Key challenges in designing CHO chassis platforms

Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines.This work has been supported the Federal Ministry for Digital and Economic Affairs (bmwd), the Federal Ministry for Transport, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, Government of Lower Austria and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. A.H. has been supported by the Portuguese NORTE-08-5369-FSE-000053 operation. Additional funding came from the PhD program BioToP (Biomolecular Technology of Proteins) of the Austrian Science Fund (FWF Project W1224) and MIT-Portugal PhD program (Bioengineering Systems). The funding agencies had no influence on the conduct of this research. Open Access Funding by the University of Vienna.info:eu-repo/semantics/publishedVersio

Beyond exponential phase: Metabolic phenotypes in the stationary phase of CHO cell cultures

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Reduced Basal Autophagy and Impaired Mitochondrial Dynamics Due to Loss of Parkinson's Disease-Associated Protein DJ-1

BACKGROUND: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2. CONCLUSIONS/SIGNIFICANCE: We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease

Investigation of ultra fast silicon detectors for ion imaging methods

MedAustron ist eine neue medizinische Einrichtung, in der Krebspatienten mithilfe von Teilchenstrahlung erfolgreich behandelt werden. Für die Bestrahlungsplanung werden bildgebende Verfahren verwendet, um ein 3D-Bild des Tumors und dessen umliegendes Gewebe zu erstellen. Die Erstellung dieser Bilder wird zurzeit mittels Computer Tomo- graphie (CT), also mit Röntgenstrahlung durchgeführt. Aufgrund der unterschiedlichen Wechselwirkung mit Materie durch Photonenstrahlung als Bildgebung und der Teilchenstrahlung zur Behandlung gibt es Unsicherheiten bei der Genauigkeit der Bestrahlung. Wenn man die Bildgebung nun auch mit Teilchenstrahlung durchführen könnte, wäre die Behandlung von Tumoren aufgrund höherer Genauigkeit medizinisch erfolgreicher. Ionen-Computer-Tomographie bzw. Protonen-Computer-Tomographie (pCT) sind sol- che bildgebenden Verfahren an denen zurzeit geforscht wird. Dabei werden Protonen (oder Ionen) mit viel höherer Energie als bei der Strahlentherapie durch den Patien- ten geschickt und verlieren durch die Wechselwirkung mit der Materie an Energie. Die Restenergie und die Ablenkung der Teilchen werden gemessen, um den Energieverlust entlang des Pfades zu rekonstruieren. Dafür werden einerseits ein Kalorimeter zur Energiebestimmung und sogenannte Tracking Detektoren für das Messen der Ablenkung der Teilchen benötigt. In dieser Arbeit wurde die Messung der Restenergie über ein Time- of-flight Kalorimeter durchgeführt, welches auf neuartiger Halbleiterdetektortechnologie basiert. In solch einem Messaufbau kann die benötigte Restenergie indirekt über eine Messung der Zeit erfolgen, die das durchtretende Teilchen für eine bestimmte Strecke benötigt. Diese Zeit wird von LGADs (Low Gain Avalanche Detector) gemessen, die aufgrund ihrer verbesserten Zeitauflösung aktuell gerade für den Einsatz für das CMS Experiment am CERN vorbereitet werden. Das Ziel dieser Diplomarbeit ist die Charak- terisierung und die Optimierung dieser Silizium Detektoren für bildgebende Verfahren in der medizinischen Anwendung.MedAustron is a new medical facility in which cancer patients are successfully treated by the use of irradiation with charged particles. For the treatment planning, medical imaging methods are used to establish a 3D image of the tumor volume and its surroundings. Currently, conventional computer tomography (CT) based on X-rays is used. Due to the different interaction mechanisms of X-rays and charged particles, uncertainties are introduced. An imaging modality based on the same charged particles could reduce these uncertainties and, as a consequence, results in a higher medical success. Such an imaging process is referred to as proton- or ion-CT. Here, protons (or ions) with energies well above the ones used for treatment pass through the patient and lose energy in the process. The residual energy and deflection of the particles are then measured to reconstruct the energy loss along the traversed path. Tracking detectors are used to measure the direction of the traversing particle and the energy is obtained by a calorimeter. In this work, the use of a time-of-flight calorimeter based on a novel semiconductor detector technology is investigated. In such a setup, the residual energy is measured indirectly by the time a particle needs to pass a fixed distance. The duration of the process is then measured by ultra fast silicon detectors, often referred to as Low Gain Avalanche Detectors (LGAD), which are currently being prepared for use at CERN’s CMS experiment. The aim of this master thesis is to characterize and optimize a time-of-flight setup for pCT, based on the above-mentioned silicon detectors.10