Process Development and cGMP Manufacturing of a Recombinant Ricin Vaccine: an Effective and Stable Recombinant Ricin A-Chain Vaccine—RVEcTM

Authors

Michael M. Meagher, University of Nebraska-LincolnFollow
Javier G. Seravalli, University of Nebraska-LincolnFollow
S. Todd Swanson, University of Nebraska-Lincoln
Roger G. Ladd, University of Nebraska-Lincoln
Yogender P. Khasa, University of Nebraska-Lincoln
Mehmet Inan, University of Nebraska-LincolnFollow
Jay C. Harner, University of Nebraska-Lincoln
Scott K. Johnson, University of Nebraska-Lincoln
Kevin E. Van Cott, University of Nebraska-LincolnFollow
Changhong Lindsey, United States Army Medical Research Institute of Infectious Diseases
Robert Wannemacher, United States Army Medical Research Institute of Infectious Diseases
Leonard A. Smith, United States Army Medical Research Institute of Infectious DiseasesFollow

Date of this Version

2011

Citation

Biotechnol. Prog., 2011, Vol. 27, No. 4; DOI 10.1002/btpr.631

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^TM) 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^TM at the 40 L scale. The average yield of the final purified bulk RVEc^TM is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEcTM was >99% pure by three HPLC methods and SDS-PAGE. The intact mass and peptide mapping analysis of RVEc^TM confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc^TM was immunoprotective against lethal ricin challenge and elicited neutralizing anti-ricin antibodies in 95–100% of the vaccinated mice.