Rapid quantification of supercoiled plasmid deoxyribonucleic acid using a monolithic ion exchanger

Mota, Élia; Sousa, Ângela; Černigoj, Urh; Queiroz, João A.; Tomaz, Cândida T.; Sousa, Fani

doi:10.1016/j.chroma.2013.03.070

Cited by 23 publications

(15 citation statements)

References 25 publications

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“…The purity and recovery yield of the sc pDNA was evaluated by an analytical method based on ion exchange chromatography using the commercial analytical monolith CIMac TM (BIA Separations) and as previously described [20]. To use this method, a calibration curve was first constructed in the pDNA concentration range of 1-25 µg/mL by considering the peak area corresponding to sc pDNA standards.…”

Section: Plasmid Quantification By Analytical Chromatographymentioning

confidence: 99%

Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

et al. 2019

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The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.

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Section: Plasmid Quantification By Analytical Chromatographymentioning

confidence: 99%

Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

et al. 2019

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“…Polyacrylate/methacrylate-based sorbents have been widely used for DNA isolation due to their high specific surface area, suitable hydrophilicity and functional groups that can create effective reversible interaction with DNA [14][15][16][17][18][19]. Methacrylate based monolith was prepared by bearing diethylaminoethyl (DEAE) and butyl groups and used as a sorbent for selective extraction of DNA [20].…”

Section: Introductionmentioning

confidence: 99%

Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation

Günal

Kip

Öğüt

et al. 2017

Artificial Cells, Nanomedicine, and Biotechnology

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Monodisperse silica microspheres with bimodal pore-size distribution were proposed as a high performance sorbent for DNA isolation in batch fashion under equilibrium conditions. The proposed sorbent including both macroporous and mesoporous compartments was synthesized 5.1 μm in-size, by a "staged shape templated hydrolysis and condensation method". Hydrophilic polymer based sorbents were also obtained in the form of monodisperse-macroporous microspheres ca 5.5 μm in size, with different functionalities, by a developed "multi-stage microsuspension copolymerization" technique. The batch DNA isolation performance of proposed material was comparatively investigated using polymer based sorbents with similar morphologies. Among all sorbents tried, the best DNA isolation performance was achieved with the monodisperse silica microspheres with bimodal pore size distribution. The collocation of interconnected mesoporous and macroporous compartments within the monodisperse silica microspheres provided a high surface area and reduced the intraparticular mass transfer resistance and made easier both the adsorption and desorption of DNA. Among the polymer based sorbents, higher DNA isolation yields were achieved with the monodisperse-macroporous polymer microspheres carrying trimethoxysilyl and quaternary ammonium functionalities. However, batch DNA isolation performances of polymer based sorbents were significantly lower with respect to the silica microspheres.

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“…Recent chromatographic procedures related to purification of pDNA vaccines include anion exchange chromatography, affinity chromatography, multimodal chromatography, size exclusion and hydrophobic interaction chromatography [ 97 , 99 , 100 , 101 , 102 ].…”

Section: Analytical Approachesmentioning

confidence: 99%

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles

et al. 2021

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In the quest for a formidable weapon against the SARS-CoV-2 pandemic, mRNA therapeutics have stolen the spotlight. mRNA vaccines are a prime example of the benefits of mRNA approaches towards a broad array of clinical entities and druggable targets. Amongst these benefits is the rapid cycle “from design to production” of an mRNA product compared to their peptide counterparts, the mutability of the production line should another target be chosen, the side-stepping of safety issues posed by DNA therapeutics being permanently integrated into the transfected cell’s genome and the controlled precision over the translated peptides. Furthermore, mRNA applications are versatile: apart from vaccines it can be used as a replacement therapy, even to create chimeric antigen receptor T-cells or reprogram somatic cells. Still, the sudden global demand for mRNA has highlighted the shortcomings in its industrial production as well as its formulation, efficacy and applicability. Continuous, smart mRNA manufacturing 4.0 technologies have been recently proposed to address such challenges. In this work, we examine the lab and upscaled production of mRNA therapeutics, the mRNA modifications proposed that increase its efficacy and lower its immunogenicity, the vectors available for delivery and the stability considerations concerning long-term storage.

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Rapid quantification of supercoiled plasmid deoxyribonucleic acid using a monolithic ion exchanger

Cited by 23 publications

References 25 publications

Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles

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