title: Process development and cGMP manufacturing of a recombinant ricin vaccine: an effective and stable recombinant ricin A-chain vaccine-RVEc™.
abstract: Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A-chain (RTA), which is an N-glycosidase enzyme, and the B-chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1-33/44-198 protein, herein denoted RVEa™) expressed in Escherichia coli using a codon-optimized gene was shown to be non-toxic, stable, and protective against a ricin challenge in mice. Here, we describe the process development and scale-up at the 12 L fermentation scale, and the current Good Manufacturing Practice (cGMP)-compliant production of RVEc™ at the 40 L scale. The average yield of the final purified bulk RVEc™ is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEc™ was >99% pure by three HPLC methods and SDS-PAGE. The intact mass and peptide mapping analysis of RVEc™ confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc™ was immunoprotective against lethal ricin challenge and elicited neutralizing anti-ricin antibodies in 95-100% of the vaccinated mice.
authors.affiliation: University of Nebraska-Lincoln Biological Process Development Facility, College of Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0668, USA.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
authors.forename: Javier G
authors.initials: JG
authors.lastname: Seravalli
authors.forename: S Todd
authors.initials: ST
authors.lastname: Swanson
authors.forename: Roger G
authors.initials: RG
authors.lastname: Ladd
authors.forename: Yogender P
authors.initials: YP
authors.lastname: Khasa
authors.forename: Mehmet
authors.initials: M
authors.lastname: Inan
authors.forename: Jay C
authors.initials: JC
authors.lastname: Harner
authors.forename: Scott K
authors.initials: SK
authors.lastname: Johnson
authors.forename: Kevin
authors.initials: K
authors.lastname: Van Cott
authors.forename: Changhong
authors.initials: C
authors.lastname: Lindsey
authors.forename: Robert
authors.initials: R
authors.lastname: Wannemacher
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D007612:Kanamycin / Q000032:analysis; D051379:Mice; D012276:Ricin / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D014614:Vaccines, Synthetic; D007612:Kanamycin; D012276:Ricin