Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 1012-1013; pp. 153 - 161
• Main Authors: Bicho, D., Caramelo-Nunes, C., Sousa, A., Sousa, F., Queiroz, J.A., Tomaz, C.T.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.02.2016
• Subjects: Agmatine; Agmatine - chemistry; Animals; Cell Line; Chromatography; Chromatography, Affinity - methods; Dynamical systems; Dynamics; Fibroblasts; Humans; Influenza; Influenza vaccine; Influenza Vaccines - genetics; Influenza Vaccines - isolation & purification; Influenza virus; Ligands; Monoliths; pDNA; Plasmids - genetics; Plasmids - isolation & purification; Purification; Transfection; Vaccines; Vaccines, DNA - genetics; Vaccines, DNA - isolation & purification; Viruses; Transfection; pDNA; Agmatine; Influenza vaccine; Monoliths; Chromatography
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2015.12.036

•Agmatine monolith allows the sc pDNA isoform purification under two strategies.•The pH manipulation affects the selectivity and retention of the pDNA isoforms.•Increasing pDNA concentration increased the DBC.•High flow rates and high pH had a diminishing effect in the DBC.•pDNA influenza vaccine purity is in accordance with FDA requirements. Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation.