DE102023111143A1 - METHOD AND DEVICE FOR MONITORING THE CONTENTS OF MIXED REACTORS - Google Patents

Abstract

The invention relates to a method and a device for monitoring the contents of mixed reactors. It is particularly applicable for monitoring the contents of mixed reactors with high demands on sterility and purity as well as in applications with reactors of complex geometry or small size and thus limited accessibility for measuring devices. Applications of the invention can thus be found, for example, in the process monitoring of cell cultures or chemical reactions, of processing, purification and formulation processes of pharmaceutical, biological or chemical products, and in the monitoring of storage processes.The object of the present invention is to provide a method by means of which the monitoring of the contents of mixed reactors with sensors inside the reactor can be carried out in a robust and easy-to-use manner, while at the same time being easy to scale to large and small reactor volumes and with the possibility of recording several properties of the reactor contents.According to the invention, the solution to the problem of monitoring the contents of mixed reactors, wherein the reactor contents have at least one property to be monitored and the at least one property to be monitored influences at least one signal of at least one sensory component and the at least one signal of the at least one sensory component is recorded by at least one measuring arrangement, is achieved by a method in which at least one sensor containing the at least one sensory component is not stationary and moves in the reactor, so that the at least one sensor is not permanently in the detection range of the at least one measuring arrangement and at least one recording of the at least one Signal of the at least one sensory component is transmitted by the at least one measuring arrangement, while the at least one sensor is located in the detection range of the at least one measuring arrangement.

Description

technical field

The invention relates to a method and a device for monitoring the contents of mixed reactors. It is particularly applicable for monitoring the contents of mixed reactors with high demands on sterility and purity as well as in applications with reactors of complex geometry or small size and thus limited accessibility for measuring devices. Applications of the invention can thus be found, for example, in the process monitoring of cell cultures or chemical reactions, of processing, purification and formulation processes of pharmaceutical, biological or chemical products as well as in the monitoring of storage processes.

Mixed reactors are used in many areas of the chemical, biological and biotechnological industry and research, in particular for carrying out chemical or biochemical reactions and syntheses, for cultivating living cells, for processing, purifying and formulating pharmaceutical, biological or chemical products and for storing various components of such processes. The contents of mixed reactors are monitored in particular for the purposes of research, process development and optimization, process control and regulation as well as for characterization and quality assurance, whereby various properties of the reactor contents or their correlates are recorded using suitable sensors and measuring arrangements and often recorded over the duration of the process.

The detection of certain properties of the reactor contents often requires the use of multi-stage measuring chains, since the properties to be monitored are not directly present as an electronically detectable signal, for example as an electromagnetic wave, electrical voltage or electrical current. Instead, sensors are used that contain at least one sensory component, which is part of at least one sensor located inside the reactor, which reacts to the property of the reactor contents to be monitored and, depending on this and possibly via intermediate steps of other sensory components, generates a signal that can be detected by a suitable measuring arrangement. The person skilled in the art is aware of a wide variety of approaches to this, in particular but not exclusively the sensory use of the dependence of optically detectable properties (e.g. absorption and emission spectra, luminescence lifetimes, quantum yields, polarization behavior, plasmon resonance) of suitable dyes on the properties of the reactor contents (e.g. pH, temperature, oxygen saturation, substrate, metabolite and product concentrations). The combination of such dyes or other sensory components that can be measured using a measuring technique with sensory components (e.g. enzymes, aptamers, antibodies) that cannot be directly measured using a suitable measuring arrangement, but that influence a sensory component that can be measured using a measuring technique, for example via by-products, co-substrates (e.g. protons as pH, oxygen), dissociation and association reactions or conformational changes, are also known to the person skilled in the art.

The combination of suitable sensors and measuring arrangements therefore basically enables the monitoring of the contents of mixed reactors, whereby the properties to be monitored are converted by suitable sensors inside the reactor into a signal that can be electronically recorded by the measuring arrangement. If the reactor contents consist of several phases (for example liquid or liquid-like reaction mixture and the overlying gas phase in the head space of the reactor), monitoring the properties of individual phases is often important in the application (for example oxygen concentration in the reaction mixture, oxygen concentration in the gas phase of the head space).

State of the art

Methods and devices are known from the prior art which enable the monitoring of the contents of mixed reactors by comprising at least one sensory component which is part of at least one sensor located inside the reactor and reacts to the property of the reactor content to be monitored and, depending on this, generates a signal which can be detected by a suitable measuring arrangement.

An overview of the state of the art for monitoring the oxygen concentration in the reactor contents is provided in the review article by Wang Xu-dong and Otto S. Wolfbeis (“Optical Methods for Sensing and Imaging Oxygen: Materials, Spectroscopies and Applications”. Chem. Soc. Rev. 43, No. 10 (2014): 3666-3761. https://doi.org/10.1039/C4CS00039K ), with Chapter 11 presenting the state of the art on sensor formats.

A comparable overview for monitoring the pH in the reactor contents is provided in the review article by Andreas Steinegger, Otto S. Wolfbeis, and Sergey M. Borisov (“Optical Sensing and Imaging of PH Values: Spectroscopies, Materials, and Applications”. Chemical Reviews 120, No. 22 (November 25, 2020): 12357-489. https://doi.org/10.1021/acs.chemrev.0c00451 ), with Chapter 14 presenting the state of the art on sensor formats.

Other comparable or derived methods and devices for monitoring other properties of the reactor contents are known to the person skilled in the art from the specialist literature.

The above-mentioned review articles describe the use of optically detectable sensor components, whereby at least one of their optical properties changes depending on the property of the reactor contents to be monitored (for example pH or oxygen concentration). Optical detection is carried out using suitable measuring arrangements, which usually have at least one light source and at least one light detector. The two review articles disclose various sensor formats and associated concepts and designs of the optical measuring arrangements. Layered sensor formats are disclosed that are applied to the inside of the reactor wall, to the inside of optical windows of immersion probes, or to optical fibers and other totally reflective light guides in contact with the reactor contents. The associated measuring arrangement is in optical contact with the sensors and the sensory components contained in them. Furthermore, micro- and nanoparticulate sensor formats are disclosed. In some designs, these particles are embedded in polymers and, similar to the aforementioned layered sensors, applied in a stationary manner to the inside of the reactor, with an associated optical measuring arrangement that is also stationary. In other embodiments, these particles are added to the reactor contents and either concentrated locally in the optical field of view of the measuring arrangement by magnetic interactions, or added in such a high concentration that such magnetic concentration is not necessary to achieve sufficiently strong optical signals.

The article by David Flitsch, Tobias Ladner, Mihaly Lukacs, and Jochen Büchs (“Easy to Use and Reliable Technique for Online Dissolved Oxygen Tension Measurement in Shake Flasks Using Infrared Fluorescent Oxygen-Sensitive Nanoparticles”. Microbial Cell Factories 15, No. 1 (December 2016): 45. https://doi.org/10.1186/s129 34-016-0444-4 ) discloses a combined design for monitoring the oxygen concentration in the reactor contents, with homogeneously distributed sensor particles in the reaction mixture and a sensor fixed to the inside of the reactor wall. The optical properties of both sensor formats are recorded using measuring arrangements with a fixed fiber outside the reactor. The article shows a serious disadvantage of sensors fixed in the reactor for applications in which the sensor is not permanently in contact with the reaction mixture. In such a case, the sensor reacts due to its inherent inertia both to properties of the reaction mixture and to properties of the gas phase in the head space of the reactor, resulting in a mixed signal that deviates from the actual signal of the respective phase.

A wide variety of methods and devices for monitoring the contents of mixed reactors are also known from the patent literature. They are all similar in terms of the fixed attachment of sensors to the inside of the reactor wall or other components of the reactor, with each sensor being assigned a suitable measuring arrangement in a fixed location, either as a complete measuring arrangement or via a component of the measuring arrangement that is fixed to the respective sensor (for example in the form of an optical fiber).

DE10101576A1 discloses an optical sensor for determining at least one parameter in a sample, which contains an indicator material with a short decay time that responds to the parameter and a reference material with a long decay time that does not respond to the parameter and serves to record a measurement signal indicating the parameter to be determined on the basis of the jointly recorded luminescence responses of the indicator and reference material. The indicator and reference material are immobilized on a common carrier and in contact with the reactor contents. The carrier can be a film, a cassette through which the sample flows, or a flat or fiber-like light guide. In the case of several sensors, the sensors can be arranged next to one another on the carrier, separated from one another, or they can also be mixed.

EP1397672A1 discloses a device for detecting oxygen, comprising a carrier with a plurality of wells for receiving samples, wherein stationary oxygen sensors are provided on the inner sides of the wells, comprising particles which contain a luminescent dye which can be erased by oxygen and a gas-permeable and essentially water-impermeable first matrix, and an essentially water-permeable second matrix, wherein the particles are dispersed in the second matrix. The device is designed as a microtiter plate and as a culture plate for cultivating cells.

EP3821230A1 , DE102018105174A1 , EP2321052A1 and US2008171383A1 reveal Devices and methods for monitoring the contents of various reactor types (channels, bags, stirred tanks), whereby the sensors are mounted in a fixed position on a sensor carrier and this sensor carrier is attached to the reactor wall in such a way that the sensor comes into contact with the reactor contents. The optical properties to be recorded by the sensors installed in a fixed position in the reactor wall are recorded using suitable fixed optical measuring arrangements or using appropriately fixed optical fibers as components of suitable optical measuring arrangements.

The methods and devices known from the prior art for monitoring the contents of mixed reactors disclose sensors which, via suitable sensor components, convert at least one property of the reactor contents to be monitored into a signal that can be electronically recorded by the measuring arrangement. The vast majority of these methods and devices use sensors that are fixedly connected to the inside of the reactor wall or the reactor internals in combination with likewise fixed measuring arrangements inside or outside the reactor to record the sensor signal.

The concept of stationary sensors and measuring arrangements is accompanied by some serious disadvantages. For example, the parallel monitoring of several properties of the reactor contents using several suitable sensors also requires parallelization or duplication of the associated measuring arrangements. The resulting increase in space required for the measuring arrangements limits the miniaturization of reactors, which is often of interest for screening and process development work, and limits the number of properties of the reactor contents in miniaturized reactors that can be monitored in parallel. Furthermore, the application of stationary sensors to internal reactor components or walls is very complex, especially under sterile conditions. Even for the simplest reactors such as shake flasks, there are currently no robust and user-friendly systems for applying stationary sensors to the inside of the reactor walls. The non-sterile application of stationary sensors to internal reactor components or walls in combination with subsequent sterilization is also disadvantageous, since many common sterilization processes have a negative impact on the behavior of sensory components.

Another disadvantage of stationary sensors in the case of measuring arrangements located outside the reactor is the required accuracy in positioning and orientation between the internal sensor and the external measuring arrangement, which can often only be achieved with complicated positioning aids. In addition, stationary sensors in mixed reactors are sometimes exposed to high shear forces, which can limit their service life or even decompose the sensors and contaminate the reaction mixture.

Some disadvantages of stationary sensors can be avoided by using micro and nano particles in the reaction mixture, as is known from the state of the art. These are either freely and evenly distributed in the reactor contents or reaction mixture, or they are magnetically focused in the detection range of the measuring arrangement for signal detection and therefore appear in both cases to the measuring arrangement like a stationary sensor. In comparison to a sensor that is fixed to the inside of the reactor wall, however, the clear phase assignment of the sensor particles means that the above-described incorrect detection of mixed signals of the properties to be monitored from different phases of the reactor contents does not occur. Furthermore, the homogeneous distribution of the sensor particles in the reactor contents or reaction mixture, as well as the magnetic focusing of the sensor particles in front of the measuring arrangement, simplifies both the introduction of the sensors into the reactor contents and the positioning and orientation of the measuring arrangement.

Nevertheless, the use of freely distributed sensor particles also has significant disadvantages. As the reactor volume increases, increasing amounts of sensor particles are required to achieve the sensor particle concentrations in the reactor contents necessary for correct signal detection by suitable measuring arrangements, so that the monitoring costs increase significantly for larger reactor volumes. In addition, the use of homogeneously distributed and magnetically focused sensor particles makes it difficult to monitor several properties of the reactor contents in parallel using several suitable sensor particles. In many cases, this is due to the mutual interaction or influence of the sensor signals (for example due to filter or scattering effects in optical sensors with absorption, luminescence or scattered light detection) of different sensor particles in the same reaction mixture. In addition, the use of identical sensor components that can be detected by suitable measuring arrangements in sensors with multi-stage measuring chains is prevented. For example, the use of sensors with the same oxygen-quenching luminophore but different oxygen-consuming enzymes for parallel monitoring of, for example, glucose, lactate and glutamine is not possible with the particle sensors known from the state of the art, since no currently available measuring arrangement could distinguish which of the different substances is to be monitored. Properties of the reactor contents influence the signal recorded by the luminophore. This disadvantage does not exist with stationary sensors, since the combination of stationary sensor and stationary measuring arrangement implicitly assigns the properties monitored in parallel and keeps them distinguishable.

DE102004017039A1 discloses a method and a device for recording process parameters of reaction liquids in several microreactors, which are continuously shaken at least until the end of the reaction in all microreactors. The process parameters in the microreactors are recorded during the reaction using at least one sensor optic as part of the measuring arrangement. In some versions, the sensor optic can be moved via a positioning unit. Versions are also disclosed in which the reaction liquids in the microreactors have at least one chemical sensor material and the chemical sensor material is attached to at least one inner surface of the microreactor. It is also disclosed that each sensor optic is not moved at least during the recording of the process parameters, so that the shaken microreactors move relative to each sensor optic. In addition, versions are also disclosed in which a flash lamp is used as the radiation source of the sensor optic, the pulse frequency of which is coordinated with the shaking movement in such a way that the light flashes always hit the microreactor at the same point during the shaking movement. An embodiment is also disclosed in which many measurement points are recorded by means of a high flash and detection frequency, which is higher than the shaking frequency of the shaker, in order to prevent the measurement signal from beating. The overall disclosed combination of movable sensor optics as a component of a measuring arrangement, chemical sensors fixedly attached to the inner wall of the reactor and a pulsed radiation source of the sensor optics synchronized to the shaking movement basically allows the detection of several stationary sensors, even using identical optical sensor components and at the same time miniaturizing the reactor. Nevertheless, the disadvantages of the complex application of stationary sensors to internal reactor components or walls under sterile conditions and the disadvantages of sterilization after application still exist here. Another disadvantage is the need to synchronize the radiation source to the shaking movement, which results from the relative movement between the shaken stationary sensor and the unshaken measuring arrangement. The disadvantage of using sensors that are fixed in place in the reactor is that there is also the risk of recording mixed signals from different phases of the reactor contents, as described above. If sensor particles that are freely distributed in the reaction mixture are used, the disadvantages described above arise with regard to the parallel recording of different properties of the reactor contents using several, or even partially identical, sensor components.

All methods and devices disclosed in the prior art for monitoring the contents of mixed reactors have at least one of the disadvantages described above with regard to scalability of the reactor volume, detection of several properties of the reactor contents or complexity in handling and compliance with sterility and purity requirements of the processes to be monitored.

task

It is therefore the object of the present invention to provide a method by means of which the monitoring of the contents of mixed reactors with sensors inside the reactor can be carried out in a robust and easy-to-use manner, while at the same time being easy to scale to large and small reactor volumes and with the possibility of recording several properties of the reactor contents. The object underlying the invention is achieved by a method according to claim 1 and a device according to claim 8; preferred embodiments emerge from the subclaims and the description.

definitions

To ensure clarity of some terms used in the description, they are defined and explained below and throughout the description.

A reactor is a container that can be used in particular for cultivating organisms or for carrying out chemical and biochemical reaction processes. Other areas of application for reactors include biocatalytic processes using organisms and/or biomolecules as well as other chemical and/or physical processes, whereby the term process includes all types of conversion, separation, combination, mixing, size change and storage of in particular chemical substances, organisms, particles, solutions, emulsions and foams. Reactors in the sense of the invention include in particular stirred tank fermenters and reactors, bubble column fermenters, shake flasks, T-flasks, microtiter plates, deep well plates, shake barrels, Fermentation bags, multi-purpose tubes and cell culture dishes.

Reactors can be closed or open to their environment.

The reactor contents include all the material that is located within the outer shell of the reactor. In the case of a reactor that is open to the environment, the reactor contents are limited by the inner wall of the reactor and by the hypothetical sealing surface that represents the transition between the interior of the reactor and the environment. The reactor contents are made up of one or more phases that are in particular designed as fluids (gases, liquids) or as phase mixtures with a fluidic character (foams, emulsions, suspensions, powder beds). In many areas of application of the invention, the reactor contents are formed by two phases, the reaction mixture in which the majority of the process to be carried out takes place, and the headspace above it, which is usually designed as a gas phase. According to the invention, each phase of the reactor contents can be a pure substance or a mixture of substances. The reactor contents and thus in particular also the reaction mixture and the headspace have physical, chemical, biological or other properties that can be the target of monitoring. In the sense of the invention, the sensor-based monitoring of one or more properties of the reactor contents, the reaction mixture, the headspace or their components can be carried out by detecting and determining the property itself or by detecting correlates of the property. Correlates in the sense of the invention are any phenomena, processes, signals, properties or environmental conditions of suitable sensors, sensory components or converting components that correlate with the property to be monitored. Properties in the sense of the invention are qualitative or quantitative quantities that are suitable for describing the state of an object, in particular but not exclusively substance and particle concentrations, environmental parameters such as temperature and pressure, optical parameters such as luminescence lifetime, emission, absorption or scattering intensity and wavelengths, other physical parameters such as emissivity, impedance, electrical capacitance, inductance and conductivity, biological parameters such as expression rates, metabolic pathway activities, division rates or viability and many more. In the sense of the invention, the monitoring of properties of the reactor contents or its components can also be carried out via the individual or joint detection of several correlates and their combination with suitable mathematical calculation and evaluation methods or with other algorithms.

Mixing of the reactor contents refers to any process that influences the reactor contents in such a way that at least two states of the distribution of the reactor contents and their components in the reactor, recorded at different times, are not the same. Common mixing processes use mechanical, thermal or thermodynamic processes in particular, but not exclusively shaking processes, stirring processes, pumping processes and diffusion processes.

Sensors in the sense of the invention are devices that are suitable for converting at least one property of the reactor contents or at least one of its components into at least one signal that can be detected by suitable measuring arrangements by means of excitation if necessary. For this purpose, sensors in the sense of the invention comprise at least one sensory component that has at least one property or a corresponding correlate that can be detected by the measuring arrangement and is influenced by the property of the reactor contents or at least one of its components to be monitored. The influence by the at least one property to be monitored can be direct or indirect, in particular also through interaction chains with various converting components or with other properties or components of the reactor contents. In the sense of the invention, the detection of properties or correlates by measuring arrangements takes place via at least one signal from at least one sensory component, which in some cases must be excited by the measuring arrangement. Sensory components within the meaning of the invention are in particular but not exclusively dyes, fluorophores, luminophores, nano- or microparticles made of metals or semiconductors and other structures that can generate or influence optical or other electromagnetic signals, as well as combinations of the aforementioned or similar components with one another and systems that contain at least one of the aforementioned components.

Sensors in the sense of the invention can further contain one or more converting components. A converting component in the sense of the invention has at least one property or a corresponding correlate which is influenced directly or indirectly (via another converting component) by the property of the reactor contents or at least one of its components to be monitored, and although it cannot itself be detected by a measuring arrangement, it directly or indirectly (via another converting component) has at least one property and thus at least one signal. nal of at least one sensory component located in the vicinity of the converting component. Converting components within the meaning of the invention can be linked via their interactions with one another or with sensory components to form interaction chains of any length in order to convert changes in at least one property of the reactor contents or at least one of its components into a signal that can be detected by at least one measuring arrangement. Converting components within the meaning of the invention advantageously have a high specificity with regard to the property to be monitored or the interacting other converting components or sensory components and are used in particular to monitor substance concentrations. Converting components within the meaning of the invention are in particular enzymes, catalysts, nucleic acids, aptamers, ribozymes, antibodies and other selectively binding proteins or structures, as well as combinations of the aforementioned or similar components with one another and systems which contain at least one of the aforementioned components. In the sense of the invention, the sensory component influenced by at least one converting component is preferably located in spatial proximity to the converting component influencing it, in particular in order to enable effective interaction between the two, for example through local changes in substance concentrations (in particular oxygen or proton concentration) or through energy, electron or proton transfer processes (in particular quenching, FRET, PET, electroluminescence, reactions).

Sensors in the sense of the invention can further contain markers which generate at least one marker signal, so that in particular the location, position and identity of the respective sensor can be determined by suitable measuring arrangements via the detection of the at least one marker signal.

Sensors within the meaning of the invention can have at least one sensor matrix which, as a structural component, provides mechanical stability, serves to maintain the internal arrangement of sensory components, converting components and markers within the sensor, can embed these in the sensor, can separate them from one another and can thus segment the sensor. A sensor matrix can also be used to give the sensor a certain shape and to maintain this. In addition, sensor matrices within the meaning of the invention can be used to set a selective material transport, for example through defined pore sizes, polarity or surface charge. In particular, but not exclusively, all types of polymers, hydrogels, membranes and amorphous, partially crystalline or crystalline solids are considered sensor matrices within the meaning of the invention. Several identical or different sensor matrices can be used in one sensor.

A measuring arrangement within the meaning of the invention is any device or combination of devices that is suitable for detecting at least one signal from at least one sensory component or at least one marker signal from at least one marker. In this respect, any property of a sensory component or marker that can be detected by a measuring arrangement or its correlate is a signal or marker signal within the meaning of the invention. For the purpose of detecting signals and marker signals, a measuring arrangement comprises at least one signal detector. If the signal or marker signal to be detected must be excited, a suitable measuring arrangement also comprises at least one signal exciter. Signals to be excited within the meaning of the invention are in particular but not exclusively optical signals such as luminescence, absorption and scattering intensities, luminescence polarization degrees, luminescence lifetimes and impedance signals such as capacitance, inductance or conductivity. Signals can be detected frequency-dependently or time-dependently. Signals can be detected by modulating the signal excitation. Derived signals (e.g. spectra, ratiometric signals, decay curves, Bode plots, time series) can be formed from individual recorded signals (e.g. intensities). Suitable electrical circuits or computers with suitable software and algorithms can be used to form derived signals, which are part of the corresponding measuring arrangement within the meaning of the invention. A computer is any electronic device that can store data (in particular arithmetic and logical) and process it on the basis of programmable rules. Computers within the meaning of the invention include in particular but not exclusively microcontrollers, microprocessors, system-on-a-chip computers (SoC), PCs and servers.

Signals that can be detected by suitable measuring arrangements are detected in the detection range of the measuring arrangement, whereby the detection range results from the detection range of at least one signal detector and, in the case of a required signal excitation, the excitation range of at least one signal exciter. In the sense of the invention, a measuring arrangement detects all signals that can be detected by it in its detection range, i.e. not only the signals of sensory components or the marker signals of markers, but also background and ambient signals, which can be eliminated if necessary by suitable methods. In this respect, measuring arrangements in the sense of the invention also detect signals, in particular ambient and background signals, when no sensor is located in their detection range.

The dwell time in the sense of the invention is the time during which a specific sensory component of a sensor is in the detection range of a measuring arrangement capable of detecting its signal.

The acquisition time in the sense of the invention is the time required by a measuring arrangement to acquire a specific signal exactly once.

A signal detector in the sense of the invention is any device or combination of devices that is suitable for detecting and digitizing at least one signal from at least one sensory component or at least one marker signal from at least one marker. Signal detectors in the sense of the invention therefore include in particular but not exclusively electrodes, antennas, photodiodes, phototransistors, photoresistors, CCD and CMOS arrays, optical elements such as filters, gratings, lenses, fibers and diaphragms, but also amplifier and signal shaping circuits, analog-digital converters, computers and combinations of all of the aforementioned elements.

A signal exciter within the meaning of the invention is any device or combination of devices that is suitable for stimulating at least one signal of at least one sensory component or at least one marker signal of at least one marker.

Signal exciters within the meaning of the invention therefore include in particular but not exclusively electrodes, antennas, LEDs, flash lamps, lasers, optical elements such as filters, gratings, lenses, fibers and diaphragms, but also driver and modulation circuits, digital-analog converters, computers and combinations of all of the aforementioned elements.

For the purposes of the invention, the addition of at least one sensor according to the invention into a reactor is deemed to be any targeted transfer of at least one sensor from outside the reactor into the interior of the reactor, in particular into the reactor contents or at least one of its components, such as, for example, into a reaction mixture or the headspace.

A storage medium within the meaning of the invention is any substance or mixture of substances with which at least one sensor according to the invention is deliberately brought into contact or kept during its storage or other storage before it is added to a reactor in order to specifically influence or maintain certain properties or behaviors of the at least one sensor. Storage media according to the invention are in particular but not exclusively air, defined gas mixtures, protective gases, aqueous solutions, buffer solutions, salt solutions, mixtures of organic and aqueous components, emulsions, foams and also powders.

Fins within the meaning of the invention can be components of sensors according to the invention. A fin within the meaning of the invention is any device that is suitable for specifically influencing the movement behavior of sensors according to the invention in the reactor contents or at least one of its components or the flow behavior of fluids inside the reactor at sensors according to the invention. Thus, fins within the meaning of the invention include in particular but not exclusively fluid-mechanical structural elements such as fins, notches, gaps, grooves and perforations, but also buoyancy elements such as weights, cavities, foams or other closed-porous structures.

An agitator within the meaning of the invention is any device in or on the reactor which can either be set in motion itself or sets the reactor in motion in order to cause a mixing movement, in particular as convective mass transport, in the reactor contents or in at least one of its components, for example in the reaction mixture or in the head space.

Solution

According to the invention, the problem of monitoring the contents of mixed reactors is solved, wherein the reactor contents have at least one property to be monitored and the at least one property to be monitored influences at least one signal of at least one sensory component and the at least one signal of the at least one sensory component is detected by at least one measuring arrangement, by a method in which at least one sensor containing the at least one sensory component is not stationary and moves in the reactor, so that the at least one sensor is not permanently located in the detection range of the at least one measuring arrangement and at least one detection of the at least one signal of the at least one sensory component is carried out by the at least one measuring arrangement while the at least one sensor is in the detection range of the at least one measuring arrangement.

According to the invention, at least one signal of the at least one sensor component that was detected while the at least one sensor was in the detection range of the at least one measuring arrangement is used to monitor or determine the at least one property of the reactor content to be monitored. According to the invention, this monitoring can also be carried out by detecting correlates of the property to be monitored.

In contrast to the micro and nanoparticle sensors known from the prior art, the sensors according to the invention are not evenly distributed in the reactor contents or reaction mixture, so that the sequential or parallel detection of several sensors with different sensory components is made possible by the same or several suitable measuring arrangements, without the sensors according to the invention influencing or interfering with each other with regard to their detection by suitable measuring arrangements. In addition, the method according to the invention of non-evenly distributed sensors moving in the reactor enables more cost-effective monitoring of larger reactors, since in order to achieve detectable measurement signals, larger quantities of sensors do not have to be added to the reactor contents, as is necessary for micro and nanoparticle sensors.

Advantageously, the use according to the invention of non-uniformly distributed sensors moving in the reactor enables sequential detection of the signals from the sensory components located in the sensors, so that, in contrast to the stationary sensors known from the prior art, the detection of different signals from different sensors and sensory components for monitoring different properties of the reactor contents is possible with a reduced number of measuring arrangements, and such monitoring can also be carried out on miniaturized reactors.

In contrast to stationary sensors, the method according to the invention also simplifies the application of sensors in the reactor, since the sensors according to the invention can simply be added during filling of the reactor and do not have to be applied beforehand to the inner walls or other internals of the reactor, as is the case with stationary sensors.

According to the invention, the volume of at least one detection area of at least one measuring arrangement that overlaps with the reactor contents is smaller than the volume of the reactor contents. In an advantageous embodiment of the invention, the volume of each detection area of each individual measuring arrangement that overlaps with the reactor contents is smaller than the volume of the reactor contents, but in particular so small that for each sensor used and detectable by the relevant measuring arrangement, the state of the method according to the invention can be achieved in which the respective sensor is not in the detection area of the relevant measuring arrangement. In a further advantageous embodiment of the invention, the detection area of each individual measuring arrangement is smaller than or equal to the volume of the reactor contents or reaction mixture minus the volume of all sensors that can be detected by the respective measuring arrangement that are currently in the reactor or reaction mixture and could enter the detection area of the respective measuring arrangement through movement.

According to the invention, the detection range of each individual measuring arrangement is at least large enough that the smallest sensor to be detected by the respective measuring arrangement can still be detected by it.

In some embodiments of the invention, at least one detection range of at least one measuring arrangement is smaller than at least one of the sensors detectable by the measuring arrangement. In some embodiments of the invention, each detection range of each measuring arrangement is smaller than the smallest sensor detectable in the corresponding detection range. Furthermore, in some embodiments of the invention, the detection range of each measuring arrangement is smaller than the smallest contiguous sensor range within a sensor that is detectable in the corresponding detection range and contains the same type of sensory components.

In an advantageous embodiment of the invention, the at least one signal or several signals or the entirety of the signals of a sensory component that can be detected by at least one suitable measuring arrangement can also be detected if the sensor does not completely fill the detection range of the at least one measuring arrangement. Such signals according to the invention are in particular but not exclusively ratiometric signals as well as lifetimes and decay times.

In some embodiments of the invention, each sensor is at least as large as or larger than the detection range of the at least one measuring arrangement which detects the at least one signal of the at least one sensory component contained in the sensor.

In an advantageous embodiment of the invention, the detection time required by a measuring arrangement for The time required to detect at least one signal of a sensory component is shorter than the residence time of the sensor containing the sensory component in the detection range of the measuring arrangement.

In some embodiments of the invention, the detection time that a measuring arrangement requires to detect at least one signal of a sensory component is shorter than the dwell time of the segment of a sensor containing the sensory component in the detection range of the measuring arrangement. This advantageously applies in particular to sensors that contain several sensory components arranged in segments.

In an advantageous embodiment of the invention, repeatedly for at least one detection time of the measuring arrangement detecting a specific sensory component, no sensors or sensor segments containing this same sensory component are in the detection range of this same measuring arrangement detecting the specific sensory component.

In an advantageous embodiment of the invention, a sensor according to the invention or several sensors according to the invention are moved passively by the movement of the reactor contents or reaction mixture that occurs during the mixing of the reactor and thus enter the detection range of at least one measuring arrangement and also exit the detection range of at least one measuring arrangement. Such methods according to the invention result in particular from the use of sensors according to the invention in shaken reactors (for example shake flasks, bags, culture plates, microtiter plates, T-flasks), in stirred tank reactors and in bubble column reactors.

In some embodiments of the invention, at least one sensor according to the invention contains at least one marker that can be detected by at least one measuring arrangement via at least one marker signal. In an advantageous embodiment of such embodiments, the marker triggers the start of the detection of at least one signal of at least one sensory component by at least one measuring arrangement via its marker signal. In an advantageous embodiment of embodiments of the invention with at least one marker, sensors that are used to monitor different properties are also equipped with markers that have different marker signals in order to trigger, in particular but not exclusively, different detection methods of the measuring arrangement or to be able to clearly assign the different sensors to the property of the reactor contents that they are monitoring. In some embodiments of the invention, at least one property of at least one sensory component that can be detected by suitable measuring arrangements is used as a marker in a sensor. For example, the luminescence intensity of a luminophore whose luminescence lifetime is detected as a signal can be detected as a marker signal, so that a suitable measuring arrangement always detects the luminescence lifetime when a luminescence marker signal is present.

In an advantageous embodiment of the invention, a measuring arrangement can detect different signals from different sensory components and/or different marker signals from different markers. This is done in particular by combining different suitable signal detectors and, where necessary, signal exciters.

In some embodiments of the invention, at least one measuring arrangement is fixed to the reactor so that no relative movement occurs between the reactor and the measuring arrangement. In other embodiments of the invention, a relative movement occurs between the reactor and at least one measuring arrangement.

In some embodiments of the invention, at least one sensor according to the invention is added to the reactor when the reactor is filled. In some embodiments of the invention, at least one sensor according to the invention is added to the reactor before the start of the process to be carried out in the reactor, for example before the start of the cultivation or incubation of cells or before the start of a chemical reaction. In other embodiments of the invention, at least one sensor according to the invention is added to the reactor before the packaging or sterilization of the reactor or while the process is already running in the reactor. In embodiments that require high sterility or purity, the addition takes place from a sterile addition system for sensors according to the invention, which are then also sterile.

In some embodiments, the sensors of the invention are stored in a storage medium prior to addition to the reactor, which ensures the durability of the sensors and keeps the sensors ready for use.

According to the invention, the shape of each sensor can be adapted to the shape and type of mixing of a reactor or its reactor contents. For example, in shake flasks or other orbitally shaken reactors, sensors in the form of ring segments or shapes derived therefrom can be used to match the radii of the reactors or orbital movements used. allow optimal movement of the sensors in the reactor. In turbulent stirred tank reactors, however, spherical or ellipsoidal sensors can be used advantageously.

In some embodiments of the invention, the sensors according to the invention have shaped elements that promote optimal alignment or movement of the sensors in the reactor contents or reaction mixture. Such shaped elements are in particular, but not exclusively, wing-, fin- or flipper-like structures.

In some embodiments of the invention, at least one sensory component or at least one converting component or at least one sensor matrix containing at least one sensory component or at least one converting component is set back or inward relative to the outer contour of the sensor in such a way that in the event of a collision or other contact between the sensor in question and the reactor (in particular the reactor wall or reactor internals), there is no direct contact between the reactor and the at least one sensory component, converting component or sensor matrix set back or inward. This advantageously reduces the mechanical load on the at least one sensory component, converting component or sensor matrix set back or inward.

In some embodiments of the invention, a sensor has a geometry that specifically hinders or prevents certain forms of movement in the reactor. In an advantageous embodiment of the invention for shaken reactors, in particular shake flasks, this is achieved by corners or edges in the geometry of the sensor, so that rolling movements are hindered and a correspondingly designed sensor preferentially moves with the reaction mixture under a wide variety of mixing conditions (in particular with regard to shaking conditions, fill level of the reactor and viscosity of the reaction mixture) instead of rolling or sliding over the reactor floor. In some such embodiments of the invention, the corners and edges are arranged in such a way and combined with suitable transitions that an overall advantageous flow condition is obtained, which also prevents undesirable rolling or sliding movements, stabilizes the position of the sensor in the reaction mixture and supports the movement of the sensor with the reaction mixture.

According to the invention, the density of sensors according to the invention can be adapted to the density of the reactor contents or the reaction mixture according to the monitoring objective in order to monitor properties of the reactor contents or reaction mixture in certain regions, for example at the bottom or at the surface of the reaction mixture.

According to the invention, several sensors of the same type and nature can be used in a reactor. This can be done in particular, but not exclusively, in large reactors in order to shorten the time between the detection of at least one signal from one or more specific sensory components by more frequent entry of corresponding sensors with these same sensory components into the detection range of suitable measuring arrangements and thus enable closer monitoring of at least one property of the reactor contents.

Sensors according to the invention can comprise one or more converting components which do not emit a signal detectable by measuring arrangements, but which interact with at least one sensory component and thus influence at least one property detectable by a measuring arrangement and thus at least one signal of the sensory component.

In some embodiments of the invention, different sensors according to the invention, each with different converting components, contain the same sensory component and different markers in order to reduce the number or complexity of the required measuring arrangements.

In some embodiments of the invention, a plurality of different signals from one or more sensory components or one or more combinations of at least one sensory component and at least one converting component are used to monitor or determine a property of the reactor contents.

In some embodiments of the invention, sensors according to the invention are used which are divided into segments, with different segments each containing different combinations of at least one marker or at least one sensory component and none, one or more converting components. In an advantageous embodiment of such embodiments, the segments are in a defined order and at a defined distance, so that only one marker is required to trigger the sequential detection of the signals of the sensory components from the respective segments of the sensor. In an advantageous embodiment of such embodiments, each segment is larger than the detection area of the sensor. In an advantageous embodiment of such embodiments, there is an area without a sensory component between adjacent segments. In some embodiments of the invention, at least one marker is located between adjacent segments or on at least one outer side of a sensor according to the invention.

In some embodiments of the invention, the movement of sensors according to the invention in the reactor contents or reaction mixture occurs periodically or approximately periodically, so that signals from the respective sensory components can be recorded at regular intervals by suitable measuring arrangements. In such embodiments, the recording of signals and marker signals can advantageously be synchronized with the movement periodicity, in particular but not exclusively on the basis of previously recorded signals and periods calculated therefrom or on the basis of the recording of periodic movement signals, for example accelerations, rotation rates or positions or distributions or movement behavior of the reactor contents or reaction mixture.

In some embodiments of the invention, the detection of at least one signal of at least one sensory component or at least one marker signal is carried out periodically. In some embodiments of the invention, in particular but not exclusively in the case of periodic mixing movements, the detection can advantageously be carried out at a frequency that is higher than the mixing frequency. Examples of mixing frequencies are stirrer speeds, shaking or rocking frequencies and volume flows.

In some other embodiments of the invention, the detection of at least one signal of at least one sensory component or at least one marker signal is non-periodic, in particular partially or completely randomized, in order to minimize or eliminate beats, oscillations, aliasing and other detection time-dependent phenomena of at least one detected signal of at least one sensory component or at least one marker signal.

In some embodiments of the invention, the reactor has devices that influence the movement of sensors according to the invention, in particular in order to influence, increase or reduce the probability of sensors according to the invention being located in certain areas of the reactor or reactor contents. In such embodiments, in particular but not exclusively, flow elements, baffles, flaps, fins, grooves, channels, nets or targeted fluid flows are used to influence the movement of sensors according to the invention.

In some embodiments of the invention, several signals of sensory components and/or marker signals of one or more sensors are detected simultaneously or in a temporally overlapping manner by several different measuring arrangements or by several different signal detectors or signal exciter-signal detector pairs on the same measuring arrangement.

In some embodiments of the invention, several detected signals are combined with one another by suitable algorithms and software on computers to produce new signals or correlates, which are then used to monitor at least one property of the reactor contents or reaction mixture.

In some embodiments of the invention, different converting components or sensory components or combinations of both are mixed with each other or otherwise embedded in spatial proximity to each other in a common segment of a sensor. In some embodiments of the invention, such mixtures of different converting components or sensory components or combinations of both are detected together by a suitable measuring arrangement, for example in the case of self-referenced sensors as part of dual-lifetime referencing or to compensate for ambient temperature or other ambient properties and parameters.

In an advantageous embodiment of the invention, the measuring arrangement is located outside the reactor and its detection area penetrates the reactor wall and part of the reactor contents. In some other embodiments of the invention, the measuring arrangement is designed as an immersion probe and is therefore located inside the reactor. In further embodiments of the invention, the measuring arrangement is located completely inside the reactor and moves with or against the reactor contents.

According to the invention, the at least one sensory component and its signal detectable by at least one measuring arrangement can be influenced directly or indirectly by the at least one property to be monitored, in particular also by interaction chains with converting components or with other properties or components of the reactor contents.

In some embodiments of the invention, the same property of the reaction to be monitored is The contents of the reactor or reaction mixture are recorded redundantly by several different combinations of sensory components and, where necessary, converting components and, if necessary, different measuring arrangements. This redundancy advantageously increases the robustness and quality of the monitoring of the property to be monitored.

In some embodiments of the invention, at least one sensor according to the invention is constructed in layers. In some embodiments of the invention, at least one sensor according to the invention has a core-shell structure, wherein a core consisting of at least one sensor matrix acts as a carrier of at least one active shell, wherein the active shell comprises at least one sensory component or at least one converting component or at least one marker or combinations of the aforementioned. In some embodiments of the invention, such a core can also comprise at least one sensory component or at least one converting component or at least one marker or combinations of the aforementioned or can itself be constructed in layers.

In some embodiments of the invention, at least one sensor according to the invention is constructed in such a way that during the dwell time of the sensor in the detection range of at least one measuring arrangement, at least one sensory component or at least one converting component or at least one marker or a combination of the aforementioned can be detected or excited or excited and detected by the measuring arrangement largely or completely independently of its orientation. In some embodiments of the invention, the sensor is therefore constructed rotationally symmetrically.

In some embodiments of the invention, at least one sensor matrix or at least one of its constituent parts or at least one component embedded by it is designed in such a way that background signals from the environment, in particular but not exclusively ambient light, electromagnetic fields or other radiation, are shielded. This reduces the interaction of such background signals with sensory or converting components or markers or with the detection of signals from sensory components or marker signals in the detection range of at least one measuring arrangement during the dwell time of the relevant sensor in the detection range of the at least one measuring arrangement. Examples of such embodiments can include in particular but not exclusively optically opaque or selectively permeable layers or dyes, scattering or absorption components and materials with suitable permeability or permittivity.

In some embodiments of the invention, at least one sensor matrix is designed such that it amplifies at least one signal of at least one sensory component or at least one marker signal or at least one signal excitation, in particular but not exclusively by means of scattering or reflection layers or scattering or reflection particles introduced into at least one sensor matrix.

The present invention is explained in more detail with reference to the figures and embodiments.

embodiments and figures

• 1 , a schematic representation of the method according to the invention using two sensors 5 with different sensory components 7.
• 2 , a schematic representation of a sensor 5 according to the invention and of a device according to the invention for carrying out the method according to the invention using the example of a T-flask as a reactor 1 for cultivating cells.
• 3 , a schematic representation of a sensor 5 according to the invention for carrying out the method according to the invention using the example of a shake flask as reactor 1 for cultivating cells with addition system 18 for sensors 5.
• 4 , a schematic representation for carrying out the method according to the invention on a shake flask as reactor 1 for culturing cells using the in 3 illustrated sensor 5 according to the invention.
• 5 , a schematic representation of two sensors 5 according to the invention and a device according to the invention for carrying out the method according to the invention in a stirred tank reactor as reactor 1.
• 6 , a schematic representation of a sensor 5 according to the invention in core-shell design with adapted geometry for use in orbitally shaken systems.

Identical or similarly functioning elements are given identical reference symbols in the figures, whereby only those reference symbols are used that are necessary for understanding the figure, including in the context of the other figures. Double designations in identical or similar components of a figure are therefore largely avoided.

1 shows a schematic representation of the method according to the invention using two sensors 5 with different sensory components 7. The method according to the invention is carried out on a reactor 1, the reactor contents 2 of which are mixed as a result of at least one mixing movement 25. In the reactor there are two sensors 5A and 5B, each containing a sensory component 7A and 7B. According to the invention, the two sensors 5A and 5B are used to monitor two properties of the reactor contents 2, wherein a property of the reactor contents 2 influences a sensory component 7A or 7B in such a way that a signal 8A or 8B emanating from the respective sensory component 7A or 7B can be detected by the measuring arrangement 6. The method according to the invention is characterized in that the sensors 5A and 5B used are not stationary and, as a result of the mixing movement 25, carry out a movement 10A or 10B in the reactor 1, so that the sensors 5A and 5B used are not permanently located in the detection area 9 of the measuring arrangement 6, but from time to time, in the case of periodic movements 10 also periodically, are in the detection area 9 of the measuring arrangement 6 for a certain residence time 24 and can be detected by the latter with regard to the signals 8 of their sensory components 7.

At the 1 In the embodiment of the method according to the invention shown, signals 8 from the detection area 9 are detected by the measuring arrangement 6 at regular intervals over a specific detection time 23. The 1 The embodiment of the method according to the invention shown uses detection times 23 that are shorter than the residence time 24 of the respective sensor 5A or 5B. As a result, and in combination with a periodically repeated detection of signals 8 by the measuring arrangement 6, it is ensured, even without the use of a marker 13, that according to the invention the detection of the respective signals 8A or 8B of the two sensory components 7A or 7B required for monitoring the reactor contents 2 takes place by the measuring arrangement 6 while the respective sensor 5A or 5B is located in the detection range 9 of the measuring arrangement 6. The measuring arrangement 6 shown is designed in such a way that it can detect different signals 8, but in particular the signals 8A or 8B of the two sensory components 7A or 7B.

1 shows in the upper and middle area four snapshots I, II, III and IV from a continuous mixing movement 25 of the reactor contents 2 in the reactor 1, which in its course over time 21 causes the non-stationary sensors 5A and 5B to carry out a movement 10 in the reactor 1. In the lower area of the 1 The course of the intensities 22 of the signals 8A and 8B of the two sensory components 7A and 7B, respectively, recorded by the measuring arrangement 6 is shown schematically over time 21, with the times of the four snapshots being marked with the respective Roman numerals I, II, III and IV.

The method according to the invention is 1 visible. In 1 , I, sensor 5A is not in the detection range 9 of the measuring arrangement 6, so that the intensity 22 of the signal 8A is so low that it is not used to determine the property of the reactor contents 2 to be monitored by sensor 5A with the sensory component 7A. The same applies to the snapshot in 1 , III in which sensor 5B is not located in the detection range 9 of the measuring arrangement 6, so that the intensity 22 of the signal 8B is so low that it is not used to determine the property of the reactor content 2 to be monitored by sensor 5B with the sensory component 7B. In the 1 , II and IV, there is no sensor 5 in the detection range 9 of the measuring arrangement 6, so that both signals 8A and 8B are so low that they cannot be used to determine the properties of the reactor contents 2 to be monitored by the sensors 5A and 5B with their sensory components 7A and 7B. In 1 , I, sensor 5B is located in the detection range 9 of the measuring arrangement 6, so that the intensity 22 of the signal 8B is so high that it is used to determine the property of the reactor contents 2 to be monitored by sensor 5B with the sensory component 7B. As a result of the short detection time 23 compared to the residence time 24B of sensor 5B in the detection range 9 of the measuring arrangement 6, the signal 8B suitable for monitoring can be identified reliably and robustly. The same applies to the 1 , III shows the situation of the sensor 5A in the detection area 9 of the measuring arrangement 6, which also produces a signal 8A suitable for monitoring the property of the reactor contents 2 to be monitored. Thus, in the embodiment shown, the respective intensity 22 of the signal 8A or 8B is used as a marker signal 14 for the respective sensor 5A or 5B.

2 shows a schematic representation of a sensor 5 according to the invention and of a device according to the invention for carrying out the method according to the invention using the example of a T-flask as a reactor 1 for cultivating cells. Shown as a side section (top left) and cross section (top right) is a capsule-shaped sensor 5 which contains a sensory component 7 which is surrounded by a converting component 11. The sensory component 7 and the converting component 11 are embedded in a sensor matrix 12. According to the invention, the sensory component 7 generates at least one signal 8 in the detection area 9 of a suitable measuring arrangement 6, which signal is dependent on at least one property of the reactor contents 2. Typical examples of such properties are known to the person skilled in the art, in particular but not exclusively these include the oxygen concentration, the pH, the polarity of the environment, the salinity, the temperature, the pressure and many more. Sensory components 7 according to the invention, which can generate signals 8 according to the invention depending on these properties, are in particular but not exclusively dyes and luminophores that generate optical signals 8, but also metallic or semiconductor-derived nano- or microparticulate structures, bioconjugates or systems combined with gels that can generate capacitive signals 8, more complex, in particular frequency-dependent detectable impedance signals 8 or magnetic signals 8.

Through the 2 In the illustrated embodiment of the invention with the sensory component 7 being coated by a converting component 11, any properties of the reactor contents 2 can be monitored according to the invention by using at least one suitable converting component 11 in combination with at least one sensory component 7. According to the invention, the at least one property of the reactor contents 2 to be monitored interacts with at least one converting component 11 and thereby influences at least one property of the sensory component 7 that can be detected by a measuring arrangement 6. Typical examples of such converting components 11 are in particular but not exclusively enzymes, antibodies, ribozymes, catalysts, nucleic acids and their derivatives as well as aptamers, wherein the interaction between the property to be monitored (for example a substance concentration) and at least one converting component 11 takes place in particular through specific association and dissociation processes or specific, often catalytically influenced, reactions. Such interactions according to the invention between at least one property of the reactor content 2 to be monitored and at least one converting component 11 locally influence, in particular in the immediate vicinity of the converting component 11, at least one property of the reactor content 2, which in turn influences at least one signal 8 of at least one sensory component 7 located in the interaction area of the converting component 11.

The in 2 The embodiment of the sensor 5 shown in FIG. 1 with a converting component 11 enveloping the sensory component 7 advantageously increases the influence of the converting component 11 on the sensory component 7. For the 2 In the embodiment shown and similar or derived embodiments of the invention, in particular but not exclusively, oxygen- or pH-dependent luminophores or luminophor systems are used as the sensory component 7 in combination with oxygen or proton-consuming or -generating enzymes and enzyme reactions as the converting component 11. Specifically binding antibodies or aptamers can also be used as the converting component 11 together with metallic or semiconducting particles or particle systems as the sensory component 7, in particular if the latter have a suitable surface plasmon resonance for optical detection or suitable magnetic, capacitive or resistive properties for, in particular frequency-dependent, impedance detection.

The in 2 The sensor matrix 12 shown not only serves to embed the converting component 11 and the sensory component 7, but also provides the sensor 5 according to the invention with mechanical stability. Furthermore, the sensor matrix 12 can also be designed as a partially or completely substance-selective structure in order to increase the specificity or sensitivity of the at least one converting component 11 or the at least one sensory component 7. Such designs of the sensor matrix 12 include in particular but not exclusively hydrogels and membranes with defined pore size and distribution or with defined polarity, hydrophobicity, oxygen or proton permeability.

2 illustrates the method according to the invention using the example of a T-flask as reactor 1, whose reactor content 2 is formed by a cell culture suspension as reaction mixture 3 and above it by a gas-filled head space 4. In the reaction mixture 3 there is a sensor 5 in the 2 embodiment shown above. Outside the reactor 1 there is a measuring arrangement 6 which comprises at least one signal exciter 16 and at least one signal detector 15. At least one combination of at least one signal exciter 16 and at least one signal detector 15 spans at least one detection area 9 of the measuring arrangement 6, which penetrates through the wall of the reactor 1 into the reactor contents 2, so that the signal excitation 17 of at least one sensor 5 according to the invention takes place there via the at least one sensory component 7 contained in it and its signal 8 or a marker signal 14 is transmitted through min at least one signal detector 15 of the measuring arrangement 6. The 2 The T-flask shown as reactor 1 is subjected to a mixing movement 25 according to the invention by shaking or weighing, which also causes a movement 10 of the sensor 5 via the resulting movement of the reaction mixture 3 and reactor 1, so that the sensor moves into and out of the detection area 9 of the measuring arrangement 6 according to the invention.

3 shows a schematic representation of a sensor 5 according to the invention for carrying out the method according to the invention using the example of a shake flask as a reactor 1 for cultivating cells with addition system 18 for sensors 5. The 3 The sensor 5 shown is designed in an arc shape in order to be able to carry out a uniform and collision-free movement 10 along the round geometry of the wall of the shake flask as reactor 1. In the front and rear areas of the sensor 5, markers 13A and 13B are embedded in the sensor matrix 12. Between the areas with markers 13A and 13B there are several areas with different sensory components 7A and 7B as well as converting component 11, whereby the components 7A and 7B or their combinations (7A surrounded by 11) are designed as particles that are embedded in the sensor matrix 12. In each area of the 3 There is a maximum of one type of particle in the sensor 5 shown. One area of the sensor 5 shown contains particles of the sensory component 7A, which generates, for example, an oxygen-dependent signal 8A. One area of the sensor 5 shown contains particles of the sensory component 7B, which generates, for example, a pH-dependent signal 8B. One area of the sensor 5 shown contains combined particles from the sensory component 7A, which are each coated with a converting component 11 and generate, for example, an oxygen-dependent signal 8A, wherein the local oxygen concentration in the area of influence of the coated sensory component 7A is influenced by an oxidoreductase (e.g. for glucose, lactate, glutamine) as the coating converting component 11. The individual areas of the sensor 5 are segmented so that the different signals 8 can be assigned locally during the recording or afterwards, thus making it possible to distinguish, for example, between the oxygen concentration and the glucose concentration in the reactor contents 2 determined using local oxygen consumption. As a result of the use of different markers 13A and 13B with different marker signals 14A and 14B in the front and rear areas of the sensor 5, the areas of the sensor 5 that contain sensory components 7 can be distinguished regardless of the orientation of the sensor 5.

The lower section of 3 shows a schematic representation of a shake flask as reactor 1 before the start of the mixing according to the invention as a result of a mixing movement 25. The reactor 1 comprises a reactor content 2, which is formed by a cell culture suspension as reaction mixture 3 and above it by a gas-filled head space 4. Above the reactor 1, an addition system 18 for sensors 5 is shown, which is divided into individual segments, each segment containing at least one sensor 5 and a storage medium 19. In some embodiments of the invention, the sensors 5 and the storage medium 19 are sterile. In the 3 In the embodiment of the invention shown, the addition 20 of at least one sensor 5 into the reactor 1 takes place as part of the method according to the invention. This also shows the advantageous simplification of the handling of sensors 5 in contrast to the prior art, since they do not have to be attached to the reactor wall. In particular in embodiments of the invention in which sterile sensors 5 are to be used under sterile conditions in the reactor 1, the inventive use of addition systems 18 for sensors 5 significantly simplifies the handling of sensors 5.

4 shows a schematic representation of the implementation of the method according to the invention on a shake flask as reactor 1 for the cultivation of cells using the 3 The sensor 5 according to the invention shown in FIG. 1 is a schematic view of a shake flask with a lid (dashed circle) as a reactor 1, the reactor contents 2 of which are formed by a cell culture suspension as a reaction mixture 3 and above it and also next to it during the shaking process by a gas-filled head space 4. In the reaction mixture 3 there is a sensor 5 in the 3 embodiment shown above, so that for the sake of clarity a numerical designation of the sensor components is omitted and 3 Outside the reactor 1 there is a measuring arrangement 6 which comprises at least one signal exciter 16 and at least one signal detector 15. In the embodiment shown, the measuring arrangement 6 comprises several signal exciters 16 and signal detectors 15 working in parallel, so that the various markers 13A and 13B as well as the sensory components 7A and 7B and the sensory component 7A coated with a converting component 11 can be detected by a single measuring arrangement 6. For the sake of clarity, 3 simplifying only one signal exciter 16 and a signal detector 15. At least one combination of at least one signal exciter 16 and at least one signal detector 15 spans at least one detection area 9 of the measuring arrangement 6, which penetrates through the wall of the reactor 1 into the reactor contents 2, so that the signal excitation 17 of at least one sensor 5 according to the invention takes place there via the at least one sensory component 7 contained in it and its signal 8 or a marker signal 14 can be detected by at least one signal detector 15 of the measuring arrangement 6.

The one in 4 The shake flask shown as reactor 1 is subjected to a mixing movement 25 according to the invention by orbital shaking, which also causes a movement 10 of the sensor 5 via the resulting periodic rotational movement of the reaction mixture 3, so that the sensor moves into and out of the detection area 9 of the measuring arrangement 6 according to the invention. In 4 , I the reaction mixture 3 is moving into the detection area 9 of the measuring arrangement 6. Since there is no sensor 5 in the detection area 9, neither a valid marker signal 14 nor a valid signal 8 of a sensory component 7 is detected by the measuring arrangement 6. If the reaction mixture 3 continues to move, the sensor 5 enters the detection area 9 of the measuring arrangement 6. First, as in 4 , II, the area of the sensor 5 with marker 13A in the detection area 9, so that the orientation of the sensor 5 can be determined based on the marker signal 14A detected by the measuring arrangement 6 with the aid of the signal excitation 17MA. In addition, the signal exciters 16 and signal detectors 15 for the following sensory components 7A and 7B are now activated, so that at the moment the respective sensory component 7 is present in the detection area 9 of the measuring arrangement 6, a valid signal 8 can be detected in each case in order to monitor the property of the reaction mixture 3 associated with the invention. Since the various particles with sensory components 7 in the sensor 5 shown are arranged in segments one behind the other and move one after the other through the detection area 9 of the measuring arrangement 6 as a result of the movement 10 of the sensor 5, the measuring arrangement 6 detects the signals 8 of the sensory components 7 one after the other, first with signal excitation 17A the signal 8A of the sensory component 7A ( 4 , II), then with signal excitation 17B the signal 8B of the sensory component 7B ( 4 , III), then with signal excitation 17A the signal 8A of the sensory component 7A coated with converting component 11 ( 4 , IV). Finally, the marker signal 14B of the marker 13B is recorded using the signal excitation 17MB ( 4 , IV), which also validates the positioning of the sensor 5 during its movement 10 through the detection area 9. The sensor 5 then moves out of the detection area 9 of the measuring arrangement 6 ( 4, V-VI ). As a result of the periodic mixing movement 25 of the shaking process shown here, the movement 10 of the sensor 5 is also subject to a certain periodicity. Based on the detection of the mixing movement 25 or the movement 10 of the sensor 5, the detection of the individual signals 8 and marker signals 14 can be synchronized in some embodiments of the invention. In addition, the use of several different markers 13 with correspondingly different marker signals 14 in different segments of a sensor 5 allows a clear assignment of different signals 8 to their respective properties of the reactor contents 2 or reaction mixture 3 to be monitored, even if the position and orientation of the sensor 5 in the detection area 9 changes as a result of the movement 10 of a sensor 5.

5 shows a schematic representation of two sensors 5 according to the invention and a device according to the invention for carrying out the method according to the invention on a stirred tank reactor as reactor 1. The sensors 5A and 5B shown contain sensory components 7 designed as particles, which are surrounded by a converting component 11 and embedded in the sensor matrix 12. Sensor 5A also contains markers 13A in two areas, the marker signal 14A of which differs from the marker signal 14B of the marker 13B contained in sensor 5B, so that the sensors 5A and 5B can be distinguished based on their marker signals 14A and 14B. Furthermore, the sensors 5A and 5B each comprise a fin 26 with a density that is higher than that of the reaction mixture 3 and a fin 27 with a density that is lower than that of the reaction mixture 3. According to the invention, these fins 26 and 27 are used in particular for the flow-dynamic stabilization and positioning of the sensors 5. Furthermore, by suitable dimensioning of the fins 26 and 27, the average density of a sensor 5 can be set and thus the probability of the sensor 5 being located in certain areas of the reactor content 2 or the reaction mixture 3 can be specifically influenced.

The one in 5 Sensor 5A shown at the top left is equipped with a large fin 27A of low density and a small fin 26A of high density, so that the average density of sensor 5A is lower than the density of the reaction mixture 3 of the 5 reactor 1 shown on the right. Therefore, even in turbulent mixing, sensor 5A is mainly located in the upper part of the reaction mixture 3 and can thus be used specifically for monitoring properties of this upper region of the reaction mixture 3. The 5 Sensor 5B shown at the bottom left is equipped with a small fin 27B of low density and a large fin 26B of high density, so that the average density of sensor 5B is higher than the density of the reaction mixture 3 of the 5 reactor 1 shown on the right. Therefore, even in turbulent mixing, sensor 5B is predominantly in the lower part of the reaction mixture 3 and can thus be used specifically to monitor properties of this lower region of the reaction mixture 3. The use of fins 26 and 27 of different densities, with the fin 26 of higher density preferably directed downwards and the fin 27 of lower density preferably directed upwards, stabilizes the position of the respective sensor 5 in the reaction mixture 3 and thus also in the detection range 9 of suitable measuring arrangements 6, whereby in some embodiments of the invention a better quality of signals 8 and marker signals 14 is achieved.

In 5 On the right is a schematic representation of an embodiment of the invention on a stirred tank reactor as reactor 1 with the 5 The sensors 5A and 5B shown on the left are shown. A stirred tank reactor is shown as reactor 1, the reactor content 2 of which is formed by a cell culture suspension as reaction mixture 3 and above it by a gas-filled head space 4. In the reaction mixture 3 there are two sensors 5A and 5B in the 5 embodiment shown on the left, with the sensors 5A being predominantly in the upper region and the sensors 5B predominantly in the lower region of the reaction mixture 3. Outside the reactor 1 there is a measuring arrangement 6 for both the upper and lower regions of the reaction mixture 3, each of which comprises at least one signal exciter 16 and at least one signal detector 15. In the embodiment shown, each measuring arrangement 6 comprises several signal exciters 16 and signal detectors 15 working in parallel, so that the various markers 13A and 13B as well as the sensory component 7A coated with a converting component 11 can be detected by a single measuring arrangement 6. For the sake of clarity, only one signal exciter 16 and signal detector 15 per measuring arrangement 6 is shown. At least one combination of at least one signal exciter 16 and at least one signal detector 15 spans at least one detection area 9 of the respective measuring arrangement 6, which penetrates through the wall of the reactor 1 into the reaction mixture 3, so that the signal excitation 17 of at least one sensor 5 according to the invention takes place there via the at least one sensory component 7 contained in it and its signal 8 or a marker signal 14 can be detected by at least one signal detector 15 of the measuring arrangement 6. The reactor 1 shown has an agitator 28 and is subjected by this to a mixing movement 25 according to the invention, which also causes a movement 10 of the sensors 5A and 5B via the resulting movement of the reaction mixture 3, so that they move into and out of the detection area 9 of the respective measuring arrangement 6 according to the invention. By assigning the measuring arrangements 6 to the upper or lower region of the reaction mixture 3, its properties can now be monitored in a spatially resolved manner using the sensors 5A and 5B via their signals 8A and 8B, whereby the use of different markers 13A and 13B via their different marker signals 14A and 14B allows a distinction to be made between the two sensors 5A and 5B if they should ever leave their target location in the reaction mixture 3.

6 shows a schematic representation of a sensor 5 according to the invention in a core-shell design with adapted geometry for use in orbitally shaken systems. The sensor matrix 12 forms the mechanically stable core of the sensor 5 and acts as a carrier of the marker 13 and the sensory component 7. 6 shows a longitudinal section of a sensor 5 according to the invention at the top and two cross sections below, on the left a cross section through the central area of the sensor 5 and on the right a cross section through one of the edge caps of the sensor 5. The sensor component 7 is set back inwards in the embodiment shown in such a way that even when the sensor 5 comes into contact with the reactor 1, it does not touch the latter and is thus largely protected from mechanical damage. In contrast, the two end caps coated with marker 13 stand out clearly and thus form the main contact points of the sensor 5 with the reactor 1. In order to prevent the sensor 5 from rolling over the bottom of the reactor 1, especially during shaking operation, and to move the sensor 5 through the reactor 1 in a controlled manner by the movement of the reaction mixture 3, the two end caps of the sensor 5 are angular, in the 6 In order to achieve an optimal and stable position and orientation of the sensor 5 in the moving reaction mixture 3 for detection by at least one suitable measuring arrangement 6, the sensor 5 is adapted to the flow conditions in the reaction mixture 3. In the embodiment shown in 6 In the embodiment shown, the sensor 5 is designed to be rotationally symmetrical and has a length-diameter ratio of more than one, preferably of at least two. Furthermore, the 6 illustrated embodiment of the sensor according to the invention 5 shows a smooth transition of the hexagonal edge structures of the two end caps carrying the marker 13 into a fluidically more advantageous edge-free geometry, here a spherical segment. The depth of and the transition to the recess of the sensory component 7 can also be advantageously selected such that in the application there is an optimal compromise between avoiding direct contact between the sensory component 7 or other mechanically sensitive components of the sensor 5 and the reactor 1 and an optimal flow of the sensor 5 for the application, in particular but not exclusively through the use of suitable geometries to avoid flow separation and vortices behind the flow-on end caps of the sensor 5. In an advantageous embodiment of the invention, the depth of the recess of the sensory component 7 is also selected to be just small enough that mechanical protection of sensitive components of the sensor 5 by avoiding contact with the reactor 1 and fluidic optimization are ensured, but at the same time as little reaction mixture 3 as possible lies between the sensory component 7 and the at least one suitable measuring arrangement 6 during the detection of at least one signal 8 of at least one sensory component 7. This is important for applications in which, in particular but not exclusively, the turbidity or absorption of the reaction mixture 3 changes over time, for example due to cell growth or the formation or dissolution of substrates, products, by-products, metabolites, foams, particles, emulsions or other phase mixtures. The 6 The sensor 5 according to the invention shown can be designed, for example, as a sensor for dissolved oxygen, wherein an oxygen-erasable luminophore is applied as a sensory component 7 as a thin layer to a core made of polyamide as a primary sensor matrix 12, wherein the end caps of this primary sensor matrix 12 also function as a carrier of another luminophore as a marker 13. The sensory component 7 and the marker 13 can also be embedded in one or more suitable sensor matrices 12 and applied by means of these to the core as a primary sensor matrix 12.

list of reference symbols

1

Reaktor

2

Reaktorinhalt

3

Reaktionsgemisch

4

Kopfraum

5

Sensor

6

Messanordnung

7

Sensorische Komponente mit mindestens einer durch die Messanordnung 6 erfassbaren Eigenschaft

8

Signal einer durch eine Messanordnung 6 erfassbaren sensorischen Komponente 7

9

Erfassungsbereich einer Messanordnung 6

10

Bewegung eines Sensors 5

11

Konvertierende Komponente, die mit mindestens einer sensorischen Komponente 7 oder mindestens einer konvertierenden Komponente interagiert

12

Sensormatrix

13

Marker

14

Markersignal

15

Signaldetektor

16

Signalanreger

17

Signalanregung

18

Zugabesystem

19

Lagermedium

20

Zugabe mindestens eines Sensors 5 in einen Reaktor 1

21

Zeit

22

Intensität von Signalen 8 beziehungsweise Markersignalen 14

23

Erfassungszeit

24

Verweilzeit

25

Mischbewegung

26

Flosse mit im Vergleich zum Reaktorinhalts 2 oder Reaktionsgemisch 3 höherer Dichte

27

Flosse mit im Vergleich zum Reaktorinhalts 2 oder Reaktionsgemisch 3 niedrigerer Dichte

28

Rührwerk

For the respective interpretation of the reference symbols, the description and claims must be observed. Reference symbols that are supplemented by letters designate separate or different elements of the same type.

1

reactor

2

reactor contents

3

reaction mixture

4

headspace

5

sensor

6

measuring arrangement

7

Sensory component with at least one property that can be detected by the measuring arrangement 6

8

Signal of a sensory component 7 detectable by a measuring arrangement 6

9

detection range of a measuring arrangement 6

10

movement of a sensor 5

11

Converting component that interacts with at least one sensory component 7 or at least one converting component

12

sensor matrix

13

marker

14

marker signal

15

signal detector

16

signal stimulator

17

signal excitation

18

addition system

19

storage medium

20

Addition of at least one sensor 5 into a reactor 1

21

Time

22

Intensity of signals 8 or marker signals 14

23

recording time

24

dwell time

25

mixing movement

26

Fin with higher density compared to reactor contents 2 or reaction mixture 3

27

Fin with lower density compared to reactor contents 2 or reaction mixture 3

28

agitator

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. 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

• DE 10101576 A1 [0012]
• EP 1397672 A1 [0013]
• EP 3821230 A1 [0014]
• DE 102018105174 A1 [0014]
• EP 2321052 A1 [0014]
• US 2008171383 A1 [0014]
• DE 102004017039 A1 [0020]

Cited non-patent literature

• Wang Xu-dong and Otto S. Wolfbeis (“Optical Methods for Sensing and Imaging Oxygen: Materials, Spectroscopies and Applications”. Chem. Soc. Rev. 43, No. 10 (2014): 3666-3761. https://doi .org/10.1039/C4CS00039K [0006]
• by Andreas Steinegger, Otto S. Wolfbeis, and Sergey M. Borisov (“Optical Sensing and Imaging of PH Values: Spectroscopies, Materials, and Applications.” Chemical Reviews 120, No. 22 (November 25, 2020): 12357-489. https://doi.org/10.1021/acs.chemrev.0c00451 [0007]
• by David Flitsch, Tobias Ladner, Mihaly Lukacs, and Jochen Büchs (“Easy to Use and Reliable Technique for Online Dissolved Oxygen Tension Measurement in Shake Flasks Using Infrared Fluorescent Oxygen-Sensitive Nanoparticles.” Microbial Cell Factories 15, No. 1 (December 2016 ): 45. https://doi.org/10.1186/s12934-016-0444-4 [0010]

Claims (10)

Method for monitoring the contents of mixed reactors 1, wherein the reactor contents 2 have at least one property to be monitored, wherein the at least one property to be monitored directly or indirectly influences at least one signal 8 of at least one sensory component 7, wherein the at least one signal 8 of the at least one sensory component 7 can be detected by at least one measuring arrangement 6, wherein the at least one sensory component 7 is a component of at least one sensor 5, characterized in that the at least one sensor 5 is not stationary and performs a movement 10 in the reactor 1, and that at least one detection of the at least one signal 8 of the at least one sensory component 7 takes place by at least one measuring arrangement 6 while the at least one sensor 5 is located in the detection range 9 of the at least one measuring arrangement 6, and that the at least one sensor 5 is not permanently located in the detection range 9 of the at least one measuring arrangement 6. Method according to the preceding claim, characterized in that the detection time 23 of the at least one signal 8 is shorter than the residence time 24 of the at least one sensor 5 in the detection area 9 of the at least one measuring arrangement 6. Method according to one of the preceding claims, characterized in that the monitoring is carried out using at least one converting component 11 contained in the sensor 5, wherein the at least one converting component 11 directly or indirectly influences at least one sensory component 7 contained in the sensor 5 and its signal 8 which can be detected by at least one measuring arrangement 6. Method according to one of the preceding claims, characterized in that several different sensors 5 are used to monitor several different properties of the reactor contents 2. Method according to one of the preceding claims, characterized in that at least one marker signal 14 of at least one marker 13 contained in the sensor 5 is detected by at least one measuring arrangement 6. Method according to one of the preceding claims, characterized in that a measuring arrangement 6 comprising several signal detectors 15 and/or several signal exciters 16 detects several signals 8 and/or several marker signals 14. Method according to one of the preceding claims, characterized in that the addition 20 of at least one sensor 5 into the reactor 1 takes place before the start of the process to be carried out in the reactor 1. Device for carrying out the method according to one of the preceding claims, characterized in that the device comprises a reactor 1 with reactor contents 2, and the device comprises at least one non-stationary sensor 5, and the device comprises at least one measuring arrangement 6 for detecting at least one signal 8 of at least one sensory component 7 contained in the sensor 5, and the volume of at least one detection area 9 of at least one measuring arrangement 6 overlapping with the reactor contents 2 is smaller than the volume of the reactor contents 2. Device according to the preceding claim, characterized in that the volume of at least one detection area 9 of at least one measuring arrangement 6 overlapping with the reactor content 2 is smaller than at least one of the sensors 5 that can be detected by the measuring arrangement 6. Device according to one of the Claims 8 until 9 , characterized in that the device comprises several non-stationary sensors 5.