title: Scale-up of the fermentation and purification of the recombinant heavy chain fragment C of botulinum neurotoxin serotype F, expressed in Pichia pastoris.
abstract: A recombinant heavy chain fragment C of botulinum neurotoxin serotype F (BoNTF(Hc)) has been expressed in Pichia pastoris for use as an antigen in a proposed human vaccine. P. pastoris cells were grown using glycerol batch, glycerol fed-batch, and methanol fed-batch methods to achieve high cell densities. The total cellular protein recovered after homogenization was 72 mg/g of cell paste. BoNTF(Hc) was purified from soluble Pichia cell lysate employing ion-exchange chromatographic (IEC) and hydrophobic interaction chromatographic (HIC) methods developed at the bench scale using 10-100 mL columns. The process was performed at the pilot scale using 1-4L columns for evaluation of scale up. The purification process resulted in greater than 98% pure product consisting of at least three forms of BoNTF(Hc) based on mass spectrometry and yielded up to 205 mg/kg cells at the bench scale and 170 mg/kg cells at the pilot scale. Full-length BoNTF(Hc) is present based on mass spectrometry and SDS-PAGE, however is postulated to be N-terminally blocked by acetylation. N-terminal sequencing showed that two of the three forms are missing the first 11 (80%) and 14 (20%) amino acids of the N-terminus from the full-length form. The ratios of the two clipped forms were consistent from the bench to pilot scales. Purified BoNTF(Hc) at the pilot scale was found to sufficiently protect mice against a high dose of BoNTF neurotoxin.
authors.affiliation: Biological Process and Development Facility, University of Nebraska-Lincoln, Lincoln, NE 68583-0919, USA.
authors.forename: Scott K
authors.initials: SK
authors.lastname: Johnson
authors.forename: Wenhui
authors.initials: W
authors.lastname: Zhang
authors.forename: Leonard A
authors.initials: LA
authors.lastname: Smith
authors.forename: Karen J
authors.initials: KJ
authors.lastname: Hywood-Potter
authors.forename: S
authors.initials: S
authors.lastname: Todd Swanson
authors.forename: Vicki L
authors.initials: VL
authors.lastname: Schlegel
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D007928:Lethal Dose 50; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D010865:Pilot Projects; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D015996:Survival Rate; D014611:Vaccination
chemical_list: D011994:Recombinant Proteins; D001905:Botulinum Toxins; C096321:botulinum toxin type F