Publikationen
Bioreactor Performance:
Insights into the Transport Properties of Aerated Stirred Tanks
Sebastian Schaepe, Andreas Lübbert, Michael Pohlscheidt, Christian Sieblist and Marco Jenzsch
Abstract
For recombinant therapeutic protein production, microbial (e.g. E.coli) and mammalian cells (e.g. CHO-cell) are the most important hosts. Progress in producing high value biologics not only depends on improving the performance of the cells but also of the efficiency of their supply in production-scale bioreactors. The activity of the cells is controlled by their immediate microscopic environment and the task of a macro-scopic bioreactor is to provide the optimal conditions to as many cells as possible for producing the desired product. This does not only mean that broth homogeneity must be approached, this also mean that the optimal conditions for the cells must precisely be adjusted in order to obtain high productivity and batch-to-batch reproducibility. The latter determines the process quality and hence the product quality as well.
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Increasing batch-to-batch reproducibility of CHO-cell cultures using a model predictive control approach
M. Aehle, K. Bork, S. Schaepe, A. Kuprijanov, R. Horstkorte, R. Simutis, A. Lübbert, 2012
Abstract:
By means of a model predictive control strategy it was possible to ensure a high batch-to-batch reproducibility in animal cell (CHO-cell) suspensions cultured for a recombinant therapeutic protein (EPO) production. The general control objective was derived by identifying an optimal specific growth rate taking productivity, protein quality and process controllability into account. This goal was approached indirectly by controlling the oxygen mass consumed by the cells which is related to specific biomass growth rate and cell concentration profile by manipulating the glutamine feed rate. Process knowledge represented by a classical model was incorporated into the model predictive control algorithm. The controller was employed in several cultivation experiments. During these cultivations, the model parameters were adapted after each sampling event to cope with changes in the process’ dynamics. The ability to predict the state variables, particularly for the oxygen consumption, led to only moderate changes in the desired optimal operational trajectories. Hence, nearly identical oxygen consumption profiles, cell and protein titers as well as sialylation patterns were obtained for all cultivation runs.
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Simplified off-gas analyses in animal cell cultures for process monitoring and control purposes
M. Aehle, A. Kuprijanov, S.Schaepe, R. Simutis, A. Lübbert, 2011
Abstract:
Batch-to-batch reproducibility of animal cell cultures can significantly be enhanced using process control procedures. Most informative signals for advanced process control can be derived from the volume fractions of oxygen and carbon dioxide in the vent line of the reactors. Here we employed simple low-cost sensors, previously not considered for off-gas analysis at a laboratory-scale cell cultures, and compared them with a simultaneously used quadrupole mass spectrometer, i.e., the standard equipment. A decisive advantage is that the sensors did not need any calibration and are easy to use. We show that monitoring and advanced control of cell cultures can significantly be simplified using the devices tested here and that the same batch-to-batch reproducibility can be obtained with much less effort than before.
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Simple control of fed-batch processes for
recombinant protein production with E. coli
S.Schaepe, A. Kuprijanov, M. Aehle, R. Simutis, A. Lübbert, 2011
Abstract:
Avery simple but effective process controltechnique is proposed that leads to a high batch-tobatchreproducibility with respect to biomass concentrationas well as the specific biomass growth rateprofiles in E. coli fermentations performed duringrecombinant protein production. It makes use of thewell-established temperature controllers in currentlyused fermenters, but takes its information from thedifference between the controlled culture temperatureTcult and the temperature Tcoolin of the coolant fed tothe fermenter’s cooling jacket as adjusted by thefermenter temperature controller. For process controlpurposes this measured difference is correctedregarding stirrer influences and cumulated before itis used as a new process control variable. As a spin-offof this control, it becomes possible to estimate onlinethe oxygen mass transfer rates and the correspondingkLa values during the real cultivation process.
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Simple and efficient control of CHO cell cultures
M. Aehle, S. Schaepe, A. Kuprijanov, R. Simutis and A. Lübbert, 2011
Abstract
Cell cultures must tightly be kept under control in order to guarantee a sufficiently small variability in the protein product quality. A simple and efficient technique for CHO-cell cultures is presented that allows keeping the viable cell count Xv and the specific growth rate μ of the cells on predefined trajectories. As Xv and μ cannot directly be measured online, they are controlled indirectly via the total mass of oxygen consumed. Online values of the latter can precisely be estimated from off gas analysis, i.e. from the O2 volume ratio measured in the vent line and air flow rate measurements. In glutamine-limited fed-batch cultivations, the glutamine feed rate can be manipulated in such a way that the viable cell density and the specific growth rate are kept on predefined profiles for nearly the entire cultivation time. The viability of the cells is not affected by the closed loop control actions. The technique was validated with CHO-cells cultured in a 2.5-L fully instrumented stirred tank bioreactor. It is shown that the controller is able to run the process exactly on predefined tracks with a high batch-to-batch reproducibility. By means of six fed-batch cultivations of CHO cells it was shown that a remarkable reproducibility of viable cell concentration could be achieved throughout 140 h cultivation time.
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Fermentation process supervision and strategies for fail-safe operation:
A practical approach
S. Gnoth, R. Simutis, A. Lübbert, 2011
Abstract:
In an industrial production environment, cultivation processes for the production of recombinant proteins run along predefined trajectories. Feedback control is the best way to keep the cultures on track. However, feedback controllers require accurate on-line values of the controlled variables. To assess whether the measurement signals are correct, process supervision techniques are required. In the case where a process failure has occurred and incorrectly measured variables have been identified, automated fail-safe techniques must be started. Here, we use
the production of a pharmaceutically relevant recombinant protein to compare different approaches to process supervision and fail-safe routines.
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A test facility for fritted spargers of production-scale-bioreactors
C. Sieblist, M. Aehle, M. Pohlscheidt, M. Jenzsch and A. Lübbert, 2011
Abstract:
The production of therapeutic proteins requires qualification of equipment components and appropriate validation procedures for all operations. Since protein productions are typically performed in bioreactors using aerobic cultivation processes air sparging is an essential factor. As recorded in literature, besides ring spargers and open pipe, sinter frits are often used as sparging elements in large scale bioreactors. Due to the manufacturing process these frits have a high lot-to-lot product variability. Experience shows this is a practical problem for use in production processes of therapeutic proteins, hence frits must be tested before they can be employed. The circumstance of checking quality and performance of frits as sparging elements was investigated and various possibilities have been compared. Criteria have been developed in order to evaluate the sparging performance under conditions comparable to those in production bioreactors. The oxygen mass transfer coefficient (k L a) was chosen as the evaluation criterion. It is well known as an essential performance measure for fermenters in the monoclonal antibody production. Therefore a test rig was constructed able to automatically test frit-spargers with respect to their k L a-values at various gas throughputs. Performance differences in the percent range could be detected.
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Increasing batch-to-batch reproducibility of CHO cultures by robust open-loop control
M.Aehle, A. Kuprijanov, S. Schaepe, R. Simutis, A.Lübbert, 2010
Abstract:
In order to guarantee the quality of recombinant therapeutic proteins produced in mammalian cell systems, the straightforward approach in industry is to run the processes as reproducible as possible. It is first shown that considerable distortions in the currently operated processes appear when the initial cell density deviates from its nominal value. Small deviations in the initial cell mass may lead to severe deviations from the desired biomass trajectory. Next, it is shown how to design a fed-batch production process in such a way that it is robust with respect to variations in the viable cell density. A simple open loop strategy is proposed for that purpose. Here we show for the first time at animal cell cultures (CHO cells) that by means of an appropriate glutamine feed rate profile F(t), which keeps the specific growth rate of the cells on a predefined value below its maximal value while maintaining the viabilities on a high level, the diverging viable cell count profiles change over into a robust converging set of profiles. The CHO cells used to validate the procedure could be focused to any specific growth rates below ìmax.
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Comparison of viable cell concentration estimation methods for a mammalian cell cultivation process
M.Aehle, R. Simutis, A.Lübbert, 2010
Abstract: Various mechanistic and black-box models were applied for on-line estimations of viable cell concentrations in fed-batch cultivation processes for CHO cells. Data from six fed-batch cultivation experiments were used to identify the underlying models and further six independent data sets were used to determine the performance of the estimators. The performances were quantified by means of the root mean square error (RMSE) between the estimates and the corresponding off-line measured validation data sets. It is shown that even simple techniques based on empirical and linear model approaches provide a fairly good on-line estimation performance. Best results with respect to the validation data sets were obtained with hybrid models, multivariate linear regression technique and support vector regression. Hybrid models provide additional important information about the specific cellular growth rates during the cultivation.
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Simple adaptive pH control in bioreactors using gain-scheduling methods
S. Gnoth, A. Kuprijanov, R. Simutis, A. Lübbert, 2010
Abstract: A simple well-performing adaptive control technique for pH control in fermentations of recombinant protein production processes is described and its design procedure is explained. First, the entire control algorithm was simulated and parameterized. Afterwards it was tested in real cultivation processes. The results show that this simple technique leads to significant reductions in the fluctuations of the pH values in microbial cultures at a minimum of expenditures. The signal-to-noise ratio and thus the information captured by the pH signal were increased by about an order of magnitude. This leads to a substantial improvement in the noise of many other process signals that are used to monitor and control the process. For instance, respiratory off-gas data of CO2 and its derived carbon dioxide production rate signals from the cultures carry much less noise as compared to those values obtained with conventional pH control. Detailed process analysis revealed that even very small pH jumps of 0.03 values during the fermentation were shown to result in pronounced deflections in CO2-volume fraction of 8% (peak to peak). The proposed controller, maintaining the pH within the interval of 0.01 around the setpoint, reduces the noise considerably.
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Advanced control of dissolved oxygen concentration in fed batch cultures during recombinant protein production
A. Kuprijanov, S. Gnoth, R. Simutis and A. Lübbert, 2009
Abstract Design and experimental validation of advanced pO2 controllers for fermentation processes operated in the fed-batch mode are described. In most situations, the presented controllers are able to keep the pO2 in fermentations for recombinant protein productions exactly on the desired value. The controllers are based on the gain-scheduling approach to parameter-adaptive proportional-integral controllers. In order to cope with the most often appearing distortions, the basic gain-scheduling feedback controller was complemented with a feedforward control component. This feedforward/feedback controller significantly improved pO2 control. By means of numerical simulations, the controller behavior was tested and its parameters were determined. Validation runs were performed with three Escherichia coli strains producing different recombinant proteins. It is finally shown that the new controller leads to significant improvements in the signal-to-noise ratio of other key process variables and, thus, to a higher process quality.
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Control of cultivation processes for recombinant protein production: a review
Gnoth S., Jenzsch M., Simutis R. and Lübbert A., 2008
The current state-of-the-art in control of cultivation processes for recombinant protein production is examined including the quantitative knowledge that can be activated for this purpose and the measurement techniques that can be employed for control at industrial manufacturing sites.
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Product formation kinetics in genetically modified E. coli bacteria: inclusion body formation
Gnoth S., Jenzsch M., Simutis R. and Lübbert A., 2008
A data-driven model is presented that can serve two important purposes. First, the specific growth rate and the specific product formation rate are determined as a function of time and thus the dependency of the specific product formation rate from the specific biomass growth rate. The results appear in form of trained artificial neural networks from which concrete values can easily be computed. The second purpose is using these results for online estimation of current values for the most important state variables of the fermentation process. One only needs online data of the total carbon dioxide production rate (tCPR) produced and an initial value x of the biomass, i.e., the size of the inoculum, for model evaluation. Hence, given the inoculum size and online values of tCPR, the model can directly be employed as a softsensor for the actual value of the biomass, the product mass as well as the specific biomass growth rate and the specific product formation rate. In this paper the method is applied to fermentation experiments on the laboratory scale with an E. coli strain producing a recombinant protein that appears in form of inclusion bodies within the cells’ cytoplasm.
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Process Analytical Technology (PAT): Batch-to-batch reproducibility of fermentation processes by robust process operational design and control
S. Gnoth, M. Jenzsch, R. Simutis and A. Lübbert, 2008
The Process Analytical Technology (PAT) initiative of the FDA is a reaction on the increasing discrepancy between current possibilities in process supervision and control of pharmaceutical production processes and its current application in industrial manufacturing processes. With rigid approval practices based on standard operational procedures, adaptations of production reactors towards the state of the art were more or less inhibited for long years. Now PAT paves the way for continuous process and product improvements through improved process supervision based on knowledge-based data analysis, “Quality-by-Design”-concepts, and, finally, through feedback control. Examples of up-to-date implementations of this concept are presented. They are taken from one key group of processes in recombinant pharmaceutical protein manufacturing, the cultivations of genetically modified Escherichia coli bacteria.
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