Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(H_c)] expressed in Pichia pastoris

Authors

Michael P. Dux, Novartis Animal Health US Inc.
Jicai Huang, Nektar Therapeutics
Rick Barent, Intervet
Mehmet Inan, Alder PharmaceuticalsFollow
S. Todd Swanson, University of Nebraska-Lincoln
Jayanta Sinha, University of Nebraska-Lincoln
John T. Ross, DynPort Vaccine Company (DVC) LLC
Leonard A. Smith, United States Army Medical Research and Materiel CommandFollow
Theresa J. Smith, United States Army Medical Research Institute of Infectious Diseases (USAMRIID)
Ian Henderson, Emergent Product Development Gaithersburg Inc.
Michael M. Meagher, University of Nebraska - LincolnFollow

Date of this Version

2011

Comments

Published in Protein Expression and Purification, 75, (2011), 177–185

Abstract

A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(H_c)], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(H_c) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(H_c) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel™), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5–100 mL columns and the process was performed at the pilot scale using 0.6–1.6 L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(H_c)/kg wet cell - weight (WCW) at the bench scale and 1.6 g rBoNTC(H_c)/kg WCW at the pilot scale. The purified rBoNTC(H_c) was stable for at least 3 months at 5 and -80 °C as determined by reverse phase-HPLC and SDS–PAGE and was stable for 24 months at -80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(H_c) was intact.