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

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. (c) 2006 Elsevier B.V. All rights reserved.