title: Development of a membrane adsorber based capture step for the purification of yellow fever virus.
abstract: Yellow fever (YF) is an endemic disease in some tropical areas of South America and Africa that presents lethality rate between 20 and 50%. There is no specific treatment and to control this disease a highly effective live-attenuated egg based vaccine is widely used for travelers and residents of areas where YF is endemic. However, recent reports of rare, sometimes fatal, adverse events post-vaccination have raised concerns. In order to increase safety records, alternative strategies should be considered, such as developing a new inactivated vaccine using a cell culture based technology, capable of meeting the demands in cases of epidemic. With this goal, the production of YF virus in Vero cells grown on microcarriers and its subsequent purification by chromatographic techniques was studied. In this work we investigate the capture step of the purification process of the YF virus. At first, virus stability was studied over a wide pH range, showing best results for the alkaline region. Considering this result and the pI of the envelope protein previously determined in silico, a strong anion exchanger was considered most suitable. Due to the easy scalability, simplicity to handle, absence of diffusional limitations and suitability for virus handling of membrane adsorbers, a Q membrane was evaluated. The amount of antigen adsorbed onto the membrane was investigated within the pH range for virus stability, and the best pH for virus adsorption was considered to be 8.5. Finally, studies on gradient and step elution allowed to determine the most adequate salt concentration for washing (0.15M) and virus elution (0.30 M). Under these operating conditions, it was shown that this capture step is quite efficient, showing high product recovery (93.2±30.3%) and efficient DNA clearance (0.9±0.3 ng/dose).
authors.affiliation: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil; Federal University of Rio de Janeiro, IQ, Biochemistry Program, Av. Athos da Silveira Ramos 149, Bloco A, 21941-909 Rio de Janeiro/RJ, Brazil.
authors.forename: Tânia P
authors.initials: TP
authors.lastname: Pato
authors.affiliation: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil.
authors.forename: Marta Cristina O
authors.initials: MC
authors.lastname: Souza
authors.affiliation: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil.
authors.forename: Andréa N M R
authors.initials: AN
authors.lastname: Silva
authors.affiliation: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil.
authors.forename: Renata C
authors.initials: RC
authors.lastname: Pereira
authors.affiliation: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil.
authors.forename: Marlon V
authors.initials: MV
authors.lastname: Silva
authors.affiliation: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil.
authors.forename: Elena
authors.initials: E
authors.lastname: Caride
authors.affiliation: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil.
authors.forename: Luciane P
authors.initials: LP
authors.lastname: Gaspar
authors.affiliation: Oswaldo Cruz Foundation (FIOCRUZ), Bio-Manguinhos, Avenida Brasil 4365, 21045-900 Rio de Janeiro/RJ, Brazil.
authors.forename: Marcos S
authors.initials: MS
authors.lastname: Freire
authors.affiliation: Federal University of Rio de Janeiro, COPPE, Cell Culture Engineering Laboratory, Cx. Postal 68502, 21941-972 Rio de Janeiro/RJ, Brazil; Federal University of Rio de Janeiro, IQ, Biochemistry Program, Av. Athos da Silveira Ramos 149, Bloco A, 21941-909 Rio de Janeiro/RJ, Brazil. Electronic address: leda@peq.coppe.ufrj.br.
authors.forename: Leda R
authors.initials: LR
authors.lastname: Castilho
mesh_terms: D000327:Adsorption; D000818:Animals; D002522:Chlorocebus aethiops; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D008566:Membranes / Q000737:chemistry; D014709:Vero Cells; D014776:Virus Cultivation / Q000379:methods*; D015005:Yellow fever virus / Q000302:isolation & purification*