Dissertação para obtenção do Grau de Mestre em
BiotecnologiaViruses are highly efficient vectors that have been used for vaccination and gene therapy applications. However, their complexity renders downstream process particularly challenging since devices and strategies especially designed for virus purification are still lacking or need
further optimization. After an introduction to the challenges of virus purification and the current strategies being employed, this dissertation presents the study of three different stages of the downstream process: clarification, ultrafiltration and chromatography.
A novel clarification procedure based on diatomaceous earth was evaluated. Small-scale
batch incubations led to the identification of Divergan RS – a synthetic non-charged material – as the most promising candidate for integration in a scalable filtration set-up.
Ultrafiltration was addressed with the evaluation of cassette and hollow fiber modules.
The results obtained show that cassette module with cut-offs in the 500 kDa range and highly hydrophilic materials enable complete recovery of infective Adenovirus while reducing process time in half when compared with the best hollow fibers. Despite the encouraging results with
Adenovirus, the experiments using Retrovirus resulted in low yields and possible optimization strategies were identified.
Membrane technology was also evaluated as an alternative to the packed-bed chromatography
columns. By using a scale-down 96-well device, the impact of ligand density, membrane structure and feed conductivity were evaluated for the purification of Adenovirus by ion exchange chromatography. The hydrogel-grafted membrane with ligand density of 2.4 μmol cm-2 operated in bind/elute mode shown the best compromise between yield and purity.
Overall, this thesis contributed to the advancement of virus purification field by exploiting innovative technologies.Fundação para a Ciência e a Tecnologia - (PTDC/EBBBIO/119501/2010
