Smart macroporous structures for the purification of viral particles

Dissertação para obtenção do Grau de Mestre em Engenharia Química e BioquímicaThe increasing application of viral particles in vaccination and gene-based therapies, has led to the development of alternative and improved purification processes. Traditional purification methods include chromatographic techniques, however the chromatographic matrices used present limitations specially when aimed at the purification of large molecules. This work presents the preparation of chitosan-based monoliths using clean processes and easy functionalization techniques intending to improve Adenovirus serotype 5 (Ad5) purification. Monoliths were prepared by blending chitosan (CHT) with glycidylmethacrylate (GMA) or poly(vinyl alcohol) (PVA), using two preparation techniques, freeze-drying and a scCO2 – assisted drying process, and were subsequently functionalized with Q ligands by three different methods. In addition, monoliths blended with magnetic nanoparticles were also prepared using the same strategies to confer them a controlled magnetic response. The monoliths produced were characterized in terms of ligand immobilization yield, and evaluated for Ad5 purification. Two types of monoliths showed potential: the CHT/PVA(50:50) prepared by freeze drying and functionalized by the alternative plasma technique (M2) and the CHT/PVA(50:50) 7% monolith prepared by scCO2 – assisted drying process and functionalized by the epoxyactivation technique (M1). The amount of ligand Q immobilized on the supports was monitored by titration assays, among which the CHT/PVA(50:50) 7% M2 prepared by scCO2 – assisted drying process exhibited the highest immobilization yield (91%). Among the results for Ad5 purification, the CHT/PVA(50:50)M2 and the CHT/PVA(50:50)7% M1 resulted in a 40% and 14% of the viral particles, respectively. Protein-binding assays were conducted using bovine serum albumin (BSA) and lysozyme, to evaluate the anionic-exchange capacity of the supports. The results make us believe in the potential of the produced monoliths to be applied in chromatographic techniques. However further improvements are necessary to enhance virus binding and recovery, to obtain an improved purification process.project PTDC/EBB-BIO/118317/201