An adsorbent monolith device to augment the removal of uraemic toxins during haemodialysis

Adsorbents designed with porosity which allows the removal of protein bound and high molecular weight uraemic toxins may improve the effectiveness of haemodialysis treatment of chronic kidney disease (CKD). A nanoporous activated carbon monolith prototype designed for direct blood contact was first assessed for its capacity to remove albumin bound marker toxins indoxyl sulphate (IS), p-cresyl sulphate (p-CS) and high molecular weight cytokine interleukin-6 in spiked healthy donor studies. Haemodialysis patient blood samples were then used to measure the presence of these markers in pre- and post-dialysis blood and their removal by adsorbent recirculation of post-dialysis blood samples. Nanopores (20–100 nm) were necessary for marker uraemic toxin removal during in vitro studies. Limited removal of IS and p-CS occurred during haemodialysis, whereas almost complete removal occurred following perfusion through the carbon monoliths suggesting a key role for such adsorbent therapies in CKD patient care

Enzymes in medical applications: investigating the enzyme horseradish peroxidase

Zsfassung in dt. SpracheIm Zentrum dieser Diplomarbeit stand das Pflanzenenzym Meerrettich-Peroxidase (MRP) sowie dessen Verwendungen in der medizinischen Diagnostik und gezielten Krebs Therapie. Aktuell wird dieses Enzym aus der Pflanze extrahiert, allerdings erhält man nur geringe Ausbeuten sowie eine Mischung aus verschiedenen Isoenzymen mit unterschiedlichen biochemischen Eigenschaften. Dies ist aber widersprüchlich zu Quality by Design sowie FDA Richtlinien. Ein weiterer Nachteil liegt in der pflanzlichen Glykosylierung des Enzyms, welche zu einem raschen Abbau im menschlichen Körper führt. Das Ziel dieser Arbeit war die rekombinante Produktion, Reinigung und Charakterisierung eines maßgeschneiderten MRP Enzyms. Da dies eine sehr vielfältige Aufgabe war, wurde die Diplomarbeit in drei Teile aufgeteilt. 1. Bis jetzt wurde ein Großteil der Studien nur am MRP Isoenzym C1A durchgeführt. Wir produzierten daher 18 neuartige MRP Isoenzyme rekombinant, charakterisierten diese und versuchten eine vorhandene Reinigungsstrategie zu optimieren. Es war möglich neue potentielle Kandidaten für spezifische Diagnose Kits zu finden und die Reinigungsstrategie zu optimieren. 2. Um die von Hefen durchgeführte Hypermannosylierung von Glykoproteinen in den Griff zu bekommen, wurde ein Glycoengineering Ansatz getestet. Hierfür wurden alle 8 N-Glykosylierungsstellen der MRP C1A einzeln mutiert und jeweils die am besten geeignetste Mutation ermittelt. Anschließend wurden alle 8 Stellen mutiert, um MRP C1A ohne N-Glykosylierung herzustellen. Obwohl dieser Mutant kaum katalytische Aktivität zeigte, stellt das Gesamtergebnis eine solide Basis für zukünftige Enzymmodifikationen dar. 3. Das dritte Projekt befasste sich mit der Herstellung von MRP in einem modifizierten P. pastoris Stamm. Hierfür wurde die Mannosyltransferase OCH1, welche Hypermannosylierung initiiert, inaktiviert. Obwohl dieser Stamm sehr schwer zu kultivieren war und einen im Wachstum gehemmten Phänotyp zeigte, war es möglich, ein homogener glykosyliertes Protein zu erzeugenThis diploma thesis focuses on the plant enzyme horseradish peroxidase (HRP) and its application in medical diagnostics and targeted cancer treatment. Currently, HRP is isolated from plant, resulting in low yields and a mixture of different HRP isoenzymes with varying biochemical properties as final enzyme preparation. This strongly contradicts Quality by Design guidelines and FDA regulations. Another major drawback lies in the foreign glycosylation of the plant enzyme leading to a rapid clearance from the human body. The objective of the present work was the recombinant production, purification and characterization of a specifically tailored HRP enzyme. Because this was a rather versatile task, the thesis was divided into three parts. 1. Until now, the majority of studies only dealt with isoenzyme HRP C1A. Therefore, we recombinantly produced 18 novel HRP isoenzymes and performed subsequent biochemical characterization as well as an optimization of the existing downstream procedure for hyperglycosylated HRP derived from yeast. We were able to identify potential candidates for specific diagnostic applications and to optimize the present downstream procedure. 2. In order to handle the tendency of yeasts for hypermannosylating glyco-proteins we tested a glycoengineering approach. Thus, all 8 N-glycosylation sites of HRP C1A were mutated and the most suitable mutation was determined. Afterwards we combined the 8 most promising mutations in order to produce active HRP C1A without N glycosylation. Although this mutant hardly showed catalytic activity the overall outcome described a useful basis for further enzyme engineering approaches. 3. The third project dealt with the production of HRP in a modified P. pastoris strain. Therefore, the mannosyltransferase OCH1, which triggers hyper-mannosylation, was knocked out. Although this strain was hard to cultivate and showed a growth-impaired phenotype, more homogeneously glycosylated protein could be produced at an adequate production rate

Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors

Ion-exchange chromatography coupled to light scattering detectors represents a fast and simple analytical method for the assessment of multiple critical quality attributes (CQA) in one single measurement. The determination of CQAs play a crucial role in Adeno-Associated Virus (AAV)-based gene therapies and their applications in humans. Today, several different analytical techniques, including size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), qPCR or ELISA, are commonly used to characterize the gene therapy product regarding capsid titer, packaging efficiency, vector genome integrity, aggregation content and other process-related impurities. However, no universal method for the simultaneous determination of multiple CQAs is currently available. Here, we present a novel robust ion-exchange chromatography method coupled to multi-angle light scattering detectors (IEC-MALS) for the comprehensive characterization of empty and filled AAVs concerning capsid titer, full-to-total ratio, absolute molar mass of the protein and nucleic acid, and the size and polydispersity without baseline-separation of both species prior to data analysis. We demonstrate that the developed IEC-MALS assay is applicable to different serotypes and can be used as an orthogonal method to other established analytical techniques

Adeno-Associated Virus-like Particles’ Response to pH Changes as Revealed by nES-DMA

Gas-phase electrophoresis on a nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) separates single-charged, native analytes according to the surface-dry particle size. A volatile electrolyte, often ammonium acetate, is a prerequisite for electrospraying. Over the years, nES GEMMA has demonstrated its unique capability to investigate (bio-)nanoparticle containing samples in respect to composition, analyte size, size distribution, and particle numbers. Virus-like particles (VLPs), being non-infectious vectors, are often employed for gene therapy applications. Focusing on adeno-associated virus 8 (AAV8) based VLPs, we investigated the response of these bionanoparticles to pH changes via nES GEMMA as ammonium acetate is known to exhibit these changes upon electrospraying. Indeed, slight yet significant differences in VLP diameters in relation to pH changes are found between empty and DNA-cargo-filled assemblies. Additionally, filled VLPs exhibit aggregation in dependence on the applied electrolyte’s pH, as corroborated by atomic force microscopy. In contrast, cryogenic transmission electron microscopy did not relate to changes in the overall particle size but in the substantial particle’s shape based on cargo conditions. Overall, we conclude that for VLP characterization, the pH of the applied electrolyte solution has to be closely monitored, as variations in pH might account for drastic changes in particles and VLP behavior. Likewise, extrapolation of VLP behavior from empty to filled particles has to be carried out with caution