Bioreactor for producing a biological medicament, and support for such a bioreactor

Abstract

The present invention relates to a bioreactor for producing a biological medicament from a biological liquid that originates from a sample taken from a patient or a donor, characterized in that the bioreactor is composed of two flexible films (10, 20) joined locally via weld zones on the inner surfaces of the two films (10, 20) in order to form leaktight barriers that mutually delimit a plurality of circulation channels (201 to 209) connecting a plurality of compartments (101 to 107), and in that at least one of the films (10, 20) comprises a plurality of interconnection vias (401 to 405), each of which opens into one of the channels (201 to 209) or compartments (101 to 107). The invention also relates to a support for such a bioreactor.

Description

Bioreactor for the production of a biological drug and support for such a bioreactor Field of invention The present invention relates to the field of the preparation of gene therapy drugs and more precisely to the field of adoptive cellular immunotherapy based on the genetic modification of T lymphocytes, immune cells of a patient, T lymphocyte, NK lymphocyte, macrophage, etc., or those of a donor so that they are able to recognize and destroy cancer cells. For such therapies, cells are isolated from a patient's blood sample that play a major role in the immune system, which controls the body's defense system by identifying and destroying cells recognized as foreign to the body, whether bacteria, viruses, or cancer cells. First, a blood sample is taken from a patient or donor in a hospital or blood transfusion center. The cells of interest are collected by a process called leukapheresis or from a whole blood sample, which consists of isolating white blood cells from other blood components. After quality control, these lymphocyte cells are sent to a specialized laboratory that performs the genetic modification. These isolated cells of interest are genetically modified so that they express a specific chimeric protein on their surface (a receptor called CAR, for Chimeric Antigen Receptor). This then allows them to recognize cancer cells, on the one hand, and to activate themselves to destroy these same cancer cells. Once modified, the cells of interest are reinjected into the patient. To achieve this modification, a new gene is introduced into the genome of the cells of interest, which will cause these cells to produce the desired chimeric protein. These cells are then cultured for cell multiplication. The personalized medication thus prepared is then administered to the patient during a single infusion. The production of these modified cells in specialized laboratories requires delicate operations of collecting the cells of interest, isolating the cells, culturing the cells in a culture dish, periodic observation of the health of the cells, aspiration, replacement, and analysis of the culture medium, etc. State of the art Patent application US2017051238 describes a rigid culture container formed by molding a resin, to form a three-dimensional structure with peripheral side faces pierced by connection orifices. The transfer between two chambers 11, 12 is done by tilting the container to ensure flow through an external conduit 112 passing through connection orifices provided on the side face of the container (paragraph

[0081] ), or by a difference in height of the orifice (paragraph

[0082] ). The container proposed by this application is therefore necessarily a thick packaging, with peripheral faces having a height greater than the section of the connection orifices. Patent application US20200231918 describes a cartridge comprising a rigid PMMA cover (paragraph

[0068] ) defining a single cell culture chamber ranging in height from 0.5 mm to 100 mm, with pillars to support the top surface, forming struts and rigid acrylate side surfaces. Patent application US2014315303 describes an apparatus for processing a biological sample. The apparatus includes a first sheet of material, a second sheet of material bonded to the first sheet of material, and a plurality of chambers defined between the first sheet of material and the second sheet of material. The chambers include a sample dissociation chamber including an inlet and an outlet; a waste collection chamber including an inlet in fluid communication with the outlet of the sample dissociation chamber, and a cell refining chamber including an inlet in fluid communication with the sample dissociation chamber and an outlet. As with patent application US2017051238, the fluid connection is made via peripheral fittings, through the peripheral edge of the device. Disadvantages of the prior art The solutions of the prior art have a three-dimensional configuration, obtained in particular by molding, and have a thick slice crossed by orifices in which supply or suction conduits are connected. The construction of such devices presents difficulties in ensuring the perfect sealing of the conduits passing through the slice. In addition, the products contained in the chamber can remain in the peripheral edges, which makes it difficult to transfer the entire chamber to the next chamber. Solution provided by the invention In order to overcome these drawbacks, the present invention relates, in its most general sense, to a bioreactor for the production of a biological drug from a biological liquid originating from a sample taken from a patient or donor having the characteristics set out in claim 1. It is constituted by two flexible films locally associated by welding zones of the interior surfaces of the two films to form sealed barriers delimiting between them a plurality of circulation channels connecting a plurality of compartments, and in that at least one of said films comprises a plurality of interconnection vias each opening into one of said channels or compartment. According to one variant, at least one of said films is made of polyolefin. According to another variant, at least one of said films is made of polyethylene. Preferably, at least one of said films is made of Fluorinated Ethylene Propylene transparent to ultraviolet rays. Advantageously, at least one of said films has an embossing locally widening the distance between said films, in a conduit zone delimited by two welds. According to one variant, the bioreactor further comprises at least one filtration zone formed by a filtering part inserted between the upper film and the lower film and welded over at least part of its periphery to at least one of said films. Advantageously, said filtering part is welded to one of said films on a part of its periphery, the non-welded part opening onto a zone surrounded by weld lines locally sealing said upper film and said lower film to form a supply zone for said filter, the film opposite that on which the filter is welded also having a zone surrounded by weld lines locally sealing said upper film and said lower film to form an outlet zone for said filter. According to a variant, said vias are extended by a tubular injection or suction pin. According to another variant, an area of ​​at least one of said films is surrounded by weld lines and has a functionalized interior surface. The invention also relates to a support for a bioreactor characterized in that it is made up of a rigid frame having means for attaching a bioreactor as well as at least one element having fluidic connectors, said element being movable between a spaced position and a position where said fluidic connectors are associated with the vias of the bioreactor. Advantageously, said frame has windows corresponding to the positions of said compartments, for the passage of an actuating means acting on the surface of the film of said bioreactor. According to one variant, the support comprises at least one sensor arranged in an area delimited by welds, said sensor being extended by wires opening onto the edge of said bioreactor. According to another variant, it includes a unique identifier readable optically or by radio frequency communication. Detailed description of a non-limiting example of embodiment The present invention will be better understood on reading the following description, concerning a non-limiting example of embodiment illustrated by the appended drawings where: represents a top view of a bioreactor according to a first exemplary embodiment of the invention represents a top view of a bioreactor support in the closed position according to a first exemplary embodiment of the invention represents a top view of a bioreactor support according to a first exemplary embodiment of the invention represents a top view of a bioreactor positioned in a support represents an enlarged perspective view of the fluid connection of the support and the bioreactor. represents an enlarged view of the formation of a channel represents a bottom view of the upper film during a first stage of formation of a filter zone represents a top view of the bioreactor during a second stage of formation of a filter zone represents a top view of the bioreactor during a third stage of formation of a filter zone represents a top view of the bioreactor during a fourth stage of formation of a filter zone represents a top view of a bioreactor according to a second exemplary embodiment of the invention. General principle of the invention The present invention relates to a bioreactor intended for carrying out a succession of physical, chemical or biochemical treatments with a view to producing Car-T, Car-NK, Car-M, bio-identical proteins, antibodies, stem cells and more generally sequences of treatments of a biological fluid obtained from a sample taken from a patient or a donor with a view to preparing a gene therapy drug. The invention is based on the principle of forming a flexible pouch, formed from two flexible films having peripheral weld lines and zones to form a pouch closed over its entire peripheral edge, as well as local weld lines and zones where the two films are assembled to form a sealed barrier separating other zones where the two films can locally be separated to form compartments or channels allowing the circulation of the fluid. The flexibility of the films makes it possible to exert an external action to expel the contents of one compartment to another compartment or to an outlet conduit by exerting pressure on the film at the area concerned, and also makes it possible to open or close circulation channels by exerting pressure on a transverse line of a channel.Some film surfaces can be functionalized to act chemically or biochemically on the fluid circulating in the bioreactor. The production in the form of a flexible multi-compartment bag defining a circulation circuit by simple local welds makes it possible to create a very economical bioreactor, usable both for the storage of a sample and for automatic processing in an automaton, which considerably reduces the production costs of personalized drugs. Description of an example of a bioreactor according to a first variant It represents a top view of a first example of a bioreactor, revealing the compartments (101 to 107) and the channels (201 to 209) through transparency. This bioreactor intended for the preparation of biological drugs in a closed system is formed by a bag composed of two main films (10, 20) hereinafter referred to by convention as "upper film (10) and lower film (20)", although the bag is perfectly reversible. These two films (10, 20) are thermally welded or by US or HF welding along lines or larger areas (300), in order to divide said bag into compartments (101 to 107) connected by channels (201 to 209). The term "welded" includes gluing or any other assembly technique making it possible to locally join the lower surface of the upper film (10) and the upper surface of the lower film (20) to form a sealed local barrier. The periphery of the bag is welded to form a sealed border whose thickness corresponds to the thickness of the two films (10, 20).Optionally, the pocket can be made from a single film folded over itself to form two superimposed panels (10, 20) connected by a common edge. When the pocket is empty, its thickness corresponds to the cumulative thickness of the films which compose it and which are arranged in parallel planes, without peripheral lateral faces. Each compartment (101 to 107) can be isolated from the others by pressing on these channels (201 to 209), for example by a system described later. Similarly, the contents of a compartment (101 to 107) can be transferred into a compartment (101 to 107) to which it is connected by a channel (201 to 209) by removing the support piece which closes the channel, and by applying pressure to this compartment (201 to 209). For example, in the example illustrated by the, by removing the support from the channel (201) and by pressing on the bag (107), the liquid can be transferred from the compartment (107) to the compartment (103), or, by maintaining the support on the channel (201) and releasing the support on the channel (203), the liquid can be transferred from the compartment (107) to the compartment (102). The bioreactor also comprises injection sites (401 to 405) distributed over the transfer channels (201, 207, 208) allowing the compartments to be filled with different liquids. These injection sites (401 to 405) are formed by vias passing through one of the films (10, 20) perpendicular to its surface, into which are inserted tubular pins constituting a fluidic connector arranged on one or two of the faces of the bag, to extend perpendicular to the median plane of the bag. These injection sites (401 to 405) correspond for example to transfusion bag standards. The bioreactor also has a welded peripheral strip (310) whose corners (311, 312, 313, 314) are pierced by holes allowing them to cooperate with lugs of a support to hold the bioreactor on a dedicated support. The materials of both films are suitable for use. For use in producing cell culture-derived factors or CarT Cells or Car-NK Cells, the upper film (10) is made of polyolefin and the lower film (20) is made of polyethylene, both medical grade. These two materials are chosen for their permeability to CO2 and O2 (for cell culture) and to UV-B to induce apoptosis if necessary. The lower polyethylene film (20) allows, thanks to the optical transparency quality of this material, to control cell proliferation or adhesion. In the case of FEP film, transparent to UV-C, it is possible to induce cell necrosis or to sterilize the contents by applying ultraviolet radiation. Description of the bioreactor support Figures 2 to 4 illustrate the configuration of the support, in the open and closed position respectively without support, and closed with support. This support (500) consists of a plate (510) made of plastic or metal, approximately 5 mm thick, and an articulated clamp (550) having a fixed branch (560) secured to the plate (510) and a pivoting branch (570). The tray (510) has on its upper surface four peripheral pins (511 to 514) for positioning the bioreactor bag and is positioned on a support (500) thanks to the four peripheral holes (311 to 314). The lower branch (560) is formed by an aluminum bar which allows the mounting of silicone pins (401 to 405) located below the transfer channels of the bioreactor. The pivoting branch (570) is formed by a second aluminum bar, secured to the fixed branch (560) via a joint (565). The pivoting branch (570) locks, in the closed position, on the front (566) to the fixed branch (560). In the upper bar, a series of ¼ turn mechanisms allows a metal anvil to be pressed onto each silicone pin as illustrated by the. The tray (510) is opaque and has cutouts (515, 516) positioned under the compartments (101 to 107) intended for an interaction between external equipment and the lower surface of the bioreactor, for example a light or optical interaction (excitation in a given wavelength or a visible or infrared spectrum, observation,) or a mechanical interaction (pressure, vibration, etc.). These cutouts (515, 516) can also be provided to transmit a vibration to a single compartment, in order to detach the adherent cells or mix the liquid contained in this compartment. The actuator used for this operation can be a surface speaker or a motor with eccentric, alternately excited electromagnet, or rotating cams. Certain cutouts (515, 516) present on the support can be filled by a plate made of materials transparent to UV-C or UV-B (FEP, PE, glass, etc.) and allow irradiation by positioning under the support a card equipped with UV-C or B LEDs for sterilization, necrosis or apoptosis of the cells present in the corresponding compartment. The compartments (101 to 103) are isolated from the four other compartments (104 to 107) by a seal (56) which allows two thermostatically controlled zones to be partitioned at two different temperatures, for example the left zone at 37°C and the right zone at 4°C. Once the bioreactor is positioned on this support (500), the transfer channels from one compartment to another can be closed by pressurizing the anvil (580 to 581) on the silicone pin by an actuator present on the machines into which these supports will be inserted. Embossments (21) made on the lower film (20), directly above the transfer channels (202), allow rapid and easy passage of the liquid from one compartment to another without applying strong pressure on the pockets. The embossing is sufficient but limited to avoid the formation of folds when they are crushed under the anvil. In the example, the channels are 8 mm wide and the embossing has a height of 0.3 mm. Filtration area The principle of production of the bioreactor according to the invention makes it easy to integrate a filtering zone. To do this, a filtering part, for example a porous membrane (600), is inserted locally between the upper film (10) and the lower film (20). This membrane (600) is, for example, made up of a rectangular part with a mesh size of 1 to 4 µm intended for cell concentration. This filter membrane (600) is positioned to separate along a transverse median plane a compartment (101 to 107), the upper volume of which, between the membrane (600) and the upper film (10), will communicate fluidly with a compartment or channel isolated from the lower volume, and the lower volume of which, between the membrane (600) and the lower film (20) will communicate with another compartment or channel, isolated from the first by a weld. The first step is to place the filter membrane (600) against the lower surface of the upper film (10). The filter membrane (600) is welded on one of the sides (620) with a thermal weld, on a strip (601) of width approximately 5 mm. The second step illustrated by consists of superimposing the lower film (20) and the upper film (10). The upper film (10) with its filter membrane (600) is turned over (filter underneath) and is positioned on the lower film (20). The third step illustrated by consists of producing local welding lines according to a configuration formed by: An open rectangular segment (651) delimiting a compartment (650) surrounding the filter membrane (600), and having an opening (652) at the level of the welding strip (601) of the filter membrane (600) A rectangular segment (681) delimiting a second compartment (680) communicating in the lower part of the filter between the membrane (600) and the lower film (20) with the compartment (650) via said opening (652) A rectangular segment (661) delimiting a third compartment (660) communicating with the second compartment (680) via a channel (662) A rectangular segment (671) delimiting a fourth compartment (670) communicating with the second compartment (680) via a channel (672). The last step is illustrated by the. A mold then allows the filter to be closed by welding the upper film (10) onto the filter membrane (600) along a peripheral line (602) completing the welding strip (601). The liquid present in the lower compartment (650) can only access one of the compartments (660, 670) by passing through the filter membrane (600). If cells are cultured in compartment (650), and channels (662) and (672) between the lower compartment (650) and the other compartments (660) and (670) respectively are clamped. Compartment (660) is empty and compartment (670) contains a different culture medium than that used in compartment (650). An automaton into which the bioreactor is introduced executes a series of actions: Step 1: it controls the opening of the channel (661) between the culture compartment (650) and the compartment (660) which is empty. Step 2: The surface of the compartment (650) concerning the culture is pressed to transfer the contents of the compartment (650) to the compartment (660). The passage must be made through the filter, the membrane (600) being interposed between the lower part of the compartment (650) and the channel (662). If the membrane (600) is sized to retain the cells (for example 0.65µm filter), the cells remain in the compartment (650), while the medium is transferred into the compartment (660). Step 3: The passage between compartments (650) and (660) is closed by clamping channel (662) and channel (672) is opened between compartment (650) and compartment (670). The medium contained in compartment (670) is transferred to resuspend the cells contained in compartment (650) which had previously been emptied of its culture medium. Steps 1 and 2 allow for cell concentration (before transduction for example). Steps 1, 2 and 3 allow for a change of medium. Bioreactor variant It represents a variant of bioreactor, the construction and technical characteristics of which, common to the previous variant, are not developed in the following. The lower part of this bioreactor shows a pocket containing the filter (600) described previously. This pocket also contains two oblong welds (191, 192) which divide this compartment into two zones (801, 802) then connected by three channels (803 to 804). Bosses made by hot embossing, form channels (803 to 804) of connections of a few tenths of a mm between these zones. Successive presses between the right and left zones allow a transfer from left to right and then from right to left to mix the liquid contained therein.

Claims

Bioreactor for the production of a biological drug from a biological fluid obtained from a sample taken from a patient or a donor characterized in that it consists of a multi-compartment flexible bag formed by two flexible films (10, 20) locally associated by welding zones of the inner surfaces of the two films (10, 20) to form watertight barriers delimiting between them a plurality of circulation channels (201 to 209) connecting a plurality of compartments (101 to 107), and in that at least one of said films (10, 20) has a plurality of interconnection vias (401 to 405) each opening into one of said channels (201 to 209) or compartment (101 to 107). Bioreactor for the production of a biological drug according to claim 1 characterized in that at least one of said films (10, 20) is made of polyolefin. Bioreactor for the production of a biological drug according to claim 1 characterized in that at least one of said films (10, 20) is made of polyethylene. Bioreactor for the production of a biological drug according to claim 1 characterized in that at least one of said films (10, 20) is made of Ethylene Propylene Fluorinated transparent to ultraviolet. Bioreactor for the production of a biological drug according to claim 1 characterized in that at least one of said films (10, 20) has an embossing locally widening the distance between said films (10, 20), in a duct area delimited by two welds. Bioreactor for the production of a biological drug according to claim 1 characterized in that it further comprises at least one filtration zone formed by a filter piece (600) interposed between the upper film (10) and the lower film (20) and welded on at least part of its periphery to at least one of said films (10, 20). Bioreactor for the production of a biological drug according to the preceding claim characterized in that said filter piece (600) is welded to one of said films (10, 20) on a part of its periphery, the unwelded part opening onto an area surrounded by weld lines (661, 662, 671, 672, 681) locally sealing said upper film (10) and said lower film (20) to form a feed zone of said filter (600), the film opposite to that on which the filter (600) is welded also having an area surrounded by weld lines locally sealing said upper film (10) and said lower film (20) to form an outlet zone of said filter (600). Bioreactor for the production of a biological drug according to claim 1 characterized in that said vias (401 to 405) are extended by a tubular injection or aspiration pin. Bioreactor for the production of a biological drug according to claim 1 characterized in that an area (650) of at least one of said films is surrounded by weld lines and has a functionalized inner surface (602). Assembly formed by a bioreactor according to claim 1 and a support characterized in that said support consists of a rigid frame having means for attaching a bioreactor and at least one element having fluidic connectors, said element being mobile between a position away from and a position where said fluidic connectors are associated with the vias of the bioreactor. Assembly according to claim 10 characterized in that said frame has windows corresponding to the positions of said compartments, for the passage of an actuation means acting on the surface of the film of said bioreactor. Assembly according to claim 10 characterized in that it comprises at least one sensor disposed in an area delimited by welds, said sensor being extended by wires opening onto the edge of said bioreactor. Assembly according to claim 10 characterized in that it comprises a unique identifier readable optically or by radio frequency communication.

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