Publication
One step towards the quantification of p53-minicircle DNA
| Summary: | 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. |
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| Main Authors: | Valente, Ana I. |
| Other Authors: | Tavares, Ana P. M.; Sousa, Fani; Freire, Mara G. |
| Subject: | Biopharmaceutical Minicircle DNA Extraction Quantification Ionic Liquids Citotoxicity |
| Year: | 2023 |
| Country: | Portugal |
| Document type: | conference output |
| Access type: | open access |
| Associated institution: | Universidade de Aveiro |
| Language: | English |
| Origin: | RIA - Repositório Institucional da Universidade de Aveiro |
| Summary: | 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. |
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Funded activities
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