Purification and Scale-Up of a Recombinant Heavy Chain Fragment C of Botulinum Neurotoxin Serotype E in Pichia Pastoris GS115

Authors

Michael P. Dux, University of Nebraska - Lincoln
Rick Barent, University of Nebraska - Lincoln
Jayanta Sinha, University of Nebraska - Lincoln
Mark Gouthro, University of Nebraska - Lincoln
Todd Swanson, University of Nebraska - Lincoln
Ardis Barthuli, University of Nebraska - Lincoln
Mehmet Inan, University of Nebraska - LincolnFollow
John T. Ross, DVC, LLC, A CSC Company, 64 Thomas Johnson Drive, Frederick, MD 21702, USA
Leonard A. Smith, United States Army Medical Research Institute of Infectious Diseases, Toxinology Division, Fort Detrick, Fredrick, MDFollow
Theresa J. Smith, United States Army Medical Research Institute of Infectious Diseases, Toxinology Division, Fort Detrick, Fredrick, MD
Robert Webb, United States Army Medical Research Institute of Infectious Diseases, Toxinology Division, Fort Detrick, Fredrick, MD
Bonnie Loveless, United States Army Medical Research Institute of Infectious Diseases, Toxinology Division, Fort Detrick, Fredrick, MD
Ian Henderson, DVC, LLC, A CSC Company, 64 Thomas Johnson Drive, Frederick, MD 21702, USA
Michael M. Meagher, Department of Chemical Engineering, University of Nebraska-LincolnFollow

Date of this Version

2006

Comments

Published in Protein Expression and Purification 45 (2006) 359–367.

Abstract

A recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(H_c), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(H_c) protein was purified from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development at the bench scale was achieved using 7– 380 mL columns and the process was performed at the pilot scale using 0.5–3.1 L columns in preparation for technology transfer to cGMP manufacturing. The purification process resulted in greater than 98% pure rBoNTE(H_c) based on HPLC and yielded up to 1.01 g of rBoNTE(H_c)/kg cells at the bench scale and 580mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(H_c) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD₅₀’s of BoNT/E.