One step towards the quantification of p53-minicircle DNA

• Autor(a) principal: Valente, Ana I.
• Data de Publicação: 2023
• Outros Autores: Tavares, Ana P. M., Sousa, Fani, Freire, Mara G.
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/41783
• topic: Biopharmaceutical; Minicircle DNA; Extraction; Quantification; Ionic Liquids; Citotoxicity

p53-minicircle DNA (mcDNA) is a biopharmaceutical with potential use in the genetic therapy of cancer. Despite the therapeutic relevance of DNA-based products, current clarification and purification steps are complex, resulting in high-cost biopharmaceuticals [1]. The existing strategies for mcDNA isolation are based on chromatographic techniques, such as size-exclusion, hydrophobic interaction, and affinity chromatography. Despite the selectivity achieved with the affinity strategy, it depends on the genetic manipulation of the vector to include specific sequences and/or in the use of enzymes to eliminate impurities [2,3]. Thus, up to date, one of the best strategies resorts to size exclusion chromatography, allowing the recovery of 66% of mcDNA with a purity of 98% [2]. Due to the lack of an efficient purification strategy, a quantification method for mcDNA is non-existent. Considering this, an analytical method based on ion exchange chromatography for mcDNA quantification is under development. Firstly, the p53-mcDNA is produced through the culture of transformed Escherichia coli. Then, the mcDNA is extracted with a commercial kit. The samples obtained after the extraction are applied in developing the new analytic method. Also, an extraction method using ionic liquid (IL)-based aqueous biphasic systems (ABS) is under evaluation for the replacement of the commercial kit used for the purpose. For that, the cytotoxicity of several analogues of glycine-betaine ILs is being also assessed.