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Item type:Publication, Access status: without files , Advanced Process and Control Strategies for BioreactorsBioreactor processes have to provide an almost optimal environment to microorganisms or cells to promote growth and product formation. The design and operation of a bioreactor as the main element of fermentation is a complex task, not only with respect to a reactor's configuration and size but to the control and mode of operation. In this chapter, some fundamentals are discussed, including process and control strategies and concepts for process development. Next, examples for the use of model-based concepts for the design of experiments, feeding strategies, seed train layout, and control strategies, including simulation tools supporting biotechnological training and education, are shown.Publicationtype: Book Part151 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Model-based strategy for cell culture seed train layout verified at lab scaleCell culture seed trains—the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale—are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HNAAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HNAAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.Publicationtype: Journal Article52 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Synchronized mammalian cell culture: part II-population ensemble modeling and analysis for development of reproducible processesThe consideration of inherent population inhomogeneities of mammalian cell cultures becomes increasingly important for systems biology study and for developing more stable and efficient processes. However, variations of cellular properties belonging to different sub-populations and their potential effects on cellular physiology and kinetics of culture productivity under bioproduction conditions have not yet been much in the focus of research. Culture heterogeneity is strongly determined by the advance of the cell cycle. The assignment of cell-cycle specific cellular variations to large-scale process conditions can be optimally determined based on the combination of (partially) synchronized cultivation under otherwise physiological conditions and subsequent population-resolved model adaptation. The first step has been achieved using the physical selection method of countercurrent flow centrifugal elutriation, recently established in our group for different mammalian cell lines which is presented in Part I of this paper series. In this second part, we demonstrate the successful adaptation and application of a cell-cycle dependent population balance ensemble model to describe and understand synchronized bioreactor cultivations performed with two model mammalian cell lines, AGE1.HNAAT and CHO-K1. Numerical adaptation of the model to experimental data allows for detection of phase-specific parameters and for determination of significant variations between different phases and different cell lines. It shows that special care must be taken with regard to the sampling frequency in such oscillation cultures to minimize phase shift (jitter) artifacts. Based on predictions of long-term oscillation behavior of a culture depending on its start conditions, optimal elutriation setup trade-offs between high cell yields and high synchronization efficiency are proposed.Publicationtype: Journal Article55 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Synchronized mammalian cell culture: part I-a physical strategy for synchronized cultivation under physiological conditions(Wiley, 2014-07-13); ; ; ; Conventional analysis and optimization procedures of mammalian cell culture processes mostly treat the culture as a homogeneous population. Hence, the focus is on cell physiology and metabolism, cell line development, and process control strategy. Impact on cultivations caused by potential variations in cellular properties between different subpopulations, however, has not yet been evaluated systematically. One main cause for the formation of such subpopulations is the progress of all cells through the cell cycle. The interaction of potential cell cycle specific variations in the cell behavior with large-scale process conditions can be optimally determined by means of (partially) synchronized cultivations, with subsequent population resolved model analysis. Therefore, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical selection methods, but not with frequently used chemical or whole-culture methods. Conventional nonsynchronizing methods with subsequent cell-specific, for example, flow cytometric analysis, can only resolve cell-limited effects of the cell cycle. In this work, we demonstrate countercurrent-flow centrifugal elutriation as a useful physical method to enrich mammalian cell populations within different phases of a cell cycle, which can be further cultivated for synchronized growth in bioreactors under physiological conditions. The presented combined approach contrasts with other physical selection methods especially with respect to the achievable yield, which makes it suitable for bioreactor scale cultivations. As shown with two industrial cell lines (CHO-K1 and human AGE1.HN), synchronous inocula can be obtained with overall synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the G2/M phase, with enrichment factors ( Ysync) of 1.71, 1.79, and 4.24 respectively. Cells are able to grow with synchrony in bioreactors over several cell cycles. This strategy, combined with population-resolved model analysis and parameter extraction as described in the accompanying paper, offers new possibilities for studies of cell lines and processes at levels of cell cycle and population under physiological conditions.Publicationtype: Journal Article51 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Process and cultivation strategies for the human industrial cell line AGE1.HN(2014); This work focuses on the development of process and cultivation strategies for a systematic characterization and optimization of mammalian cell cultures, especially for the novel human industrial cell line AGE1.HN. This cell line was studied within the collaborative project SysLogics -Systems Biology of Cell Culture for Biologics- with the final goal of generating quantitative data on mammalian cell metabolism. The strategies developed in this work establish a solid basis for systematic studies with mammalian production cell lines, by allowing for their characterization from an engineering point of view, and the implementation of results in systems biology research.Publicationtype: Doctoral Thesis495 1642 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , A New Integrated Lab-on-a-Chip System for Fast Dynamic Study of Mammalian Cells under Physiological Conditions in Bioreactor(Multidisciplinary Digital Publishing Institute (MDPI), 2013-05-27); ; ; ; For the quantitative analysis of cellular metabolism and its dynamics it is essential to achieve rapid sampling, fast quenching of metabolism and the removal of extracellular metabolites. Common manual sample preparation methods and protocols for cells are time-consuming and often lead to the loss of physiological conditions. In this work, we present a microchip-bioreactor setup which provides an integrated and rapid sample preparation of mammalian cells. The lab-on-a-chip system consists of five connected units that allow sample treatment, mixing and incubation of the cells, followed by cell separation and simultaneous exchange of media within seconds. This microsystem is directly integrated into a bioreactor for mammalian cell cultivation. By applying overpressure (2 bar) onto the bioreactor, this setup allows pulsation free, defined, fast, and continuous sampling. Experiments evince that Chinese Hamster Ovary cells (CHO-K1) can be separated from the culture broth and transferred into a new medium efficiently. Furthermore, this setup permits the treatment of cells for a defined time (9 s or 18 s) which can be utilized for pulse experiments, quenching of cell metabolism, and/or another defined chemical treatment. Proof of concept experiments were performed using glutamine containing medium for pulse experiments. Continuous sampling of cells showed a high reproducibility over a period of 18 h.Publicationtype: Journal ArticleScopus© Citations 12 377 239 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Batch-to-batch variability of two human designer cell lines - AGE1.HN and AGE1.HN.AAT : carried out by different laboratories under defined culture conditions using a mathematical model(Wiley-VCH, 2013-03-18); ; ; ; Systems biology approaches involve collaboration of a larger number of research groups. Experiments are being performed in different laboratories dealing with different aspects of the topic of interest. Therefore, comparability of data collected for further analysis and modeling needs critical assessment. Here, growth and product formation of two human designer cell lines (AGE1.HN and its α1-antitrypsin producing clone AGE1.HN.AAT) were investigated by four research laboratories. Cell lines were cultivated in shake flasks and stirred tank bioreactors operated at standardized conditions using a chemically defined medium, and a simple mathematical model was used to estimate characteristic process parameters. Results obtained for 35 batches showed that neither the initial viable cell concentration nor the initial concentration of glucose and glutamine showed significant differences between laboratories. For these measurements with low variations, specific growth rate and yields varied between 8.5 and 26% (relative standard error), indicating comparability of cultivations between laboratories for exponential growth. Higher variations of fitted parameters related to measurements with high initial variation. Comparing the nonproducing with the producing cell line, no significant differences were found regarding growth dynamics and metabolism. Overall, it seems justified to draw conclusions based on the entire experimental dataset of this systems biology project. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.Publicationtype: Journal Article235 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Protein-protein-interaction network organization of the hypusine modification system(American Society for Biochemistry and Molecular Biology, HighWire Press, 2012-08-10); ; ; ; Hypusine modification of eukaryotic initiation factor 5A (eIF-5A) represents a unique and highly specific post-translational modification with regulatory functions in cancer, diabetes, and infectious diseases. However, the specific cellular pathways that are influenced by the hypusine modification remain largely unknown. To globally characterize eIF-5A and hypusine-dependent pathways, we used an approach that combines large-scale bioreactor cell culture with tandem affinity purification and mass spectrometry: "bioreactor-TAP-MS/MS." By applying this approach systematically to all four components of the hypusine modification system (eIF-5A1, eIF-5A2, DHS, and DOHH), we identified 248 interacting proteins as components of the cellular hypusine network, with diverse functions including regulation of translation, mRNA processing, DNA replication, and cell cycle regulation. Network analysis of this data set enabled us to provide a comprehensive overview of the protein-protein interaction landscape of the hypusine modification system. In addition, we validated the interaction of eIF-5A with some of the newly identified associated proteins in more detail. Our analysis has revealed numerous novel interactions, and thus provides a valuable resource for understanding how this crucial homeostatic signaling pathway affects different cellular functions.Publicationtype: Journal ArticleScopus© Citations 15 286 371