Process development and optimization towards the production of a non-antibody scaffold-based biotherapeutic

• Autor(a) principal: Matos, Manuel João de Almeida Albuquerque Brandão
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/50529
• topic: biotechnology; upstream; downstream; nanofitin; pharmaceutical industry; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

In a context of “green” biotechnology the tendency is to increase investment in methods that rely on microrganisms for the production of biological compounds. The use of these methodologies, contrary to those that rely on the use of chemical components, translate into a higher sustainability in the economical, social and environmental sectors. The upstream development is deeply connected with the cultivation of microrganisms expressing all kind of recombinant proteins. Regarding this work, special attention was given to a specific type of recombinant protein, nanofitins. Nanofitins are artificial proteins created through the technique of ribosome display which consists in several cycles of selection with the goal of creating a synthetic protein with affinity to a specific target. The objective in this experimental work was to implement a protocol for the production and purification of a nanofitin at a pilot scale. Regarding upstream analysis, different specific growth rates were tested during fed-batch administration on a 30L bioreactor, as well as operation times and small scale growth conditions were optimized. At upstream it was concluded that the use of a specific growth rate of 0,175h-1 during fed-batch is sufficient to maximize the microbial growth and its biomass production, as well as the use of only one resistance antibiotic, ampicillin, managed to optimize these parameters. At the downstream level different conditions were studied, namely the techniques of ion exchange chromatography and hydrophobic interaction. The technique of tangential flow filtration was also tested, showing that the use of this technique with two cutoffs, 30kDa and 5kDa, followed by a cationic exchange chromatography is the combination of techniques which presented higher productivities with higher purity ratio. To fulfill GMP requirements, the remotion of the content of endotoxins in the final product was also assessed. The use of Sartobind STIC nano successfully removed up to 97% of endotoxins content on the final product.