Im Rahmen des beantragten Projektes wird die systematische Aufklärung von Struktur-Funktionszusammenhängen des zielgerichteten Metabolitentransfers in Multienzymkomplexen -- insbesondere auf Basis des Metabolic Channeling -- angestrebt. Eine wesentliche Rolle während aller Arbeitsschritte spielen multiskalige Modelingmethoden: Von der Nanostruktur auf Molekülebene, über die Mesostruktur in Mikrokompartimenten bis hin zur Makroskala, d.h. zum Gesamtprozess und dessen Optimierung. Ziel ist die Realisierung der notwendigen Basistechnologien für effiziente zellfreie enzymkatalysierte synthetische Reaktionskaskaden in vitro unter Ausnutzung dieser Channelingeffekte, um bisher auftretende effizienzmindernde Schwachpunkte, wie z.B. Intermediat-Diffusion und Feedback-Inhibierung, zu vermeiden. Die zu erreichenden Ziele stehen im Einklang mit den während der Fachgespräche von Oktober 2010 bis Januar 2012 formulierten Meilensteinen des BMBF-Strategieprozesses ''Biotechnologie 2020+''.

Fundamentals and process development of cell cultures for application in pharmaceutical industry

Development of general methods and tools of bioprocess and biosystems engineering and their applications for bioprocess development in industrial and pharmaceutical biotechnologies.

Many biological problems (drug discovery, toxicity testing, etc.) cannot be answered with 2D cell cultures adequately. As a consequence, test results obtained in 2D culture are only par-tially transferable to the situation in 3D or in vivo. Even if there are a number of well-described 3-D models, still an on-line characterization of the 3D culture is difficult. Therefore goal of the project is to develop a scaffold with a defined geometric structure and integrated sensors for measuring the impedance as indicator for cell growth activity of the 3D culture of animal and human cells. Such a scaffold with integrated sensors would be a valuable tool for drug screening, e.g. for evaluation of anticancer drugs. Here, different cell culture models growing in 3D come into consideration, such as tumor cell models or liver models. The defined geometric structure of the intended scaffold is designed to allow for substantially uniform flow conditions, as well as to ensure a supply of the cells within the scaffolds. By impedance measurements, growth and distribution of the cells will be evaluated. The results of these measurements will be compared with 2D models.

A rapid sampling and the following sample processing are critical for a reliable metabolomic analysis. Due to the complicated procedure and the high quantity of parameters there are many possibilities for variations and errors. The effective and sufficient separation of the exo- and endometabolome is a specific challenge in this context. Our aim is to develop new technologies and methods to minimize the errors and to design new experiments to investigate and describe cellular metabolism and its regulation. A new fully automated rapid sampling unit with on-chip filtration has been successfully developed which greatly improves the accuracy of intracellular metabolites, especially to overcome the problem of metabolic leakage. The concept combines flexibility and accuracy. The rapid sampling unit is based on a modular system. A specific valve system assures definite sample volumes and efficient mixing of the sample with the quenching solution. A special on-line and fast filtration system was implemented which can handle high cell density culture (up to OD value near 100). This is the first fast sampling system which can work under such extrem conditions more relevant to industrial bioprocesses. The system has been use to study different metabolic regulation in microorganisms such as E. coli and Corynebacterium glutamicum.

The proposal is based on the joint project -Development and characterization of a microfluidic dynamically cultured full-thickness skin model-. The aim of the project is the development of human skin models in dynamic culture conditions as well as their characterization with regard to substance transport, tissue formation and microfluidic. It is based on a multi-organ chip (MOC) as a new technology for modeling of segments of the human organism on a chip. This offers a unique platform for the development of physiologically approximated skin equivalents. By the adaptable construction method and quick design cycles, modular skin models can be developed stepwise including components like fatty tissue, hair follicle or a vascular system. In the current project this skin models were successfully developed and permeability coefficients for the simulation of mass transport and distribution effects with respect to the nutrients and the resulting metabolites in the supply and disposal of cell components as well as for large molecule ingredients were elaborated. Whereas so far the focus was on understanding the penetration of active ingredients, in the follow-up project penetration and permeation of industrially manufactured nanomaterials in human skin models will be studied. For this, new and meaningful study methods for the detection of penetration and permeation of nanomaterials in and through human skin will be established. The chip-based long term skin culture technology developed at TU Berlin will be optimized with respect to nanomaterial exposure and compared to other methods by in vitro and in vivo data.

Ziel des Verbundprojekts ist es, eine Plattform-Technologie für die modell-gestützte Auslegung neuer Bioprozesse zur Nutzung minderwertiger organischer Abfälle für die Herstellung von flüchtigen Fettsäuren (VFA, wie Essig-, Propion-, Butter- und Valeriansäure) mittels Mischkulturfermentation zu entwickeln und in eine Semi-Pilotanlage zu implementieren. Dazu kommen folgende Methoden und Technik im Einsatz: Entwicklung eines bioenergetisch basierten kinetischen Models für die Vorhersagebevorzugter metabolischer Routen von Mischpopulationen; Unterstützung der experimentellen Untersuchungen durch gezielte Optimierung der Betriebsbedingungen anhand des Modells und Werkzeuge der Bioprozesstechnik; Optimierung der mikrobiellen Gemeinschaft für ein gewünschtes Produktspektrum und Produktausbeute durch gezielte Manipulation der Betriebsbedingungen; Erhöhung der Produktivität und Überwindung der Produktinhibierung durch Einsatz einer in-situ Produkttrennungstechnik; Entwicklung einer virtuellen Anlage zur Prozessauslegung für ausgewählte Produkte aus verschiedenen Susbtraten. Kooperationen: Universidade de Santiago de Compostela, Spanien VTT Technical Research Centre of Finland Ltd.

The development and implementation of bio-refinery processes is of crucial importance to build a bio-based economy. The EuroBioRef project will develop a new highly integrated and diversified concept including multiple feedstocks (non-edible), multiple processes (chemical, biochemical, thermochemical), and multiple products (aviation fuels and chemicals). The project has a specific aim to overcome the fragmentation in the biomass industry. As efficiency is the key to the bio-refinery processes, this implies to take decisive actions to facilitate better networking, coordination and cooperation among a wide variety of actors. New synergies, cost efficiencies and improved methods will be achieved by involving the stakeholders at all levels: large and small (bio)chemical industries, academics and researchers from the whole biomass value chain, as well as European organisations. Large-scale research, testing, optimisation and demonstrations of processes in the production of a range of products design adapted to large- and small-scale production units, which will be easier to install in various European areas. The overall efficiency of this approach will be a vast improvement of the existing situation, and will ensure the production of aviation fuels and multiple chemical products in a flexible and optimized way. It will also take advantage of the differences in biomass components and intermediates. The target is also to improve cost efficiency by as much as 30 per cent through improved reaction and separation effectiveness, reduced capital investments, improved plant and feedstock flexibility, and reduction of production time and logistics. Further, we expect to reduce by 30 per cent the energy used and produce zero waste. Raw material management will also mean that a reduction of feedstock consumption will be possible to the tune of at least 10 per cent. The EuroBioRef concept achieves integration across the whole system from feedstock to product diversification and adapts to regional conditions, integrating into existing infrastructures, minimizing risks to investors. The flexible approach means widening bio-refinery implementation to the full geographical range of Europe, and offers opportunities to export bio-refinery technology packages to more local markets and feedstock hotspots. The impact of the project in terms of environment, social and economic benefits is important and could give a serious advantage for European bio-industry. The techno-economic evaluation of the whole integrated biorefinery will be carried out. Moreover, the environmental life cycle assessment studies will be performed in line with the requirements of the International Reference Data System (ILCD) Handbook and the LCI data will be made available via the ILCD Data Network. The approach on social sustainability will be based on the recently developed UNEP guidelines for social life cycle assessment of products, allowing for the required modifications to meet the requirements of respective analysis on biorefinery chains.

Both biology and engineering are entering new areas owing to rapid advances in enabling technologies such as genome sequencing, functional genomics, computation, microfluidics, nanotechnology, systems and synthetic biology. The cluster SynBio studies biological and technological fundamentals of synthetic biology as an emerging new field. In addition to better understanding natural bioprocesses synthetic biology particularly aims at generating efficient and interchangeable parts by molecular-biological and engineering tools or directly from natural biology by screening and assembling them into technologically artificial but useful biological systems. Synthetic biology has thus a high potential for applications such as targeted synthesis of biopharmaceuticals, sustainable chemical industry and energy generation, and production of smart (bio)materials. Parallels have been drawn between the design and manufacture of semiconductor chips in information and communication technologies (ICTs) and the construction of standardized biological parts (also called biobricks) in synthetic biology. Whereas semiconductor and microelectronics have revolutionized ICTs, it is expected that synthetic biology in combination with microfluidics and nanotechnology has similar impacts for biotechnology and life sciences in the near future. The structural and scientific objectives of SynBio are to establish an interdisciplinary and excellent research cluster in Hamburg with focus on studying fundamentals for developing novel synthetic biocatalytic pathways and systems with applications in biotechnology and life sciences.

Die Entwicklung und Optimierung biobasierter Produkte ist noch stark empirisch geprägt. Dazu werden häufig „Design of Experiment“ (DoE)-Methoden eingesetzt, die eine hohe Anzahl an zeitintensiven Experimenten erfordern. Die Konzeption, insbesondere die Eingrenzung des Parameterraumes, ist schwierig und die Aussagekraft häufig begrenzt. Die zentrale Projektidee besteht darin, modulare Software-Tools anzubieten, die DoE-Strategien mit mathematischen Prozessmodellen verknüpfen und somit den experimentellen Aufwand signifikant reduzieren und die Aussagekraft eines DoE entscheidend erhöhen. In der Sondierungsphase wurde der hohe Bedarf des Software-Tools abgeschätzt und in einer on-line Umfrage bestätigt. In der Machbarkeitsphase soll die Software-Toolbox zusammen mit den Partnern schrittweise entwickelt, evaluiert und ihre Leistungsfähigkeit an relevanten bioökonomischen Beispielprozessen demonstriert werden.

Upscaling of bioprocesses is often accompanied by loss in productivity compared to well-mixed lab-scale processes. One reason is the formation of population heterogeneity whose mechanistic understanding is still comparably low. During the first funding period (project ProPHet) quantitative understanding of population heterogeneity originating from cell-bioreactor interaction in fed-batch processes with E. coli FUS4 (pF81kan) converting glycerol to L-phenylalanine (L-Phe) was raised. Experimental investigation was done with a quadruple reporter strain monitoring growth, general stress response, oxygen availability and product formation of single cells. Cultivations in a well-mixed stirred-tank bioreactor (STR) were compared with large-scale bioprocess conditions simulated in a scale-down reactor system consisting of a STR and a novel by-pass reactor that is designed as a coiled flow inverter (CFI). Theoretical investigation was done with population balance equations and agent based modeling after generating a non-segregated coarse-grained model. Building upon results from ProPHet, ProPHet2Con is aiming at controlling population heterogeneity induced variations in process performance in the fed-batch processes for L-Phe production. A population heterogeneity level that is beneficial for process performance will be maintained by promoting the abundance of subpopulations with advantageous characteristics. A special focus will be put on the process in the STR-CFI. Prio to implementation of process control, the L-Phe production process will be optimized applying the coarse-grained model and stoichiometric modeling to improve final product titer and productivity, especially in the STR-CFI setup. As a prerequisite for influencing the level of population heterogeneity by automated process control, manual at-line flow cytometry measurement will be replaced by automated real-time flow cytometry (ART-FCM). The ART-FCM setup will be advanced by the previously published process concept segregostat, that allows to control the degree of diversification of a bioreactor population at a predefined setpoint by single-cell physiology-based feeding of the carbon source. Furthermore, model-based soft sensors will implemented to estimate quantities cannot be measured directly. Then, based on the outcome of experimental investigations, strategies will be formulated to influence the level of population heterogeneity by model-based process feedback control following ART-FCM evaluation of single cell physiology. To our knowledge such a coupling between ART-FCM and an advanced model-based process control has so far never been realized. The formulated strategies will be experimentally validated in the fed-batch process for L-Phe production in the STR and the STR-CFI.Once process control for L-Phe production is realized, scale-up potential of the process to pilot-scale will be investigated.

Schwere Durchfallerkrankungen (Diarrhöe) sind mit über 40 % die häufigste Todesursache bei Kälbern. Die Behandlung ist sehr pflegeintensiv, da die betroffenen Tiere trotz Behandlung mit Antibiotika über lange Zeit sehr geschwächt sind. Eine antibiotika-freie, natürliche Behandlung kann nach neuesten wissenschaftlichen Erkenntnissen durch Zugabe von natürlichen Milcholigosacchariden (MOS) im Futter erfolgen. Allerdings fehlt es derzeit an Technologien zur industriellen Produktion von MOS. Die chemische Synthese von MOS ist sehr aufwändig und wirtschaftlich uninteressant. Eine wirtschaftliche Produktion von Milcholigosacchariden erfordert daher einen neuen biokatalytischen Ansatz auf Basis eines GRAS-Organismus (generally-regarded-as-safe). Die Steuerung von Fermentationsprozessen erfolgt heute meist empirisch und beruht häufig auf Erfahrungswerten. Dies erschwert die Optimierung und kann besonders bei neuen Verfahren sehr langwierig und damit teuer sein. Es fehlen geeigneten Modelle, die die komplexen Prozesse abbilden und Vorhersagen für eine optimale Steuerung machen. Die Weiterentwicklungen der Prozessleitsystemsoftware WinErs durch IB Schoop wird neue Einsatzmöglichkeiten in der Biotechnologie eröffnen und an dem Prozess beispielhaft die Möglichkeiten einer intelligenten modellgestützten Prozessführung zeigen. Ziel des Projektes ist die biotechnologische Produktion von natürlichen Milcholigosacchariden mit Hilfe modellgestützter Prozessentwicklungssysteme auf der Basis des Prozessleit- und Simulationssystems WinErs, um so der biopharmazeutischen Industrie Zugang zu therapeutisch relevanten Grundstrukturen wie Sialyllactose für die Behandlung schwerer Darmerkrankungen zur Verfügung zu stellen. Durch die Kombination von wissenschaftlicher Exzellenz der TU Hamburg und der Innovationskraft kleiner und mittlerer Unternehmen wollen die Antragsteller dazu beitragen, Hamburg zu einem national und international führenden Standort der industriellen Biotechnologie zu entwickeln.

prot P.S.I. ist eine unternehmerisch geführte Forschungs- und Entwicklungsallianz im Rahmen des Förderprogramms „Innovationsinitiative industrielle Biotechnologie“. Das übergeordnete Ziel der strategischen Innovationsallianz prot P.S.I. ist, die industrielle Biotechnologie und somit eine Biologisierung in der Feinchemie durch branchenübergreifende Nutzbarmachung des Prozessparameters „Druck“ voranzutreiben. Hierzu haben sich die Allianzpartner durch koordinierte Zusammenarbeit entlang einer Wertschöpfungskette zur Aufgabe gesetzt, die kritische Stabilitätsgrenze von Proteinen und somit ihre Reaktivität bei (hohem) Druck zu identifizieren und für verfahrenstechnische Prozesse, primär im Bereich der industriellen Feinchemie, nutzbar zu machen. Durch die gewonnenen Erkenntnisse soll eine Steigerung von Prozess-Effizienzen erreicht werden, welche branchenübergreifend auch in anderen Bereichen der industriellen Bioprozesstechnologie und biokatalytischen Produktionsverfahren Anwendung finden kann. Das wesentliche Ziel des Teilprojektes C2 im Rahmen des Verbundprojektes prot P.S.I. ist die Entwicklung von softwarebasierten Werkzeugen für die Prozessentwicklung für Prozesse unter Druck, speziell enzymatische Prozesse. Hierzu werden von der Fa. IB Schoop sowie den AG´s Hass und Pörtner zunächst die Werkzeuge zum schnellen Prozesstransfer druckbeeinflusster biotechnischer Prozesse entwickelt und für Prozessleitsysteme (wie z.B. WinErs, WinCC) nutzbar gemacht. Hierzu gehört auch die Entwicklung neuer Regelungsstrategien und ggf. Algorithmen für biotechnische Prozesse unter Druck. In der ersten Phase wird der Schwerpunkt bei der Modellierung und Einbindung der Modellierung in WinErs (inkl. Parameterschätzung etc.) liegen, in der 2. Phase Fokus auf modellgestützter Prozessführung. Für das komplexe enzymatische System, das bei verschiedenen Bedingungen (Druck niedrig, hoch, suspendiert, immobilisiert, Strömungsrohr) ablaufen soll, stellt dies eine große Herausforderung und wurde in der Form in der Literatur auch noch nicht beschrieben. 1.) Enzymsysteme (einzeln und in Kombination gemäß C1) unter Normalbedingungen in Suspension und immobilisiert an suspendierten Trägern (experimentelle Umsetzung bei GALAB); 2.) Enzymsysteme (einzeln und in Kombination gemäß C1) unter Druck in Suspension und immobilisiert an suspendierten Trägern (experimentelle Umsetzung bei AG Liese, C1), 3.) Enzymsysteme in UHPLC gemäß C1) unter Druck (experimentelle Umsetzung in C1 bei AG Liese und GALAB) Die in C1 eingesetzten Enzyme werden von GALAB hergestellt. In C2 werden von GALAB reaktionskinetische Daten unter Normalbedingungen generiert und an AG´s Pörtner und Hass weitergegeben. Reaktionskinetische Daten unter Druck (Suspension und immobilisiert in UHPLC) werden von AG Liese in C1 ermittelt. Kooperationen: GALAB GmbH, HamburgIngenieurbüro Dr.-Ing. Schoop GmbHProf. Dr.-Ing. Volker C. Hass, HS Furtwangen

Motivation Targeted formation of stable enzyme clusters and agglomerates is of highest interest for the construction of efficient and adjustable bioreaction cascades Nowadays no suitable methodology available to predict formation and stability of clusters and agglomerates Impact of process parameters – pH, temperature, mechanical stress – on cluster and aggregate formation and functionality yet hardly investigated Objectives of this project: Identify mechanisms and determinants responsible for building functional and efficient agglomerates. Develop and validate solid model framework to describe and predict dynamic formation and reformation of multi-enzymatic complex clusters and agglomerates Influence of process conditions on formation and catalytic efficiency

tIt is conventionally assumed that mammalian cell cultures cultivated in bioreactors exhibit a homogeneous and indifferent kinetic behavior. However, real cell cultures consist of a mixture of multiple subpopulations with variable composition that interact with each other via the culture medium. A major cause for the occurrence of such subpopulations is the progress of individual cells within the cell cycle. Potential metabolic and regulatory variations of the behavior of mammalian cell cultures during the different cell cycle phases have not been systematically examined in literature. Experimental results are often incomplete and contradictory, leading to intense disputes with a spectrum of opinions ranging from the assumption of completely eventless metabolism and regulation in the cell cycles towards extremely complex and not mechanistically explained cell-cycle dependent regulation cascades. A main reason for these discrepancies lies in incompletely validated or non-physiological synchronization methods and insufficient statistic analysis and proper model description. During the last few years, our group has established the necessary framework regarding process control, cell culture techniques and modeling in order to conduct systematic kinetic and well validated experimental examinations of cell-cycle related metabolic processes and regulations in cell cultures under widely undisturbed, that is process relevant physiological conditions. First analyses already indicated cell cycle dependent variations of cell mass specific substrate turnover rates. In the proposed project, a systematic elucidation of relevant cell cycle specific metabolic processes in cell cultures under process conditions, especially at high cell density, shall be performed utilizing population based analysis and statistic evaluation methods. The focus is especially laid on the regulation kinetics of the pyruvate metabolism under stress conditions and the influence of high cell density on growth kinetics and central metabolism. Furthermore, we intend also to explore the possibility of using such population-based kinetic relationships for optimal control of process stability under variable cultivation conditions.