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 Pichict 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(Hc) 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 7380 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(Hc) based on HPLC and yielded up to 1.01 g of rBoNTE(H-c)/kg cells at the bench scale and 580 mg 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. (c) 2005 Elsevier Inc. All rights reserved.