID NO: 1 covalently linked to an O-antigen polysaccharide of E. coli serotype O4.

[0271] The O4-EPA bioconjugate can be monoglycosylated with one O-antigen polysaccharide linked to EPA, or polyglycosylated with two, three, or four O-antigen polysaccharides linked to EPA, such that the bioconjugate composition is a mixture of monoglycosylated and polyglycosylated forms of the O4-EPA bioconjugate, at least 10% of which is polyglycosylated. The percentages of polyglycosylated and monoglycosylated O-EPA are determined by cGE as described above.

[0272] In one embodiment, the bioconjugate composition (i) of the O-EPA bioconjugate of E. coli serotype O4 comprises at least 10% polyglycosylated O-EPA and no more than 60% polyglycosylated O-EPA. Thus, in certain embodiments, the bioconjugate composition (i) comprises 10-60% polyglycosylated O-EPA, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 44%, 45%, 50%, 55%, or 60% polyglycosylated O-EPA. Furthermore, in certain embodiments, the bioconjugate composition (i) comprises at least 40% monoglycosylated O-EPA and no more than 90% monoglycosylated O-EPA. Thus, in certain embodiments, the bioconjugate composition (i) comprises 40-90% monoglycosylated O-EPA, e.g., 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% monoglycosylated O-EPA.

[0273] Preferably, the bioconjugate composition (i) is at least 90% pure, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% pure, as measured by SE-HPLC, or at least 95% pure, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% pure, as measured by RP-HPLC. Typically, the bioconjugate composition (i) is at least 95% pure, e.g., at least 96%, 97%, 98%, 99% pure, as measured by SE-HPLC or RP-HPLC.

[0274] In certain embodiments, the pharmaceutical composition described above comprises each of bioconjugate compositions (a), (b), (c), (d), (e), (f), (g), and (i), or each of bioconjugate compositions (a), (b), (c), (d), (e), (f), (g), (h), and (i), and in each case in a volume of about 0.5 mL to about 10 L, e.g., about 1 mL to about 1 L, and a total concentration of the bioconjugate of about 90 μg / mL to about 600 μg / mL, e.g., about 170 μg / mL to about 300 μg / mL (based on the amount of polysaccharide measured as conventionally practiced in the art; typically the polysaccharide / protein (w / w) ratio of the bioconjugate in the composition is about 0.1 to about 0.5, e.g., about 0.15 to about 0.45, e.g., about 0.2 to about 0.4).

[0275] In certain embodiments, the pharmaceutical composition as defined above further comprises a bioconjugate composition (j) of an O-EPA bioconjugate of E. coli serotype O8. In certain embodiments, the O8-EPA bioconjugate composition comprises an O-EPA bioconjugate comprising a carrier protein (EPA) having the amino acid sequence of SEQ ID NO: 1 covalently linked to an O-antigen polysaccharide of E. coli serotype O8. Preferably, the bioconjugate composition (j) has a purity of at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9% or 100%, as measured by SE-HPLC, or a purity of at least 95%, such as 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9% or 100%, as measured by RP-HPLC. Typically, the bioconjugate composition (j) has a purity of at least 95%, e.g., at least 96%, 97%, 98%, or 99%, as measured by SE-HPLC or RP-HPLC. Thus, in certain embodiments, the pharmaceutical composition described above comprises each of the bioconjugate compositions (a), (b), (c), (d), (e), (f), (g), (h), (i), and (j) in a volume of about 0.5 mL to about 10 L, e.g., about 1 mL to about 1 L, and the total bioconjugate concentration is about 90 μg / mL to about 600 μg / mL, e.g., about 170 μg / mL to about 300 μg / mL (based on the amount of polysaccharide, as conventionally measured in the art; typically, the polysaccharide / protein (w / w) ratio of the bioconjugate in the composition is about 0.1 to about 0.5, e.g., about 0.15 to about 0.45, e.g., about 0.2 to about 0.4).

[0276] In a further embodiment, conjugates of different E. coli serotypes (i.e., O-antigen polysaccharides covalently coupled to a carrier protein) can be added, for example, to obtain a pharmaceutical composition comprising 10-20 conjugates (e.g., O-EPA conjugates). Such conjugates of different E. coli serotypes can also be bioconjugates or bioconjugate compositions and can also have been purified according to the methods described herein.

[0277] Sequence Description

[0278] SEQ ID NO: 1 (EPA carrier protein including four N-linked glycosylation consensus sequences)

[0279]

[0280] SEQ ID NO: 2 (Example PglB oligosaccharyltransferase)

[0281]

[0282] SEQ ID NO: 3 (Example signal sequence of EPA carrier protein)

[0283]

[0284] Exemplary glycosylation consensus sequences

[0285] Asn-X-Ser(Thr), where X can be any amino acid except Pro.

[0286] Exemplary optimized glycosylation consensus sequences

[0287] Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro.

[0288] The following of the present invention Example It is intended to further illustrate the nature of the present invention. It should be understood that the following examples do not limit the present invention and that the scope of the present invention is determined by the appended claims.

[0289] Example 1: New Production Method for O-EPA Bioconjugates

[0290] This is an example of the inventive method of producing the ExPEC O-EPA bioconjugate.

[0291] Bioconjugate production strains

[0292] Methods for constructing different bioconjugate production strains have been previously described, for example in Example 6 of WO2020 / 191082. For some bioconjugates, previously described strains were used, while for some other bioconjugates, new production strains were constructed to improve yields. The parent cell was Escherichia coli strain W3110. In the production strains, the rfb locus encoding the enzyme responsible for the production of the relevant E. coli O-antigen polysaccharide (serotype-specific, the sequence of the corresponding rfb locus is provided in Table 2 herein by reference to the sequences provided in WO 2020 / 191082 and WO 2022 / 208430) replaces the deleted endogenous W3110 rfb locus; in each of these production strains, the same EPA carrier protein (having four N-linked glycosylation sequences; carrier protein having SEQ ID NO: 1) is encoded by the plasmid; and in these production strains, the oligosaccharyltransferase that transfers the O-antigen to Asn in the N-linked glycosylation sequence of the carrier protein is also encoded by the plasmid and is Campylobacter jejuni PglB having SEQ ID NO: 2 or a variant thereof, which can increase the yield of specific E. coli O-antigen bioconjugates and differs from SEQ ID NO: 2 by the mutations shown in the last column of Table 2 below.

[0293] Table 2. Relevant characteristics of the production strain of ExPEC9V bioconjugate.

[0294]

[0295] Note: In strains producing E. coli O4 bioconjugates, a nucleic acid encoding the E. coli O4-specific GtrS protein is also present in the genome (e.g., the amino acid sequence of the GtrS protein and the nucleic acid sequence encoding the protein are shown in SEQ ID NO: 4 and SEQ ID NO: 5 of WO 2020 / 191082, respectively) to allow the production of bioconjugates having Glc-branched O4 polysaccharides as shown in structure (O4) in Table 1 (see also WO 2020 / 191082)).

[0296] These strains are used to produce bioconjugates by the methods of the invention described herein.

[0297] The method of the present invention implemented in this example consists of the following steps: AEX1 (capture AEX chromatography); particle reduction filtration 1; mixed mode chromatography; HIC buffer adjustment; particle reduction filtration 2; HIC; TFF1; particle reduction filtration 3; AEX2 (polishing chromatography); TFF2; bioburden filtration.

[0298] The buffers involved in this example and the following examples (including their compositions) are listed in Table 3 below.

[0299] Step i: Providing a filtered periplasmic fraction containing the O-EPA bioconjugate. The filtered periplasmic fraction containing the O25B-EPA bioconjugate is used as the starting material for the purification method described below. The production of O-EPA bioconjugates has been previously described (see, for example, WO2009 / 104074, WO2015 / 124769, and WO2020 / 191082) and is carried out similarly to these protocols. Specifically, as previously described [WO2020191082], after incubation in a 200 L bioreactor (fermenter), the periplasmic fraction of the E. coli host cells containing the O25B-EPA bioconjugate is obtained by osmotic shock treatment. The filtered periplasmic fraction is obtained essentially as follows. The incubation in the 200 L bioreactor is equivalent to 165 L harvest equivalents (HE; LHE).

[0300] In more detail:

[0301] As a harvesting step, 165 L of culture broth was cooled to below 20°C and then harvested by a disc centrifuge (DSC) using a separator with a constant flow rate (eg, about 160 L / h). The biomass was collected and the centrifuge (supernatant) was discarded.

[0302] Following steps are osmotic shock, and it uses about 6-10 ℃ solution to carry out.Determine the cell wet weight (CWW) of concentrated harvest, and based on CWW, cell suspension is diluted to the target CWW of for example about 390g / L with 1 / 3TBS (pH 7.4), and determine cumulative volume.60% sucrose solution is added in the harvest of dilution to 25% target concentration, and hatches 1h with cell at 6-10 ℃, mixing solution simultaneously.After hatching with sucrose, with the 10mM Tris-HCL (pH 8.0) of cell / sucrose solution and 4x volume, at 6-10 ℃, online mixing.Mixing is carried out by static mixer.Product is discharged in supernatant (being called periplasmic fraction, PF) from periplasmic space, and collects PF.

[0303] Subsequently, the material was clarified: cell debris in the PF was removed by separation in a disc centrifuge. A constant flow rate was applied and the flow rate was adjusted based on the turbidity of the centrifuge. The supernatant was collected and called the centrifuged periplasmic fraction (CPF).

[0304] The CPF still contains cell debris and is filtered through depth and bioburden reduction filters and collected as filtered periplasmic fraction (FPF). The FPF is used to further purify O-EPA starting from the AEX1 step (step ii), as further described below.

[0305] Step ii: Capture AEX1 chromatogram

[0306] ii-1: No conductivity adjustment required for the filtered fermentor harvest. The periplasmic fraction of the osmotically shocked and filtered fermentor harvest was loaded onto Q Ceramic HyperD F resin in bind-elute mode for a first anion exchange chromatography run (AEX1; 20 cm bed height, 0.34 L resin / L harvest equivalent). After equilibration of the column with low-salt buffer A, the filtered periplasmic fraction containing the O-EPA bioconjugate was loaded onto the column. The column was then washed with 3 column volumes (CV) of buffer A.

[0307] ii-2: Elution was performed with a step gradient using 20% ​​buffer BV2 in buffer A. The eluate was divided into four 0.25 CV fractions, wherein collection of the eluted fractions began when the UV absorbance increased at a slope greater than 1.0 AU / min.

[0308] ii-3: Create an "AEX1 pool" (AEX1 eluate) by combining fractions 1 and 2.

[0309] Step iii: Mixed-mode chromatography

[0310] iii-1: Particle reduction filtration was performed using a PES membrane (Sartopore) with a cut-off of 0.45+0.2 μm and a filtration area of ​​0.45 m2 / 165 LHE.

[0311] iii-2: Mixed mode chromatography was performed in bind elution mode (Capto adhesion multimodal resin, 20 cm bed height, 6.3 L / 165 LHE harvest equivalent).

[0312] After pre-equilibration with 3CV of buffer CC, the column was equilibrated with 5CV of buffer AA and the AEX1 eluate was applied to Capto Adhere multimodal resin (bind-elute mode). The column was washed with 5CV of buffer AA, then 5CV of buffer BB and finally 2CV of buffer AA.

[0313] iii-3: Elution was performed using 3.5 CV of buffer CC. The eluate was collected after the UV slope (measured at 280 nm) exceeded 0.05 mAU (MMR eluate).

[0314] iii-4: Two fractions of 1 CV each were collected and combined without prior analysis by SDS-PAGE.

[0315] Step iv: HIC chromatography

[0316] iv-1: Add adjustment buffer (buffer Q) to the combined MMR eluates until the added weight reaches 3 times the initial weight of the combined MMR eluates.

[0317] iv-2: Particle reduction filtration was performed using a PES membrane (Sartopore) with a cut-off of 0.45+0.2 μm and a filtration area of ​​0.45 m2 / 165 LHE.

[0318] iv-3: HIC was performed in bind-elute mode. An HIC capsule (Sartobind Phenyl Jumbo 5L, 0.8 cm bed height) was equilibrated with 3 CV of Buffer Q and the MMR eluent was applied to adjust the load. The HIC capsule was then washed with 1 CV of Buffer Q, followed by 4 CV of a buffer mixture containing 70% Buffer Q and 30% Buffer R.

[0319] IV-4: Elute the O-EPA bioconjugate by applying 4 CV of a buffer mixture containing 30% Buffer Q and 70% Buffer R. The eluate was divided into four 0.8 CV fractions. Collection of eluate fractions began when the UV absorbance increased at a slope greater than 0.5 AU / min and the UV-adsorption value was greater than 0.05 AU.

[0320] iv-5: A "HIC pool" was created by pooling fractions 1-3 without prior analysis by SDS-PAGE.

[0321] Step v: Polishing AEX2 chromatography

[0322] v-1: Tangential flow filtration (TFF1) was performed to prepare the HIC eluate for the polishing step (i.e. AEX2). The HIC eluate was filtered by TFF (mPES KrosFlo filter module Q, 10 kDa, 1.25 m 2 The HIC eluate was diafiltered with buffer U until the target diafiltration volume of 5-6 DV was reached.

[0323] v-2: Then use a filter with a cutoff value of 0.45±0.2μm and a cutoff value of 0.45m 2 The adjusted HIC eluate was filtered through a PES membrane with a filtration area of ​​100 nm using Sartopore 2 Capsule Size 0.

[0324] v-3: Polishing AEX chromatography (AEX2) was performed in bind-elute mode. The column (Source 15Q, 20 cm bed height, 6.3 L / 165 LHE) was equilibrated with 3 CV of Buffer U. The adjusted HIC eluate was applied to the column.

[0325] Elution was performed first with 7.5 CV of 21% buffer V in buffer U (step gradient), followed by 7.5 CV of a linear gradient from 21 to 56% buffer V in buffer U. Fraction collection began 1.2 CV after the start of elution and 30 fractions of 0.5 CV were subsequently collected.

[0326] v-4: Individual fractions were analyzed by SDS-PAGE and stained with Coomassie stain. An AEX2 eluate pool was created by pooling fractions starting with the first fraction showing a strong product band and only weak to moderately intense impurity bands, up to the first fraction showing faded product bands. Fractions showing faded product bands typically show an additional band of increased intensity corresponding to unglycosylated-EPA. In this example, fractions 11-30 were pooled. In this example, an O25B-EPA bioconjugate with a purity of 99.4% as measured by SE-HPLC was obtained.

[0327] Step vi: Adjust to a pharmaceutically acceptable buffer and concentration

[0328] vi-1: Apply a second TFF (mPES KrosFlo filter module Q, 10 kDa, 1.25 m 2 / 165LHE) to obtain an O-EPA bioconjugate concentration of OD280 = 0.5 ± 0.1 using an excipient buffer containing 6.19 mM KH2PO4, 3.81 mM Na2HPO4, 5% (w / w) sorbitol, 10 mM methionine, pH 7.0 (buffer XV2). The AEX2 eluate was then diafiltered with excipient buffer for 5-6 diafiltration volumes and then concentrated to a target concentration of OD280 = 1.40 ± 0.15. Polysorbate-80 (Tween-80) was then added to the excipient buffer to a final concentration of 0.02% (w / w) to obtain the O25B-EPA conjugate in a pharmaceutically acceptable buffer (Tween-adjusted buffer XV2) containing 6.19 mM KH2PO4, 3.81 mM Na2HPO4, 5% (w / w) sorbitol, 10 mM methionine, 0.02% (w / w) polysorbate-80, pH 7.0 (see, e.g., WO 2018 / 077853).

[0329] vi-2: Use a PES membrane with a cut-off value of 0.45 + 0.2 μm and a 0.2 μm 2 Bioburden filtration was performed using Sartopore 2 capsules size 9 with a filtration area of ​​100 nm.

[0330] vi-3: The resulting product (drug substance) was filled into vials (drug substance) and frozen in a primary freezer at -70°C + / - 10°C. After 72 hours, the DS vials were taken out of the primary freezer and placed directly into a final storage freezer at -70°C + / - 10°C without allowing any thawing of the DS.

[0331] In this example, the purity of the obtained O25B-EPA bioconjugate measured by SE-HPLC was 99.6%, and the yield was 24 mg PS / L HE, corresponding to an estimated overall yield of about 23% (relative to the O-EPA conjugate present in the FPF of step i above).

[0332] The same methods of the present invention as described above were used to purify a wide range of bioconjugates. Specifically, O-EPA bioconjugates from E. coli serotypes O1A, O2, O4, O6A, O15, O16, O18A, O25B, and O75 were purified. Each of these was typically at least 96% pure as measured by SE-HPLC, and in most cases, 98-100% pure as measured by SE-HPLC. The estimated overall yield of the method varied from strain to strain and, in particular, depended on the starting expression level, ranging from approximately 5-35%, with an average of approximately 20%. This demonstrates that the methods of the present invention are broadly applicable to a variety of different O-EPA conjugates and are suitable for economical, large-scale production of any O-EPA conjugate at a purity sufficient for administration to humans.

[0333] The E. coli O-antigen bioconjugates thus prepared as drug substance (DS) exhibited comparable critical quality attributes:

[0334] (1) Purity (measured by SE-HPLC or RP-HPLC) is higher than 95%.

[0335] (2) The polysaccharide / protein ratio ranges from about 0.1 to 0.5, mainly from 0.15 to 0.45.

[0336] (3) Bacterial endotoxin (European Pharmacopoeia 2.2.3) is less than 0.5 EU / μg polysaccharide.

[0337] (4) The average chain length of a single polysaccharide is typically about 10-20 repeat units (measured using high-resolution SDS-PAGE).

[0338] Example 2 (comparative): method according to WO 2022 / 214620

[0339] This is a comparative example of a previously preferred method for producing the ExPEC O-EPA bioconjugate (see WO 2022 / 214620). Only the differences from the above-described method of the present invention are described. The most significant change in the method of the present invention compared to the previously described method (e.g., WO 2022 / 214620) involves replacing the hydroxyapatite step (step iii) with a mixed-mode chromatography step.

[0340] Step iii: Hydroxyapatite chromatography

[0341] iii-1: The pooled AEX1 fractions were adjusted to a target pH of 7.2±0.2 using buffer Y, and in a subsequent step, the pooled AEX1 fractions were adjusted to a target conductivity of 7.5-9 mS / cm using buffer Z.

[0342] iii-2: Particle reduction filtration is performed as described above for the method of the present invention.

[0343] iii-3: Ceramic hydroxyapatite chromatography (cHA, 20 cm bed height, 0.34 L resin / L harvest equivalent) was performed in bind-elute mode.

[0344] The loaded AEX1 eluate was applied to the cHA resin after pre-equilibration with 6 CV of buffer N to adjust the pH and then equilibrated with 6 CV of buffer J. The column was washed with 1 CV of a low conductivity buffer consisting of 5% buffer K in buffer J.

[0345] iii-4: Product elution was performed using a salt gradient (i.e., a sodium chloride and potassium phosphate gradient). First, a 2.5 CV linear gradient of 20-45% buffer K in buffer J was applied, followed by a 2.5 CV step gradient of 70% buffer K in buffer J. After applying a 1.9 CV linear gradient, 14 fractions of 15.565 L / 165 L HE were collected.

[0346] iii-5: Analyze the individual fractions by SDS-PAGE. Select fractions to create a "cHA pool" based on the intensity of the band corresponding to the O-EPA bioconjugate relative to the band corresponding to the impurities. Then, create a combined cHA eluate by combining the selected fractions.

[0347] Step iv: HIC

[0348] IV-1: In this previous method, the load of the eluate from the previous step was adjusted until the target conductivity of 118 ± 2 mS / cm was reached. In the method of the present invention, adjustment buffer was added until the weight added reached three times the initial weight of the combined eluate from the previous chromatography step. Therefore, this step has been simplified in the method of the present invention.

[0349] Step v: Polishing AEX2 chromatography

[0350] v-1: Tangential flow filtration (TFF1) was performed to prepare the HIC eluate for the polishing step (i.e. AEX2). First, the HIC eluate was filtered through a TFF (mPES KrosFlo filter module Q, 10 kDa, 1.25 m 2 / 165LHE, transmembrane pressure about 0.8 bar), the HIC eluate was adjusted (diluted or concentrated) with buffer T to an OD280 of 1.35±0.15, and then diafiltered with buffer T to achieve a target conductivity of 5-6 mS / cm.

[0351] In some cases, the pH of the HIC eluate requires additional adjustment prior to the subsequent chromatography step. In this case, buffer Y is used to lower the pH to a target pH of 6.5 ± 0.2. In a further step, the conductivity is adjusted to 8.7 ± 0.3 mS / cm using buffer V. Thus, adjustment of the HIC eluate includes at least two steps (concentration adjustment and diafiltration), and in some cases three steps (including pH adjustment). In contrast, for the method of the present invention (Example 1), adjustment of the HIC eluate includes only one step (diafiltration).

[0352] v-3: The adjusted HIC eluate was applied to the column (same column as in Example 1) and a wash step was performed using 1.5 CV of buffer U. In the second wash, a linear gradient of 15-21% buffer V in buffer U was applied. In contrast, for the method of the present invention (Example 1), no wash step was required after applying the HIC eluate to the AEX2 column.

[0353] v-4: Elution was performed initially using 3 CV of 21% Buffer V in Buffer U (step gradient), followed by a 7.5 CV linear gradient from 21% to 56% Buffer V in Buffer U. Fraction collection began 1.25 CV after the start of elution, and 20 fractions of 0.4975 CV were subsequently collected. Thus, compared to this previous method, the step gradient in the method of the present invention was extended (7.5 CV instead of 3 CV).

[0354] v-5: Individual fractions were analyzed by SDS-PAGE and stained with Coomassie staining. The AEX2 eluate pool was created by combining the first fractions with an OD280 of at least 0.04 AU. Thus, in this comparative method, fractions were pooled based on an OD280 value of at least 0.04 AU, whereas in the method of the present invention (Example 1), OD measurement was not required. Instead, fractions were pooled based solely on SDS-PAGE analysis.

[0355] In this example, the purity of the obtained O25B-EPA bioconjugate measured by SE-HPLC was 99.7%.

[0356] Step vi: TFF2

[0357] vi-1: Compared to the method of the present invention (Example 1), in this comparative method it is necessary to adjust the pH of the load of the AEX2 eluate before TFF2 (which is only optional and not usually performed in the method of the present invention).

[0358] Table 3. Buffer

[0359]

[0360]

[0361] Example 3: Increased polyglycosylation of O-EPA bioconjugates obtained by the method of the present invention

[0362] This example describes the degree of glycosylation (DOGY) of three O-EPA bioconjugate compositions (corresponding to E. coli O-antigen polysaccharides of serotypes O1A, O2, and O25B, each conjugated to an EPA carrier protein having SEQ ID NO: 1) produced by the method of the present invention (Example 1) and the comparative method according to WO 2022 / 214620 (Example 2).

[0363] For each pair of O-EPA bioconjugates shown in Table 4, the same production strain (characteristics shown in Table 2 above) and fermentation conditions were used.

[0364] DOGY was determined by cGE according to the following scheme:

[0365] The O-EPA bioconjugate was concentrated to a target concentration of 3 mg / mL and separated in a bare fused silica capillary (total capillary length 30.2 cm, inner diameter 50 μm) containing SDS-gel buffer (pH 8.0, 0.2% SDS, 1% PEG6000). Sample separation was performed at -15 kV, and the absorbance of the bioconjugate was measured at 220 nm. The relative amounts of mono-, di-, tri-, and tetra-glycosylated forms were determined by integration of the corresponding peaks.

[0366] For each measurement, the corresponding O-EPA bioconjugate drug substance from a previous production batch was used as a reference substance and analyzed under the same conditions to ensure correct performance of the method (external control).

[0367] Table 4. Degree of glycosylation (measured by cGE) of O-EPA bioconjugate compositions comprising the O-antigen polysaccharide of E. coli serotypes O1A, O2, or O25B, respectively, coupled to an EPA carrier protein having SEQ ID NO: 1, produced according to the present invention (Example 1; "Inventive") compared to the prior art (Example 2; "Comparative").

[0368]

[0369] For each of the three different bioconjugates, the production strain and fermentation conditions were identical between the method according to WO 2022 / 214620 (Comparative) and the inventive method described herein, and therefore any differences in the products obtained can be attributed to differences in the purification methods. Compared to the comparative and previously preferred methods described in WO 2022 / 214620, the inventive method of the present invention surprisingly resulted in a decrease in monoglycosylated O-EPA bioconjugates and an increase in polyglycosylated O-EPA bioconjugates in the final purified product. This increase in polyglycosylation in the bioconjugate composition is advantageous because more available glycosylation sites in the EPA carrier protein are utilized in the product thus obtained, resulting in more available immunogenic glycans per carrier protein molecule.

[0370] In addition to improvements in the percentage of polyglycosylated O-EPA, the novel method of the present invention (see Example 1) generally resulted in even lower levels of transaldolase B (the most abundant residual host cell protein) in the purified O-EPA bioconjugate composition for several serotypes compared to the levels of transaldolase B obtained with a comparative method (i.e., having a cHA step instead of an MMC step as the second chromatographic step; see Example 2).

[0371] Furthermore, for several serotypes, the novel method of the invention described above (Example 1) resulted in a significant reduction in buffer consumption compared to the previously preferred method (Example 2), typically operating the second column with a reduction in buffer consumption of approximately 81%.

[0372] Example 4: Glycosylation Degrees of 9 O-EPA Bioconjugate Compositions

[0373] This example describes the degree of glycosylation (DOGY) of several O-EPA bioconjugate compositions (corresponding to E. coli O-antigen polysaccharides of serotypes O1A, O2, O4, O6A, O15, O16, O18A, O25B, and O75, each conjugated to an EPA carrier protein having SEQ ID NO: 1) produced by the methods of the present invention (Example 1). Because the bioconjugate production strain of individual bioconjugates may also influence DOGY, depending, for example, on the genes in the rfb locus and / or the PglB variant used, these relevant characteristics of the identified production strains are listed in Table 2 above. Several batches of each bioconjugate composition were produced, and some variation in DOGY was observed. Table 5 shows the results obtained for material produced in the context of a clinical trial production campaign. DOGY was measured by cGE as described in Example 3.

[0374] Table 5. The degree of glycosylation (measured by cGE) of O-EPA bioconjugate compositions comprising the O-antigen polysaccharide of E. coli serotype O1A, O2, O4, O6A, O15, O16, O18A, O25B, or O75, respectively, coupled to the EPA carrier protein having SEQ ID NO: 1, produced according to the production strains of Table 2 and according to the methods of the invention described herein (Example 1).

[0375]

[0376] Due to rounding effects, the sum of the mono-, di-, and tri-glycosylated O-EPA bioconjugate compositions may deviate slightly from 100%.

[0377] For O4, the composition thus obtained showed a reduction in polyglycosylated O-EPA relative to monoglycosylated O-EPA compared to a composition obtained using a previous production strain (WO 2020 / 191082) and a previous production method (Example 2). For O15, the proportion of polyglycosylated forms in the composition thus obtained was comparable to that obtained using a previous production strain (WO 2020 / 191082) and a previous production method (Example 2). However, other of these bioconjugate compositions had polyglycosylation levels not previously described and not achieved in bioconjugates of the same O-serotype coupled to the same carrier protein prepared according to prior art methods, i.e., in most cases, the novel bioconjugates of the present invention had increased polyglycosylation levels.

[0378] Example 5: Immunogenicity of the Nine-Valent Drug Product in Rabbits

[0379] Nine monovalent drug substances are mixed (according to step (vii) of the present invention, counting as follows from Example 1) to obtain a nine-valent drug product composition suitable for human use (see, for example, WO 2022 / 058945), wherein each of the nine monovalent drug substances corresponds to one of the O-EPA bioconjugates, wherein the O-EPA bioconjugate comprises an EPA carrier protein having SEQ ID NO: 1 coupled to an O-antigen polysaccharide of Escherichia coli serotype O1A, O2, O4, O6A, O15, O16, O18A, O25B or O75, and the O-EPA bioconjugate is obtained according to the method of the present invention described herein (Example 1; the identified production strains are listed in Table 2).

[0380] As described above, for several serotypes, the new method generally resulted in even lower levels of transaldolase B in the purified O-EPA bioconjugate composition compared to the levels of transaldolase B obtained using the comparative method (i.e., having a cHA step instead of an MMC step as the second chromatographic step; see Example 2). Overall, the remaining amount of transaldolase B impurity in the nine-valent drug product composition prepared using the new method according to the present invention was further reduced by about 2-3 times compared to a ten-valent drug product composition comprising the same dose of polysaccharide prepared using the comparative method. Such lower levels of this impurity are advantageous for drug products.

[0381] Administration of a nine-valent pharmaceutical product composition prepared using the novel method of the present invention to rabbits induced antibodies against the E. coli serotype O-antigens (O1A, O2, O4, O6A, O15, O16, O18A, O25B, and O75) present in the composition. Thus, it was demonstrated that the bioconjugates and compositions according to the present invention are suitable for inducing an immune response against E. coli.

Claims

1. A pharmaceutical composition comprising one or more bioconjugate compositions, wherein each bioconjugate composition comprises Pseudomonas aeruginosa exoprotein A carrier protein (EPA) having SEQ ID NO: 1 covalently linked to an O-antigen polysaccharide of a specific Escherichia coli (E. coli) serotype (O-EPA bioconjugate), wherein the O-EPA bioconjugate can be monoglycosylated with one O-antigen polysaccharide linked to EPA, or polyglycosylated with two, three, or four O-antigen polysaccharides linked to EPA, such that each of the one or more bioconjugate compositions is a mixture of monoglycosylated and polyglycosylated forms of the O-EPA bioconjugate, It is characterized by The pharmaceutical composition comprises one or more of the following: (a) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O1A, at least 50% of which is polyglycosylated; (b) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O2, at least 44% of which is polyglycosylated; (c) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O6A, at least 64% of which is polyglycosylated; (d) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O16, at least 65% of which is polyglycosylated; (e) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O18A, at least 70% of which is polyglycosylated; (f) a bioconjugate composition of an O-EPA bioconjugate of E. coli serotype O25B, at least 50% of which is polyglycosylated; and / or (g) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O75, at least 50% of which is polyglycosylated, The percentages of polyglycosylated and monoglycosylated O-EPA were each determined by capillary gel electrophoresis.

2. The pharmaceutical composition of claim 1, comprising at least one of the bioconjugate compositions (a), (b), and (f).

3. The pharmaceutical composition of claim 1 or 2, comprising all seven bioconjugate compositions (a)-(g).

4. The pharmaceutical composition according to any one of claims 1 to 3, It also includes: (h) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O15, and (i) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O4; or It also includes: (h) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O15, (i) a bioconjugate composition of an O-EPA bioconjugate of Escherichia coli serotype O4, and (j) Bioconjugate composition of O-EPA bioconjugate of E. coli serotype O8.

5. A method for producing a purified bioconjugate from a Gram-negative bacterial host cell, preferably an E. coli host cell, wherein the bioconjugate comprises a bacterial O-antigen polysaccharide covalently coupled to an exoprotein A carrier protein (O-EPA) from Pseudomonas aeruginosa, the method comprising: i. providing a filtered periplasmic fraction (FPF) of the host cell expressing the bioconjugate, the FPF comprising the bioconjugate; ii. subjecting the optionally loaded FPF to a first anion exchange chromatography (AEX 1) step to obtain a first AEX eluate (AEX1) comprising the bioconjugate; iii. subjecting the AEX1 eluate, optionally with adjusted loading, to a mixed mode chromatography (MMC) step on a multimodal resin (MMR) to obtain an MMR eluate comprising the bioconjugate, the MMR comprising an anion exchange function and a hydrophobic function; iv. subjecting the MMR eluate having an adjusted load to a hydrophobic interaction chromatography (HIC) step to obtain a HIC eluate comprising the bioconjugate; and v. subjecting the HIC eluate adjusted for loading to a second anion exchange chromatography (AEX 2) step to obtain an AEX2 eluate comprising the bioconjugate, wherein in purification steps (ii) to (v), conditions are first adjusted to allow the bioconjugate to bind to the chromatography medium, and subsequently adjusted to allow the bioconjugate to elute from the chromatography medium.

6. The method of claim 5, wherein the MMR comprises a ligand of formula (I), in RES represents the resin of MMR, preferably compressible resin; R 1 represents a C1-C4 alkyl group, preferably a methyl group; R 2 represents a C1-C4 alkyl group substituted by a phenyl, tolyl or xylyl group, preferably a benzyl group; R 3 represents a C1-C4 alkyl group substituted by a hydroxyl group or a mercapto group, preferably a 2-hydroxyethyl group.

7. The method of claim 6, wherein the ligand of formula (I) is N-benzyl-N-methylethanolamine.

8. The method according to any one of claims 5 to 7, wherein the O-polysaccharide is specific for a Gram-negative bacterium selected from the list of Escherichia and Shigella, preferably Escherichia coli.

9. The method of any one of claims 5 to 8, wherein the Gram-negative bacterial host cell from which the bioconjugate is obtained comprises genetic information encoding: (a) Bacterial O-antigen polysaccharide; (b) recombinant Pseudomonas aeruginosa exoprotein A protein (EPA), comprising at least one glycosylation site; and (c) a metabolic apparatus that carries out N-glycosylation of EPA with the O-antigen polysaccharide, The bioconjugate is thereby produced in vivo in the periplasm of the host cell.

10. The method of any one of claims 5 to 9, wherein the recombinant EPA comprises four N-glycosylation sites having the amino acid sequence Asn-X-Ser(Thr), preferably having the amino acid sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro.

11. The method of any one of claims 5-10, wherein the recombinant EPA comprises the amino acid sequence of SEQ ID NO:

1.

12. The method of any one of claims 5 to 11, wherein the bacterial O-antigen polysaccharide is an Escherichia coli O-antigen polysaccharide, preferably selected from the group consisting of Escherichia coli O-antigen polysaccharides O1A, O2, O4, O8, O6A, O15, O16, O18A, O25B and O75, more preferably selected from the group consisting of Escherichia coli O-antigen polysaccharides O1A, O2, O6A, O16, O18A, O25B and O75, and most preferably selected from the group consisting of Escherichia coli O-antigen polysaccharides O1A, O2 and O25B.

13. The method of any one of claims 5 to 12, wherein the host cells have been cultured in a bioreactor having a volume of 100 L to 20,000 L, such as 150 L to 5,000 L.

14. The method of any one of claims 5 to 13, wherein in an additional step (vi) the load of the AEX2 eluate is adjusted to a pharmaceutically acceptable buffer and concentration, thereby obtaining the purified O-EPA conjugate as a drug substance.

15. The method of claim 14, wherein in an additional step (vii), several purified bioconjugate drug substances are combined to obtain a multivalent drug product, wherein optionally in step (vii), the multivalent drug product comprises at least four, preferably at least eight O-antigen polysaccharides, And wherein the O-antigen polysaccharide is preferably selected from Escherichia coli O-antigen polysaccharide O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B and O75.

16. An O-EPA bioconjugate composition obtainable by or obtained by the method of any one of claims 5 to 15.

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