title: Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115.
abstract: 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(Hc), was produced intracellular in Pichia 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 7-380 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 1.01g of rBoNTE(Hc)/kg cells at the bench scale and 580mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(Hc) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50's of BoNT/E.
authors.affiliation: Biological Process Development Facility, Department of Chemical Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0466, USA.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Dux
authors.forename: Rick
authors.initials: R
authors.lastname: Barent
authors.forename: Jayanta
authors.initials: J
authors.lastname: Sinha
authors.forename: Mark
authors.initials: M
authors.lastname: Gouthro
authors.forename: Todd
authors.initials: T
authors.lastname: Swanson
authors.forename: Ardis
authors.initials: A
authors.lastname: Barthuli
authors.forename: Mehmet
authors.initials: M
authors.lastname: Inan
authors.forename: John T
authors.initials: JT
authors.lastname: Ross
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
authors.forename: Theresa J
authors.initials: TJ
authors.lastname: Smith
authors.forename: Robert
authors.initials: R
authors.lastname: Webb
authors.forename: Bonnie
authors.initials: B
authors.lastname: Loveless
authors.forename: Ian
authors.initials: I
authors.lastname: Henderson
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005260:Female; D005285:Fermentation; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D001905:Botulinum Toxins; C096326:botulinum toxin type E