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Item type:Publication, Access status: with files , Regulation of pyruvate dehydrogenase complex related to lactate switch in CHO cellsThe metabolism of Chinese hamster ovary (CHO) cell lines is typically characterized by high rates of aerobic glycolysis with increased lactate formation, known as the ”Warburg” effect. Although this metabolic state can switch to lactate consumption, the involved regulations of the central metabolism have only been partially studied so far. An important reaction transferring the lactate precursor, pyruvate, into the tricarboxylic acid cycle is the decarboxylation reaction catalyzed by the pyruvate dehydrogenase enzyme complex (PDC). Among other mechanisms, PDC is mainly regulated by phosphorylation–dephosphorylation at the three sites Ser232, Ser293, and Ser300. In this work, the PDC phosphorylation in antibody-producing CHO DP-12 cell culture is investigated during the lactate switch. Batch cultivations were carried out with frequent sampling (every 6 h) during the transition from lactate formation to lactate uptake, and the PDC phosphorylation levels were quantified using a novel indirect flow cytometry protocol. Contrary to the expected activation of PDC (i.e., reduced PDC phosphorylation) during lactate consumption, Ser293 and Ser300 phosphorylation levels were 33% higher compared to the phase of glucose excess. At the same time, the relative phosphorylation level of Ser232 increased steadily throughout the cultivation (66% increase overall). The intracellular pyruvate was found to accumulate only during the period of high lactate production, while acetyl-CoA showed nearly no accumulation. These results indicate a deactivation of PDC and reduced oxidative metabolism during lactate switch even though the cells undergo a metabolic transition to lactate-based cell growth and metabolism. Overall, this study provides a unique view on the regulation of PDC during the lactate switch, which contributes to an improved understanding of PDC and its interaction with the bioprocess.Publicationtype: Journal ArticleScopus© Citations 5 200 193 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Scopus© Citations 33 376 401 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Quantification of the dynamics of population heterogeneities in CHO cultures with stably integrated fluorescent markers(Springer, 2020-03-04); ; ; ; Cell population heterogeneities and their changes in mammalian cell culture processes are still not well characterized. In this study, the formation and dynamics of cell population heterogeneities were investigated with flow cytometry and stably integrated fluorescent markers based on the lentiviral gene ontology (LeGO) vector system. To achieve this, antibody-producing CHO cells were transduced with different LeGO vectors to stably express single or multiple fluorescent proteins. This enables the tracking of the transduced populations and is discussed in two case studies from the field of bioprocess engineering: In case study I, cells were co-transduced to express red, green, and blue fluorescent proteins and the development of sub-populations and expression heterogeneities were investigated in high passage cultivations (total 130 days). The formation of a fast-growing and more productive population was observed with a simultaneous increase in cell density and product titer. In case study II, different preculture growth phases and their influence on the population dynamics were investigated in mixed batch cultures with flow cytometry (offline and automated). Four cell line derivatives, each expressing a different fluorescent protein, were generated and cultivated for different time intervals, corresponding to different growth phases. Mixed cultures were inoculated from them, and changes in the composition of the cell populations were observed during the first 48 h of cultivation with reduced process productivity. In summary, we showed how the dynamics of population heterogeneities can be characterized. This represents a novel approach to investigate the dynamics of cell population heterogeneities under near-physiological conditions with changing productivity in mammalian cell culture processes.Publicationtype: Journal ArticleScopus© Citations 4 281 183 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation(Springer, 2020-01-31); ; ; ; The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils. In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.Publicationtype: Journal ArticleScopus© Citations 10 172 139 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Data‐driven multiscale modeling of self‐assembly and hierarchical structural formation in biological macromolecular systemsMacromolecular systems are present inmany applications of biotechnology andprocess engineering and the physical phenomena involved therein often spreadover vast scales of size and time. To gaininsight, generally applicable models are developed to transfer the essential dynamics (including directional dependency)[1] and complex interaction of biologicalmacromolecules from MD [2] to DEM ina modeling methodology termed by usthe ‘‘molecular discrete element method’’(MDEM). The models are parameterized bottom-up and validated top-down bycomparison with experimental data, whichis obtained from BLI and DLS. As a modelsystem the multi-enzyme pyruvate dehy-drogenase complex (PDC) is used, whichfeatures organized self-structuring pro-cesses and a highly regulated multi-enzymatic machinery dependent upon thestructure.Obtained results for the PDC compo-nent E2 show that the continuous formation and breakup of enzymatic agglomerates can be predicted using the developedMDEM methodology. This approach requires no experimental data fitting andproduces accurate scale-bridging kineticsas well as agglomerate sizes matching corresponding dynamic light scattering data.Publicationtype: Conference Poster not in Proceedings102 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Near-physiological cell cycle synchronization with countercurrent centrifugal elutriationThe bioreactor conditions and cell diversity in mammalian cell cultures are often regarded as homogeneous. Recently, the influence of various kinds of heterogeneities on production rates receives increasing attention. Besides spatial gradients within the cultivation system, the variation between cell populations and the progress of the cells through the cell cycle can affect the dynamics of the cultivation process. Strong metabolic up- and down-regulations leading to variable productivities, even in exponentially growing cell cultures, have been identified in CHO cell cultivations. Consequently, scientific studies of cell cycle-related effects and metabolic regulations require experiments utilizing cell cycle-enriched subpopulations. Importantly, the enrichment procedure itself must not strongly interfere with the cell culture under investigation. Such subpopulations can be generated by near-physiological countercurrent centrifugal elutriation, which is described in the following chapter. At first, a brief overview regarding the cell cycle, currently identified effects and commonly used methods, and their applicability is outlined. Then, the experimental setup and the synchronization itself are explained.Publicationtype: Book Part130 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Investigation on the influence of high protein concentrations on the thermal reaction behaviour of β-lactoglobulin by experimental and numerical analyses(2019-10); ; ; ; In this study, treatments at various temperature–time profiles were performed for β-lactoglobulin samples at different concentrations (50–70%) using a special rheometer as processing device. Rheological measurements, offline protein chemical analyses, and molecular dynamics analyses were performed to investigate the influence of high protein concentrations and treatment temperature on the denaturation and aggregation behaviour of β-lactoglobulin. Under these conditions, the degree of denaturation and aggregation decreased with increasing protein concentration. This corresponded to a strongly decreased diffusion and increased stability of exposed surface protein regions at high concentrations. Irreversible denaturation was observed for temperatures above 60 °C. Increasing thermal treatment intensity resulted in an increase of aggregation. Depending on the thermal treatment conditions, different protein–protein interactions were measured. By increasing the treatment temperature, the resulting aggregates were increasingly stabilised by covalent bonds. In addition to disulphide bonds, non-disulphide covalent cross-links were formed at temperatures above 100 °C.Publicationtype: Journal Article205 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Direct and highly sensitive measurement of fluorescent molecules in bulk solutions using flow cytometry(2019-04); ; ; Utilizing flow cytometry to monitor progress of bulk biochemical reactions and concentration of chemical species normally relies on the utilization of cells carrying intrinsic fluorescence or modified beads. We present a method for a simple measurement of the fluorescent marker molecule fluorescein and GFPuv in bulk solutions with high sensitivity using a CytoFLEX flow cytometer and without the need for modified beads. Polystyrene beads were used to trigger measurements based on their high scatter signal, to detect the fluorescence signal from two different fluorophores present in the sample solution. We report sensitivities of 33 pg/mL for fluorescein and 50 ng/mL for GFPuv. This method is comparable in sensitivity to a typical spectrometric fluorescence assay tested with fluorescein, and approximately ten times more sensitive for the measurement of GFPuv. PEG was added to the sample at a low concentration of 0.001% (w/v) to block unspecific GFPuv binding to the beads. The method was further applied to measure the GFPuv concentration in crude cell lysate samples used for cell free protein expression. An advantage of this method over spectrometric assays is the ability to differentiate signal subpopulations in the sample based on their individual fluorescence intensities.Publicationtype: Journal Article205 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Toward Multiscale Modeling of Proteins and Bioagglomerates: An Orientation-Sensitive Diffusion Model for the Integration of Molecular Dynamics and the Discrete Element Method(2019-01-28); ; ; ; Most processes involved in biological and biotechnological systems spread over many scales in space and time. For example, the interaction of multiple enzymes in heterogeneous enzymatic agglomerates or clusters, necessary for efficient enzymatic conversion, is of high interest for research and enzyme engineering. In order to understand and predict their overall behavior and performance, it is important to describe these scales as completely as possible, known as multiscale modeling. While many different approaches have been presented in recent years, knowledge about protein formation and bioagglomeration at the micro scale is still very limited. In an attempt to address such systems, we propose a bottom- up multiscale modeling methodology, bridging the gaps between molecular dynamics (MD) with an explicit solvent and the larger scale discrete element method (DEM) using an implicit solvent and abstracting macromolecules (e.g., proteins) as objects with anisotropic properties. We term this approach the molecular discrete element method (MDEM). For this, we present an orientation-sensitive diffusion model for DEM, which describes the dynamics of anisotropic translational and rotational diffusion, while implicitly considering solvent molecules and enforcing a canonical ensemble. A general-purpose model and parametrization approach is presented, which can be used to simulate any process involving diffusion of discrete particles. Effects of temperature and viscosity changes can be considered, and guidance is provided concerning time step selection. This model is generally applicable and serves as a precondition to enforce the proper dynamics (i.e., diffusion characteristics and canonical ensemble, similar to a thermostat in MD) for the proposed multiscale modeling methodology with anisotropic properties. Thereby, it presents a first step toward modeling at the micro scale and is integral to enforcing dynamics of such systems and therefore extensively validated. As a next step, interaction models are to be defined and added to the presented model. In comparison to atomistic and coarse-grained (CG) MD, a speedup of 5-7 orders of magnitude can be achieved. The approach is demonstrated on multiple components of the pyruvate dehydrogenase enzyme complex, a multienzymatic machinery that involves very different types of enzymes and is of high value to further elucidate the mechanisms of bioagglomeration and metabolic channeling.Publicationtype: Journal Article124 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: with files , Process-induced cell cycle oscillations in CHO cultures: Online monitoring and model-based investigation© 2019 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc. The influence of process strategies on the dynamics of cell population heterogeneities in mammalian cell culture is still not well understood. We recently found that the progression of cells through the cell cycle causes metabolic regulations with variable productivities in antibody-producing Chimese hamster ovary (CHO) cells. On the other hand, it is so far unknown how bulk cultivation conditions, for example, variable nutrient concentrations depending on process strategies, can influence cell cycle-derived population dynamics. In this study, process-induced cell cycle synchronization was assessed in repeated-batch and fed-batch cultures. An automated flow cytometry set-up was developed to measure the cell cycle distribution online, using antibody-producing CHO DP-12 cells transduced with the cell cycle-specific fluorescent ubiquitination-based cell cycle indicator (FUCCI) system. On the basis of the population-resolved model, feeding-induced partial self-synchronization was predicted and the results were evaluated experimentally. In the repeated-batch culture, stable cell cycle oscillations were confirmed with an oscillating G1 phase distribution between 41% and 72%. Furthermore, oscillations of the cell cycle distribution were simulated and determined in a (bolus) fed-batch process with up to 25 × 106 cells/ml. The cell cycle synchronization arose with pulse feeding only and ceased with continuous feeding. Both simulated and observed oscillations occurred at higher frequencies than those observable based on regular (e.g., daily) sample analysis, thus demonstrating the need for high-frequency online cell cycle analysis. In summary, we showed how experimental methods combined with simulations enable the improved assessment of the effects of process strategies on the dynamics of cell cycle-dependent population heterogeneities. This provides a novel approach to understand cell cycle regulations, control cell population dynamics, avoid inadvertently induced oscillations of cell cycle distributions and thus to improve process stability and efficiency.Publicationtype: Journal ArticleScopus© Citations 13 341 343