Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H_c): Antigen E) by Pichia pastoris

Authors

Jayanta Sinha, University of Nebraska-Lincoln
Mehmet Inan, University of Nebraska-LincolnFollow
Sarah Fanders, University of Nebraska-Lincoln
Shinichi Taoka, University of Nebraska-LincolnFollow
Mark Gouthro, University of Nebraska-Lincoln
Todd Swanson, University of Nebraska-Lincoln
Rick Barent, University of Nebraska-Lincoln
Ardis Barthuli, University of Nebraska-Lincoln
Bonnie M. Loveless, United States Army Medical Research Institute of Infectious Diseases, Toxicology Division, Fort Detrick, Fredrick, MD
Leonard A. Smith, United States Army Medical Research Institute of Infectious Diseases, Toxicology Division, Fort Detrick, Fredrick, MDFollow
Theresa Smith, United States Army Medical Research Institute of Infectious Diseases, Toxicology Division, Fort Detrick, Fredrick, MD
Ian Henderson, DynPort Vaccine Company LLC, Frederick, MD
John Ross, DynPort Vaccine Company LLC, Frederick, MD
Michael M. Meagher, University of Nebraska-LincolnFollow

Date of this Version

2007

Comments

Published in Journal of Biotechnology 127 (2007), pp. 462–474. doi:10.1016/j.jbiotec.2006.07.022 This article is a U.S. government work and is not subject to copyright in the United States.

Abstract

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H_c)in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H_c) gene inserted into pHILD4 Escherichia coli—P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H_c) sequence. Expression of rBoNTE(H_c) from the Mut⁺ HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H_c). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H_c) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H_c) per gram wet cell mass as determined by HPLC and Western blot analysis.