Cultivation system and container attachment for a cultivation container

Description

[0001] The invention relates to a cultivation system with a container attachment for a cultivation container, in particular a handheld one, e.g. for a shaker flask.

[0002] Bioreactors and shake flasks are used to cultivate microorganisms, animal and plant cells, thus opening up a wide range of applications for biotech production processes. There is a general need to further optimize these processes. Particularly in the production of biopharmaceuticals, improvements in product yield and thus increased profits are being pursued. Various approaches are available for controlling and monitoring production processes, increasing product yield, and reducing costs.

[0003] Generally, processes can be controlled and monitored by determining substrate and product concentrations. However, this is time-consuming and usually requires resource-intensive offline analysis. Furthermore, the sampling process carries a significant risk of contamination.

[0004] When using conventional bioreactors, process control and thus yield can be optimized by real-time process control of key parameters. In particular, in-situ monitoring of parameters such as temperature, metabolism- or product-formation-relevant substances, as well as real-time control of cultivation conditions, is advantageous for increasing product yield.

[0005] In contrast, when shake flasks or other handheld cultivation containers are used, a sample draw is still required, which poses a contamination risk due to the handling involved with the cultivation unit open. Furthermore, the sample draw is time-consuming, as is the determination of substrate and product concentrations using offline analysis.

[0006] A sample withdrawal can also mean that the interruption of mixing via a shaking incubator brings the mass exchange, especially of dissolved gases, to a standstill, inhibiting culture. Additionally, the multi-hour reaction time required for pH adjustments or substrate additions (feed), which is due to the duration of the off-line analysis, can be disruptive. Furthermore, the risk of contamination increases with each opening of the shake flask.

[0007] Metabolism typically produces pH-lowering metabolic products. Attempts are sometimes made to counteract this by buffering the medium. Particularly during process development, pH monitoring and additions to adjust the pH may be necessary when the buffering capacity is exhausted. Medium components should not be introduced at arbitrarily high concentrations at the start of cultivation, as this can lead to undesirable substrate inhibition.

[0008] To reduce the number of sample draws when using a shake flask, approaches include measuring the optical density and luminescence, e.g. for pH and pO2, of glued-in spots through the shake flask.

[0009] However, the optical quality of the shake flask wall (e.g., transmission, scattering) limits this principle. Furthermore, it must be considered that measuring cell growth via optical density requires a constant extinction coefficient. This coefficient, however, varies with the changing medium composition during cultivation. This results in a difference between viable and lysed cells. With embedded spots, the limitations of leaching and drift are further compounded.

[0010] DE202016000554U1 relates to a disposable connection device for insertion into a connection opening of a top plate of a bioreactor and / or into a connection opening of a bag bioreactor, with multiple passages and a fastening section, wherein the disposable connection device is made in one piece from plastic and the fastening section has a fastening structure on an outer circumferential surface for fastening the disposable connection device in the connection opening.

[0011] WO2024044612A1 relates to a reactor tank designed as a disposable element with a lid and / or opto-electronically readable sensor patches attached inside, a reactor comprising the reactor tank and a reactor tank receiving periphery, itself comprising a reactor tank holder and optionally an opto-electronic measuring system for reading sensor patches, wherein the reactor tank holder is coupled to a drive unit for generating a rotary-oscillating movement of the reactor tank about its own central vertical axis, and the use of this device for cultivating cells and / or microorganisms.

[0012] EP3109314A1 relates to a cell cultivation device comprising a culture tank for receiving a culture solution containing a cell, a shaft element that is at least partially arranged in the culture tank, a stirring mechanism that is supported by the shaft element, is arranged in the culture tank and has at least one pair of stirring blades configured to be rotatable about the shaft element, and a filter that is arranged in contact with the shaft element to draw the culture solution from the culture tank and / or to supply the culture solution to the culture tank.

[0013] CN201658978U relates to a bioexperimentation apparatus comprising at least one bottle body; the bottle body is provided with a bottle opening; a bottle stopper is arranged over the bottle opening; a bottle cap is screwed onto the bottle opening; the inner side wall of the bottle cap and the outer circumferential wall of the bottle opening are threaded together; the bottle cap presses the bottle stopper onto the bottle opening; the upper surface of the bottle cap is provided with an opening that causes the bottle stopper to be partially exposed; the exposed part of the bottle stopper is partially inserted into an air inlet tube, an air outlet tube, and a sampling tube; and the upper end of the sampling tube is provided with a sealing cover for sealing the sampling tube.

[0014] WO2018097510A1 relates to a bioreaction vessel comprising: a culture chamber which accommodates a culture solution and an organism within its interior and has an open upper end; a chamber closure part coupled to the upper end of the culture chamber and comprising on one side of it a projection tube which communicates with the interior; a filter cap which is detachably coupled to the projection tube and opens / closes the projection tube; a gas injection part which penetrates the chamber closure part and communicates with the interior to supply a predetermined gas to the interior; and an acid / basicity adjustment part which is provided on the chamber closure part in a state in which an adjustment solution for adjusting the pH of the culture solution is contained within it, and which allows the adjustment solution to be dispensed into the interior by atmospheric pressure.

[0015] CN202297585U relates to a tank for the cultivation of liquid microorganisms comprising a tank body and further a tank body cover, an air compressor, an agitator, a bottle containing acidic material, a bottle containing alkaline material, a temperature sensor and a pH sensor, wherein the tank body cover covers an end opening of the tank body; a water inlet is formed in the tank body cover; a cushion opening is formed in the center of the tank body cover; the rear end of the agitator penetrates through the cushion opening and the tank body cover axially positions the agitator; the temperature sensor and the pH sensor are arranged on the inner side wall of the tank body; an outlet opening is formed in the lower part of the side wall of the tank body; the air compressor is connected to the inner bottom part of the tank body by a pipe;and the bottle containing the acidic material and the bottle containing the alkaline material are each connected to the interior of the tank body by pipes.

[0016] WO1990008816A1 relates to an apparatus, device, and method for the sequential enrichment of a specific microorganism up to a threshold value to enable its detection. Using the device for the sequential enrichment of microorganisms reduces the time normally required to enrich samples to allow the growth of a sufficient number of microorganisms to detect the specific microorganism of interest.

[0017] JP002007202542A relates to a cell culture vessel in which a culture solution reservoir is attached to the bottom of the vessel, and the tip of a drain tube for draining the culture solution from the inside of the vessel is positioned to the outside of the vessel in such a way that it comes into contact with the liquid surface of the culture solution reservoir. This removes the bubbles from the culture solution reservoir.

[0018] WO2018037402A1 relates to a bioreactor comprising an interior space and a perforated barrier within the vessel through which a liquid can flow, while preventing cells or microorganisms from passing through the perforated barrier. The perforated barrier divides the interior space of the bioreactor into a first chamber and a second chamber. Cells are cultivated in the second chamber and can be perfused through the bioreactor by recirculating the liquid, e.g., a growth medium. Various inlet and outlet openings allow for the control of the growth medium's flow parameters.

[0019] The JPH06141850A relates to a culture tank equipped with an agitator and connected via appropriate pumps to a temporary cell reservoir and a waste liquid tank. During cultivation, the agitation is temporarily stopped to allow the resulting cell aggregates to settle, and the supernatant culture liquid containing the cells to be harvested is drawn into the tank where the culture liquid is temporarily stored. The cell aggregates adhering to the tank wall are then detached, for example, by vigorous agitation or the application of ultrasonic vibration, and discharged into the waste liquid tank along with the cell aggregates remaining in the culture tank.

[0020] Therefore, one object of the invention is to make cultivations using shake flasks or other handheld cultivation containers as efficient as bioreactors, while simultaneously minimizing the requirements for peripheral equipment, e.g., avoiding the need for a sterile workbench. A general aspect of the invention's objective is to make biotech production processes more cost-efficient and to enable more agile application.

[0021] The invention discloses a container attachment for mounting on a cultivation container, particularly a handheld one, which will be discussed in more detail below. The invention further relates to a cultivation system comprising a cultivation container, particularly a handheld one, for holding a culture medium and a

[0022] Container attachment which can be placed on the cultivation container. The invention is a container attachment according to claim 1, a cultivation system according to claim 10, and a method for propagating or cultivating biological material according to claim 13.

[0023] The cultivation vessel, which is preferably designed as a shake flask, has a neck and an opening extending through the neck. Preferably, the opening forms the only access to the interior of the cultivation vessel; that is, it is preferably not an additional port on the cultivation vessel.

[0024] The container attachment is placed on the neck of the cultivation container to close the opening, ensuring a sterile seal. When the container attachment is placed on the neck of the cultivation container, one inner side of the attachment faces the interior of the cultivation container, i.e., the sterile area, and one outer side of the attachment faces the exterior of the cultivation container, i.e., the non-sterile area.

[0025] The container attachment is characterized by the fact that it includes at least one sensor unit, in particular at a port for installing a sensor unit, and a dispensing unit, in particular at a port for installing a dispensing unit.

[0026] The sensor unit is positioned, at least partially, on the inside of the container attachment to enable parameter measurement inside the cultivation container. The sensor unit can, for example, be permanently attached to the inside of the container attachment.

[0027] On the other hand, the sensor unit can also be arranged or installed on the inside of the cultivation container, thus enabling modular replacement, for example by installing a sensor component or a multi-sensor component in a corresponding port of the container attachment. Reference is made in particular to German patent application 10 2019 117 446.5.

[0028] The dispensing unit is also located, at least partially, on the inside of the container attachment to allow liquid to be dispensed into the interior of the cultivation container. The dispensing unit can, in turn, be permanently attached to the container attachment, particularly completely or at least partially on its inside, to enable the dispensing of liquid into the interior of the container.

[0029] On the other hand, the dispensing unit can also be arranged or installed at least partially, i.e., at least with a part of the dispensing unit, on the inside of the cultivation container, so that in particular a modular exchange is possible, e.g. by installing the dispensing unit in a corresponding port of the container attachment.

[0030] While a sensor unit can capture and, if necessary, process key parameters for process control in real time, a dispensing unit allows for active corrections, such as pH adjustment or feed rate. If parameter deviations from the target value lie outside the intervention limits, actuators can be actively controlled, for example, for pH adjustment or feed rate. By incorporating a sensor unit or a corresponding port and / or a dispensing unit or a corresponding port into the container attachment, the integration of sensors and / or actuators is enabled, making autonomous cultivation systems with closed-loop control for optimized cultivation conditions possible. If both a sensor unit and a dispensing unit are included or installed, a CAP system (Controlled and Adjusted Process) is created, which, in particular, enables the demand-oriented use of sensors and actuators within a single system.

[0031] With the invention, for example, a CAP cultivation system comprising, in particular, a handheld cultivation vessel or a shake flask, the requirements for peripheral equipment are drastically reduced, especially compared to conventional bioreactors. In some cases, a shake incubator and an autoclave may suffice. Even a sterile workbench can potentially be avoided, particularly through the use of sterile connectors, as explained in more detail below. These aspects increase cost-efficiency and enable broader and more agile application. The invention ensures that cultivation conditions are continuously maintained within the optimal range. Parameter deviations can be counteracted instantly. The invention provides a cultivation system with comparable performance to a bioreactor.

[0032] The cultivation vessel can, in principle, have any shape, for example, like a classic bioreactor (possibly with flow breakers) with straight sidewalls. However, handheld cultivation vessels, especially shake flasks, are preferred. Reasons for this include availability and design, with mass and heat transfer being a key design feature, which can be influenced in particular by the number of baffles.

[0033] The cultivation container has a neck, which is preferably annular, and in particular cylindrical. Accordingly, the container attachment can have an annular, and in particular cylindrical, slot to accommodate the neck of the cultivation container.

[0034] Furthermore, the container attachment may have an outer rim, which belongs to the outside of the container attachment and radially surrounds the neck of the cultivation container when the container attachment is placed on the neck of the cultivation container. The container attachment may also have an inner projection, which belongs to the inside of the container attachment and projects into the opening extending through the neck of the cultivation container when the container attachment is placed on the neck of the cultivation container. It may be provided that the outer rim and the inner projection of the container attachment are monolithic. In other words, the outer rim is specifically designed not to be rotatable relative to the inner projection.

[0035] The cultivation container can have a flat base for placement on a surface. The neck of the cultivation container can, for example, extend vertically upwards, particularly when the container is placed on the surface. Accordingly, the container attachment can be placed vertically from above onto the neck of the cultivation container and preferably further designed to be held in place by gravity on the neck of the cultivation container.

[0036] The container attachment, which can be placed on the cultivation container, preferably has a stop surface that comes into contact with the cultivation container when the container attachment is placed on it. The stop surface is located, in particular, inside a gap provided in the container attachment, especially an annular one, for receiving the neck of the cultivation container. In other words, the container attachment rests, in particular with its stop surface, on the end face of the neck when the container attachment is placed on the cultivation container.

[0037] The cultivation vessel has a volume of less than 2801 milliliters, preferably less than 1801 milliliters, more preferably less than 501 milliliters, more preferably less than 251 milliliters and most preferably less than 126 milliliters.

[0038] Furthermore, the cultivation container can have a diameter of less than 22 centimeters or less than 21 centimeters, preferably less than 14 centimeters or less than 12 centimeters, particularly preferably less than 9 centimeters or less than 8 centimeters.

[0039] The cultivation container can have a height of less than 31 centimeters, preferably less than 23 centimeters, more preferably less than 19 centimeters, particularly preferably less than 17 centimeters, and most particularly preferably less than 14 centimeters.

[0040] The container extension, which can be placed on the cultivation container, preferably has a height, measured from the contact surface, of less than 10 centimeters, preferably less than 5 centimeters, more preferably less than 4 centimeters, even more preferably less than 3 centimeters, and most preferably less than 2 centimeters. In other words, when placed on the container, the extension results in a maximum increase in the height of the cultivation container by the specified values.

[0041] As already described, the container attachment preferably comprises one or more dispensing units which serve to control process parameters. The at least one dispensing unit of the container attachment comprises a reservoir for liquid storage, wherein the reservoir has a lower reservoir section with an outlet opening for liquid dispensing and an upper reservoir section with a reflow opening for gas reflow.

[0042] The lower reservoir section is located on the inside of the container attachment to allow liquid to be dispensed through the outlet opening into the interior of the cultivation container, and the upper reservoir section is located on the outside of the container attachment and is wider to form a stop when the dispensing unit and / or reservoir is installed on a port of the container attachment.

[0043] The container attachment and / or the dispensing unit may have a connection, in particular comprising a hose, between the inflow opening of the upper reservoir section and the inside of the container attachment to allow, in particular sterile, inflow of gas from the interior of the cultivation container when the container attachment is placed on the neck of the cultivation container.

[0044] The reservoir, in particular the upper reservoir section, may further have a refill opening for refilling with liquid, wherein the container attachment and / or the dispensing unit preferably has a connection, in particular comprising a hose, between the refill opening of the reservoir and a valve arranged or arrangable on the outside of the container attachment to enable, in particular sterile, refilling of liquid into the reservoir. The container attachment and / or the dispensing unit may further have a bracket, e.g. radially mounted on the outside, for releasably fixing the valve.

[0045] To facilitate the addition or removal of material, particularly sterile material, from the interior of the culture vessel when the vessel attachment is placed on the neck of the culture vessel, the vessel attachment may also have a connection, particularly comprising a tube, between the inside of the vessel attachment and a valve located or arrangable on the outside of the vessel attachment. In this respect, the vessel attachment may also have a holder, e.g., radially mounted on the outside, for the releasable fixation of the valve.

[0046] At least one sensor unit of the container attachment can be designed, in particular, as a biosensor unit for analyte-specific parameter measurement. Analyte-specific parameter measurement can imply that a specific measurand, the analyte, is selectively detected, and in particular, that multiple parameters, e.g., different analytes, are not detected simultaneously.

[0047] The at least one or more sensor units of the container attachment, which are specifically not designed as biosensor units, can alternatively be designed as luminophore units for luminescence-based parameter measurement. In luminescence-based parameter measurement, analyte-dependent signal quenching can be performed. The excitation wavelength can be shifted in wavelength and / or phase by interaction with the measured quantity.

[0048] At least one or more sensor units can also be configured as alternating field units for dielectric-based parameter measurement. When charges shift in an analyte within an electric field, a dipole is induced. In dielectric-based measurement, this frequency-dependent interaction can be utilized within a sensor unit.

[0049] At least one or more sensor units can also be configured as transistor units for field-effect-based parameter measurement. In this process, ionic analytes can induce a conductivity-generating mirror charge in a semiconductor element, particularly by reversibly adhering to a sensor chip in an ion-selective manner.

[0050] With regard to the sensor unit, reference is made to German patent application 10 2019 117 446.5. In particular, the at least one sensor unit can be arranged or arranged on a front housing section of a sensor component, especially a multi-sensor component, such that the at least one sensor unit can be arranged on the inside of the container attachment by installing the sensor component at a port of the container attachment to enable parameter measurement inside the cultivation container when the container attachment is placed on the neck of the cultivation container. In the case of a multi-sensor component, several sensor units can be installed simultaneously.

[0051] Preferably, the container attachment comprises a plurality of sensor / dispensing units, in particular at corresponding ports, and most preferably selectively at least one sensor unit and at least two dispensing units.

[0052] In addition to the cultivation system described above with cultivation container and container attachment, the invention also relates to the container attachment for placing on a cultivation container, in particular a handheld one, preferably on a shaker piston.

[0053] The container attachment comprises an inner side, which faces the inside of the cultivation container when the container attachment is placed on the cultivation container, and an outer side, which faces the outside of the cultivation container when the container attachment is placed on the cultivation container.

[0054] Furthermore, the container attachment comprises at least one sensor unit, in particular at a port for installing a sensor unit, and at least one dispensing unit, in particular at a port for installing a dispensing unit. The sensor unit is arranged at least partially on the inside of the container attachment to enable parameter measurement inside the cultivation container. The dispensing unit is also arranged at least partially on the inside of the container attachment to enable liquid dispensing into the interior of the cultivation container.

[0055] The container attachment may have an annular, particularly cylindrical, slot to accommodate the neck of the cultivation container. Furthermore, the container attachment may have an outer rim that radially surrounds the neck of a cultivation container when the attachment is mounted on it. Additionally, the container attachment may have an inner projection that extends into the neck of a cultivation container when the attachment is mounted on it. The outer rim and the inner projection may be monolithic.

[0056] The container attachment is specifically designed to be placed vertically from above onto a cultivation container and is preferably designed to be held in place on the cultivation container by gravity.

[0057] The container attachment preferably has a stop surface which comes into contact with the cultivation container when the container attachment is placed on the cultivation container, wherein the stop surface is located in particular inside a gap provided in the container attachment, in particular annular, for receiving the neck of the cultivation container.

[0058] The container attachment, which can be placed on the cultivation container, preferably has a height of less than 10 centimeters measured from the stop surface, preferably less than 5 centimeters, more preferably less than 4 centimeters, even more preferably less than 3 centimeters, and particularly preferably less than 2 centimeters.

[0059] The at least one dispensing unit of the container attachment comprises a reservoir for liquid storage, wherein the reservoir has a lower reservoir section with a discharge opening for liquid dispensing and an upper reservoir section with a reflow opening for gas reflow.

[0060] The lower reservoir section is located on the inside of the container attachment to allow liquid to be dispensed through the outlet opening into the interior of the cultivation vessel. The upper reservoir section is located on the outside of the container attachment and is wider to form a stop when the dispensing unit and / or reservoir is installed on a port of the container attachment.

[0061] The container attachment and / or the dispensing unit may have a connection between the reflow opening of the upper reservoir section and the inside of the container attachment to allow gas to reflow from the interior of the cultivation container when the container attachment is placed on the cultivation container.

[0062] Furthermore, the reservoir can have a refill opening for refilling with liquid, wherein the container attachment and / or the dispensing unit preferably has a connection between the refill opening of the reservoir and a valve arranged or arrangable on the outside of the container attachment to allow refilling with liquid into the reservoir. The container attachment and / or the dispensing unit can have a bracket for releasably fixing the valve.

[0063] To allow for the addition or removal of material from the interior of the cultivation container when the container attachment is placed on the cultivation container, the container attachment may furthermore have a connection between its inner surface and a valve located, or arrangable, on the outer surface of the container attachment. For this purpose, the container attachment may also include a holder for the detachable fixation of the valve.

[0064] The at least one sensor unit of the container attachment can be configured as a biosensor unit for analyte-specific parameter measurement. Alternatively, the at least one or a further sensor unit of the container attachment, which is specifically not configured as a biosensor unit, can be configured as a luminophore unit for luminescence-based parameter measurement. The at least one or a further sensor unit can also be configured as an alternating field unit for dielectric-based parameter measurement. Furthermore, the at least one or a further sensor unit can also be configured as a transistor unit for field-effect-based parameter measurement. This is based in particular on German Patent Application 10 2019 117 446.5. The at least one sensor unit can be arranged or arranged on a front housing section of a sensor component, such that the at least one sensor unit can be arranged on the inside of the container attachment by installing the sensor component on a port of the container attachment.

[0065] In a preferred embodiment, the container attachment has a plurality of sensor / dispensing units, in particular at corresponding ports, most preferably at least one sensor unit and at least two dispensing units.

[0066] The disclosure further relates to a dispensing unit for installation on a port of a container attachment, in particular as described above, wherein the dispensing unit comprises a reservoir for liquid storage, the reservoir having a lower reservoir section with an outlet opening for liquid dispensing and preferably an upper reservoir section with a refill opening for gas flow. The reservoir may have a refill opening for refilling with liquid, and a connection may also be provided between the refill opening of the reservoir and a valve to enable, in particular sterile, refilling of liquid into the reservoir.

[0067] The liquids in the reservoirs and the medium are preferably matched to cover the full range of cultivations in the life sciences. For example, a feed solution in the reservoir can contain the substrates required for product formation, such as saccharides. Since cell metabolism can produce pH-lowering components of the medium, such as organic acids, a pH-raising correction solution, e.g., containing NaOH, can preferably be provided. Thus, a pH correction solution can be provided in the reservoir. A pH correction solution in the reservoir can, in particular, form a buffer system with the medium. For example, cell metabolism produces CO₂ and thus H₂CO₃, and the HCO₃⁻ correction solution creates a buffer system with a regulated buffer capacity and a stable pH.

[0068] Furthermore, a feed solution containing pH correction can also be provided. Stoichiometry, in particular, can be used to determine the amount of pH-lowering substances produced by the metabolism of the feed. Accordingly, the pH correction can be added to the feed solution, taking into account the substrate turnover rate and the buffering capacity of the medium. This allows, for example, biomass to be generated by dosing from a first reservoir, while product formation can be optimized by dosing from a second reservoir.

[0069] The disclosure further relates to a process for the propagation or cultivation of biological material, preferably for the production of pharmaceuticals, in particular biopharmaceuticals, comprising the following process steps: providing a cultivation system, in particular as described above, comprising a cultivation vessel and at least one reservoir, each with an outlet opening for dispensing liquid into the interior of the cultivation vessel; providing a feed solution, in particular containing the substrates required for product formation, e.g., saccharides, wherein the feed solution is preferably provided in the reservoir; providing a pH correction solution, in particular for raising the pH value, e.g., containing NaOH, wherein the pH correction solution is preferably provided in a further reservoir and / or is provided in the feed solution.

[0070] The disclosure further relates to a method for the propagation or cultivation of biological material, preferably for the production of pharmaceuticals, in particular biopharmaceuticals, comprising the following process steps: providing several cultivation systems, in particular as described above, connecting the several cultivation systems to a pump module via an injection / withdrawal tube, and preferably automating the inoculation of the several cultivation systems.

[0071] Extended use of the described cultivation system can be particularly beneficial for modularization and process intensification. In particular, several of the described cultivation systems can be used, each representing a standardized bioprocess module that allows for flexible adaptation to desired requirements. For example, in addition to parallel cultivations, these systems serve to implement process intensification strategies that include an efficient increase in space-time yields. This is preferably achieved by replacing a conventional cryostock vail with a cryostock syringe, which allows for the direct, and especially sterile, inoculation of a prepared cultivation system via an injection / withdrawal tube (more precisely: an associated valve). This advantageously eliminates industry-standard handling and peripheral requirements.Within the framework of a Controlled and Adjusted Process (CAP) in a cultivation system of the type described, cell revitalization and logical proliferation can be facilitated. Additional cultivation systems can be automatically inoculated via an addition / removal tube (transfer tube) and, if necessary, a pump module. This allows, for example, the processing of various personalized biopharmaceuticals in a single incubator. By connecting further cultivation systems via an automated transfer and replenishing the medium in the initial bioprocess module as needed, continuous bioprocesses and thus process intensification can be achieved. Even in the event of contamination, the loss is minimal compared to conventional bioprocesses.

[0072] The following are some specific, non-exhaustive, embodiments of the invention explained with reference to the accompanying drawings. These show: Fig. 1 a three-dimensional view of a cultivation system, Fig. 2 further three-dimensional views of the cultivation system made of Fig. 1 , Fig. 3 a sectional view of the cultivation system made of Fig. 1 , Fig. 4 another sectional view of the cultivation system made of Fig. 1 , Fig. 5 a three-dimensional view of a cultivation system, Fig. 6 a three-dimensional view of a container attachment, Fig. 7 a three-dimensional view of an electronics module, Fig. 8 three-dimensional view of two dispensing units.

[0073] Referring to Fig. 1 bis 3 A cultivation system 10 comprises a cultivation container 50, in particular made of glass or polymer, and a container attachment 100.

[0074] The container attachment 100 is mounted vertically from above onto the neck 52 (see Fig. 3 ) of the cultivation container 50, wherein the cultivation container 50 has a flat bottom 60 in order to be placed on a base, so that the neck 52 of the container extends vertically upwards.

[0075] The container attachment 100 has an inner side 102 and an outer side 104, wherein the inner side 102 projects into the opening 54 of the cultivation container 50 extending through the neck 52 and is thus facing the interior of the container 50, and the outer side 104 is facing the exterior of the container 50.

[0076] The container attachment 100 has an annular gap 106 into which the neck 52 of the cultivation container 50 projects. This enables a sterile closure of the cultivation container 50. The gap 106 separates the outer rim 105, belonging to the outer surface 104, from the inner projection 103 of the container attachment 100, belonging to the inner surface 102. The base body of the container attachment 100 is monolithic, i.e., formed in one piece, so that in particular the outer rim 105 and the inner projection 103 are formed from a single piece of material.

[0077] In the example shown, the container attachment includes a port 110 for installing a sensor unit 112, which in this case is housed in a modular multi-sensor component 114 that can be installed on the port 110, as well as two ports 120 for installing two dispensing units 122.

[0078] Based on the Fig. 5 bis 8 It can be seen that the cultivation system 10 is preferably modular in design, so that, for example, the sensor unit 112 and / or the dispensing unit 122 can be installed modularly on the container attachment 100. Fig. 6 Figure 1 shows a container attachment 100 designed as a base body 101 and including ports 110 and 120 for installing sensor units and dispensing units. The container attachment shown also has a port 130 for installing an electronics module 132 (see Figure 1). Fig. 7 ), which in turn forms part of port 110 for the installation of a sensor unit 112 or a (multi-)sensor component 114.

[0079] Referring to Fig. 8 The dispensing unit 122 of the container attachment 100 includes a reservoir 140, specifically for feed and pH adjustment reagents. The reservoir 140 comprises a lower reservoir section 142, which has a discharge opening 144, and an upper reservoir section 146, which has a reflow opening 148 for gas replenishment. The upper reservoir section 146 is wider than the lower reservoir section 142 and thus forms a stop 147 for the modular installation of the dispensing unit 122. The upper reservoir section 146 also has a refill opening 149 for refilling the reservoir 140 with liquid.

[0080] How best to use the following: Fig. 4 and Fig. 5 As can be seen, a hose 160 is provided, which leads from the inflow opening 148 to the inside 102 of the container attachment 100 and thus into the interior of the cultivation container 50. This enables a sterile inflow of gas from the inside of the container. Furthermore, this allows for pressure-regulated dosing of the feed or pH setting, as will be explained below.

[0081] A further tube 162 is provided to connect the refill opening 149 to a valve 164 attached to a bracket 163, enabling sterile refilling of the reservoir (Luer-lock valves for sterile reservoir filling). Additionally, a tube 170, designed specifically as an addition / withdrawal tube, is provided, leading from a valve 174 attached externally to a bracket 173 to the inside of the container attachment 100 to allow sterile addition or withdrawal from inside the culture container 50 (Luer-lock valve for sterile inoculation and / or withdrawal).

[0082] In a further development of the invention (not shown), an additional addition and / or removal opening leading to the inside of the container attachment can optionally be provided, in particular with a hose (addition / removal hose), preferably with a valve, which may optionally be attachable to a holder, and particularly preferably in conjunction with a transfer pump, especially to couple a plurality of cultivation systems 10. A transfer pump can be designed as a module comprising several (e.g., four) peristaltic pumps, which may, for example, be arranged on a base body, such that the arrangement fits onto a shaker piston holder corresponding to a cultivation system or a cultivation container.

[0083] The cultivation system 10 according to the invention is also referred to as a CAP system (Controlled and Adjusted Processes). CAP means that, regardless of the shape of the cultivation vessel 50, both the in-situ sensors and the actuators are integrated into the container attachment 100, which is designed as a cap. This creates a closed control loop with which the key process parameters are kept sterile and safely within the optimal range in real time. A multi-sensor component according to German Patent Application 10 2019 117 446.5 is particularly suitable for the in-situ sensors.

[0084] With the CAP cultivation system according to the invention, the requirements for the peripheral equipment are advantageously reduced compared to conventional bioreactors. For example, to avoid the need for a sterile workbench, the valve 174 can be configured with a syringe to form a sterile connector. In this embodiment, a cover film with a tab can be provided under the protective cap of both the valve and the syringe. The connector faces can be disinfected and pressed together. The valve and syringe can, for example, have a male / female configuration, allowing the cover films to be pulled out without contamination by pressing them together over the tabs, thus opening the transfer path. The same principle can be used for the connection via the valves 164 of the reservoirs. In other words, at least one valve 164, 174 can generally be configured as a sterile connector, e.g.It must be sealed with a protective film to ensure a sterile connection. This allows inoculation via a syringe or transfer using sterile fluid / air, even after storage at, for example, -80°C.

[0085] The cultivation vessel 50 itself can be shaped like a classic bioreactor (possibly with flow breakers) with straight sidewalls. Shaker flasks are preferred due to their design and availability. Mass and heat transfer is a key design feature, and this can be influenced particularly by the number of baffles.

[0086] The CAP system can be prepared for cultivation using an autoclave and a sterile workbench. Furthermore, with the present invention, this is advantageously possible even without a sterile workstation.

[0087] When using sterile workbenches and autoclaves, the medium can be placed in a standard shake flask 50 and the CAP base 101 and reservoirs 140 attached. The CAP base 101 is temperature-stable up to 141°C, optionally including the previously mentioned tubing 160, 162, 170, which can be made of PP, for example, reservoirs 140 or dispensing units 122, and sensor components 114 or sensor units 112. The container attachment can thus seal the cultivation vessel, i.e., in particular the shake flask, just as sterile and reliably as a conventional shake flask cap.

[0088] Preferably, two delivery units 122 with differently sized reservoirs 140 are available, which can be used flexibly adapted to the cultivation.

[0089] Temperature-resistant feed and pH correction solutions can also be placed in reservoirs 140 and covered with lids 146D (see Fig. 8 The containers should be sealed. Temperature-resistant media can be placed in the shake flask. Once prepared in this way, autoclaving can then be carried out, during which the reservoir tubing can be clamped with wire clamps, followed by inoculation in a sterile workbench.

[0090] Special Luer-lock ports can be provided to enable sterile operation without a dedicated sterile workstation. These Luer-lock ports include a Luer-lock valve and, optionally, a 160 or 162 mm tube (e.g., made of PP) that can be routed through the 146D lid of the respective reservoir 140. The reservoirs can be filled sterilely with filtered pH correction and feed solution via these ports or valves.

[0091] Another port, through which tubing 170 (e.g., made of PP) is inserted into the shake flask, allows for sterile filling with sterile medium, as well as sterile inoculation. Tubing 170 can also be inserted directly into the medium, e.g., for sampling, repeated-batch, or continuous cultivations, especially in conjunction with another addition / removal tube (not shown) that leads to the inside of the container attachment.

[0092] The invention is particularly suitable for cultivation in an orbital shaking incubator. This allows for the adjustment of mixing, temperature, and atmosphere (e.g., humidity or CO2 concentration).

[0093] Holders 163 and 173 can be designed as clip holders. This allows for easy handling and fixation during cultivation in the shaking incubator, particularly via the flexible PP hose connection.

[0094] Mixing for mass and heat transfer is also possible in combination with a magnetic stirrer and, in particular, a system-compatible stir bar.

[0095] At the in Fig. 5 In the configuration shown, sterility in the cultivation unit is already ensured. Subsequently, an electronic module 132 (see below) can be installed, free from the requirement of a sterile environment. Fig. 7 ) can be attached. A sensor unit 112 can then be installed. This can be connected to the electronic module via a cable and a connector. The module has a connection 180 for power supply and data transmission.

[0096] The connection can be made to the interface of the shaking incubator or a central unit. Such a central unit can integrate the cultivation units, a power supply (mains or battery), and / or a process control system. The process control system can be visualized via a mobile app. It is also possible to integrate the functions of this connection into the electronic module 132; however, this can be omitted to keep the electronic module 132 compact and lightweight, and to avoid the risk of stability loss in the orbital shaker.

[0097] To implement a closed-loop control system that allows key process parameters to be kept sterile and safely within their optimal range in real time, actuators 190 for pressure-regulated dosing can be integrated into the electronic module. In other words, the container attachment 100, the dispensing unit 122, and / or the electronic module 132 can include an actuator 190 to control the flow of gas through a connection, in particular a hose 160, between the reservoir and the inside of the container attachment. Such an actuator 190 is used specifically for pressure-regulated dosing and can include a coil and a return spring to transmit force to the PP hoses.

[0098] The PP tubes 160 lead from the lid 146D of the reservoirs to the shaker flask 50 as described. Atmospheric pressure can prevail in the shaker flask via the industry-standard sterile gap 106 between the shaker flask 50 and the container attachment 100. The outlet 144 of the reservoirs 140 can be significantly tapered.

[0099] When filling the reservoirs 140 via the Luer-Lock valves 164, these are preferably filled first, and the shaking piston 50 is tilted so that displaced air can escape via the reservoir outlet 144.

[0100] To regulate the key parameters, which can be detected in real time in situ via the sensor unit 112, the dosing of the solutions held in the reservoirs 140 can now be controlled by actuating the actuators 190. When an actuator 190 is reset, dosing occurs via the reservoir outlet 144. If the PP tube is pinched shut by the actuators 190, no feed or pH correction solution is dispensed, as a negative pressure develops in the reservoir. An embodiment (not shown) provides for one actuator to control the reservoir outlet 144. This may require a smaller reservoir volume due to space constraints.

Claims

1. A container attachment (100) for being placing on a cultivation container(50), in particular a hand-held one, preferably on a shake flask, the cultivation container (50) having a neck (52) and an opening (54) extending through said neck, wherein the container attachment (100) comprises: an inner side (102) facing the interior of the cultivation container when the container attachment is placed on the cultivation container, and an outer side (104) facing the exterior of the cultivation container when the container attachment is placed on the cultivation container; at least one sensor unit (112), said sensor unit being arranged on the inner side (102) of the container attachment, at least a section thereof, to allow for a parameter measurement in the interior of the cultivation container (50); characterized by at least one dispensing unit (122), said dispensing unit being arranged on the inner side (102) of the container attachment, at least a section thereof, to enable liquid to be dispensed into the interior of the cultivation container (50); and wherein the dispensing unit (122) comprises a reservoir (140) for holding liquid, said reservoir (140) comprising a lower reservoir section (142) having an outlet opening (144) for dispensing liquid and an upper reservoir section (146) having an inflow opening (148) for inflow of gas; wherein the upper reservoir section (146) is arranged on the outer side (104) of the container attachment (100) and has a larger width so as to define a stop (147) when the dispensing unit (122) and / or the reservoir (140) is installed on a port of the container attachment.

2. The container attachment (100) according to the preceding claim, wherein the container attachment has an annular, in particular cylindrical slot (106) for receiving a neck (52) of the cultivation container; and / or wherein the container attachment has an outer collar (105) radially surrounding the neck (52) of the cultivation container (50) when the container attachment is placed on the cultivation container (50); and / or wherein the container attachment has an inner projection (103) protruding into the neck (52) of the cultivation container when the container attachment is placed on the cultivation container (50); and wherein the outer collar (105) and the inner projection (103) are preferably formed monolithically.

3. The container attachment (100) according to any one of the preceding claims, wherein the container attachment can be placed onto the cultivation container (50) vertically from above and is preferably designed so as to be held on the cultivation container (50) by gravity.

4. The container attachment (100) according to any one of the preceding claims, wherein the container attachment (100) has an abutment surface (108) that is in particular located at the upper end of the slot (106), which engages on the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50), and wherein the container attachment (100) has a height of less than 10 centimeters measured from said abutment surface, preferably less than 5 centimeters, more preferably less than 4 centimeters, yet more preferably less than 3 centimeters, even more preferably less than 2 centimeters, most preferably less than 14 centimeters.

5. The container attachment (100) according to any one of the preceding claims, wherein the container attachment and / or the dispensing unit (122) comprise a connection between the inflow opening (148) of the upper reservoir section and the inner side of the container attachment so as to allow gas to flow in from the interior of the cultivation container when the container attachment is placed on the cultivation container.

6. The container attachment (100) according to any one of the preceding claims, wherein the reservoir (140) has a refill opening (149) for refilling liquid; and wherein the container attachment and / or the dispensing unit (122) preferably comprise a connection between the refill opening (149) of the reservoir and a valve (164) that is arranged or arrangeable on the outer side of the container attachment, to enable the reservoir to be refilled with liquid; and wherein the container attachment and / or the dispensing unit (122) preferably comprise a bracket (163) for releasably fixing the valve (164).

7. The container attachment (100) according to any one of the preceding claims, wherein the container attachment comprises a connection between the inner side (102) of the container attachment and a valve (174) that is arranged or arrangeable on the outer side of the container attachment, to provide for feed into or extraction from the interior of the cultivation container when the container attachment is placed on the cultivation container; and wherein the container attachment preferably comprises a bracket (173) for releasably fixing the valve (174).

8. The container attachment (100) according to any one of the preceding claims, wherein the at least one sensor unit (112) is in the form of a biosensor unit for analyte-specific parameter measurement; and / or wherein the at least one or a further sensor unit (112) of the container attachment, which in particular is not a biosensor unit, is in the form of a luminophoric unit for luminescence-based parameter measurement; and / or wherein the at least one or a further sensor unit (112) of the container attachment is in the form of an alternating field unit for dielectric-based parameter measurement; and / or wherein the at least one or a further sensor unit (112) of the container attachment is in the form of a transistor unit for field effect-based parameter measurement; and / or wherein the at least one sensor unit (112) is arranged or arrangeable on a front housing section of a sensor component (114) such that the at least one sensor unit is arrangeable on the inner side of the container attachment by installing the sensor component on a port (110) of the container attachment.

9. The container attachment (100) according to any one of the preceding claims, wherein the container attachment comprises at least one sensor unit and at least two dispensing units.

10. A cultivation system (10), comprising: a container attachment (100) according to any one of claims 1 to 9; and a cultivation container (50), in particular a hand-held one, preferably in the form of a shake flask, for holding a culture medium, the cultivation container having a neck (52) and an opening (54) extending through said neck; wherein the container attachment (100) can be placed on the neck (52) of the cultivation container (50) so as to close the opening (54) of the cultivation container, in particular in a sterile manner, the container attachment (100) having an inner side (102) and an outer side (104), with the inner side (102) facing the interior of the cultivation container and the outer side (104) facing the exterior of the cultivation container when the container attachment (100) is placed on the neck (52) of the cultivation container.

11. The cultivation system (10) according to claim 10, wherein the cultivation container (50) has a flat base (60) for being placed on a supporting surface; and / or wherein the neck (52) of the cultivation container (50) extends vertically upwards, in particular when the cultivation container is placed on the supporting surface; and / or wherein the container attachment (100) can be placed onto the neck (52) of the cultivation container (50) vertically from above and is preferably designed so as to be held on the neck (52) of the cultivation container (50) by gravity.

12. The cultivation system (10) according to any one of claims 10 or 11, wherein the cultivation container (50) has a volume of less than 2801 milliliters, preferably less than 1801 milliliters, more preferably less than 501 milliliters, even more preferably less than 251 milliliters, most preferably of less than 126 milliliters; and / or wherein the cultivation container (50) has a diameter of less than 22 centimeters or less than 21 centimeters, preferably less than 14 centimeters or less than 12 centimeters, most preferably less than 9 centimeters or less than 8 centimeters; and / or wherein the cultivation container (50) has a height of less than 31 centimeters, preferably less than 23 centimeters, more preferably less than 19 centimeters, even more preferably less than 17 centimeters, most preferably less than 14 centimeters.

13. A method for propagation or cultivation of biological material, preferably for producing pharmaceuticals, in particular biopharmaceuticals, comprising: providing a cultivation system (10) according to any one of claims 10 to 12; providing a feed solution, in particular containing the substrates required for product formation, e.g. saccharides, wherein said feed solution is provided in the reservoir of the dispensing unit (122); providing a pH correction solution, especially for raising the pH value, e.g. containing NaOH, wherein said pH correction solution is preferably provided in a further reservoir and / or is provided included in said feed solution; and wherein the sensor unit (112) is used to capture, in real time, a parameter for process control, and the dispensing unit (122) is used to accomplish an active correction, e.g. pH correction, or in feed rate.