Evaluation of novel chemistries on porous supports for plasmid DNA polishing chromatography

• Autor(a) principal: Luís, Marco Ângelo
• 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/10400.6/13622
• topic: Agmatina; Dna Plasmídico; Isoforma Superenrolada; Suporte de Resina; Terapêuticos de Dna; Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia

DNA-based therapeutics are one of the main applications aiming to find a course of treatment for hereditary diseases as well as cancer. They provide additional benefits when compared to traditional therapeutics. Plasmid DNA (pDNA) is one of the most utilized species of DNA-based therapeutics due to its ability to induce significant expression of a trans-gene and a comparably easy manufacturing process, without any of the disadvantages viral vectors show. This form of DNA presents linear, open circle and supercoiled isoforms, while being possible to switch between them, based on the amount and location of nicks in the sequence. The supercoiled isoform is the most biologically significant one since it allows for higher efficiencies of trans-gene expression. In order to use pDNA for human treatment, it needs to be purified so that the final product consists of a homogeneous supercoiled pDNA pool. To obtain this, a chromatography process with a combination of several interactions presents the best course of action, which is demonstrated by multiple products currently on the market. Nevertheless, the existing supports that are focused on the chromatographic step specific for the isolation of the relevant pDNA isoform (polishing step) do not present the best performance characteristics, both in terms of the purity obtained and the yield. Therefore, the present work aims to develop a novel polishing chromatographic material to produce supercoiled pDNA with higher purity and recovery. To do this, firstly, static binding experiments were conducted to confirm the results previously obtained by another working collaboration. Then, the promising prototypes were subjected to dynamic binding conditions, with the objective of assessing their capabilities to isolate the supercoiled isoform. This was followed by the development of several new agmatine-based prototypes where elution buffer composition and pH were optimized to achieve the highest resolution and purity of supercoiled pDNA. Afterwards, a step-elution method and optimization were conducted with a 3rd generation agmatine prototype, since it presented the best results in terms of supercoiled purity. With the optimized method, a complex sample of RNA and pDNA was applied to the prototype to evaluate if it was capable of separating supercoiled pDNA from other isoforms and similar molecules. The results indicated that the agmatine prototype is efficient in the purification of supercoiled pDNA from complex samples, with a high degree of purification, and in accordance with the specifications of regulatory agencies.