Pharmaceutical composition comprising clostridioides difficile vaccine

Abstract

The present invention relates to a vaccine formulation comprising a polysaccharide-protein conjugate of formula (I), an adjuvant comprising a pharmaceutically acceptable aluminum salt, a pharmaceutically acceptable buffer, and water; and its use in therapy, in particular for prevention and / or treatment of diseases associated with Clostridioides difficile.

Description

[0001] Pharmaceutical Composition comprising Clostridioides Difficile Vaccine

[0002] The present invention relates to novel vaccine formulation comprising a polysaccharide- protein conjugate of formula (I), an adjuvant comprising a pharmaceutically acceptable aluminum salt; a pharmaceutically acceptable buffer; and water; and to its use in prevention or therapy. In particular, the present invention relates to novel formulations for prevention and / or treatment of diseases associated with Clostridioides difficile. formula (I)

[0003] Clostridioides difficile (formerly Clostridium difficile) is a Gram-positive, spore-forming, toxinproducing, strictly anaerobic bacterium, and is a leading cause of antibiotic-associated diarrhea in developed countries [Bouza 2012, Kelly 2008], It is found widely in the mammalian gastrointestinal tract and can cause toxin-mediated C. difficile infection (CDI) with symptoms that range from mild diarrhea to pseudomembranous colitis, which can lead to death primarily in the elderly [Kelly 2008, Kuijper 2007, Oughton 2008], CDI recurrence is common and reaches an incidence of up to 35% in treated patients [Venugopal 2012], C. difficile is seen as a major public health threat in patients of all ages [Bouza 2012], Older age has been identified as a risk factor for both initial and recurrent CDI [Cioni 2016, Finn 2021],

[0004] The incidence of CDI cases in Europe was estimated by the European Centre for Disease Prevention and Control as a mean hospital incidence density of 3.19 per 10,000 patient days [ECDC 2018], In the United States, C. difficile caused over 500,000 infections per year in the year 2017, resulting in an estimated 28,000 deaths [Lessa 2015, Guh 2020], Of these infections, 30-50% were community-acquired, contrary to the common assumption that CDI is almost exclusively nosocomial [CDC 2020, Guh 2020, Roldan 2018], CDI places a significant economic burden on the healthcare system. The acute-care direct costs of CDI were estimated to be $5.4-6.3 billion per year in the United States in 2014- 2015 [Desai 2016, Zhang 2016] and €3.0 billion per year in Europe [Kuijper 2006], Hospital stays during which CDI was a secondary diagnosis lasted more than twice as long as those with CDI as a principal diagnosis (16.0 vs 6.9 days) and costs were more than three times higher ($31 ,500 vs $10,100) [Gerding 2015],

[0005] Vancomycin, metronidazole, and fidaxomicin are the standard treatments of CDI and are highly efficacious in clearing primary CDI. For later recurrences, fecal microbiota transplantation is also recommended [McDonald 2018], One of the reasons for high recurrence rates is that C. difficile spores may still be present in the patient’s gut and germinate to the vegetative form after completion or discontinuation of antibiotic treatment. Poor host immune response to C. difficile and frequent disruption of the normal gut microbiota due to antibiotic treatment may also contribute to the high recurrence rate [DuPont 2011],

[0006] C. difficile toxins A and B are significant mediators of intestinal damage and pathology [Hussack 2010], However, the contribution of non-toxin virulence factors to C. difficile colonization and disease is becoming increasingly appreciated [Jank 2008], Cell-wall glycopolymers, including capsular polysaccharides and teichoic acids, are known virulence factors in other pathogenic bacteria [Chu 2016], Several C. difficile surface polysaccharides have been structurally characterized, including the water-soluble PS-I and PS-II and phenol- soluble PS-Ill (a lipoteichoic acid) [Broecker 2016, Kirk 2017, Martin 2013], PS-I is expressed only at low levels on the cell surface, and it is unclear if it is present across diverse C. difficile strains. PS-II and PS-Ill, on the other hand, appear to be conserved surface polysaccharide antigens among all C. difficile strains tested [Kirk 2017], Moreover, published data show that synthetic PS-II glycan molecules are immunogenic after conjugation to a protein carrier and induce anti-PS-ll antibodies [Adamo 2012, Broecker 2019, Monteiro 2016, Oberli 2011].

[0007] Glycoconjugates of synthetic oligosaccharides are a promising alternative to vaccines derived from isolated polysaccharides [Kaplonek 2018],

[0008] The international patent application WO 2009 / 033268 A1 discloses the isolation of the PS- I and PS-II cell-surface saccharide of C. difficile from C. difficile bacteria of strains ribotype 027, MOH900 and MOH718. A synthetic approach to PS-II cell-surface saccharide of C. difficile was followed by Danieli et al. (Org. let. 2011 , 13, 378-381), Costantino et al. (WO 2012 / 085668 A2), Seeberger (WO 2012 / 119769 A1) and Oberli et al. (Chemistry & Biology 2011 , 18, 580). Monteiro (Meth Mol. Biol. 2016, 397-408) reports on the isolation of water- soluble PS-I and PS-II as well as water- and phenol-soluble PS-Ill polysaccharide from C. difficile biomass by hot water - phenol treatment. The preparation and medical use of formula (I) is described in W02020 / 104697 A1.

[0009] WO 2012 / 085668 A2 and Adamo et al. (ACS Chem. Biol. 2012, 7, 1420-1428) disclose formulations comprising C. difficile PS-II saccharide with MF59, an oil-in-water emulsion as preferred adjuvant. Further, Broecker et al. (ACS Chem. Biol. 2019, 14, 2720-2728) discloses a vaccine formulation to protect mice from C. difficile infections with the use of AddaVax as adjuvant. AddaVax is a water-in-oil emulsion adjuvant similar to MF59. The same paper shows that AddaVax was also used because Alum did not support induction of detectable anti-compound IgG.

[0010] It has been now surprisingly found that formulation with an aluminum salt as adjuvant provides increased survival compared to a formulation with AddaVax as adjuvant in the mouse challenge / infection model.

[0011] Description of Figure 1 :

[0012] Figure 1 shows a survival plot of mice infected with a lethal dose of C. difficile spores at day 0. Before infection, the mice received three injections with a PS-I l-CRM 197 glycoconjugate either in the presence of Alum or AddaVax adjuvant, or adjuvant in buffer (Placebo), at days -35, -21 and -7.

[0013] Description of the invention:

[0014] Surprisingly, it has been found that that formulation with an aluminum salt as adjuvant provides increased survival compared to a formulation with AddaVax as adjuvant in the mouse challenge / infection model.

[0015] 1) A first embodiment relates to a pharmaceutical composition comprising:

[0016] • a polysaccharide-protein conjugate of formula (I) formula (I) wherein n represents an integer selected from 0, 1 , 2, and 3; i represents an integer between 5 and imax, wherein imax is an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP (preferably i represents an integer between 5 and 25);

[0017] Z represents *-CH2-O-P(O)(OH)-, or *-O-P(O)(OH)-, wherein the star represents the attachment point to the anomeric carbon atom (C-1 carbon atom) of the attached mannose;

[0018] L-T represents a linker L and a spacer T which together form a bridge between the oxygen atom of the phosphate group and a nitrogen atom of a lysine residue of the carrier protein CP; wherein the bridge consists of a chain of 2 to 50 groups independently selected from -CH2-, -NRN-, -O-, -S-, -CRC1RC2-, -CH=CH-, -C C-, (C3-C8)cycloalkylene, and (C3-C8)heterocycloalkylene, wherein the (C3- 8)carbocyclene and the (C3-8)heterocyclene are unsubstituted or independently substituted with 1 to 4 substituents independently selected from the group consisting of (Ci-4)alkyl, (Ci-4)alkoxy, (C3-8)cycloalkyl, (Ci-4)fluoroalkyl, halogen and oxo; and wherein at least 1 group is different from -CH2-;

[0019] RNrepresents hydrogen or (Ci-4)alkyl (preferably hydrogen);

[0020] RC1and RC2represent independently of each other (Ci-4)alkyl, (Ci-4)alkoxy, (C3- 8)cycloalkyl, (Ci-4)fluoroalkyl, hydroxy, or halogen; or RC1and RC2together represent oxo;

[0021] CP represents a carrier protein selected from the group consisting of CRM 197; diphtheria toxoid; tetanus toxoid; cholera toxin B subunit; Neisseria meningitidis outer membrane protein (OMP); capsid protein of bacteriophage Q|3; oligomers or virus-like particles prepared with capsid protein of bacteriophage Q|3; detoxified Exotoxin A of Pseudomonas aeruginosa (EPA); maltose-binding protein (MBP); the He fragment of tetanus toxin (TetHc); detoxified hemolysin A of Staphylococcus aureus, clumping factor A (ClfA) and clumping factor B (ClfB) of Staphylococcus aureus: Escherichia coll FimH; Escherichia coll FimHC; detoxified variants of Escherichia coll heat labile enterotoxin; detoxified variants of cholera toxin; Escherichia coll Sat protein; the passenger domain of Escherichia coll Sat protein; detoxified variants of Streptococcus pneumoniae pneumolysin;

[0022] Campylobacter jejuni AcrA; Pseudomonas PcrV protein; Campylobacter jejuni natural glycoproteins; bovine serum albumin (BSA); pilus protein GBS80 from Group B Streptococcus’ Escherichia coll heat-labile enterotoxin; tetanus toxin; cholera toxin; and Streptococcus pneumoniae pneumolysin; or a pharmaceutically acceptable salt thereof;

[0023] • an adjuvant comprising a pharmaceutically acceptable aluminum salt;

[0024] • a pharmaceutically acceptable buffer; and

[0025] • water.

[0026] Preferably, i represents an integer between 5 and imax, wherein imaxis an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP; more preferably, i represents an integer between 5 and 75% of the number of lysine residues contained in the carrier protein CP; even more preferably, i represents an integer between 5 and 40% of the number of lysine residues contained in the carrier protein CP. As an example, in case the carrier protein CP contains 39 lysine residues, the range “between 5 and 40% of the number of lysine residues contained in the carrier protein CP” means that i ranges from 5 to 16.

[0027] Preferably, n represents an integer selected from 0, 1 , 2, and 3, more preferably n is 0 or 1. In case n is 0 (zero), the corresponding part of the polysaccharide-protein conjugate of formula (I) is absent as shown on the figure below:

[0028]

[0029] The term "cycloalkyl", used alone or in combination, refers especially to a saturated monocyclic hydrocarbon ring containing three to seven carbon atoms. The term "Cx.y- cycloalkyl" (x and y each being an integer), refers to a cycloalkyl group as defined before containing x to y carbon atoms. For example, a (C3-C8)cycloalkyl group contains from three to eight carbon atoms. Representative examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term “-Cx.y-cycloalkylene-”, used alone or in combination, refers to bivalently bound cycloalkyl group as defined before containing x to y carbon atoms.

[0030] The term "heterocycloalkyl”, used alone or in combination, and if not explicitly defined in a broader or more narrow way, refers to a saturated or unsaturated non-aromatic monocyclic hydrocarbon ring containing one or two ring heteroatoms independently selected from nitrogen, sulfur, and oxygen (especially one oxygen atom, one sulfur atom, one nitrogen atom, two nitrogen atoms, two oxygen atoms, or one nitrogen atom and one oxygen atom). The term “x- to y-membered heterocycloalkyl” refers to such a heterocycle containing a total of x to y ring atoms. The term “heterocycloalkylene” used alone or in combination, refers to bivalently bound heterocycloalkyl group as defined before.

[0031] The term “alkyl”, used alone or in combination, refers to a saturated straight or branched chain hydrocarbon group containing one to six carbon atoms. The term “Cx.y-alkyl” (x and y each being an integer), refers to an alkyl group as defined before, containing x to y carbon atoms. For example, a (Ci-4)alkyl group contains from one to four carbon atoms. Examples of “(Ci-4)alkyl” groups are methyl, ethyl, n-propyl, / so-propyl, n-butyl, / so-butyl, sec.-butyl and te / Y.-butyl. The term “alkoxy”, used alone or in combination, refers to an alkyl-O- group wherein the alkyl group is as defined before. The term “Cx.y-alkoxy” (x and y each being an integer) refers to an alkoxy group as defined before containing x to y carbon atoms. Examples of “(Ci-4)alkoxy” groups are methoxy, ethoxy, n-propoxy, and n-butoxy.

[0032] The term "cycloalkyl", used alone or in combination, refers especially to a saturated monocyclic hydrocarbon ring containing three to seven carbon atoms. The term "Cx.y- cycloalkyl" (x and y each being an integer), refers to a cycloalkyl group as defined before containing x to y carbon atoms. For example, a (C3-8)cycloalkyl group contains from three to eight carbon atoms. Representative examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0033] The term "fluoroalkyl”, used alone or in combination, refers to an alkyl group as defined before containing one to four carbon atoms in which one or more (and possibly all) hydrogen atoms have been replaced with fluorine. The term “Cx.y-fluoroalkyl” (x and y each being an integer) refers to a fluoroalkyl group as defined before containing x to y carbon atoms. For example, a Ci.4-fluoroalkyl group contains from one to four carbon atoms in which one to nine hydrogen atoms have been replaced with fluorine. Examples of “(Ci-4)fluoroalkyl” groups are fluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2-difluoroethyl, and 2,2,2- trifluoroethyl.

[0034] The term “halogen” means fluorine, chlorine, or bromine; preferably fluorine.

[0035] The term “oxo” relates to the functional group =0, i.e. a substituent oxygen atom connected to another atom (preferably carbon atom) by a double bond.

[0036] “CRM197” refers to Cross Reactive Material 197, which is a nontoxic mutant version of the diphtheria toxin, wherein the single amino acid exchange of a glycine (Gly, G) in position 52 to a glutamic acid (Glu, E) renders the protein non-toxic.

[0037] CRM-197 is produced by C. diphtheriae infected by the nontoxigenic phage pi97tox created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida et al, J. Biol. Chem., 1973, Vol. 245, No. 11 , pp. 3838-3844). The CRM197 protein is a safe and effective T-cell dependent carrier for saccharides. CRM197 is for instance described by Giannini et al. in Nucleic Acids Research, Vol 12, No. 10, 1984, pp. 4063-4069. Further details about CRM197 and production thereof can be found e.g. in US5,614,382, which are incorporated herein by reference. CRM197 may be produced in various expression systems, for instance in Corynebacterium diphtheria , Escherichia coli or Pseudomonas fluorescens (Hickey et al, J. Pharm. Sci., 2018, 107, 1806-1819).

[0038] In the present invention, the term “CRM197” encompasses a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9% identity to amino acid sequence SEQ ID NO: 1 (preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 1 ; and notably at least 95%, 96%, 97%, 98%, 99% or 99.9% identity to amino acid sequence SEQ ID NO: 1), which optionally comprises an additional methionine (Met, M) at the N-terminus, and / or optionally includes residues resulting from functionalizing CRM 197 at lysine sites, which residues may be in a capped (i.e. deactivated) form.

[0039] The phrase “residues resulting from functionalizing CRM197 at lysine sites” means that CRM197 is functionalized at lysine sites with functional groups suitable for forming a covalent bond to the linker and / or spacer part attached to the antigen, i.e. the oligosaccharide-linker part of the conjugate. Such lysine-functionalized CRM197 is known to the skilled person. The functional groups are particularly suitable for linking thiols or for performing clickchemistry. For instance, such functional groups are groups containing a bromo-acetamide, a iodo-acetamide, a maleimide, an azido, or an alkyne group. This means that CRM 197 optionally includes lysine residues functionalized with a bromo-acetamide, a iodoacetamide, a maleimide, an azido, or an alkyne group (preferably a bromo-acetamide, a iodo-acetamide, a maleimide group), which groups may be in a capped form.

[0040] Preferred functionalized CRM 197 contains groups carrying bromo-acetamide, iodoacetamide or maleimide groups, all of them being suitable for reaction with thiol-groups provided by the oligosaccharide / linker moiety. Unreacted functional groups at CRM 197 may subsequently be quenched with any pharmaceutically acceptable thiol, such as for instance L-cysteine or cysteamine (2-aminoethane-1 -thiol) to give the “capped form”.

[0041] Preferred lysine-functionalized CRM197 is selected from the group consisting of: wherein Z is Br or I, q is 2 or 3, and t is from 1 to 28; wherein Z is Br or I, and t is from 1 to 28.

[0042] In a preferred embodiment, CRM197 is not functionalized in the above-described way. This means that there is no “pre-functionalization”, but rather, the “natural” lysine residues, i.e. the non-modified amino groups of the lysine residues, are used for directly attaching the oligosaccharide / linker / spacer part thereto.

[0043] The amino acid sequence of CRM197 is known to the skilled person and is outlined below as SEQ ID NO:1.

[0044] The use of CRM197 for the synthesis of saccharide conjugates and preferred conjugation sites on CRM197 has been reported (e.g. Mbginger et al., Sci. Rep. 6, 20488; doi:10.1038 / srep20488 (2016)), which is incorporated herein by reference.

[0045] For avoidance of any doubt, throughout the present invention, the term “lysine residue” and “lysine site” are used synonymously; the term “immunogenic compound” is used synonymously to “oligosaccharide-carrier protein conjugate”.

[0046] It is further to be understood that the pH value of the aqueous pharmaceutical composition may be adjusted to the target value with a pharmaceutically acceptable base. The pharmaceutically acceptable base used in the preparation of the aqueous pharmaceutical composition may be a pharmaceutically acceptable strong base, a pharmaceutically acceptable weak base different from the basic component of the pharmaceutically acceptable buffer, or the basic component of the pharmaceutically acceptable buffer that is used in the aqueous pharmaceutical composition. It is understood that the basic component of the pharmaceutically acceptable buffer may be in essentially pure form or in a mixture with the acidic component of the pharmaceutically acceptable buffer, wherein the ratio between the basic and the acidic component is sufficiently high to adjust the pH value of the aqueous pharmaceutical composition to the target value. In the context of adjusting the pH value of the aqueous pharmaceutical composition, the term "pharmaceutically acceptable base" refers to a Bransted base whose reaction products from the adjustment of the pH value (e.g. a sodium salt and water if sodium hydroxide is used as base) exhibit minimal undesired toxicological effects. The term "pharmaceutically acceptable strong base" refers to a "pharmaceutically acceptable base" that is completely or almost completely ionized or dissociated if dissolved in water. Examples of pharmaceutically acceptable strong bases are hydroxide salts of alkali or alkaline earth metals; especially sodium hydroxide or potassium hydroxide; and notably sodium hydroxide. In a preferred embodiment, the pharmaceutically acceptable base is a pharmaceutically acceptable strong base, or the basic component of the pharmaceutically acceptable buffer that is used in the aqueous pharmaceutical composition. In a most preferred embodiment, the pharmaceutically acceptable base is a pharmaceutically acceptable strong base (especially sodium hydroxide).

[0047] To adjust the pH value of the aqueous pharmaceutical composition, the pharmaceutically acceptable base may be used in essentially pure form without a solvent, or as a solution in a pharmaceutically acceptable solvent or solvent mixture (especially a solution in water). In a most preferred embodiment, a solution of sodium hydroxide in water is used as the pharmaceutically acceptable base.

[0048] Only in the case that the pH value of the aqueous pharmaceutical composition before pH adjustment is higher than the targeted pH value, a pharmaceutically acceptable acid, especially a pharmaceutically acceptable strong acid, such as aqueous hydrochloride acid, may be used for pH adjustment. In the context of adjusting the pH value of the aqueous pharmaceutical composition, the term "pharmaceutically acceptable acid" refers to a Bransted acid whose reaction products from the adjustment of the pH value (e.g. a sodium salt and water if hydrochloric acid is used as an acid) exhibit minimal undesired toxicological effects. The term "pharmaceutically acceptable strong acid" refers to a "pharmaceutically acceptable acid" that is completely or almost completely ionized or dissociated if dissolved in water. Examples of pharmaceutically acceptable strong acids are hydrogen salts of non- metal elements; Sulfuric acid, Hydrochloric acid, Phosphoric acid, Hydrofluoric acid, Phosphoric acid, Citric acid; and notably hydrochloric acid.

[0049] It is known in the art that the pH value of an aqueous pharmaceutical composition may change over time, especially if stored for one or several years. Thus, if an aqueous pharmaceutical composition is described and / or claimed to have a specific pH value, this means that the pH value of the pharmaceutical composition has the specific pH value at least immediately (for instance 1 hour or 2 hours) after final preparation of the pharmaceutical composition (i.e. after all ingredients are added to the pharmaceutical composition). Preferably the pH value remains at the specific pH value or within the specified range for at least 6 months (preferably at least 12 months and most preferably at least 18 months) after final preparation of the pharmaceutical composition.

[0050] The term “pharmaceutically acceptable buffer” refers to a mixture of a weak Bransted base (the basic component of the pharmaceutically acceptable buffer) and its conjugated acid (the acidic component of the pharmaceutically acceptable buffer), wherein both, the weak base and the conjugated acid exhibit minimal undesired toxicological effects. The weak base may be an inorganic or organic base and may be in the form of a neutral molecule or in form of a salt comprising a protonatable anion. For avoidance of doubt, in the present application boric acid is understood to be a weak Bransted acid and a boric acid buffer is thus comprised in the definition of a pharmaceutically acceptable buffer. The pharmaceutically acceptable buffer is selected from the group of pharmaceutically acceptable buffer with a pKa value in water at 25°C in the range of the targeted pH value ±1.5 (pH-1.5 < pKa < pH+1.5); preferred are pharmaceutically acceptable buffer with a pKa value in water at 25°C in the range of the targeted pH value ±1 (pH-1 < pKa < pH+1). For instance, if the target value for the aqueous pharmaceutical composition is pH = 9.0, the pharmaceutically acceptable buffer is selected from pharmaceutically acceptable buffer with a pKa value in water at 25°C in the range between 7.5 and 10.5 (and preferably between 8.0 and 10.0). Examples of pharmaceutically acceptable buffer are ammonium buffer, boric acid buffer, carbonate buffer, phosphate buffer, arginine buffer, glycine buffer, histidine buffer, meglumine buffer (also known as N-methyl-D-glucamine buffer), 2-aminoethanol buffer (also known as monoethanolamine buffer), bis(2-hydroxyethyl)amine buffer (also known as diethanolamine buffer), tris(hydroxymethyl)aminomethane buffer (also known as tromethamine or Tris buffer), and tris(hydroxymethyl)aminomethane-hydrochloride (also known as Tris-HCI, Tris-hydrochloride, and tromethan-hydrochloride). Most preferred is Tris-HCI buffer.

[0051] The pharmaceutically acceptable buffer may be obtained from: ammonium chloride, ammonium bromide or ammonia (e.g. as solution in water) for ammonium buffer; boric acid or sodium tetraborate for boric acid buffer; sodium hydrogencarbonate or sodium carbonate for carbonate buffer; sodium dihydrogenphosphate or disodium hydrogenphosphate for phosphate buffer; arginine or arginine hydrochloride for arginine buffer; glycine or glycine hydrochloride for glycine buffer; histidine or histidine hydrochloride for histidine buffer; N- methyl-D-glucamine or N-methyl-D-glucamine hydrochloride for meglumine buffer; 2- aminoethanol or 2-aminoethanol hydrochloride for 2-aminoethanol buffer; sodium citrate or citric acid for citrate buffer; sodium succinate for succinate buffer; bis(2-hydroxyethyl)amine or bis(2-hydroxyethyl)amine hydrochloride for bis(2-hydroxyethyl)amine buffer; tris(hydroxymethyl)aminomethane or tris(hydroxymethyl) aminomethane hydrochloride for tris(hydroxymethyl)aminomethane buffer; 3-( / V-morpholino)propanesulfonic acid for 3-( / V- morpholino) propanesulfonic acid buffer; or from any other suitable starting material.

[0052] It is understood that any reference to one of the amino acids arginine or histidine refers to the respective amino acid in its D-form, its L-form or any mixture (including a racemic mixture) thereof; and especially to the respective amino acid in its enantiomerically essentially pure L-form.

[0053] The term adjuvant refers to a substance that enhances immune response through physical or chemical associations with antigens. In particular, adjuvants assist in boosting specific immune responses against antigens contained in the vaccine.

[0054] The term “pharmaceutically acceptable aluminum salt” when used for adjuvants refers to aluminum salts such as hydroxides, phosphates, sulphates or mixtures thereof. Most preferred adjuvant comprising a pharmaceutically acceptable aluminum salt is Alum. The term Alum as used in the context of the present invention, refers to aluminum hydroxide (AI(OH)3).

[0055] The term "pharmaceutically acceptable salt" refers to salts that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological effects. Such salts include inorganic or organic acid and / or base addition salts depending on the presence of basic and / or acidic groups in the subject compound. For reference see for example ‘Handbook of Pharmaceutical Salts. Properties, Selection and Use.’, P. Heinrich Stahl, Camille G. Wermuth (Eds.), Wiley-VCH, 2008 and ‘Pharmaceutical Salts and Co-crystals’, Johan Wouters and Luc Quere (Eds.), RSC Publishing, 2012.

[0056] The term “target value” in relation to the pH value of the aqueous pharmaceutical composition (or alternatively “targeted pH value”) means the pH value to which the aqueous pharmaceutical composition should be adjusted. Preferably, the pH value of the aqueous pharmaceutical composition immediately after its final preparation is equal to the target value.

[0057] The term “essentially”, for example when used in a term such as "essentially pure" is understood in the context of the present invention to mean especially that the respective composition I component I compound I stereoisomer etc. consists in an amount of at least 90, especially of at least 95, and notably of at least 99 per cent by weight of the respective pure composition I component I compound I stereoisomer etc. Especially, the term “consisting essentially of’ is understood in the context of the present invention to mean that the respective composition consists in an amount of at least 90, especially of at least 95, notably of at least 99, and in particular in an amount of 100 per cent by weight (i.e. in the meaning of "consisting of') of the respective composition in the amounts as explicitly stated in the respective embodiment.

[0058] The total weight percent of the aqueous pharmaceutical composition as defined in embodiment 1), and embodiments 2) to 68) below is 100.

[0059] For avoidance of any doubt, it is well understood that pharmaceutical compositions as defined in embodiment 1) and 2) to 68) may additionally comprise further conventional ingredients and / or additives, which may be used alone or in combination.

[0060] Reference is made to the extensive literature on the subject for these and other pharmaceutically acceptable excipients and procedures mentioned herein, see for example R.C. Rowe, P.J. Seskey, S.C. Owen, Handbook of Pharmaceutical Excipients, 5th edition, Pharmaceutical Press 2006; Remington, The Science and Practice of Pharmacy, 21st Edition (2005), Part 5, “Pharmaceutical Manufacturing” [published by Lippincott Williams & Wilkins]; and Nema, Brendel, Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions, PDA J Pharm Sci and Tech (2011), 65, 287-332.

[0061] Examples of such conventional ingredients or additives are tonicity modifiers such as salts (e.g. NaCI, KCI, MgCI2, CaCI2). Preferred tonicity modifier is NaCI. Further conventional ingredients or additives are for example antimicrobial preservatives such as used e.g. in bacteriostatic water for injection. Examples of pharmaceutically acceptable antimicrobial preservatives includes Phenol, Benzalkonium chloride, Phenoxyethanol, Propylparaben, m- Cresol, Benzyl alcohol; and most notably benzyl alcohol.

[0062] It is understood that the aqueous pharmaceutical composition may in addition to water further comprise up to 20 ww% (especially up to 10 ww% and notably up to 5 ww%) of a pharmaceutically acceptable solvent or a mixture of pharmaceutically acceptable solvents. The term "pharmaceutically acceptable solvent" refers to a solvent that exhibits minimal undesired toxicological effects. Representative examples of pharmaceutically acceptable solvents are polyethylene glycol, polysorbate, DMSO and NMP. In a preferred embodiment, the aqueous pharmaceutical composition does not contain a pharmaceutically acceptable solvent in addition to water (other than trace amounts of residual solvent present in an ingredient used in the preparation of the aqueous pharmaceutical composition).

[0063] The term “pharmaceutical composition” is interchangeable with the term “formulation”.

[0064] Unless used regarding temperatures, the term “about” placed before a numerical value “X” refers in the current application to an interval extending from X minus 10% of X to X plus 10% of X, and preferably to an interval extending from X minus 5% of X to X plus 5% of X (wherein it is well understood that the lower limit is equal or above 0% and the higher limit is equal or below 100%). In the particular case of concentrations, the term “about” placed before a concentration “Y” refers in the current application to an interval extending from the concentration Y minus 10 % to Y plus 10 %; and preferably to an interval extending from Y minus 5 % to Y plus 5 %.

[0065] Whenever the word “between” or "to" is used to describe a numerical range, it is to be understood that the end points of the indicated range are explicitly disclosed and included in the range. For example: if a pH value is described to be between 8.2 and 12.0 (or 8.2 to 12.0), this means that the end points 8.2 and 12.0 are included in the range; or if a variable is defined as being an integer between 1 and 4 (or 1 to 4), this means that the variable is the integer 1 , 2, 3, or 4.

[0066] The expression “ww%” (or % (w / w)) refers to a percentage by weight compared to the total weight of the composition considered. If not explicitly stated otherwise, the considered total weight is the total weight of the pharmaceutical composition. In case a certain value is given as % value, in absence of further specification such value refers to ww%. The expression (wt / wt) relating to a ratio refers to a ratio by weight of the respective components.

[0067] The expression “w / v%” (or % (w / v)) refers to a percentage by weight compared to the total volume of the composition considered. If not explicitly stated otherwise, the considered total volume is the total volume of the pharmaceutical composition.

[0068] Further embodiments of the invention are presented hereinafter:

[0069] 2) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 1), wherein n represents 0.

[0070] 3) In another embodiment, the invention relates to a pharmaceutical composition according to embodiments 1) or 2), wherein i represents an integer between 5 and 25, 5 and 20, 5 and 18, 6 and 23, 6 and 20, 6 and 18, or 6 and 15.

[0071] The variable i describes the loading of antigens, i.e. polysaccharides on the carrier protein (preferably CRM197) and is an integer in respect of one single molecule. However, when considering the polysaccharide-protein conjugate as a product of more than one single molecule, it has to be noted that the loading can be described as a statistical distribution, i.e. essentially a Gaussian distribution. The chemical process of producing the product results in a mixture of molecules with such statistical distribution of the loading, and the loading is then provided as the mean of the statistical distribution, in particular the Gaussian distribution.

[0072] It is to be understood that for i > 5, five or more polysaccharides are attached via -L-T- to the carrier protein CP (preferably CRM197). The linker-spacer unit -L-T- is identical for the i polysaccharides of a specific polysaccharide-carrier protein conjugate. 4) In another embodiment, the invention relates to a pharmaceutical composition according to embodiments 1) or 2), wherein i represents an integer between 5 and 25.

[0073] 5) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 4), wherein Z represents *-CH2-O-P(O)(OH)-, wherein the star represents the attachment point to the anomeric carbon atom (C-1 carbon atom) of the attached mannose.

[0074] 6) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 5), wherein CP represents a carrier protein selected from the group consisting of CRM197; diphtheria toxoid; tetanus toxoid; and bovine serum albumin (BSA).

[0075] 7) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 5), wherein CP is CRM197.

[0076] 8) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 7), wherein

[0077] L-T represents a linker L and a spacer T which together form a bridge between the oxygen atom of the phosphate group and a nitrogen atom of a lysine residue of the carrier protein CP; wherein the bridge consists of a chain of 2 to 25 groups independently selected from - CH2-, -NRN-, -O-, -S-, -CRC1RC2-, -CH=CH-, -C C-, (C3-C8)cycloalkylene, and (C3- C8)heterocycloalkylene, wherein the (C3-8)carbocyclene and the (C3-8)heterocyclene are unsubstituted or independently substituted with 1 to 4 substituents independently selected from the group consisting of (Ci-4)alkyl, (Ci-4)alkoxy, (C3-8)cycloalkyl, (Ci-4)fluoroalkyl, halogen and oxo; and wherein at least 1 group is different from -CH2-;

[0078] RNrepresents hydrogen or (Ci-4)alkyl (preferably hydrogen);

[0079] RC1and RC2represent independently of each other (Ci-4)alkyl, (Ci-4)alkoxy, (C3-8)cycloalkyl, (Ci-4)fluoroalkyl, hydroxy, or halogen; or RC1and RC2together represent oxo.

[0080] 9) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 7), wherein

[0081] L represents:

[0082] *-(C2-io)alkylene-NH-;

[0083] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1 , 2 or 3;

[0084] -CH2CH2S-CH2CH2NH-; -(C2-io)fluoroalkylene-NH-;

[0085] *-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;

[0086] *-(CH2)dNHC(O)NH(CH2)d-NH-, wherein d and d’ are independently from each other from 2 to 6;

[0087] *-(Ci-io)alkylene-C(0)-NH-(C2.io)alkylene-NH-; or

[0088] *-(C2.io)alkylene-0-NH-;

[0089] T represents

[0090] -C(0)-(CO-10)alkylene-C(O)-;

[0091] -C(O)-CH2CH2-(OCH2CH2)j-C(O)-, wherein j is from 1 to 5;

[0092] -C(O)-CH2(CH2)k-(SCH2(CH2)k)k -C(O)-, wherein k is 0 or 1 , k’ is 0 or 1 , and k” is 1 , 2, or 3; , wherein p is from 1 to 4, and p’ is 1 or 2; or

[0093] L-T represents

[0094] *-(C2.io)alkylene-S-R1; and

[0095] R1represents

[0096] , wherein q is 2 or 3;

[0097] The “*” appointed in the linker L means that at this location, the linker is attached to the oligosaccharide.

[0098] The “*” appointed in the spacer T means that at this location, the spacer is attached to the linker L.

[0099] The appointed in R1means that at this location, R1is attached to the sulphur.

[0100] 10) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 9), wherein

[0101] L represents

[0102] *-(CH2)a-NH-; wherein a is from 2 to 10;

[0103] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1 , 2 or 3;

[0104] *-CH2CH2S-CH2CH2NH-;

[0105] *-(C2-io)fluoroalkylene-NH- with fluoroalkylene being a saturated straight chain;

[0106] *-(CH2)CNHC(O)(CH2)C-NH-, wherein c and c’ are independently from each other from 2 to 6;

[0107] *-(CH2)dNHC(O)NH(CH2)d-NH-, wherein d and d’ are independently from each other from 2 to 6;

[0108] *-(CH2)e-C(O)-NH-(CH2)e’-NH-; wherein e is from 1 to 10 and e’ is from 2 to 10; or

[0109] *-(CH2)f-O-NH-, wherein f is from 2 to 10; or

[0110] L-T represents

[0111] *-(CH2)g-S-R1, wherein g is from 2 to 10.

[0112] 11) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 9), wherein

[0113] L represents

[0114] *-(CH2)a-NH-; wherein a is from 2 to 10;

[0115] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1 , 2 or 3; *-(C2-io)fluoroalkylene-NH- with fluoroalkylene being a saturated straight chain;

[0116] *-(CH2)e-C(O)-NH-(CH2)e’-NH-; wherein e is from 1 to 10 and e’ is from 2 to 10; or

[0117] *-(CH2)f-O-NH-, wherein f is from 2 to 10; or

[0118] L-T represents

[0119] *-(CH2)g-S-R1, wherein g is from 2 to 10.

[0120] 12) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 9), wherein

[0121] L represents

[0122] *-(CH2)a-NH-; wherein a is from 2 to 10, preferably from 2 to 6; *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1 , 2 or 3, preferably 1 or 2;

[0123] *-(CH2)e-C(O)-NH-(CH2)e’-NH-; wherein e is from 1 to 10, preferably from 1 to 6 and e’ is from 2 to 10, preferably from 2 to 6; or

[0124] *-(CH2)f-O-NH-, wherein f is from 2 to 10, preferably from 2 to 6; or

[0125] L-T represents

[0126] *-(CH2)g-S-R1, wherein g is from 2 to 10, preferably from 2 to 6.

[0127] 13) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 9), wherein

[0128] L represents

[0129] *-(CH2)a-NH-; wherein a is from 2 to 10, preferably from 2 to 6;

[0130] *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1 , 2 or 3, preferably 1 or 2; or *-(CH2)f-O-NH-, wherein f is from 2 to 10, preferably from 2 to 6.

[0131] 14) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 9), wherein

[0132] L represents

[0133] *-(CH2)a-NH-; wherein a is from 2 to 10, preferably from 2 to 6; or *-(CH2CH2O)b-CH2CH2NH-, wherein b is 1 , 2 or 3, preferably 1 or 2.

[0134] 15) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 9), wherein L represents *-(CH2)a-NH-; wherein a is from 2 to 10, preferably from 2 to 6. 16) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 9), wherein L represents *-(CH2)2-NH-, *-(CH2)3-NH-, *-(CH2)4- NH-, *-(CH2)5-NH-, or *-(CH2)6-NH-.

[0135] 17) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 9), wherein L represents *-(CH2)s-NH-.

[0136] 18) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 9) to 17), wherein

[0137] T represents

[0138] -C(O)-(CH2)h-C(O)-, wherein h is from 0 to 10, preferably from 0 to 6;

[0139] -C(O)-CH2CH2-(OCH2CH2)j-C(O)- , wherein j is from 1 to 5, preferably from 1 to 3, more preferably 1 ;

[0140] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)-, wherein k is 0 or 1 , k’ is 0 or 1 , and k” is 1 , 2, or 3, preferably 1 ; or

[0141] 19) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 8) to 17), wherein

[0142] T represents

[0143] -C(O)-(CH2)h-C(O)-, wherein h is from 0 to 6;

[0144] -C(O)-CH2CH2-(OCH2CH2)j-C(O)- , wherein j is 1 or 2; or

[0145] -C(O)-CH2(CH2)k-(SCH2(CH2)k’)k”-C(O)-, wherein k is 0 or 1 , k’ is 0 or 1 , and k” is 1 , preferably wherein k and k’ are 0 and k” is 1 .

[0146] 20) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 9) to 17), wherein

[0147] T represents -C(O)-(CH2)h-C(O)-, wherein h is 0, 1 , 2, 3, 4, 5, or 6, preferably 4.

[0148] 21) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 8), wherein L represents *-(CH2)s-NH- and T represents -C(O)-(CH2)4-C(O)-. 22) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 1), wherein the polysaccharide-protein conjugate has the formula: formula (la) wherein i represents an integer between 5 and 25.

[0149] For avoidance of any doubt, the polysaccharide-protein conjugate of formula (la) according to this embodiment can also be schematically drawn as follows: formula (la’) wherein i represents an integer between 5 and 25.

[0150] CRM197’ means CRM197 as defined herein, with the only difference in that in formula (la’), the amino-group of the lysine residue is specifically shown as the attachment position of the linker / spacer part -L-T-.

[0151] 23) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 22), wherein the adjuvant comprises aluminum hydroxide. 24) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 23), wherein the concentration of the adjuvant is 188 - 313 pg / mL.

[0152] 25) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 23), wherein the concentration of the adjuvant is 250 pg / mL.

[0153] 26) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 25), wherein the pharmaceutically acceptable buffer has a pH value between 5.5 and 6.4, between 6.8 and 8.0, or between 5.5 and 7.4.

[0154] 27) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 25), wherein the pharmaceutically acceptable buffer has a pH value between 6.8 and 8.0.

[0155] 28) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 25), wherein the pharmaceutically acceptable buffer has a pH value of 6.8, 7.4, or 8.0.

[0156] 29) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 25), wherein the pharmaceutically acceptable buffer has a pH value of 7.4.

[0157] 30) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 25), wherein the pharmaceutically acceptable buffer is selected from sodium succinate, Tris-HCI, histidine, and citrate.

[0158] 31) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 25), wherein the pharmaceutically acceptable buffer is Tris-HCI.

[0159] 32) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 31), wherein the concentration of the pharmaceutically acceptable buffer is about 10 mM.

[0160] 33) In a preferred embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 25), wherein the pharmaceutically acceptable buffer is Tris-HCI at a concentration of 10 mM and at pH of 7.4.

[0161] 34) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 33), wherein said pharmaceutical composition comprises a surfactant.

[0162] The term surfactant refers to an organic compound that is amphiphilic, meaning it contains both hydrophobic groups and hydrophilic groups. Surfactants may be cationic, anionic, or non-ionic surfactants. Examples of cationic surfactants suitable for compositions of the invention include quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Anionic surfactants suitable for the composition of the present invention may be selected from the group consisting of alkoxylated tristyryl phenol phosphates, oleoyltaurate salts, polyacrylates, alkylarylsulfonic acid salts, dialkylsulfo Succinate salts, ether Sulfates and phosphate esters.

[0163] In a preferred embodiment of the invention, one or more non-ionic surfactants are selected from the group consisting of alkoxylated triglycerides, alkoxylated fatty alcohols, alkoxylated tristyryl phenols, ethoxylated fatty acids, alkyl polyglycosides, fatty acid PEG esters, alkoxylated sorbitan esters, polyoxyethylene polyoxypropylene block polymers, poly oxyethylene polyoxypropylenealkylaryl ethers, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene polyoxypropylene polyaryl ethers, polyoxyethylene fatty acid esters, and fatty acid esters of polyoxyethylene polyoxypropylene block polymers. Preferred surfactants are non-ionic surfactants. Examples of non-ionic surfactants suitable for compositions of the invention include polysorbate 20 (PS20), polysorbate 80 (PS80), sorbitol, poloxamer 188 (P188), and trehalose.

[0164] 35) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 34), wherein the surfactant is polysorbate 80 (PS80).

[0165] 36) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 35), wherein the concentration of the surfactant in the composition is 0.03 w / v %.

[0166] 37) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 36), wherein said pharmaceutical composition comprises a tonicity modifier.

[0167] 38) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 37), wherein the pharmaceutical composition comprises NaCI.

[0168] 39) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 38), wherein the concentration of NaCI is 140 mM.

[0169] 40) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 39), wherein the concentration of the polysaccharide is about 3, 7, or 14 pg / mL (preferably 14 pg / mL). 41) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 39), wherein the concentration of the polysaccharide is about 28 pg / mL.

[0170] For avoidance of any doubt, the term “polysaccharide”, “antigen” and “glycan” are used synonymously throughout the present application. The term “antigen”, “polysaccharide” or “glycan” refers to the polysaccharide part of formula (I) i.e. excluding linker L, spacer T, and carrier protein.

[0171] 42) In another embodiment, the invention relates to a pharmaceutical composition comprising:

[0172] • a polysaccharide-protein conjugate of formula (I) which is also a polysaccharide-protein conjugate of formula (II)

[0173] Formula (II) wherein i represents an integer between 5 and imax, wherein imax is an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP (preferably i represents an integer between 5 and 25);

[0174] • an adjuvant comprising a pharmaceutically acceptable aluminum salt;

[0175] • a pharmaceutically acceptable buffer; and

[0176] • water.

[0177] 43) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 42), wherein said pharmaceutical composition further comprises:

[0178] • a surfactant; and

[0179] • a tonicity modifier. 44) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 43), wherein said pharmaceutical composition comprises:

[0180] • a polysaccharide-protein conjugate of formula (I) which is also a polysaccharide-protein conjugate of formula (II)

[0181] Formula (II) wherein i represents an integer between 5 and imax, wherein imax is an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP (preferably i represents an integer between 5 and 25);

[0182] • Alum adjuvant;

[0183] • Tris-HCI;

[0184] • water;

[0185] • polysorbate 80; and

[0186] • NaCI.

[0187] 45) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 42) to 44), wherein the concentration of the polysaccharide is about 3, 7, or 14 pg / mL (preferably 14 pg / mL).

[0188] 46) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 42) to 44), wherein the concentration of the polysaccharide is about 28 pg / mL.

[0189] 47) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 45) or 46), wherein the concentration of the Alum adjuvant is about 250 pg / mL, the concentration of Tris-HCI is about 10mM at pH of 7.4, the concentration of polysorbate 80 is about 0.03 w / v%, and the concentration of NaCI is about 140 mM.

[0190] 48) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 47), wherein the water is water for injection (WFI).

[0191] 49) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 48), wherein the pharmaceutical composition is in a container.

[0192] 50) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 49), wherein the container is selected from a vial, an ampoule, a cartridge, or a syringe.

[0193] 51) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 49), wherein the container is a vial.

[0194] 52) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 49) to 51), wherein the container is a glass container (preferably a glass vial, more preferably borosilicate glass vial).

[0195] 53) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 52), wherein the volume of the pharmaceutical composition in the container is between 0.5 mL and 0.8 ml_, or between 1 .0 mL and 1 .4 mL.

[0196] 54) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 52), wherein the volume of the pharmaceutical composition in the container is 0.7 mL or 1 .2 mL.

[0197] 55) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 49) to 54), wherein the pharmaceutical composition is withdrawn from the container with a syringe.

[0198] 56) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 55), wherein the amount of the pharmaceutical composition withdrawn from the container with the syringe corresponds to the amount of the unit dose.

[0199] The term “unit dose” means the amount of the pharmaceutical composition that is administered and / or is to be administered to a patient in a single dose.

[0200] 57) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 56), wherein the amount of the pharmaceutical composition withdrawn with the syringe corresponds to 0.5 mL. 58) In another embodiment, the invention relates to a pharmaceutical composition according to embodiment 56), wherein the amount of the pharmaceutical composition withdrawn with the syringe corresponds to 1 .0 mL.

[0201] The term “corresponds to”, as used in the context of embodiment 57) and 58), means that the amount of the pharmaceutical composition withdrawn with the syringe equals to 0.5 mL (embodiment 57)) or 1.0 mL (embodiment 58)) plus a small addition to compensate for a loss of pharmaceutical composition during administration, such loss being caused for instance by the dead volume of the syringe.

[0202] 59) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 56) or 57), wherein the amount of the polysaccharide in the unit dose is between 1 pg and 8 pg.

[0203] 60) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 56) or 57), wherein the amount of the polysaccharide in the unit dose is 1.5 pg, 3.5 pg, or 7 pg.

[0204] 61) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 56) or 57), wherein the amount of the polysaccharide in the unit dose is 14 pg.

[0205] 62) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 61), wherein the pharmaceutical composition is an emulsion, a suspension, or a solution.

[0206] 63) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 61), wherein the pharmaceutical composition is a suspension.

[0207] 64) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 63), wherein the pharmaceutical composition is sterile.

[0208] 65) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 64), wherein the pharmaceutical composition is stored at 2 to 8 °C.

[0209] 66) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 65), wherein the amount of polysaccharide- protein conjugate of formula (I) in the pharmaceutical composition after storage for 1 year at 2 to 8 °C is at least 75% (preferably at least 85% and most preferably at least 90%) of the amount of polysaccharide-protein conjugate of formula (I) in the pharmaceutical composition immediately (for instance 1 hour or 2 hours) after final preparation of the pharmaceutical composition (i.e. after all ingredients are added to the pharmaceutical composition).

[0210] 67) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 65), wherein the amount of polysaccharide- protein conjugate of formula (I) in the pharmaceutical composition after storage for 2 years at 2 to 8 °C is at least 75% (preferably at least 85% and most preferably at least 90%) of the amount of polysaccharide-protein conjugate of formula (I) in the pharmaceutical composition immediately (for instance 1 hour or 2 hours) after final preparation of the pharmaceutical composition (i.e. after all ingredients are added to the pharmaceutical composition).

[0211] 68) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 65), wherein the amount of polysaccharide- protein conjugate of formula (I) in the pharmaceutical composition after storage for 6 months at 2 to 8 °C is at least 75% (preferably at least 85% and most preferably at least 90%) of the amount of polysaccharide-protein conjugate of formula (I) in the pharmaceutical composition immediately (for instance 1 hour or 2 hours) after final preparation of the pharmaceutical composition (i.e. after all ingredients are added to the pharmaceutical composition).

[0212] The pharmaceutical composition according to the invention may be used as a medicament, it may be used in a method of raising an immune response in a patient, comprising a step of administering the composition to the patient. The pharmaceutical compositions of the present invention may be administered before a subject (notably a mammal, especially a human) is exposed to C. difficile and / or after a subject is exposed to C. difficile bacteria.

[0213] 69) In another embodiment, the invention relates to a pharmaceutical composition according to any one of embodiments 1) to 68), for the use as medicament, in particular as vaccine. In other words, the invention relates to a vaccine comprising the pharmaceutical composition according to any one of embodiments 1) to 68). Preferably, the vaccine is used for active vaccination.

[0214] 70) A further aspect of the present invention relates a pharmaceutical composition according to any one of embodiments 1) to 68), for the use in the prevention and / or treatment of C. difficile infection (GDI). 71) In another embodiment, the pharmaceutical composition according to any one of embodiments 1) to 68), may be used for the preparation of a medicament, in particular vaccine for the prevention and / or treatment of C. difficile infection (CDI).

[0215] The terms “prevention”, “preventing” and / or “prophylaxis” are used synonymously and refer to inhibiting the initial onset of a pathologic process, such that the pathologic process that could eventually lead to development of symptoms never develops or that symptoms develop in lower, non-dangerous intensity (i.e. preventing the development of a disease, disorder, or condition in a prophylactic manner).

[0216] For avoidance of any doubt, if an aqueous pharmaceutical composition is described as useful for the preparation of a medicament for the prevention / prophylaxis or treatment of certain diseases or disorders, or as suitable for use in the prevention or treatment of certain diseases or disorders, such pharmaceutical composition is likewise suitable for use in a method of preventing or treating said diseases or disorders comprising administering to a subject (notably a mammal, especially a human) in need thereof a pharmaceutically active amount of said aqueous pharmaceutical composition.

[0217] The pharmaceutical composition according to the invention is suitable for parenteral administration (such as subcutaneous or intramuscular administration); especially for intramuscular administration.

[0218] 72) In a further embodiment, the pharmaceutical composition according to the invention is administered and / or is to be administered by parenteral administration.

[0219] 73) In a further embodiment, the pharmaceutical composition according to the invention is administered and / or is to be administered by subcutaneous or intramuscular administration.

[0220] 74) In a further embodiment, the pharmaceutical composition according to the invention is administered and / or is to be administered by intramuscular administration.

[0221] Experimental Part

[0222] The polysaccharide-protein conjugate of formula (I) (PS-II-CRM197 glycoconjugate) may be prepared according to the procedures as disclosed in WO 2020 / 104697 A1 .

[0223]

[0224] Polysaccharide-protein conjugate used in the Experimental part

[0225] To identify a suitable adjuvant for the polysaccharide-protein conjugate of formula (I), which throughout the experimental part is also named as PS-II-CRM197 glycoconjugate, eitherwith Aluminum hydroxide (Alum) adjuvant (Alhydrogel) or with AddaVax (InvivoGen), a squalene-based oil-in-water adjuvant similar to MF59, which is an adjuvant used in some human seasonal influenza vaccines was used for the formulation. Alum has a favorable safety profile and weak associated adverse events. However, a previous study showed that Alum did not support the induction of PS-II specific antibodies in mice immunized with a C. difficile PS-II-CRM197 glycoconjugate (ACS Chem. Biol. 2019, 14, 2720-2728). AddaVax / MF59 was shown previously as favorable adjuvant for C. difficile PS-II containing vaccines, including glycoconjugate vaccines with the carrier protein CRM 197 (patent WO 2012 / 085668 A2; Adamo et al., ACS Chem. Biol. 2012, 7, 1420-1428; Broecker et al., ACS Chem. Biol. 2019, 14, 2720-2728). The vaccine formulations were prepared as follows:

[0226] *The glycan concentration of the glycoconjugate stock was 507 pg / mL.

[0227] For the PS-II-CRM197 + Alum formulation, the antigen was adsorbed to the Alum adjuvant by incubating the formulation for 2 hours at room temperature on a shaking plate. This formulation was kept at 2-8 °C until use. A volume of 100 pL of this formulation corresponds to a glycan dose of 2.5 pg. Adsorption of greater than 90% of the PS-II-CRM197 glycoconjugate to the Alum adjuvant was confirmed by bicinchoninic acid (BCA) assay protein quantification of whole formulation and the supernatant after centrifugation of the formulation which leads to sedimentation of the particulate Alum adjuvant to the bottom of reaction tubes. The PS-II-CRM197 + AddaVax formulation was first prepared without adjuvant and at a glycan concentration of 50 pg / mL. The adjuvant was added to this formulation just prior to the immunization at a 1 :1 volume-by-volume ratio, i.e., 50 pL of the formulation were combined with 50 pL of AddaVax for each mouse dose, and a homogenous emulsion was prepared by pipetting up and down with a micropipette. The PS- II-CRM197 + AddaVax (without the adjuvant) and the Placebo formulations were also stored at 2-8 °C until use for immunization studies in mice.

[0228] Immunization of mice with PS-II-CRM197 glycoconjugate with different adjuvants followed by lethal infection with C. difficile spores

[0229] Groups of 10 mice (C57BL / 6, female, 7-9 weeks old at study start) were immunized three times, at days -35, -21 and -7 relative to infection, subcutaneously (100 pL injection volume) with PS-II-CRM197 glycoconjugate (2.5 pg glycan dose) in the presence of either Aluminum hydroxide (Alum) adjuvant (25 pg Aluminum per dose) or AddaVax adjuvant (50 pL per dose, formulated as an emulsion). A placebo group of mice received adjuvant (Alum) only. Starting at day -12 relative to infection, mice were pre-conditioned for 10 days by treatment with cefoperazone (Apollo Scientific Ltd) prepared in sterile drinking water (0.5 mg / mL, starting on day -12 until day -2 relative to infection). Mice were allowed to drink the antibiotic containing water ad libitum. The antibiotic preparation was replaced every 48 hours to minimize deterioration of the cefoperazone. Mice were returned to drinking water free of cefoperazone 48 hours prior to infection for the remaining duration of the study. At 48 hours prior to infection a single dose of clindamycin (10 mg / kg, Focus Pharmaceuticals Ltd) was administered by intraperitoneal injection. The mice were infected with a lethal dose of C. difficile strain ATCC 43255 (VPI 10463). C. difficile spores were prepared using a modified protocol described by Edwards & McBride (Edwards & McBride, Methods Mol. Biol. 2016, 1476, 117-128). Bacteria were streaked from frozen bead stock onto pre-reduced brain heart infusion-supplemented (BHIS) agar and incubated anaerobically at 37°C overnight to 48 hours. 10 mL pre-reduced BHIS + 0.1% taurocholate and 0.2% fructose was inoculated from a single colony and incubated anaerobically at 37°C overnight followed by back- dilution to optical density at 600 nm wavelength (OD6oo) of 0.5 with BHIS. 250 pL of culture was spread onto fresh, pre-reduced agar plates and incubated anaerobically at 37°C for 24 hours. The entire bacterial lawn was scraped off the plates using a sterile loop and resuspended in 10 mL sterile phosphate-buffered saline (PBS). Cells were washed in 10 mL PBS and centrifuged at 4700 g for 10 minutes at room temperature. The pellet was resuspended in 10 mL PBS, 10 mL of 95% ethanol were added to the re-suspended cell pellet and incubated at room temperature for 1 hour. Cells were washed three times with 20 mL sterile PBS and the final pellet was re-suspended in a minimum volume of sterile-filtered PBS + 1% (w / v) bovine serum albumin to give a homogenous suspension. Spore stock was heated to 70°C for 20 minutes and then allowed to cool to room temperature. 100 pL aliquots of spore stock were frozen in a dry ice / ethanol bath before storing at -80°C. Spore numbers of each preparation were enumerated on pre-reduced BHIS + 0.1% taurocholate agar by performing ten-fold serial dilutions in pre-reduced PBS + 1% (w / v) bovine serum albumin (BSA). Plates were incubated at 37°C anaerobically for ~24 hours to allow spore germination and sufficient growth for quantification before colonies were counted. 100 pL of inoculum was used to infect the mice by oral gavage. The inoculum was quantified following preparation and was ~1.3 x 106spores / mouse. The mice were observed for survival over a period of 10 days after infection. Surprisingly, survival of mice immunized with PS-II-CRM197 in the presence of aluminum hydroxide (Alum) adjuvant showed significantly (P < 0.05, Log Rank test) better survival (7 / 10 mice), compared to 2 / 10 mice in the placebo group, while those mice immunized with PS-II-CRM197 with AddaVax did not show better survival compared to placebo (P > 0.05, Log Rank test) (Figure 1).

[0230] Figure 1 shows a Survival plot of mice infected with a lethal dose of C. difficile spores at day 0. Before infection, the mice received three injections with a PS-II-CRM197 glycoconjugate either in the presence of Alum or AddaVax adjuvant, or adjuvant in buffer (Placebo), at days -35, -21 and -7. Statistical significance was evaluated with the Log Rank test, with one asterisk indicating statistical significance (P < 0.05) of the indicated comparison, whereas ‘n.s.’ indicates no statistical significance (P > 0.05) of the indicated comparison.

[0231] Vaccine formulation comprising a polysaccharide-protein conjugate of formula (I)

[0232] The chemical stability of the aqueous pharmaceutical composition may be tested in conventional manner, e.g. by measurement of Polysaccharide-protein conjugate and its degradation products (such as free glycans especially GalNAc). The amounts of Polysaccharide-protein conjugate and its degradation products in a sample may be for instance evaluated via HPLC. The aqueous pharmaceutical composition according to any one of embodiments 1) to 68) may be formulated in containers. For example, a batch size corresponding to 320 g of Polysaccharide-protein conjugate, may lead to at least 3000 vials (especially glass vials) of about 0.7 g of aqueous pharmaceutical composition per vial. Starting from Polysaccharide- protein conjugate, the following amounts of ingredients may be used:

[0233] The amounts may be adjusted for the concentration of the Polysaccharide-protein conjugate in the drug substance. The amount of Polysaccharide-protein conjugate in the Drug substance formulation may be adjusted such that the amount of Polysaccharide-protein conjugate in the unit dose is 1.5 - 14 pg. The content of conjugated antigen is calculated based on the measured total protein concentration (determined by UV method) and the loading factor calculated based on the average molecular mass determined by MALDI-TOF. The following formula is used: Content of conjugated antigen =

[0234] It is understood that the amount of Polysaccharide-protein conjugate in the unit dose may vary within the limits accepted by the health authorities and especially within a range of plus or minus 25% of the corresponding amount; this variation may come along with a respective change in the ratio between the ingredients or with a respective change in the total amount of the unit dose or with a combination of both.

[0235] The process for the preparation of an aqueous pharmaceutical composition as described before and filled in a container according to the present invention, may comprise the following steps:

[0236] • add specific amount of sodium chloride (40.950 g) to a glass bottle,

[0237] • add specific amount of Tris-HCI (e.g. 6.050 g) to a glass bottle,

[0238] • fill up with WFI to final weight,

[0239] • add specific amount (e.g. 100 mL) of aluminum hydroxide stock to a stainless-steel bottle under stirring,

[0240] • add the solution containing sodium chloride, Tris-HCI and WFI to the aluminum hydroxide stock in the stainless-steel bottle,

[0241] • heat sterilize the formulate aluminum hydroxide,

[0242] • add Polysaccharide-protein conjugate to a solution of Tris Base (e.g. 6.050 g), sodium chloride (e.g. 40.950 g), Polysorbate 80 (e.g. 3.000 g), and WFI, at pH 7.4 to a glass bottle to formulate the drug substance,

[0243] • adjust the pH to 7.4 with sodium hydroxide solution,

[0244] • filter the formulated drug substance by one bioburden reduction filter size 0.22 pm and one sterilizing filter size 0.22 pm,

[0245] • transfer the filtered sterile formulated drug substance using sterile-to-sterile connections to the sterile formulated aluminum adjuvant suspension with a concomitant sterilizing filtration of the drug substance under stirring,

[0246] • fill the suspension into vials (e.g. 0.7 g ±2% per vial),

[0247] • stopper the vial, and

[0248] • cap the vial.

[0249] Example 1

[0250] Preparation and composition of an aqueous pharmaceutical composition comprising a Tris-HCI buffer: Table 1 : Tris-HCI buffer

[0251] Preparation of the composition

[0252] The Polysaccharide-protein conjugate is diluted in 10 mM Tris-HCI, 140 mM NaCI, 0.06% Polysorbate 80, and WFI, and adjusted to pH 7.4. The formulated bulk DS is filtered by x2 0.22pm sterilizing filters. Formulated aluminum was prepared by adding aluminum hydroxide to formulation buffer containing NaCI, Tris-HCI, and WFI. This suspension bottle was heat sterilized and then mixed continuously to create the formulated aluminum suspension. The formulated Drug substance bulk is transferred to the formulated aluminum adjuvant suspension bottle in a 1 :1 ratio via peristaltic pump and sterile connections. The bulk drug product at final concentration suspension is kept homogenous by constant stirring.

[0253] Preparation of the primary packaging

[0254] 0.7 mL of the sterile filtered final Drug Product suspension formulation was filled into a sterile vial that is then stoppered, capped, and crimped. The filling takes place under sterile and particle free conditions.

[0255] Example 2

[0256] Preparation and composition of an aqueous pharmaceutical composition comprising a Histidine-HCI buffer:

[0257] Table 2: Histidine HCI buffer Preparation of the composition

[0258] The Polysaccharide-protein conjugate is diluted in 10 mM Histidine-HCI, 140 mM NaCI, 0.06% Polysorbate 80, and WFI, and adjusted to pH 7.4. The formulated bulk DS is filtered by x2 0.22pm sterilizing filters. Formulated aluminum was prepared by adding aluminum hydroxide to formulation buffer containing NaCI, Histidine-HCI, and WFI. This suspension bottle was heat sterilized and then mixed continuously to create the formulated aluminum suspension. The formulated Drug Substance bulk is transferred to the formulated aluminum adjuvant suspension bottle in a 1 :1 ratio via peristaltic pump and sterile connections. The bulk drug product at final concentration suspension is kept homogenous by constant stirring.

[0259] Preparation of the primary packaging

[0260] 0.5 mL of the sterile filtered final formulation was filled into a sterile vial that is then stoppered, capped, and crimped. The filling takes place under sterile and particle free conditions.

[0261] Example 3

[0262] Preparation and composition of an aqueous pharmaceutical composition comprising a Citrate buffer:

[0263] Table 3: Citrate buffer

[0264] Preparation of the composition

[0265] The Polysaccharide-protein conjugate is diluted in 10 mM Citrate, 300 mM Sorbitol, 0.06% Polysorbate 80, and WFI, and adjusted to pH 7.4. The formulated bulk Drug Substance is filtered by x2 0.22pm sterilizing filters. Formulated aluminum was prepared by adding aluminum hydroxide to formulation buffer containing Sorbitol, Citrate, and WFI. This suspension bottle was heat sterilized and then mixed continuously to create the formulated aluminum suspension. The formulated Drug Substance bulk is transferred to the formulated aluminum adjuvant suspension bottle in a 1 :1 ratio via peristaltic pump and sterile connections. The bulk drug product at final concentration suspension is kept homogenous by constant stirring.

[0266] 0.7 mL of the sterile filtered final formulation was filled into a sterile vial that is then stoppered, capped, and crimped. The filling takes place under sterile and particle free conditions.

[0267] SEQ ID No. 1 : amino acid sequence of CRM197:

[0268] GADDVVDSSK SFVMENFSSY HGTKPGYVDS 30

[0269] IQKGIQKPKS GTQGNYDDDW KEFYSTDNKY 60

[0270] DAAGYSVDNE NPLSGKAGGV VKVTYPGLTK 90

[0271] VLALKVDNAE TIKKELGLSL TEPLMEQVGT 120

[0272] EEFIKRFGDG ASRVVLSLPF AEGSSSVEYI 150

[0273] NNWEQAKALS VELEINFETR GKRGQDAMYE 180

[0274] YMAQACAGNR VRRSVGSSLS CINLDWDVIR 210

[0275] DKTKTKIESL KEHGPIKNKM SESPNKTVSE 240

[0276] EKAKQYLEEF HQTALEHPEL SELKTVTGTN 270

[0277] PVFAGANYAA WAVNVAQVID SETADNLEKT 300

[0278] TAALSILPGI GSVMGIADGA VHHNTEEIVA 330

[0279] QSIALSSLMV AQAI PLVGEL VDIGFAAYNF 360

[0280] VESI INLFQV VHNSYNRPAY SPGHKTQPFL 390

[0281] HDGYAVSWNT VEDSI IRTGF QGESGHDIKI 420

[0282] TAENTPLPIA GVLLPTI PGK LDVNKSKTHI 450

[0283] SVNGRKIRMR CRAIDGDVTF CRPKSPVYVG 480

[0284] NGVHANLHVA FHRSSSEKIH SNEI SSDSIG 510

[0285] VLGYQKTVDH TKVNSKLSLF FEIKS 535

Claims

Claims1 . A pharmaceutical composition comprising:• a polysaccharide-protein conjugate of formula (I)formula (I) wherein n represents an integer selected from 0, 1 , 2, and 3; i represents an integer between 5 and imax, wherein imax is an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP;Z represents *-CH2-O-P(O)(OH)-, or *-O-P(O)(OH)-, wherein the star represents the attachment point to the anomeric carbon atom (C-1 carbon atom) of the attached mannose;L-T represents a linker L and a spacer T which together form a bridge between the oxygen atom of the phosphate group and a nitrogen atom of a lysine residue of the carrier protein CP; wherein the bridge consists of a chain of 2 to 50 groups independently selected from -CH2-, -NRN-, -O-, -S-, -CRC1RC2-, -CH=CH-, -C C-, (C3-C8)cycloalkylene, and (C3-C8)heterocycloalkylene, wherein the (C3- 8)carbocyclene and the (C3-8)heterocyclene are unsubstituted or independently substituted with 1 to 4 substituents independently selected from the group consisting of (Ci-4)alkyl, (Ci-4)alkoxy, (C3-8)cycloalkyl, (Ci-4)fluoroalkyl, halogen and oxo; and wherein at least 1 group is different from -CH2-;RNrepresents hydrogen or (Ci-4)alkyl;RC1and RC2represent independently of each other (Ci-4)alkyl, (Ci-4)alkoxy, (C3- 8)cycloalkyl, (Ci-4)fluoroalkyl, hydroxy, or halogen; or RC1and RC2together represent oxo;CP represents a carrier protein selected from the group consisting of CRM 197; diphtheria toxoid; tetanus toxoid; cholera toxin B subunit; Neisseria meningitidisouter membrane protein (OMP); capsid protein of bacteriophage Q|3; oligomers or virus-like particles prepared with capsid protein of bacteriophage Q|3; detoxified Exotoxin A of Pseudomonas aeruginosa (EPA); maltose-binding protein (MBP); the He fragment of tetanus toxin (TetHc); detoxified hemolysin A of Staphylococcus aureus, clumping factor A (ClfA) and clumping factor B (ClfB) of Staphylococcus aureus: Escherichia coll FimH; Escherichia coll FimHC; detoxified variants of Escherichia coll heat labile enterotoxin; detoxified variants of cholera toxin; Escherichia coll Sat protein; the passenger domain of Escherichia coll Sat protein; detoxified variants of Streptococcus pneumoniae pneumolysin;Campylobacter jejuni AcrA; Pseudomonas PcrV protein; Campylobacter jejuni natural glycoproteins; bovine serum albumin (BSA); pilus protein GBS80 from Group B Streptococcus’ Escherichia coll heat-labile enterotoxin; tetanus toxin; cholera toxin; and Streptococcus pneumoniae pneumolysin; or a pharmaceutically acceptable salt thereof;• an adjuvant comprising a pharmaceutically acceptable aluminum salt;• a pharmaceutically acceptable buffer; and• water.

2. A pharmaceutical composition according to claim 1 , wherein n represents 0.

3. A pharmaceutical composition according to any one of claims 1 or 2, wherein i represents an integer between 5 and 25.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein Z represents *-CH2-O-P(O)(OH)-, wherein the star represents the attachment point to the anomeric carbon atom (C-1 carbon atom) of the attached mannose.

5. A pharmaceutical composition according to any one of claims 1 to 4, wherein CP is CRM197.

6. A pharmaceutical composition according to any one of claims 1 to 5, wherein L represents *-(CH2)a-NH-, wherein a is from 2 to 10.

7. A pharmaceutical composition according to claim 6, wherein T represents -C(O)-(CH2)h- C(O)-, wherein h is 0, 1 , 2, 3, 4, 5, or 6.

8. A pharmaceutical composition according to any one of claims 1 to 7, wherein the adjuvant comprises aluminum hydroxide.

9. A pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutically acceptable buffer has a pH value between 6.8 and 8.0.

10. A pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutically acceptable buffer is selected from sodium succinate, Tris-HCI, histidine, and citrate.1 1. A pharmaceutical composition according to any one of claims 1 to 10, wherein said pharmaceutical composition comprises a surfactant.

12. A pharmaceutical composition according to any one of claims 1 to 1 1 , wherein said pharmaceutical composition comprises a tonicity modifier.

13. A pharmaceutical composition according to claim 1 , wherein said pharmaceutical composition comprises:• a polysaccharide-protein conjugate of formula (II)Formula (II) wherein i represents an integer between 5 and imax, wherein imax is an integer corresponding to 90% of the number of lysine residues contained in the carrier protein CP;• Alum adjuvant;• Tris-HCI;• water;• polysorbate 80; and• NaCI.

14. A pharmaceutical composition according to any one of claims 1 to 13, wherein the pharmaceutical composition is in a container and wherein said container is selected from a vial, an ampoule, a cartridge, or a syringe.

15. A pharmaceutical composition according to any one of claims 1 to 14 for use in the prevention or treatment of C. difficile infection (CDI).

16. A pharmaceutical composition according to any one of claims 1 to 14 for use according to claim 15, wherein said pharmaceutical composition is administered and / or is to be administered by intramuscular administration.

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