Compositions, in particular pharmaceutical compositions of bacteriophages of the class Caudoviricetes, process and associated uses

Abstract

Title: Compositions, in particular pharmaceutical compositions of bacteriophages of the class Caudoviricetes, process and associated uses. The present invention relates to compositions, and in particular pharmaceutical compositions, comprising or even consisting of a suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, at a titer of at least 10⁸ PFU / mL, in an aqueous solution comprising or even consisting of: - at least one non-ionic surfactant, and in particular a single non-ionic surfactant, selected from the polysorbate family, at a molar concentration above its critical micellar concentration, but not exceeding 2 mg / mL, - one or more neutral salts formed of a pair of monovalent ions, the total molar concentration of which is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5,typically selected from the pairs hydrogen phosphate / dihydrogen phosphate, citric acid / citrate, and citric acid / hydrogen phosphate, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, water, said composition comprising less than 0.9 units of bacterial endotoxins per mL, a total protein concentration of less than 0.09 mg / mL, and said bacteriophage suspension in said aqueous solution having an osmolality between 150 mOsm / kg and 600 mOsm / kg, as well as associated processes and uses. Figure for the abstract: none.

Description

Title of the invention: Compositions, in particular pharmaceutical compositions of bacteriophages of the class Caudoviricetes, process and associated uses. Technical field

[0001] The present invention relates to the field of therapy. More specifically, it relates to novel pharmaceutical compositions containing an aqueous suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, and in particular, bacteriophages directed against at least one strain of Staphylococcus aureus; such pharmaceutical compositions for their use as a medicinal product and in various medical applications; as well as their preparation method. Prior art

[0002] In July 2022, the European Medicines Agency (EMA) published a "list of key therapeutic groups in crisis preparedness" in response to the increasing number of drug shortages exacerbated by the COVID-19 crisis (European Medicines Agency 2022). This list includes "systemic antibacterials." However, the growing threat of antibiotic resistance is compromising the effectiveness of this therapeutic group. Indeed, in April 2019, the World Health Organization predicted that, at the current rate, 10 million people could die each year from multidrug-resistant bacteria by 2050 (Interagency Coordination Group on Antimicrobial Resistance 2019). New approaches to treating bacterial infections are therefore being sought, and older ones, such as phage therapy, are being re-evaluated.

[0003] Bacteriophages are viruses that infect bacteria. They specifically recognize a bacterium, inject their genetic material into it, and hijack the host bacterium's metabolic pathway to form new virions, leading to lysis of the bacterial cell wall and the release of phage particles into the immediate environment of the lysed bacterium (Stone et al. 2019). When present at the site of infection of the targeted bacteria, they can significantly reduce the bacterial load and combat bacterial infections. In 1919, Félix d'Hérelle successfully treated bacillary dysentery with phages (d'Hérelle 1931), and phage-based products were marketed in France from 1930 to 1976 (Dublanchet 2014; Vidal 1943).However, conflicting clinical results, the advent of mass antibiotic production, and a bilateral geopolitical context ultimately led to the demise of this therapeutic strategy. Nevertheless, some... countries (e.g., Georgia, Russia, Poland) have maintained this therapy for over a hundred years (Miçdzybrodzki et al. 2018; Zaczek et al. 2022).

[0004] The European Pharmacopoeia, which establishes standards for pharmaceutical products in Europe, includes a general chapter “5.31. MEDICINAL PRODUCTS USED IN PHAGOTHERAPY”. It contains a set of requirements relating to active substances and medicinal products for human or veterinary use used in phage therapy, as well as their production and control. These standards are essential to ensure the quality and consistency of bacteriophage-based products throughout Europe. However, the diversity of production methods reported in the literature is subject to significant quantitative and qualitative fluctuations, making it difficult to apply the usual Good Manufacturing Practice standards (Ferry et al. 2021; Tanir et al. 2021). In 2018, at the initiative of the Belgian authorities, a generic monograph was developed on how to safely prepare and test phages intended for patients (Pimay et al.2018; Verbeken and Pirnay 2022; Tanir et al. 2021). However, these texts do not specify any formulations that allow for prolonged storage of phage suspensions, purified or not, with and without agitation.

[0005] Over the past fifty years, more than 5,100 bacteriophages have been identified and studied, over 90% of which have a tail and belong to the myovirus, siphovirus, and podovirus morphotypes (Harada et al. 2018). Virus taxonomy was completely revised in 2021, and the order Caudovirales, as well as the families Myoviridae, Siphoviridae, and Podoviridae, were abolished. The taxonomy used in this patent application is defined in Walker et al. 2021 (Walker et al. 2021). Phages, although specific to bacterial types, exhibit similarities in protein structures and nucleic acids. Schematically, a prototypical bacteriophage particle is composed of one or two strands of nucleic acid protected by the protein icosahedral capsid, which is attached to the contractile sheath terminated at its base by a hexagonal-shaped plate coordinating the movement of the tail fibers.

[0006] The stability of bacteriophage preparations is affected by the external protein structure, which can undergo unfolding that promotes aggregation, reducing the infectivity of the phages. These aggregation phenomena depend on a large number of factors, including the nature of the dispersing phase (water, water-co-solvent mixture) (Mendez et al. 2002), the concentrations of dissolved substances (mono- and divalent cations) (Drab 2018), the pH (Meyer et al. 2017), mechanical agitation (Hoe et al. 2014; Francius et al. 2021), the production and storage temperature (Ergin 2022), and the packaging (glass, polypropylene) (Richter et al. 2021). The higher the bacteriophage titer, the more difficult it is to obtain bacteriophage suspensions that are stable over time. The bacteriophage titer is a key element of their stability and activity in suspension (Duyvejonck et al. 2021).

[0007] Furthermore, in order to be used in compositions administered to humans, bacteriophages must undergo a purification process, notably to reduce the presence of endotoxins and residual bacterial proteins. The purification steps disrupt the interactions between phages and the dispersing medium and between phages and the packaging material, and are a source of instability for purified phage suspensions. The more purified the bacteriophage suspensions, the more the bacteriophages tend to aggregate, which can impair their lytic activity on target bacteria. Moreover, in order to limit instability phenomena (including aggregation) in bacteriophage suspensions, they are often sold unpurified with bacteriophages frozen inside their host cell.

[0008] The chemical structure (protein and nucleic) of bacteriophages greatly differentiates them from proteins and antibodies, which makes the preparation of bacteriophage formulations with good stability and preserved anti-infective properties very specific (Batinovic et al. 2019).

[0009] In this context, the objective of the invention is to provide compositions comprising a high bacteriophage titer and a low level of endotoxins and residual bacterial proteins, which are administrable to humans or animals, particularly in the form of an injectable composition, and therefore usable as a raw material for pharmaceutical, medical, and veterinary use. These compositions must be stable over time, reduce bacteriophage aggregation, and ensure the maintenance of antibacterial lytic activity. Description of the invention

[0010] The present invention relates to compositions comprising a suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, at a concentration of at least 10⁸ PFU / mL, preferably at a concentration of 10⁸ to 10¹⁰ PFU / mL, and in particular at a concentration of 10⁹ to 10¹⁰ PFU / mL, in an aqueous solution. Said aqueous solution comprises: - at least one non-ionic surfactant, and in particular a single non-ionic surfactant, selected from the polysorbate family, at a molar concentration above its critical micellar concentration, but not exceeding 2 mg / mL (corresponding to 0.2% (w / v)), - one or more neutral salts formed of a pair of monovalent ions, the total molar concentration of which is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water. Said composition comprises less than 0.9 units of bacterial endotoxins (EU) per mL, a total protein concentration of less than 0.09 mg / mL and said bacteriophage suspension in said aqueous solution exhibits an osmolality between 150 mOsm / kg and 600 mOsm / kg.

[0011] Advantageously, in the compositions according to the invention, the aqueous solution in which the bacteriophages are suspended consists of: - of at least one non-ionic surfactant, and in particular a single non-ionic surfactant, chosen from the polysorbate family, at a molar concentration higher than its critical micellar concentration (CMC), but not exceeding 2 mg / mL (or 0.2% (w / v)), - one or more neutral salts formed from a pair of monovalent ions, the total molar concentration of which is in the range of 75 to 160 rnM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically chosen from the pairs hydrogen phosphate / dihydrogen phosphate, citric acid / citrate and citric acid / hydrogen phosphate, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water.

[0012] Also, in particular, the present invention relates to compositions consisting of a suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, the titer of said bacteriophages being at least 10⁸ PFU / mL, preferably from 10⁸ to 10¹⁰ PFU / mL, and in particular from 10⁹ to 10¹⁰ PFU / mL, in an aqueous solution consisting of: - of at least one non-ionic surfactant, and in particular a single non-ionic surfactant, chosen from the polysorbate family, at a molar concentration higher than its critical micellar concentration, but not exceeding 2 mg / mL (which corresponds to 0.2% (w / v)), - one or more neutral salts formed from a pair of monovalent ions, with a total molar concentration in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably 7.0 to 7.5, typically chosen from the following ion pairs hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, said aqueous suspension comprising less than 0.9 units of bacterial endotoxins (EU) per mL, a total protein concentration of less than 0.09 mg / mL and said bacteriophage suspension in said aqueous solution having an osmolality between 150 mOsm / kg and 600 mOsm / kg.

[0013] In the definition of the compositions according to the invention, the total molar concentration given in the range from 75 to 160 mM corresponds to the molar concentration of the neutral salt(s) formed from a pair of monovalent ions present in the composition.

[0014] The pH and osmolality are chosen to be compatible with administration in humans and animals, and in particular to allow parenteral or enteral administration. The osmolality of the suspension is chosen to maintain blood and tissue isotonicity.

[0015] Preferably, the compositions according to the invention are pharmaceutical compositions. In particular, the present invention relates to pharmaceutical compositions comprising a bacteriophage suspension as described herein, and especially to pharmaceutical compositions consisting of such a bacteriophage suspension.

[0016] In the compositions according to the invention, the bacteriophages form a colloidal suspension in the dispersing medium, which corresponds to the selected aqueous solution. That is to say, the bacteriophages are dispersed in the aqueous solution in the form of particles whose size, at least in one direction, is between 1 nanometer and 1 micrometer.

[0017] The inventors have shown, in a non-obvious manner, that the compositions according to the invention make it possible to obtain a stable suspension of bacteriophages. Stability (shelf-life) is, in particular, assessed, after a period of storage, without agitation, under refrigerated conditions (temperature in the range of 2°C to 8°C), by maintaining the bacteriophage titer at a value of 10⁸ PFU / mL or higher. "PFU" stands for "plaque-forming unit," according to a definition well known to those skilled in the art. The bacteriophage titer can, in particular, be determined by spot titration. Bacteriophages with lytic activity lyse the host cell, causing a clearing zone (or plaque) on a bed of bacteria targeted by the lytic activity of the bacteriophages, and it is these zones that are counted for the determination of the titer.

[0018] In the examples given below, tests were carried out highlighting other characteristics of the compositions according to the invention that are related to the observed stability, including (i) a pH variation of less than 1, and / or (ii) maintenance of the suspension's osmolality (error less than 10%), and / or (iii) an aggregation index, determined by UV spectrophotometry, of less than 7. The compositions according to the invention exhibit long-term stability, in particular over a period exceeding one year, and preferably two years or more, under refrigerated conditions (temperature in the range of 2°C to 8°C) and one month at room temperature (temperature in the range of 15°C to 25°C). In particular, it was found that for the compositions in the examples illustrating the invention, stability at 5°C for a period of at least one year could be achieved.

[0019] The aqueous solution, or dispersing medium, present in the compositions according to the invention was selected to optimize the stability of the bacteriophage suspensions obtained. To this end, stability studies were conducted (i) under stress conditions (i.e., temperature, pH, ionic strength, horizontal and rotary agitation promoting ventilation and cavitation) in order to induce aggregation and fragmentation phenomena of the bacteriophages that inactivate them; (ii) in different packaging formats for which the wettability of the compositions was studied; (iii) under storage conditions associated with shelf life.Thus, the present invention reports the unexpected stabilizing effect of a particular combination of a neutral surfactant and a specific buffered saline solution on suspensions of bacteriophages of the class Caudoviricetes, and in particular bacteriophages of the class Caudoviricetes belonging to the genus Silviavirus, having, notably, a myovirus morphotype and lytic activity against at least one strain of Staphylococcus aureus. Furthermore, the stabilizing effect against pH variations and mechanical stress, which are known to inactivate bacteriophages during storage, has been demonstrated.

[0020] In the compositions according to the invention, it has been demonstrated that aggregation between bacteriophages was reduced and that antibacterial activity was preserved over time.

[0021] In particular, in the compositions according to the invention, and especially in the pharmaceutical compositions according to the invention, the bacteriophages of the class of Caudoviricetes belong to the genus Silviavirus, and have, in particular, a myovirus morphotype, said bacteriophages having lytic activity against at least one strain of Staphylococcus aureus.

[0022] In the context of the invention, for the sake of simplicity, the term bacteriophages will be used to designate bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram positive bacteria, and in particular bacteriophages of the class Caudoviricetes belonging to the genus Silviavirus, and having, in particular, a myovirus morphotype, and having lytic activity against at least one strain of Staphylococcus aureus.

[0023] According to advantageous embodiments, in the compositions according to the invention, and in particular in the pharmaceutical compositions according to the invention, the aqueous solution, or dispersing medium, has an ionic strength of 150 to 400 mM. The ionic strength is chosen (i) to further limit the aggregation phenomena induced by electrostatic interactions (attraction / repulsion) related to the charges carried by the bacteriophages, (ii) to facilitate the destabilization of the bacterial cell wall of the target bacteria.

[0024] Advantageously, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are stable for at least one year at a temperature in the range of 2 to 8°C, i.e., they maintain a bacteriophage titer greater than or equal to 10⁸ PFU / mL. Within the scope of the invention, storage at a temperature in the range of 2 to 8°C is carried out without agitation. In particular, such storage of the compositions according to the invention is carried out in a type I or type II glass container.

[0025] In particular, before the storage period of at least one year at a temperature in the range of 2 to 8°C, said compositions according to the invention, and in particular said pharmaceutical compositions according to the invention, have a bacteriophage titer in the range of 10⁹ to 10¹⁰ PFU / mL. Also, preferably, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are stable for at least one year at a temperature in the range of 2 to 8°C, that is to say, they have a bacteriophage titer of the class Caudoviricetes belonging to the genus Silviavirus, and which have, in particular, a myovirus mophotype, and which have lytic activity against at least one strain of Staphylococcus aureus which remains greater than or equal to 10⁸ PFU / mL.In particular, prior to a storage period of at least one year at a temperature within the range of 2 to 8°C, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, have a bacteriophage titer of the class Caudoviricetes belonging to the genus Silviavirus, and which, in particular, have a myovirus mophotype, and which have lytic activity against at least one strain of Staphylococcus aureus, which falls within the range of 10⁹ to 10¹⁰ PFU / mL. In particular, no decrease in the bacteriophage titer of more than one log is observed over the period considered.

[0026] It can be noted that in the compositions according to the invention, and in particular in the pharmaceutical compositions according to the invention, the bacteriophages are neither in conjugated form nor in complexed form. Thus, the compositions according to the invention are simple to prepare.

[0027] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, comprise polysorbate 80, as a non-ionic surfactant, at a molar concentration greater than 0.013 mM (equivalent to 0.017 mg / mL, which corresponds to the CMC of polysorbate 80) and less than or equal to 1.6 mM (equivalent to 2.0 mg / mL), preferably in the range from 0.16 mM (equivalent to 0.2 mg / mL) to 0.38 mM (equivalent to 0.5 mg / mL), or polysorbate 20, at a molar concentration greater than 0.05 mM (equivalent to 0.06 mg / mL, which corresponds to the CMC of polysorbate 20) and less than or equal to 1.77 mM (equivalent to 2.0 mg / mL), preferably in the range from 0.18 mM (equivalent to 0.2 mg / mL) to 0.44 mM (equivalent to 0.5 mg / mL).

[0028] According to advantageous embodiments which can be combined with the preceding ones, in the compositions according to the invention, and in particular in the pharmaceutical compositions according to the invention, the buffer mixture is a KH2PO4 / Na2HPO4 mixture, typically KH2PO4 at a molar concentration of 1 to 15 mM and Na2HPO4 at a molar concentration of 3 to 56 mM.

[0029] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, comprise only NaCl, as a neutral salt, which is present at a molar concentration of 75 to 160 mM, typically at a molar concentration of 150 to 160 mM.

[0030] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, do not contain any compounds of animal origin. Therefore, in this case, the polysorbates present in the composition will be of synthetic origin.

[0031] Advantageously, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are sterile, pyrogen-free and isotonic or hypertonic.

[0032] Furthermore, due to the presence of the non-ionic surfactant and the adjustment of its concentration, it is possible to modulate the contact angle of the composition with different types of surfaces. This is advantageous both for packaging and for the preparation of the composition according to the invention, particularly its sterilization, which requires a final filtration operation, as it broadens the range of usable filters. In particular, the compositions according to the invention exhibit adsorption at gas-liquid and liquid-material interfaces. classically used in the medical field in particular, low enough to reduce surface adsorption phenomena.

[0033] Also, according to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are packaged in a container such as a bottle, a bag or a syringe delimited by walls which are made of type I glass, type II glass, polystyrene, polypropylene, polyethylene, polyethylene terephthalate, treated or not with silicone oil, in particular closed with a stopper made of a rubber material (in particular, bromobutyl or chlorobutyl) perfluorinated or not.

[0034] In particular, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, exhibit a wettability of the walls of their container corresponding to a contact angle of 20 to 60°.

[0035] In particular, the container is closed by a cap and the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, exhibit a wettability of the cap closing their container corresponding to a contact angle of less than 90°.

[0036] According to particular embodiments that can be implemented regardless of the embodiment of the compositions described in this description, in the compositions according to the invention, and in particular in the pharmaceutical compositions according to the invention, said bacteriophage suspension in said aqueous solution, or dispersing medium, is supplemented with a viscous solution or hydrogel of cellulose ether - preferably, of hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, polyethylene glycol, polyvinyl alcohol, sodium alginate, PVA-sodium alginate, acrylate, methacrylate, polyacrylate, hyaluronic acid, acrylamide, polyacrylamide (e.g., poly(N-isopropylacrylamide), poly(N-isopropylacrylamide-co-allylamine)), polyethylene glycol-maleimide, polyethylene oxide, poloxamers, guar gum, agarose, gelatin, collagen, chitosan, nanohydroxyapatite, silicate, or hydrated aluminum silicate. That is to say, the bacteriophage suspension in the aqueous solution selected according to the invention is mixed with said viscous solution or hydrogel. It is then the resulting mixture that is administered as a medicinal product.

[0037] According to advantageous embodiments, the compositions according to the invention, and in particular the pharmaceutical compositions according to the invention, are adapted for enteral administration, or adapted for administration by injection, in particular by parenteral route, including intravenous, intraarterial, intramuscular, subcutaneous and / or intra-articular administrations.

[0038] The invention also relates to compositions according to the invention, and in particular to pharmaceutical compositions according to the invention, for their use as a medicinal product, in particular for their use in the treatment of osteoarticular infections.

[0039] Furthermore, the compositions according to the invention are very simple to prepare, use widely available and inexpensive components, and are compatible with various applications, particularly for use as pharmaceutical compositions. The excipients used exhibit excellent safety and are compatible with different routes of administration, including enteral and parenteral administration, and are readily available as pharmaceutical raw materials (PRMs). Their molarity and osmolality are also suitable for administration in humans or animals, while ensuring the stability and activity of the bacteriophages present, which was a real challenge in the current context.

[0040] The invention also relates to a method for preparing a composition, and in particular a pharmaceutical composition, according to the invention, comprising introducing into an aqueous solution comprising, or even consisting of: - at least one non-ionic surfactant, and in particular a single non-ionic surfactant, selected from the polysorbate family, at a molar concentration greater than its critical micellar concentration, but not exceeding 2 mg / mL (which corresponds to 0.2% (w / v)), - one or more neutral salts formed from a pair of monovalent ions, the total molar concentration of which is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically selected from the hydrogen phosphate / dihydrogen phosphate, citric acid / citrate, and citric acid / hydrogen phosphate pairs, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water,- of purified bacteriophages in the quantity necessary to obtain in the final composition a titer of at least 108 PFU / mL, and typically from 108 to 1010 PFU / mL for bacteriophages, and in particular from 109 to 1010 PFU / mL for bacteriophages.

[0041] Due to the purification of the bacteriophages, said suspension of bacteriophages in aqueous solution obtained and therefore the composition which comprises it or which is made up of said suspension of bacteriophages in aqueous solution, comprises less than 0.9 units of bacterial endotoxins (EU) per mL and a total protein concentration of less than 0.09 mg / mL.

[0042] Furthermore, said aqueous solution, and therefore said suspension of bacteriophages in said aqueous solution, has an osmolality between 150 mOsm / kg and 600 mOsm / kg. Indeed, the presence of bacteriophages has no influence on the osmolality obtained.

[0043] In particular, in the preparation process according to the invention, after the introduction of the bacteriophages into the aqueous solution, or dispersing medium, the resulting suspension is sterilized by implementing a terminal filtration operation, by passing through a filter made of polyethersulfone, polystyrene, poly(styrene-butadiene) or polyvinylidene fluoride.

[0044] According to another aspect of it, the invention relates to the use of an aqueous solution or dispersing medium comprising, or even consisting of: - at least one non-ionic surfactant, and in particular a single non-ionic surfactant, selected from the polysorbate family, at a molar concentration above its critical micellar concentration, but not exceeding 2 mg / mL (or 0.2% (w / v)), - one or more neutral salts formed from a pair of monovalent ions, the total molar concentration of which is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably 7.0 to 7.5, typically selected from the hydrogen phosphate / dihydrogen phosphate, citric acid / citrate, and citric acid / hydrogen phosphate pairs, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, to improve storage stability at a temperature in the range of 2 to 8°C, bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria,said bacteriophages being suspended in said aqueous solution at a concentration of at least 10⁸ PFU / mL, and typically from 10⁸ to 10¹⁰ PFU / mL for bacteriophages, and in particular from 10⁹ to 10¹⁰ PFU / mL, and said composition comprising less than 0.9 bacterial endotoxin units (EU) per mL, a total protein concentration of less than 0.09 mg / mL, and said bacteriophage suspension in said aqueous solution having an osmolality between 150 mOsm / kg and 600 mOsm / kg.

[0045] In particular, according to this use, the stability obtained corresponds to a bacteriophage titer which remains greater than or equal to 108 PFU / mL, after storage of the composition at a temperature belonging to the range of 2 to 8°C, for at least one year.

[0046] In the processes and uses according to the invention, the selected aqueous solution is used as a dispersing medium for bacteriophages.

[0047] All the characteristics described in this description, in connection with the compositions according to the invention, apply to the preparation processes and uses according to the invention.

[0048] The more detailed description that follows, with reference to the accompanying figures and the examples given for illustrative purposes only, will provide a better understanding of the invention. The compositions according to the invention comprise a number of features and components which will be described in more detail.

[0049] Generally, unless otherwise specified, the terms used in the present invention have the meanings conventionally recognized by those skilled in the art, and in particular are as defined by the European Pharmacopoeia. References to the European Pharmacopoeia (Ph. Eur.) refer to the version applicable on January 1, 2024.

[0050] Osmolality is determined according to Ph. Eur. 2.2.35. Osmolality in mOsm / kg is measured by cryoscopic lowering with a cryoscopic osmometer.

[0051] The pH is determined according to Ph. Eur. 2.2.3.

[0052] Sterility is determined according to Ph. Eur. 2.6.1.

[0053] The CMC is determined by the abrupt change (bend) in the surface tension curve (in mN / m) as a function of the log of the surfactant concentration in the water. Surface tension can usefully be measured by a tensiometer, for example a ring tensiometer.

[0054] The concentration of bacterial endotoxins present, expressed in endotoxin units (EU) per mL, is determined according to Ph. Eur. 2.6.14.

[0055] The total protein concentration in the composition is determined according to the Bradford method (method 3 of Ph. Eur 01 / 2008: 20533 on the determination of total protein). A protein is defined as a macromolecule formed of one or more polypeptide chains, said polypeptide chains corresponding to a sequence of more than 20 amino acids linked together by peptide bonds.

[0056] The ionic strength is calculated using the following formula: x^2] with ci the molar concentration of the ionic species i, and z; the charge of the ion considered. The charges z; of the ions considered are as follows: Na+=1; Cl =-1; K+=1; H2PO4=-1; HPO42=-2.

[0057] In particular, the more detailed methods described in the examples may be used.

[0058] Bacteriophages

[0059] Bacteriophages of the class Caudoviricetes are phages with a head-tail structure (Zhu et al. 2022) that can exhibit lytic activity against bacteria. By lytic activity against a strain of bacteria, we mean that the Bacteriophages cause the lysis of at least some of the bacteria of said strain. The lytic activity against the target bacteria can be evaluated using any technique known to those skilled in the art, in particular in vitro by counting the plaques of lysis during titrations of bacteriophages on a bed of at least one bacterial strain targeted by the phage. The titration can be performed, for example, by spot test.

[0060] In the context of the invention, bacteriophages of the class Caudoviricetes that exhibit lytic activity against at least one strain of Gram-positive bacteria, and in particular against at least one strain of Staphylococcus aureus, are particularly advantageous and belong predominantly to the family Herelleviridae, the subfamily Twortvirinae, and the genus Silviavirus (ICTV 2023).In particular, phages exhibiting lytic activity against various strains of Gram-positive bacteria, and especially against at least one strain of Staphylococcus aureus, will be preferred. A tailed phage consists of a head containing its viral genome (in the context of this invention, it is often an icosahedral capsid containing double-stranded DNA), and a tail that plays a crucial role in the recognition and infection of target bacteria. The tail is composed of specific proteins that selectively interact with receptors on the surface of bacteria, thus enabling the phage to attach to and efficiently penetrate bacterial cells. Once inside the bacterium, the tailed phage releases its genetic material, thereby initiating the process of viral replication and ultimately leading to the lysis of the host bacterium.

[0061] Bacteriophages, and in particular those of the class Caudoviricetes (Turner, Kropinski, and Adriaenssens 2021), and especially of the genus Silviavirus, exhibiting the following morphotypes, can be used within the framework of the invention (Harada et al. 2018): - Myovirus: Myoviruses have a contractile tail. - Siphovirus: Siphoviruses have a long, non-contractile tail. - Podovirus: Podoviruses have a short, non-contractile tail.

[0062] By way of example of bacteriophages that can be used in the context of the invention, the following may be cited: - Bacteriophages directed against Staphylococcus aureus, corresponding, for example, to PP1493 and PP1815 (https: / / www.hygienes.net / actualite / focus / lansm-autor ise-un-acces-compassionnel-pour-des-bacteriophages-dans-les-infections-osteo-articulaires), to one of the bacteriophages listed in the following documents: Plumet et al. 2022, Malik et al. 2017, WO 2018 / 162566, or to one of those used in the examples that follow, namely vB_SauM-VlSA19 and vB_SauM-VlSA20 filed in GenBank under numbers ON814134.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / ON814134) and ON814135.1 (https: / / www.ncbi.nlm.nih.gov / nuccore / ON814135). These Two recent bacteriophages have also been the subject of a publication presenting TEM images and comparisons of their genomes, notably (C. Kolenda, 2022). Any other bacteriophage with lytic activity against at least one strain of Staphylococcus aureus can be used. - Bacteriophages directed against Streptococcus pneumoniae, corresponding, for example, to phage Dp-1 (siphovirus, Taxonomy ID: 59241), Bacteriophage SOCP (podovirus, Taxonomy ID: 1498213) listed in the following document: (Qadir and Sajjad 2017). - Bacteriophages directed against Enterococcus faecalis, corresponding, for example, to Ben (myovirus), Bill (myovirus), Cari (myovirus), CCS1 (myovirus), CCS2 (siphovirus), SDS1 (siphovirus) and UMP (podovirus) listed in the following document: (Wandro et al. 2022). - Bacteriophages directed against Cutibacterium acnes, corresponding, for example, to phage Y3Z (siphovirus), deposited in GenBank under number OQ411034 (https: / / www.ncbi.nlm.nih.gov / nuccore / OQ411034) (Xuan et al. 2023), or to phage PA6 (siphovirus) deposited in GenBank under number DQ431235 (https: / / www.ncbi.nlm.nih.gov / nuccore / DQ431235).

[0063] The compositions according to the invention may contain a single type of bacteriophage or a combination of different types of bacteriophages, and in particular a combination of different types of bacteriophages of the class Caudoviricetes, notably of the genus Silviavirus, having, in particular, a myovirus morphotype, which exhibit lytic activity against at least one strain of Staphylococcus aureus bacteria. The use of a combination of different types of bacteriophages may be chosen to increase the spectrum of action leading to the lysis of a greater number of target bacteria of the compositions according to the invention.

[0064] In the compositions according to the invention, the bacteriophages in suspension were purified after in vitro culture and isolation from their natural environment. The in vitro culture can be carried out using any conventional technique, particularly those described in the aforementioned publications, on host cells corresponding to one of the bacteria against which lytic activity is sought.

[0065] Purified bacteriophage suspensions can be obtained using methods described in the literature that employ filtration and / or centrifugation steps, notably those described in Kolenda et al. 2022. In particular, bacteriophages are collected from the environment (e.g., upstream of wastewater treatment plants, from cattle effluents) or are derived from clinical strains. So-called lytic bacteriophages are then isolated by successive spotting of lysis plaques isolated from bacterial agar plates and subsequently genetically characterized; the bacteriophages are trained, if necessary, to increase their activity, spectrum, and... affinity, etc. (e.g., Appelmans protocol or other technique promoting recombination events) (Burrowes, Molineux, and Fralick 2019); produced on a bacterial strain of the species against which they are directed, free of prophages, resistance factors, major virulence; purified by ultracentrifugation with a cesium chloride gradient (densities of 1.6, 1.5 and 1.3) following which they are collected between the rings corresponding to the densities of 1.6 and 1.5 then they are dialyzed several times in a PBS type buffer (from the English "phosphate-buffered saline"), finally a polishing step allows them to be resuspended in the aqueous solution (used as a bacteriophage dispersing medium) selected to form the composition according to the invention.

[0066] After such a purification step, the concentrations of bacterial endotoxins and residual bacterial proteins are considerably reduced. Thus, it is possible to obtain compositions comprising such purified bacteriophages as a therapeutic active ingredient with a bacterial endotoxin concentration of less than 0.9 EU / mL per composition. Advantageously, in the compositions according to the invention, the total protein concentration is less than 0.09 mg / mL, which corresponds almost exclusively to the constituent proteins of the bacteriophages.

[0067] The compositions according to the invention comprise or consist of a suspension of bacteriophages of the class Caudoviricetes in an aqueous solution (used as a dispersing medium for the bacteriophages) as defined within the scope of the invention. This aqueous solution is composed of water, at least one nonionic surfactant, at least one neutral salt consisting of monovalent ions, and a buffer mixture, the details of which are given below.

[0068] Non-ionic surfactants

[0069] The compositions according to the invention comprise one or more nonionic surfactants selected from polysorbates, said nonionic surfactant being present at a concentration higher than its critical micelle concentration (CMC). Its presence helps to limit the impact of cavitation, ventilation, and bacteriophage-bacteriophage aggregation phenomena. Polysorbates are known esters of fatty acids and polyoxyethylene sorbitan. In addition to ensuring the stability of the composition and maintaining bacteriophage activity, in combination with the other components present, the polysorbate(s) present allow for adjusting the wettability of the composition and the contact angles between the composition and air, and between the composition and the walls of the containers or caps. Furthermore, the polysorbate(s) present also help to limit aggregation phenomena at the gas-liquid and liquid-solid interfaces.The non-ionic surfactant of the polysorbate type, at the chosen concentration, is compatible with . administration in humans or animals, and in particular, by injection, notably with parenteral administration.

[0070] Advantageously, the quantity of polysorbate, and in particular the quantity of polysorbate 80, is chosen to ensure wettability of the packaging materials (container and cap when a cap is present) of the composition according to the invention corresponding to a material / composition contact angle of 20° to 60° for the walls of the containers and to a contact angle less than or equal to 90° for the caps.

[0071] Polysorbates are esters of fatty acids and polyoxyethylene sorbitan. In the context of the invention, polysorbate 80 (PS80 or Tween® 80) or polysorbate 20 (PS20 or Tween® 20) will preferably be used. The numbers 20, 40, 60, and 80 following the term "polysorbate" correspond to the main type of fatty acid associated with the polyoxyethylene sorbitan residue of the molecule; that is, monolaurate is indicated by 20, monopalmitate by 40, monostearate by 60, and monooleate by 80.

[0072] Neutral salts

[0073] The composition according to the invention comprises one or more neutral salts formed of monovalent ions. Typical examples of neutral salts include NaCl and KCl. The total molar concentration of the neutral salt is 75 to 160 mM. If only one neutral salt formed of monovalent ions, and in particular NaCl, is present in the composition according to the invention, its concentration in the composition is 75 to 160 mM. If several neutral salts formed of monovalent ions are present (typically NaCl and KCl) in the composition according to the invention, their total concentration in the composition is 75 to 160 mM. Advantageously, the composition according to the invention comprises only NaCl as a neutral salt formed of monovalent ions.

[0074] Buffer mixture

[0075] The composition according to the invention has a pH of 6.0 to 7.9, and preferably 7.0 to 7.5. This pH is adjusted and kept stable by the presence of a buffer mixture. The buffer mixture may consist of a weak base and its conjugate acid, or even of an acid other than the conjugate acid, or alternatively, of a weak acid and its conjugate base, or even of a base other than its conjugate base. Examples of buffer mixtures that may be used in the compositions according to the invention include the hydrogen phosphate / dihydrogen phosphate, citric acid / citrate, and citric acid / hydrogen phosphate pairs. The molarity of the buffer mixture (i.e., the sum of the molarities of the base and the acid constituting the buffer mixture) is in the range of 4 mM to 100 mM.

[0076] The buffer mixture preferably has a buffer capacity in the range of 8 mM / ApH to 30 mM / ApH.

[0077] The buffer mixture is advantageously a KH2PO4 / Na2HPO4 mixture, typically KH2PO4 at a molar concentration of 1 to 15 mM and Na2HPO4 at a molar concentration of 3 to 56 mM. With such concentrations, the molarity of the buffer mixture is in the range of 4 to 71 mM.

[0078] The molarity of the buffer mixture can be adjusted according to the desired route of administration. More specifically, the molarity of the buffer mixture can be chosen to allow for intravenous and intramuscular administration: in this case, it will be less than or equal to 25 mM.

[0079] In the context of the invention, the water used is water suitable for pharmaceutical use, in particular water for injectable preparation, in particular as defined in Ph. Eur.

[0080] Packaging

[0081] The composition according to the invention can be packaged in any suitable container, in particular in the form of a bottle, bag or syringe, delimited by one or more walls in contact with the composition which advantageously shall be made of type I glass (commonly used in the medical field and which generally has a contact angle with water of 40°), type II glass, polystyrene (which generally has a contact angle with water of 68°), polypropylene (which generally has a contact angle with water of 102°), polyethylene (which generally has a contact angle with water of 96°), polyethylene terephthalate (which generally has a contact angle with water of 64°), the walls being able or not to be coated with a silicone oil (which when present leads to a contact angle with water of 90°).In particular, for packaging the compositions according to the invention, type I or II glass containers are preferred. Type I and II glasses are, in particular, defined in Ph. Eur. 3.2.1.

[0082] Preferably, the composition exhibits a wettability of the packaging materials corresponding to a material / liquid medium contact angle of 20° to 60° with the container walls, and less than or equal to 90° with a cap capable of closing said container. Examples of materials that can constitute the cap include silicones, polypropylene, high-density polyethylene, rubbers, particularly chlorobutyl or perfluorinated bromobutyl, etc.

[0083] With such wettability in particular, the compositions according to the invention can be filtered, particularly during sterilization operations, through a polyethersulfone filter (which generally has a contact angle with water of 60°), a polystyrene filter (which generally has a contact angle with water of 68°), or a poly(styrene-butadiene) filter (which generally has a contact angle with water of 69°). Preferably, the composition ensures wettability of the filter materials such that the contact angles between the filter material and the liquid medium are less than 60°. This makes it possible to use polyvinylidene fluoride filters (contact angle with water of 145°-89°).

[0084] Further details on the compositions, and in particular the pharmaceutical compositions according to the invention and their medical uses

[0085] The composition according to the invention can directly be, as such, a pharmaceutical composition administrable to humans or animals. A pharmaceutical composition administrable to humans or animals can therefore consist of the suspension of bacteriophages of the class Caudoviricetes in the aqueous solution (used as a dispersing medium for the bacteriophages) defined within the scope of the invention. It can be adapted for enteral and topical administration (e.g., cutaneous, mucosal, buccal, ophthalmic) or for administration by injection, via the parenteral route, including intravenous, intra-arterial, intramuscular, subcutaneous, and / or intra-articular administration.

[0086] Depending on the route of administration for which a pharmaceutical composition according to the invention is intended, according to other embodiments, the latter may also comprise one or more components, and in particular one or more pharmaceutically acceptable excipients, making it suitable for the intended route of administration. In such a case, the pharmaceutical composition administrable to humans or animals comprises a composition according to the invention consisting of a suspension of bacteriophages of the class Caudoviricetes in the aqueous solution (used as a bacteriophage dispersing medium) defined within the scope of the invention, mixed with one or more other pharmaceutically acceptable excipients or one or more other active ingredients.

[0087] The pharmaceutical compositions according to the invention are intended for the treatment of conditions related to the presence of Gram-positive bacteria, and in particular Staphylococcus aureus. They will be administered to a subject, who may be a human or an animal, in an effective quantity. An "effective" quantity in the context of a treatment refers to a treatment that results in a decrease in the number of Gram-positive bacteria, and in particular Staphylococcus aureus, in a subject after said treatment compared to the number of Gram-positive bacteria, and in particular Staphylococcus aureus, before said treatment.

[0088] In the case of a pharmaceutical composition suitable for enteral and topical (e.g., cutaneous, mucosal, buccal, ophthalmic) administration, preferably, a pharmaceutical composition comprises a composition according to the invention consisting of a suspension of bacteriophages of the class Caudoviricetes in the aqueous solution (or dispersing medium) defined within the scope of the invention, mixed with a viscous solution or hydrogel of cellulose ether - preferably, of hydroxypropyl ethylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, polyethylene glycol, polyvinyl alcohol (PVA), sodium alginate, PVA-sodium alginate, acrylate, methacrylate, polyacrylate, hyaluronic acid, acrylamide, polyacrylamide (e.g., poly(N-isopropylacrylamide), poly(N-isopropylacrylamide-co-allylamine)), polyethylene glycol-maleimide, polyethylene oxide, poloxamers, guar gum, agarose, gelatin, collagen, chitosan, nanohydroxyapatite, silicate, hydrated aluminum silicate.

[0089] A pharmaceutical composition administrable to humans or animals may include a composition according to the invention consisting of a suspension of bacteriophages of the class Caudoviricetes in aqueous solution (used as a dispersing medium for the bacteriophages) defined within the scope of the invention, mixed with an antibacterial agent exhibiting synergy and / or complementarity of action enabling the improvement, increase, reinforcement and / or expansion of the antibacterial activity of the composition.For example, in the context of bacteriophage suspensions directed against Staphylococcus aureus, compounds that can potentiate the antibacterial effect of bacteriophages can be chosen from the group of M penicillins (oxacillin, cloxacillin, flucloxacillin), A penicillins (amoxicillin / clavulanic acid), synergistins (pristinamycin), fusidic acid, mupirocin, cefazolin, gentamicin, macrolides, lincosamines, trimethoprim-sulfamethoxazole, rifampicin, glycopeptides (vancomycin), ceftaroline or linezolid.

[0090] Depending on the intended application, the composition according to the invention may have a bactericidal effect or a bacteriostatic effect and may be chosen according to the condition.

[0091] By way of example, a composition of bacteriophages of the class Caudoviricetes according to the invention is a pharmaceutical composition, in particular suitable for enteral administration or an injectable pharmaceutical composition for parenteral administration and, in particular, intended for the treatment of an osteoarticular infection, septicemia or an infection in a diabetic person.

[0092] The pharmaceutical compositions according to the invention are in particular intended and adapted for use in humans.

[0093] All preferred characteristics described in this description apply to the pharmaceutical compositions, medical uses and treatment methods described in this description.

[0094] The compositions according to the invention can be used for the manufacture of a medicinal product, in particular for the manufacture of a medicinal product for the treatment of a bone and joint infection, septicemia or an infection in a person diabetic, of a drug having a bactericidal or bacteriostatic effect against Staphylococcus aureus. Treatment methods comprising the administration in humans or animals of an effective quantity of a composition according to the invention, for the treatment of an osteoarticular infection, septicemia or an infection in a diabetic person, or for obtaining a bactericidal or bacteriostatic effect against Staphylococcus aureus, are also part of the present invention.

[0095] Examples

[0096] The following examples, with reference to the attached Figures, are given for illustrative purposes only.

[0097] Figure 1A shows the UV-visible spectra of a bacteriophage suspension used in the examples during the purification steps. The UV-visible spectrum of a phage suspension obtained after the ultrafiltration step (“post-polishing”) formulated in medium F4 (KH2PO4: 1.06 mM; Na2HPO4: 2.97 mM; NaCl: 155.17 mM), shows two characteristic wavelengths (256 nm and 244 nm) corresponding to the absorbances of the protein-nucleic acid complex of the phages.

[0098] Fig. 1B shows the UV-visible spectra of a suspension of bacteriophages obtained after successive dilutions in medium F4 (KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM, initial titer: 26.7 x 108 PFU / mL).

[0099] Figure 1C shows the absorbance curve (256 nm) of a bacteriophage suspension formulated in medium F4 (KH2PO4: 1.06 mM; Na2HPO4: 2.97 mM; NaCl: 155.17 mM) as a function of the bacteriophage titer (absorbance vs. PFU / mL). The limits of detection and quantification (i.e., 3 x standard deviation / slope and 9 x standard deviation / slope) were 1.6 x 10⁸ PFU / mL and 4.7 x 10⁸ PFU / mL, respectively. The impact of the filtration used for terminal sterilization on the bacteriophage titer is approximately 10% (non-significant variation).

[0100] Figure 2 shows the wettability characterization of (A) stoppers (1. BIIR: perfluorinated bromobutyl; 2. CIIR: chlorobutyl), (B) type I glass bottles, (C) polystyrene bottles, (D) polyethylene terephthalate bottles, and (E) poly(styrene-butadiene) filters as a function of the PS80 concentration (%). The wettability of the materials is determined from the average of 3 (t) or 4 (¢) measurements of the solution / material contact angles. The error bars show the standard deviation of these measurements. The composition of the buffer mixture and neutral salts in the Fl is KH2PO4: 14.78 mM, Na2HPO4: 56.38 mM, NaCl: 154 mM. The composition of buffer mixture and neutral salts of formulation F4 is KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM. The variations in contact angle between suspensions supplemented or not with PS80 are compared by a Student-t test: NS: not significant. * p < 0.05; ** p < 0.01; *** p < 0.001.

[0101] Figure 3 shows the evolution of the aggregation index (AI) as a function of pH. The F4 bacteriophage suspension (KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM) exhibited strong bacteriophage aggregation at pH < 4. Each value corresponds to an experimental determination. AI > 30 indicates strong aggregation; AI < 7 indicates a stability threshold.

[0102] Figure 4 shows (A) the evolution of the bacteriophage titer (initial titer: 109 PFU / mL) of formulation F1-PS80 (KH2PO4: 14.78 mM; Na2HPO4: 56.38 mM; NaCl: 154 mM; PS80: 0.05% (corresponding to 0.38 mM)) packaged in type I glass bottles (5 mL filled to 3 mL; headspace: 40%) stored under shelf-life conditions (i.e., 4°C and 25°C). (B) Detail of the evolution at 5°C and (C) Detail of the evolution at 25°C. Each value is the mean ± standard deviation of three experimental determinations. The line represents the threshold below which the suspension no longer meets the stability criterion (titration <108 PFU / mL). Analysis of variance over all groups followed by multiple comparisons using Post Hoc tests (Tukey HSD): NS: not significant. * p<0.05; ** p<0.01 ; *** pcO.OOL ND : not detectable.

[0103] Figure 5 shows (A) the evolution of the titer (initial titer: 109 PFU / mL) of a Suspension of myovirus morphotype bacteriophages, formulated in F4 (KH2 PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM), after 21 days of horizontal shaking (25 Hz) in polystyrene bottles (15 mL filled to 10.6 mL; headspace: 30%) as a function of PS80 concentration. Each value is the mean ± standard deviation of two experimental determinations. (B) Analysis of variance over all groups followed by multiple comparisons using post-hoc tests (Tukey HSD or Bonferroni): NS: not significant. * p<0.05; ** p<0.01; *** pcO.OOL

[0104] Figure 6 shows the evolution of the titer (initial titer >109 PFU / mL) of a lysate Phage (unpurified; hatched bars) and a purified suspension (white bars) in formulation F4 without PS80 (KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM), packaged in polystyrene bottles (15 mL filled to 5 mL; 67% headspace) and subjected to rotary shaking (0.7 Hz) for 28 days. Each value represents the mean ± standard deviation of 3 experimental determinations. Analysis of variance was performed across all groups followed by multiple comparisons using post-hoc tests (Tukey HSD). NS: not significant. * p < 0.05; ** p < 0.01; *** p < 0.001; ND: not detected.

[0105] LIST OF RAW MATERIALS, MATERIALS AND EQUIPMENT USED IN THE EXAMPLES

[0106] The excipients used are as follows: sodium chloride (NaCl) FRESENIUS 0.9% (Fresenius Kabi, Sevres, France); water for injections Lavoisier (Laboratoires Chaix et du Marais, Gailletrous, France); PBS (phosphate-buffered saline) IX at pH 7.4 (GibcoTM, Life Technologies Corporation, Grand Island, NY, USA and Life TechnologiesTM, Paisley, England); monobasic potassium phosphate KH2PO4, CAS: 7778-77-0 (ReagentPlus®, Sigma-Aldrich®, Saint Louis, MO, USA); dibasic anhydrous sodium phosphate Na2HPO4, CAS: 7558-79-4 (CARLO ERBA Reagents SAS, Val de Reuil, France); Tween® 80 (polysorbate 80 or PS80) (EMPROVE® ESSENTIAL, Merck KGaA, Darmstadt, Germany); Tween® 20 (polysorbate 20 or PS20) (BioXtra, Merck KGaA, Darmstadt, Germany).

[0107] A 1 M hydrochloric acid (HCl) solution was prepared extemporaneously to force bacteriophage aggregation at acidic pH (see [Fig. 3]): 10.8 g HCl R (CARLO ERBA Reagents SAS, Val de Reuil, France) diluted in demineralized water, sufficient quantity to make (qsp.) 100 mL. It was used to modify the pH of the solution, in relation to the results presented [Fig. 3].

[0108] The packaging used is listed below: 5 mL type I white glass vials (SGD Pharma, Puteaux, France); chlorobutyl, Teflon-coated bromobutyl and silicone stoppers (Gravis Trelazé, Trélazé, France); Raydylyo® fluorinated polymer-coated bromobutyl stoppers (ARaymond® Life SASU, St Egreve, France); 15 mL polystyrene conical centrifuge tubes with polypropylene stoppers (Corning™ Falcon™, Fischer Scientific SAS, Illkirch, France).

[0109] GENETIC CHARACTERISTICS OF BACTERIOPHAGES USED OF MYOVIRUS MORPHOTYPE

[0110] vB_SauM-VlSA19 - of myovirus morphotype (Kolenda et al. 2022) used in all the examples that follow: • Lineage: Viruses; Duplodnaviria; Heunggongvirae; Uroviricota; Caudoviricetes', Herelleviridae; Twortvirinae; Silviavirus', unclassified Silviavirus • Taxonomy ID: 2972385 • GenBank partial genome: ON814134.1 • 245 proteins; genome size 1,138,507 bp. [YES] MATERIALS AND METHODS:

[0112] OBTAINING PURIFIED BACTERIOPHAGE SUSPENSIONS:

[0113] Bacteriophages were isolated as described in (Kolenda et al. 2022). The S. aureus strain P2SA225 (CC6 clonal complex; methicillin-susceptible; isolated from a nasal swab obtained during clinical trial ERS 1242607) was incubated for The culture was incubated for 2 hours at 37°C with shaking at 180 rpm in 1 L of Superior Broth™ culture medium (Athena Enzyme Systems™, Baltimore, MD, USA) in a 2.5 L Frenbach culture flask (Avantor™ VWR™, Radnor, PA, USA). Bacteriophage vB_SauM-VlSA19 was added to achieve a multiplicity of infection (MOI) of 102, and the mixture was then incubated for approximately 3 hours (Kolenda et al. 2022). The amount of double-stranded DNA in the Frenbach medium was then quantified using a Quantus™ Fluorometer (Promega, Madison, WI, USA) with the Quantifluor® dsDNA System kit (Promega, Madison, WI, USA). For each pg of DNA, (i) 1 unit of Benzonase® Nuclease enzyme (Sigma-Aldrich Chimie Sarl, Saint-Quentin de Fallavier, France) and (ii) 2 mL of 1 M MgCl2 (Invitrogen™, ThermoFischer Scientific Baltic UAB, Vilnius, Lithuania) were added to the Fernbach, an amount sufficient to achieve a concentration in the Fembach of 2 mM.The mixture was incubated for 1 hour at 37°C, with shaking at 180 rpm.

[0114] The product was collected and filtered through a Polycap TC 150 progressive filtration cartridge (0.8 µm - 0.22 µm) (Cytiva, Buckinghamshire, England) and packaged in 1 L uLDPE FlexFilm® bags (Avantor™, VWR™, Radnor, PA, USA). The resulting product is called phage lysate. The bacterial endotoxin concentration of this phage lysate was typically between 1 and 50 EU / mL, depending on the production lot. The total protein concentration of this phage lysate was typically greater than 0.2 mg / mL.

[0115] The product was then purified by ultracentrifugation (Boulanger 2009). Into an ultraclear, thin-walled tube (Beckman Coulter France SAS, Villepinte, France), 9 mL of (i) cesium chloride (CICs) solution (Sigma-Aldrich Chimie Sarl, Saint-Quentin de Fallavier, France) at 1.6 g / L in 0.9% Fresenius NaCl (Fresenius Kabi, Sevres, France); (ii) CICs solution at 1.5 g / L; (iii) CICs solution at 1.3 g / L; and (iv) bacteriophage suspension to be purified were added. The tube was ultracentrifuged at 120,000 g (SW32Ti rotor, Beckman, Brea, CA, USA) for 2 hours at 4°C. The purified bacteriophages were then harvested between the density rings of 1.5 and 1.6 g / L before being dialyzed (10K MWCO cassettes, Serva Electrophoresis GmbH, Heidelberg, Germany) twice in 3 L of PBS IX buffer at pH 7.4 (Gibco™, Life Technologies Corporation, Grand Island, NY, USA and Life Technologies™, Paisley, England).

[0116] After purification, the quantities of endotoxins and total protein were evaluated. The concentration of bacterial endotoxins in the formulations was between 0.05 and 0.5 EU / mL, depending on the production batch, and their Total protein concentrations were <0.01-0.08 mg / mL, depending on the production batch.

[0117] PREPARATION OF BACTERIOPHAGE COMPOSITIONS:

[0118] A 15 mL Amicon® Ultra ultrafiltration unit made of Ultracel-100 regenerated cellulose membrane (100 kD, Merck-Millipore, Sigma-Aldrich Chimie SarL, Saint-Quentin de Fallavier, France) was filled with the purified bacteriophage suspension obtained and centrifuged (Centrifuge 5810 with rotor A-4-81 Eppendorf SE, Hamburg, Germany) for 15 minutes at 3100 g. This step is commonly referred to as polishing. The retentate was then resuspended in the selected formulation aqueous solution (bacteriophage dispersing medium) (detailed below), and the resulting composition was stored in 125 mL polyethylene terephthalate (PET) bottles (PharmaTainer™, SaniSure®, Camarillo, CA, USA).

[0119] Then the formulation was sterilized by terminal filtration. More specifically, this filtration consists of filtering and aliquoting the suspension in the packaging of interest using a 3-piece 10 mL BD Plastipak™ syringe (Becton Dickson France SAS, Grenoble, France) connected to a 0.22 µm polyethersulfone filtration unit (Sterivex-GP, Millipore Merck KGaA, Damstadt, Germany).

[0120] Fig. 1A shows the evolution of the UV-visible spectrum of the bacteriophage suspension as a function of the purification steps. The ultrafiltration step during the preparation of the bacteriophage compositions, called polishing, is characterized by a UV-visible spectrum with two absorbances at 244 nm and 256 nm corresponding to the protein-nucleic acid complex (Morag et al. 2018). Determining the absorbance at 350 nm allows for the assessment of the aggregation index (AI) of the suspended bacteriophages (Pignataro, Herrera, and Dodero 2020). A linear relationship between the maximum absorbance and the bacteriophage titer was demonstrated. Fig. 1B shows the evolution of the UV spectrum as a function of dilution, and Fig. 1C illustrates the non-significant reduction in the bacteriophage titer following the final sterilizing filtration.

[0121] STABILITY TEST UNDER STIRRING

[0122] The prepared compositions were placed either in (i) vertical rotary stirring at 40 rpm (Cole-Parmer® TR-200D Stuart Series, Saint Neots, England); or in (ii) horizontal shaking with vibrating motion between 150 and 2000 rpm (3 mm orbit, Multi Reax, Heidolph Instruments, Schwabach, Germany).

[0123] MEASURES TAKEN

[0124] Absorbance measurements were performed using a V-730 UV-Visible spectrophotometer (Jasco, Lisses, France). The aggregation index (AI) was calculated based on the measures absorbance and according to the formula y _ x 100 (Pignataro, Herrera, and Dodero 2020) with Xmax measured at 255 nm. With AI > 30: strong aggregation; AI < 7: stability threshold. pH and osmolality measurements were carried out respectively with the InLab Ultra-Micro ISM probe linked to the SevenCompact™ Duo S213 pH meter (Mettler-Toledo SAS, Viroflay, France) and the OsmoPRO (Radiometer SAS, Neuilly-Plaisance, France).

[0125] Bacteriophage titer measurements (PFU / mL) were performed by spot titration on a reference bacterial strain (in this case, P2SA225 for bacteriophage vB_SauM-VlSA19). P2SA225 was cultured the previous day at 180 rpm and 37°C in 9 mL of Trypto Casein Soy culture medium (TSB, bioMérieux SA, Marcy-L'Etoile, France). After approximately 20 hours of incubation, 500 pL of bacterial broth was transferred to a 120 x 120 mm square polystyrene Petri dish (Greiner Bio-one, Courtaboeuf, France). Approximately 30 mL of liquefied TSB-soft (30 g / L solid TSB BD-Difco™ - 7.56 g / L granulated agar BD-Difco™ (FisherScientific SAS, Illkirch, France)) was poured over the mixture and homogenized before drying to form an agar plate. The suspension to be titrated was serially diluted 1:10 in TSB (bioMérieux SA, Marcy-L'Etoile, France) in a 96-well plate, and then 5 µL from each well was loaded onto the agar plate.Each measurement was performed in triplicate in bacterial broth. Bacteriophage titers were read after incubation for 20 h at 37 °C.

[0126] Contact angle measurements were performed by depositing 10 pL of liquid on the surface of interest, then a photo was taken and the angle at the interface was measured with the Image! software (Nih.gov).

[0127] Statistical analyses were carried out, as indicated in the figure legends, by ANOVA followed by multiple comparisons by Post Hoc (Tukey HSD or Bonferroni) or Student's test with a=0.05, using KaleidaGraph software (version 3.6, Synergy Software).

[0128] METHOD FOR PREPARING AQUEOUS SOLUTIONS (BACTERIOPHAGE DISPERSING MEDIUM) OF THE COMPOSITIONS:

[0129] The formulations were prepared as detailed below, at the latest the day before the bacteriophages were suspended: • Fl: KH2PO4: 14.78 mM; Na2HPO4: 56.38 mM; NaCl: 154 mM. In two sterile 180 mL polypropylene powder containers (CORNING® GOSSELIN™, Borre, France), weigh out 800 mg of Na2HPO4 and 200 mg of KH2PO4, respectively. Dissolve both powders in less than 100 mL of 0.9% NaCl added using a 30 mL BD Plastipak™ three-piece syringe (Becton Dickson France SAS, Grenoble, France), then transfer the liquid into the container holding the Na2HPO4, which was tareed before adding the powder. Finally, add 0.9% NaCl, q.s. 100 g. If the If dissolution is not complete, place the powder container in an ultrasonic bath for 10 minutes at 40°C (FB15051, Fischerbrand®, FisherScientific SAS, Illkirch, France). • F2: water for injection preparation. • F1-PS80 (composition according to the invention): KH2PO4: 14.78 mM; Na2HPO4: 56.38mM; NaCl: 154 mM; PS80: 0.05% (corresponding to 0.38 mM) Same formulation as Fl. Then add 47 pL of PS80 to 100 mL of Fl. • F1-PS20 (composition according to the invention): KH2PO4: 14.78 mM; Na2HPO4: 56.38mM; NaCl: 154 mM; PS20: 0.05% (corresponding to 0.44 mM) Same formulation as Fl. Then add 46 pL of PS20 to 180 mL of Fl. • F2-PS80: water for injection (WFI); PS80: 0.05% (corresponding to 0.38 mM) Weigh 100 mg of EPPI into a sterile 180 mL powder container (CORNING® GOSSELIN™, Borre, France). Add 47 pL of PS80. • F3-PS80: NaCl 0.9%; PS80: 0.05% (corresponding to 0.38 mM) Weigh 100 mg of 0.9% NaCl (corresponding to 154 mM) into a sterile 180 mL powder container (CORNING® GOSSELIN™, Borre, France). Add 47 pL of PS80. • F4-PS80 (composition according to the invention): KH2PO4: 1.06 mM; Na2HPO4: 2.97mM; NaCl: 155.17 mM; PS80: 0.05% (corresponding to 0.38 mM) Weigh 100 mg of PBS IX at pH 7.4 (GibcoTM, Life Technologies Corporation, Grand Island, NY, USA and Life TechnologiesTM, Paisley, England) into a sterile 180 mL powder container (CORNING® GOSSELIN™, Borre, France). Add 47 pL of PS80.

[0130] The % are mass percentages relative to the volume of the aqueous solution. The resulting compositions were then immediately packaged in the selected container.

[0131] The concentration of bacterial endotoxins in all formulations was between 0.05 and 0.5 EU / mL, and their total protein concentration was <0.01 to 0.08 mg / mL.

[0132] RESULTS OBTAINED

[0133] In the examples according to the invention, highly concentrated (>10⁸ PFU / mL) suspension compositions of Caudoviricetes phages were obtained and proved resistant to pH variations, adsorption onto packaging materials, and mechanical stress known to reduce bacteriophage activity during (i) the production process, (ii) storage, and (iii) transport. The suspension composition was chosen based on intrinsic biochemical and biophysical properties evaluated during forced stress tests (mechanical agitation, pH, ionic strength, wettability of packaging materials). (Nowak et al. 2017; Dumas, Huille, and Prades 2019) to reduce (i) the exposure of hydrophobic regions of bacteriophage proteins, (ii) the rate of non-covalent (reversible hydrophobic and / or electrostatic interactions) and covalent (disulfide bridge formation) bacteriophage (potentially immunogenic) aggregates.

[0134] To assess the stability of bacteriophage suspension compositions, and in particular to evaluate resistance to aggregation and pH variations, an aggregation test induced (i) by rotary agitation (40 rpm, 0.7 Hz), (ii) by horizontal agitation (1500 rpm, 25 Hz) of the final packaging was carried out for 14–21 days at room temperature (15°C–25°C) (Kopp et al. 2020). Measurement of the UV-visible spectrum recorded during the test made it possible to determine the aggregation index (AI) of the bacteriophages. A spot titration of the bacteriophages was performed during and at the end of the test.

[0135] In order to further limit the aggregation phenomena (i) bacteriophage-bacteriophage, (ii) at gas-liquid and liquid-solid interfaces, a primary packaging (bottle and cap) was chosen taking into account: the filling volume in relation to the total volume offered by the packaging; the wettability of the primary packaging.

[0136] The selected packaging met the following criteria: - Polystyrene bottle. The wettability of the polystyrene bottle was significantly improved, achieving a value below 70°, by the presence and quantity of selected non-ionic surfactant, as shown in [Fig. 2] ©, compared to a solution without surfactant. The hydrophilic surface area of ​​the bottle (assessed by a contact angle with water of less than 90°) helps to limit aggregation between the hydrophobic parts of the bacteriophage proteins and the bottle walls. - Type I pharmaceutical glass bottle. The wettability of the type I glass bottle was significantly improved, achieving a value below 32°, by the presence and quantity of a selected non-ionic surfactant, as shown in [Fig. 2] (B). The hydrophilic surface area of ​​such bottles (assessed by a contact angle with water of less than 90°) helps to limit aggregation between the hydrophobic parts of bacteriophage proteins and the bottle walls. - Stopper material. The wettability of bromobutyl stoppers coated with a perfluorinated polymer (BIIR) and chlorobutyl (CIIR) was significantly improved, achieving a value below 90°, by the presence and quantity of a selected non-ionic surfactant, as shown in [Fig. 2], parts A-1 and A-2.

[0137] By way of comparison, after 14 days of rotary agitation at 0.7 Hz, as detailed in Table 1, the bacteriophage placed in surfactant-free suspensions (F1: buffered saline solution and F2: water for injection) exhibits a drop in its bacteriophage titer of at least 2 log, associated with a high aggregation index measured by UV spectrophotometry (25 and a negative value, respectively), indicating that the aggregates formed significantly disrupt the incident light. Thus, the presence of nonionic surfactant at the recommended concentration is necessary to maintain the titer and prevent aggregation. However, again according to Table 1, the simple addition of surfactant is not sufficient to reduce aggregation to an acceptable level (AI of 12 at day 14 for formulation F2-PS80; AI of 7 at day 14 for formulation F3-PS80).

[0138] By way of illustration, [Fig. 3] shows the evolution of the aggregation rate of the F4-PS80 bacteriophage suspension as a function of pH (the pH is modified by the addition of HCl). The presence of the phosphate saline buffer in the compositions used in the examples allows for maintaining a pH close to neutral. Unexpectedly and unpredictably, the addition of neutral salts formed from a monovalent pair such as NaCl (which are in the first elements of the Hofmeister series and therefore likely to induce aggregation (Okur et al. 2017)) enhanced the antiaggregatory power and the stability to shaking of the bacteriophage suspensions. Indeed, according to Table 1: - With comparable packaging (5 mL type I glass filled to 3 mL), the reduction in bacteriophage titer is greater in formulation Fl (bacteriophage in 338 mM phosphate saline buffer) than in formulation F2 (bacteriophages in water for injection). Thus, while bacteriophages exhibit relative stability in water under agitation, bacteriophage-water interactions are destabilized by the addition of kosmotropic anions (present in Fl). The Fl formulation without surfactant is insufficient to maintain the titer under agitation. -The F1-PS80 and F4-PS80 compositions according to the invention, containing the electrolytes described above, preserve the bacteriophage titer and have an aggregation index close to 3, while the F2-PS80 and F3-PS80 formulations free of phosphate buffer have a preserved titer but an aggregation index greater than or equal to 7, which is predictive of instability.

[0139] The results in Table 1 illustrate that one without the other, i.e., (i) saline solution containing a buffer mixture, of the phosphate type in the illustrated example and (ii) PS80, are insufficient to maintain the antibacterial activity of bacteriophages as well as acceptable aggregation rates throughout the stages through which a finished product passes, e.g., shelf-life storage; transport.

[0140] Table 1 shows the particle size, electrokinetic, and bacteriophage titer analyses after 14 days of rotary agitation (0.7 Hz) at room temperature 15°C–25°C for compositions outside the invention and according to the invention, corresponding to myovirus morphotype bacteriophage suspensions comprising a buffer mixture and a neutral surfactant, selected from the polysorbate (PS) series, packaged (i) in polystyrene bottles (20 mm diameter, 2 or 5 mL fill volume in a 15 mL bottle closed with an HDPE cap), (ii) in type I bottles (20 mm diameter, 3 mL fill volume in a 5 mL bottle closed with a perfluorinated bromobutyl cap). The initial bacteriophage titer was 10⁹ PFU / mL. Each value is the mean ± standard deviation of 3 (t) or 4 (¢) experimental determinations. ND: not determined.

[0141] Aggregation indices of 7 or more are predictive of a decline in stability over time.

[0142] [Tables 1] Formulations and conditioning Concentration of buffer mixture (m M) Buffer capacity mM / Ap H Ionic strength (mM) Osmolality (mOsm / kg )• PH AI+ Titration 108* (PFU / mL) ' Fl KH2PO4: 14.78 m M; Na2HPO4: 56.38 mM; NaCl: 154 mM 3 mL - Type I glass bottle 71 28 338 431 ± 1 7.3 -32 ± 15 0.01 ±<0.01 F2 Water for injections 2 mL - Polystyrene bottle 0 0 0 ND ND ND <0.01 3 mL - Type I glass bottle 0 0 0 4 ±< 1 6.7 25 ±<1 0.6 ± 0.2 F2- PS8 Water for injections; PS80: 0.05% 0 2 mL - Polystyrene bottle 0 0 0 ND ND ND 0.06 ±0.07 3 mL - glass vial of type I 0 0 0 4 + < 1 6.6 12 + 1 2+1 F3- PS8 0 NaCl : 154 mM ; PS80 : 0.05% 3 mL - glass vial of type I 0 0 154 287 + 1 6.5 7 + 1 3+1 F4- PS8 0 KH2PO4 : 1.06 m M ; Na2HPO4: 2.97 mM NaCl : 155.17 m M ; PS80: 0.05% 5 mL - polystyrene vial 4 8 165 294+1 7.4 4 +<1 2 + <0.01 Fl- PS8 0 KH2PO4: 14.78 m M ; Na2HPO4: 56 .38 mM ; NaCl: 1 54 mM ; PS80: 0 .05% 3 mL - glass bottle of type I 71 28 338 433 + 1 7.3 2 + 1 5 + 3 5 mL - polystyrene bottle 71 28 338 423 + 1 7.3 3 + 1 0.47 + 0.47 + PS - 0.002 KH2PO4: 14.78 m M ;Na2HPO4: 56.38 mM ; NaCl: 1 54 mM ; PS20: 0.05% 5 mL - polystyrene vial 71 28 338 423 + 1 7.3 6 + 7 0.65 + 0.09

[0143] A measurement of the bacteriophage titer (PFU / mL) after rotary shaking (0.7 Hz) confirms the link between aggregation and antibacterial activity (Table 2). In an initial early phase (< 5 days), in the absence of PS80, significant aggregation is observed, followed, between 5 and 14 days, by probable irreversible aggregation and then fragmentation of the bacteriophages resulting in a lack of antibacterial activity (Table 2).

[0144] Table 2 shows: the bacteriophage titer of bacteriophage suspensions formulated in F4 (KH2PO4: 1.06 mM, Na2HPO4: 2.97 mM, NaCl: 154 mM), packaged in a polystyrene bottle (15 mL filled to 6 mL; headspace: 60%) with a high-density polyethylene (HDPE) stopper, as a function of the PS80 concentration (%) after 14 days of rotary agitation (initial titer: 109 PFU / mL, 40 rotations per minute, 0.7 Hz).

[0145] Each value is the mean ± standard deviation of 3 or 4 experimental determinations. Analysis of variance on all groups followed by multiple comparisons using post-hoc tests (Tukey HSD or Bonferroni): NS: not significant. * p < 0.05; *** p < 0.0001.

[0146] [Tables2] Composition [1] PS80 (%) Contact angle PFU / mL [l] / polystyrene (bottle) [1] / PEHD (cap) a: 0 74° 97° nd b: 0.001 72° 97° 2 %102±4 x 10W;“ c: 0.005 63° 92° 6x 106±2x 10” 0.05 56° 70° 2 x 10% 1 x 10

[0147] For comparison, the aggregation rates and titers of bacteriophage suspensions of the class Caudoviricetes subjected (i) to rotary shaking (14 days, 40 rotations per minute, 0.7 Hz), (ii) maintained in two buffered aqueous solutions of different ionic strengths (F1-PS80 and F4-PS80), (iii) containing 0.05% PS80, (iv) packaged in type I glass bottles (5 mL filled to 3 mL, headspace: 40%), and (v) closed with a perfluorinated BIIR stopper are shown in Table 1. The compositions F1-PS80 and F4-PS80, having concentrations of sodium chloride (155 mM) - phosphate buffer (approximately 4 and 71 mM, respectively) - 0.05% PS80 - type I borosilicate glass closed with a perfluorinated BIIR stopper, preserve bacteriophage titer under conditions of intense mechanical stress.

[0148] In the absence of agitation and surfactant, the composition of sodium chloride (155 mM) - phosphate buffer (4-71 mM) - type I borosilicate glass closed with a perfluorinated BIIR stopper maintains the bacteriophage titer as a function of storage temperature ([Fig. 4]-A and [Fig. 4]-B). At 5°C, the bacteriophage titer does not show a significant decrease at one year compared to day 0 ([Fig. 4]-B), whereas a decrease of approximately 4 log from the initial titer is observed for a storage temperature of 25°C ([Fig. 4]-C). These differences are explained, in part, by the The reduction of Brownian motion at low temperatures reduces material-bacteriophage and bacteriophage-bacteriophage collisions (i.e., bacteriophages are non-mobile colloidal particles (Kasman and Porter 2022)), leading to adsorption onto materials, aggregation / agglomeration between bacteriophages, and then fragmentation of viral particles. It should be noted that after one year, the titer of a bacteriophage suspension stored at 5°C is still above the stability threshold of 10⁸ PFU / mL (8 log).

[0149] In order to distinguish the effects of cavitation and / or ventilation (produced by the rotation and falling of the suspension) from purely mechanical effects, a bacteriophage aggregation test induced by horizontal agitation (1500 rotations per minute, 25 Hz) was carried out for 21 days at room temperature (15°C–25°C) in a polystyrene bottle (15 mL filled to 10.6 mL, headspace: 30%). The results of the reduction in bacteriophage titer over time for different concentrations of PS80 are shown in [Fig. 5] (A) and [Fig. 5] (B). It should be noted that the tests at 0.02% and 0.05% are above the CMC of polysorbate 80.

[0150] The instability under shaking of a purified suspension formulated without PS80, compared to a phage lysate (unpurified), is shown in [Fig. 6]. Indeed, after only two days of shaking, the titer of the purified suspension is less than 108 PFU / mL, whereas the titer of the unpurified suspension is still greater than 108 PFU / mL after one month of shaking. References

[0151] Boulanger, Pascale. 2009. “Purification of Bacteriophages and SDS-PAGE Analysis of Phage Structural Proteins from Ghost Particles.” In Bacteriophages: Methods and Protocols, Volume 2 Molecular and Applied Aspects, edited by Martha RJ Clokie and Andrew M. Kropinski, 227-38. Methods in Molecular Biology™. Totowa, NJ: Humana Press, https: / / doi.org / 10.1007 / 978-l-60327-565-l 13.

[0152] Batinovic, Steven, Flavia Wassef, Sarah A. Knowler, Daniel TF Rice, Cassandra R. Stanton, Jayson Rose, Joseph Tucci, et al. “Bacteriophages in Natural and Artificial Environments”. Pathogens 8, no. 3 (July 12, 2019): 100. https: / / doi.org / 10.3390 / pathogens8030100.

[0153] Burrowes, Ben H., Lan J. Molineux, and Joe A. Fralick. “Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol”. Viruses 11, no. 3 (March 11, 2019): 241, https: / / doi.org / 10.3390 / vl 1030241.

[0154] Drab, Marek. 2018. “Phage Aggregation-Dispersion by Ions: Striving beyond Antibacterial Therapy.” Prendres in Biotechnology 36 (9): 875-81. https: / / d0i.0rg / l 0.1016 / j.tibtech.2018.03.002.

[0155] Dublanchet, Alain. 2014. “What is phage therapy?” » Hegel 4 (4): 354-70. https: / / doi.org / 10.4267 / 2042 / 54390.

[0156] Dumas Jacques, Sylvain Huille, et Catherine Prades. 2019. « Développabilité ». médecine / sciences 35 (12): 1163-70. https: / / doi.org / 10.105 l / medsci / 2019231.

[0157] Duyvejonck, Hans, Maya Merabishvili, Mario Vaneechoutte, Steven de Soir, Rosanna Wright, Ville-Petri Friman, Gilbert Verbeken, et al. « Evaluation of the Stability of Bactériophages in Different Solutions Suitable for the Production of Magistral Préparations in Belgium ». Viruses 13, n° 5 (8 mai 2021): 865. https: / / doi.org / 10.3390 / vl305Q865.

[0158] Ergin, Firuze. 2022. « Effect of Freeze Drying, Spray Drying and Electrospraying on the Morphological, Thermal, and Structural Properties of Powders Containing Phage Félix 01 and Activity of Phage Félix 01 during Storage ». Powder Technology 404 (mai): 117516. https: / / doi.Org / 10.1016 / j.powtec.2022.117516.

[0159] European Medicines Agency. 2022. « List of the “Main Therapeutic Groups” (MTGs) in Crisis Preparedness EMA / 243660 / 2022”.https: / / www.ema.e uropa.eu / en / documents / other / list-main-therapeutic-groups-mtgs-crisis-preparedness en.pdf.

[0160] Ferry, Tristan, Camille Kolenda, Thomas Briot, Aubin Souche, Sébastien Lustig, Jérôme Josse, Cécile Batailler, et al. 2021. “Past and Future of Phage Therapy and Phage-Derived Proteins in Patients with Bone and Joint Infection”. Viruses 13 (12): 2414. https: / / doi.org / 10.3390 / vl3122414.

[0161] Francius, Grégory, Manon Cervulle, Eloïse Clément, Xavier Bellanger, Saeid Ekrami, Christophe Gantzer, and Jérôme FL Duval. 2021. “Impacts of Mechanical Stiffness of Bacteriophage-Loaded Hydrogels on Their Antibacterial Activity.” ACS Applied Bio Materials 4 (3): 2614-27. https: / / doi.org / 10.1021 / acsabm.0c01595.

[0162] Harada, Liliam K., Erica C. Silva, Welida F. Campos, Fernando S. Del Fiol, Marta Vila, Krystyna Dqbrowska, Victor N. Krylov, et Victor M. Balcâo. 2018. « Biotechnologie al Applications of Bactériophages: State of the Art ». Microbiological Research 212-213 (juillet): 38-58. https: / / doi.Org / 10.1016 / j.micres.2018.04.007.

[0163] Herelle, F. d’. 1931. « Bactériophage as a Treatment in Acute Medical and Surgical Infections ». Bulletin ofthe New York Academy of Medicine 7 (5): 329-48.

[0164] Hoe, Susan, Mohammed A. Boraey, James W. Ivey, Warren H. Finlay, et Reinhard Vehring. 2014. « Manufacturing and Device Options for the Delivery of Biotherapeutics ». Journal of Aérosol Medicine and Pulmonary Drug Delivery 27 (5): 315-28. https: / / doi.org / 10.1089 / jamp,2013.1090.

[0165] ICTV. « Genus: Silviavirus », 2023. https : / / ictv. global / report / chapter / herelleviridae / h erelleviridae / silviavirus.

[0166] Interagency Coordination Group on Antimicrobial Resistance. “No Time to Wait: Securing the Future from Drug-Resistant Infections.” » WHO Antimicrobial Resistance Division, 2019. https: / / www.who.int / antimicrobial-resistance / interagency-coordinatio n-group / IACG final summary EN.pdf.

[0167] Kasman, Laura M., and La Donna Porter. 2022. “Bacteriophages”. In StatPearls. Treasure Island (FL): StatPearls Publishing. http: / / www.ncbi.nlm.nih.gov / books / NBK493185 / .

[0168] Kolenda, Camille, Mathieu Médina, Mélanie Bonhomme, Floriane Laumay, Tiphaine Roussel-Gaillard, Patricia Martins-Simoes, Anne Tristan, et al. 2022. “Phage Therapy against Staphylococcus Aureus: Selection and Optimization of Production Protocols of Novel Broad-Spectrum Silviavirus Phages.” Pharmaceutics 14 (9): 1885. https: / / doi.org / 10.3390 / pharmaceuticsl4Q91885.

[0169] Kopp, Marie R.G., Adriana-Michelle Wolf Pérez, Marta Virginia Zucca, Umberto Capasso Palmiero, Brigitte Friedrichsen, Nikolai Lorenzen, et Paolo Arosio. « An accelerated surface-mediated stress assay of antibody instability for developability studies ». mAbs 12, n° 1 (2020): 1815995. https: / / doi.org / 10.1080 / 1942086 2.2020.1815995.

[0170] Malik, Danish J., Ilya J. Sokolov, Gurinder K. Vinner, Francesco Mancuso, Salvatore Cinquerrui, Goran T. Vladisavljevic, Martha R.J. Clokie, Natalie J. Garton, Andrew G.F. Stapley, et Anna Kirpichnikova. « Formulation, Stabilisation and Encapsulation of Bactériophage for Phage Therapy ». Advances in Colloid and Interface Science 249 (novembre 2017): 100-133. https: / / doi.Org / 10.1016 / j.cis.2017.05.014.

[0171] Mendez, J, J Jofre, F Lucena, N Contreras, K Mooijman, et R Araujo. 2002. « Conservation of Phage Reference Materials and Water Samples Containing Bactériophages of Enteric Bacteria ». Journal ofVirological Methods 106 (2): 215-24. https: / / doi.org / 10.1016 / S0166-0934(02)00163-5.

[0172] Meyer, Abigail, Melissa Greene, Chad Kimmelshue, et Rebecca Cademartiri. 2017. « Stabilization of T4 Bactériophage at Acidic and Basic PH by Adsorption on Paper ». Colloids and Surfaces B: Biointerfaces 160 (décembre): 169-76. https: / / doi.org / ! 0.1016 / j.colsurfb.2017.09.002.

[0173] Miçdzybrodzki, Ryszard, Naomi Hoyle, Fikria Zhvaniya, Marzanna Lusiak-Szelachowska, Beata Weber-Dqbrowska, Malgorzata Lobocka, Jan Borysowski, et al. 2018. « Current Updates from the Long-Standing Phage Research Centers in Georgia, Poland, and Russia ». Bactériophages: Biology, Technology, Therapy, édité par David R. Harper, Stephen T. Abedon, Benjamin H. Burrowes, et Malcolm L. McConville, 1-31. Cham: Springer International Publishing. https: / / doi.org / 10.1007 / 978-3-319-40598-8 31-1.

[0174] Morag, Omry, Nikolaos G. Sgourakis, Gili Abramov, et Amir Goldbourt. 2018. « Filamentous Bactériophage Viruses: Préparation, Magic-Angle Spinning Solid-State NMR Experiments, and Structure Détermination ». Methods in Molecular Biology (Clifton, N.J.) 1688: 67-97. https: / / doi.org / 10.1007 / 978-l-4939-7386-6 4.

[0175] Nowak, Christine, Jason K. Cheung, Shara M. Dellatore, Amit Katiyar, Ram Bhat, Joanne Sun, Gomathinayagam Ponniah, et al. “Forced Degradation of Recombinant Monoclonal Antibodies: A Practical Guide”. mAbs 9, No. 8 (30 August 2017): 1217-30. https: / / doi.org / 10.1080 / 19420862.2017.13686Q2.

[0176] Okur, Halil I., Jana Hladilkovâ, Kelvin B. Rembert, Younhee Cho, Jan Heyda, Joachim Dzubiella, Paul S. Cremer, and Pavel Jungwirth. 2017. “Beyond the Hofmeister Series: lon-Specific Effects on Proteins and Their Biological Functions”. The Journal ofPhysical Chemistry B 121(9): 1997-2014. https: / / doi.org / 10.1021 / acs.jpcb.6bl0797.

[0177] Pignataro, Mary Florence, Mary Georgina Herrera, and Veronica Elizabeth Dodero. 2020. “Evaluation of Peptide / Protein Self-Assembly and Aggregation by Spectroscopic Methods”. Molecules 25(20):4854.

[0178] Pirnay, Jean-Paul, Gilbert Verbeken, Pieter-Jan Ceyssens, Isabelle Huys, Daniel De Vos, Charlotte Ameloot, et Alan Fauconnier. 2018. « The Magistral Phage ». Viruses 10 (2): 64. https: / / doi.org / 10.3390 / vlQ020064.

[0179] Plumet Lucile, Ahmad-Mansour Nour, Dunyach-Remy Catherine, Kissa Karima, Sotto Albert, Lavigne Jean-Philippe, Costechareyre Denis et Molle Virginie, 2022, «Bactériophage Therapy for Staphylococcus Aureus Infections: A Review of Animal Models, Treatments, and Clinical Trials » Frontiers in Cellular and Infection, Volume 12, Article 907314, 1-16.

[0180] Qadir, Muhammad Imran, et Sahir Sajjad. « Phage Therapy against Streptococcus Pneumoniae: Modem Tool to Control Pneumonia ». Critical Reviews in Eukaryotic Gene Expression 27, n° 4 (2017): 289-95. https: / / doi.org / 10.1615 / CritRevEuk aryotGeneExpr.2017019527.

[0181] Richter, Eukasz, Karolina Ksiçzarczyk, Karolina Paszkowska, Marta Janczuk-Richter, Joanna Niedziôlka-Jônsson, Jacek Gapinski, Marcin Los, Robert Holyst, et Jan Paczesny. 2021. « Adsorption of Bactériophages on Polypropylene Labware Affects the Reproducibility of Phage Research ». Scientific Reports 11 (1): 7387. https: / / doi.org / 10.1038 / s41598-Q21-86571-x.

[0182] Stone, Edel, Katrina Campbell, Irene Grant, et Olivia McAuliffe. 2019. « Understanding and Exploiting Phage-Host Interactions ». Viruses 11 (6): 567. https: / / doi.org / 10.3390 / vl 1060567.

[0183] Tanir, Tayfun, Marvin Orellana, Aster Escalante, Carolina Moraes de Souza, et Michael S. Koeris. 2021. « Manufacturing Bactériophages (Part 1 of 2): Cell Line Development, Upstream, and Downstream Considérations ». Pharmaceuticals 14 (9): 934. https: / / doi.org / 10.3390 / phl4090934.

[0184] Turner, Dann, Andrew M. Kropinski, and Evelien M. Adriaenssens. “A Roadmap for Genome-Based Phage Taxonomy”. Viruses 13, No. 3 (March 2021): 506. https: / / doi.org / 10.3390 / vl30305Q6.

[0185] Verbeken, Gilbert, and Jean-Paul Pirnay. 2022. “European Regulatory Aspects of Phage Therapy: Magistral Phage Preparations.” Current Opinion in Virology 52 (February): 24-29. https: / / doi.Org / 10.1016 / j.coviro.2021.ll.005.

[0186] Vidal, Louis. 1943. Dictionary of pharmaceutical specialties. Paris: Office de vulgarisation pharmaceutique, https: / / www.biusante.parisdescartes.fr / histoire / medica / resultats / index.php?do=chapitre&cote=pharma pl 1247x1943.

[0187] Walker, Peter J., Stuart G. Siddell, Elliot J. Lefkowitz, Arcady R. Mushegian, Evelien M. Adriaenssens, Poliane Alfenas-Zerbini, Andrew J. Davison, et al. « Changes to Virus Taxonomy and to the International Code of Virus Classification and Nomenclature Ratified by the International Committee on the Taxonomy of Viruses (2021) ». Archives of Virology 166, No. 9 (September 1, 2021): 2633-48. https: / / doi.org / l 0.1007 / s00705-021-05156-1

[0188] Wandro, Stephen, Pooja Ghatbale, Hedieh Attai, Clark Hendrickson, Cyril Samillano, Joy Suh, Sage JB Dunham, David T. Pride, and Katrine Whiteson. “Phage Cocktails Constrain the Growth of Enterococcus”. mSystems 7, No. 4 (2022): eOOO 19-22. https: / / doi.org / 10.1128 / msystems.00019-22.

[0189] WO 2018 / 162566

[0190] Xuan, Guanhua, Yixian Wang, Yinfeng Wang, Hong Lin, Changyuan Wang, et Jingxue Wang. « Characterization of the newly isolated phage Y3Z against multi-drug résistant Cutibacterium acnés ». Microbial Pathogenesis 180 (1 juillet 2023): 10611L https: / / doi.Org / 10.1016 / j.micpath.2023.106111.

[0191] Zaczek, Maciej, Andrzej Gôrski, Beata Weber-Dqbrowska, Slawomir Letkiewicz, Wojciech Fortuna, Pawel Rogôz, Edyta Pasternak, et Ryszard Miçdzybrodzki. 2022. « A Thorough Synthesis of Phage Therapy Unit Activity in Poland—Its History, Milestones and International Récognition ». Viruses 14 (6): 1170. https: / / doi.org / ! 0.3390 / v 14061170.

[0192] Zhu, Yilin, Jiayu Shang, Cheng Peng, et Yanni Sun. « Phage family classification under Caudoviricetes-. A review of current tools using the latest ICTV classification framework ». Frontiers in Microbiology 13 (2022). https: / / www.frontiersin.org / articl es / 10.3389 / fmicb.2022.1032186.

Claims

Demands

1. A composition, and in particular a pharmaceutical composition, comprising or consisting of a suspension of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, at a titer of at least 10⁸ PFU / mL, typically at a concentration of 10⁸ to 10¹⁰ PFU / mL, and in particular 10⁹ to 10¹⁰ PFU / mL, in an aqueous solution comprising or consisting of: - at least one non-ionic surfactant, and in particular a single non-ionic surfactant, selected from the polysorbate family, at a molar concentration above its critical micellar concentration, but not exceeding 2 mg / mL, - one or more neutral salts formed of a pair of monovalent ions, the total molar concentration of which is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably from 7.0 to 7.5,typically selected from the pairs hydrogen phosphate / dihydrogen phosphate, citric acid / citrate and citric acid / hydrogen phosphate, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, said composition comprising less than 0.9 units of bacterial endotoxins (EU) per mL, a total protein concentration of less than 0.09 mg / mL and said bacteriophage suspension in said aqueous solution having an osmolality between 150 mOsm / kg and 600 mOsm / kg.

2. Composition according to claim 1, characterized in that the bacteriophages of the class Caudoviricetes belong to the genus Silviavirus, having lytic activity against at least one strain of Staphylococcus aureus.

3. Composition according to claim 1 or 2, characterized in that said bacteriophages have a myovirus morphotype.

4. Composition according to any one of the preceding claims, characterized in that the aqueous solution has an ionic strength of 150 to 400 mM.

5. Composition according to any one of the preceding claims, characterized in that it is stable for at least one year at a temperature in the range of 2 to 8°C, i.e. it has a bacteriophage titer which remains greater than or equal to 108 PFU / mL.

6. Composition according to any one of the preceding claims, characterized in that the bacteriophages are neither in conjugated form nor in complexed form.

7. Composition according to any one of the preceding claims, characterized in that it comprises polysorbate 80, as a non-ionic surfactant, at a molar concentration greater than 0.013 mM and less than or equal to 1.6 mM, preferably in the range of 0.16 mM to 0.38 mM, or polysorbate 20, as a non-ionic surfactant, at a molar concentration greater than 0.05 mM and less than or equal to 1.77 mM, preferably in the range of 0.18 mM to 0.44 mM.

8. Composition according to any one of the preceding claims, characterized in that the buffer mixture is a KH2PO4 / Na2HPO4 mixture, typically KH2PO4 at a molar concentration of 1 to 15 mM and Na2HPO4 at a molar concentration of 3 to 56 mM.

9. Composition according to any one of the preceding claims, characterized in that it comprises only NaCl, as a neutral salt, which is present at a molar concentration of 75 to 160 mM, typically at a molar concentration of 150 to 160 mM.

10. Pharmaceutical composition according to any one of the preceding claims, characterized in that it contains no compound of animal origin.

11. Composition according to any one of the preceding claims, characterized in that it is sterile, pyrogen-free and isotonic or hypertonic.

12. Composition according to any one of the preceding claims, characterized in that it is packaged in a container such as a bottle, a bag or a syringe delimited by walls which are of type I glass, type II glass, polystyrene, polypropylene, polyethylene, polyethylene terephthalate, treated or not with silicone oil, in particular closed with a stopper of a perfluorinated or non-perfluorinated rubber material.

13. Composition according to claim 12, characterized in that it exhibits a wettability of the container walls corresponding to a contact angle of 20 to 60°.

14. Composition according to claim 12 or 13, characterized in that the container is closed by a stopper and the composition has a stopper wettability corresponding to a contact angle of less than 90°.

15. Composition according to any one of the preceding claims, characterized in that said bacteriophage suspension in said aqueous solution is supplemented with a viscous solution or hydrogel of cellulose ether, polyethylene glycol, polyvinyl alcohol, sodium alginate, PVA-sodium alginate, acrylate, methacrylate, polyacrylate, hyaluronic acid, acrylamide, polyacrylamide, polyethylene glycol-maleimide, polyethylene oxide, poloxamers, guar gum, agarose, gelatin, collagen, chitosan, nanohydroxyapatite, silicate, or hydrated aluminum silicate.

16. Composition according to any one of the preceding claims, characterized in that it is suitable for enteral administration, or suitable for administration by injection, in particular by parenteral route, including intravenous, intraarterial, intramuscular, subcutaneous and / or intra-articular administrations.

17. Composition according to any one of the preceding claims, for its use as a medicinal product, in particular for its use in the treatment of osteoarticular infections.

18. A method for preparing a composition, and in particular a pharmaceutical composition, according to any one of claims 1 to 16, comprising introducing into an aqueous solution comprising, or even consisting of: - at least one nonionic surfactant, and in particular a single nonionic surfactant, selected from the polysorbate family, at a molar concentration above its critical micellar concentration, but not exceeding 2 mg / mL, - one or more neutral salts formed from a pair of monovalent ions, the total molar concentration of which is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably of 7.0 to 7.5, typically selected from the hydrogen phosphate / dihydrogen phosphate ion, citric acid / citrate ion and citric acid / hydrogen phosphate ion pairs, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, purified bacteriophages in the quantity necessary to obtain in the final composition a titer of at least 108 PFU / mL, and typically 109 to 1010 PFU / mL in bacteriophages.

19. A preparation method according to claim 18 characterized in that after the introduction of the bacteriophages into the aqueous solution (or dispersing medium), the resulting suspension is sterilized by carrying out a filtration operation, by passing through a filter made of polyethersulfone, polystyrene, poly(styrene-butadiene) or polyvinylidene fluoride.

20. Use of an aqueous solution comprising, or even consisting of: - at least one nonionic surfactant, and in particular a single nonionic surfactant, selected from the polysorbate family, at a molar concentration above its critical micellar concentration, but not exceeding 2 mg / mL, - one or more neutral salts formed from a pair of monovalent ions, the total molar concentration of which is in the range of 75 to 160 mM, - a buffer mixture giving the composition a pH of 6.0 to 7.9, and preferably 7.0 to 7.5, typically selected from the hydrogen phosphate / dihydrogen phosphate, citric acid / citrate, and citric acid / hydrogen phosphate pairs, the molarity of the buffer mixture being in the range of 4 mM to 100 mM, - water, to improve storage stability at a temperature within the range of 2 to 8°C,of bacteriophages of the class Caudoviricetes having lytic activity against at least one strain of Gram-positive bacteria, said bacteriophages being suspended in said aqueous solution at a titer of at least 108 PFU / mL, and typically from 108 to 1010 PFU / mL in bacteriophages, and in particular from 109 to 1010 PFU / mL and said composition comprising less than 0.9 units of endotoxins, bacterial per mL, less than 0.09 mg / mL or 90 nm / mL of total protein and the osmolality of said bacteriophage suspension in said aqueous solution being in the range of 150 mOsm / kg to 600 mOsm / kg.

21. Use according to claim 20, characterized in that the stability obtained corresponds to a bacteriophage titer which remains greater than or equal to 108 PFU / mL, after storage of the composition at a temperature in the range of 2 to 8°C for at least one year.

Images