BE1029567B1 - METHOD FOR PRODUCTION OF BACTERIOPHAGES - Google Patents

Abstract

A method of producing bacteriophages comprising: providing at least one cell culture chamber; providing a first reservoir containing a culture medium being in first closed circuit connection with said at least one cell culture chamber, the culture medium comprising at least one strain of phages and at least one strain of bacteria; transferring the culture medium from the first reservoir via the first closed circuit connection into the said at least one cell culture chamber to deposit the culture medium as a culture layer in the said at least one culture chamber, incubating the culture medium for propagating phages; providing a container which is in second closed circuit connection with said at least one cell culture chamber, the second closed circuit connection comprising a filtration system between said at least one cell culture chamber and the container; and transferring the culture medium comprising the propagated phages from said at least one culture chamber to the second closed circuit connection and purifying said propagated phages via the filtration system, the culture medium being in liquid form during transfer from said at least least one culture chamber to the second closed circuit connection and purification.

Description

METHOD FOR PRODUCTION OF BACTERIOPHAGES

Technical area

The present invention allows the production on an intermediate scale of bacteriophages for therapeutic purposes.

Technology background

Bacteriophages or phages are viruses that only infect bacteria. A phage has genetic material encapsulated in a protein structure usually consisting of a head and a tail. For more than 95% of known phages, this material is a double-stranded DNA molecule with a length of 5 to 650 kbp (kilobases) and their size varies from 24 to 200 nm. The infection of a bacterial cell by a phage can cause its lysis with the release of a few tens or even hundreds of phage particles. Each phage particle thus released will infect a new bacterium and restart the lytic cycle. Bacteriophages, unable to reproduce on their own, inject their genetic material into host bacteria. Thanks to the host's enzymes and ribosomes (and to certain viral proteins depending on the case), the viral genome can be replicated and translated to form numerous copies of the virus which are released with the lysis of the host bacterium: we speak of lytic cycle or production cycle. Some bacteriophages behave differently, their genetic material is replicated and integrates into the bacterium's chromosome (or exists as a plasmid). The virus is then designated by the term prophage, which is transmitted to the offspring of the infected bacterium (lysogenic line) and we then speak of lysogeny or lysogenic cycle. In some cases (eg, bacterial stress), the lysogenic infection can potentially switch to a lytic cycle.

This natural ability of phages to infect and kill bacteria and the specific interactions between phages and bacteria form the basis of phage therapy.

In recent years, phage therapy is gaining interest as a promising solution to effectively treat bacterial infectious diseases, particularly in view of the many resistances to antibiotic therapies.

Antibiotics have significantly reduced mortality associated with infectious diseases during the 20th century. The remarkable effectiveness of antibiotics has motivated their massive and repeated use in human and animal health. However, their massive and repeated use has led to the appearance of resistant bacterial strains.

Punctual at first, this resistance has become massive and worrying. Some strains are multi-resistant, i.e. resistant to several antibiotics. Others have even become toto-resistant, that is to say resistant to almost all the antibiotics available. This phenomenon, which is constantly increasing, places doctors in a therapeutic impasse: they no longer have any solution to fight against the infection.

Some bacteria are naturally resistant to antimicrobials. Of more concern, acquired resistance refers to the appearance of resistance to one or more antibiotics in a previously susceptible bacterium. These resistances can arise via a genetic mutation affecting the bacterium's chromosome, or be linked to the acquisition of foreign genetic material (plasmid, transposon) carrying one or more resistance genes from another bacterium. Chromosomal resistance generally concerns only one antibiotic or a family of antibiotics.

Plasmid resistance can concern several antibiotics, or even several families of antibiotics. They represent the most common resistance mechanism, representing 80% of acquired resistance.

Phage therapy or phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infection. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science and agriculture.

Bacteriophages are much more specific than antibiotics. They are typically harmless not only to the host organism, but also to other beneficial bacteria, such as the intestinal flora, reducing the chances of opportunistic infections. Moreover, phages are specific enough only to target certain strains of bacterial species, while they are ineffective against other strains of these same species. They have a high therapeutic index, i.e. phage therapy would be expected to result in few side effects. Because phages replicate in vivo, a small effective dose can be used.

Furthermore, the specificity of phages constitutes an obstacle for the development of phage-based therapies. In view of their specificity, it is generally necessary to access a library of different phages and to test the effectiveness of the phages against the bacterial infection of a specific patient. As such, phage therapy tends to be personalized, and current phage production methods are generally too time-consuming and resource-intensive to be commercially viable. Furthermore, current phage production methods are still often intended for production for scientific research and are not suitable for generating compositions that can be safely administered as a drug.

To this end, it is crucial to develop methods for producing bacteriophage compositions which are efficient, i.e. which are sufficiently virulent, non-toxic, host-specific, and practically usable.

A method for producing bacteriophage compositions based on a double layer culture method is known. Document WO2004/052274 discloses a method for producing strains of phages active against Pseudomonas aeruginosa. A solid phase comprising agar at a concentration of 1.5% is spread on dishes and a mixture of phages and bacteria is then added to a semi-solid layer comprising 0.27% agar.

After incubation, the semi-solid phase is scraped from the dish and subjected to an extraction protocol repeated several times including the addition of fresh medium (extraction solution) to the semi-solid phase to obtain a mixture and the centrifugation of the mixture to obtain a supernatant comprising a crude extract with bacteriophages, the crude extract then being purified.

The method for producing bacteriophage compositions according to this document has adapted the techniques used on a laboratory scale, namely a double layer culture method, for a production of bacteriophage compositions on a commercial scale. The process according to document WO2004/052274 aims to create favorable conditions for the cultivation of phages leading to the obtaining of a high titer, by reducing the operating volumes during the production process, which makes it possible to eliminate the use of high-volume fermenters. Precisely, according to this document, the initial operating volume, namely the volume of the semi-solid phase in which the phages are propagated, is of the order of 5 to 10 liters, or even 10 to 20 liters. Furthermore, the volume of the extraction solution corresponds to 20 to 100 times the volume of the semi-solid phase, which leads to a total operating volume per production cycle which is between 100 and 2000 liters. The methods according to this document also require several steps during which the cultured phages are transferred from one container to another, which leads to significant risks of contamination.

Thus, the methods for producing compositions of bacteriophages of the state of the art, such as that disclosed in document WO2004/052274, today encounter significant limits characterized by the absence of a method for producing compositions of bacteriophages intended for phage therapy which is rapid, mobile, easy to implement, and presenting lower risks of contamination.

Description of the invention

The invention makes it possible to solve the drawbacks of the state of the art by providing a method for producing compositions of bacteriophages which is rapid, transposable, easy to implement and more economical, reducing the risks of contamination, and this by guaranteeing obtaining compositions of bacteriophages which can be used in phage therapy, that is to say which are sufficiently virulent, non-toxic, host-specific, and which can be used in practice. In particular, firstly, the inventor discovered that it was possible to produce bacteriophages by connecting, via a first closed circuit connection, a first reservoir containing a culture medium comprising a strain of phage and a strain of bacteria with a cell culture chamber and, via a second closed circuit connection including a filtration system, the cell culture chamber with a container. In this way, the culture medium comprising the bacteria strain and the phage strain to be propagated is transferred from the first reservoir via the first closed circuit connection to the cell culture chamber in which the phages are then propagated, and then transferred to the second connection in closed circuit and purified via the filtration system. Secondly, the inventor has discovered that it is possible to transfer the propagated phages to the second connection in a closed circuit and to purify them via the filtration system, thanks to a culture medium which is in liquid form during the transfer from the culture chamber to the second closed circuit connection and purification. While maintaining high bacteriophage titers, the methods according to the invention make it possible to considerably reduce the operating volumes required,

can be implemented easily, are transposable while maintaining sterile conditions, are fast and reduce the number of steps which also carry a risk of contamination.

More particularly, a process for producing compositions of bacteriophages 5 according to the invention makes it possible to obtain compositions of bacteriophages having a titre which is at least 10"° pfu/ml, preferably which is between 10% and 10 % pfu/ml, preferably which is between 10"° and 10? pfu/ml, and this with a total operating volume less than or equal to 80 liters, preferably less than or equal to 50 liters, preferably less than or equal to 25 liters, more preferably less than or equal to 10 liters, preferably less than or equal to 5 liters in a total time that is less than or equal to 20 hours.

To solve this problem, there is provided according to the invention a process for the production of bacteriophage compositions comprising: - providing at least one cell culture chamber; - providing a first reservoir containing a culture medium being in first connection in closed circuit with said at least one cell culture chamber, the culture medium comprising at least one strain of phages and at least one strain of bacteria; - transferring the culture medium from the first reservoir via the first closed circuit connection into said at least one cell culture chamber to deposit the culture medium in the form of a culture layer in said at least one culture chamber, - incubating the culture medium to propagate phages; - providing a container which is in a second closed circuit connection with said at least one cell culture chamber, the second closed circuit connection comprising a filtration system between said at least one cell culture chamber and the container; and - transferring the culture medium comprising the propagated phages from said at least one culture chamber to the second closed-circuit connection and purifying said propagated phages via the filtration system, the culture medium being in liquid form during transfer from said at least one culture chamber to the second closed circuit connection and purification.

By the term “bacteriophage”, it is understood within the meaning of the present invention a virus which infects only bacteria. The term "bacteriophage" and the term "phage" are used interchangeably in the present invention. According to the invention, the bacteriophages are preferably lytic bacteriophages.

In the context of the present invention, all percentages are percentages by weight by volume, unless otherwise indicated.

For the purposes of the present invention, all ratios are volume to volume ratios, unless otherwise indicated.

By the terms “total operating volume”, it is understood within the meaning of the present invention, the volume of the culture layer added to the volume necessary for the extraction of the propagated phages from the culture layer.

In the context of the present invention, it has been surprisingly observed that the method for producing bacteriophages according to the invention has several advantages, while making it possible to obtain high-titer bacteriophage compositions that can be used in phage therapy.

Firstly, the method for producing bacteriophages according to the invention can be carried out with a total operating volume less than or equal to 80 liters, preferably less than or equal to 50 liters, preferably less than or equal to 25 liters, even more preferably less than or equal to equal to 10 liters, preferably less than or equal to 5 liters. Depending on the production need sought, the total operating volume is preferably between 40 and 80 liters or, preferably, between 1 and 5 liters.

It has been observed, surprisingly, that this effect is obtained in particular thanks to a culture medium which is in liquid form during the transfer from the culture chamber to the second connection in a closed circuit and during purification. The implementation of the method for producing bacteriophage compositions with a total operating volume according to the invention makes it possible to obtain a method for producing bacteriophage compositions which is more practical, more easily carried out, faster and more economical. Indeed, a total operating volume such as that of the present invention provides a process which does not require large installations, unlike the prior art for which installations adapted to large volumes (hundreds or even thousands of liters) must be used, which can be transposed, that is to say which can be carried out in particular in hospitals or laboratories, which requires fewer consumables and which is better suited to the technician who will more easily and more quickly handle small volumes.

Secondly, the method for producing bacteriophage compositions according to the invention is a direct culture method which comprises fewer steps than the methods known from the state of the art, in particular document WO2004/052274. Indeed, according to this document, the phages propagated in the culture layer are first extracted according to the following steps: - harvesting the culture layer (semi-solid culture medium); - add fresh medium to the culture layer to obtain a mixture; - mix the mixture intensely; and - centrifuging the mixture to obtain a supernatant which comprises a crude extract of bacteriophages.

According to document WO2004/052274, the crude extract of bacteriophages is then purified using methods such as, for example, column chromatography.

Contrary to the process disclosed in this document, the process for producing phage compositions according to the invention makes it possible to optimize/reduce the number of steps, which makes it faster, more practical, easier and more economical than the known methods of the state of the art. Indeed, in the context of the present invention, after

The incubation of the culture medium to propagate phages, the culture medium comprising the propagated phages is directly transferred and filtered to purify the propagated phages, a centrifugation step used in the methods according to the prior art therefore not being necessary in the scope of the present invention.

Thirdly, the method according to the invention is carried out in a closed system, which improves the efficiency of the production of bacteriophage compositions because such a closed system reduces the risk of external contamination and therefore the risk of failure of the production cycle. Further, the use of a first closed circuit connection and a second closed circuit connection which includes filters to purify the propagated phages according to the method of the present invention, makes it possible to provide a method which is transposable, c that is to say which can be carried out in particular in hospitals or laboratories, and which does not need to be carried out in a sterile environment. Indeed, the first and second closed circuit connections make it possible to maintain sterile conditions throughout the production process according to the present invention. In particular, it is not necessary to open or close the culture chamber to carry out the transfers of the culture medium throughout the production process according to the invention. Therefore, it is not necessary to carry out the production method according to the invention in a sterile environment, as is generally the case for cell culture.

Unlike the production process according to the invention, the process according to the document

WO2004/052274 requires a sterile environment because the culture dish is first opened to spread a solid phase comprising agar therein and then to add a semi-solid layer also comprising agar and a mixture phages and bacteria. After incubation, the culture dish is opened again and the semi-solid phase is scraped from the dish and subjected to an extraction protocol repeated several times including the addition of fresh medium (extraction solution) to the phase. semi-solid to obtain a mixture and centrifuging the mixture to obtain a supernatant comprising a crude extract with bacteriophages, the crude extract then being purified. Several stages of the process according to document WO2004/052274, in particular during which the culture dishes are opened and during which the cultured phages are transferred from one container to another, lead to significant risks of contamination.

According to a particularly advantageous embodiment of the invention, the culture layer formed by depositing the culture medium in said at least one culture chamber has a thickness less than or equal to 10 cm, preferably less than or equal to 5 cm , again preferably less than or equal to 2 cm.

The inventor has observed surprisingly that the advantageous effects of the method according to the invention are particularly good when the culture layer has a thickness less than or equal to 10 cm and are even better when it is less than or equal to 5 cm, and further improved when it is less than or equal to 2 cm. In particular, a method according to this preferred embodiment makes it possible to obtain compositions of bacteriophages with a particularly high titer while being easy and economical.

According to a preferred embodiment, the culture layer is incubated for a sufficient time to have a multiplication of the bacteriophage.

Preferably, the culture layer is incubated for a sufficient time to obtain confluence of the multiplied bacteriophages.

Advantageously, the incubation temperature of the culture layer is between 25 and 45°C, preferably around 37°C.

Preferably, the incubation period of the culture layer is between 15 and 20 hours, preferably about 18 hours.

According to a particular embodiment, the production method according to the invention further comprises: - depositing a layer of substrate comprising a first polymer capable of gelling it in said at least one cell culture chamber, prior to the transfer of the culture medium of the first reservoir in said at least one cell culture chamber, such that the culture medium is deposited in the form of a culture layer on the gelled substrate layer.

The inventor has surprisingly observed that the advantages of the process according to the invention are also obtained when a layer of substrate comprising a first polymer capable of gelling it is deposited beforehand on the culture medium in the cell culture chamber, in such a way so that the culture medium is deposited in the form of a culture layer on the gelled substrate layer.

According to a particular embodiment, the method according to the invention further comprises, prior to the step of supplying the first reservoir containing the culture medium, - supplying a second reservoir containing the substrate comprising a first polymer which is in first connection in closed circuit with said at least one cell culture chamber; and - transferring the substrate from the second reservoir via the first closed-circuit connection into said at least one cell culture chamber to deposit the layer of substrate in said at least one culture chamber.

According to a particular embodiment, the method according to the invention comprises: - providing said at least one cell culture chamber; - providing the second reservoir containing the substrate comprising the first polymer capable of gelling it which is in first connection in a closed circuit with said at least one cell culture chamber; - transferring the substrate from the second reservoir via the first closed-circuit connection into said at least one culture chamber; - depositing the substrate layer in said at least one cell culture chamber;

- providing the first reservoir being in first connection in a closed circuit with said at least one cell culture chamber and containing the culture medium comprising at least one strain of phages and at least one strain of bacteria; - transferring the culture medium from the first reservoir via the first closed-circuit connection into said at least one cell culture chamber to deposit the culture medium in the form of a culture layer in said at least one culture chamber on the gelled substrate layer; - incubate the culture medium to propagate phages; - providing the container which is in second closed circuit connection with said at least one cell culture chamber, the second closed circuit connection comprising the filtration system between said at least one cell culture chamber and the container; and - transferring the culture medium comprising the propagated phages from said at least one culture chamber to the second closed-circuit connection and purifying said propagated phages via the filtration system, the culture medium being in liquid form during transfer from said at least one culture chamber to the second closed circuit connection and purification.

According to a particular embodiment, the first reservoir containing the culture medium and the second reservoir containing the substrate are identical. According to this particular embodiment, the reservoir first contains the substrate which is transferred via the first closed-circuit connection into said at least one culture chamber. The tank no longer containing the substrate which has been transferred is then filled with the culture medium which is then also transferred via the first closed-circuit connection into said culture chamber to deposit the culture medium in the form of a layer of culture in said at least one culture chamber on the gelled substrate layer. Preferably, during the filling of the reservoir with the culture medium, the reservoir is isolated from said at least one culture chamber so that the system remains in a closed circuit. Still preferably, the tank is cleaned and disinfected between the transfer of the substrate and the filling with the culture medium.

According to another particular embodiment, the first reservoir containing the culture medium and the second reservoir containing the substrate are different. According to this particular embodiment, the second reservoir is in first connection in a closed circuit with said at least one culture chamber. After transferring the substrate into said at least one culture chamber to deposit a layer of substrate, the second reservoir is disconnected from the first closed-circuit connection and the first reservoir containing the culture medium is connected via the first circuit connection to said at least one culture chamber for transferring culture medium into said at least one culture chamber and depositing culture medium in the form of a culture layer in said at least one culture chamber on the gelled substrate layer . Preferably, during the disconnection of the second reservoir and the connection of the first reservoir, said at least one culture chamber is isolated so that the system remains in a closed circuit. Alternatively, the first reservoir and the second reservoir are connected in parallel to the first closed circuit connection and the transfers of the substrate and of the culture medium are carried out sequentially via the first closed circuit connection.

According to a particular embodiment of the method according to the invention, the culture medium further comprises a second polymer capable of gelling it, such that phages are propagated in the gelled culture medium, and in that the method comprises furthermore, after the incubation of the culture medium to propagate phages and prior to the transfer of the culture medium comprising the propagated phages from the said at least one culture chamber to the second connection in a closed circuit, a step comprising a liquefaction of the medium gelled culture so as to obtain a liquefied culture medium. In a particularly advantageous manner, the liquefaction of the gelled culture medium comprises: - bringing an extraction solution into contact with the gelled culture medium according to a volume ratio of between 5:1 and 1:5; and - dissolving the gelled culture medium in the extraction solution to obtain a liquefied culture medium.

According to a particular embodiment, the bringing into contact of the extraction solution with the gelled medium is carried out by a transfer of the extraction solution from a third tank containing the extraction solution via the first connection in a closed circuit with said at least one cell culture chamber.

According to this particular embodiment, the liquefaction of the gelled culture medium is also carried out in a closed circuit, which further improves the efficiency of the production of bacteriophage compositions because such a closed system reduces the risk of external contamination and therefore the risk of failure of the production cycle.

According to a particular embodiment, the third reservoir is identical to the first and/or to the second reservoir. According to this particular embodiment, the reservoir optionally contains first the substrate which is transferred via the first closed-circuit connection into said at least one culture chamber. The tank is then filled with the culture medium which is then also transferred via the first closed circuit connection into said culture chamber to deposit the culture medium in the form of a culture layer in said at least one culture chamber , optionally, on the gelled substrate layer. Finally, the tank is filled with the extraction solution which is then also transferred via the first closed circuit connection in the said culture chamber, and this for bringing the extraction solution into contact with the gelled medium.

Preferably, during the filling of the reservoir with the culture medium or with the extraction solution, the reservoir is isolated from said at least one culture chamber so that the system remains in a closed circuit. Still preferably, the reservoir is cleaned and disinfected before filling with the culture medium or with the extraction solution.

According to another particular embodiment, the third reservoir is different from the first and/or from the second reservoir. According to this particular embodiment, the method comprises an optional step according to which the second reservoir is in first closed-circuit connection with said at least one culture chamber, the substrate is transferred into said at least one culture chamber to deposit a layer of substrate and the second reservoir is disconnected from the first closed circuit connection. Then, the method comprises connecting the first tank containing the culture medium via the first circuit connection to said at least one culture chamber to transfer the culture medium into said at least one culture medium, depositing the culture medium in the form of a culture layer in said at least one culture chamber, optionally, on the gelled substrate layer and the disconnection of the first reservoir from the first closed circuit connection. Finally, the method comprises the connection of the third reservoir containing the extraction solution which is then also transferred via the first connection in a closed circuit into the said culture chamber, and this for bringing the extraction solution into contact with the gel medium. Preferably, optionally during the disconnection of the second reservoir and the connection of the first reservoir, and during the disconnection of the first reservoir and the connection of the third reservoir, said at least one culture chamber is isolated so that the system remains in closed circuit. Alternatively, the first reservoir, optionally the second reservoir, and the third reservoir are connected in parallel to the first connection in a closed circuit and the transfers of the substrate, the culture medium and the extraction solution are carried out sequentially via the first closed circuit connection.

Preferably, the extraction solution is contacted with the culture layer in a volume ratio of between 4:1 and 1:4, preferably between 3:1 and 1:3, preferably between 2: 1 and 1:2, preferably between 1:1 and 1:2, preferably which is about 1:1.

Advantageously, the step of dissolving the gelled culture layer in the extraction solution to obtain a liquefied culture medium comprises incubating the extraction solution directly on the culture layer.

According to a particular embodiment, the step of dissolving the gelled culture layer in the extraction solution comprises an extraction of the phages propagated in the extraction solution.

Preferably, incubation of the extraction solution is about 1 hour or less, more preferably about 30 minutes or less, more preferably about 20 minutes.

According to a preferred embodiment, the culture chamber is stirred during the incubation of the extraction solution.

Thus, according to the invention, it is not necessary for the culture layer to be harvested, for example by scraping, prior to the step of transferring the culture medium comprising the phages propagated from said at least one culture chamber to the second closed circuit connection. In the context of the present invention, the extraction solution can be added directly to the still-spread culture layer.

After incubation, once the liquefied solution is obtained, it is subjected to the following process steps.

Preferably, the steps of transferring the culture medium, and optionally the substrate and the extraction solution, are carried out by pumping.

Preferably, the step of purification of the culture medium comprising the phages propagated via the filtration system is carried out by pumping or by gravity, more preferably by pumping.

The use of a pump to transfer the culture medium, and optionally the substrate and the extraction solution, in particular for the transfer of the culture medium comprising the propagated phages from said at least one culture chamber to the second connection in a closed circuit, and for the purification is particularly advantageous in the case where the culture medium comprises a second polymer capable of gelling it because this makes it possible to reduce the risk of contamination linked to a step of harvesting the culture layer gelled, for example by scraping with a spatula, requiring the opening of the culture chamber comprising the culture layer.

Preferably, the extraction solution is a solution of DPBS (Dulbecco's Phosphate - buffered Saline).

Advantageously, in the context of the present invention, the phages are present in a volume of at most 5 times the volume of the culture layer throughout the production method, preferably in a volume of at most 4 times the volume of the culture layer, preferably in a volume of at most 3 times the volume of the culture layer, more preferably in a volume of at most 2 times the volume of the culture layer, more preferably the phages are present in a volume of at most the volume of the culture layer.

In a particular embodiment of the present invention, said first polymer is selected from the group consisting of agar, ionotropic polymers, slow-curing thermoplastic polymers and mixtures thereof.

Preferably, said first polymer is selected from the group consisting of ionotropic polymers, slow-polymerizing thermoplastic polymers and mixtures thereof.

According to a particular embodiment, said second polymer is selected from the group consisting of ionotropic polymers, slow-polymerizing thermoplastic polymers and mixtures thereof.

According to a particular embodiment of the process according to the invention, said first and second polymers are selected independently from the group consisting of ionotropic polymers, slow-polymerizing thermoplastic polymers and mixtures thereof.

It has been observed, in the context of the present invention, that the method according to

The invention in which the first and/or the second polymer(s) is an ionotropic polymer and/or a slow polymerizing thermoplastic polymer is optimized.

Ionotropic polymers are polymers whose polymerization is triggered by an ionic reaction. Unlike conventional thermoplastic polymers, ionotropic polymers remain in a liquid form at room temperature. It is the addition of ions, for example sodium ions in the case of alginate, which will be responsible for the polymerization.

Within the meaning of the present invention, thermoplastic polymers with slow polymerization are thermoplastic polymers, that is to say polymers which become in a liquid form when they are heated, for example at a temperature of 60° C., but which , unlike conventional thermoplastic polymers, exhibit slow polymerization. By the terms "slow polymerization" is meant within the meaning of the present invention, a polymerization which is not complete when the polymer reaches a temperature which allows safe handling, for example a temperature of 36° C. or less, preferably at room temperature. Preferably, within the meaning of the present invention, the slow-polymerizing thermoplastic polymers comprise polymers which have a solidification point of between 5 and 40° C., preferably between 15 and 30° C., preferentially at room temperature.

As indicated above, the process according to the invention in which these types of polymers are chosen is optimized because their handling is easier, the risk of burns is virtually eliminated, the support is not deformed during their application due to a high temperature, unlike conventional thermoplastic polymers. Indeed, conventional thermoplastic polymers polymerize rapidly and must therefore be applied while they are still at a high temperature, namely a temperature between 60 and 70°C, which can cause burns during handling, melting of the support, but also the clogging of the material due to its rapid polymerization or even the deterioration of the substrate layer during the application of the culture layer at a high temperature. The use of such polymers according to this particular mode of the invention is useful with plastic support materials, in particular with plastic cell culture chambers comprising multiple parallel supports, as explained in more detail below. The use of substrate and/or culture solutions which remain fluid at reduced temperatures for a sufficient time, makes it possible to deposit the layers on the support without having the risk of burns for the operator, the risk of deformation of the support or the risk of a hardening of part of the solution arriving too soon. It has also been surprisingly observed that such polymers according to this particular mode of the invention allow a contact zone between the substrate layer and the culture layer which is more regular and which therefore allows better culture of phages and bacteria on the substrate layer. In particular, a more regular contact area between the substrate layer and the culture layer means that the contact surface between the substrate layer and the culture layer is larger than the contact surface between the substrate layer and the culture layer when such polymers according to this particular embodiment of the invention are not used.

Preferably, the substrate layer and the culture layer are deposited at a temperature of 45°C or less, preferably 40°C or less, 36°C or less, or 30°C or less.

Advantageously, the ionotropic polymers are chosen from the group consisting of alginate, chitosan, pullulan and mixtures thereof.

Advantageously, the ionotropic polymers are chosen from the group consisting of alginate, pullulan and mixtures thereof.

In a particularly advantageous manner, the ionotropic polymer is alginate.

In a particular embodiment of the method of the invention, the culture layer formed by depositing the culture medium in said at least one culture chamber comprises beads of polymerized alginate.

In a particularly advantageous form of the process according to the invention, the slow-polymerizing thermoplastic polymers are selected from the group consisting of collagen, gelatin, cellulose and mixtures thereof.

Preferably, the slow-polymerizing thermoplastic polymer is collagen.

Preferably, said first and second polymers are independently selected from the group consisting of alginate and collagen.

Indeed, it has been observed that the advantages of the process according to the invention are improved when said first and second polymers are selected independently from the group consisting of alginate and collagen.

According to a preferred embodiment, said first and second polymers are identical.

According to another preferred embodiment, said first and second polymers are identical and are selected from the group consisting of alginate and collagen.

In a particularly advantageous embodiment of the process according to the invention, the first polymer is present at a concentration of between 2 and 5% of the layer of substrate, preferably between 3 and 4% of the layer of substrate. It has been observed that a concentration of the first polymer of between 2 and 5% of the substrate layer, preferably between 3 and 4% of the substrate layer, is particularly suitable in the context of the present invention and allows a improvement of the culture of the phages and bacteria present in the culture layer, in particular by further improving the contact surface between the two layers, and therefore makes it possible to obtain a titer of the bacteriophage compositions produced that is acceptable in phage therapy while involving a low total operating volume.

Advantageously, the second polymer is present at a concentration lower than the concentration of the first polymer, in particular between 1 and 2% of the culture medium of the culture layer. It has been observed, in the context of the present invention, that a concentration of the second polymer of between 1 and 2% of the culture layer is particularly suitable and allows an improvement of the method according to the invention, in particular by improving the propagation phages, the ability of bacteria to form colonies but also by improving the efficiency of the extraction of the propagated phages, and therefore makes it possible to obtain a titer of the bacteriophage compositions produced which is acceptable in phage therapy while involving a small volume of total exploitation.

According to one embodiment, the compositions of bacteriophages according to the invention can be prepared by cultivating the phages in the presence of a microorganism including, for example, staphylococci, hemophili, helicobacter, mycobacterium, streptococci, neisseria, klebsiella, enterobacter, proteus, bacteroides, pseudomonas, borrelia, citrobacter, escherichia, salmonella, propionibacterium, treponema, shigella, enterococci and leptospirex.

Preferably, the microorganism includes, for example, Staphylococcus aureus,

Staphylococcus epidermidis, Helicobacter pylori, Streptococcus pneumomae, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguis, Streptococcus pyogenes,

Streptococcus viridans, Group A streptococcus and anaerobic streptococcus, Hemophilus influenzae, Shigella dysenteriae, Mycobacterium tuberculosis, Mycobacterium leprae,

Mycobacterium asiaticum, Mycobacterium intracellulare, Mycoplasma pneumoniae,

Mycoplasma hominis, Neisseria meningitidis, Neisseria gonorrhea, Klebsiella pneumoniae,

Pseudomonas aeruginosa, Propionibacterium acnes, Treponema pallidum, Treponema pertanue, Treponema carateum, Escherichia coli, Salmonella typhimurium, Borrelia burgdorferi and Leptospirex such as Leptospirex hemoragia Citrobacter fruendii.

Preferably, the microorganism is selected from Staphylococci, Streptococci, Citrobacter, Escherichia and Klebsiella, and even more preferably, the microorganism is selected from Staphylococcus aureus, Streptococcus pyogenes, Klebsielia oxytoca,

Escherichia coli and Citrobacter fruendii.

The specific bacterium-bacteriophage combination is then selected according to the intended use.

Preferably, the volume of the substrate layer is similar to the volume of the culture layer. In particular, the ratio of the volume of the substrate layer to the volume of the culture layer is approximately 1:1.

According to a preferred embodiment, the layers of substrate and culture are incubated for a sufficient time to have multiplication of the bacteriophage.

Advantageously, the incubation temperature of the culture and substrate layers is between 25 and 45°C, preferably around 37°C.

Preferably, the incubation period of the culture and substrate layers is less than or equal to 20 hours, preferably between 15 and 20 hours, preferably approximately 18 hours.

According to a preferred embodiment, the substrate layer is polymerized before the deposition of the culture layer, preferably the substrate layer is polymerized in 30 minutes or less.

In a particular embodiment of the present invention, the filtration system comprises at least two filters in cascade. The term “cascaded” means that the filters are connected in series. The term "cascade" and the term "serial" are used interchangeably in the present invention.

According to another embodiment, at least three filters placed in series are used.

According to a particular mode, the first filter (that is to say the first filter according to the direction of filtration) is a particle filter with a pore size of between approximately 0.5 and

2.0 µm, preferably between about 0.8 and 1.6 µm, more preferably between 1.0 and 1.4 µm. Such a filter is also referred to as an "in-depth" filter.

According to another particular embodiment, the second filter (that is to say which is placed after the first filter according to the direction of filtration) is a sterilizing filter, in particular a sterilizing filter with a pore size less than 0 .5 μm, more particularly less than 0.4 μm. In another embodiment, the sterilizing filter has a pore size less than 0.3 µm. In yet another embodiment, the pore size of the second filter is 0.01 µm or greater. Preferably, the pore size of the second filter is between 0.1 and 0.3 µm, such as 0.2 µm. Such a filter is also referred to as a "bioburden" filter.

According to another particular embodiment, the third filter (that is to say which is placed after the second filter according to the direction of filtration) is a filter which removes viral particles and endotoxins. Preferably, the third filter is an anion exchanger, in particular a filter comprising an anion exchange membrane which binds plasma DNA and negatively charged proteins. Such a filter is also referred to as an "endotoxin" filter.

The “in-depth” type filter separates the components of the culture medium according to size and will only allow the bacteriophages to pass as well as the components which have a size less than or equal to the bacteriophages. In a particular embodiment, the "in-depth" type filters used in the context of the present invention have a pore size of between approximately 0.1 and 1.5 μm, preferably between approximately 1 and 1 .5 μm and, more preferably still around 1.2 μm. The "endotoxin" type filter purifies the culture medium of endotoxins which may be responsible for side effects during phage therapies. The “bioburden” type filter sterilizes the culture medium of any micro-organisms still present in the solution.

According to a preferred embodiment, the method according to the invention does not include centrifugation.

Preferably, said at least one culture chamber comprises a bottom. According to this preferred embodiment, the culture medium is deposited on the bottom of said at least one culture chamber to form a culture layer on the bottom of said at least one cell culture chamber.

The term “bottom” within the meaning of the present invention corresponds to the lower wall of the cell culture chamber which is intended to rest on its support, for example a table.

According to a preferred embodiment of the invention, the bottom of said at least one cell culture chamber has a surface adapted to allow optimal exchanges of Oz between bacteriophages and bacteria.

Advantageously, the bottom surface of said at least one cell culture chamber is between 50 and 800 cm³.

According to a particularly advantageous embodiment, the culture layer formed by depositing the culture medium in said at least one culture chamber has a thickness less than or equal to 10 cm, preferably less than or equal to 5 cm, even more preferably less or equal to 2 cm, and the surface of the bottom of said at least one cell culture chamber is between 50 and 800 cm”, preferably between 75 and 650 cm”, preferably between 250 and 650 cm”, even more preferably between 600 and 650 cm?.

Preferably, the culture layer formed by depositing the culture medium in said at least one culture chamber has a volume of between 50 and 750 ml, preferably between 250 and 500 ml, preferably approximately 500 ml.

Preferably, the cell culture chamber is plastic. In a particular embodiment, the cell culture chamber is disposable.

According to another particular embodiment, the cell culture chamber comprises a culture bag, preferably the culture bag is made of plastic.

Preferably, the culture bag rests on a rigid support. According to this embodiment, the rigid support on which the culture bag rests provides a bottom to the culture bag. Thus, the culture medium is deposited on the bottom of said culture bag provided by the rigid support on which said culture bag rests to form a layer of culture on the bottom of said culture bag.

Advantageously, the culture medium is deposited on multiple parallel bottoms of a cell culture chamber, such as CellStack® culture chambers from Corning® or Nunc"“EasyFill""Cell Factory""Systems from Thermofisher. use of a cell culture chamber having multiple parallel bottoms in the method according to the invention makes it possible to increase the yield of bacteriophages produced per production cycle and therefore to further improve the speed of the production method according to the invention.

According to one embodiment of the invention, the cell culture chamber can be composed of several units which are stacked to form a chamber with multiple parallel bottoms.

The cell culture chamber according to the invention makes it possible to create a sterile chamber with a large support surface for the culture layer and/or the substrate layer.

In particular, each parallel bottom has a surface of between 250 and 650 cm? and has a volume between 250 and 500 ml.

According to a preferred embodiment, the cell culture chamber comprises a single inlet and outlet, such that the deposition of the culture layer, and optionally of the substrate layer and of the extraction solution is carried out on the multiple parallel bottoms via the first closed circuit connection and that the harvesting of the liquid or liquefied culture medium deposited on the multiple parallel bottoms is carried out via the second closed circuit connection.

Preferably, the method according to the invention comprises: - providing at least one cell culture chamber comprising multiple parallel bottoms; - providing a first reservoir containing a culture medium being in first connection in a closed circuit with said at least one cell culture chamber, the culture medium comprising at least one strain of phages and at least one strain of bacteria; - transferring the culture medium from the first reservoir via the first closed circuit connection into said at least one cell culture chamber to deposit the culture medium in the form of culture layers on the multiple parallel bottoms of said at least one cell culture chamber culture; - incubate the culture medium to propagate phages; - providing a container which is in second closed circuit connection with said at least one cell culture chamber, the second closed circuit connection comprising a filtration system between said at least one cell culture chamber and the container; And

- transferring the culture medium comprising the propagated phages from said at least one culture chamber to the second closed-circuit connection and purifying said propagated phages via the filtration system, the culture medium being in liquid form during transfer from said at least one least one culture chamber to the second closed circuit connection and purification.

Preferably, the cell culture chamber comprises 2 to 20 parallel bottoms, preferably 5 to 15 parallel bottoms, preferably 10 parallel bottoms.

Advantageously, the titre of the purified phages is between 10"° and 10% pfu/ml, in particular between 10"° and 1022 pfu/ml.

According to a preferred embodiment, the method according to the invention further comprises: - combining the purified bacteriophages with a pharmaceutically acceptable vector to produce a pharmaceutical formulation comprising the bacteriophages.

Preferably, the pharmaceutical formulation is administered by injection, for example by intravenous injection, orally in the form of a drink, liposomes, lipophilic microcapsules or dendrimers, intranasally, for example via a spray, an aerosol or an aerosol powder, rectally, or transdermally, for example via a cream or gel.

According to a preferred embodiment, the method according to the invention further comprises: - administering a composition comprising the purified bacteriophages to a person in need thereof.

The composition is administered in a dosage and for a period to treat the bacterial infection. By the terms "treating the bacterial infection", it is understood within the meaning of the present invention, to kill or inhibit enough bacterial microorganisms to render the microorganisms incapable of infecting the host.

These and other embodiments of the present invention are set forth in the appended claims.

The invention will now be described in more detail in the following examples which describe non-limiting embodiments of various aspects of the present invention.

Description of figures

Figure 1: graph representing the production titers of the ICP phage strain according to the double agar method (comparative example) and according to different production methods according to the invention.

Figure 2: graph representing the production titers of the phage strain 14/1 according to the double agar method (comparative example) and according to different production methods according to the invention. The light gray bars correspond to the initial densities and the dark gray bars represent the densities at 24 h.

Figure 3: graph representing the production titers of the PNM phage strain according to the double agar method (comparative example) and according to different production methods according to the invention. The light gray bars correspond to the initial densities and the dark gray bars represent the densities at 24 h.

Examples

Example 1: Preparation of different alginate solutions

The gelatinization potentials of different types of alginate were compared. Alginate solutions were prepared using 4 different powders of pure alginate (BR-

GM, BR-W, BR-L and an alginate:pullulan premix). In addition, sodium chloride (0.9% final concentration), LB (/ysogeny broth) medium (1x standard) and calcium carbonate (15 mM final concentration) were added to the different powders before sterilization at the autoclave of the solutions obtained.

The protocol for making 400 ml of a 5% alginate solution is as follows: - heat 400 ml of pure water to 65°C on a hot plate; - add 3.6 g of NaCl and mix to dissolve; - slowly add 20 g of the alginate powder, mix constantly until completely dissolved (up to 20 minutes); - add 8 g of LB medium and continue mixing until dissolved; - add 0.6 g of calcium chloride (will remain in suspension in the solution for up to 30 minutes); - pour the solution into a 500 ml Duran bottle, then sterilize at 121°C for 20 minutes;

- After cooling, mix completely by inversion to resuspend the calcium carbonate; - Add 12 ml of GDL (glucono delta-lactone, 1M), invert to mix; and - Pour immediately into a culture chamber.

At 5% concentration, BR-GM and BR-W alginate powders produced very thick solutions, which were not usable. In contrast, the alginate:pullulan premix and BR-L powders produced finer solutions, however, plates produced using these 5% solutions were still too soft compared to standard agar. BR-L is described as a low viscosity, low freeze resistance product, and therefore not suitable for phage production requirements.

At lower concentrations, BR-GM and BR-W solutions are finer, but the gelation time remains fast for all concentrations (less than 5 minutes for pouring), with variation in the strength of the gelled product dependent alginate concentration. BR-GM and BR-W alginates produce firm gels at 2% concentration, suitable for spreading bacteria or serving as a bottom layer for phage plating. To minimize wetness and improve diaper adhesion, it is best to pour diapers the day before and leave overnight on the bench before use.

For double layer alginate plates, 1.5% alginate (BR-GM, Table 1) provides a suitable top layer (culture layer), adhering well to the bottom layer (substrate layer).

For the preparation of a 2% alginate solution, in single or double layer, the alginate solutions are prepared according to the above procedure, adjusting the proportions to correspond with the desired alginate concentration (Table 1) .

Table 1: composition of a 2% and 1.5% alginate solution

For a double alginate layer culture, the bottom (substrate) layer is prepared using a 2% alginate solution as above and left to dry overnight. For the top layer (culture layer): - add 100 µl of the appropriate host culture to a 15 ml Falcon® tube and add phages as required; - invert the 1.5% alginate solution to resuspend the calcium chloride and add to the bacteria/phage mixture until a solution with a volume of 14 ml is obtained; and - add 420 µl of GDL, invert to mix and pour immediately to deposit the layer on top of the substrate layer.

Example 2: Evaluation of bacterial growth according to different production conditions

Comparison of S. aureus growth on alginate:pullulane plates and standard agar plates revealed no difference in density, with similar colony morphology on all media. However, due to the humidity on the alginate plates, colonies of P. aeruginosa scatter much more on the alginate plates than on the agar and, although the densities are similar, cannot be counted with accuracy. This suggests that if alginate is an appropriate medium for bacterial growth, a firmer setting alginate (i.e. BR-GM alginate) is preferable to the alginate:pullulan premix.

Example 3: Evaluation of the production of bacteriophages according to the method in double layer of agar (comparative example) and according to the method of the invention

The following bacteriophage production conditions were tested: - double layer of agar (comparative example) — 50 ml 1.2% agar in LB medium for the lower layer (of substrate); 15ml 0.6% agar in LB medium for top (culture) layer - single layer agar (LBA, comparative example) — 15ml 0.6% agar in medium

LB (culture layer) - agar layer on collagen (comparative example) — 15 ml 0.6% agar in medium

LB (culture layer); 2.4 µg/cm? collagen (substrate layer)

- double layer of alginate — 50 ml 2% BR-GM alginate in LB medium for the lower layer (substrate); 15 ml 1.5% BR-GM alginate in LB medium for top (culture) layer - liquid medium (LB) — 30 ml LB medium (culture layer) - liquid medium on collagen — 30 ml of LB medium (culture layer); 2.4 µg/cm? of collagen (substrate layer) - single layer of alginate — 15 ml 1.5% BR-GM alginate in LB medium (culture layer) - alginate layer on collagen — 15 ml 1.5% d BR-GM alginate in LB medium (culture layer); 2.4 µg/cm? of collagen (substrate layer) - alginate beads — 15 ml 1.5% alginate beads

Three phage strains (ISP, PNM and 14/1) were tested according to each of the production conditions mentioned above, and this in triplicate. Starting densities were calculated to maximize phage production (i.e. suitable phage dilution to obtain an array pattern): PNM at 10° units and ISP and 14/1 at 10° units.

The bacteria are prepared beforehand (see Example 2) and harvested in exponential phase in DPBS with calcium and magnesium. The phages to be produced are also suspended in DPBS with calcium and magnesium.

The substrate and culture layers are prepared according to the corresponding protocols indicated above and are deposited in the culture chamber according to the different experimental conditions tested, namely, double agar (comparative example, double agar), double layers of alginate (double_alginate), LB liquid medium (LB), LB liquid medium on collagen (LB _collagen), single layer of agar (comparative example, LBA), single layer of agar on collagen (comparative example, LBA collagen), single layer — of alginate (alginate), single layer of alginate on collagen (alginate_collagen) and single layer of alginate comprising alginate beads (beads_alginate).

The substrate and/or culture layers are then incubated at 37°C for 12 hours (for the ISP strain) or 24 hours (for the 14/1 and PNM strains) until bacterial lysis is observed.

In case LB liquid medium is not used for the culture layer, an extraction buffer consisting of 130 ml of DPBS with calcium and magnesium is then added to the culture chamber on the culture layer comprising the phages propagated and incubated with shaking at room temperature for 3 hours.

The liquefied culture layer or the LB liquid medium comprising the propagated phages is then successively filtered through a filter of the “Bioburden” type HDC II 1.2 μm (Pall,

KA1)012P2), an “In depth” type filter STRL 0.2 um (Pall) and an “Endotoxin” type filter

Mustang Q (Pall).

The results of the production titers obtained for the ISP, 14/1 and PNM strains are respectively represented in Figures 1, 2 and 3. For Figures 2 and 3, the light gray bars correspond to the initial densities and the dark gray bars represent the densities at 24 h.

These results revealed, firstly, that the production of bacteriophage strains ISP, 14/1 and PNM in a closed circuit and using small operating volumes (15 ml culture layer) was efficient and made it possible to obtain high phage titers using (1) a culture layer comprising alginate, (2) a culture layer comprising alginate and a substrate layer comprising collagen, (3) a double layer (of culture and substrate) of alginate or (4) a layer of culture comprising alginate in the form of beads (Figures 1, 2 and 3). Indeed, the production processes according to these conditions make it possible to obtain production titles comparable to those obtained according to the known methods, for example the method in double layer of agar (comparative example), and this by reducing the risk of contamination. thanks to the system (in particular the first and second connections) in closed circuit of the method according to the invention and by involving low operating volumes (only 15 ml for the culture layers comprising alginate), thus making the method production according to the invention faster, more economical, easier to implement and transposable, for example in hospitals. In particular, the use of 15 ml of culture medium comprising alginate as the culture layer in the method according to the invention provides production titers reaching 1022 pfu/ml (Figure 3).

In addition, the inventor has observed quite surprisingly that higher production titers are achieved when LB liquid medium is used as culture medium (culture layer) that it is deposited directly in the culture chamber cellular (LB, without a layer of substrate deposited beforehand) or that it is deposited on a layer of substrate comprising collagen (LB_collagen). Indeed, production titers of between 10% pfu/mI (ISP and PNM strains) and 107 pfu/mI (strain 14/1) are obtained by carrying out the method of the invention in a closed circuit in only 30 ml of medium LB liquid (culture layer). In particular, the use of 30 ml of LB liquid medium as a culture layer in the method according to the invention provides production titers up to 100 times higher than the known method in double agar (Figure 1).

In conclusion, the results obtained demonstrate that the method according to the invention makes it possible to obtain high phage titers (comparable or even higher than the phage titers obtained with the known method in double agar), and this by reducing the risk of contamination thanks to the closed circuit system and by involving small operating volumes, thus making the production method according to the invention faster, more economical, easier to implement and transposable, for example in hospitals.

Claims (17)

29 BE2021/5524 CLAIMS 1. A method for producing bacteriophages comprising: - providing at least one cell culture chamber: - providing a first reservoir containing a culture medium being in first closed circuit connection with said at least one cell culture chamber, the culture comprising at least one strain of phages and at least one strain of bacteria: - transferring the culture medium from the first reservoir via the first closed-circuit connection into said at least one cell culture chamber to deposit the culture medium in the form a culture layer in said at least one culture chamber, - incubating the culture medium to propagate phages; - providing a container which is in second closed circuit connection with said at least one cell culture chamber, the second closed circuit connection comprising a filtration system between said at least one cell culture chamber and the container; and - transferring the culture medium comprising the propagated phages from said at least one culture chamber to the second closed-circuit connection and purifying said propagated phages via the filtration system, the culture medium being in liquid form during transfer from said at least one cultivation chamber to the second closed circuit connection and purification, wherein the filtration system comprises, in the filtration direction, a first particle filter with a pore size of between about 0.5 and 2.0 µm, a second sterilizing filter with a pore size between 0.1 and 0.3 µm and a third anion exchange filter. 2. The method for producing phages according to claim 1, characterized in that it further comprises - depositing a layer of substrate comprising a first polymer capable of gelling it in said at least one cell culture chamber, prior to the transfer of the culture medium from the first reservoir in said at least one culture chamber 30 BE2021/5524 cells, so that the culture medium is deposited in the form of a culture layer on the gelled substrate layer. 3. The method for producing phages according to claim 1 or 2, characterized in that the culture medium further comprises a second polymer capable of gelling it, such that phages are propagated in the gelled culture medium, and in that the method further comprises, after the incubation of the culture medium for propagating phages and prior to the transfer of the culture medium comprising the propagated phages from the said at least one culture chamber to the second closed circuit connection, a step comprising liquefying the gelled culture medium so as to obtain a liquefied culture medium. 16 4. The process for producing phages according to claim 3, characterized in that the liquefaction of the gelled culture medium comprises: - bringing an extraction solution into contact with the gelled culture medium according to a volume ratio of between 5: 1 and 1:5; and - dissolving the gelified culture medium in the extraction solution to obtain a liquified culture medium. 5. The method for producing phages according to any one of the preceding claims, characterized in that the phages are present in a volume of at most 5 times the volume of the culture medium throughout the method of production, of preferably in a volume of at most 3 times the volume of the culture medium, preferentially, the phages are present in a volume of at most the volume of the culture medium. 6. The method for producing phages according to any one of claims 2 to 5, characterized in that said first polymer is selected from the group consisting of agar, ionotropic polymers, slow-polymerizing thermoplastic polymers and their mixtures. 7. The process for producing phages according to any one of claims 3 to 6, characterized in that said second polymer is selected from the group consisting of ionotropic polymers, slow polymerizing thermoplastic polymers and mixtures thereof. 31 BE2021/5524 8. The process for producing phages according to any one of the preceding claims, characterized in that the substrate layer and/or the culture medium are deposited at a temperature of 36°C or less. 9. The process for producing phages according to any one of claims 2 to 8, characterized in that the first polymer is present at a concentration of between 2 and 5% of the substrate layer. 10. The process for producing phages according to any one of claims 3 to 9, characterized in that the second polymer is present at a concentration of between 1 and 2% of the culture medium. 16 11. The process for producing phages according to any one of the preceding claims, characterized in that the layer of substrate and/or the culture medium are deposited on multiple parallel bottoms of a cell culture chamber. 12. The method for producing phages according to any one of claims 6 to 11, characterized in that the ionotropic polymers are chosen from the group consisting of alginate, chitosan, pullulan, and mixtures thereof. 13. The method for producing phages according to any one of claims 6 to 11, characterized in that the slow-polymerizing thermoplastic polymers are selected from the group consisting of collagen, gelatin, cellulose, and mixtures thereof. . 14. The method for producing phage according to any of the preceding claims, comprising: - providing at least one cell culture chamber comprising multiple parallel bottoms; - providing a first reservoir containing a culture medium being in first connection in a closed circuit with said at least one cell culture chamber, the culture medium comprising at least one strain of phages and at least one strain of bacteria: - transferring the medium culture from the first reservoir via the first closed circuit connection in said at least one cell culture chamber to deposit the 32 BE2021/5524 culture medium in the form of culture layers on multiple parallel bottoms of said at least one culture chamber: - incubating the culture medium to propagate phages; - providing a container which is in second closed circuit connection with said at least one cell culture chamber, the second closed circuit connection comprising a filtration system between said at least one cell culture chamber and the container; and - transferring the culture medium comprising the propagated phages from said at least one culture chamber to the second closed circuit connection and purifying 16 said propagated phages via the filtration system, the culture medium being in liquid form during the transfer from said at least one culture chamber to the second closed circuit connection and purification. 15. The process for producing phages according to any one of the preceding claims, in which the titre of the purified phages is between 10° and 10% pfu/ml, in particular between 10° and 102 pfu/ml. 16. The method of producing phages according to any preceding claim, further comprising: - combining the purified hacteriophages with a pharmaceutically acceptable carrier to produce a pharmaceutical formulation comprising the bacteriophages. 17. The process for producing phages according to any one of the preceding claims, characterized in that the culture layer formed by depositing the culture medium in said at least one culture chamber has a thickness less than or equal to 10 cm , preferably less than or equal to 5 cm.