Method and device for tuning optical measurements on continuously mixed reactors

Abstract

A method for tuning optical measurements on continuously mixed reactors, wherein a content of the reactor has at least one optically detectable measured variable, which is carried out by at least one optical measuring arrangement, wherein at least one optical measurement is tuned to a form, distribution, or movement state of at least one phase of the reactor content, wherein mixing the reactor content causes local changes in the permittivity within the reactor, which is detected at at least one location having a known distance from the at least one optical measuring arrangement to be tuned based on a permittivity signal, and wherein the detected permittivity signal of at least one location having a known distance from the at least one optical measuring arrangement to be tuned is used to tune at least one optical measurement to the form, distribution, or movement state of the reactor content.

Claims

1. A method for tuning optical measurements on continuously mixed reactors, wherein a reactor content of the reactor has at least one optically detectable measured variable, wherein at least one optical measuring arrangement carries out optical measurements of at least one of the optically detectable measured variables, wherein the at least one optical measurement is to be tuned to a form, distribution, or movement state of at least one phase of the reactor content, wherein mixing the reactor content causes local changes in the permittivity within the reactor, characterized in that, a local permittivity is detected at at least one location having a known distance from the at least one optical measuring arrangement to be tuned based on a permittivity signal, and a detected permittivity signal of at least one location having a known distance from the at least one optical measuring arrangement to be tuned is used to tune the at least one optical measurement to the form, distribution, or movement state of the reactor content.

2. The method according to claim 1, characterized in that the change in at least one local permittivity or some other value derived therefrom is utilized to tune the at least one optical measurement to the form, distribution, or movement state of the at least one phase of the reactor content.

3. The method according to claim 1, characterized in that the tuning of the at least one optical measuring arrangement takes place as activation and deactivation of the optical measuring arrangement as a function of the form, distribution, or movement state of at least one phase of the reactor content in a visual field of the optical measuring arrangement.

4. The method according to claim 1, characterized in that the tuning of the at least one optical measuring arrangement takes place as correction or normalization of the optical measuring signal of the optical measuring arrangement as a function of the form, distribution, or movement state of at least one phase of the reactor content in a visual field of the optical measuring arrangement.

5. The method according to claim 1, characterized in that the detection of the form, distribution, or movement state of the reactor content takes place via multiple permittivity sensors.

6. The method according to claim 1, characterized in that the detection of the at least one local permittivity takes place via at least one electrical field that locally interacts with the reactor content and at least one permittivity sensor.

7. The method according to claim 1, characterized in that the detection of the at least one local permittivity takes place via a capacitive measuring method based on a capacitive permittivity sensor.

8. A device for carrying out the method according to claim 1, characterized in that the device includes at least one permittivity sensor at at least one location having a known distance from the at least one optical measuring arrangement to be tuned, the permittivity sensor being configured for detecting changes in the local permittivity, and the device includes at least one control unit that is configured for carrying out the tuning of the optical measurement to the form, distribution, or movement state of the reactor content or at least one of its phases, based on the at least one permittivity signal of the at least one permittivity sensor.

9. The device according to claim 8, characterized in that the device includes at least one capacitive permittivity sensor that is configured for detecting the at least one local permittivity.

10. The device comprising a control unit configured for controlling a method according to claim 1.

11. The device according to claim 10, characterized in that the device includes at least one capacitive permittivity sensor that is configured for detecting the at least one local permittivity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic block diagram of the method according to the invention, using a device according to the invention.

(2) FIG. 2 shows a schematic illustration of a device according to the invention for carrying out the method according to the invention in a shake flask as a reactor for culturing cells.

(3) FIG. 3 shows a schematic illustration of a device according to the invention for carrying out the method according to the invention with multiple permittivity sensors.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A. Definitions

(4) To ensure clarity of several terms used in the description, these terms are defined and explained below and over the course of the description.

(5) A reactor is in particular a container that may be used in particular for culturing organisms or for carrying out chemical and biochemical reaction processes. Further fields of application of reactors include, among others, biocatalytic processes using organisms and/or biomolecules, and other chemical and/or physical processes, wherein the term process encompasses all types of conversion, separation, combination, mixing, changing the size of in particular chemical substances, organisms, particles, solutions, emulsions, and foams. Within the meaning of the invention, reactors include in particular stirred tank fermenters, bubble column fermenters, shake flasks, T flasks, microtiter plates, deep well plates, shake vessels, fermentation bags, multipurpose vials, and cell culture dishes. Reactors may be closed or open to their surroundings.

(6) The reactor content encompasses all matter that is present within the outer shell of the reactor. The reactor content encompasses one or more phases and/or is composed of one or more phases.

(7) Mixing a reactor or the reactor content refers in particular to any method for influencing the reactor content in such a way that at least two states, recorded at different times, of the distribution of the reactor content and its components in the reactor are not the same. Common mixing methods make use in particular of mechanical or thermal/thermodynamic processes, in particular but not limited to shaking processes, stirring processes, and diffusion processes. For periodically repeated mixing methods (orbital shaking, rocker shaking, stirring), the mixing frequency is the number of mixing periods per unit time.

(8) The coexistence of incompletely miscible material systems in a reactor content results in the formation of phases. A phase is a material system that is characterized by a dominant state of aggregation and that is not completely miscible with other phases. Typical examples of such are gas phases, liquid phases, and solid phases. Within the meaning of the invention, however, the term phase is used not only for pure states of aggregation, but also for material systems characterized by one or more at least substantially homogeneously miscible, suspendable, or emulsifiable material systems or substances. Examples of pure phases are liquid water or gaseous nitrogen. Examples of phases in the expanded scope of definition of the invention according to the above-described criteria are air (gaseous homogeneous mixture), culture medium with cells (virtually homogeneous suspension of cells in a homogeneous aqueous solution of media components such as salts, proteins, carbohydrates, etc.), or a stainless steel mixing turbine (virtually homogeneous mixing of iron and alloying elements). Homogeneity is scale-dependent on the resolution of the permittivity sensors used, so that a homogeneous phase is considered to be present when the permittivity sensor used can no longer detect local changes in permittivity as inhomogeneous. A phase-specific permittivity may be associated with each phase on account of its homogeneity, resulting in a volumetric and molar weighted average value over the relative permittivities of all components of the phase. Named as an example here is the culture medium with cells phase, whose average permittivity, as a function of the frequency, results in a weighted average value of the relative permittivities of the main component water in addition to all other components (ions, cells, dissolved molecules, dissolved gases, etc.). Another example of a phase is air, whose average permittivity, as a function of the frequency, results in a weighted average value of the relative permittivities of its components, in particular nitrogen, oxygen, water vapor, carbon dioxide, noble gases, etc.

(9) The combination of phase-specific permittivities with the mixing-related change in the form, distribution, or movement state of at least one phase of the reactor content results in local permittivities and differences in permittivity. Thus, the local permittivity at a location in the reactor where water is present differs significantly from the local permittivity at a location where air is present. Consequently, the detection of local permittivities and local changes in permittivity allows an assessment and/or quantitative recording of the form, distribution, or movement state of the reactor content or at least one phase of the reactor content.

(10) Permittivity sensors detect at least one local permittivity. This takes place in particular via at least one electrical field that interacts with at least one permittivity sensor and also with the reactor content at the location to be examined. The volume of the examined location depends in particular, but not exclusively, on the shape, polarity, and frequency of the electrical field, and on the composition and phase distribution of the reactor content in the interaction region of the electrical field. Permittivity sensors may be implemented in particular as capacitive sensors having one or more electrodes. The electrical field customarily required for the detection of permittivities may be generated either via the sensor electrode itself, or by additional electrodes that interact with the sensor electrode. The positioning of permittivity sensors at a known distance from at least one optical measuring arrangement to be tuned allows detection of the local permittivity with spatial reference to the optical measuring position, so that tuning of the optical measurement to the form, distribution, or movement state of the reactor content or of at least one phase of the reactor content relative to the optical measuring arrangement may take place as a result.

(11) The tuning of an optical measurement to the form, distribution, or movement state of the reactor content encompasses all methods which provide the planning, implementation, processing, evaluation, or display of the optical measurement in conjunction with the form, distribution, or movement state of the reactor content. Within the meaning of the invention, tuning of optical measurements is in particular, but not exclusively, the synchronization of the optical measurement to at least one state of the form, distribution, or movement state of the reactor content, and the incorporation of information concerning the form, distribution, or movement state of the reactor content into the evaluation of the optical measuring data (by correction or normalization, for example). Criteria or limit values concerning detected permittivity signals or values derived therefrom may be used for the purpose of tuning.

(12) Within the meaning of the invention, methods for tuning an optical measurement to the form, distribution, or movement state of the reactor content are carried out or implemented by at least one control unit. In this regard, control units are all devices that associate permittivity signals or values and data derived therefrom concerning the form, distribution, or movement state of the reactor content with the planning, implementation, processing, evaluation, or display of the optical measurement to be tuned. However, within the meaning of the invention, depending on the tuning method, control units in particular are not exclusively operational amplifiers, comparators, PID controllers, Schmitt triggers, and computers. A computer is considered to be any electronic device that can store data (in particular arithmetic and logic data) and process it based on programmable rules. Within the meaning of the invention, computers are considered as, but not limited to, microcontrollers, microprocessors, system on a chip (SoC) computers, PCs, and servers.

B. Description with Reference to the Drawings

(13) FIG. 1 shows a schematic block diagram of the method according to the invention, using a device according to the invention. The device includes a reactor 1 having a reactor content 2. In the schematic illustration in FIG. 1, the reactor content 2 includes two incompletely miscible phases 2A and 2B having different phase-specific permittivities; in particular the reactor content is composed of the two phases. FIG. 1 shows four snapshots from a continuous mixing movement of the reactor 1, which in its course causes a change in the form, distribution, or movement state of the reactor content 2. Situated outside the reactor 1 is at least one optical measuring arrangement 3 whose measurements are to be tuned to the form, distribution, or movement state of the reactor content 2. In one advantageous embodiment of the invention, at least one optical measuring arrangement 3 includes at least one light source 3Q for irradiating light into the reactor content 2, and/or at least one light sensor 3S for detecting light that is emitted from the reactor content 2. The detection of the local permittivity takes place in a specified area of the reactor content 2 via at least one permittivity sensor 4 that interacts with at least one electrical field 5, resulting in at least one permittivity signal 6 that is relayed to a control unit 7. The control unit in turn takes over the tuning 8 of at least one optical measuring arrangement 3. In some embodiments of the invention, the tuning 8 may be an activation signal for at least one optical measuring arrangement 3, as also shown in FIG. 1.

(14) The method according to the invention is apparent in FIG. 1. As the result of mixing, the local permittivity in the reactor 1 changes, so that, by means of the electrical field 5 that interacts with the reactor content 2 and the permittivity sensor 4, and the accompanying detection of the local permittivity in the interaction region of the permittivity sensor 4, information concerning the form, distribution, or movement state of the reactor content 2 is transmitted in the form of a permittivity signal 6 to the control unit 7, which carries out tuning of the optical measurements of at least one optical measuring arrangement 3.

(15) For the embodiment of the invention illustrated in FIG. 1, the tuning 8 takes place in particular as activation of the optical measuring arrangement 3 when the reactor content 2 having dominant phase 2B is situated in front of the optical measuring arrangement 3. The change in the local permittivity which accompanies this movement state and distribution state results in a strong permittivity signal 6, as the result of which the control unit 7 activates the optical measuring arrangement 3 (tuning 8, partial illustration in the lower left corner). In the illustrated embodiment, the tuning 8 takes place by activation of at least one light source 3Q of the optical measuring arrangement 3 to be tuned, so that light 9 is now irradiated from the light source 3Q into the reactor content 2, in particular into phase 2B, where it interacts with components of the reactor content 2 and is subsequently detected as incident light 10 on the light sensor 3S. The optical measurement of the optical measuring arrangement 3 is thus tuned to the form, distribution, or movement state of the reactor content 2, and in the illustrated embodiment, in particular by synchronizing the optical measurement to the presence of phase 2B in front of the optical measuring arrangement 3.

(16) FIG. 2 shows a schematic illustration of a device according to the invention for carrying out the method according to the invention in a shake flask as a reactor 1 for culturing cells. The top left portion of FIG. 2 shows a side view of the measuring arrangement according to the invention. Five exemplary states of the embodiment according to the invention during the course of the shaking movement 11 as an orbital shaking movement are illustrated in the lower portion of FIG. 2. A schematic view of the embodiment from the top is shown. The upper right portion of FIG. 2 shows a schematic diagram that illustrates the particular associated permittivity signal 6.1/6.2 for each of the five movement states discussed below.

(17) A shake flask is used as the reactor 1. The reactor content 2 includes two phases 2A and 2B, 2A being formed by air as a gaseous phase, and 2B, as an overall liquid phase, being composed predominantly of water and various culture media components and cells (culture broth as a suspension of cells in culture medium). For mixing of the reactor content 2, the flask as the reactor 1 carries out a shaking movement 11 that is responsible for a continuous change in the form, distribution, and movement state of the reactor content 2.

(18) Situated beneath the reactor 1 is an optical measuring arrangement 3 whose measurements are to be tuned to the form, distribution, or movement state of the reactor content 2. Within the meaning of the invention and in the field of application of the embodiment of the invention illustrated in FIG. 2, optical measuring arrangements are in particular, but not exclusively, measuring arrangements for detecting the turbidity, cell density, or biomass concentration of phase 2B via scattered light or absorption measurements, as well as measuring arrangements for exciting and/or detecting fluorescence or bioluminescence of certain components of phase 2B or certain fluorescence/luminescence sensors that are at least temporarily in contact with phase 2B (GFP, NADH, proteins, luciferin, and fluorescent pH, oxygen, CO.sub.2, glucose, lactate, or metal ion sensors, for example).

(19) The optical measuring arrangement 3 is flanked, tangentially with respect to the shaking movement, by two permittivity sensors 4.1/4.2 which locally interact with the reactor content 2 and its components via a respective associated electrical field 5.1/5.2. In one advantageous embodiment of the invention, as illustrated in FIG. 2, at least two permittivity sensors 4.1/4.2 are situated along the movement path of the mixed reactor content 2. The permittivity sensors 4.1/4.2 detect local changes in permittivity in their vicinity which are caused by the movement of the reactor content 2 and in particular its phases 2A and 2B, and which thus allow an assessment of the local form, distribution, or movement state of the reactor content 2. The resulting permittivity signals 6.1 (solid diamond) and 6.2 (circular ring) are relayed to a control unit 7 which carries out the tuning 8 of the measurements of the optical measuring arrangement 3.

(20) The method according to the invention is implemented in the embodiment in FIG. 2 as follows. Within a shaking period, the permittivity sensors 4.1/4.2 detect the local permittivity of the reactor content 2 multiple times (five times here, as an example). Since the aqueous phase 2B has a much higher relative permittivity than does the air of the gaseous phase 2A, the movement of the liquid for each permittivity sensor 4.1/4.2 results in a characteristic permittivity signal 6.1/6.2 as a function of the particular movement state of the reactor content 2. The greater the local portion of phase 2B in the interaction region of the particular electrical field 5.1/5.2, the greater the permittivity signal 6.1/6.2 of the particular permittivity sensor 4.1/4.2. Based on the known distances between the optical measuring arrangement 3 and the permittivity sensors 4.1/4.2, the control unit 7 can detect the form, distribution, or movement state of the reactor content 2 in spatial reference to the optical measuring arrangement 3, in particular when a certain quantity of aqueous phase 2B is situated above the optical measuring arrangement 3.

(21) The resulting movement-dependent local permittivity signals 6.1/6.2 are schematically illustrated in the upper right portion of FIG. 2. When the tuning 8 of the optical measuring arrangement 3 takes place as activation or deactivation, in one advantageous embodiment of the invention the introduction of at least one boundary condition 12 may be beneficially used, wherein the activation signal as the form of tuning 8 is output by the control unit 7 only when the local permittivity signals 6.1/6.2 meet the boundary condition 12. The boundary condition 12 illustrated in FIG. 2 is formed by a line above which the local permittivity signals 6.1/6.2 must lie in order for activation 8 of the optical measuring arrangement 3 by the control unit 7 to take place. In the illustrated embodiment of the invention, this boundary condition is met only by state 5, so that activation 8 of the optical measuring arrangement 3 takes place only when it is completely covered with phase 2B (culture medium with cells) at a certain minimum height. Tuning 8 of the measurements of the optical measuring arrangement 3 to the form, distribution, and movement state of the reactor content 2 and in particular the aqueous phase 2B is thus achieved by detecting two local permittivities at a known distance from the optical measuring arrangement 3.

(22) Such tuning of optical measurements may be beneficially used, in particular but not exclusively, in continuously agitated reactor systems (shake flasks as well as microtiter plates, shake vessels, T flasks, and shake bags) in order to tune optical measurements directly to the maximum liquid level via at least one optical measuring arrangement, and thus eliminate or reduce external interfering factors such as foam, inhomogeneous phase distributions, or undesirable reflections and scatterings on the reactor wall. The direct detection of the form, distribution, or movement state of the reactor content via the detection of local permittivities or their change is superior to the methods known from the prior art with regard to accuracy, temporal and spatial resolution, calculation effort, and susceptibility to process-related changes in the fluid characteristic of the reactor content 2.

(23) FIG. 3 shows a schematic illustration of a device according to the invention for carrying out the method according to the invention with multiple permittivity sensors. Two identical devices according to the invention, having two shake flasks as the reactor 1, are illustrated, with different positioning above the optical measuring arrangement 3 and the permittivity sensors 4.1/4.2/4.3.

(24) With regard to the composition of the reactor content 2, the mixing movement, the basic interaction of the permittivity sensors 4.1/4.2/4.3 with the reactor content 2, and the characteristics of the optical measuring arrangement 3, the embodiment of the invention illustrated in FIG. 3 corresponds to the descriptions for FIG. 2. However, in a departure therefrom, three permittivity sensors 4.1/4.2/4.3 are situated at known distances from the optical measuring arrangement 3, the permittivity sensors 4.1/4.3 flanking the optical measuring arrangement radially with respect to the shaking movement, and the permittivity sensor 4.2 being situated between the light source 3Q and the light sensor 3S. Due to the radial arrangement, the permittivity sensors 4.1/4.2/4.3 detect a radial volume range of the reactor content 2, so that the respective permittivity signals 6.1/6.2/6.3 represent a height profile or volume profile of a radial section through the reactor content 2. This is illustrated in the lower left diagram in FIG. 3 for the movement and distribution states of the reactor content 2 shown at the top in FIG. 3; the permittivity signals 6.1/6.2/6.3 for flask 1 are illustrated as solid diamonds, and for flask 2 are illustrated as a circular ring.

(25) The movement state illustrated in FIG. 3 corresponds to state 5 from FIG. 2, in which an activation 8 of the optical measuring arrangement 3 takes place. It is apparent from FIG. 3 that, due to the differences in relative positioning between the optical measuring arrangement 3 and the reactor 1 and reactor content 2, the volume of phase 2B, which is relevant for the optical measurement, changes in the visual field of the optical measuring arrangement, which, despite identical conditions in phase 2 (identical cell density, for example), results in differences in the signal of the optical measuring arrangement 3 (see the bottom center diagram in FIG. 3: solid triangle for flask 1, blank triangle for flask 2).

(26) The tuning 8 according to the invention of optical measurements on continuously mixed reactors 1 therefore also includes the correction of optical signals by means of the permittivity signals 6.1/6.2/6.3 obtained by the local permittivity measurements, and the information derived therefrom concerning the form, distribution, or movement state of the reactor content 2. As illustrated in the lower left diagram in FIG. 3, different local permittivity profiles result, depending on the positioning or filling volume of the reactor 1. According to the invention, these may be utilized, in particular but not exclusively, to derive volume profiles, and to use these to calculate the volume, situated in the visual field of the optical measuring arrangement, of the components of the reactor content 2 (shown as phase 2B in FIG. 3) that are relevant for the optical measurement. On the basis of such calculations, corrective tuning and in particular normalization of the optical measurement to the measurement-relevant volume in the visual field of the optical measuring arrangement 3 are then carried out by the control unit 7. The result is a tuned measuring signal of the particular optical measuring arrangement 3 which is independent of the form, distribution, or movement state, as illustrated in the lower right portion of FIG. 3 (solid triangle for flask 1, blank triangle for flask 2, both overlapping due to tuning with the same Y axis value).

(27) Such normalizing tunings of optical measurements on continuously mixed reactors 1 find application according to the invention in particular for tuning to positioning differences between at least two reactors 1 to be compared, for tuning to different filling volumes or phase portions of the reactor contents 2 of at least two reactors 1 to be compared, and for tuning to different mixing conditions, and thus, differences in the form, distribution, or movement state of the reactor contents 2 of at least two reactors 1 to be compared.

LIST OF REFERENCE NUMERALS

(28) Reference is made to the description and claims for interpretation of the reference numerals.

(29) TABLE-US-00001 1 reactor 2, 2A, 2B reactor content and phases 2A, 2B with different phase- specific permittivities 3, 3Q, 3S optical measuring arrangement with light source 3Q and light sensor 3S 4, 4.1, Permittivity sensor, multiple permittivity sensors 4.1, 4.2, 4.3 4.2, 4.3 5, 5.1, electrical field in interaction with permittivity sensor 4 and 5.2, 5.3 reactor content 2, multiple electrical fields 5.1, 5.2, 5.3 in interaction in each case with reactor content 2 and with permittivity sensor 4.1, 4.2, 4.3 6, 6.1, permittivity signal, signals 6.1, 6.2, 6.3 of the respective 6.2, 6.3 permittivity sensors 4.1, 4.2, 4.3 7 control unit 8 tuning of at least one optical measuring arrangement 3 9 emitted light from light source 3Q 10 incident light on light sensor 3S 11 movement for mixing the reactor content 2 12 boundary condition for activating (as tuning 8) the optical measuring arrangement 3