Method and device for automating the foam control in a bioreactor

Abstract

The invention relates to a method for automating the foam regulation during a bioprocess for cultivating cells in a bioreactor, said method comprising the following steps: determining values of one or more process variables of the bioprocess which is / are relevant for the formation of foam in the bioreactor; transferring the determined values as input signals to a controller; generating, by the controller, output signals for at least one foam regulation device depending on the input signals; and controlling the at least one foam regulation device in a requirements-oriented manner by means of the output signals output by the controller.

Description

[0001] Method and device for automating foam control in a bioreactor

[0002] The invention relates to a method for automating foam control in a bioreactor during a bioprocess for cultivating cells in a bioreactor. The invention further relates to a device for carrying out this method.

[0003] A well-known problem in bioprocesses is disruptive foam formation, which can have various causes. Foam is undesirable because, among other things, it can interfere with the function of sensors and lead to sticking and fouling of the reactor walls, requiring increased cleaning effort. Furthermore, foam can penetrate fluid lines leading into the headspace of the bioreactor. The use of larger containers only partially eliminates the problem and is also uneconomical. Apart from that, foam is disruptive in downstream processes.

[0004] Various techniques are known for detecting foam in a bioreactor, such as capacitive measurements in a disposable bioreactor using a patch sensor unit (see, e.g., EP 3 872 162 A1) or conductivity measurements in a stainless steel bioreactor using a ceramic foam probe. Camera or image-based systems are also used for foam detection (see, e.g., WO 2020 / 198518 A1, US 11 327 064 B2, EP 3 957 712 A1), as are optical systems that measure light scattering or transmission.

[0005] In principle, foam can be controlled chemically, mechanically, and thermally. Chemical antifoam agents, such as silicone oils, can be introduced in various concentrations. They are typically added in the headspace of the bioreactor, from where the antifoam drops onto the surface of the medium in the bioreactor. However, submerged addition is also possible. A device with an antifoam reservoir in the headspace of the bioreactor, which releases antifoam upon contact with foam, is known from WO 2017 / 029259 A1. Devices for mechanical foam destruction are primarily used in glass or stainless steel bioreactors. However, there are also solutions for disposable bioreactors, e.g., using a spray nozzle or ultrasound (see DE 202020 003 748 U1).

[0006] The use of foam-destroying or foam-inhibiting measures can be carried out according to a predetermined time-based profile that has been created using empirical values ​​for the respective process (subjective optical control and previous experience regarding the required amount of antifoam agent). For example, 1 mL of antifoam agent of a certain concentration can be added every 60 seconds. Antifoam measures can also be sensor-based. If, for example, an optical system detects foam, antifoam agent is pumped into the bioreactor at a predetermined fixed pumping rate until the foam level falls below a critical value, which can be determined, for example, using a foam probe. Alternatively, antifoam agent can be pumped into the bioreactor over a predetermined time interval, and then a control loop is used to check whether the addition was sufficient. If the sensor signal is still present oris still too high, another pumping interval is started.

[0007] WO 2021 / 011484 A1 discloses a fermenter control system comprising a gas volume fraction (GVF) measuring device, a controller, and one or more venting mechanisms. The controller is configured to determine an appropriate amount of antifoam based on the inputs received from the GVF measuring device. The fermenter control system may further comprise one or more auxiliary measuring devices, with the controller then generating a control signal for the venting mechanism based on the inputs from the GVF measuring device and the further auxiliary measuring devices. The auxiliary measuring devices may comprise a temperature sensor, a pH sensor, a mixing rate sensor, or a flow sensor for the process, inlet, or recirculation lines of the fermenter.This technology, which originates from the brewing industry, is hardly suitable for cell cultivation given the specific requirements of pharmaceutical biotechnology. Regardless, the gas volume fraction in a culture medium in a fermentation vessel is not important for foam control during cell cultivation. US 2022 / 290090 A1 discloses an automated system designed to detect and predict the presence of foam and / or a foam formation level using a machine learning-based detection mechanism. Foam formation and / or other parameters of a fermentation process are designed to be automatically controllable based on an estimated process state. A trained model can be used to predict foam formation. In any case, however, image data from an image capture device is required.In addition, real-time parameters determined by sensors can also be used, such as pH value, dissolved oxygen concentration, optical density, or temperature.

[0008] The object of the invention is to enable more effective foam control in a bioprocess for cultivating cells in a bioreactor.

[0009] This object is achieved by a method having the features of claim 1 and by a device having the features of claim 13. Advantageous and expedient embodiments of the method according to the invention and the device according to the invention are specified in the associated subclaims.

[0010] The method according to the invention serves to automate foam control during a bioprocess for cultivating cells in a bioreactor. The term "cells" here includes, in particular, mammalian cells, fungal cells, yeast cells, and bacteria. The method according to the invention comprises the following steps: repeatedly determining values ​​of one or more process variables of the bioprocess that are relevant to foam formation in the bioreactor; transmitting the determined values ​​as input signals to a controller; generating output signals by the controller for at least one foam control device depending on the input signals; and controlling the at least one foam control device as needed using the output signals output by the controller.

[0011] The step "determining values ​​of one or more process variables of the bioprocess that are relevant for foam formation in the bioreactor" is performed repeatedly, with the time intervals being specified by a process control system. A process variable relevant for foam formation in the bioreactor is understood here to be a process parameter related to the specific bioprocess that has a significant influence on foam formation and is measured, calculated, or determined in some other way. Such a process variable must be distinguished from a signal from a dedicated foam detection device. In particular, the method according to the invention, contrary to the technology described in US 2022 / 290090 A1, does not require the acquisition and evaluation of images for passive detection of foam.

[0012] "Demand-based" control of at least one foam control device using the output signals issued by the controller means, for example, that as little antifoam as possible is used during chemical foam control, or that, during mechanical foam destruction, such as by stirring in the headspace, the corresponding measure is not carried out with a higher intensity than necessary for the planned continuation of the bioprocess. In this respect, demand-based control differs from simply activating a measure.

[0013] The invention is based on the realization that the intensity of the currently customary measures for combating foam in bioprocesses cannot be controlled automatically, but rather - if at all - only manually, which can be time-consuming and inaccurate, and furthermore there is no provision for coordination with the respective process or process event. The method according to the invention allows for finer, more precise and thus better control. This ultimately leads to significant advantages. For example, the consumption of antifoam agents can be reduced, which is important because antifoam agents have a negative impact on downstream processing. Furthermore, thanks to the invention, antifoam measures are used in a targeted manner and at a suitable time, which increases effectiveness, because if intervention is made too early, the effect would be reduced or even diminished. All of this is also advantageous for the performance and / orLongevity of the entire exhaust air system and improved and more reproducible dissolved oxygen flow / control. Another significant advantage is that the method according to the invention enables foam control without a dedicated foam sensor.

[0014] According to a further development of the invention, the values ​​of several different process variables are transmitted to the controller as several input signals, with the controller then generating and outputting the output signals depending on the separate input signals for controlling the at least one foam control device. This further development is useful when several process variables relevant to foam formation in the bioreactor can be determined during the bioprocess. Appropriate processing (evaluation algorithm) can generally provide more precise conclusions about foam formation from the plurality of input signals, and these findings can be incorporated into targeted foam control measures.

[0015] The following biological process variables have proven to be particularly suitable for foam formation in the bioreactor: cell density; viable cell volume; viable cell density; cell growth rate; protein concentration; protein quantity; oxygen consumption; volume-related mass transfer coefficient (kLa value); carbon dioxide concentration; glucose consumption; nutrient solution consumption; turbidity. Accordingly, the value of one or more of these process variables and / or their temporal change is preferably transmitted as input to the controller.

[0016] In addition to the one or more biological process variables, values ​​of one or more of the following procedural process variables can also be transmitted to the controller as input signals: perfusion rate, bleed rate (cell density correction rate), aeration rate.

[0017] The time from the start of the process (batch time) or the time from the start of a specific process step or the time from a specific process event can also be used as controller input.

[0018] The use of a sparger can also have a significant impact on foam formation. Depending on the sparger type (e.g., microsparger, macrosparger), the number and size of the holes, and / or the gassing rate through the sparger, more or less foam is produced. The type of foam produced by the respective sparger type—fine-pored or coarse-pored, moist or dry—also plays a key role in tailored control. Accordingly, a preferred embodiment of the invention also considers these aspects as process variables in foam regulation. This means that the sparger type(s) currently operating in the process and / or their gassing rate (each) is a process variable in the controller, just like cell density, for example. For example, the use of a microsparger generally results in more foam than a macrosparger, requiring a higher foam correction intensity.With regard to one or more spargers currently in operation, it should be noted that a sparger can be replaced during a running process, especially in typical processes that use a combination sparger. For example, you can choose between a macro sparger (large air bubbles) and a micro sparger (small air bubbles) or a similar device – or a combination of both. Since the sparger in operation has a direct influence on the foam behavior in the bioreactor, it is advisable to include it in the control system. Furthermore, the currently operated sparger can be traced back to certain bioprocess parameters (oxygen consumption, cell count, cultivation time, etc.).

[0019] The values ​​of the process variable(s) whose value(s) are transmitted to the controller as input are, where possible, measured directly by existing sensors, or they are derived from the process control protocol (e.g., sparger type used, gassing rate). However, the value(s) used to control the foam control device(s) can also be determined indirectly, in particular by further processing measured values ​​from one or more sensors, such as an exhaust gas sensor. It should be noted here that these sensors are not image-capturing sensors or sensors for the direct detection of foam.

[0020] According to a particularly advantageous embodiment of the invention, the controller comprises a master controller, to which the values ​​of the one or more process variables are transmitted, and at least one slave controller that controls the at least one foam control device. The at least one foam control device is assigned a control profile that depends on the output signal of the master controller. Such operation can be referred to as "polygon control." The control profile can be fixed or preset by the user.

[0021] In this way, several foam control devices can be controlled by the controller's output signals, either simultaneously or in cascade.

[0022] According to a further development of the invention, the values ​​of several different process variables are each transmitted as separate input signals to several controllers. Each controller generates output signals depending on its input signals for controlling at least one foam control device. Depending on the selected strategy, one or more output signals are selected to control one or more foam control devices.

[0023] The one or more foam control devices preferably carry out at least one of the following measures: addition of antifoam, preferably in an adjustable concentration, by means of a pump controlled by the output signals of the controller; mechanical foam destruction; alteration of a gas supply by one or more spargers. It should be noted that the respective measure - as previously explained - is carried out as needed, i.e. is not simply switched on and off again according to a fixed specification. If antifoam is added, this can be added depending on cell density, process time, etc. in such a way that the antifoam is applied to the surface of the medium in the bioreactor as a type of protective layer. If it is to be expected that more foam will form with increasing cell density, this will be responded to as needed.

[0024] Additionally, the controller's input signals can be based on signals from sensors that directly detect or monitor foam, its growth, and / or the fill level in the bioreactor. This additional information can potentially lead to even better foam control. Likewise, a previously created model can be incorporated into the demand-based control of at least one foam control device. The model can be used as the basis for further controller input or to monitor or adjust the other input variable(s).

[0025] As already indicated, it is particularly advantageous that the foam regulation can be carried out by the demand-based control of at least one foam regulation device without using a dedicated foam detection device.

[0026] The invention also provides a device for automating foam control during a bioprocess for cultivating cells in a bioreactor, comprising a device for determining values ​​of one or more process variables of the bioprocess that are relevant to foam formation in the bioreactor, a controller to which the determined values ​​are transmitted as input signals, and a foam control device controlled by output signals generated by the controller as a function of the input signals. The device is configured to carry out the method according to the invention.

[0027] Further features and advantages of the invention will become apparent from the following description and the accompanying drawings, to which reference is made. In the drawings:

[0028] - Figure 1 is a schematic sketch of a bioreactor plant with sensors and foam control devices;

[0029] - Figure 2 shows a basic control loop of an automated foam control system;

[0030] - Figure 3 shows a first example of a control profile combination;

[0031] - Figure 4 shows a second example of a control profile combination;

[0032] - Figure 5 shows a third example of a control profile combination; and

[0033] - Figure 6 shows a fourth example of a control profile combination. Figure 1 shows, by way of example, a bioreactor system 10 with a container 12 containing a medium. The container contains, among other things, several sensors 14, 16 and foam control devices 18, 20, 22.

[0034] The sensors 14, 16 can each be used to measure or indirectly determine one of the following process variables: cell density; viable cell volume; viable cell density; cell growth rate; protein concentration; protein quantity; oxygen consumption; value for volume-related mass transfer coefficient (kLa value); carbon dioxide concentration; glucose consumption; nutrient solution consumption; perfusion rate, bleed rate, gassing rate; turbidity.

[0035] The foam control devices 18, 20, 22 provided here are a device for adding antifoam 18, in particular by means of a pump, and a device for mechanical foam destruction 20. In addition, the spargers, here a combination sparger 22 with two differently designed hole configurations (microsparger and macrosparger), are also used as foam control devices.

[0036] The sensors 14, 16 and the foam control devices 18, 20 are connected to a controller 24 (measurement and control system) located outside the container 12, which also has a timer for timing. The controller 24 comprises one or more master controllers, to which the sensors 14, 16 are assigned, as well as one or more slave controllers that control the foam control devices 18, 20, 22. Control profiles are stored in the controller 24, which will be discussed in more detail later. The control profiles can be predefined or user-selectable or individually configured.

[0037] During an ongoing bioprocess for cell cultivation, foam forms, which is symbolically represented here and designated by the reference numeral 26. The foam 26 can take on various shapes and structures. For example, the foam 26 can be coarse- or fine-pored, have a different moisture or moisture gradient, be distributed locally or extensively over the medium (distributed centrally by an impeller), etc. These parameters depend on the process variables mentioned above. Below, some basic relationships between some process variables and their influence on foam formation are briefly explained, without claiming to be exhaustive:

[0038] A lot of foam 26 can generally be expected if

[0039] - the process has been going on for a long time;

[0040] - the cell density is high (determinable with a cell density sensor or live cell volume sensor);

[0041] - there is a lot of protein in the medium (determinable with a cell density sensor or live cell volume sensor), for example due to certain media components, product formation, "contamination" by dead cells);

[0042] - the cell growth rate is high;

[0043] - oxygen consumption or carbon dioxide production is high (indicates a high growth rate or high cell density);

[0044] - the pH change is strong (indicates strong growth or change in metabolic state);

[0045] - glucose consumption or medium consumption is high (indicates strong growth);

[0046] - an induction or a temperature shift is present (indicates a change in the metabolic state of the cells, e.g. product formation is molecularly activated; depending on the type of process and other circumstances, foam formation could also be reduced because cell growth is reduced);

[0047] - the perfusion rate or bleed rate is high (indicates a high growth rate and high medium exchange; the effect can go in both directions, ie foam formation can increase or decrease depending on the influence of the fresh medium, although the effects can also balance each other out);

[0048] - a sparger with small holes is in operation or the aeration rate is high. Figure 2 shows a basic control loop for foam control in the bioreactor system 10 shown as an example in Figure 1. It should be noted that all controller input signals (if several are available) and all controller output signals controlling the foam control devices 18, 20, 22 (if several are available) are summarized.

[0049] As already indicated, the foam control devices 18, 20, 22 can take one or more of the following measures to combat foam:

[0050] Antifoam is added, preferably in an adjustable concentration, by means of a pump controlled by the output signals of the controller 24, wherein the pump rate (pump speed) can be increased or decreased and / or the time interval for the antifoam addition can be extended or shortened and / or the concentration of the antifoam can be increased or decreased.

[0051] The existing foam 26 is destroyed mechanically, whereby the intensity (speed or rate) of the measure can be increased or decreased.

[0052] The gas supply through the sparger(s) 22 is changed (selection of the microsparger or the macrosparger, adjustment of the gassing rate).

[0053] In principle, it is also possible to use several measures to combat foam in a cascaded manner (one after the other).

[0054] Figures 3 to 6 show examples of various polygon combinations for foam control. Figure 3 combines cell density and batch time, Figure 4 combines cell density and reactor volume, Figure 5 combines glucose consumption / concentration and dissolved gas concentration (e.g., oxygen), and Figure 6 combines turbidity and reactor volume. The diagrams each indicate which output signal, given as a percentage value relative to the control range of the master controller (the range of interest in the examples shown is between 0% and 50%, but can also be 0% to 100% or another interval), is output to control one or more foam control devices 18, 20, 22. The foam control can provide for one or more output signals to be used to control one or more foam control devices 18, 20, 22.From an application perspective, foam control based on a single process variable is generally preferable to a combination of process variables, although the use of a combination depends on the chosen strategy. Foam control can be supplemented by real-time spectroscopy measurements and their evaluation using stored models and / or additional level measurements (e.g., using an existing camera). The higher the level, the more intensive the foam control should be. However, at very low levels, for example, immediate addition of antifoam agent can be omitted (for the time being).

[0055] Likewise, the properties of the foam 26, in particular porosity (coarse-pored or fine-pored), strength, moisture, moisture gradient, expansion above the medium, can be determined using suitable devices and incorporated into the foam regulation.

[0056] List of reference symbols

[0057] 10 Bioreactor plant

[0058] 12 containers

[0059] 14 first sensor

[0060] 16 second sensor

[0061] 18 first foam regulation device

[0062] 20 second foam regulation device

[0063] 22 third foam regulation device / combi-sparger

[0064] 24 controllers

[0065] 26 foam

Claims

Patent claims 1. A method for automating foam control during a bioprocess for cultivating cells in a bioreactor, the method comprising the following steps: - repeated determination of values ​​of one or more process variables of the bioprocess that are relevant for foam formation in the bioreactor; - Transmitting the determined values ​​as input signals to a controller; - generating output signals by the controller for at least one foam regulating device depending on the input signals; - demand-based control of at least one foam regulating device by means of the output signals issued by the controller.

2. Method according to claim 1, characterized in that the values ​​of several different process variables are transmitted to the controller as several input signals and that the controller generates and outputs the output signals as a function of the separate input signals for controlling the at least one foam regulating device.

3. Method according to claim 1 or 2, characterized in that the one or more process variables is / are one or more of the following and / or a temporal change in one or more of the following biological process variables: cell density; viable cell volume; viable cell density; cell growth rate; protein concentration; protein amount; oxygen consumption; carbon dioxide concentration; glucose consumption; nutrient solution consumption; turbidity.

4. Method according to claim 3, characterized in that in addition to the one or more biological process variables, values ​​of one or more of the following process variables are transmitted to the controller as input signals: Value for volume-related mass transfer coefficient (kLa value); perfusion rate; bleed rate; gassing rate.

5. The method according to claim 3 or 4, characterized in that, in addition to the one or more biological process variables, the time from the start of the process, the time from the start of a specific process step, or the time from a specific process event; and / or the type of a currently operated sparger and / or the gassing rate introduced via a sparger are transmitted as input signals to the controller.

6. Method according to one of the preceding claims, characterized in that the determination of the values ​​of the one or more process variables is carried out indirectly by further processing of measured values ​​from a sensor, such as an exhaust gas sensor.

7. Method according to one of the preceding claims, characterized in that the controller comprises a master controller to which the values ​​of the one or more process variables are transmitted, and at least one slave controller which controls the at least one foam regulating device, wherein the foam regulating device is assigned a control profile which is dependent on the output signal of the master controller.

8. Method according to one of the preceding claims, characterized in that several foam regulating devices are controlled by the output signals of the controller.

9. The method according to claim 8, characterized in that the output signals of the controller are used to cascade control a plurality of foam regulating devices.

10. Method according to one of the preceding claims, characterized in that the values ​​of several different process variables are each transmitted as separate input signals to several controllers and that each controller generates output signals depending on its input signals for controlling at least one foam regulating device.

11. Method according to one of the preceding claims, characterized in that the one or more foam regulating devices are less- At least one of the following measures must be carried out: addition of antifoam agent, preferably in an adjustable concentration, by means of a pump controlled by the controller's output signals; mechanical foam destruction; modification of a gas supply by one or more spargers.

12. Method according to one of the preceding claims, characterized in that the input signals of the controller are additionally based on signals from sensors which directly detect or monitor foam or its growth and / or the fill level in the bioreactor.

13. Method according to one of the preceding claims, characterized in that a previously created model is included in the demand-based control of the at least one foam regulating device.

14. Method according to one of the preceding claims, characterized in that the foam regulation is carried out by the demand-based control of the at least one foam regulation device without using a dedicated foam detection device.

15. A device for automating foam control during a bioprocess for cultivating cells in a bioreactor, comprising a device for determining values ​​of one or more process variables of the bioprocess that are relevant for foam formation in the bioreactor, a controller to which the determined values ​​are transmitted as input signals, and a foam control device that is controlled by output signals generated by the controller as a function of the input signals, characterized in that the device is designed to carry out the method according to one of the preceding claims.

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