Effects of process factors on the performance of electrochemical disinfection for wastewater in a continuous-flow cell reactor.

Although electrochemical disinfection has been shown to be an effective approach to inactivate bacteria in saline water, the effects of process parameters and reactor design for its application in low-salinity water have not been well understood. In this study, factorial experiments were performed to investigate the direct and confounded effects of applied current (5-20 mA), contact time (2.5-20 min), anode surface area (185-370 cm2), and chloride concentration (50-400 mg L-1) on the disinfection efficiency in fresh water and the secondary effluent of municipal wastewater. An electrochemical disinfection reactor cell with an internal volume of 75 cm3 was designed and fabricated. Residence time distribution analysis showed that the internal mixing of the reactor is similar to that of a dispersed plug-flow reactor. All studied process parameters showed significant effect on the kill efficiency, with the applied current and contact time having the most dominant effect. Although the effect of chloride concentration, which is responsible for electrochemical production of free chlorine in water, is statistically significant, it is not as prominent as those reported for high salinity water. A synergistic effect between chloride concentration and anode surface area was identified, leading to high kill efficiency (99.9%, 3 log kill) at low current density (0.0135 mA cm-2). Response surface modeling results suggested that a scaled-up disinfection reactor can be designed using large anode surface area with long contact time for high chloride water (400 mg L-1) or high current density with short contact time for low chloride water (50 mg L-1). The power requirement of a portable system treating 37.85 m3 day-1 (10,000 gpd) of municipal wastewater was estimated to be 1.9 to 8.3 kW to achieve a 3 log kill, depending on the reactor design.

Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

Optimization and revision of the production process of the Necator americanus glutathione S-transferase 1 (Na-GST-1), the lead hookworm vaccine recombinant protein candidate.

Infection by the human hookworm Necator americanus is a leading cause of anemia and disability in the developing countries of Africa, Asia, and the Americas. In order to prevent childhood hookworm disease in resource poor settings, a recombinant vaccine is under development by the Sabin Vaccine Institute and Texas Children's Hospital Center for Vaccine Development, a Product Development Partnership (PDP). Previously, we reported on the expression and purification of a highly promising hookworm vaccine candidate, Na-GST-1, an N. americanus glutathione s-transferase expressed in Pichia pastoris (yeast), which led to production of 1.5 g of 95% pure recombinant protein at a 20L scale. (1) (,) (2) (,) (3) This yield and purity of Na-GST-1 was sufficient for early pilot manufacturing and initial phase 1 clinical testing. However, based on the number of doses which would be required to allow mass vaccination and a potential goal to deliver a vaccine as inexpensively as possible, a higher yield of expression of the recombinant antigen at the lowest possible cost is highly desirable. Here we report on modifications to the fermentation (upstream process) of the antigen expressed in P. pastoris, and to the purification (downstream process) of the recombinant protein that allowed for a 2-3-fold improvement in the final yield of Na-GST-1 purified protein. The major improvements included upstream process changes such as the addition of a sorbitol pulse and co-feed during methanol induction as well as an extension of the induction stage to approximately 96 hours; downstream process changes included modifying the UFDF to flat sheet with a 10 kDa Molecular Weight cut-off (MWCO), adjusting the capacity of an ion-exchange chromatography step utilizing a gradient elution as opposed to the original step elution, and altering the hydrophobic interaction chromatography conditions. The full process, as well as the purity and stability profiles of the target Na-GST-1, and its formulation on Alhydrogel(®), is described.