WO2013037686A1 - Method for monitoring a fermentation process - Google Patents

Abstract

The invention relates to a method for monitoring a fermentation process in which one or more monitored parameters that represent the state of the fermentation process are measured. The method according to the invention is characterized in that a fermentation container (1) with a solution (L), which is contained in said container and in which a fermentation takes place, is at least partly introduced into a nuclear magnetic resonance spectrometer (2). The nuclear magnetic resonance spectrum (S) of the solution (L) is measured as a monitored parameter using the nuclear magnetic resonance spectrometer (2) at one or multiple points in time.

Description

description

Method for monitoring a fermentation process

The invention relates to a method and a device for monitoring a fermentation process.

It is known from the prior art to obtain various types of active ingredients in the context of a fermentation process. For this purpose, so-called. Bioreactors are used, which comprise a fermentation tank, which is filled with a corresponding solution in which then runs a fermentation, which leads to the formation of an active ingredient in the solution. Under fermentation process is to understand the process engineering implementation or control of the course of the fermentation.

When carrying out fermentation processes in bioreactors, it is necessary that certain process parameters are technically recorded. In particular, the solution should be analyzed at regular intervals to determine whether the active substance to be produced is already present in sufficient quantity or whether the fermentation is already completed. To monitor a fermentation process measuring probes are nowadays used, which are introduced into the fermentation tank. The measurement with measuring probes is complex and involves the risk of contamination of the fermentation solution with foreign cells. Alternatively, it is possible that the composition of the fermentation solution is determined by taking samples of the solution and examining them outside in a laboratory. However, this is time-consuming and expensive, so that only comparatively slowly to measurement results by changing parameters of the fermentation process can be reacted.

The object of the invention is therefore to provide a method and a device for monitoring a fermentation process to measure parameters of the fermentation process in a simple way.

This object is achieved by the method according to claim 1 and the device according to claim 11. Further developments of the invention are defined in the dependent claims.

In the context of the method according to the invention, one or more monitoring parameters representing the state of the fermentation process are measured for monitoring a fermentation process. For this purpose, a fermentation vessel with solution contained therein, in which a fermentation takes place, is at least partially introduced into a nuclear magnetic resonance spectrometer. The fermentation tank is preferably part of a bioreactor with which the fermentation process is controlled. According to the invention, the nuclear magnetic resonance spectrum of the solution is measured with the nuclear magnetic resonance spectrometer at one or more points in time as a monitoring parameter.

The invention is based on the finding that the nuclear magnetic resonance spectroscopy known per se, which is used primarily in the medical field for analyzing the human or animal body, can be used to analyze a fermentation process. For this purpose, a known nuclear magnetic resonance spectrometer can be used. Such a spectrometer has a magnetic field generating device for generating a main magnetic field, in which the object to be examined, which is a solution within the scope of the invention, is introduced. The spectrometer further comprises a high-frequency signal transmitting device and a high-frequency signal receiving device, which may optionally be combined in one device. With the high-frequency signal transmitting device, a high-frequency alternating field is irradiated perpendicular to the main magnetic field. As a result, certain atomic nuclei are excited resonantly in the solution, this resonant excitation in the Radio frequency signal receiving means for generating electrical signals leads. In this way, a dependent on the frequency of the radiated alternating field spectrum can be determined, wherein the peaks contained in the spectrum represent certain nuclei or components of the solution. For efficient performance of nuclear magnetic resonance spectroscopy, the wall of the fermentation vessel is at least in the region which is introduced into the nuclear magnetic resonance spectrometer, preferably of dielectric material, in particular plastic.

The method according to the invention has the advantage that a fermentation process can be monitored in a simple manner by means of the nuclear magnetic resonance spectroscopy known per se. This monitoring is non-contact, and it is therefore not necessary to introduce appropriate probes in the fermentation tank, which can lead to contamination of the solution.

In order to detect signals having a high signal-to-noise ratio with the nuclear spin resonance spectrometer, in a preferred embodiment the fermentation vessel is introduced into the nuclear magnetic resonance spectrometer such that the high-frequency signal receiver of the nuclear magnetic resonance spectrometer at least in a partial area of 10 mm or less from the wall of the fermentation tank and, in particular, in at least a partial area in contact with the wall of the fermentation tank.

The measurement of the spectrum by the nuclear magnetic resonance spectrometer can be made based on known methods. In a particularly preferred embodiment, high-frequency pulses are emitted via the high-frequency signal transmitting device, whereby the lying in the frequency band of the pulse nuclear spin transitions are excited. With the aid of a Fourier transformation, the corresponding spectrum can then be determined. In a further preferred embodiment, the measured nuclear magnetic resonance spectrum is a proton spectrum which is particularly suitable for determining constituents of a fermentation solution.

With the method according to the invention, any fermentation processes can be analyzed. In a particularly preferred embodiment, a medicinal active substance is obtained with the fermentation process. In the context of carrying out the fermentation process, the solution which forms a nutrient medium is preferably mixed with mammalian cells.

A particularly hygienic implementation of the fermentation process is achieved in the process according to the invention in that the fermentation takes place in a disposable fermentation container, which preferably has a flexible wall and thus can be introduced particularly well into the nuclear magnetic resonance spectrometer. Disposable fermentation containers are known from the prior art and are preferably made of plastic.

In a further embodiment of the method according to the invention, the determined nuclear magnetic resonance spectrum is output via a user interface. A person monitoring the fermentation process can then draw conclusions about the fermentation process via the spectrum and initiate appropriate measures, in particular adjusting process parameters.

In a further embodiment of the method according to the invention, it is determined as a further monitoring parameter for the nuclear magnetic resonance spectrum of the solution whether and / or in which concentration one or more predetermined constituents, in particular one or more metabolites, are present in the solution. This embodiment is based on the recognition that certain metabolites allow meaningful conclusions about the state of the fermentation process. In a preferred variant is dependent on the presence and / or concentration of the predetermined component (s) in the solution as a further monitoring parameter determines whether the fermentation has ended, the completion of the fermentation being determined, in particular, when the concentration of the metabolite lactate in the solution exceeds a predetermined value , One uses the knowledge that a large amount of lactate indicates that the metabolic processes in the solution are deficient in oxygen, which in turn is an indicator that the cells are in poor constitution or dying.

In a further embodiment of the method according to the invention, one or more adjustment parameters of the fermentation process, in particular the supply of oxygen to the solution and / or the temperature of the solution, are automatically changed and / or become dependent on the presence and / or concentration of the predetermined constituents a proposed change in the setting parameter (s) is output via a user interface. If e.g. An increased concentration of lactate may increase the oxygen supply to the solution as the increased production of lactate may also be due to an oxygen deficiency. If the increase in the oxygen supply does not lead to a reduction in the lactate concentration, the termination of the fermentation is finally determined in a preferred embodiment.

In addition to the method described above, the invention further relates to a device for monitoring a fermentation process, comprising a fermentation vessel for filling with a solution in which a fermentation takes place, and a measuring device for measuring one or more monitoring parameters representing the state of the fermentation process. The fermentation tank is preferably part of a bioreactor, so that in a preferred embodiment be the bioreactor belongs to the device. The device according to the invention is characterized in that the measuring device comprises a nuclear magnetic resonance spectrometer, in which the fermentation container can be introduced with solution contained therein, wherein the nuclear magnetic resonance spectrometer is designed such that it operates as a monitoring parameter, the nuclear magnetic resonance spectrum of Measure solution in the introduced into the nuclear magnetic resonance spectrometer fermentation tank. The device is preferably designed such that one or more of the above-described preferred variants of the method according to the invention can be carried out with the device.

Embodiments of the invention are described below in detail with reference to the accompanying drawings.

Show it:

Fig. 1 is a schematic representation of an arrangement for

 Carrying out the monitoring of a fermentation process according to the invention; and

Fig. 2 is a nuclear magnetic resonance spectrum, which in a

 Embodiment of the method according to the invention can be added.

Hereinafter, an embodiment of the method according to the invention using a disposable bioreactor (English: Disposable Bioreactor) is described, which comprises a disposable fermenter in the form of a flexible bag of dielectric material and in particular of plastic. Such bioreactors are known per se from the prior art and are sold, for example, by the company Sartorius Stediril Biotech. The one-time use of the fermenter ensures that the fermentation process can take place under particularly hygienic conditions. The invention can be used to monitor any fermentation processes. In the following, the production of a medicinal active substance will be described as an example of a fermentation process. The medicinal substance is a monoclonal antibody used in cancer immunotherapy. The bioreactor thereby controls the fermentation of a solution introduced into the fermentation vessel, wherein the reactor regulates certain parameters of the fermentation process, in particular the supply of oxygen or other components as well as the temperature of the process. The fermentation process is divided into an upstream process, which relates to the actual fermentation process, and a downstream process, which relates to the recovery of the medicinal active ingredient from the fermented solution. In the example described here, as part of the upstream process, a nutrient medium of glucose, vitamins, amino acids and water is introduced into the fermentation vessel. In the nutrient medium CHO cell culture (CHO = Chinese hamster ovaries) is set. Subsequently, the fermentation proceeds, wherein the solution in the course of the fermentation process glucose, vitamins and oxygen are supplied. The process temperature is kept at 37 ° C and the pH of the solution at 7. The process duration varies between 20 and 100 days. Finally, at the end of the upstream process, a yield of 1 to 5 g of medicinal active ingredient per liter of the fermentation batch in the fermentation tank is obtained.

This is followed by the downstream process, followed by centrifugation of the solution, various filtrations (e.g., deep, ultra, slide, cascade), chromatography, and other filtration. The embodiment of the invention described below is used throughout the upstream process and ultimately determines the process duration. In particular, it is measured when the fermentation is stopped.

1 shows an arrangement according to the invention with which the fermentation process just explained can be monitored. In this case, the flexible one-way fermentation tank 1 already described above is shown in a schematic plan view, in which the solution L is located, in which the fermentation takes place. It is now essential to the invention that within the scope of the fermentation process using a nuclear magnetic resonance spectrometer, which is also referred to as NMR spectrometer (NMR = Nuclear Magnetic Resonance), a nuclear magnetic resonance spectrum of the solution L is taken up in the fermentation vessel 1 at regular time intervals. The nuclear magnetic resonance spectrometer has a known structure. It uses the principle of nuclear magnetic resonance of atoms to determine the components of solution L. The spectrometer comprises in known manner an electromagnet with the two poles 301 and 302, with which a homogeneous magnetic field with a high magnetic field strength, for example in the range of one to several Tesla, is generated. Usually, a superconducting electromagnet is used to generate this magnetic field.

In the field of the electromagnet, the flexible fermentation tank 1 is introduced via a corresponding opening in the spectrometer. During measurement with the nuclear magnetic resonance spectrometer, ensure that the solution is at rest. Any intended shaking table for mixing the fermentation mixture is therefore to be switched off during the measurement. In order to determine the spectrum, a high-frequency electromagnetic alternating field perpendicular to the static magnetic field of the electromagnet is emitted via a transmitting coil 4, which is controlled by means of a transmitting unit 5. In the variant described here, the alternating field is emitted based on short pulses. Depending on the application, the frequency of the alternating field is preferably between 300 and 1000 MHz. The pulse bandwidth of the emitted pulses is inversely proportional to the pulse duration. Depending on the choice of pulse duration and pulse power, all nuclear spin transitions in the solution in the frequency band of the pulse are excited by atoms in the solution. In the embodiment described here, by appropriate adjustment of the alternating Selfeldes nuclear spin transitions excited by protons, ie it is carried out with the spectrometer, a proton spectroscopy. This spectroscopy is a further development of magnetic resonance tomography and derives from conventional NMR spectroscopy of in vivo samples.

After switching off the high-frequency field of the transmitting coil 4, the nuclear spins of the protons oscillate briefly with their individual Lamor frequency. The sum of these oscillations induces a current in the receiving coil 6 arranged adjacent to the transmitting coil 4. In a receiving unit 7 assigned to the receiving coil, the measured current is digitized and subjected to a Fourier transformation. In this way, one obtains a nuclear magnetic resonance spectrum of the solution, which represents a monitoring parameter within the meaning of the claims. In the embodiment of FIG. 1, an evaluation unit 8 is furthermore provided which, based on the spectrum, influences certain parameters of the fermentation process or determines the end of the fermentation taking place in container 1.

An example of a spectrum that can be detected with the MNR spectrometer of FIG. 1 is shown in FIG. This figure shows the proton spectrum S of the metabolites of cells. Metabolites are metabolic intermediates in living cell tissue. These metabolites are also usually produced in the fermentation in bioreactors, since the solutions are usually added to live cell cultures. In the spectrum of FIG. 1, the deviation of the resonance frequencies from a predetermined main frequency is indicated along the abscissa in a manner known per se. This deviation is given in ppm (ppm = parts per million). The measured amplitudes A are plotted along the ordinate. The respective peaks at the corresponding frequencies represent a resonant excitation of metabolites. In the spectrum S shown, the peak CHO at 3.25 ppm represents the metabolite choline, the peak CRE at 2.95 ppm the metabolite creatine and the peak NAA at 2.0 ppm the metabolite NAA (NAA = N-acetylaspartate). The spectrum also contains a very small peak at 1.3 ppm, which represents lactate. The position of this peak is indicated by arrow LAC. The metabolite lactate can be used in the process according to the invention to control parameters of the fermentation process or to determine the end of the fermentation. This is because the concentration of lactate is an indicator of metabolic processes in cells. Lactate occurs only when the metabolic processes are anaerobic (ie under oxygen deficiency). A high lactate value with good oxygen supply indicates that the cell metabolism is no longer optimal. As a result, cells produce lactate before they die off, so the lactate concentration is an indicator of the end of the fermentation. With the arrangement of FIG. 1, the lactate concentration can now be determined from the spectrum of FIG. 2 via the evaluation unit 8. The determination of the concentration is effected by forming the integral of the amplitude of the corresponding peak over the half-width of the peak and placing it in proportion to the integration of the other peaks, thus enabling a relative determination of the concentration of the metabolite lactate and also of the other metabolites , If the concentration of lactate exceeds a predetermined threshold value, this can be evaluated in the evaluation unit in that the fermentation is ended. Following this, appropriate steps can then be taken to proceed to the downstream process described above for obtaining the medical drug.

Optionally, in the measurement of an increased lactate concentration, it may also be initially attempted to change the process parameters. In particular, the increased lactate concentration Stir also concentration that the solution L is supplied to less oxygen. Consequently, in the case of an increased lactate concentration in a first step, first the supply of oxygen to the solution can be increased. If the increased oxygen supply does not lead to a reduction in the lactate concentration, then in a second step, the end of the fermentation can be determined.

In the context of the method according to the invention, a conventional NMR spectrometer, which is used for example for the examination of human or animal body, can be used for the analysis of a fermentation process. It is not even necessary that the concentration of the components in the fermentation tank is determined in a spatially resolved manner. That is, a homogeneous magnetic field in combination with an alternating electromagnetic field is sufficient for carrying out the spectroscopy. Nevertheless, the nuclear magnetic resonance spectrometer may also determine the concentration of the individual components in a spatially resolved manner for different volume elements of the solution. For this purpose, a nuclear magnetic resonance spectrometer is used, in which the homogeneous main magnetic field is superimposed with additional magnetic gradients. Such spectrometers are also known in the medical field, but have not hitherto been used for the analysis of fermentation processes.

The embodiments of the method according to the invention described above have a number of advantages. A direct contactless analysis of a fermentation process, eg based on the lactate concentration of cells, is made possible by means of a nuclear magnetic resonance spectrum, which is detected by an NMR spectrometer. By means of this the vitality of the cells in the solution can be controlled and thus a control of the fermentation process can be made possible or a statement can be made directly at the end time of the fermentation. The NMR spectrometer used in the monitoring of the fermentation process can be simple, because - in contrast to medical applications - a local dissolution of the constituents of the solution in which the fermentation takes place is not absolutely necessary. In particular, the solution in the fermentation tank can be regarded as homogeneously mixed. A high signal-to-noise ratio of the spectrum measured with the spectrometer can be achieved by placing the receiver coil very close to the liquid in the fermentation tank. In this case, the wall of the fermentation vessel may optionally be brought into direct contact with the receiving coil, ie the distance between the liquid and the receiving coil is determined by the wall thickness of the fermentation vessel.

In contrast to medical technology, there is also the possibility that a transmission signal with high signal strength is irradiated, which may exceed the valid human SAR limit value of 10 W / kg (SAR = Specific Absorption Rate). Thus, it is not necessary to observe the requirements for the quality of the transmission field valid for medical NMR spectrometers. The NMR spectrometer used in the invention can thus be realized relatively inexpensively. In addition, due to the measuring principle, a wide variety of bioreactors can be monitored with the NMR spectrometer without having to make any changes to the spectrometer.

The rate of obtaining the analytical data relating to the fermentation process is essentially only dependent on the execution of the signal processing and / or signal processing, and can be accomplished in less than a second using modern digital signal processing processors. Thus, the state of a fermentation process can be monitored in near-real time.

Claims

claims 1. A method for monitoring a fermentation process in which one or more monitoring parameters representing the state of the fermentation process are measured, characterized in that  a fermentation vessel (1) with solution (L) contained therein, in which a fermentation takes place, is at least partially introduced into a nuclear magnetic resonance spectrometer (2);  is measured as a monitoring parameter, the nuclear magnetic resonance spectrum (S) of the solution (L) with the nuclear magnetic resonance spectrometer (2) at one or more times. 2. The method according to claim 1, characterized in that the fermentation container (1) is introduced into the nuclear magnetic resonance spectrometer (2) such that a high-frequency signal receiving means (6) of the nuclear magnetic resonance spectrometer (2) at least in a partial region Has a distance of 10 mm or less from the wall of the fermentation vessel (1) and in particular in at least a partial area in contact with the wall of the fermentation vessel (1). 3. The method according to claim 1 or 2, characterized in that the measured nuclear magnetic resonance spectrum (S) is a proton spectrum. 4. The method according to claim 1, 2 or 3, characterized in that the fermentation process, a medicinal active ingredient is obtained. 5. The method according to any one of the preceding claims, characterized indicates that for carrying out the fermentation process, the solution (L), which forms a nutrient medium, is mixed with mammalian cells. 6. The method according to any one of the preceding claims, characterized in that the fermentation takes place in a one-way fermentation container (1), which preferably has a flexible wall. 7. The method according to any one of the preceding claims, characterized in that the nuclear magnetic resonance spectrum (S) of the solution (L) is output via a user interface. 8. The method according to any one of the preceding claims, characterized in that for the nuclear magnetic resonance spectrum (S) of the solution (L) is determined as a further monitoring parameters, whether and / or in which concentration one or more predetermined components, in particular one or more metabolites (LAC, NAA, CRE, CHO) in which solution (L) is present. 9. The method according to claim 8, characterized in that is determined depending on the presence and / or concentration of the predetermined components or in the solution (L) as a further monitoring parameters, if the fermentation is completed, the termination of the fermentation in particular is then determined when the concentration of the metabolite lactate in the solution (L) exceeds a predetermined value. 10. The method according to claim 8 or 9, characterized in that, depending on the presence and / or concentration of the predetermined components or in the solution (L) one or more adjustment parameters of the fermentation process, in particular the supply of oxygen to the solution (L ) and / or the temperature of the solution (L) are automatically changed and / or a proposed change of the setting parameter (s) is output via a user interface. 11. A device for monitoring a fermentation process, comprising a fermentation tank (1) for filling with a solution (L) in which a fermentation takes place, and a measuring device for measuring one or more monitoring parameters representing the state of the fermentation process, characterized in that the measuring device comprises a nuclear magnetic resonance spectrometer (2) into which the fermentation vessel (1) with solution (L) contained therein can be introduced, the nuclear magnetic resonance spectrometer (2) being designed in operation as a monitoring parameter measures the nuclear magnetic resonance spectrum (S) of the solution (L) in the fermentation tank (1) introduced into the nuclear magnetic resonance spectrometer (2). 12. The device according to claim 11, characterized in that the device for carrying out a method according to one of claims 2 to 10 is configured.