title: Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.
abstract: 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.
authors.affiliation: a Departments of Pediatrics and Molecular Virology and Microbiology; National School of Tropical Medicine; Baylor College of Medicine ; Houston , TX , USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Seid
authors.forename: Elena
authors.initials: E
authors.lastname: Curti
authors.forename: R Mark
authors.initials: RM
authors.lastname: Jones
authors.forename: Elissa
authors.initials: E
authors.lastname: Hudspeth
authors.forename: Wanderson
authors.initials: W
authors.lastname: Rezende
authors.forename: Jeroen
authors.initials: J
authors.lastname: Pollet
authors.forename: Lori
authors.initials: L
authors.lastname: Center
authors.forename: Leroy
authors.initials: L
authors.lastname: Versteeg
authors.forename: Sonya
authors.initials: S
authors.lastname: Pritchard
authors.forename: Konstantin
authors.initials: K
authors.lastname: Musiychuk
authors.forename: Vidadi
authors.initials: V
authors.lastname: Yusibov
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hotez
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Bottazzi
mesh_terms: D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D015870:Gene Expression; D017221:Necator americanus / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D013678:Technology, Pharmaceutical / Q000379:methods; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D000947:Antigens, Helminth; D014614:Vaccines, Synthetic; D016282:Aspartic Acid Endopeptidases; C100354:aspartic proteinase-I
keywords: product development partnership, vaccine, human hookworm, process development, tobacco plant expression, aspartic protease