Cultivation system and container attachment for a cultivation container

Abstract

Container attachment (100) for mounting on a cultivation container (50), preferably on a shake flask, wherein the cultivation container (50) has a neck (52) and an opening (54) extending through the neck, wherein the container attachment (100) comprises: an inner side (102) which faces the interior of the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50) and an outer side (104) which faces the exterior of the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50), characterized in that the container attachment (100) comprising at least one sensor unit (112), wherein the sensor unit (112) is arranged at least partially on the inside (102) of the container attachment (100) to enable parameter measurement inside the cultivation container (50), and at least one dispensing unit (122) with a reservoir (140) for liquid storage, wherein the dispensing unit (122) is arranged at least partially on the inside (102) of the container attachment (100) to enable liquid dispensing into the interior of the cultivation container (50).

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. In particular, improvements in product yield and thus profit increases are being driven for the production of biopharmaceuticals. 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] 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.

[0011] The invention discloses a cultivation system comprising a cultivation container, in particular a handheld container, for receiving a culture medium and a container attachment which can be placed on the cultivation container.

[0012] 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.

[0013] The container attachment is placed on the neck of the culture container to close the opening, ensuring a sterile seal. When the container attachment is placed on the neck of the culture container, one inner side of the attachment faces the interior of the culture container, i.e., the sterile area, and one outer side of the attachment faces the exterior of the culture container, i.e., the non-sterile area.

[0014] The container attachment is characterized by the fact that it includes at least one sensor unit or a port for installing a sensor unit and / or a dispensing unit or a port for installing a dispensing unit.

[0015] The sensor unit, if included, is located, or can be located, 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.

[0016] 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. In this regard, particular reference is made to German patent application 10 2019 117 446.5, which is hereby incorporated by reference.

[0017] The dispensing unit, if included, is also located, or can be 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.

[0018] 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.

[0019] 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.

[0020] 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 by utilizing sterile connectors, as will be explained in more detail below. These aspects increase cost-efficiency and enable both 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.

[0021] 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.

[0022] The cultivation container has a neck, which is preferably ring-shaped, particularly cylindrical. Accordingly, the container attachment can have a ring-shaped, particularly cylindrical, slot to accommodate the neck of the cultivation container.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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 preferably comprises a reservoir for liquid storage, wherein the reservoir has a lower reservoir section with an outlet opening for liquid dispensing and preferably an upper reservoir section with a reflow opening for gas reflow.

[0031] The lower reservoir section is preferably arranged or can be arranged 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 preferably arranged or can be arranged on the outside of the container attachment and is particularly preferably wider to form a stop when the dispensing unit and / or the reservoir is installed on a port of the container attachment.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Preferably, the container attachment comprises a plurality of sensor / dispensing units or corresponding ports, in particular at least one sensor unit or corresponding port and at least one dispensing unit or corresponding port, particularly preferably selectively at least one sensor unit or corresponding port and at least two dispensing units or corresponding ports.

[0041] 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.

[0042] 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.

[0043] Furthermore, the container attachment includes at least one sensor unit or a port for installing a sensor unit and / or at least one dispensing unit or a port for installing a dispensing unit. A sensor unit is located, or can be located, at least partially on the inside of the container attachment to enable parameter measurement inside the cultivation container. A dispensing unit is also located, or can be located, at least partially on the inside of the container attachment to enable liquid dispensing into the interior of the cultivation container.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The at least one dispensing unit of the container attachment can comprise a reservoir for liquid storage, wherein the reservoir has a lower reservoir section with a discharge opening for liquid dispensing and preferably an upper reservoir section with a reflow opening for gas reflow.

[0049] The lower reservoir section is preferably arranged, or can be arranged, 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 preferably arranged, or can be arranged, on the outside of the container attachment and is particularly preferably wider to form a stop when the dispensing unit and / or the reservoir is installed on a port of the container attachment.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] In a preferred embodiment, the container attachment has a plurality of sensor / dispensing units or corresponding ports, in particular at least one sensor unit or corresponding port and at least one dispensing unit or corresponding port, more preferably at least one sensor unit or corresponding port and at least two dispensing units or corresponding ports.

[0055] The invention 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, wherein the reservoir has a lower reservoir section with an outlet opening for liquid dispensing and preferably an upper reservoir section with a reflow opening for gas reflow.

[0056] The reservoir may have a refill opening for refilling with liquid, and may also have a connection between the refill opening of the reservoir and a valve to allow, in particular sterile, refilling of liquid into the reservoir.

[0057] 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 pH-lowering components of the medium, such as organic acids, can be produced through cell metabolism, a pH-raising correction solution, e.g., containing NaOH, can preferably be provided. Thus, a pH correction solution can be provided in the reservoir. In particular, a pH correction solution in the reservoir can form a buffer system with the medium. For example, cell metabolism produces CO2 and thus H2CO3, and the H2CO3 correction solution creates a buffer system with a regulated buffer capacity and a stable pH.

[0058] 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.

[0059] The invention further relates to a method for propagating or cultivating 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.

[0060] The invention further relates to a method for propagating or cultivating 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.

[0061] 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.

[0062] 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 from Fig. 1, Fig. 3 a cross-sectional view of the cultivation system Fig. 1, Fig. 4 another cross-sectional view of the cultivation system 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 electronic module, Fig. 8 three-dimensional two delivery units.

[0063] Referring to Fig. 1 to 3 a cultivation system 10 comprises a cultivation container 50, in particular made of glass or polymer, and a container attachment 100.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Based on the Fig. From figures 5 to 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. Figure 6 shows, for example, a container attachment 100 designed as a base body 101 and including ports 110, 120 for installing sensor units and dispensing units. The container attachment shown also has a port 130 for installing an electronic module 132 (see Figure 6). Fig. 7), which in turn forms part of port 110 for the installation of a sensor unit 112 or a (multi-)sensor component 114.

[0069] Referring to Fig. The dispensing unit 122 of the container attachment 100 comprises a reservoir 140, specifically for feed and pH adjustment reagents. The reservoir 140 includes 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 reflow. 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.

[0070] How best to use the following: Fig. 4 and Fig. As can be seen in Figure 5, 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 interior of the container. Furthermore, this allows for pressure-regulated dosing of the feed or pH setting, as will be explained below.

[0071] 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).

[0072] 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.

[0073] 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.

[0074] 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 ensuring an open transfer path. The same principle can be applied to 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Preferably, two delivery units 122 with differently sized reservoirs 140 are available, which can be used flexibly adapted to the cultivation.

[0079] Temperature-resistant feed and pH correction solutions can also be placed in reservoirs 140 and covered with lids 146D (see Fig. 8) be sealed. Temperature-resistant media can be placed in the shake flask. Once prepared in this way, autoclaving can then be carried out, whereby the tubing of the reservoirs can be clamped with wire clamps, and subsequently inoculation can take place in a sterile workbench.

[0080] Special Luer-lock ports can be provided to enable sterile operation without a dedicated sterile workstation. The Luer-lock ports include a Luer-lock valve and, if necessary, 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.

[0081] 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.

[0082] 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).

[0083] 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.

[0084] Thorough mixing for mass and heat transfer is also possible using a combination of a magnetic stirrer and, in particular, a system-compatible stir bar. In the case of the Fig. In the configuration shown in section 5, sterility in the cultivation unit is already ensured. Subsequently, an electronic module 132 can be installed, free from the requirement of a sterile environment (see...). 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] DE 10 2019 117 446.5 [0016, 0039, 0053, 0073]

Claims

[1] Container attachment (100) for mounting on a cultivation container (50), preferably on a shake flask, wherein the cultivation container (50) has a neck (52) and an opening (54) extending through the neck, wherein the container attachment (100) comprises: an inner side (102) which faces the interior of the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50) and an outer side (104) which faces the exterior of the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50), characterized by , that the container attachment (100) comprising at least one sensor unit (112), wherein the sensor unit (112) is arranged at least partially on the inside (102) of the container attachment (100) to enable parameter measurement inside the cultivation container (50), and at least one dispensing unit (122) with a reservoir (140) for liquid storage, wherein the dispensing unit (122) is arranged at least partially on the inside (102) of the container attachment (100) to enable liquid dispensing into the interior of the cultivation container (50). [2] Container attachment (100) according to the preceding claim, wherein the container attachment (100) has an annular, in particular cylindrical, gap (106) for receiving a neck (52) of the cultivation container (50) and / or wherein the container attachment has an outer ring (105) which radially surrounds the neck (52) of the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50) and / or wherein the container attachment (100) has an internal projection (103) which extends into the neck (52) of the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50) and wherein the outer ring (105) and the inner projection (103) are preferably monolithic. [3] Container attachment (100) according to one of the preceding claims, wherein the container attachment (100) can be placed vertically from above onto the cultivation container (50) and is preferably designed to be held on the cultivation container (50) by gravity. [4] Container attachment (100) according to one of the preceding claims, wherein the container attachment (100) has a stop surface (108) located, in particular at the upper end of the gap (106), which comes into contact with 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 measured from the stop surface of less than 10 centimeters, preferably less than 5 centimeters, more preferably less than 4 centimeters, more preferably less than 3 centimeters, more preferably less than 2 centimeters, and most preferably less than 14 centimeters. [5] Container attachment (100) according to one of the preceding claims, wherein the reservoir (140) has an upper reservoir section (146) and a lower reservoir section (142), wherein the upper reservoir section (146) has a reflow opening (148) for the reflow of gas and wherein the lower reservoir section (142) is arranged on the inside (102) of the container attachment and has a discharge opening (144) to allow liquid to be discharged through the discharge opening (144) into the interior of the cultivation container (50). [6] Container attachment (100) according to one of the preceding claims, wherein the reservoir (140) has an upper reservoir section (146) and a lower reservoir section (142), wherein the upper reservoir section (146) is wider than the lower reservoir section (142) and wherein the upper reservoir section (146) is preferably arranged on the outside (104) of the container attachment (100) and is particularly preferably wider to form a stop (147) when the dispensing unit (122) and / or the reservoir (140) is installed at a port of the container attachment (100). [7] Container attachment (100) according to the preceding claim, wherein the container attachment (100) and / or the dispensing unit (122) has a connection between the inflow opening (148) of the upper reservoir section (146) and the inside of the container attachment (100) to allow gas to flow in from the interior of the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50). [8] Container attachment (100) according to one of the preceding claims, wherein the reservoir (140) has a refill opening (149) for refilling with liquid and wherein the container attachment (100) and / or the dispensing unit (122) preferably has a connection between the refill opening (149) of the reservoir (140) and a valve (164) arranged or arrangable on the outside of the container attachment (100) to allow refilling of liquid into the reservoir (140) and wherein the container attachment (100) and / or the dispensing unit (122) preferably has a holder (163) for the releasable fixing of the valve (164). [9] Container attachment (100) according to one of the preceding claims, wherein the container attachment (100) has a connection between the inside (102) of the container attachment (100) and a valve (174) arranged or arrangable on the outside (104) of the container attachment (100) to allow addition or removal from the interior of the cultivation container (50) when the container attachment (100) is placed on the cultivation container (50) and wherein the container attachment (100) preferably has a holder (173) for the releasable fixing of the valve (174). [10] Container attachment (100) according to one of the preceding claims, wherein at least one sensor unit (112) is designed as a biosensor unit for analyte-specific parameter measurement and / or wherein at least one or one further sensor unit (112) of the container attachment (100), which in particular is not designed as a biosensor unit, is designed as a luminophore unit for luminescence-based parameter measurement and / or wherein at least one or another sensor unit (112) of the container attachment (100) is designed as an alternating field unit for dielectric-based parameter measurement and / or wherein at least one or another sensor unit (112) of the container attachment (100) is designed as a transistor unit for field-effect-based parameter measurement and / or wherein the at least one sensor unit (112) is arranged or can be arranged on a front housing section of a sensor component (114) such that the at least one sensor unit (112) can be arranged on the inside of the container attachment (100) by installing the sensor component on a port (110) of the container attachment (100). [11] Container attachment (100) according to one of the preceding claims, wherein the container attachment (100) comprises a plurality of sensor / dispensing units (112, 122), preferably at least one sensor unit (112) and at least two dispensing units (122). [12] Cultivation system (10) comprising: a container attachment (100) according to one of claims 1 to 11, as well as a cultivation container (50), in particular a handheld container, preferably designed as a shaker flask, for receiving a culture medium, wherein the cultivation container has a neck (52) and an opening (54) extending through the neck, wherein the container attachment (100) can be placed on the neck (52) of the cultivation container (50) in order to close the opening (54) of the cultivation container (50), in particular to close it sterilely, wherein the container attachment (100) has an inner side (102) and an outer side (104), wherein the inner side (102) faces the interior of the cultivation container (50) and the outer side (104) faces the exterior of the cultivation container (50) when the container attachment (100) is placed on the neck (52) of the cultivation container (50). [13] Cultivation system (10) according to claim 12, wherein the cultivation container (50) has a flat bottom (60) for placement on a surface and / or wherein the neck (52) of the cultivation container (50) extends vertically upwards, particularly when the cultivation container (50) is placed on the ground, and / or wherein the container attachment (100) can be placed vertically from above onto the neck (52) of the cultivation container (50) and is preferably designed to be held on the neck (52) of the cultivation container (50) by gravity. [14] Cultivation system (10) according to one of claims 12 or 13, wherein the cultivation vessel (50) has a volume of less than 2801 milliliters, preferably less than 1801 milliliters, more preferably less than 501 milliliters, again more preferably less than 251 milliliters, and particularly preferably 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, particularly 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, particularly preferably less than 17 centimeters, and most preferably less than 14 centimeters.

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