title: Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography.
abstract: The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.
authors.affiliation: Departamento de Ingeniería Química y Metalurgia, Universidad de Sonora, Blvd. Luis Encinas y Rosales s/n, 83000, Hermosillo, SON, México.
authors.forename: Diana Ivonne
authors.initials: DI
authors.lastname: Franco-Medrano
authors.affiliation: Departamento de Ingeniería Química y Metalurgia, Universidad de Sonora, Blvd. Luis Encinas y Rosales s/n, 83000, Hermosillo, SON, México. pguerrero@iq.uson.mx.
authors.forename: Patricia
authors.initials: P
authors.lastname: Guerrero-Germán
authors.affiliation: Departamento de Matemáticas, Universidad de Sonora, Hermosillo, SON, México.
authors.forename: Rosa María
authors.initials: RM
authors.lastname: Montesinos-Cisneros
authors.affiliation: Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico NacionalAv. Instituto Politécnico Nacional # 2508, Cd. De, 07360, México, México.
authors.forename: Jaime
authors.initials: J
authors.lastname: Ortega-López
authors.affiliation: Departamento de Investigaciones Científicas y Tecnológicas, Universidad de Sonora, Blvd. Luis Encinas s/n, 83000, Hermosillo, SON, México.
authors.forename: Armando
authors.initials: A
authors.lastname: Tejeda-Mansir
mesh_terms: D000327:Adsorption; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D007218:Industrial Microbiology / Q000379:methods; D007891:Leishmania / Q000378:metabolism*; D046911:Macromolecular Substances; D008567:Membranes, Artificial; D010477:Perfusion; D010957:Plasmids / Q000378:metabolism*; D014612:Vaccines / Q000737:chemistry
chemical_list: D046911:Macromolecular Substances; D008567:Membranes, Artificial; D014612:Vaccines; D004247:DNA
keywords: Leishmaniasis; Perfusion chromatography; Plasmid DNA; Vaccines