Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(H(c))] expressed in Pichia pastoris.
Protein Expr Purif
; 75(2): 177-85, 2011 Feb.
Article
em En
| MEDLINE
| ID: mdl-20933089
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pichia
/
Toxinas Botulínicas
/
Proteínas Recombinantes
/
Clostridium botulinum
Limite:
Animals
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Estados Unidos