Accelerating Virus-Based Biopharmaceuticals Manufacturing

• Autor(a) principal: Nascimento, Sara Colares Costa Gaspar do
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/145752
• topic: flow-through chromatography; capillary electrophoresis; flaviviruses; viral vectors; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Chimeric virus technology has been an explored alternative for vaccination in order to find a safer solution for viral diseases whose virus do not offer the same safety and stability. This is the case of Yellow Fever Virus (YFV) being used as a viral vector against other diseases, such as Dengue fever, Zika infection and Japanese encephalitis. However, this could be difficult due to long process development time span. Hence, the construction of a universal workflow for the purification of YFV is of high interest, so that it is possible to apply it to different chimeric variants. The first part of this thesis presents the evaluation of a purification strategy for YFV involving chromatography in the flow-through mode, performed with a multimodal resin, and a Tangential Flow Filtration step with a 300 kDa cassette. This showed promising results, obtaining a virus recovery yield of 69% and DNA and protein impurity clearance of around 90%. During process development, it is important to have robust analytical tools that allow for fast decision making. The development of an analytical tool for impurity DNA fragment size detection in-process was the second goal of this thesis. Capillary gel electrophoresis conjugated with laser-induced fluorescence (CGE-LIF) was used, and the technique was optimized to identify whether samples are within regulatory authorities’ guidelines for impurity DNA fragment size, i.e., below 200 bp. This technology was used in this work for impurity assessment, both protein and DNA, and for viral protein identification, proving to be a useful analytical not only for process development, but also for final product characterization. Overall, the work presented in this thesis contributed for the optimization of YFV purification process, and for the development of a complementary, highly sensitive, versatile analytical tool for in-process analysis.