Bioreactor with a breached wall through which a receiving tube with a single-use sensor device passes

Description

[0001] The present patent application describes single-use biomass sensor devices.

[0002] The present invention relates to a bioreactor or container according to the generic term of claim 1. The development of new molecules by biotechnological means is still considered the main axis of pharmaceutical development. Today, the pharmaceutical industry recognizes significant advantages associated with single use: safety, due to the limitation of the risk of cross-contamination, and productivity gains obtained (i) by reducing operating costs, in particular personnel costs; (ii) by implementing high-density cultivation helping to limit cultivation times and land use; and (iii) by faster and less expensive industrial scale-up.

[0003] Traditionally, the bioreactors used are reusable, made of glass or stainless steel tanks, requiring constant cleaning, sterilization, and revalidation. These operations, increasingly costly due to the continuous growth of regulatory and quality requirements, are being eliminated by the implementation of single-use solutions.

[0004] There is a growing demand for equipment such as single-use “upstream & downstream” fermenter bioreactors, storage tanks and preparation systems. However, such systems are necessarily equipped with sensors designed to measure physicochemical, biochemical or biophysical parameters. Examples include sensors performing impedance spectroscopy, in particular the capacitive technology sensors designed by the present applicant.

[0005] These capacitive sensors can detect relative variations in capacitance of 10 -5 < in low resistance media (a few tens of Ohms). This technique also allows, thanks to impedance spectroscopy, to determine the parameters of the β dispersion curves (radio frequency). The measurements then provide not only a concentration of living cells but also indications on the morphology and physiological state of the cells. The usual measurement probes are macroscopic in size and intended for stainless steel fermentation tanks. They typically use 4-electrode configurations to overcome the effects of double polarization layer.

[0006] Examples include capacitive sensors specifically dedicated to conductance measurements, particularly for downstream purification applications. These conductance sensors can have the same geometries and electrode structures as capacitive sensors, allowing complete impedance measurements with capacitance and conductance measurements. The use of an aseptic connector is also known, which allows solid sensors to be inserted into a disposable fermenter provided that the environment is very protected and under a laminar flow hood. This solution is generally more expensive due to the cost and operating costs of solid sensors: decontamination, disassembly, cleaning, and sensor assembly.Furthermore, solid sensors intended to equip single-use bioreactors must be sterilized before each use, which does not in itself limit the risk of contamination linked to the sequence of necessary tasks.

[0007] The article Davey, CL et al.: "Real-time monitoring of the accretion of Rhizopus oligosporus biomann during the solid-substrate tempe fermentation" in World Journal of Microbiology and Biotechnology, Rapid Communications of Oxford, Oxford (GB), 1991, pp. 248-249, presents an incubator in which Petri dishes are placed. Sensors, such as temperature sensors, a pH sensor and a Bugmeter sensor with four electrodes to measure capacitance, are placed in the Petri dishes. The walls of the incubator are crossed by the sensor signal lines, which transmit the signals detected by the sensors to data processing devices located outside the incubator.

[0008] From WO 2005 / 068059 A1 a mixing bladder for use in biological technical processes is known. In the mixing bladder, which has a flexible outer shell, a fluid is received and stirred using a stirring element arranged in the fluid bladder. The stirring element is driven by a drive device located outside the mixing bladder. The bladder may have an integral gas distributor and a sensor housing.

[0009] Document FR 2 867 278 A1 discloses a method and device for online measurement and online characterization of biomass in a fermentation process with lactic acid bacteria. FR 2 867 278 A1 discloses in this regard the arrangement of a capacitive sensor in a container directly receiving a liquid.

[0010] The article Carvell, John P., Dowd, Jason E.: "On-line measurements and control of viable cell density in cell culture manufacturing processes using radio-frequency impedance" in Springer-Verlag: "Cytotechnology" (2006) 50: p. 35-48, present, at the figure 5 , a bioreactor whose housing wall is traversed by individual sensors that immerse in the liquid contained in the bioreactor. In this disclosure, the bioreactor itself serves as a container to receive the liquid to be treated.

[0011] WO 02 / 102961 A2 discloses a combination of continuous and batch fermentation processes. The fermentation processes described therein are used to produce potable alcohol. The print discloses a bioreactor head plate to which read and select heads are welded. In addition, a membrane valve for taking samples from the bioreactor is welded to the wall of the bioreactor.

[0012] The aim of the present invention is to overcome these drawbacks by proposing a single-use bioreactor or container integrating a capacitive sensor which has sufficient performance for bioreactor applications, while having a minimized production cost and operating cost.

[0013] The present invention achieves this objective with a bioreactor or container having all the characteristics of claim 1. An embodiment of a single-use bioreactor or container according to the present invention comprises an enclosure intended to contain a biological medium, and at least one device for capturing at least one impedance measurement in a physical, physicochemical and / or biological process in said medium, characterized in that this sensor device is fixed through a wall of said enclosure so as to be an integral part thereof and comprises a capture part in direct contact with the interior of said bioreactor or container and a connection part extending towards the outside of said enclosure.

[0014] To carry out the invention, a single-use device is proposed for capturing at least one impedance measurement in a physical, physicochemical and / or biological process, comprising means for carrying out at least one impedance measurement in a physical, physicochemical and / or biological process in said medium. This device is arranged to be fixed through a wall of an enclosure intended to contain a biological medium, so as to be an integral part thereof, and in that it comprises a capture part in direct contact with said biological medium and a connection part extending towards the outside of said enclosure.

[0015] According to the present invention, this single-use capacitive device is specifically dedicated to the measurement of living biomass in a biological environment, by means of capacitance measurement.

[0016] The single-use sensor device in connection with the invention can be associated online with other single-use physical, physicochemical or biochemical sensors such as sensors for temperature, pH, dissolved oxygen concentration, dissolved CO2 concentration, optical density, pressure, level, ammonium concentration, glycerol concentration, lactate and glucose levels, and more generally any other single-use sensor with the aim of improving knowledge and control of an organic crop.

[0017] An optical sensor using a single-use fluorescent pellet is an advantageous coupling technique and concerns the online measurement of parameters such as pH, dissolved oxygen concentration and dissolved CO2 concentration measurement. It can thus integrate several single-use sensors on the same support.

[0018] Thus, with the invention, it becomes possible to produce the first online, biocompatible and sterilizable single-use multi-sensor system, measuring the key stages of a cell culture, with a view to controlling it according to quantitative and qualitative production criteria and objectives at any scale. It will also make it possible to couple biodiagnostics and microtechnologies for the characterization of various biological environments.

[0019] In a particular embodiment, the sensor device is arranged to be installed in a sterile envelope with multiple applications.

[0020] The sensor device may also be arranged to be inserted and immersed in an enclosure intended to contain biological cells.

[0021] According to the invention, the single-use sensor device is arranged to be installed in a bioreactor made for use in a fixed bed, i.e. any type of cell culture on an adherent, micro-carrier and macro-carrier support or any other application in which the desired measurements come from an adherent-type culture process. It can, for example, be inserted into a basket immersed in an enclosure designed to contain biological cells whose growth model is considered static, for example, for the culture of stem cells for the purpose of medical applications.

[0022] In another variant of the invention, the single-use sensor device according to the invention is arranged to be installed in a single-use bioreactor.

[0023] The sensor device according to the invention can also be inserted into a passage provided in a wall of the bioreactor and have a capture face oriented and in contact with the interior of this bioreactor.

[0024] In an advantageous embodiment, the single-use sensor device comprises electrodes extending over the sensing portion.

[0025] It can be produced in the form of a machined mechanical part arranged to receive the electrodes on a capture part and on a connection part the measurement signal output means.

[0026] It can also be produced in the form of an injection-molded part including the electrodes on a capture part and the measurement signal output means on a connection part.

[0027] The measurement signal output means may comprise a connector or a cable or hose.

[0028] The electrodes can be made from metal parts or by metal deposition on an insulating support which can be flexible.

[0029] This flexible support can be produced in the form of two insulating films, at least one of which supports an electrode produced by deposition and electrically connected to a conductor placed between the two insulating films.

[0030] The single-use sensor device may be arranged to be installed in a sterile multi-application envelope.

[0031] In a particular embodiment, the single-use sensor device comprises on-board means for locally processing impedance measurement signals. These on-board means may be provided to produce an alert signal in response to an anomaly detection by the on-board processing means.

[0032] According to another aspect of the invention, a method is proposed for producing a single-use sensor device according to the invention, implemented in a production environment meeting the asepsis and sterility criteria required for the use of a bioreactor.

[0033] In fact, all the techniques implemented (welding, washing, cleaning, bacterial and viral decontamination, mechanical tools, analysis means) represent a new production methodology which until now had not been implemented for the production of solid and traditional sensors.

[0034] The materials used to produce the single-use sensor device must be biocompatible. These materials can advantageously be compatible with sterilization by gamma irradiation before and / or after installation of the sensor device on the bioreactor.

[0035] The present invention is also applicable to offline measurement methods such as medical diagnostics, biological, medical, environmental quality control, or specific instrumented packaging for biological control, and is therefore not limited to sterile enclosures with online measurement.

[0036] In another application of a single-use biomass sensor, it is mounted on an aseptic enclosure and is optionally associated with a physical or biological sensor, to create a disposable biological medium storage container on which disposable sensors are integrated. These sensors allow the living biomass and other physicochemical parameters to be monitored at any time with other associated sensors.

[0037] This storage method can be made using the same sensors and enclosure materials as for the bioreactor application. It allows any biological incident to be detected during storage of the material, without the risk of loss of sterility during this control because there is no sampling.

[0038] This application is particularly suitable for industrial control of biological or chemical materials before and after delivery as part of product quality monitoring. It also provides a definite advantage for any long-term storage where regular quality control is required or the expiry date of a product such as food is to be estimated.

[0039] It thus becomes possible to create intelligent packaging that can detect, using its integrated sensors, any anomaly or storage accident, and provide traceability for the transport of compositions, products or foodstuffs involving safety and quality requirements.

[0040] Other advantages and characteristics of the invention will appear on examining the detailed description of a non-limiting embodiment, and the appended drawings in which: There figure 1 is a schematic view of a single-use sensor implemented in a fixed-bed single-use reactor; The figure 2 is a schematic view of a single-use bioreactor, which does not belong to the present invention, for suspension cell cultures equipped with single-use sensors; The figure 3 illustrates a practical example of a single-use bioreactor equipped with single-use sensors; The figure 4A is a sectional view of a four-electrode single-use sensor; The figure 4B is a top view of the single-use sensor of the figure 4A ; There figure 5A is a sectional view of a first method of connecting the electrodes of a single-use sensor made from a machined part and using a connector coated with glue; The figure 5B is a sectional view of a second method of connecting the electrodes of a single-use sensor made from a machined part and using a cable or a flexible hose; figure 6A is a sectional view of a single-use sensor having injection-molded electrodes and connector; figure 6B is a sectional view of a single-use sensor provided with injection-molded electrodes and a cable or hose; The figures 7A, 7B et 7C illustrate a particular example of a single-use sensor in which the electrodes are deposited directly on a flexible tube; The figure 8 illustrates a combination of a single-use impedance measuring sensor and a single-use fluorescence measuring sensor; in a separate assembly, and The figure 9 illustrates the combination of a single-use impedance measuring sensor and a single-use fluorescence measuring sensor, mounted on the same support.

[0041] We will now describe, with reference to the aforementioned figures, several examples of single-use sensors, in different implementation configurations.

[0042] We will first describe an example of implementation of a single-use impedance sensor 10 implemented in a reusable bioreactor 1, with reference to the figure 1 This bioreactor 1 may, by way of non-limiting example, be of the type marketed by the companies WAVE, SARTORIUS or ATMI.

[0043] The impedance sensor implemented may be of the capacitive type such as that marketed by the present applicant and disclosed in document FR2 812 725. It may also be of the “multi-sensor” type, such as those including optical sensors which are disclosed in document FR2 867 279 in the name of the present applicant.

[0044] The bioreactor 1 comprises, in an enclosure 3, a fixed bed 5 containing biological cells and equipped with a stirrer 4, a cover 7 fixed to the enclosure 3 by welds S and pierced with an orifice 2 in which is inserted a receiving tube 9 intended to receive a single-use impedance sensor 10 provided at its end with a set of electrodes 6. These electrodes may be two, four or more in number, depending on the measurement technique implemented. The impedance sensor 10 comprises conductors 8 for delivering the measurement signals generated by this sensor.

[0045] When it comes to equipping a single-use bioreactor 20, with reference to the figure 2 , single-use sensors 21, 22, 23 can be installed respectively on a side face, the bottom and the front face of the tank of this bioreactor. Thus, as illustrated in the figure 3 , a single-use bioreactor 30 implemented on a permanent use control apparatus 36 can be provided fully equipped with single-use sensors 31-35.

[0046] In reference to the figures 4A et 4B , a single impedance measuring sensor 40 can be arranged in an orifice of a wall 42 of a bioreactor, with a capture part of which a capture face 41 is arranged inside the bioreactor and fixed by welding S to the inner face of this wall. The impedance measuring sensor then forms an integral part of the bioreactor.

[0047] Other fixing methods such as screwing, fitting or gluing can also be provided. The sensor 40 is provided on the capture face 41 with four electrodes E1-E4, two of which provide an excitation function and two a measurement function.

[0048] The connection of the electrodes of a single-use sensor to a measuring or control device can be carried out either by means of a connector ( figures 5A And 6A ), either by means of a cable or flexible ( figures 5B And 6B ). These connection means constitute the connection part of the single-use sensor.

[0049] The single-use sensor can be made either from a machined plastic part ( figures 5A et 5B ), or by injection with overmolding of the electrodes and connection means ( figures 6A et 6B ).

[0050] The electrodes of a single-use impedance measuring sensor can be made: either in the form of metal parts that can be glued ( figures 4A, 4B, 5A, et 5B ) or overmolded ( figures 6A et 6B ), or by deposition, for example by vacuum deposition techniques, by screen printing, lithography, or electrolysis.

[0051] In a first configuration illustrated by the figure 5A , the single-use sensor 50 inserted into an orifice of a bioreactor is made from a machined part 52 in which four electrodes E1 - E4 and a connector 51 fixed to the machined part by gluing C have been arranged.

[0052] In a second configuration illustrated by the figure 5B , the single-use sensor 60, made from a machined part 62, is equipped with a flexible tube or cable 61 electrically connected to the four electrodes E1 - E4.

[0053] In a third configuration illustrated by the figure 6A , the single-use sensor 55 is produced by injection of plastic material with overmolding of the four electrodes E1 - E4 and a connector 53.

[0054] In a fourth configuration, the single-use sensor 62 is produced by injection of plastic material with overmolding of the four electrodes E1 - E4 and a flexible tube 63.

[0055] In another embodiment illustrated by the figures 7A, 7B et 7C , the electrodes E'1 - E'4 of a single-use sensor 70 are produced by metal deposition directly on a flexible tube 71. This sensor 70 can then be fixed by welding S on the wall 42 of a bioreactor.

[0056] In reference to the figure 7C , the flexible tube 71 is produced by superimposing two insulating flexible tubes 71.1, 71.2 between which a conductive track 72 is produced, connected to a deposited electrode E by means of a via 73 passing through the insulating flexible tube 71.1 on the outer face of which the electrode E is deposited.

[0057] A single-use impedance measuring sensor 40 may be combined with another single-use sensor, for example a fluorescence sensor for measuring pH, oxygen (O 2 ) or carbon dioxide (CO 2 ).

[0058] The two single-use sensors can either be mounted separately ( figure 8 ), or arranged on the same support ( figure 9 ). In the first mode, each single-use sensor 40, 82 is provided on a separate support 41, 81, while in the second mode, the active elements of the two sensors - the four electrodes E1 - E4 of the impedance measurement sensor, and the fluorescence sensor 82 - are arranged on the same patch or support 91 and constitute a double single-use sensor 90.

[0059] We will now describe the constraints for producing single-use sensors according to the invention. According to the rules of good manufacturing practices (cGMP), solvents and all pollution must be prohibited from manufacturing processes to avoid polluting the biomaterials used in the industrial production of the sensor. In particular, the "leachables and extractables" standard (21 CFR part 600.11) must be followed.

[0060] The sterilization chain must not be broken between the manufacture of a sensor and its implantation at a subcontractor. Single-use sensors are sent in sterile packaging and are mounted under laminar flow on the bag.

[0061] Once all the elements are assembled, the bag is sterilized by gamma radiation.

[0062] The materials used must of course be biocompatible and resistant to the dose of gamma rays used.

[0063] The single-use sensor must be pre-decontaminated, i.e. be free of any pollutants below a threshold accepted by the application. These pollutants are mainly captured during the processes used for manufacturing the sensor (machining, assembly, welding, bonding, etc.). The packaging of single-use sensors is designed to maintain the sterility of the sensor and to be compatible with the requirements and constraints for use in a clean room. Therefore, double-walled packaging is recommended.

[0064] The sensor is mounted on the bioreactor and gamma sterilization is preferably carried out after the sensor and all components (pipes, caps, fittings, etc.) have been mounted. For other applications, sterilization can be carried out immediately after the sensor has been packaged. Other sterilization methods can also be considered.

[0065] Single-use sensors can be connected, wired or wireless, to electronic impedance measurement equipment available on the market or to equipment more specific to biomass measurement such as that disclosed in document FR2 812 725.

[0066] It is also possible to provide for all or part of the measurement and processing electronics to be embedded in the bioreactor, the packaging or the smart container, using integration technologies, possibly with self-power supply. In particular, optical or audible warning devices can also be associated with the single-use sensors according to the invention. For example, in the case of smart packaging intended to store a biological medium, an alert signal can be generated when an abnormal change in the capacitance or a physical or physicochemical quantity is detected. Reference may in particular be made to document FR2 874 26 in the name of the present applicant, which discloses a method and device for determining biomass in a medium.

[0067] Manufacturing techniques from electronics and microelectronics could be implemented for large-scale manufacturing of electrodes of a single-use sensor according to the invention. In particular, a capacitive detection integrated circuit could be produced by diffusion techniques, for example on polymer substrates or substrates commonly used for the production of microsystems.

Claims

1. Bioreactor or container (1) comprising an enclosure (3) intended to contain a biological medium and comprising at least one disposable device for capturing at least one impedance measurement in a physical, physico-chemical and / or biological process in said medium, this sensor device being specifically dedicated to the measurement of living biomass in a biological medium, by means of capacitance measurement, this sensor device being fixed through a wall (7) of said enclosure (3) so as to be an integral part thereof and comprising a collection portion in direct contact with the inside of said bioreactor or container (1) and a connection portion extending to the outside of said enclosure, this wall (7) being pierced by an orifice (2) into which is inserted a receiving tube (9) designed to receive the disposable impedance sensor device (10) provided at its end with a set of electrodes (6), the wall (7) to which the sensor device (10) is attached being a cover (7), characterized in that the bioreactor or container (1) additionally comprises in the enclosure (3) a fixed bed (5) spaced from the enclosure, and in that the cover (7) is attached to the remaining enclosure by welds (S).

2. Bioreactor or container according to claim 1, characterized in that the receiving tube (9) is secured to the wall (7), particularly to the cover (7), by welds (S).

3. Bioreactor or container according to any of the preceding claims, characterized in that the sensor device (10) comprises conductors (8) for outputting the measurement signals generated by the sensor device (10).

4. Bioreactor or container according to any of the preceding claims, characterized in that the fixed bed (5) contains biological cells, in particular living cells.

5. Bioreactor or container according to any of the preceding claims, characterized in that it is a reusable bioreactor or container.

6. Bioreactor or container according to any one of claims 1 to 4, characterized in that it is a single-use bioreactor or container.

7. Bioreactor or container according to any of the preceding claims, characterized in that all or part of the measurement and processing electronics is on-board the bioreactor.