Methods and systems for determining an actual PH in a liquid phase of a tank

Abstract

The present invention relates to a method for determining an actual pH liquid phase of a tank. The method measures an exhaust CO2 concentration in a gaseous phase in or from the tank. In the method, a signal from a pH measuring device in the liquid phase of the tank is obtained. A CO2 concentration is measured in the gaseous phase. The signal from the pH measuring device and the CO2 concentration are obtained for a plurality of consecutive time points. The next step comprises determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase. The measured CO2 concentration is representative of the actual pH when a predetermined correlation between the pH values measured in the liquid phase and the measured CO2 concentration in the gaseous phase is observed. Then, the actual pH in the liquid phase may be calculated from the measured CO2 concentration. The measurements of the CO2 concentration and signal from the pH probe are performed before an equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank.

Description

F. Hoffmann-La Roche AGMethods and systems for determining an actual pH in a liquid phase of a tank

[0001] The present invention relates to methods and systems for determining an actual pH in a liquid phase of a tank.

[0002] Bioreactors may be used for chemical and / or biological processes, for example processes performed by living organisms, in a controlled manner, e.g., in order to obtain a chemical compound, e.g., a particular peptide, protein, or other kind of chemical or biological substance. A common goal is to operate the bioreactor in a way that the microorganisms or cells are able to perform their desired function with limited production of impurities and / or in a time- and cost-efficient manner. Environmental conditions inside the bioreactor, such as temperature, nutrient concentrations, pH, and dissolved gases, among other parameters, are typically chosen such that the growth and productivity of the organisms is optimized. One parameter that is of particular importance is the pH value in a liquid phase of the bioreactor. The pH value may need to be closely and precisely monitored and controlled to obtain the desired chemical or biochemical compound.

[0003] Figure 1 shows an embodiment of an example known bioreactor 100. The bioreactor 100 includes a tank 110, a gas inlet 101, and a liquid inlet 102. The liquid inlet 102 is connected to a liquid feed line 103 (e.g. a pipe) and the gas inlet 101 is connected to a gas feed line 104. The gas feed line 104 (e.g. a pipe) may feed CO2 111 and / or other gases (e.g. nitrogen, air) into the tank 110. The bioreactor 100 may include a mixer 105. The bioreactor 100 may be filled with a fresh liquid medium through the liquid feed line 103 and the liquid inlet 102. Further liquids may be added through the liquid feed line 103 or through one or more further inlets (not shown). The bioreactor 100 also includes an outlet 106 with an outlet feedline 107 (e.g. a pipe) for removing contents from the bioreactor 100 and a gas outlet 108 with an off-gas feed line 109. Further, the bioreactor 100 includes a pH probe 112 that measures the pH of a liquid phase within the bioreactor.

[0004] The bioreactor 100 may be repeatedly used. Alternatively, the bioreactor may be for a single use. Before a new media can be added, e.g. via liquid inlet 102 and liquid feedline 103,the tank 110 must be sterilized. The sterilization may include the application of steam and / or the application of aggressive chemical agents. Sterilization in place (sometimes abbreviated as SIP in the art) substantially prevents any contamination of a product of the bioreactor. SIP may mean that all surfaces of the tank that come into contact with the product are sterilized without significant disassembly, e.g. without removal of the pH probe. However, while sterilization in place may allow for a particularly sterile bioreactor, the sterilization may also affect the measurement accuracy of the pH probe 112. For example, since SIP temperature is above the working range of the pH probe, a change in the asymmetry of the probe and therefore a change in the measured pH occurs. Single Use Bioreactors may be gamma radiated for sterilization which also may change measuring characteristics of the pH sensor. Further, the pH probe may be offset by a pH value. Thus, after sterilization, the probe needs to be recalibrated.

[0005] In some applications with less strict requirements, such as in food processing, the pH probe may simply be removed and recalibrated. However, in particular in pharmaceutical applications, this is not possible as a removal of the pH probe may impact the sterilization of the bioreactor.

[0006] In the art, after a media fill and before inoculation of the bioreactor, the bioreactor pH probe signals may be verified and adjusted. A user may draw a liquid sample from the bioreactor and perform a pH measurement in a separate lab. This is called an off-line pH measurement. This off-line pH measurement may then be used to recalibrate the pH probe 112 of the bioreactor 100 by correcting an offset between the pH probe signal and the pH measurement in the lab. This may include adjusting a zero point of the original calibration function. This may be called a product calibration in the measuring amplifiers. Alternatively, the off-line pH measurement may be used in a one-point calibration to recalibrate the pH probe 112 of the bioreactor 100. A one-point calibration may include adjusting a slope of the pH probe 112 to a theoretical Nernst slope and correcting a zero point (or other point on the slope) on the basis of the measured value.

[0007] However, these methods of recalibration require the removal of a liquid sample probe (e.g. a sampling procedure), require a sample hold time, a CO2 degassing, an estimation of sample properties, e.g. using viable cell density, VCD, and an estimation of effects of sample properties, e.g. using VCD. Further, the sample must be transported to a lab. Thereafter, usingthis pH, the sensor is recalibrated. The time needed for this process is relatively long which leads to undesirable offsets in the calibration. Further, local differences in sample processing may lead to deviations in the determined pH. As a result, the calibration and thus the measured pH of the pH probe may be inaccurate, e.g. by 0.1 delta pH.

[0008] WO 2017 / 072346 Al (filed by the present applicant) proposes measuring a CO2 concentration in the outlet feedline of a tank when the tank is in a PH-CO2 equilibrium. In particular, an equilibrium CO2 concentration in the off-gas feed line. However, this requires a user to wait until a PH-CO2 equilibrium is achieved. This may, depending on the reaction, require a hold time of tens of hours or even days. In some examples this may require a hold time of 21 hours or more. Further, the waiting time may depend on various factors such as aeration rates, bioreactor / tank total volume, start pH, media configuration and so on. Inoculation should only occur after the pH probe is accurately calibrated as accuracy of the off-gas CCh / pH determination is limited after inoculation. This is a major limitation especially in a manufacturing setting where, e.g., bioreactors tend to have high volumes, and manufacturing schedules may not allow for long waiting times (media hold times).

[0009] Consequently, it is an object of the present invention to provide a system which provides an improvement over the known processes. In particular, it is an object to provide a faster calibration of pH probes and / or a determination of a pH within a medium that is preferably on-line and may be in real time.

[0010] This object is achieved with the features of the independent claims. Dependent claims refer to preferred embodiments.

[0011] According to a first aspect, a method for determining an actual pH liquid phase of a tank is provided. The method measures an exhaust CO2 concentration in a gaseous phase in or from the tank. In the method, a signal from a pH measuring device in the liquid phase of the tank is obtained (step a)). A CO2 concentration is measured in the gaseous phase (step b)). The CO2 concentration may be measured in the tank or in a feed line connected to the tank. The signal from the pH measuring device and the CO2 concentration are obtained for a plurality of consecutive time points (step c)). Said differently, the signal from the pH measuring device and the CO2 concentration may be repeatedly obtained. In a further step (step d)), it is determined that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase. The measured CO2 concentration is representative of the actual pHwhen a predetermined correlation between the pH values measured in the liquid phase and the measured CO2 concentration in the gaseous phase is observed. Then (step e)), the actual pH in the liquid phase may be calculated from the measured CO2 concentration. According to the invention, the steps a) to c) are performed before an equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank.

[0012] "Correlation" as mentioned herein may refer to any relationship between the pH values measured in the liquid phase and the measured CO2 concentration in the gaseous phase (including, but not limited to a change in difference of the pH values measured in the liquid phase and the measured CO2 concentration in the gaseous phase).

[0013] Contrary to the art, the present invention allows for the determination of a pH using a CO2 concentration without having to wait for an equilibrium in the liquid phase. Thus, the present invention may allow for a faster determination of a pH and thus a faster calibration of a pH probe. In some embodiments, this may allow for a calibration of the pH sensor within 6 hours rather than 21 hours as in the art.

[0014] The term actual pH as used herein refers to the pH of the liquid phase. The signal from the pH probe may also indicate a pH value. However, as outlined above, this pH value may deviate from the actual pH value. Hereafter, the signal measured from the probe may also be denoted as probe pH. The liquid phase of a tank may refer to the liquid that is held within the tank. The tank may or may not include a gaseous phase, which may refer to a gas in the tank. In another example, in case the tank is filled, the gaseous phase may be only present in a gas outlet of the tank.

[0015] The signal from the pH measuring device may be obtained by a controller. The controller may be a computing device. In alternative embodiments, the signal from the pH measuring device may also be read out by a user. The CO2 concentration in the gaseous phase (e.g., in an exhaust line or in the tank) may be measured by a CO2 concentration sensor as outlined below. The CO2 concentration may also be obtained by the controller or, alternatively, by a user. The measurements with the pH measuring device and of the CO2 concentrations are repeated to obtain a trend of the values measured by the pH measuring device and the CO2 concentration. If these trends satisfy a predetermined correlation, then it is determined that a pH value that may be obtained from the CO2 concentration is accurate.This allows directly calculating the pH from the CO2 concentration without having to wait for an equilibrium. Rather, the predetermined correlation indicates that the CO2 concentration is already sufficiently representative without needing to wait for the equilibrium.

[0016] While the claim numbers the method steps with Latin letters (a), b), etc.) this is not intended to provide for a determined order other than technically necessary. For example, step b) may occur, or at least partially occur, before step a) or the order of steps d) and e) may be reversed.

[0017] Preferably, the signal from the pH measuring device and the CO2 concentration in the gaseous phase may be obtained simultaneously, i.e., at the same time. Simultaneous as mentioned herein may mean that the results are obtained within seconds (10, 20, 30 or 45) or few minutes (1, 2, 5) of each other. Alternatively, the measurements may also be obtained at different times. If the signal and the CO2 concentration are obtained at the same time, a difference between a pH calculated from the CO2 concentration and the pH of the measuring device may be directly calculated. Otherwise, the method may require an interpolation or other fitting between the data points to calculate the predetermined correlation.

[0018] Preferably, step d) comprises calculating an exhaust CO2 derived pH value from the measured CO2 value. Thereby, the pH values that may be obtained from the CO2 measurement and the pH values obtained from the pH measuring device may be directly compared. The method may further comprise the determination of a change over time of the difference between the pH of the measuring device and the pH derived from the exhaust CO2.

[0019] Preferably, step d) further comprises determining that the exhaust CO2 derived pH value is sufficiently representative of the pH in the liquid phase when the change of the difference is within a predetermined range. Preferably, the predetermined range is around 0, more preferably when the difference is approximately 0. A change in difference of the pH values measured in the liquid phase and the measured CO2 concentration in the gaseous phase may be less than 0.1, 0.05, 0.03, 0.02, 0.01, 0.005 (plus or minus) pH units. The change in difference may be a change per time, e.g. per second, minute, or hour.

[0020] The change of the difference may also be described as a first derivative of the difference between the exhaust CO2 derived pH values and the measured pH values. A derivative as described herein may mean a derivative in the sense of a change of a function's output with respect to an input. However, a derivative may also encompass calculating achange between two consecutive points in time, e.g., calculating the difference between a pH calculated and / or measured at a first time and a second pH calculated and / or measured at a second point in time.

[0021] Preferably, the method comprises fitting a function (also "curve" herein) to the difference between the signal from the pH measuring device and the exhaust CO2 derived pH value. The fitting may include calculating a regression over time. A time derivative of the function may be determined. It may be determined that the exhaust CO2 derived pH value is sufficiently representative of the pH in the liquid phase (see step d) in claim 1) when the time derivative is within the predetermined range. In particular, the time derivative may be within a predetermined range around zero, more preferably when the difference is approximately zero. The range may include less than 0.1, 0.05, 0.02, 0.01, or 0.005 change in pH difference between a pH calculated from the CO2 concentration and the pH of the measuring device.

[0022] Preferably, the method comprises a step of sparging the liquid phase with a gas mixture that contains carbon dioxide. The sparging may lead to an adjustment in pH, as the CO2 may be dissolved in the liquid phase or removed from the liquid phase, in particular if the liquid phase contains water.

[0023] Preferably, the method comprises the step of obtaining the predetermined correlation from an electronic storage. The correlation may encompass a regression or a relationship or a curve or function. The function may be an e-function. The function (also "curve" herein) may be, inter alia, a limited growth function, a saturation function, or a regression function. The electronic storage may be part of a controller of the tank. In alternative embodiments, the electronic storage may also be included in an external computing device and / or may be provided prior to, during or after filling liquid into the tank.

[0024] Preferably, the method comprises a step of predetermining the correlation prior to step a) of claim 1 by measuring a CO2 concentration in the gaseous phase of the tank (e.g. in the tank or in the gas exhaust line) for a set biological or chemical process in the tank and by measuring a corresponding actual pH in the liquid phase of the tank of the set biological or chemical process. The method may further include calculating the correlation from the measurements. More precisely the method may include determining a function (e.g. one of the functions mentioned above) under less sterile conditions, e.g., by exchanging and recalibrating the probe prior to the process. While such a process may not necessarily lead toa product that is usable for further manufacturing, this process may be used to obtain a correlation prior to manufacturing.

[0025] A second aspect of the present invention relates to a method for determining an actual pH in a liquid phase of a tank by measuring an exhaust CO2 concentration in a gaseous phase in or from the tank comprising the following steps:

[0026] A CO2 concentration in the gas phase is measured (step a)). The measurement is repeated for a plurality of consecutive time points (step b)). A target CO2 concentration in the gas phase is predicted (step c)). The target CO2 concentration may include a target saturation concentration. Preferably, the target CO2 concentration is calculated as a target saturation curve for the CO2 concentration in the gas phase. The saturation curve may be a limited growth function. The target CO2 concentration is compared to the measured CO2 concentration and the method comprises determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when the measured CO2 concentration is within a predetermined error margin from the target CO2 concentration (step d)). The predetermined error margin may be obtained over a defined time interval. The method also comprises calculating the actual pH in the liquid phase from the measured CO2 concentration (step e)). The measurement steps may be performed before the equilibrium of the pH in the liquid phase is reached. The exhaust CO2 concentration in the gaseous phase is established in the tank, e.g., by measuring the gas phase directly in the tank or by measuring a CO2 concentration in an off-gas from the tank.

[0027] Similar to the first aspect of the present invention, the second aspect of the invention is directed to determining the point in time when the CO2 concentration becomes a reliable indicator of the actual pH in a liquid phase. According to the second aspect, the CO2 concentration becomes a reliable indicator of the actual pH in a liquid phase once the measured CO2 concentration is within the predetermined error margin from the target CO2 concentration.

[0028] The following embodiments may be used in conjunction with the first and / or second aspect.

[0029] Preferably, the measurements, i.e., the measurements of CO2 concentrations, are performed online and in real-time. Thereby, it is ensured that the pH concentration can be obtained as quickly as possible.

[0030] Preferably, the liquid phase consists of or comprises a liquid medium, preferably a sterile or autosterile medium. The medium may be a buffered medium, preferably a carbonate buffered medium. In alternative embodiments, the medium may be unbuffered.

[0031] Preferably, the tank has been sterilized prior to measurement steps of the first or second aspect. The sterilization may be a sterilization in place.

[0032] Preferably, the tank is a bioreactor, fermenter, cultivation tank and / or a cultivation vessel. In such tanks, the present invention is particularly advantageous as a precise determination of a pH is particularly important in bioreactors, fermenters and cultivation tanks and / or vessels.

[0033] Preferably, the pH measuring device is a tank internal pH measuring device. The pH measuring device may be selected from the group consisting of an optical pH measuring device and electrochemical pH electrode, an antimon electrode, and an ISFET pH electrode. The pH measuring device may also be described as a pH probe herein. These are particularly suitable pH measurement devices for tanks. The pH measuring device may be removable or fixedly installed in the tank.

[0034] Preferably, the CO2 concentration is measured with a mass spectrometer, an optical CO2 probe, an opto-chemical CO2 sensor, a Severinghaus electrode, or an off-gas analyzer. These are particularly suitable CO2 concentration measurement devices / sensors.

[0035] Preferably, the pH measuring device is calibrated or recalibrated to the determined actual pH. In case the pH measured by the pH measuring device (i.e. the pH probe in the tank) deviates from the actual pH as determined from the CO2 concentration, the pH measuring device must be calibrated or recalibrated. The presently proposed methods allow for a calibration that can be made online without requiring liquid samples from the tank. Further, the presently proposed methods allow for a more precise calibration since deviations due to transport of the liquid samples and timing differences may be avoided.

[0036] A further aspect of the present invention relates to a use of a tank for the preparation of a composition comprising a protein. The tank comprises a pH measuring device calibrated or recalibrated according to the methods described above.

[0037] A further aspect of the invention relates to a method for preparing a composition comprising a protein the method comprises the following steps:a) providing a tank comprising a pH measuring device calibrated or re-calibrated according to the method described above; b) culturing cells capable of producing the protein; c) recovering a composition comprising the protein.

[0038] The tank may comprise a sterile medium and / or the tank may be sterile. Thus, the bioreactors can be more efficiently used, and the output of a single bioreactor may be increased. Further, the precise measurement and precise calibration of a pH sensor, improves the process and may lead to a higher output as the process parameter pH can be controlled more precisely.

[0039] Preferably, the composition comprising the protein is a solution comprising a protein, preferably an aqueous solution. The solution may be a buffered solution, i.e. may be buffered prior to insertion into the tank. The method may comprise the step of inoculating a liquid phase in the tank with a cell capable of producing the protein. The inoculating may occur after calibration or recalibration of the sensor and / or before culturing cells capable of producing the protein. The protein may be an antibody. The antibody may be a monoclonal antibody. In a particular embodiment, the antibody is a human or humanized antibody. The term protein as mentioned herein also encompasses protein fragments. The protein fragment may have substantially the same activity as the full-length protein. The term protein may also encompass biological cells (e.g., bacterial, prokaryotic or eukaryotic cells) or sub-cellular fragments, biological tissues, viral particles, virus-like particles or viruses and cellular organelles, lipid nanoparticles (LNPs) and the like. In examples, protein encompasses viral vectors (e.g. adenoviruses, adeno-associated viruses (AAV), retroviruses) and the like. Protein may also encompass amino acids, proteins, peptides, mono- and disaccharides, polysaccharides, lipids, glycolipids, fatty acids, sterols, vitamins, neurotransmitter, nucleotides, metabolites, nucleic acids, and combinations thereof.

[0040] Further example proteins may be selected from the group consisting of enzymes (e.g., carbonic anhydrase, beta lactamase TEM1, or kinases such as MEK1 and p38), transporter proteins (e.g., MBP), inhibitory proteins (e.g., beta lactamase inhibitory protein BLIP, Anakinra), structural proteins, signaling proteins, ligand-binding proteins, chaperones (e.g., heat shock protein HSP90), antibodies (e.g., Trastuzumab), membrane proteins, and receptors (e.g., interleukin 1 receptor).

[0041] Nucleic acids include DNA, RNA (e.g. mRNA, tRNA, rRNA and the like), LNA, plasmids, and PNA. Also, modifications (e.g. chemical modifications) of said nucleic acids fall under the scope of the present disclosure.

[0042] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as the above possess the desired antigen-binding activity.

[0043] An "antibody fragment" may refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv), and multispecific antibodies formed from antibody fragments.

[0044] In particular embodiments, the protein may be any one of the following antibodies: anti-CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an anti-IL6 antibody, an anti-lgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-Ll antibody, an anti-VEGF-A antibody, an anti-VEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-factor D antibody, an anti-factor IX antibody, an anti-factor X antibody, an anti-abeta antibody, an anti-tau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti- FGFR1 / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, ocrelizumab, pertuzumab, trastuzumab, tocilizumab, faricimab, polatuzumab, gantenerumab, cibisatamab, crenezumab, mosunetuzumab, crovalimab, trontinemab tiragolumab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lebrikizumab, omalizumab ranibizumab, emicizumab, selicrelumab, prasinezumab, glofitamab, simlukafusp alfa, and RG7827.

[0045] A further aspect of the present invention relates to a method for preparing a protein formulation. The method may comprise the steps of the method for preparing a composition comprising a protein outlined above. Further, the method for preparing a protein formulation may comprise any one or all of the following steps: adding a buffer diluent, excipient, stabilizer, surfactant and / or carrier to the composition obtained or obtainable by the method for preparing a composition. In one embodiment, the protein concentration, preferably theantibody concentration is at least 0,1 mg / ml and up to 250 mg / ml. The protein formulation may be a pharmaceutical composition. In some embodiments the protein concentration may be 0,1 mg / ml to 1 mg / ml or 10 mg / ml to 100 mg / ml. In other embodiments, the protein concentration maybe 100 mg / ml to 250 mg / ml. The protein may be a hormone such as EPO or any suitable antibody. In a further example the protein formulation may be a diagnostic composition. In the example of erythropoetin (EPO), the concentration may be 0,79 mg / ml.

[0046] A further aspect of the present invention relates to a protein composition that is obtained by or obtainable by the method for preparing a composition comprising a protein described above. A further aspect of the present invention relates to a protein formulation obtained or obtainable by the method for preparing a protein formulation described above. The protein formulation may be for use as a medicament or maybe for use in medicine. Alternatively, the protein formulation may be for use as a drug or as a medicine.

[0047] The protein formulation may be a pharmaceutical or diagnostic protein formulation.

[0048] While the present invention can be used in pharmaceutical applications and prior to inoculation, it should be noted that the present application is not limited to such applications. The present invention may be used in conjunction with any (non-)buffered solution that is aerated or gassed. The present invention may require an off-gas as well as a gassing of the liquid phase and may thus be applied in chemical processes in which pH measurement is desirable as well. Examples include, but are not limited to, a calibration / elution of a chromatography column. Calibration can be done, for example, in the liquid used for elution. The pH adjustment could be done, e.g., by means of CO2

[0049] A further aspect of the present invention relates to a method for preparing a tank comprising a calibrated or recalibrated pH measuring device. The method includes the steps of the method of determining a pH as described above.

[0050] A further aspect of the present invention relates to a tank that is obtained or obtainable by the method for preparing a tank.

[0051] A further aspect of the present invention relates to a system that comprises a bioreactor with the tank, a pH measuring device and a CO2 measurement device. The pH measuring device may be configured to measure a pH value in the liquid phase. The CO2 measurement device may be configured to measure a CO2 concentration in an off-gas fromthe tank. In an alternative embodiment, the CO2 measurement device may also be configured to measure a CO2 concentration in the tank. The system additionally comprises a controller that is configured to perform the method of determining an actual pH as described above. In particular, the controller may comprise a processor and a memory comprising instructions for making the controller perform the method as described above.

[0052] A further aspect of the present invention relates to a computer-implemented method for determining an actual pH in a liquid phase of a tank from a measured CO2 concentration in a gaseous phase in or from the tank. The computer-implement method comprises the following steps: a) receiving first data indicative of a measured pH value in the liquid phase; b) receiving second data indicative of a measured CO2 concentration in the gaseous phase; c) repeating steps a) and b) for a plurality of consecutive time points; d) determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when a predetermined correlation between the pH values measured in the liquid phase and the measured CO2 concentrations in the gaseous phase is observed; e) calculating the actual pH in the liquid phase from the measured CO2 concentration,

[0053] Steps a) to e) are performed before an equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank.

[0054] A further aspect of the present invention relates to another computer-implemented method for determining an actual pH in a liquid phase of a tank from a measured exhaust CO2 concentration in a gaseous phase in orfrom a tank. The method comprises the following steps: a) receiving data indicative of a measured CO2 concentration in the gas phase; b) repeating step a) for a plurality of consecutive time points; c) predicting a target CO2 concentration in the gas phase, preferably wherein the target CO2 concentration includes a target saturation concentration, more preferably wherein the target CO2 concentration is calculated as a targetsaturation curve for the CO2 concentration in the gas phase, even more preferably wherein the curve is a limited growth function; d) comparing the target CO2 concentration to the measured CO2 concentration and determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when the measured CO2 concentration is within a predetermined error margin from the target CO2 concentration, preferably wherein the predetermined error margin is obtained over a defined time interval; e) calculating the actual pH in the liquid phase from the measured CO2 concentration.

[0055] Steps a) to b) include measurements that were performed before the equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank.

[0056] Further aspects of the present invention relate to a data processing apparatus comprising means for carrying out the steps of the methods described above, a computer program comprising instructions which, when the program is executed by a computer cause the computer to carry out the methods described above, and a computer readable medium comprising instructions, which, when executed by a computer cause the computer to carry out the methods described above.

[0057] The subject matter of the invention will be explained in more detail in the following text with reference to preferred exemplary and non-limiting embodiments which are illustrated in the attached schematic drawings. These figures disclose embodiments of the invention for i II ustrationa I purposes only. In particular, the disclosure provided by the figures and description is not meant to limit the scope of protection conferred by the invention.

[0058] Figure 1 shows an example bioreactor that can be used in conjunction with the present invention.

[0059] Figure 2 shows a graph of example measured and calculated pH values.

[0060] Figure 3 shows a graph of an example limited growth function which may be fitted to measured CO2 concentrations.

[0061] The present invention is based on the example bioreactor as shown in figure 1 andprovides for an improved calibration and / or pH measurement without requiring additional hardware. Before the bioreactor 100 can be used, it needs to be sterilized. As outlined above, the bioreactor 100, in particular the tank 110 may be sterilized using sterilization in place with steam. Due to the steam, a calibration of the pH probe may be offset. For example, an electrolyte within the probe may liquify due to the high temperature of the steam and an asymmetry and glass of the pH probe may change.

[0062] In the first step of manufacturing a product, a liquid media is filled into the tank 110 through liquid feed line 103 and liquid inlet 102. As illustrated in figure 1, gas may be fed into the liquid media using the gas feed line 104 and the gas inlet 101. The gas contains CO2. In addition, other gases such as nitrogen or oxygen may be added. In some embodiments, the device may include a mix of CO2 and O2 and / or N2. As can be seen in figure 1, the gas 111 (e.g. CO2) traverses the liquid medium in the tank 110 and exits the tank through gas outlet 108. The CO2 may diffuse into the liquid medium. As a result of the CO2 diffusing into the liquid medium, the pH of the liquid medium changes. Thus, using the feed rate and concentration of CO2 in the gas inlet, a pH in the liquid medium may be adjusted. If the concentration of CO2 in the inlet gas is higher than in the liquid medium, CO2 diffuses into the liquid medium until an equilibrium is reached.

[0063] The medium may contain 2.5 g / L sodium bicarbonate buffer system. Thereby a suitable medium for an off-gas CO2 pH determination is provided (see also description above).

[0064] In an alternative example, the medium includes Kaighn's Modification of Ham's F-12 Medium comprising, for example, putrescine, thymidine, hypoxanthine, zinc, and higher levels of all amino acids and sodium pyruvate. These additions allow the medium to be supplemented with very low levels of serum or defined components, for some cell types. Ham's F-12K (Kaighn's) Medium contains no proteins or growth factors, and is therefore often supplemented with growth factors and Fetal Bovine Serum (FBS) that may be optimized for a particular cell line. Ham's F-12K (Kaighn's) Medium uses a sodium bicarbonate buffer system (2.5 g / L). The medium alternatively or additionally include an LB medium (Lysogeny Broth).

[0065] Returning to figure 1, the gas leaving the tank 110 (also described as "off-gas" herein) is analyzed. The off-gas can be analyzed to determine a pH in the liquid medium. In particular, the carbon dioxide content in the off-gas is analyzed and the corresponding pH is determined based on a medium-dependent correlation. Cell cultures often include a carbonate buffersystem that may be used to control pH via carbon dioxide in the gas supply. Carbon dioxide is thereby used as acidic pH correction agent. It reacts with water forming carbonic acid. The latter is further converted to bicarbonate and carbonate. Since CChfaq) is almost entirely converted to carbonic acid at room temperature, both species can be combined to the "compound carbonic acid".

[0066] The off-gas CO2 concentration thus depends on the inlet gas, in particular the CO2 concentration, feed rate, and the particular medium. In a saturated system, i.e., in equilibrium, there is a linear relationship between carbon dioxide in the gas phase and in the liquid phase in equilibrium. Then, the measured carbon dioxide in the gas phase can be used to determine the concentration in the liquid phase and therefore calculate pH by means of a medium specific correlation. With increasing carbon dioxide concentration, pH is decreasing. As long as pressure and temperature are both identical, the exhaust carbon dioxide concentration has to be identical at identical pH, as long as an equilibrium is existent.

[0067] The present inventors found that the off-gas CO2 concentration may also be used to determine a pH of a liquid medium in non-equilibrium conditions. While the relationship may no longer be linear, the pH can still be obtained.

[0068] In non-equilibrium, the conversion of aqueous CO2 to carbonic acid and the conversion of carbonic acid to bicarbonate and carbonate (and vice versa) is still occurring. For pH determination to be reliable, CO2 off-gas must represent the liquid phase. Directly after media fill, this may not be the case due to high mass transfer, in particular if concentration difference is high, and back mixing of bioreactor headspace that consists of air only after media fill occurs. Furthermore, pH probe signals directly after media fill may drift until they are adapted to cell culture media which is also detected by proposed approaches. Nevertheless, in this dynamic environment the trend in pH change as obtained from the CO2 measurement may be indicative of the actual pH trend and actual pH value.

[0069] Figure 2 shows a schematic graph. The graph indicates curves of an off-gas carbon dioxide concentration 21 as detected by the carbon dioxide sensor in the off-gas stream, a pH value 22 calculated based on the carbon dioxide concentration in the off-gas stream as well as a pH value 23 as measured from the pH probe. A difference between curve 22 and 23 is indicated as curve 24.

[0070] As outlined above, the reading of the pH probe may not be accurate. Nevertheless,while the absolute value of the pH probe may not reflect the pH of the liquid medium accurately, the trend of the signal of the pH may still be accurate. In other words, the pH as measured by the pH probe may be offset from the accurate value. Thus, the measurement curve of the probe represents an accurate measurement of the change in pH in the liquid medium.

[0071] The off-gas carbon dioxide concentration measurements may be used to calculate a pH value. An example calculation is shown in WO 2017 / 072346 Al on pages 7 to 11 and the further disclosure of WO 2017 / 072346 Al. While this calculation is made under an equilibrium assumption, the inventors found that the pH calculation according to this scheme may already lead to reliable and precise pH values prior to reaching equilibrium.

[0072] In order to verify at which point in time the pH calculation from the CO2 off-gas starts to provide reliable and precise measurement results, the pH calculated from the CO2 off-gas is compared to the pH measured from the probe signal. In one embodiment, curves may be fitted through both, the off gas derived pH and the bioreactor pH probe signal. Once a difference between the bioreactor pH probe signal and the off gas derive pH remains substantially constant (or within a predetermined range), it can be assumed that the measurement results from the off-gas derived pH is accurate. The point in time from which a difference between the off gas derived pH and the bioreactor pH probe signal does not change substantially anymore is indicated as tl in figure 2. This point is found by determining the pH repeatedly with using the pH probe and CO2 concentration and then by obtaining the difference 24 between the two pH values. Then the currently obtained difference is compared to the previously obtained difference. Once the difference in pH is not changing anymore, the curve 24 becomes constant as can be seen in figure 2. This indicates that the pH calculated from the CO2 signal is accurate.

[0073] In one example, the point in time may be calculated by calculating a derivative of the difference between the off-gas derived pH and the bioreactor pH probe signal. Once the derivative becomes sufficiently close to 0, it can be assumed that the pH value is derived from the off-gas is accurate. As outlined above, using the first derivative of the difference is one example of comparing the trends of the CO2 derived pH and the bioreactor pH probe signal. Any other mathematical or empirical method for comparing the trends of the CO2 derive pH and the bioreactor pH probe signal are also suitable.

[0074] In a second embodiment, a determination that a CO2 measurement can be used to calculate an accurate pH is made using only the CO2 measurement. Figure 3 shows an example graph illustrating CO2 concentration measurements using a CO2 concentration sensor, e.g. one of the CO2 concentration sensor described above. In a given bioreactor having a given size, a given liquid phase, and a given sparging, the CO2 concentration will follow a characteristic curve, i.e. a saturation curve. This curve can be modeled using a limited growth function which is indicated as curve 31 in figure 3. Curve 31 is in general similar to curve 21 shown in figure 2 and may be fitted through measurement results of a CO2 sensor.

[0075] The limited growth function may be formulated as follows c (t) = cmax (1 - ekAt), in which c(t) is a measured CO2 concentration, cmax is a saturation CO2 concentration, k is the time constant for the respective curve and At being the time period between t + toffset, wherein t is the elapsed time and toffset is a time offset. The saturation CO2 concentration cmax is the CO2 concentration that is reached in equilibrium. The saturation CO2 concentration cmax, the time constant k, and the time offset toffset can be obtained in one or more test runs. For example, these parameters may be obtained by measuring c(t) and t and fitting (e.g. using a Solver and a Nealder Mead Algorithm) the data to the function. Furthermore, it is also possible to set corresponding restrictions for the function, e.g. if it is known that only a maximum concentration of 26% CO2 can occur and / or if it is known that the CO2 concentration cannot be negative.

[0076] As such, in equilibrium, the maximal CO2 concentration may be similar to the CO2 concentration in the gas feed line 104.

[0077] In this example, at least two consecutive measurements of c(t) are obtained as well as the corresponding measurement times. If the measured data fits the limited growth function well enough, i.e. if the left hand side and the right and side match (or do so within a predetermined range, e.g. using mean squared error as an assessment criterion), the measured CO2 concentration is considered sufficiently representative of the actual pH in the liquid phase. The limited growth function may also be used to predict a third CO2 concentration at a third time. If the measured CO2 concentration at the third point in time is within a certain error margin, the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase. Said differently, in the second embodiment, the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase once themeasured data can be accurately modelled using a limited growth function. Thereafter, the pH may be calculated from the CO2 concentration as described above.Aspects of the present invention may include the following:1. A method for determining an actual pH in a liquid phase of a tank by measuring an exhaust CO2 concentration in a gaseous phase in or from the tank comprising the steps: a) obtaining a signal from a pH measuring device in the liquid phase; b) measuring a CO2 concentration in the gaseous phase; c) repeating steps a) and b) for a plurality of consecutive time points; d) determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when a predetermined correlation between the pH values measured in the liquid phase and the measured CO2 concentrations in the gaseous phase is observed; e) calculating the actual pH in the liquid phase from the measured CO2 concentration, wherein at least steps a) to c) are performed before an equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank.2. The method according to aspect 1, wherein steps a) and b) are performed substantially simultaneously.3. The method according to aspect 1 or 2, wherein step d) comprises calculating an exhaust CCh-derived pH value from the measured CO2 value.4. The method according to aspect 3, wherein step d) further comprises calculating a difference between the signal from the pH measuring device and the exhaust CO2- derived pH value and determining a change over time of the difference.5. The method according to aspect 4, wherein step d) further comprises determining that the exhaust CCh-derived pH value is sufficiently representative of the pH in the liquid phase when the change of the difference is within a predetermined range, preferablywithin a predetermined range around zero, more preferably when the difference is approximately zero.6. The method according to aspect 4, further comprising: i) fitting a function to the difference between the signal from the pH measuring device and the exhaust CCh-derived pH value, wherein the fitting preferably includes calculating a regression over time; ii) determining a time derivative of the function of step i); and wherein it is determined that the exhaust CCh-derived pH value is sufficiently representative of the pH in the liquid phase when the time derivative is within the predetermined range, preferably within a predetermined range around zero, more preferably when the difference is approximately zero.7. The method according to one of the preceding aspects, wherein the method comprises a step sparging the liquid phase with a gas mixture that contains carbon dioxide.8. The method according to one of the preceding aspects, wherein the method comprises a step of obtaining the predetermined correlation from an electronic storage.9. The method according to aspect 8, wherein the method comprises a step of predetermining the correlation prior to step a) by measuring a CO2 concentration in the gaseous phase of the tank for a set biological or chemical process in the tank and by measuring a corresponding actual pH in the liquid phase of the tank of the set biological or chemical process and calculating the correlation from the measurements.10. A method for determining an actual pH in a liquid phase of a tank by measuring an exhaust CO2 concentration in a gaseous phase in or from a tank comprising the steps: a) measuring a CO2 concentration in the gaseous phase in or from tank; b) repeating step a) for a plurality of consecutive time points; c) predicting a target CO2 concentration in the gas phase, preferably wherein the target CO2 concentration includes a target saturation concentration, more preferably wherein the target CO2 concentration is calculated as a targetsaturation curve for the CO2 concentration in the gas phase, even more preferably wherein the curve is a limited growth function; d) comparing the target CO2 concentration to the measured CO2 concentration and determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when the measured CO2 concentration is within a predetermined error margin from the target CO2 concentration, preferably wherein the predetermined error margin is obtained over a defined time interval; e) calculating the actual pH in the liquid phase from the measured CO2 concentration; wherein steps a) to b) are performed before the equilibrium of the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank.11. The method according to any one of the preceding aspects, wherein the measurements are performed online and in real-time.12. The method according to any one of the preceding aspects, wherein the liquid phase consists of a medium, preferably a sterile medium.13. The method according to aspect 12, wherein the medium is a buffered medium, preferably a carbonate buffered medium, more preferably a carbonate buffered medium.14. The method according to any one of the preceding aspects, wherein the tank has been sterilized prior to step a).15. The method according to any one of the preceding aspects, wherein the tank is a bioreactor, fermenter, cultivation tank and / or cultivation vessel.16. The method according to any one of the preceding aspects, wherein the pH measuring device is a tank-internal pH measuring device.17. The method according to any one of the preceding aspects, wherein the pH measuring device is selected from the group consisting of an optical pH measuring device, an electrochemical pH electrode, an antimon electrode and ISFET pH electrode.18. The method according to any one of the preceding aspects, wherein the CO2 concentration is measured with a mass spectrometer, an optical CO2 probe, an optochemical CO2 sensor, a Severinghaus electrode, or an off-gas analyzer.19. The method according to any one of the preceding aspects, further comprising a step f) wherein the pH measuring device is calibrated or re-calibrated to the determined actual pH.20. The method according to aspect 19, wherein the pH measuring device is calibrated or re-calibrated without removing and re-inserting the pH measuring device from and to the tank.21. Use of a tank for the preparation of a composition comprising a protein, wherein the tank comprises a pH measuring device calibrated or re-calibrated according to the method of any one of aspects 1 to 20.22. A method for preparing a composition comprising a protein, the method comprising the steps(a) providing a tank comprising a pH measuring device calibrated or re-calibrated using a pH measurement according to the method of any one of aspects 1 to 20;(b) culturing cells capable of producing the protein;(c) recovering a composition comprising the protein.23. The method according to aspect 22, wherein the composition comprising a protein is a solution comprising a protein, preferably an aqueous solution.24. The method according to aspect 23, wherein the solution is a buffered solution.The method according to any one of aspects 22 to 24, wherein the method comprises a step of inoculating a liquid phase in the tank with a cell capable of producing the protein. The method according to any one of aspects 22 to 25, wherein the protein is an antibody. The method according to aspect 26, wherein the antibody is a monoclonal antibody. The method according to aspect 26 or 27, wherein the antibody is a human or a humanized antibody. The method according to any one of aspects 22 to 28, wherein the protein is an anti- CD20 antibody, an anti-CD40 antibody, an anti-HER2 antibody, an a nti-l L6 antibody, an anti-lgE antibody, an anti-IL13 antibody, an anti-TIGIT antibody, an anti-PD-Ll antibody, an anti-VEGF-A antibody, an antiVEGF-A / ANG2 antibody, an anti-CD79b antibody, an anti-ST2 antibody, an anti-factor D antibody, an anti-factor IX antibody, an anti-factor X antibody, an anti-abeta antibody, an antitau antibody, an anti-CEA antibody, an anti-CEA / CD3 antibody, an anti-CD20 / CD3 antibody, an anti-FcRH5 / CD3 antibody, an anti-Her2 / CD3 antibody, an anti-FGFRl / KLB antibody, a FAP-4-1 BBL fusion protein, a FAP-IL2v fusion protein, ocrelizumab, pertuzumab, trastuzumab, tocilizumab, faricimab, polatuzumab, gantenerumab, cibisatamab, crenezumab, mosunetuzumab, tiragolumab, bevacizumab, rituximab, atezolizumab, obinutuzumab, lampalizumab, lebrikizumab, omalizumab ranibizumab, emicizumab, selicrelumab, prasinezumab, glofitamab, simlukafusp alfa, and RG7827. A method for preparing a protein formulation, the method comprising the steps of the method according to any one of aspects 22 to 29, wherein the method for preparing the protein formulation further comprises adding a buffer, diluent, excipient, stabilizer, surfactant and / or carrier to the composition.31. A method for preparing a protein formulation, wherein the method for preparing the protein formulation further comprises adding a buffer, diluent, excipient, stabilizer, surfactant and / or carrier to the composition obtained by or obtainable by the method according to any one of aspects 22 to 29.32. The method according to any one of aspects 22 to 31, wherein the protein concentration, preferably the antibody concentration, is at least 0,1 mg / ml and up to 250 mg / ml.33. The method according to any one of aspects 30 to 32, wherein the protein formulation is a pharmaceutical composition.34. A protein composition obtained by or obtainable by the method according to any one of aspects 22 to 29.35. A protein formulation obtained by or obtainable by the method according to any one of aspects 30 to 33.36. The protein formulation of aspect 35 for use as a medicament or for use in medicine.37. A method for preparing a tank comprising a calibrated or re-calibrated pH measuring device, the method comprising the method of aspect 19.38. A tank obtained or obtainable by the method of aspect 37.39. A system comprising a bioreactor with a tank; a pH measuring device configured to measure a pH value in the liquid phase; a CO2 measurement device configured to measure a CO2 concentration in an off-gas from the tank or in the tank; a controller configured to perform the method according to any of aspects 1 to 29.A computer-implemented method for determining an actual pH in a liquid phase of a tank from a measured exhaust CO2 concentration in a gaseous phase in or from the tank comprising the steps: a) receiving first data indicative of a measured pH value in the liquid phase; b) receiving second data indicative of a measured CO2 concentration in the gaseous phase; c) repeating steps a) and b) for a plurality of consecutive time points; d) determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when a predetermined correlation between the pH values measured in the liquid phase and the measured CO2 concentrations in the gaseous phase is observed; e) calculating the actual pH in the liquid phase from the measured CO2 concentration, wherein steps a) to e) are performed before an equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank. A computer-implemented method for determining an actual pH in a liquid phase of a tank from a measured exhaust CO2 concentration in a gaseous phase in or from a tank comprising the steps: a) receiving data indicative of a measured CO2 concentration in the gas phase; b) repeating step a) for a plurality of consecutive time points; c) predicting a target CO2 concentration in the gas phase, preferably wherein the target CO2 concentration includes a target saturation concentration, more preferably wherein the target CO2 concentration is calculated as a target saturation curve for the CO2 concentration in the gas phase, even more preferably wherein the curve is a limited growth function; d) comparing the target CO2 concentration to the measured CO2 concentration and determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when the measured CO2 concentration is within a predetermined error margin from the target CO2concentration, preferably wherein the predetermined error margin is obtained over a defined time interval; e) calculating the actual pH in the liquid phase from the measured CO2 concentration; wherein steps a) to b) relate to data that was obtained before the equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank.

Claims

Claims1. A method for determining an actual pH in a liquid phase of a tank (110) by measuring an exhaust CO2 concentration in a gaseous phase in or from the tank (110) comprising the steps: a) obtaining a signal from a pH measuring device (112) in the liquid phase; b) measuring a CO2 concentration in the gaseous phase; c) repeating steps a) and b) for a plurality of consecutive time points; d) determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when a predetermined correlation between the pH values measured in the liquid phase and the measured CO2 concentrations in the gaseous phase is observed; e) calculating the actual pH in the liquid phase from the measured CO2 concentration, wherein at least steps a) to c) are performed before an equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank (110).

2. The method according to claim 1, wherein steps a) and b) are performed simultaneously.

3. The method according to claim 1 or 2, wherein step d) further comprises calculating a difference between the signal from the pH measuring device (112) and the exhaust CO2-derived pH value and determining a change over time of the difference.

4. The method according to any one of the preceding claims, wherein the method comprises a step of obtaining the predetermined correlation from an electronic storage wherein the method comprises a step of predetermining the correlation prior to step a) by measuring a CO2 concentration in the gaseous phase of the tank (110) for a set biological or chemical process in the tank (110) and by measuring a corresponding actual pH in the liquid phase of the tank (110) of the set biological or chemical process and calculating the correlation from the measurements.

5. A method for determining an actual pH in a liquid phase of a tank (110) by measuring an exhaust CO2 concentration in a gaseous phase in or from a tank (110) comprising the steps: a) measuring a CO2 concentration in the gaseous phase in or from tank (110); b) repeating step a) for a plurality of consecutive time points; c) predicting a target CO2 concentration in the gas phase, preferably wherein the target CO2 concentration includes a target saturation concentration, more preferably wherein the target CO2 concentration is calculated as a target saturation curve for the CO2 concentration in the gas phase, even more preferably wherein the curve is a limited growth function; d) comparing the target CO2 concentration to the measured CO2 concentration and determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when the measured CO2 concentration is within a predetermined error margin from the target CO2 concentration, preferably wherein the predetermined error margin is obtained over a defined time interval; e) calculating the actual pH in the liquid phase from the measured CO2 concentration; wherein steps a) to b) are performed before the equilibrium of the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank (110).

6. The method according to one of the preceding claims, wherein step d) of claim 1 or step e) of claim 5 comprises calculating an exhaust CCh-derived pH value from the measured CO2 value.

7. The method according to one of the preceding claims, wherein the method comprises a step sparging the liquid phase with a gas mixture that contains carbon dioxide.

8. The method according to any one of the preceding claims, wherein the tank (110) has been sterilized prior to step a).

9. The method according to any one of the preceding claims, wherein the pH measuring device (112) is a tank-internal pH measuring device (112).

10. The method according to any one of the preceding claims, further comprising a step further step, wherein the pH measuring device (112) is calibrated or re-calibrated to the determined actual pH.

11. The method according to any one of the preceding claims, wherein the measurements are performed online and in real-time.

12. The method according to any one of the preceding claims, wherein the liquid phase is a buffered solution.

13. A system comprising a bioreactor (100) with a tank (110); a pH measuring device (112) configured to measure a pH value in the liquid phase; a CO2 measurement device configured to measure a CO2 concentration in an off-gas from the tank (110) or in the tank (110); a controller configured to perform the method according to any of claims 1 to 12.

14. A computer-implemented method for determining an actual pH in a liquid phase of a tank (110) from a measured exhaust CO2 concentration in a gaseous phase in or from the tank (110) comprising the steps: a) receiving first data indicative of a measured pH value in the liquid phase; b) receiving second data indicative of a measured CO2 concentration in the gaseous phase; c) repeating steps a) and b) for a plurality of consecutive time points; d) determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when a predetermined correlation between the pH values measured in the liquid phase and the measured CO2 concentrations in the gaseous phase is observed;e) calculating the actual pH in the liquid phase from the measured CO2 concentration, wherein steps a) to e) are performed before an equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank (110).

15. A computer-implemented method for determining an actual pH in a liquid phase of a tank (110) from a measured exhaust CO2 concentration in a gaseous phase in or from a tank (110) comprising the steps: a) receiving data indicative of a measured CO2 concentration in the gas phase; b) repeating step a) for a plurality of consecutive time points; c) predicting a target CO2 concentration in the gas phase, preferably wherein the target CO2 concentration includes a target saturation concentration, more preferably wherein the target CO2 concentration is calculated as a target saturation curve for the CO2 concentration in the gas phase, even more preferably wherein the curve is a limited growth function; d) comparing the target CO2 concentration to the measured CO2 concentration and determining that the measured CO2 concentration is sufficiently representative of the actual pH in the liquid phase when the measured CO2 concentration is within a predetermined error margin from the target CO2 concentration, preferably wherein the predetermined error margin is obtained over a defined time interval; e) calculating the actual pH in the liquid phase from the measured CO2 concentration; wherein steps a) to b) relate to data that was obtained before the equilibrium between the pH in the liquid phase and the exhaust CO2 concentration in the gaseous phase is established in the tank (110).

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