title: A baseline process for the production, recovery, and purification of bacterial influenza vaccine candidates.
abstract: The current commercial system for influenza vaccine production depends on the culture of virus in embryonated eggs--a strategy that is both costly and poorly scalable. Consequently, a sudden pandemic event with a demand for millions of vaccine doses in a short time could readily overwhelm the available world production capacity. In this communication, we present a process that uses Escherichia coli for scalable production of recombinant vaccine candidates against influenza. A monomeric and a dimeric fragment of hemagglutinin of the influenza A H1N1/2009 virus were successfully expressed in a BL21 (DE3) pLysS variety of C41 E. coli. We present results from batch processes where induction is made with isopropyl thiogalactoside and from fed-batch experiments where expression is induced using lactose/glucose pulses. Concentrations in the range of 1.188-0.605 g/L of recombinant protein were observed in 2-L stirred tank bioreactors. The genetic construct included an N-terminal histidine tag sequence that facilitated recovery, purification, and proper refolding of the vaccine candidate by affinity chromatography in columns loaded with Ni(+2) . The proteins produced by this strategy selectively and specifically recognizes antibodies from patients diagnosed as positive to influenza A H1N1/2009. Overall protein recovery yields between 30.0 and 34.7% were typically observed. Based on these yields, a production of 4.6 × 10(3) doses L(-3) day(-1) is feasible.
authors.affiliation: Centro de Biotecnología-FEMSA, Tecnológico de Monterrey at Monterrey, Monterrey, N.L. México, C.P. 64849.
authors.forename: Pamela B
authors.initials: PB
authors.lastname: Sánchez-Arreola
authors.forename: Salvador
authors.initials: S
authors.lastname: López-Uriarte
authors.forename: Pável A
authors.initials: PA
authors.lastname: Marichal-Gallardo
authors.forename: Juan C
authors.initials: JC
authors.lastname: González-Vázquez
authors.forename: Roberto
authors.initials: R
authors.lastname: Pérez-Chavarría
authors.forename: Pedro
authors.initials: P
authors.lastname: Soto-Vázquez
authors.forename: Felipe
authors.initials: F
authors.lastname: López-Pacheco
authors.forename: Alicia
authors.initials: A
authors.lastname: Ramírez-Medrano
authors.forename: María R
authors.initials: MR
authors.lastname: Rocha-Pizaña
authors.forename: Mario M
authors.initials: MM
authors.lastname: Alvarez
mesh_terms: D019149:Bioreactors; D003001:Cloning, Molecular; D006388:Hemagglutinins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007252:Influenza Vaccines / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D007251:Influenza, Human / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D006388:Hemagglutinins; D007252:Influenza Vaccines; D011994:Recombinant Proteins
keywords: Escherichia coli; Influenza; affinity chromatography; bioreactors; bioseparation; vaccine production