Antibody degradation in tobacco plants: a predominantly apoplastic process.

BACKGROUND: Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem. RESULTS: In this study, we investigated the dynamics of the assembly and breakdown of a human IgG(1)κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space. CONCLUSIONS: The results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

Die Anwendung des Convenience-Konzepts auf den Betriebstyp Vending

Die Arbeit wurde mit dem Rudolf Sallinger Preis und dem Preis der Dr. Maria Schaumayer-Stiftung ausgezeichnet. Die am Institut für Handel & Marketing der Wirtschaftsuniversität Wien verfasste Arbeit beschäftigt sich mit dem bisher in der Handelswissenschaft vernachlässigten Betriebstyp Vending, dem Verkauf von Waren über Automaten. Eine umfassende Aufarbeitung des Vending nach terminologischen, historischen, marktrelevanten und betriebstypenspezifischen Aspekten zeigt, dass Konsumenten bei der Automatenverwendung Convenience als wichtige Größe erachten. An eine theoretische Beschäftigung mit Convenience schließt eine kombinierte qualitative und quantitative Untersuchung des Konstrukts im empirischen Teil der Arbeit an. Die Ergebnisse der empirischen Untersuchung bestätigen die große Bedeutung von Convenience beim Vending und sind als Empfehlung für die Betreiber von Automaten gedacht, entsprechende Maßnahmen zur Erhöhung der Convenience ihrer Automaten zu ergreifen

Urban Climate Futures Lab (UCFL): Advancing Inter- and Transdisciplinary Research for Sustainable Urban Development

The Urban Climate Future Lab (UCFL) is a multidisciplinary research initiative designed to investigate and address the complex interplay between urban development, climate change impact as well as mitigation and adaption, with a particular focus on Lower Saxony and its transferability to wider global contexts. It is funded by zukunft.niedersachsen, a funding program of the Lower Saxony Ministry of Science and Culture and the Volkswagen Foundation. UCFL’s unique value lies in bridging the gap between fundamental and transformative research, aligning academic excellence with real-world implementation and stakeholder engagement, reflected within a global perspective

Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications

[EN] Although plant expression systems used for production of therapeutic proteins have the advantage of being scalable at a low price, the downstream processing necessary to obtain pure therapeutic molecules is as expensive as for the traditional Chinese hamster ovary (CHO) platforms. However, when edible plant tissues (EPTs) are used, there is no need for exhaustive purification, because they can be delivered orally as partially purified formulations that are safe for consumption. This economic benefit is especially interesting when high doses of recombinant proteins are required throughout the treatment/prophylaxis period, as is the case for antibodies used for oral passive immunization (OPI). The secretory IgA (SIgA) antibodies, which are highly abundant in the digestive tract and mucosal secretions, and thus the first choice for OPI, have only been successfully produced in plant expression systems. Here, we cover most of the up-todate examples of EPT-produced pharmaceuticals, including two examples of SIgA aimed at oral delivery. We describe the benefits and drawbacks of delivering partially purified formulations and discuss a number of practical considerations and criteria to take into account when using plant expression systems, such as subcellular targeting, protein degradation, glycosylation patterns and downstream strategies, all crucial for improved yield, high quality and low cost of the final product.The authors would like to thank Annick Bleys for assistance with the manuscript preparation. P.J. would like to express gratitude towards the Spanish Ministry of Economy and Competiveness for her FPU fellowship and towards the International Society for Plant Molecular Farming for their generous bursaries for attending the PBVAB 2015. This work was supported by grants from Research Foundation Flanders (FWO project G0C9714N), from the European Commission (H2020-MSCA-IF-2014 Proposal 658701-ImmunoFarm) and from the Spanish Ministry of Economy and Competiveness (Plan Nacional I+D Grant BIO2013-42193R).Juarez, P.; Virdi, V.; Depicker, A.; Orzáez Calatayud, DV. (2016). Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications. Plant Biotechnology Journal. 14(9):1791-1799. https://doi.org/10.1111/pbi.12541S1791179914

Timber-hybrid-infill : urban redensification in Vienna by resource-efficient multilevel timber-hybrid construction for gaps between buildings

Abweichender Titel nach Übersetzung der Verfasserin/des VerfassersDie vorliegende Arbeit untersucht, inwiefern ressourceneffiziente Holzhybridbauweise eine Antwort auf die Nachverdichtung hinsichtlich innerstädtischer Lückenschließung angesichts ökologischer, ökonomischer und gesellschaftlicher Herausforderungen bietet. Des Weiteren wird die Konkurrenzfähigkeit von Holzhybridbauweise überprüft.Der Vergleich von Holz, Beton und Stahl zeigt materialspezifische Eigenschaften sowie Stärken und Schwächen auf. Anhand existierender Beispiele zwischen bebauten Grundstücken, die einen hohen Anteil an erneuerbaren beziehungsweise ökologischen Materialien nachweisen, wird recherchiert, warum welche Baustoffe zum Einsatz kommen und welche Besonderheiten sich für die Nutzer*innen daraus ergeben. Im weiteren Schritt folgt die Festlegung der heutigen und zukünftigen technischen Anforderungen.Die gewonnenen Erkenntnisse finden in einen Entwurf für eine existierende innerstädtische Baulücke zwischen bebauten Grundstücken in Wien Anwendung. Ein Bauträgerwettbewerbsbeispiel des Wohnfonds Wien dient dabei als Grundlage. Es werden Vorteile und Qualitäten des mehrgeschossigen Bauens in Holzhybridbauweise im Rahmen der Lückenschließung erörtert. Die wirtschaftliche Konkurrenzfähigkeit eines Gebäudes durch den hohen Einsatz des Baustoffes Holz, aber auch dessen Kombination mit anderen Baustoffen in einem Entwurf dargelegt. Das Ergebnis ist eine Konstruktion, welche verschiedene Stufen der Nutzungsflexibilität, einen hohen Vorfertigungsgrad sowie raschen Montageablauf bei gleichzeitiger Ressourcenschonung als auch Kohlenstoffspeicherung aufweist.Zudem werden Alternativlösungen für den Einsatz von Holz zugunsten des Brandschutzes dargestellt. Durch das sichtbar Belassen des Konstruktionsmaterials Holz wird zum einen die Signalwirkung nach außen hin gesteigert, während zum anderen ein hoher Wohlfühlfaktor im Inneren des Gebäudes erzielt wird.The following paper examines resource efficient timber-hybrid construction as an answer to redensification of urban infills, facing upcoming ecological, economical and social challenges. In addition the thesis evaluates the competitive abilities of timber-hybrid construction.A comparison of timber, concrete and steel highlights properties specific to each material as well as their strengths and weaknesses. It is followed by an analysis of existing buildings that make up urban infills using a high amount of renewable and ecological materials and what kind of profits their users are gaining from it. As a following step current as well as future technical requirements are determined.The gained knowledge is used to design a building for an urban infill in Vienna. A competition of the Wohnfonds Wien is forming the projects basis.The Design shows advantages and qualities of multilevel timber-hybrid construction used for redensification. The usage of a high amount of timber reveals lifecycle orientated competitive capacities - also in combination with other materials. The result is a structure that shows multiple levels of flexibility, a high grade of prefabrication as well as a quick assembly process while providing resource-efficiency and carbon storage capabilities. Alternative solutions in favor of the usage of timber for fire safety are demonstrated which hold the same level of quality. By the exposing the timber, a signaling effect is achieved on the outside while contributing to a living comfort on the inside of the building.17