Accelerating Complex Biopharmaceuticals Manufactoring

• Autor(a) principal: Ruiz, Afonso de Brito
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/160882
• topic: affinity chromatography; biolayer interferometry; biopharmaceuticals; downstream processing; extracellular vesicles; lentiviral vectors; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Extracellular vesicles (EV) are phospholipid involucrum of cytosol, secreted by cells, implicated in inter-cellular communication, and enacting important roles in physiological and pathological processes. It is speculated they should give rise to a new class of gene therapy products, with clinical trials already under way. Viral vectors, on the other hand, are already used in gene and oncolytic therapy, as well as, for vaccines. Yet, the manufacturing process of such biopharmaceuticals is challenged by their complexity and heterogeneity. For EV as for lentiviral vectors (LV) the establishment of scalable and compatible with good manufacturing practices (cGMP) downstream processes (DSP) is still ongoing. The similarity between EV’s and LV’s chemical/physical properties inspired, in the present work, the devise of an affinity chromatography (AC) technology with two applications: one aimed at capturing EV; the other focused on the removal of product related impurities from LV feedstocks. Concomitantly, analytical tools based on fluorescence NTA (F-NTA) and biolayer interferometry (BLI) were developed. Using a CD81 ligand, intermediate scale experiments demonstrated the enrichment in EV, for which ~30% recoveries were obtained using vesicles from HEK293 and hiPSC. F-NTA proved to be unsuited for estimating the ratio of EV per total particles (TP), yet BLI showed potential for phenotype specific EV quantification, with <30% deviation from Interferometric Reflectance Imaging Sensor (SP-IRIS) results. Regarding LV purification, in a small-scale screening study, a 4-fold reduction in the ration between TP and transduction units (TU) was observed with CD81 and CD63 ligands. Furthermore, decrease in the TP/TU ratio was observed when utilizing purified material as load for AC, showcasing it could be added as a polishing step to current DSP processes. Altogether, this thesis contributed to the development of specific and efficient purification technologies which could potentiate the advent of EV-based gene therapies and/or complement current LV manufacturing pipelines.