A baseline process for the production, recovery, and purification of bacterial influenza vaccine candidates

作者：Sanchez Arreola Pamela B; Lopez Uriarte Salvador; Marichal Gallardo Pavel A; Gonzalez Vazquez Juan C; Perez Chavarria Roberto; Soto Vazquez Pedro; Lopez Pacheco Felipe; Ramirez Medrano Alicia; Rocha Pizana Maria R; Alvarez Mario M^*

来源：Biotechnology Progress, 2013, 29(4): 896-908.

DOI：10.1002/btpr.1749

• vaccine production
• Influenza
• Escherichia coli
• bioreactors
• affinity chromatography
• bioseparation

The current commercial system for influenza vaccine production depends on the culture of virus in embryonated eggsa 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 4.6x10(3) doses L(-3)day(-1) is feasible.

• 出版日期2013-7