4 research outputs found
Continuous processing of viral gene therapy vectors
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Standardized Production of Anti-Desmoglein 3 Antibody AK23 for Translational Pemphigus Vulgaris Research.
No full textAntibody-mediated receptor activation is successfully used to develop medical treatments. If the activation induces a pathological response, such antibodies are also excellent tools for defining molecular mechanisms of target receptor malfunction and designing rescue therapies. Prominent examples are naturally occurring autoantibodies inducing the severe blistering disease pemphigus vulgaris (PV). In the great majority of patients, the antibodies bind to the adhesion receptor desmoglein 3 (Dsg3) and interfere with cell signaling to provoke severe blistering in the mucous membranes and/or skin. The identification of a comprehensive causative signaling network downstream of antibody-targeted Dsg3 receptors (e.g., shown by pharmacological activators or inhibitors) is currently being discussed as a basis to develop urgently needed first-line treatments for PV patients. Although polyclonal PV IgG antibodies have been used as proof of principle for pathological signal activation, monospecific anti-Dsg3 antibodies are necessary and have been developed to identify pathological Dsg3 receptor-mediated signal transduction. The experimental monospecific PV antibody AK23, produced from hybridoma cells, was extensively tested in our laboratory in both in vitro and in vivo models for PV and proved to recapitulate the clinicopathological features of PV when generated using the standardized production and purification protocols described herein. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Bovine IgG stripping from FBS and quality control Basic Protocol 2: AK23 hybridoma expansion and IgG production Basic Protocol 3: AK23 IgG purification Basic Protocol 4: AK23 IgG quality control Support Protocol 1: Detection of endotoxin levels Support Protocol 2: Detection and removal of mycoplasma
Continuous purification of a parvovirus using two aqueous two-phase extraction steps
No full textAqueous two-phase systems (ATPS) are a liquid–liquid extraction method that offers low-cost, continuous-adaptable virus purification. A two-step ATPS using polyethylene glycol (PEG) and sodium citrate that recovered 66% of infectious porcine parvovirus with 2.0 logs of protein removal and 1.0 logs of DNA removal in batch has now been run continuously. The continuous system output of \u3c 10 ng/mL DNA regardless of starting DNA titer agreed with batch studies. However, the continuous system had a five-fold higher contaminating protein titer than batch studies, likely because of incomplete mixing or settling. Turbidity was tested as a measure of mixing and settling efficiency. Monitoring in-line absorbance at 880 nm directly after mixing and before collection in the settling reservoir could track both mixing and settling during operation. Settling time was reduced by changing the settling line material from PVC to PTFE, which is more hydrophobic. A flow-through AEX filter tested to make impurity removal more robust recovered 90% of PPV and removed an additional 87% of host cell DNA. The filter did not add any additional protein removal. In the future, in-line absorbance sensors could be implemented along with conductivity sensors to measure salt concentration, refractive index sensors to track the PEG-citrate interface, and scales to track mixer and reservoir volumes to enable automated, continuous ATPS. Our vision is to integrate continuous ATPS into a fully continuous end-to-end production for viral vectors
Co-creation, innovation, decision-making, tech-transfer, and sustainability actions
No full textFunding Information: Open access funding provided by FCT|FCCN (b-on). This work was funded by the European Union’s Horizon 2020 program [H2020-SC5-2019–2]—869520 NextLand, [H2020-SPACE-202]—101004362 NextOcean, Fundação para a Ciência e a Tecnologia (UIDB/00124/2020 and Social Sciences DataLab, PINFRA/22209/2016), POR Lisboa and POR Norte (Social Sciences DataLab, PINFRA/22209/2016). Publisher Copyright: © 2023, The Author(s).European Community (EC) Horizon-funded projects and Earth Observation-based Consortia aim to create sustainable value for Space, Land, and Oceans. They typically focus on addressing Sustainable Development Goals (SDGs). Many of these projects (e.g. Commercialization and Innovation Actions) have an ambitious challenge to ensure that partners share core competencies to simultaneously achieve technological and commercial success and sustainability after the end of the EC funds. To achieve this ambitious challenge, Horizon projects must have a proper governance model and a systematized process that can manage the existing paradoxical tensions involving numerous European partners and their respective agendas and stakeholders. This article presents the VCW-Value Creation Wheel (Lages in J Bus Res 69: 4849–4855, 2016), as a framework that has its roots back in 1995 and has been used since 2015 in the context of numerous Space Business, Earth Observation, and European Community (EC) projects, to address complex problems and paradoxical tensions. In this article, we discuss six of these paradoxical tensions that large Horizon Consortia face in commercialization, namely when managing innovation ecosystems, co-creating, taking digitalization, decision-making, tech-transfer, and sustainability actions. We discuss and evaluate how alliance partners could find the optimal balance between (1) cooperation, competition, and coopetition perspectives; (2) financial, environmental, and social value creation; (3) tech-push and market-pull orientations; (4) global and local market solutions; (5) functionality driven and human-centered design (UX/UI); (6) centralized and decentralized online store approaches. We discuss these challenges within the case of the EC H2020 NextLand project answering the call for greening the economy in line with the Sustainable Development Goals (SDGs). We analyze NextLand Online Store, and its Business and Innovation Ecosystem while considering the input of its different stakeholders, such as NextLand’s commercial team, service providers, users, advisors, EC referees, and internal and external stakeholders. Preliminary insights from a twin project in the field of Blue Economy (EC H2020 NextOcean), are also used to support our arguments. Partners, referees, and EC officers should address the tensions mentioned in this article during the referee and approval processes in the pre-grant and post-grant agreement stages. Moreover, we propose using the Value Creation Wheel (VCW) method and the VCW meta-framework as a systematized process that allows us to co-create and manage the innovation ecosystem while engaging all the stakeholders and presenting solutions to address these tensions. The article concludes with theoretical implications and limitations, managerial and public policy implications, and lessons for Horizon Europe, earth observation, remote sensing, and space business projects.publishersversionpublishe
