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

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 (TM)) 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 06-1 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 degrees C as determined by reverse phase-HPLC and SDS-PAGE and was stable for 24 months at -80 degrees C based on mouse potency bioassay N-Terminal amino acid sequencing confirmed that the N terminus of the purified rBoNTC(H-c) was intact (C) 2010 Elsevier Inc All rights reserved