title: Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith.
abstract: 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.
authors.affiliation: CICS-UBI- Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: D
authors.initials: D
authors.lastname: Bicho
authors.affiliation: CICS-UBI- Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Caramelo-Nunes
authors.affiliation: CICS-UBI- Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: A
authors.initials: A
authors.lastname: Sousa
authors.affiliation: CICS-UBI- Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: F
authors.initials: F
authors.lastname: Sousa
authors.affiliation: CICS-UBI- Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal.
authors.forename: J A
authors.initials: JA
authors.lastname: Queiroz
authors.affiliation: CICS-UBI- Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal; Department of Chemistry, University of Beira Interior, Rua Marquês d'Ávila e Bolama, 6201-001 Covilhã, Portugal. Electronic address: ctomaz@ubi.pt.
authors.forename: C T
authors.initials: CT
authors.lastname: Tomaz
mesh_terms: D000376:Agmatine / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D005347:Fibroblasts; D006801:Humans; D007252:Influenza Vaccines / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection; D019444:Vaccines, DNA / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D007252:Influenza Vaccines; D019444:Vaccines, DNA; D000376:Agmatine
keywords: Agmatine; Chromatography; Influenza vaccine; Monoliths; Transfection; pDNA