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

Autor: Dux MP; Novartis Animal Health US Inc., 1447 140[th] Street, Larchwood, IA 51241, USA., Huang J, Barent R, Inan M, Swanson ST, Sinha J, Ross JT, Smith LA, Smith TJ, Henderson I, Meagher MM
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2011 Feb; Vol. 75 (2), pp. 177-85. Date of Electronic Publication: 2010 Oct 07.
DOI: 10.1016/j.pep.2010.09.016
Abstrakt: 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™), 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 5-100mL columns and the process was performed at the pilot scale using 0.6-1.6L 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°C as determined by reverse phase-HPLC and SDS-PAGE and was stable for 24 months at -80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(H(c)) was intact.
(Copyright © 2010 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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