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EP-4095626-B1 - METHOD FOR INDUSTRIAL BIOREACTOR OPTIMIZED WITH MUTUALLY DEPENDENT, COUPLED PROCESS CONTROL LOOPS

EP4095626B1EP 4095626 B1EP4095626 B1EP 4095626B1EP-4095626-B1

Inventors

  • BUCHMANN, Leandro

Dates

Publication Date
20260506
Application Date
20210615

Claims (15)

  1. Method for an industrial bioreactor (1) with a dual cycle-controlled process providing a cultivation process (21) for cell cultures, cell components or metabolic products of the cells in a nutrient medium (11), the bioreactor comprising a reactor vessel (12) providing controlled bioreactor conditions for the cultivation process (21), and a control unit connected to sensory devices (14) measuring sensory parameter (141) values comprising at least measures related to the composition (1411) of the nutrient medium (11) and/or concentration (1412) of the nutrient medium (11) and/or oxygen (1413) and/or temperature (1414) and/or pH-value (1415) and/or sterility (1416), and transmitting them to the control unit, wherein the control unit controls and/or steers the cultivation process (21) by adjusting operational parameters of the bioreactor (1) affecting the measured sensory parameter (141) values, the dual cycle-controlled process characterized by the steps of adjusting, by a cultivation optimization cycle (2) optimizing cultivation performance (241) of the cultivation process (21), operational first parameters of the bioreactor (1) by identifying a biologically optimized window for cultivation and/or treatment, and optimizing, by a treatment optimization cycle (3), a treatment process (32) applied to the cell culture during the cultivation process (21) within the optimized treatment window by applying operational second parameters of the bioreactor (1), measuring (22) first sensory parameters by first sensory devices of the sensory devices (14), in the cultivation optimization cycle (2) (from before), by capturing the cultivation performance of the cultivation process (21), wherein the first sensory parameters are transmitted to a fist analyzer (23) reconciling between a target cultivation performance and the measured cultivation performance and if the target cultivation performance is not met, the operational first parameters are adjusted and the measuring (22) and reconciliation (24) is reiterated, measuring bioimpedance by a measuring device of the first sensory devices measuring the cultivation performance of the cultivation process (21) by means of the first sensory parameters by detecting their response to electric excitation, where by means of electrodes a current- or potential-based excitation signal is applied to the cell culture and the response is measured converting the charge to ionic charge and vice versa providing detection of at least cell number and/or cell size and/or cell viability of the cells, and triggering, if the target cultivation performance is met, the treatment optimization cycle (3), the treatment optimization cycle (3) comprising measuring (32) second sensory parameters by second sensory devices of the sensory devices (14) capturing the treatment performance of the treatment process (31) by measuring the treatment induced deviation in the cultivation performance, wherein the second sensory parameters are transmitted to a second analyzer (33) reconciling between a target treatment performance and the measured treatment performance and if the target treatment performance is not met, the operational second parameters are adjusted and the measuring (32) and reconciliation (34) is reiterated, otherwise the dual cycle-controlled optimization process is completed.
  2. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to claim 1, characterized in that a phase angle of the cell culture is measured by the bioimpedance measurement (22), the phase angle being correlated with a cell viability, wherein with increasing measured phase angle the treatment (31) is applied while with decreasing measured phase angle, the treatment (31) is stopped.
  3. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 1 or 2, characterized in that the treatment process (31) comprises applying nanosecond pulsed electric fields (nsPEF) to the cell culture using at least two applied electrodes, the electric fields being applied by coupling one electrode to higher voltage and one electrode to ground or lower voltage, and the pulsed electric fields having a definable shape and/or frequency and/or strength.
  4. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to claim 3, characterized in that the control unit comprises predefined basic nanosecond pulsed electric fields settings for each possible cell type.
  5. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 1 to 4, characterized in that the treatment performance is measured by means of dielectric spectroscopy system measuring dielectric properties of the cells as a function of frequency, wherein the frequency-dependent permittivity in a target range is measured, and wherein the measured amplitude or signal intensity serving as a measured target parameter value for the performance of the treatment.
  6. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to claim 5, characterized in that the target range having 0.1-30 MHz.
  7. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 3 to 6, characterized in that the treatment performance is measured by means of a flow cytometer as at least one of the second sensory devices measuring metabolic activity based on a fluorescence assay, a conversion of a fluorescent dye and a signal intensity serving as a measured target parameter value for the performance of the treatment.
  8. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to claim 7, characterized in that the fluorescence assay is fluorescein diacetate (FDA).
  9. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 7 or 8, characterized in that the flow cytometer comprises at least a measuring system and a detector and an amplification unit, the flow cytometer being connected to and transferring measuring signals to the control unit for analysis of the transmitted signals.
  10. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to claim 9, characterized in that the measuring system measures impedance or conductivity using optical systems emitting light signals.
  11. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 9 or 10, characterized in that the detector comprises an analog-to-digital conversion (ADC) system converts analog measurements of forward-scattered light (FSC), side-scattered light (SSC), and dye-specific fluorescence signals into digital signals being processable by the control unit.
  12. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 9 to 11, characterized in that the amplification unit is linear or logarithmic realized.
  13. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 1 to 12, characterized in that the treatment performance is optimized if an acceleration of the cultivation process (21) and/or a targeted biological growth is measured in the bioreactor (1) based on the measured second sensory parameter values.
  14. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 1 to 13, characterized in that the bioreactor (1) is realized as a fermenter and/or a germination box in case of seed germination applications.
  15. The method for an industrial bioreactor (1) with a dual cycle-controlled process according to one of the claims 1 to 14, characterized in that the control unit comprises a machine-learning or artificial-intelligence based unit capturing the measured treatment performance measured by means of dielectric spectroscopy system and/or the measured treatment performance measured by means of the flow cytometer, wherein the operational first parameters and/or operational second parameters are automatically adapted by the control unit (13), wherein at least the first and secondary sensory parameter are applied as input values to the machine-learning or artificial-intelligence based unit, and wherein the output of the machine-learning or artificial-intelligence based unit triggers the adjustment of the operational first parameters and/or operational second parameters until the target cultivation performance with the applied treatment process (32) is reached.

Description

Field of the invention The present invention relates generally to the industrial production of biomass, and in particular to industrial processes for generating biomass by providing a controlled cultivation process for cell cultures, components of cells or metabolic products of the cell cultures using bioreactors. Even more particular, the present invention relates to industrial processes for generating biomass by cell cultivation and treatment within bioreactors e.g. by applying a pulse-regulated electric field potential. Background of the invention (i) Bioreactors Bioreactors (in the context of microbial or enzymatic conversion of organic substances into acid, gases or alcohol also referred to as fermenters), typically are containers in which certain microorganisms, cells or small plants are cultivated (fermented) under applied, technically controlled conditions. The operation of a bioreactor is thus an application of the field of biotechnology that uses or harnesses biological processes (bioconversion, biocatalysis) in technical facilities. Important factors, characteristics and/or operating parameters that are controllable or steerable in most bioreactors, are, for example, the composition of the nutrient medium (also nutrient solution or substrate), oxygen supply, temperature, pH value, sterility and others. The purpose of cultivation in a bioreactor can be the controlled recovery of the cells or components of the cells or the recovery of metabolic products or the controlled metabolism of cell cultures as such. The cells, components of cells or the metabolic products can be used, for example, as active ingredients in the pharmaceutical industry, as basic chemicals in the chemical industry, or in the context of cell cultivation, for example, in the cultured food production, as e.g. cultured meet production or fermented food, e.g. tempeh, miso, koji, and soy sauce etc., or in the medical applications in organ or tissue replacement treatments etc.. The degradation of chemical compounds can also take place in bioreactors, as in the treatment of wastewater in sewage treatment plants. The production of beer, wine and other products that have been produced historically also takes place in bioreactors (fermenters). Today, a wide variety of organisms are cultivated in bioreactors for different purposes. Several reactor variants are available in different designs. Examples are stirred tank reactors made of metal, which can have a volume of a few to thousands of liters and are filled with nutrient solution. However, widely differing variants, such as fixed-bed reactors, photobioreactors etc. are also used. The main purpose of bioreactors are to deliver the highest possible product yields. This is achieved in particular by creating optimal conditions for the organism or cells used in the specific case. The conditions in the bioreactor are adapted to various parameters that prevail in the natural habitat or the cultivated organisms or the natural environment of the cultivated cells. Typically, cultivation process parameters as the type and concentration of nutrients, temperature, oxygen content, pH value, etc. are important or critical to achieve an optimized cultivation. The control of the parameters is realized using technical devices as e.g. agitators or other devices necessary to ensure a homogeneous adjustment of these parameters over the reactor chamber. In addition to the requirements of the organisms or cells, other technical, organizational, and economic factors often are to be considered that influence the choice of operating parameters. Examples can be, inter alia, the prevention of foam formation and the choice of either a continuous or a batch mode of operation. Using probes or sensors, many of these parameters can e.g. be measured directly in the nutrient medium or in the exhaust air. In addition, the course of the process can usually also be assessed or monitored via these parameters. The cell density can be determined by measuring the absorbance (optical density), which in turn allows conclusions to be drawn about the product quantity. An alternative is often the measurement of the concentration of a characteristic chemical compound, e.g. the increase in concentration of a metabolic product or the decrease in substrate concentration, for example, by optical sensors or other appropriate sensors. At the beginning of a cultivation or fermentation process, typically a small amount of the microorganisms or cells to be cultivated and obtained from a pre-cultivation are added to the culture medium. This amount is called inoculum, and the process is often referred to as inoculation. The suspension obtained from the cultivation process is prepared in several process steps during so-called downstream processing. The nutrient medium must provide the organisms/cells with all the nutrients they need for growth. These include e.g. main nutrients (macronutrients) required in larger quantities, such as carbon, nitr