Method and apparatus for foam control in an automated bioreactor

Abstract

A method for automated foam conditioning during a biological process for culturing cells in a bioreactor, the method comprising the steps of: determining values of one or more process variables of the biological process associated with foam formation in the bioreactor; transmitting the determined value as an input signal to a controller; generating, by the controller, an output signal for the at least one foam adjustment device as a function of the input signal; and controlling the at least one foam adjusting device as needed by means of the output signal output by the controller.

Description

Technical Field

[0001] This invention relates to a method for automating foam control in a bioreactor during a bioprocess of culturing cells in a bioreactor. The invention also relates to an apparatus for performing this method. Background Technology

[0002] In bioprocessing, a known problem is the disruptive formation of foam, which can have various causes. Therefore, foam is particularly undesirable because it can interfere with sensor function and cause adhesion and fouling of reactor walls, leading to increased cleaning costs. Furthermore, foam can enter the fluid lines leading to the top space of the bioreactor. Using larger containers only partially overcomes these problems and is also uneconomical. In addition, foam is disruptive in downstream processes.

[0003] Various techniques are known for detecting foam in bioreactors, such as capacitive measurements using patch sensor units in disposable bioreactors (see, for example, EP 3 872 162 A1) or conductive measurements using ceramic foam probes in stainless steel bioreactors. Camera- or image-based systems are also used for foam identification (see, for example, WO 2020 / 198518 A1, US 11 327 064 B2, EP 3 957 712 A1), as well as optical systems that measure light scattering or transmission.

[0004] In principle, foam can be suppressed chemically, mechanically, and thermally. Chemical defoamers, such as silicone oil, can be introduced in varying concentrations. In particular, addition is made in the headspace of the bioreactor, from which the defoamer drips onto the surface of the substrate within the bioreactor. However, underwater addition is also feasible. A device with a defoamer reservoir in the headspace of a bioreactor is known from WO 2017 / 029259 A1, which releases the defoamer upon foam contact. Devices for mechanical foam destruction are particularly used in glass or stainless steel bioreactors. However, solutions also exist for single-use bioreactors, such as those using nozzles or ultrasound (see DE 202020 003 748 U1).

[0005] The use of foam-destructive or foam-blocking measures can be based on a time-based preset curve, created based on empirical values ​​of the corresponding process (subjective visual inspection and prior experience regarding the required defoaming dosage). For example, it can be proposed to add 1 mL of a specific concentration of defoamer every 60 seconds. Defoaming measures can also be sensor-based. For example, if an optical system detects foam, defoamer is delivered to the bioreactor at a preset fixed pumping rate until the foam level drops below a critical value, which can be determined, for example, using a foam probe. Alternatively, defoamer can be pumped into the bioreactor at preset time intervals and then checked by means of a control loop to see if the addition is sufficient. If the sensor signal is still present or still too high, then another pumping interval is started.

[0006] WO 2021 / 011484 A1 discloses a fermenter control system having a device for measuring gas volume fraction (GVF), control instruments, and one or more venting mechanisms. The control is configured such that it determines an appropriate amount of defoamer based on input received from the GVF measuring device. The fermenter control system may also include one or more auxiliary measuring devices, wherein the control instruments then generate control signals for the venting mechanisms based on inputs from the GVF measuring device and the additional auxiliary measuring devices. The auxiliary measuring devices may include temperature sensors, pH sensors, mixing rate sensors, or flow sensors for the fermenter's process lines, inlet lines, or recirculation lines. Given the specific requirements of biopharmaceutical technology, this technology from the brewing industry is largely unsuitable for cell culture. In contrast, the gas volume fraction in the culture medium within the fermentation vessel is not critical for foam suppression during cell culture.

[0007] US 2022 / 290090 A1 discloses an automated system designed to detect and predict the presence and / or level of foam formation using a machine learning-based recognition mechanism. Foam formation and / or other parameters of the fermentation process should be automatically controlled based on estimated process conditions. A trained model can be used to predict foam formation. However, image data from an image recognition device is required in all cases. Additionally, real-time parameters derived from sensors, such as pH, dissolved oxygen concentration, optical density, or temperature, can also be used. Summary of the Invention

[0008] The purpose of this invention is to achieve more effective foam control in bioprocesses used for culturing cells in bioreactors.

[0009] This objective is achieved by a method having the features of claim 1 and an apparatus having the features of claim 13. Advantageous and suitable designs of the method and apparatus according to the invention are described in the dependent claims.

[0010] The method according to the invention is used for automating foam control during a bioprocess of culturing cells in a bioreactor. The term "cell" herein includes, in particular, mammalian cells, fungal cells, yeast cells, and bacteria. The method according to the invention comprises the steps of: repeatedly determining values ​​of one or more process variables related to foam formation in the bioreactor; transmitting the determined values ​​as input signals to a controller; generating an output signal for at least one foam control device by the controller based on the input signals; and controlling at least one foam control device as needed by means of the output signal output by the controller.

[0011] Repeat the step “Determine the values ​​of one or more process variables related to foam formation in the bioreactor”, where the time interval is preset by process control.

[0012] Here, "process variables related to foam formation in a bioreactor" are understood as process parameters of a specific bioprocess that have a decisive influence on foam formation and are measured, calculated, or otherwise determined. These process variables are distinct from the signals of dedicated foam detection devices. In particular, unlike the technology described in US 2022 / 290090 A1, the method according to the invention does not require the detection and evaluation of images used for passive foam detection.

[0013] Controlling at least one foam adjustment device "as needed" by means of an output signal from the controller means, for example, using as little defoamer as possible in the case of chemical foam suppression, or not performing the corresponding measures at an intensity higher than required for the planned further progress of the biological process in the case of mechanical foam suppression, for example by stirring in the headspace. In this sense, control as needed differs from simple activation measures.

[0014] This invention is based on the understanding that currently used foam suppression measures in bioprocesses cannot be automatically controlled in terms of intensity, but are (if any) only manually controlled, which is time-consuming and defective, and furthermore, lacks coordination with the corresponding process or process events. The method according to the invention allows for finer, more precise, and thus better regulation. As a result, this leads to significant advantages. For example, it is possible to reduce the consumption of defoamers, which is important because defoamers have a negative impact on downstream treatment. Furthermore, due to the invention, targeted and timely application of defoaming measures improves efficiency, as premature intervention will reduce or subsequently diminish the effect. All of these are also beneficial for the performance or durability of the entire exhaust gas path and for better and more reproducible dissolved oxygen variation processes / control. Another important advantage is that the method according to the invention enables foam regulation without the need for dedicated foam sensors.

[0015] According to an improved embodiment of the invention, the values ​​of multiple different process variables are transmitted as multiple input signals to a controller, which then generates and outputs output signals based on the individual input signals to control at least one foam adjustment device. This improved embodiment is useful if multiple process variables related to foam formation in a bioreactor can be determined during the bioprocess. Through corresponding processing (evaluation algorithms), it is theoretically possible to derive more accurate conclusions about foam formation from multiple input signals and to incorporate this knowledge into targeted foam suppression.

[0016] The following bioprocess variables have proven to be particularly suitable for process variables related to foam formation in bioreactors: cell density; viable cell volume; viable cell density; cell growth rate; protein concentration; protein mass; oxygen consumption; volume-dependent mass transfer coefficient (kLa value); carbon dioxide concentration; glucose consumption; nutrient solution consumption; and turbidity. Correspondingly, it is preferable to transmit one or more values ​​of these process variables and / or their time variations as inputs to the controller.

[0017] In addition to one or more bioprocess variables, the values ​​of one or more of the following process variables in terms of method technology can also be transmitted as input signals to the controller: perfusion rate; bleed rate (cell density correction rate); ventilation rate.

[0018] Additionally, the time from the start of the process (batch processing time), the time from the start of a specific process step, or the time from the start of a specific process event can also be used as controller input.

[0019] The use of sprayers can also have a significant impact on foam formation. Foam is formed more or less depending on the type of sprayer (e.g., micro-sprayers, large sprayers) or the number and size of orifices and / or the aeration rate through the sprayer. The type of foam produced by the corresponding sprayer type (fine or coarse pores, wet or dry) also plays an important role in the required suppression. Correspondingly, a preferred embodiment of the invention proposes that these aspects also be considered as process variables in foam adjustment. This means that one or more sprayer types currently operating in the process and / or their aeration rates (respectively) are process variables in the controller, just like cell density. Therefore, using a micro-sprayer, for example, typically produces more foam compared to a large sprayer, which requires a higher foam correction intensity. Regarding one or more “currently” operating sprayers, it should be noted that sprayers can be changed in continuous processes, especially in typical processes using combined sprayers. For example, it is possible to choose between large sprayers (large bubbles) and micro-sprayers (small bubbles), or a combination of both. Because individually operating sprayers have a direct impact on foam performance in the bioreactor, it is meaningful to combine them into the regulation process. Furthermore, the currently operating sprayers can be used to track certain bioprocess parameters (oxygen consumption, cell count, culture duration, etc.).

[0020] The values ​​of process variables, which are transmitted as input to the controller (where feasible), are measured directly by existing sensors or derived from process control protocols (e.g., the type of sprayer used, aeration rate). However, it is also possible to indirectly determine one or more values ​​used to control the foam adjustment equipment, particularly by further processing measurements from one or more sensors, such as exhaust sensors. It should be noted that these sensors are not image-detection sensors or sensors used for directly detecting foam.

[0021] According to a particularly advantageous embodiment of the invention, the controller includes a main controller and at least one secondary controller, the values ​​of one or more process variables being transmitted to the main controller, and the secondary controller controlling at least one foam adjustment device. The at least one foam adjustment device is equipped with a control curve correlated to the output signal of the main controller. This operation can be termed "polygonal control." The control curve can be a fixed preset or a user-defined preset.

[0022] In this way, multiple foam adjustment devices can also be controlled either simultaneously or in a cascade manner via the controller's output signal.

[0023] According to an improved embodiment of the invention, the values ​​of multiple different process variables are transmitted as separate input signals to multiple controllers. Each controller generates an output signal based on its input signal to control at least one foam adjustment device. Depending on the selected strategy, one or more output signals are selected to control one or more foam adjustment devices.

[0024] Preferably, one or more foam adjustment devices perform at least one of the following measures: adding defoamer via a pump controlled by an output signal from a controller, preferably at a settable concentration; mechanically breaking up the foam; and altering the gas delivery through one or more sprayers. It is important to note that in this case, the corresponding measures (as described above) are performed as needed, that is, not simply switched on and off after a fixed preset time. When adding defoamer, it can be added according to cell density, process time, etc., so that the defoamer is applied as a protective layer to the surface of the matrix in the bioreactor. That is, if more foam is expected to be generated with increasing cell density, it is reacted to in this way as needed.

[0025] Additionally, it can be suggested that the controller's input signals are further based on signals from sensors that directly detect or monitor the foam or its growth and / or the liquid level in the bioreactor. This additional information may enable better foam control.

[0026] Similarly, the pre-created model can be incorporated into controlling at least one foam adjustment device as needed. Here, the model can serve as a basis for inputs to other controllers or for controlling or balancing other input parameters.

[0027] As has been shown, it is particularly advantageous that foam adjustment can be performed by controlling at least one foam adjustment device as needed, without the need for a dedicated foam identification device.

[0028] The present invention also provides an apparatus for automated foam control during a bioprocess of culturing cells in a bioreactor, the apparatus comprising: means for determining the values ​​of one or more process variables related 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 an output signal generated by the controller based on the input signals. The apparatus is configured to perform the method according to the invention. Attached Figure Description

[0029] Other features and advantages of the invention will become apparent from the following description and the accompanying drawings. These are illustrated in the drawings:

[0030] Figure 1 A schematic diagram of a bioreactor facility with sensors and foam adjustment equipment is shown.

[0031] Figure 2 The basic control loop for automated foam control is shown;

[0032] Figure 3 This shows a first example of a combination of control curves;

[0033] Figure 4 This shows a second example of a combination of control curves;

[0034] Figure 5 A third example of a combination of control curves is shown; and

[0035] Figure 6 The fourth example of a combination of control curves is shown. Detailed Implementation

[0036] exist Figure 1 The image exemplarily illustrates a bioreactor facility 10 having a container 12 containing a substrate. In particular, a plurality of sensors 14, 16 and foam adjustment devices 18, 20, 22 are provided within the container.

[0037] Sensors 14 and 16 can be used to measure or indirectly determine one of the following process variables: cell density; live cell volume; live cell density; cell growth rate; protein concentration; protein mass; oxygen consumption; volume-related mass transfer coefficient (kLa value); carbon dioxide concentration; glucose consumption; nutrient solution consumption; perfusion rate; hemorrhage rate; ventilation rate; turbidity.

[0038] As foam conditioning devices 18, 20, and 22, there is a device 18 for adding defoamer, especially by means of a pump, and a device 20 for mechanical foam destruction. In addition, a sprayer, here a combined sprayer 22 (micro sprayer or large sprayer) having two differently configured orifices, is also used as foam conditioning device.

[0039] Sensors 14 and 16, along with foam adjustment devices 18 and 20, are connected to a controller 24 (measurement and regulation system) located outside the container 12. This controller also includes a timer for time measurement. The controller 24 includes one or more main controllers associated with sensors 14 and 16, and one or more secondary controllers controlling the foam adjustment devices 18, 20, and 22. Control curves, which will be discussed in more detail later, are stored in the controller 24. These control curves can be fixed presets or can be selected or individually configured by the user.

[0040] Foam, symbolically shown here and labeled with reference numeral 26, is formed during the continuous bioprocess used to culture cells. Foam 26 can have different forms and structures. For example, foam 26 can be coarse-porous or fine-porous, have different humidity levels or humidity gradients, be distributed locally or planarly on the substrate (centered on the impeller), etc. These parameters all depend on the process variables mentioned above.

[0041] The following is a brief, not exhaustive, explanation of some basic relationships between process variables and their impact on foam formation:

[0042] In principle, a large amount of bubble is expected under the following circumstances:26

[0043] - The process has been ongoing for a long time;

[0044] - High cell density (which can be determined using a cell density sensor or live cell volume sensor).

[0045] - A large amount of protein is present in the matrix (which can be determined using cell density sensors or live cell volume sensors), for example, due to specific matrix components, product formation, or "contamination" caused by dead cells);

[0046] - High cell growth rate;

[0047] - High oxygen consumption or carbon dioxide formation (indicating high growth rate or high cell density).

[0048] - Dramatic pH changes (indicating rapid growth or altered metabolic state);

[0049] - High glucose or substrate consumption (indicating rapid growth);

[0050] - The presence of induction or temperature shift (indicating a change in the metabolic state of the cells, such as product formation being activated by molecules; depending on the type of process and other factors, it may also be that foam formation is reduced due to decreased cell growth).

[0051] - High perfusion or exfusion rate (indicates high growth rate and high matrix exchange, an effect that can be bidirectional, i.e., foam formation may increase or decrease depending on the effect of the fresh matrix, and the aforementioned effects may also be balanced).

[0052] - Sprayers with small orifices or high ventilation rates are used.

[0053] exist Figure 2 The diagram shows a basic control loop for foam conditioning, in which foam conditioning... Figure 1The diagram exemplarily illustrates bioreactor facility 10. It is noteworthy in the diagram that all controller input signals (if multiple input signals are available) and all controller output signals (if multiple output signals are available) controlling foam adjustment devices 18, 20, 22 are summarized.

[0054] As already indicated, foam control devices 18, 20, and 22 are capable of suppressing foam by taking one or more of the following measures:

[0055] The defoamer is preferably added at a settable concentration by means of a pump controlled by the output signal of the controller 24, wherein the pumping rate (pumping speed) can be increased or decreased and / or the time interval between defoamer additions can be extended or shortened and / or the concentration of the defoamer can be increased or decreased.

[0056] The existing foam 26 is mechanically destroyed, which can increase or decrease the intensity (speed or rate) of the measure.

[0057] Change the gas delivery through one or more sprayers 22 (select micro sprayers or large sprayers, set the ventilation rate).

[0058] Using multiple measures for foam suppression in a cascading manner (in sequence) is, in principle, feasible.

[0059] Figures 3 to 6 Examples of different polygon combinations used for foam adjustment are shown. Figure 3 The combination of cell density and batch time in Figure 4 The combination of cell density and reactor volume in Figure 5 The combination of glucose consumption / concentration and dissolved gas concentration (e.g., oxygen), as well as in Figure 6 The diagram combines turbidity and reactor volume. These graphs illustrate which output signal is used to control one or more foam conditioning devices 18, 20, 22, expressed as a percentage of the main controller's adjustment range (in the example shown, the range of interest is between 0% and 50%, but it could also be between 0% and 100% or other intervals). Foam conditioning can be proposed to use one or more output signals to control one or more foam conditioning devices 18, 20, 22. From an application perspective, foam conditioning based on only one process variable is generally preferred over a combination of process variables, where the use of combinations depends on the chosen strategy.

[0060] Foam adjustment can be supplemented by real-time spectral measurements and assessments using stored models, and / or by additional liquid level measurements (e.g., using an existing camera). Higher liquid levels require stronger foam suppression, while in cases of very low levels, it may be possible to (temporarily) forgo immediate addition of defoamer. The properties of foam 26, particularly its porosity (coarse or fine), strength, humidity, humidity gradient, and extensibility within the matrix, can also be determined using suitable equipment and incorporated into foam adjustment.

[0061] List of reference numerals

[0062] 10 Bioreactor Facilities

[0063] 12 containers

[0064] 14 First Sensor

[0065] 16 Second Sensor

[0066] 18 First Foam Adjustment Equipment

[0067] 20 Second foam adjustment equipment

[0068] 22 Third foam adjustment equipment / combination sprayer

[0069] 24 controllers

[0070] 26 bubbles

Claims

1. A method for automating foam control during a bioprocess of culturing cells in a bioreactor, wherein the method comprises the following steps: - Repeatedly determine the values ​​of one or more process variables of the bioprocess, which are relevant to foam formation in the bioreactor; - The determined value is transmitted to the controller as an input signal; - The controller generates an output signal for at least one foam adjustment device based on the input signal; - The at least one foam adjustment device is controlled as needed by means of the output signal output by the controller.

2. The method according to claim 1, characterized in that, The values ​​of multiple different process variables are transmitted to the controller as multiple input signals, and the controller generates and outputs the output signal based on the individual input signals to control the at least one foam adjustment device.

3. The method according to claim 1 or 2, characterized in that, The one or more process variables It is the time variation of one or more of the following bioprocess variables and / or one or more of the following bioprocess variables: cell density; viable cell volume; viable cell density; cell growth rate; protein concentration; protein mass; oxygen consumption; carbon dioxide concentration; glucose consumption; nutrient solution consumption; turbidity.

4. The method according to claim 3, characterized in that, In addition to the aforementioned one or more bioprocess variables, the values ​​of one or more of the following process variables related to method technology are transmitted to the controller as input signals: Volume-dependent mass transfer coefficient (kLa value); perfusion rate; pleuropneumonia rate; ventilation rate.

5. The method according to claim 3 or 4, characterized in that, In addition to the one or more bioprocess variables mentioned above, the following will also be transmitted to the controller as input signals: The time from the start of the process, or the time from the start of a specific process step, or the time from the start of a specific process event; and / or The type of sprayer currently in operation and / or the ventilation rate introduced via the sprayer.

6. The method according to any one of the preceding claims, characterized in that, The values ​​of the one or more process variables are determined indirectly by further processing the measurements from sensors, such as exhaust gas sensors.

7. The method according to any one of the preceding claims, characterized in that, The controller includes a main controller and at least one secondary controller. The values ​​of the one or more process variables are transmitted to the main controller. The secondary controller controls the at least one foam adjustment device, wherein the foam adjustment device is equipped with a control curve that depends on the output signal of the main controller.

8. The method according to any one of the preceding claims, characterized in that, The controller outputs signals to control multiple foam adjustment devices.

9. The method according to claim 8, characterized in that, The controller's output signal is used to control multiple foam adjustment devices in a cascade manner.

10. The method according to any one of the preceding claims, characterized in that, The values ​​of multiple different process variables are transmitted as separate input signals to multiple controllers, and each controller generates an output signal based on its input signal to control at least one foam adjustment device.

11. The method according to any one of the preceding claims, characterized in that, One or more of the foam adjustment devices perform at least one of the following measures: adding defoamer by means of a pump controlled by the output signal of the controller, preferably adding defoamer at a settable concentration; performing mechanical foam destruction; changing the gas delivery through one or more sprayers.

12. The method according to any one of the preceding claims, characterized in that, The input signal of the controller is additionally based on the signal from the sensor, which directly detects or monitors the foam or its growth and / or the liquid level in the bioreactor.

13. The method according to any one of the preceding claims, characterized in that, The pre-created model is incorporated into the required control of the at least one foam adjustment device.

14. The method according to any one of the preceding claims, characterized in that, Foam adjustment is performed by controlling the at least one foam adjustment device as needed, without the need for a dedicated foam identification device.

15. An apparatus for automating foam control during a bioprocess of culturing cells in a bioreactor, the apparatus comprising: means for determining values ​​of one or more process variables relating to foam formation in the bioreactor; a controller, the determined values ​​being transmitted as input signals to the controller; and a foam control device controlled by an output signal generated by the controller based on the input signals, characterized in that, The device is configured to perform the method according to any one of the preceding claims.

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